Introduction 1
Laboratory Diagnosis
Tuberculosis
Dr. Kiarash Ghazvini
Department for bacteriology and virology,
Mashhad University of medical Sciences
Mycobacterium tuberculosis
long, slender, straight or curved, acid fast bacilli
slow growers, obligate aerobes,
intracellular bacterium
structure composed of high
molecular weight acidic waxes,
mycolic acid, cord factor
Diagnosis
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Direct Microscopy identification
M. tuberculosis is a acid–fast
bacterium, rod-shaped bacterium
measuring 2-4 x 0.2-0.5 μm. They
appear as bright red rods against a
contrasting background.
The Ziehl-Neelsen stain is used to
demonstrate the presence of the bacilli
in a smear. The technique is simple,
inexpensive and detects those cases of
tuberculosis who are infectious.
M. tuberculosis appearing as bright red bacilli
(rods) in a sputum smear stained with the
Ziehl-Neelsen stain
Reporting on AFB Microscopy
Number of bacilli seen
Result reported
None per 100 oil immersion fields
Negative
1-9 per 100 oil immersion fields
Scanty, report
exact number
10-99 per 100 oil immersion fields
1+
1-10 per oil immersion field
2+
> 10 per oil immersion field
3+
AFB MICROSCOPY
Advantages
-Rapid
- High specificity (AFB in sputum = TB)
• All mycobacterium are acid fast, no exception ;
• > 98% for AFB in high burden countries
- Accurate diagnoses
- Using simple and available equipment
Disadvantage
Low sensitivity; Reported sensitivity ranging 25 to 65% when compared to culture
Species differentiation impossible. False positive; Saprophytic mycobacteria.
Three sputum smears are optimal
100%
93%
Cumulative Positivity
100%
81%
50%
0%
First
Second
Third
AFB MICROSCOPY: SENSITIVITY

Quality of sputum, morning sputum 10-100% more positives

Number of samples

Quality of smear, staining, quality of microscopes,

Effort in examining number of fields
3 smears = sensitivity of 1 culture
About 95% of infectious cases
Smear-positive
patients are 4-20
times more infectious
Untreated,
a
patient
may
persons/year
smear-positive
infect
10-15
Smear-positive
patients are much
more likely to die if untreated
Rouillon A. Tubercle 1976;57:275-99
Direct Microscopy identification
• Fluorescent dye (Auramine O and Rhodamine B)
• Good for labs with high workload.
• Auramine O- Bright yellow
• Auramine O-Rhodamine B- Yellow orange.
Appropriate collection techniques
– Time
•
Sputum -3 consecutive samples.
–
Early morning complete sputum.
• Urine -3 or more consecutive samples
–
24 h pooled ?
– Volume
– Pleural, peritoneal fluids
– Cerebrospinal fluid
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Culture
M.
tuberculosis
grows
in
Lowenstein Jensen medium, which
contains inhibitors to keep
contaminants from outgrowing the
organism.
Because of its slow growth, it takes
4-6 weeks before small buffcoloured colonies are visible on the
medium.
Typical small, buff coloured colonies of M.
tuberculosis on Lowenstein Jensen
medium
Culture

more sensitive, but relative
• low income countries : only 25% gain over smear ?
• and in these settings less specific ?

main problem: DELAYS
• on classical media (Lowenstein-Jensen....)
• newer commercial media (BACTEC..)
» faster (about 10 days)
» but expensive++, technical demands

first step for susceptibility testing
Other Methods for
Laboratory Diagnosis of
Tuberculosis
BACTEC –TB System
 based
on the principal that the organisms
multiply in the broth and metabolize C 14containing
palmatic–acid,
producing
radioactively labeled 14CO2.
*.Containing
• Middle Brook 7H12 containing palmatic acid
• PANTA (Polymyxin B , Amphotericin B , Nalidic acid , trimethoprim
,Azlocillin )
• OADC enrichment
Mycobacterium Growth Indicator Tubes (MGIT)

A fluorescent compound is embedded in
silicone on the bottom of 16 x 100 mm
round-bottom tubes.
Tests based on Immune responce
PPD test
Gama interferone response
Invitro Blood Test (ELISPOT)
Serodiagnostic Test (ELISA)
Blood Tests for TB

New blood tests examine immune response to TB antigens

Draw blood from patient, expose white cells to TB antigens,
look for signs of immune response

Two most common methods are ELISA and ELISPOT
Lancet 356: 1099 2000
Blood Tests for TB

One-time blood draw

Less inter-reader variability than PPD

Looks at response to 2 TB-specific antigens: ESAT-6 and
CFP-10

Antigens are not found in BCG, most non-TB mycobacteria

ELISA and ELISPOT technologies commercially available
(Pai M et al, Lancet Inf Dis 4: 761 2004)
T-SPOT (ELISPOT) test
Meier T et al, Eur J Clin Microbiol Infect Dis 24:529 (2005)
TST
Blood tests

Test itself cheap

More expensive

Clearly correlated with
development of TB

Unclear correlation with
development of TB

Can be done anywhere

Blood must be sent quickly to
lab

Reliability fair

Reliable

Cross-reacts with BCG,
NTM

Does not cross-react with
BCG, most NTM
TST vs. Blood Tests
Sensitivity
TST
Blood Tests
60-80%
67-96%
As low as 35%
95-100%
(in pts with active TB)
Specificity
(in low-risk pts)
Summary

Have great potential to reduce false-positive results

Likely more sensitive for active TB, but clinical utility in this
setting less clear

Logistical and cost barriers to implementation

Ideally will replace TST in many settings
Adenosine deaminase (ADA)

Adenosine deaminase (ADA) and interferon gamma studied
for dx of extrapulmonary TB

Both are markers of immune response to TB

Not that specific
Immune dx of TB

ADA and interferon gamma may be useful in endemic areas

In low-incidence areas, specificity and sensitivity are not
good enough for routine use

More specific markers needed
PCR
polymerase chain amplification
1) Diagnose tuberculosis rapidly by identifying DNA from M.
tuberculosis in clinical samples.
2) Determine rapidly whether acid-fast organisms identified by
microscopic examination in clinical specimens are M.tuberculosis
3) Identify the presence of genetic modifications known to be
associated with resistance to some anti mycobacterial agents.
4) Determine whether or not isolates of M.tuberculosis from
different patients have a common origin in the context of
epidemiological studies.
Molecular method
Sensitivity and Specificity of PCR

Incase of smear and culture positive the sensitivity is
ranging 80% to 90% with specificity of 97%-99%.

Incase of smear negative and culture positive the
sensitivity is ranging 60% to 80% with specificity of
97%-99%.
Disadvantages:
– Identification of the target sequence of DNA doesn't imply organism viability.
– Contamination of samples by product from previous PCR experiments
NAA Summary

NAA is useful to distinguish TB from NTM in smear +
specimens

Less sensitive in smear – specimens

Clinical judgement must always take priority

Relatively expensive tests; need data to support costeffectiveness
Real time PCR
Rapid analysis (typically under 90 min)
No post-amplification processing (no gels or autoradiographs)
Automated (data collection and analysis)
Objective (controls & standards can be built-in)
Precise, sensitive and reproducible
TB/HIV
MDR/XDR-TB