Diagnosis of Tuberculosis

an update DrTVRao MD

DrTVRao MD 1

Testing for TB Infectionbull Tuberculosis (TB) is a disease that is

spread through the air from one person to another There are two kinds of tests that are used to determine if a person has been infected with TB bacteria the tuberculin skin test and other direct and indirect Tests

DrTVRao MD 2

Diagnosis of TB Diseasebull People suspected of having TB disease should

be referred for a medical evaluation which will include

Medical history Physical examination Test for TB infection (TB skin test or TB blood test)bull Chest radiograph (X-ray) andbull Appropriate laboratory tests

DrTVRao MD 3

Who Should Get Tested for TBbull TB tests are generally not needed for people

with a low risk of infection with TB bacteriabull Certain people should be tested for TB

bacteria because they are more likely to get TB disease including

bull People who have spent time with someone who has TB disease People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 4

Who Should Get Tested for TB

bull People who have spent time with someone who has TB disease

bull People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 5

Who Should Get Tested for TBbull People from a country where TB disease is

common (most countries in Latin America the Caribbean Africa Asia Eastern Europe and Russia)

bull People who live or work somewhere in the United States where TB disease is more common (homeless shelters prison or jails or some nursing homes)

bull People who use illegal drugsDrTVRao MD 6

X ray of Chest a Commonly used Diagnostic tool

bull Acute pulmonary TB can be easily seen on an X-ray However the picture it presents is not specific and a normal chest X-ray cannot exclude extrapulmonary TB Also in countries where resources are more limited there is often a lack of X-ray facilitiesDrTVRao MD 7

Other testsbull Other tests such

as a chest x-ray and a sample of sputum are needed to see whether the person has TB disease

DrTVRao MD 8

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Testing for TB Infectionbull Tuberculosis (TB) is a disease that is

spread through the air from one person to another There are two kinds of tests that are used to determine if a person has been infected with TB bacteria the tuberculin skin test and other direct and indirect Tests

DrTVRao MD 2

Diagnosis of TB Diseasebull People suspected of having TB disease should

be referred for a medical evaluation which will include

Medical history Physical examination Test for TB infection (TB skin test or TB blood test)bull Chest radiograph (X-ray) andbull Appropriate laboratory tests

DrTVRao MD 3

Who Should Get Tested for TBbull TB tests are generally not needed for people

with a low risk of infection with TB bacteriabull Certain people should be tested for TB

bacteria because they are more likely to get TB disease including

bull People who have spent time with someone who has TB disease People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 4

Who Should Get Tested for TB

bull People who have spent time with someone who has TB disease

bull People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 5

Who Should Get Tested for TBbull People from a country where TB disease is

common (most countries in Latin America the Caribbean Africa Asia Eastern Europe and Russia)

bull People who live or work somewhere in the United States where TB disease is more common (homeless shelters prison or jails or some nursing homes)

bull People who use illegal drugsDrTVRao MD 6

X ray of Chest a Commonly used Diagnostic tool

bull Acute pulmonary TB can be easily seen on an X-ray However the picture it presents is not specific and a normal chest X-ray cannot exclude extrapulmonary TB Also in countries where resources are more limited there is often a lack of X-ray facilitiesDrTVRao MD 7

Other testsbull Other tests such

as a chest x-ray and a sample of sputum are needed to see whether the person has TB disease

DrTVRao MD 8

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Diagnosis of TB Diseasebull People suspected of having TB disease should

be referred for a medical evaluation which will include

Medical history Physical examination Test for TB infection (TB skin test or TB blood test)bull Chest radiograph (X-ray) andbull Appropriate laboratory tests

DrTVRao MD 3

Who Should Get Tested for TBbull TB tests are generally not needed for people

with a low risk of infection with TB bacteriabull Certain people should be tested for TB

bacteria because they are more likely to get TB disease including

bull People who have spent time with someone who has TB disease People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 4

