Tuberculosis Care , ISTC training Module

Coordinator - Dr.T.V.Rao MDInstitution/organization Travancore

Medical College, Kollam India

International Standards 2-6, 10, 11

Microbiologic Diagnosis of Tuberculosis

ISTC TB Training Modules 2009

I request all the Medical, Nursing and Health Care workers go through this informative programme on Tuberculosis created by

International standards for Tuberculosis Please send to as many professionals as

Possible to work with Tuberculosis on Scientific basis

Dr.T.V.Rao MD Travancore Medical College, Kollam India

Copyright of ISTC TB trainingCopyright of ISTC TB training


Microbiologic Diagnosis of TB

Objectives: At the end of this presentation, participants will be able to: Understand the important role of sputum

smear microscopy in the diagnosis of TB Describe the various methods of sputum

staining and processing, and identify methods that may enhance results

Recognize the role of culture and drug sensitivity testing in the diagnosis and management of TB


Microbiologic Diagnosis of TBOverview: Significance of microbiologic testing in TB care Sputum staining and processing

•Direct smears, unconcentrated• Fluorochrome staining and fluorescence

microscopy•Concentration and chemical processing• Specimen collection and transport

Culture and drug-susceptibility testing Rapid diagnostic testing

International Standards 2, 3, 4, 5, 6, 10, and 11


Standards for Diagnosis



Significance of microbiologic testing forpublic health goals and patient care: WHO global target of 70% case detection of new

smear-positive cases Rapid and accurate case detection coupled with

effective treatment is essential to reduce the incidence of TB

Failure to perform a proper diagnostic evaluation before initiating treatment potentially:• Exposes the patient to the risks of unnecessary or

wrong treatment • May delay accurate diagnosis and proper treatment


Sputum Smear Microscopy Sputum smear microscopy

is the most important test for the diagnosis of pulmonary TB in many areas of the world

Direct smears (unconcentrated specimen) are most common

Fluorescence microscopy and chemical processing can increase sensitivity

Assessment of laboratory quality is essential


Sputum Microscopy: Direct Smears

Direct smears of unconcentrated sputum:

Fast, simple, inexpensive, widely applicable

Extremely specific for M. tuberculosis in high-incidence areas

Ziehl-Neelsen staining (carbol fuchsin type) most common


Sputum Smear Microscopy

Carbolfuchsin-based stains Utilize a regular light microscope Must be read at a higher magnification Two types: Ziehl-Neelsen and Kinyoun. Both

use carbolfuchsin/phenol as the primary dye Smear is then decolorized with acid (HCI)

alcohol and counter-stained with methylene blue


Ziehl-Neelsen (ZN) Stain


Auramine-rhodamine Stain


Fluorescence Microscopy

Advantages: More accurate: 10% more

sensitive than light microscopy, with specificity comparable to ZN staining

Faster to examine = less technician time

Disadvantages: Higher cost and technical

complexity, less feasible in many areas

Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (9):570-81


Sputum processing for optimizing smear results (vs. direct smear of unconcentrated sputum): Concentration by centrifugation and/or

sedimentation Chemical pretreatment (e.g., bleach, NaOH,

NaLC) for decontamination and digestion Usually both

Higher sensitivity (15-20% increase) and higher smear positive rate

Sputum Processing

Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (10):664-74


Collect specimens in a laboratory-approved, leak-proof container

Label all containers (name and date collected)

Collect specimens prior to initiation of therapy

Infection Control: Consider the safety of other patients and healthcare workers•Collect sputum in well-ventilated area,

preferably outdoors

Specimen Collection and Transport


Minimize contamination of specimens by:

• Instructing the patient to rinse mouth with water before collection

•Transport the specimen to the lab as soon as feasible after collection

Keep specimens refrigerated if possible

Specimen Collection and Transport


Mase SR, Int J tuberc Lung Dis 2007;11(5): 485-95

Average yield of single early morning specimen: 86.4%Average yield of single spot specimen: 73.9%

Specimen Number

Incremental Yield of smear specimens

(of all smear-positive)

Incremental Sensitivity of smear specimens

(compared with culture)

1 85.8% 53.8%

2 11.9% 11.1%

3 2.4% 3.1%

Total 100% 68.0%

Performance of Sputum Microscopy


Standard 2: Sputum Microscopy

Standard 2: All patients (adults, adolescents, and children who are capable of producing sputum) suspected of having pulmonary TB should have at least two sputum specimens obtained for microscopic examination in a quality-assured laboratory. When possible, at least one early morning specimen should be obtained.


