Microbiologic
Diagnosis of
Tuberculosis
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International Standards 2-6, 10, 11
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 TB
Overview:
 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 for
public 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
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
Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (10):664-74
ISTC TB Training Modules 2009
Specimen Collection and Transport
 Collect specimens in a laboratoryapproved, 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
ISTC TB Training Modules 2009
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
ISTC TB Training Modules 2009
Performance of Sputum Microscopy
Incremental Yield of Incremental Sensitivity
Specimen smear specimens
of smear specimens
Number
(of all smear-positive) (compared with culture)
1
85.8%
53.8%
2
11.9%
11.1%
3
2.4%
3.1%
Total
100%
68.0%
Average yield of single early morning specimen: 86.4%
Average yield of single spot specimen: 73.9%
Mase SR, Int J tuberc Lung Dis 2007;11(5): 485-95
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 qualityassured 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
Culture and Drug Susceptibility Testing
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.
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
Culture: Liquid Media
 More sophisticated equipment
 Faster detection of growth
 Higher sensitivity than solid
media
 Can also be used for drugsusceptibility testing
 Two examples:
BACTEC
• BACTEC
• MGIT
MGIT
ISTC TB Training Modules 2009
MGIT Incubator
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
Culture: Identification of Mycobacteria
Visual assessment of colony morphology:
Smooth, buff-colored
colonies suggestive
of Mycobacterium
avium complex
ISTC TB Training Modules 2009
Rough, buff-colored colonies
suggestive of Mycobacterium
tuberculosis
Culture: Cross-Contamination
 Be aware that faulty
technique can lead to
laboratory crosscontamination 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 SmearStandard 5: The diagnosis of sputum smearnegative 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
TB Training
Modules
Modules
2008
2009
(1 of 3)
Standard 6: Culture in children
(2 of 3)
 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
TB Training
Modules
Modules
2008
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.
ISTC TB Training Modules 2009
(3 of 3)
Culture: Drug Susceptibility Testing
Methods for 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
ISTC TB Training Modules 2009
Standard 11: Drug Susceptibility
(1 of 2)
Standard 11: An assessment of the likelihood of drug
resistance,
• based on history of prior treatment,
• exposure to a possible source case having drugresistant 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
ISTC TB Training Modules 2009
Standard 11: Drug Susceptibility
(2 of 2)
 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
ISTC TB Training Modules 2009
Rapid Diagnostic Testing
Nucleic acid probe tests (non-amplified) to
identify 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
*GenoType MTDBRplus strips
(Hain Lifescience)
 Bacteriophage-based assays
*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 drugsensitivity 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
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.
* Abbreviated versions
ISTC TB Training Modules 2009
Summary: ISTC Standards Covered*
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.
* Abbreviated versions
ISTC TB Training Modules 2009
Summary: ISTC Standards Covered*
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.
* Abbreviated versions
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
ISTC: Key Points
 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 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
ISTC TB Training Modules 2009