Who Should Get Tested for TB

bull People who have spent time with someone who has TB disease

bull People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 5

Who Should Get Tested for TBbull People from a country where TB disease is

common (most countries in Latin America the Caribbean Africa Asia Eastern Europe and Russia)

bull People who live or work somewhere in the United States where TB disease is more common (homeless shelters prison or jails or some nursing homes)

bull People who use illegal drugsDrTVRao MD 6

X ray of Chest a Commonly used Diagnostic tool

bull Acute pulmonary TB can be easily seen on an X-ray However the picture it presents is not specific and a normal chest X-ray cannot exclude extrapulmonary TB Also in countries where resources are more limited there is often a lack of X-ray facilitiesDrTVRao MD 7

Other testsbull Other tests such

as a chest x-ray and a sample of sputum are needed to see whether the person has TB disease

DrTVRao MD 8

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Who Should Get Tested for TBbull TB tests are generally not needed for people

with a low risk of infection with TB bacteriabull Certain people should be tested for TB

bacteria because they are more likely to get TB disease including

bull People who have spent time with someone who has TB disease People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 4

Who Should Get Tested for TB

bull People who have spent time with someone who has TB disease

bull People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 5

Who Should Get Tested for TBbull People from a country where TB disease is

common (most countries in Latin America the Caribbean Africa Asia Eastern Europe and Russia)

bull People who live or work somewhere in the United States where TB disease is more common (homeless shelters prison or jails or some nursing homes)

bull People who use illegal drugsDrTVRao MD 6

X ray of Chest a Commonly used Diagnostic tool

bull Acute pulmonary TB can be easily seen on an X-ray However the picture it presents is not specific and a normal chest X-ray cannot exclude extrapulmonary TB Also in countries where resources are more limited there is often a lack of X-ray facilitiesDrTVRao MD 7

Other testsbull Other tests such

as a chest x-ray and a sample of sputum are needed to see whether the person has TB disease

DrTVRao MD 8

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Who Should Get Tested for TB

bull People who have spent time with someone who has TB disease

bull People with HIV infection or another medical problem that weakens the immune system

bull People who have symptoms of TB disease (fever night sweats cough and weight loss)

DrTVRao MD 5

Who Should Get Tested for TBbull People from a country where TB disease is

common (most countries in Latin America the Caribbean Africa Asia Eastern Europe and Russia)

bull People who live or work somewhere in the United States where TB disease is more common (homeless shelters prison or jails or some nursing homes)

bull People who use illegal drugsDrTVRao MD 6

X ray of Chest a Commonly used Diagnostic tool

bull Acute pulmonary TB can be easily seen on an X-ray However the picture it presents is not specific and a normal chest X-ray cannot exclude extrapulmonary TB Also in countries where resources are more limited there is often a lack of X-ray facilitiesDrTVRao MD 7

Other testsbull Other tests such

as a chest x-ray and a sample of sputum are needed to see whether the person has TB disease

DrTVRao MD 8

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Who Should Get Tested for TBbull People from a country where TB disease is

common (most countries in Latin America the Caribbean Africa Asia Eastern Europe and Russia)

bull People who live or work somewhere in the United States where TB disease is more common (homeless shelters prison or jails or some nursing homes)

bull People who use illegal drugsDrTVRao MD 6

X ray of Chest a Commonly used Diagnostic tool

bull Acute pulmonary TB can be easily seen on an X-ray However the picture it presents is not specific and a normal chest X-ray cannot exclude extrapulmonary TB Also in countries where resources are more limited there is often a lack of X-ray facilitiesDrTVRao MD 7

Other testsbull Other tests such

as a chest x-ray and a sample of sputum are needed to see whether the person has TB disease

DrTVRao MD 8

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

X ray of Chest a Commonly used Diagnostic tool

bull Acute pulmonary TB can be easily seen on an X-ray However the picture it presents is not specific and a normal chest X-ray cannot exclude extrapulmonary TB Also in countries where resources are more limited there is often a lack of X-ray facilitiesDrTVRao MD 7

Other testsbull Other tests such

as a chest x-ray and a sample of sputum are needed to see whether the person has TB disease

DrTVRao MD 8

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Other testsbull Other tests such

as a chest x-ray and a sample of sputum are needed to see whether the person has TB disease

DrTVRao MD 8

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Types of specimens-Sputum- BAL-Pleural effusions- Urine- Stool-CSF-Aspiration ( gastric ndash cold abscess)- Blood in case of haematogenous TB

