Multi Drug Resistant Tuberculosis

Basics Concerns and Diagnosis

DrTVRao MD

A Tribute to Robert Koch Discoverer of Mycobacterium

Tuberculosis

DrTVRao MD 2

Nobody is absolutely

Immune to Tuberculosis

DrTVRao MD 3

Basic concepts ndash Keep facts

Primary (Initial) resistance

TB patientrsquos initial Mycobacterium tuberculosis population

resistant to drugs

Secondary (Acquired) resistance

Drug-resistant M tuberculosis in initial population

selected by inappropriate drug use (inadequate

treatment or non-adherence)

DrTVRao MD 4

Changing Definition of

MDR TB

1950s-1970s

M tb resistant to INH

streptomycin andor PAS

1980s-current

M tb resistant to at least INH

and Rifampin

Definition of MDR - TBMDR-TB caused by strains of

Mycobacterium Tuberculosis resistant

both Rifampicin and Isoniazid with or

without resistance to other drugs

Single Isoniazid or Rifampicin resistance

is not MDR - TB

MDR TB is a laboratory diagnosis Not

a Clinical assumption

DrTVRao MD 7

Why INH and RifampinMost potent and bactericidal drugs for Tuberculosis

Tb can be treated effectively with INH+Rif alone

Mono-resistance to one of them can be treated effectively with a regimen containing the other agent with very low failure rate (25-5)

Failure rate when INH+Rif resistant is 44 in non-HIV and 70 in HIV patients

Duration required for cure doubles to triples

DrTVRao MD 8

MDR-TB amp XDR-TB

THE 2008 REPORT

of MDR-TB among new TB cases 1994-2007

DrTVRao MD 11

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

A Tribute to Robert Koch Discoverer of Mycobacterium

Tuberculosis

DrTVRao MD 2

Nobody is absolutely

Immune to Tuberculosis

DrTVRao MD 3

Basic concepts ndash Keep facts

Primary (Initial) resistance

TB patientrsquos initial Mycobacterium tuberculosis population

resistant to drugs

Secondary (Acquired) resistance

Drug-resistant M tuberculosis in initial population

selected by inappropriate drug use (inadequate

treatment or non-adherence)

DrTVRao MD 4

Changing Definition of

MDR TB

1950s-1970s

M tb resistant to INH

streptomycin andor PAS

1980s-current

M tb resistant to at least INH

and Rifampin

Definition of MDR - TBMDR-TB caused by strains of

Mycobacterium Tuberculosis resistant

both Rifampicin and Isoniazid with or

without resistance to other drugs

Single Isoniazid or Rifampicin resistance

is not MDR - TB

MDR TB is a laboratory diagnosis Not

a Clinical assumption

DrTVRao MD 7

Why INH and RifampinMost potent and bactericidal drugs for Tuberculosis

Tb can be treated effectively with INH+Rif alone

Mono-resistance to one of them can be treated effectively with a regimen containing the other agent with very low failure rate (25-5)

Failure rate when INH+Rif resistant is 44 in non-HIV and 70 in HIV patients

Duration required for cure doubles to triples

DrTVRao MD 8

MDR-TB amp XDR-TB

THE 2008 REPORT

of MDR-TB among new TB cases 1994-2007

DrTVRao MD 11

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Nobody is absolutely

Immune to Tuberculosis

DrTVRao MD 3

Basic concepts ndash Keep facts

Primary (Initial) resistance

TB patientrsquos initial Mycobacterium tuberculosis population

resistant to drugs

Secondary (Acquired) resistance

Drug-resistant M tuberculosis in initial population

selected by inappropriate drug use (inadequate

treatment or non-adherence)

DrTVRao MD 4

Changing Definition of

MDR TB

1950s-1970s

M tb resistant to INH

streptomycin andor PAS

1980s-current

M tb resistant to at least INH

and Rifampin

Definition of MDR - TBMDR-TB caused by strains of

Mycobacterium Tuberculosis resistant

both Rifampicin and Isoniazid with or

without resistance to other drugs

Single Isoniazid or Rifampicin resistance

is not MDR - TB

MDR TB is a laboratory diagnosis Not

a Clinical assumption

DrTVRao MD 7

Why INH and RifampinMost potent and bactericidal drugs for Tuberculosis

Tb can be treated effectively with INH+Rif alone

Mono-resistance to one of them can be treated effectively with a regimen containing the other agent with very low failure rate (25-5)

Failure rate when INH+Rif resistant is 44 in non-HIV and 70 in HIV patients

Duration required for cure doubles to triples

DrTVRao MD 8

MDR-TB amp XDR-TB

THE 2008 REPORT

of MDR-TB among new TB cases 1994-2007

DrTVRao MD 11

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Basic concepts ndash Keep facts