Standards 3 & 4: Sputum Microscopy

Standard 3: For all patients (adults, adolescents, and children) suspected of having extrapulmonary TB, appropriate specimens from the suspected sites of involvement should be obtained for microscopy, culture, and histopathological examination.

Standard 4: All persons with chest radiographic findings suggestive of tuberculosis should have sputum specimens submitted for microbiological examination


Standard 10: Sputum Microscopy

Standard 10*: Response to therapy in patients with pulmonary tuberculosis should be monitored by follow-up sputum smear microscopy (2 specimens) at the time of completion of the initial phase of treatment (2 months).

If the sputum smear is positive at completion of the initial phase, sputum smears should be examined again at 3 months and, if possible, culture and drug susceptibility testing should be performed.

(* Partial Standard 10)


Although sputum microscopy is the first bacteriologic diagnostic test of choice, both culture and drug susceptibility testing (DST) can offer significant advantages in the diagnosis and management of TB.

Culture and Drug Susceptibility Testing


Culture: Advantages

Higher sensitivity than smear microscopy (culture can make diagnosis despite fewer bacilli in specimen)

If TB suspected and sputum smears are negative, culture may provide diagnosis

Allows for identification of mycobacterial species

Allows for drug susceptibility testing


Culture: Disadvantages

Cost Technical complexity May take weeks to get results Requires ongoing quality assurance

Therefore, more likely to be found in major referral centers. Avoid delaying appropriate TB treatment in suspicious cases while awaiting results.


Culture: Solid Media

Solid media have the advantage that organisms (colonies) can be seen on the surface of the medium

Types most commonly used are:• Lowenstein-Jensen:

egg-based

• Middlebrook 7H 10 or 7H11: agar-based

• Ogawa


MGIT Incubator

Culture: Liquid Media

More sophisticated equipment Faster detection of growth Higher sensitivity than solid

media Can also be used for drug-

susceptibility testing Two examples:

• BACTEC

• MGIT

MGIT

BACTEC


Culture: Identification of Mycobacteria

Growth characteristics (preliminary ID) Preliminary indication of M.tb can be determined

from colony characteristics• Rate of growth• Colonial morphology • Pigmentation

Biochemical tests There is a battery of 8 – 12 biochemical tests

used to differentiate M.tb within the genus Nitrate reduction and niacin production are

definitive for M.tb


Smooth, buff-colored colonies suggestive of Mycobacterium avium complex Rough, buff-colored colonies

suggestive of Mycobacterium tuberculosis

Culture: Identification of Mycobacteria

Visual assessment of colony morphology:


Culture: Cross-Contamination

Be aware that faulty technique can lead to laboratory cross-contamination of specimens (difficult to verify without access to more technical testing).

Adequate quality control is an essential component of any mycobacteriology laboratory.


Standard 5: Culture in Smear-Standard 5: The diagnosis of sputum smear-

negative pulmonary TB should be based on the following criteria: • At least two negative sputum smears (including

at least one early morning specimen) • Chest radiography findings consistent with TB • Lack of response to a trial of broad-spectrum

antimicrobial agents (*avoid fluoroquinolones)For such patients, sputum cultures should be obtained. In persons are seriously ill or have known or suspected HIV infection, the diagnostic evaluation should be expedited and if clinical evidence strongly suggests TB, a course of antituberculosis treatment should be initiated.