DrTVRao MD 9

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFB

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Sputum Collectionbull Sputum specimens

are essential to confirm TBndash Specimens should be

from lung secretions not saliva

bull Collect 3 specimens on 3 different days

bull Spontaneous morning sputum more desirable than induced specimens

bull Collect sputum before treatment is initiated

11

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Interpretation of sputum stained by Z N Stain (WHO )

More than 10 bacilli field ------- +++From 1 ndash 10 bacilli field ------- ++From 10 ndash 99 bacilli 100 fields ----- +From 1 -9 bacilli100 fields ------ write the noNo bacilli seen ---------- negative

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by auramine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Acid Fast Bacilli under Fluorescent Microscope

DrTVRao MD 14

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Culturebull Used to confirm

diagnosis of TBbull Culture all

specimens even if smear is negative

bull Initial drug isolate should be used to determine drug susceptibility

15

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Laboratory Diagnosis1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB

Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons

may not produce cavities amp sputum containing AFBDrTVRao MD 16

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

2- Detecting AFB by fluorochrome stain using fluorescence microscopy

The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope

Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

DrTVRao MD 17

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

LowensteinndashJensen mediumbull When grown on LJ

medium M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis DrTVRao MD 18

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media

with addition of salts 5 glycerol Malachite green

Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100

ml) - Can differentiate between TB complex amp

NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 19

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Recent Methods for DiagnosisI ndashBACTEC 460 ( rapid radiometric

culture system ) specimens are cultured in a liquid medium

(Middle brook7H9 broth base )containing C14 ndash labelled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base with OADC enrichment amp PANTA antibiotic mixture

All types of clinical specimens pulmonary as well as extra-pulmonary ( except blood) could be cultured on this type of media

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

The MGIT 960 System The MGIT 960 system is a non-radiometeric

automated system that uses the MGIT media amp sensors to detect the fluorescence

Advantages -The system holds 960 plastic tubes which are

continuously monitored- Early detection as the machine monitoring amp

reading the tubes every hour

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Detection and identification of mycobacteria directly

from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB

DrTVRao MD 23

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown

Polymerase Chain Reaction (PCR)

DrTVRao MD 24

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens

Quantiferon-GOLD

DrTVRao MD 26

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

What Positive Skin Test ( Tuberculin Test means) bull A positive TB skin test

or TB blood test only tells that a person has been infected with TB bacteria It does not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease

DrTVRao MD 27

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Tuberculin TestInterpretation A positive test indicates previous exposure and carriage

of TB A negative tuberculin test excludes infection in

suspected persons Tuberculin positive persons may develop reactivation

type of TB Tuberculin negative persons are at risk of gaining new

infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria

DrTVRao MD 28

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

False negative reactions may be due to -bull False negative reactions may be due to

- Sever tuberculosis infection (Miliary TB) - Hodgkinrsquos disease bull Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no

exposure historybull Positive test must be regarded suspicious

DrTVRao MD 29

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Tuberculin Test( Mantoux Test )

bull Test to be interpreted in relation to clinical evaluation

bull Even the induration of 5 mm to be considered positive when tested on HIV patients

bull Lacks specificity

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Positive Test Means bull This means the personrsquos

body was infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 31

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Negative Test Means bull This means the

personrsquos body did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 32

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Tuberculin Testing - Limitations

bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -

Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -

AIDSbull Children below 5 years of age with no exposure

historybull - Positive test must be regarded suspicious

DrTVRao MD 33

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )

Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture

Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument

The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium

DrTVRao MD 34

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

TB blood testsbull TB blood tests (also called interferon-gamma

release assays or IGRAs) measure how the immune system reacts to the bacteria that cause TB An IGRA measures how strong a personrsquos immune system reacts to TB bacteria by testing the personrsquos blood in a laboratory

bull Two IGRAs are approved by the US Food and Drug Administration (FDA) and are available in the United States

DrTVRao MD 35

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Positive IGRAbull This means that the

person has been infected with TB bacteria Additional tests are needed to determine if the person has latent TB infection or TB disease A health care worker will then provide treatment as needed