Primary (Initial) resistance

TB patientrsquos initial Mycobacterium tuberculosis population

resistant to drugs

Secondary (Acquired) resistance

Drug-resistant M tuberculosis in initial population

selected by inappropriate drug use (inadequate

treatment or non-adherence)

DrTVRao MD 4

Changing Definition of

MDR TB

1950s-1970s

M tb resistant to INH

streptomycin andor PAS

1980s-current

M tb resistant to at least INH

and Rifampin

Definition of MDR - TBMDR-TB caused by strains of

Mycobacterium Tuberculosis resistant

both Rifampicin and Isoniazid with or

without resistance to other drugs

Single Isoniazid or Rifampicin resistance

is not MDR - TB

MDR TB is a laboratory diagnosis Not

a Clinical assumption

DrTVRao MD 7

Why INH and RifampinMost potent and bactericidal drugs for Tuberculosis

Tb can be treated effectively with INH+Rif alone

Mono-resistance to one of them can be treated effectively with a regimen containing the other agent with very low failure rate (25-5)

Failure rate when INH+Rif resistant is 44 in non-HIV and 70 in HIV patients

Duration required for cure doubles to triples

DrTVRao MD 8

MDR-TB amp XDR-TB

THE 2008 REPORT

of MDR-TB among new TB cases 1994-2007

DrTVRao MD 11

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Changing Definition of

MDR TB

1950s-1970s

M tb resistant to INH

streptomycin andor PAS

1980s-current

M tb resistant to at least INH

and Rifampin

Definition of MDR - TBMDR-TB caused by strains of

Mycobacterium Tuberculosis resistant

both Rifampicin and Isoniazid with or

without resistance to other drugs

Single Isoniazid or Rifampicin resistance

is not MDR - TB

MDR TB is a laboratory diagnosis Not

a Clinical assumption

DrTVRao MD 7

Why INH and RifampinMost potent and bactericidal drugs for Tuberculosis

Tb can be treated effectively with INH+Rif alone

Mono-resistance to one of them can be treated effectively with a regimen containing the other agent with very low failure rate (25-5)

Failure rate when INH+Rif resistant is 44 in non-HIV and 70 in HIV patients

Duration required for cure doubles to triples

DrTVRao MD 8

MDR-TB amp XDR-TB

THE 2008 REPORT

of MDR-TB among new TB cases 1994-2007

DrTVRao MD 11

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Definition of MDR - TBMDR-TB caused by strains of

Mycobacterium Tuberculosis resistant

both Rifampicin and Isoniazid with or

without resistance to other drugs

Single Isoniazid or Rifampicin resistance

is not MDR - TB

MDR TB is a laboratory diagnosis Not

a Clinical assumption

DrTVRao MD 7

Why INH and RifampinMost potent and bactericidal drugs for Tuberculosis

Tb can be treated effectively with INH+Rif alone

Mono-resistance to one of them can be treated effectively with a regimen containing the other agent with very low failure rate (25-5)

Failure rate when INH+Rif resistant is 44 in non-HIV and 70 in HIV patients

Duration required for cure doubles to triples

DrTVRao MD 8

MDR-TB amp XDR-TB

THE 2008 REPORT

of MDR-TB among new TB cases 1994-2007

DrTVRao MD 11

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Why INH and RifampinMost potent and bactericidal drugs for Tuberculosis

Tb can be treated effectively with INH+Rif alone

Mono-resistance to one of them can be treated effectively with a regimen containing the other agent with very low failure rate (25-5)

Failure rate when INH+Rif resistant is 44 in non-HIV and 70 in HIV patients

Duration required for cure doubles to triples

DrTVRao MD 8

MDR-TB amp XDR-TB

THE 2008 REPORT

of MDR-TB among new TB cases 1994-2007

DrTVRao MD 11

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

MDR-TB amp XDR-TB

THE 2008 REPORT

of MDR-TB among new TB cases 1994-2007

DrTVRao MD 11

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

DrTVRao MD 12

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Epidemiology of MDR TB

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Genesis of MDR TBResistance is a man-made amplification of a

natural phenomenon

Inadequate drug delivery is main cause of

secondary drug resistance

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of

resistant strains

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for

anti TB are unlinkedDrTVRao MD 14

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

When to suspect MDR

TBRe-treatment patients whorsquos sputum smear remains positive after three monthsrsquo of intensive therapy

Treatment failure and interruption cases

Close contacts of MDR tuberculosis cases

Positive diagnoses with

TB culture and susceptibility testing

DrTVRao MD 16

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Factors Contributing to

Development

and Spread of MDR and XDR TB

Weak TB programs (DOTS)