Standard 6: Culture in Children

Standard 6: In all children suspected of having intrathoracic (i.e., pulmonary, pleural, and mediastinal or hilar lymph node) TB, bacteriological confirmation should be sought through examination of sputum (by expectoration, gastric washings, or induced sputum) for smear microscopy and culture.

ISTC Training Modules 2008

(1 of 3)


Standard 6: Culture in children

In the event of negative bacteriological results, a diagnosis of TB should be based on:• The presence of abnormalities

consistent with TB on chest radiography• A history of exposure to an infectious

case, evidence of TB infection (positive tuberculin skin test or interferon gamma-release assay), and

• Clinical findings suggestive of TB

ISTC Training Modules 2008

(2 of 3)


Standard 6: Culture in Children

For children suspected of having extrapulmonary tuberculosis, appropriate specimens from the suspected sites of involvement should be obtained for microscopy and for culture and histopathological examination.

(3 of 3)


Culture: Drug Susceptibility Testing

Agar proportion method: Compares growth on solid agar media with and without one of the four primary drugs (on discs)

Broth based (BACTEC, MGIT): Liquid broth is inoculated with each test drug; growth in vial indicates resistance to that drug

Methods for susceptibility testing


Standard 11: Drug Susceptibility

Standard 11: An assessment of the likelihood of drug resistance, • based on history of prior treatment, • exposure to a possible source case having drug-

resistant organisms, • and the community prevalence of drug resistance,

should be obtained for all patients. Drug susceptibility testing should be performed at the

start of therapy for all previously treated patients Patients who remain sputum smear-positive at

completion of 3 months of treatment and patients who have failed, defaulted from, or relapsed following one or more courses of treatment should always be assessed for drug resistance

(1 of 2)


Standard 11: Drug Susceptibility

For patients in whom drug resistance is considered to be likely, culture and testing for susceptibility/resistance to at least isoniazid and rifampicin should be performed prompltly

Patient counseling and education should begin immediately to minimize the potential for transmission

Infection control measures appropriate to the setting should be applied

(2 of 2)


Rapid Diagnostic Testing

Nucleic acid probe tests (non-amplified) toidentify organisms grown in culture: DNA probe tests are species or complex

specific• Commercial probes are available for M.tb complex,

MAC, M. kansasii and M. gordonae

Nucleic acid amplification tests (NAAT): These tests are designed to amplify and detect

DNA specific to M.tb Enables direct detection of M.tb in clinical

specimens


Other Rapid Diagnostic Tests

Loop-mediated isothermal amplification (LAMP)•Rapid, simplified NAAT still under

investigation

•May be more feasible in lower resource settings

Immunological tests

•Serologic tests for antibody, antigens, and immune complexes; not currently accurate enough to replace microscopy and culture.


Other Rapid Diagnostic Tests

High performance liquid chromatography (HPLC)

•HPLC uses a liquid chromatography method to identify mycobacteria based on their mycolic acid profiles (cell wall composition)

• The equipment is expensive and is usually reserved for larger, specialized, reference laboratories


Rapid Drug Susceptibility Tests

Line-probe assays• Identifies M.tb and

genetic mutations associated with INH and RIF resistance

•Can be used directly on sputum specimens, results within 1-2 days

Molecular beacons

Bacteriophage-based assays

*GenoType MTDBRplus strips (Hain Lifescience)

*Barnard et al. Am. J. Respir. Crit. Care Med 2008; 177: 787-792



Summary: Smear microscopy

plays a central role in the diagnosis and management of tuberculosis.

It is important to understand the aspects of specimen handling and processing that can ensure or enhance accurate results.



Summary (cont.): Culture and drug-

sensitivity testing should be obtained, when feasible, for smear-negative TB and treatment failure.

Quality assurance is essential for all TB diagnostic procedures


* Abbreviated versions

Summary: ISTC Standards Covered*

Standard 2: All TB suspects should have at least 2 sputum specimens obtained for microscopic examination (at least one early morning specimen if possible).

Standard 3: Specimens from suspected extrapulmonary TB sites should be obtained for microscopy, culture and histopathological exam.