DrTVRao MD 36

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Negative IGRAbull This means that

the personrsquos blood did not react to the test and that latent TB infection or TB disease is not likely

DrTVRao MD 37

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

IGRAs are the preferred method of TB infection testing for the following

People who have received bacilli ChalmettendashGueacuterin (BCG) BCG is a vaccine for TB disease People who have a difficult time returning for a second appointment to look for a reaction to the TSTThere is no problem with repeated IGRAs

DrTVRao MD 38

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

III Polymerase Chain Reaction (PCR) amp Gene probe

Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells

Advantages - Rapid procedure - High sensitivity (1-10 ( 3 ndash 4 hours) bacilli ml sputum)

DrTVRao MD 39

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Molecular DR testing is particularly useful for

bull Patients suspected or at high risk of having drug-resistant TBvery ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 40

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Detection of MDR Tuberculosis

bull Very ill patients for whom drug-susceptibility information might alter case management decisions such as patients who do not get better while taking standard first-line therapy

DrTVRao MD 41

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Detection of MDR Tuberculosis

bull outbreak or contact investigations when drug resistance is suspected in the source case or in some severely immunocompromised persons such as HIV-infected persons or those receiving dialysis in which knowledge of drug-susceptibility would be a significant benefit and affect preventive therapy decisions

DrTVRao MD 42

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Tuberculosis and HIV infection

bull HIV association has become a threat to the developed countries too

bull HIV association will lead to rapid spread of tuberculosis

DrTVRao MD 43

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

HIV Considerationsbull HIV is the strongest risk factor for

progression to active diseasebull HIV kills CD4+ T Helper cells which

normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with

rifampin

DrTVRao MD 44

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is

effectedbull 15 million suffer the diseasebull 3 million are highly infective

DrTVRao MD 45

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

MDR tuberculosisbull Multidrug resistant tuberculosis has become a

global threatbull In 1993 WHO declared Tuberculosis a Global

emergencybull Animals shed the bacilli in Milk humanrsquos get

infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of

Bovine tuberculosis

DrTVRao MD 46

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

Why Tuberculosis continues to be Important

bull Someone in the world is newly infected with TB bacilli every second

bull Overall one-third of the worlds population is currently infected with the TB bacillus

bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB

DrTVRao MD 47

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48

bull Programme Created by DrTVRao MD for Medical and Paramedical

Students in Developing world bull Email

bull doctortvraogmailcom

DrTVRao MD 48

• Diagnosis of Tuberculosis an update
• Testing for TB Infection
• Diagnosis of TB Disease
• Who Should Get Tested for TB
• Who Should Get Tested for TB (2)
• Who Should Get Tested for TB (3)
• X ray of Chest a Commonly used Diagnostic tool
• Other tests
• Slide 9
• Laboratory Diagnosis
• Sputum Collection
• Interpretation of sputum stained by Z N Stain (WHO )
• Slide 13
• Acid Fast Bacilli under Fluorescent Microscope
• Culture
• Laboratory Diagnosis (2)
• Slide 17
• LowensteinndashJensen medium
• 3- Cultures on L J media Lowenstein ndash
• Recent Methods for Diagnosis
• Mycobacteria Growth Indicator Tube (MGIT)
• The MGIT 960 System
• Detection and identification of mycobacteria directly from clin
• Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR) amp Gene probe
• Quantiferon-GOLD
• What Positive Skin Test
• Tuberculin Test
• False negative reactions may be due to -
• Tuberculin Test ( Mantoux Test )
• Positive Test Means
• Negative Test Means
• Tuberculin Testing - Limitations
• Recent Methods for Diagnosis (2)
• TB blood tests
• Positive IGRA
• Negative IGRA
• IGRAs are the preferred method of TB infection testing for the
• III Polymerase Chain Reaction (PCR) amp Gene probe
• Molecular DR testing is particularly useful for
• Detection of MDR Tuberculosis
• Detection of MDR Tuberculosis (2)
• Tuberculosis and HIV infection
• HIV Considerations
• Epidemiology
• MDR tuberculosis
• Why Tuberculosis continues to be Important
• Slide 48