Low completioncure rates

Lack of treatment follow up and patient support

Unreliable drug supply

Diagnostic delay

Absent or inadequate infection control measures

Uncontrolled use of 2nd line drugs

DrTVRao MD 17

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Mechanism of resistanceINH

Chromosomally mediated

Loss of catalaseperoxidase

Mutation in mycolic acid synthesis

Regulators of peroxide response

DrTVRao MD 18

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Mechanism of resistance

Rifampin

Reduced binding to RNA polymerase

Clusters of mutations at ldquoRifampin Resistance Determining Regionrdquo (RRDR)

Reduced Cell wall permeability

DrTVRao MD 19

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

LABORATORY DIAGNOSIS

OF MDR-TB AND XDR-TBThe laboratory is an essential component in TB

control programs and broader access to DST is

a priority for most countries Early choice of

appropriate treatment is an essential

determinant of favourable outcome and rapid

determination of drug resistance can allow a

customized approach to treatment early in the

course of the disease and can potentially reduce

morbidity mortality and infectiousness

DrTVRao MD 20

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

CONVENTIONAL CULTURE-BASED

METHODSUsing standardized DST procedures with

conventional methods eight to 12 weeks

are required to identify drug-resistant

microorganisms on solid media (ie

Lowenstein-Jensen medium) In general

such methods assess inhibition of M

tuberculosis growth in the presence of

antibiotics to distinguish between

susceptible and resistant strains

DrTVRao MD 21

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Proportion method The proportion method allows precise

determination of the proportion of resistant

mutants to a certain drug the resistance ratio

method compares the resistance of an unknown

strain with that of a standard laboratory strain

While relatively inexpensive and undemanding of

sophisticated equipment results usually take

weeks and this is challenging inappropriate

choice of treatment regimen may result in death

within weeks of initiation such as in the case of

XDR-TB (especially in HIV-infected patients)

DrTVRao MD 22

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Conventional Methods

are OutdatedIn addition delayed

identification of drug

resistance results in

inadequate treatment

which may generate

additional drug

resistance and

continued

transmission in the

community

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

LIQUID CULTURE-BASED

METHODS

The BACTEC 460 TB radiometric system (Becton Dickinson USA) was considered to be a major advancement when it was introduced but has been replaced by the Mycobacteria Growth Indicator Tube system (Becton Dickinson USA) Several published studies have shown the excellent performance of the Mycobacteria Growth Indicator Tube system for the rapid detection of resistance to first- and second-line anti-TB drugs Detection of drug resistance can be accomplished in days rather than weeks although still constrained by high cost (equipment and consumables)

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Diagnosis of MDR-TB

and XDR-TB

The diagnosis of MDR-TB and XDR-TB is hampered by the absence of effective and affordable rapid diagnostic techniques for drug sensitivity Several approaches phenotypic and molecular have been explored to develop rapid reliable and accurate methods for the rapid detection of drug resistance in M tuberculosis These methods should also be evaluated and applied in high-incidence areas

DrTVRao MD 25

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Susceptibility Testing

1048708 Direct and indirect testing

1048708 Primary Drugs testing

1048708 Isoniazid

1048708 Rifampicin

1048708 Ethambutol ()

1048708 Pyrazinamide ()

DrTVRao MD 26

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Drug susceptibility testing

(DST)

DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment

Accuracy is more important than speed

DST results should come from a small number of well-equipped experienced laboratories who participate and perform well in an international DST quality control scheme

The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage

DrTVRao MD 27

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Drug susceptibility Testing

Assessment of growth inhibition on solid media containing various dilutions of the drug in comparison with the test strains

As the method depend observation of growth Results are not available until several weeks after isolation of the organism

DrTVRao MD 28

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

MODSMicroscopic Observation

of Drug Susceptibility

Testing

DrTVRao MD 29

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

MODS affordable

Technically Feasible

MODS arose during experiments conducted by

Luz Caviedes under the guidance of Professor

Robert Gilman at Universidad Peruana

Cayetano Heredia in Lima Peru in the late

1990s in which a colorimetric test for TB growth

was being investigated The observation that

micro colonies could be seen under the

microscope long before a colour change

occurred prompted the development of MODS

DrTVRao MD 30

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Observation of Growth in

liquid Media

MODS depends upon three key principles

(which have been known for decades) (1)

Mycobacterium tuberculosis grows faster in

liquid (broth) than on solid media (2) in liquid

cultures M tuberculosis grows in a visually

characteristic manner (tangles cording) which

can be observed under the microscope long

before the naked eye could visualize colonies

on solid agar

DrTVRao MD 31

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Least time required for detection of

MDRIncorporation of

anti-TB drugs

into broth

cultures at the

outset enables

direct

susceptibility

testing from

sputum samplesDrTVRao MD 32

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Advantages of MODS

methodology in MDR detection

bull All the chemical ingredients are available locally except few which can be acquired easily

bull Existing infrastructure in District and Teaching hospital can be adopted for implementation of

