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International Standards 2-6, 10, 11
Microbiologic Diagnosis of Tuberculosis
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
Standards for Diagnosis
ISTC TB Training Modules 2009
Microbiologic Diagnosis of TB
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
Ziehl-Neelsen (ZN) Stain
ISTC TB Training Modules 2009
Sputum Microscopy: Fluorochrome Stain
Fluorochrome stain Fluorochrome stained smears require a
fluorescent microscope Generally read at 250X-450X magnification
which allows rapid scanning of the smear Auramine-rhodamine is an example of such a
stain where the AFB appear yellow against a black background
ISTC TB Training Modules 2009
Auramine-rhodamine Stain
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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.
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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)
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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.
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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:
ISTC TB Training Modules 2009
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.
ISTC TB Training Modules 2009
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.
ISTC TB Training Modules 2009
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)
ISTC TB Training Modules 2009
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)
ISTC TB Training Modules 2009
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)
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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)
ISTC TB Training Modules 2009
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)
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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.
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
Microbiologic Diagnosis of TB
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.
ISTC TB Training Modules 2009
Microbiologic Diagnosis of TB
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
ISTC TB Training Modules 2009
* 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.
ISTC TB Training Modules 2009
Summary: ISTC Standards Covered*
* Abbreviated versions
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.
ISTC TB Training Modules 2009
Summary: ISTC Standards Covered*
* Abbreviated versions
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.
ISTC TB Training Modules 2009
Summary: ISTC Standards Covered*
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
* Abbreviated versions
ISTC TB Training Modules 2009
Alternate Slides
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
Resource Contact Information
Laboratory testing: Microscopy/culture/DST
[Name, addresses, e-mail, etc.]
Specimen transport:
[Name, addresses, e-mail, etc.]
ISTC TB Training Modules 2009
Purpose of ISTC
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
Questions
ISTC TB Training Modules 2009
Microbiologic Diagnosis of TB
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
ISTC TB Training Modules 2009
Microbiologic Diagnosis of TB
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
ISTC TB Training Modules 2009
Microbiologic Diagnosis of TB
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