Standard 4: All persons with chest radiographic findings suggestive of TB should have sputum specimens submitted for microbiological examination.




Standard 5: The diagnosis of smear-negative pulmonary TB should be based on the following: at least two negative sputum smears (including at least one early morning specimen); CXR finding consistent with TB; lack of response to broad-spectrum antibiotics (avoid fluoroquinolones), and culture data. Empiric treatment if severe illness.

Standard 6*: In all children suspected of having intrathoracic and extrapulmonary TB, specimens (sputum, extrapulmonary tissue) should be obtained for microscopy, culture, and histopathological (tissue) examination. TB diagnosis should be based on chest radiography, history of TB exposure, positive TB test, and suggestive clinical findings if bacteriologic studies are negative.




Standard 10 (partial): Response to therapy in patients with pulmonary tuberculosis should be monitored by follow-up sputum smear microscopy (2 specimens) at the time of completion of the initial phase of treatment (2 months). If the sputum smear is positive at completion of the initial phase, sputum smears should be examined again at 3 months and, if possible, culture and drug susceptibility testing should be performed.



Standard 11: An assessment of the likelihood of drug resistance, based on history of prior treatment, exposure to a possible source case having drug-resistant organisms, and the community prevalence of drug resistance, should be obtained. • DST should be performed at the start of therapy for

all previously treated patients• For patients in whom drug resistance is considered to

be likely, culture and testing for susceptibility/resistance to at least isoniazid and rifampicin should be performed promptly

• Patient counseling and education should begin immediately to minimize the potential for transmission

• Infection control measures appropriate to the setting should be applied



Alternate Slides


Resource Availability

The following tests are available at _________: Smear microscopy

• Direct smear / light microscopy

• Fluorescence microscopy

• Concentration/chemical processing

Culture• Solid media

• Liquid media

Drug susceptibility testing Other


Resource Contact Information

Laboratory testing: Microscopy/culture/DST

[Name, addresses, e-mail, etc.]

Specimen transport:

[Name, addresses, e-mail, etc.]


Purpose of ISTC


ISTC: Key Points

21 Standards (revised/renumbered in 2009) Differ from existing guidelines: standards

present what should be done, whereas, guidelines describe how the action is to be accomplished

Evidence-based, living document Developed in tandem with Patients’ Charter

for Tuberculosis Care Handbook for using the International

Standards for Tuberculosis Care


Audience: all health care practitioners, public and private

Scope: diagnosis, treatment, and public health responsibilities; intended to complement local and national guidelines

Rationale: sound tuberculosis control requires the effective engagement of all providers in providing high quality care and in collaborating with TB control programs

ISTC: Key Points


Questions



1. All of the following can increase sensitivity of sputum smear microscopy except:

A. Fluorescence microscopy

B. Sputum collection after the start of anti-tuberculosis treatment

C. Concentration by centrifugation and/or sedimentation

D. Chemical pretreatment



2. A 37 year-old man with diabetes presents with clinical symptoms highly suspicious for TB. Two sputum smears are negative. The patient collected the specimens ten days before he brought them back and kept them in a cool area of the house (no refrigeration). Which of the following statements is most correct?

A. Two negative smears predict that a culture would be negative, and therefore a culture offers no further diagnostic advantage and need not be obtained

B. A lack of response to broad-spectrum antimicrobial agents and a chest film suggestive of TB, would together suggest a diagnosis of smear-negative TB

C. The delay in transport and lack of refrigeration for the sputum specimens are unlikely to have a negative effect on results

D. Six sputum specimens for smear microscopy would have tripled the sensitivity for diagnosing TB compared to two specimens



3. Advantages of culture for TB compared to sputum microscopy alone include all of the following except:

A. Obtaining a positive culture can allow for drug-susceptibility testing

B. Culture can allow for identification of non-tuberculous mycobacterium species

C. Culture has a higher sensitivity than smear microscopy for diagnosing TB.

D. Culture, particularly by liquid media, can be faster than smear microscopy

Tuberculosis Care , ISTC training Module

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