MODS

bull Risk to technician handling the specimens is minimal there is no absolute need to obtain

grade III safety cabinets

Technology transfer is easier all the new technical manpower can be trained easily

DrTVRao MD 33

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Other accredited

MethodsRadiometric and Non

radiometric methods

Nucleic acid

technology ndash

effective up to 95 in

mutations to

rifampicin resistance

to gene rpoB gene

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Drug susceptibility testing

(DST)

As a minimum laboratories supplying DST data should correctly identify resistance to isoniazid and rifampicin in over 90 of quality control samples in two out of the last three quality control rounds

DrTVRao MD 35

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Detection of Rifampicin Drug

susceptibility testing (DST) is more

important

Early identification of mycobacterial growth as M tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB

Laboratories should aim to identify isolates as M tuberculosis complex and perform rifampicin resistance in 90 of isolates within 1-2 working days This is technologically feasible

DrTVRao MD 36

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Drug susceptibility testingFor DST laboratories

modern molecular

techniques permit the

successful

identification of

isoniazid resistance in

at least 75 of

mycobacterial cultures

within 1-2 working

days and are useful

preliminary screens for

isoniazid resistance

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Secondary Drugs testing

[lack of standardized methods]

Ofloxacin quinolones

Ethionamide

Kanamycin

Capreomycin

Ensure quality control and

quality assurance

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Other WHO-Endorsed

ToolsLiquid culture (eg MGIT BacTALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from culture

Molecular line probe assays (eg Genotype MTBDRplus INNO-LiPA RifTB)

Strip test for detection of TB and drug-resistance conferring mutations

DrTVRao MD 39

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

NOVEL RAPID

MOLECULAR METHODS

The identification of specific mutations responsible for

drug resistance has facilitated the development of novel

rapid molecular tools for DST The detection of RIF

resistance is traditionally used as a predictor of MDR-TB

ndash its positive predictive value is a function of the

sensitivity and specificity of RIF resistance testing and

the prevalence of MDR and non-MDR RIF resistance

which is highest among previously treated cases in

settings with high MDR prevalence and low non-MDR

RIF resistance

DrTVRao MD 40

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Xpert MTBRIF

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

The Xpert MTBRIF

The Xpert MTBRIF is a

cartridge-based

automated diagnostic

test that can identify

Mycobacterium

tuberculosis (MTB)DNA

and resistance to

rifampicin (RIF)by

nucleic acid

amplification

technique(NAAT

DrTVRao MD 42

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

WHO Endorses Xpert MTBRIF

In December 2010

the World Health

Organization (WHO)

endorsed the Xpert

MTBRIF for use in

TB endemic

countries[2] and

declared it a major

milestone for global

TB diagnosis

DrTVRao MD 44

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Xpert MTBRIF detects DNA

sequences

The Xpert MTBRIF detects

DNA sequences specific

for Mycobacterium

tuberculosis and rifampicin

resistance by polymerase

chain reaction It is based

on the Cepheid GeneXpert

system a platform for

rapid and simple-to-use

nucleic acid amplification

tests (NAAT)

DrTVRao MD 45

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

How Xpertreg MTBRIF

Works The Xpertreg MTBRIF purifies and concentrates Mycobacterium tuberculosis bacilli from sputum samples isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR

DrTVRao MD 46

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Xpertreg MTBRIF Helps in Faster

Diagnosis of Resistance to

Rifampicin

The process identifies all the clinically relevant Rifampicin resistance inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real time format using fluorescent probes called molecular beacons Results are obtained from unprocessed sputum samples in 90 minutes with minimal biohazard and very little technical training required to operateThis test was developed as an on-demand near patient technology which could be performed even in a doctors office if necessary

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Summary

Drug resistant TB

Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB if identified early can be treated and cured but experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics treatments and vaccines

DrTVRao MD 49

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

No testing method replaces clinical

assessment for Tuberculosis

DrTVRao MD 50

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Progress in diagnosing multidrug-

resistant tuberculosis20 March 2014 | Geneva - Almost half a million people fell ill with multidrug-resistant tuberculosis (MDR-TB) in 2012 yet less than one in 4 of these people was diagnosed mainly due to a lack of access to quality diagnostic services

DrTVRao MD 51

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Koch failed to conquer tuberculosis which still

causes enormous health problems worldwide 100

years after his Nobel award

The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures first of all the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91 which severely damaged Kochs reputation

Medical History 2001 45 1-32 CHRISTOPH GRADMANN

DrTVRao MD 52

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Tuberculosis is a Concern

for Everyone

DrTVRao MD 53

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

MDR Tuberculosis is Global

Emergency

DrTVRao MD 54

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Visit me for more Articles of

Interest on Infectious Diseases

DrTVRao MD 55

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom

Programme Created by DrTVRao MD for

Medical and Health Professionals in the

Developing World

Email

doctortvraogmailcom