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

E p i d e m i o l o g y 2

Direct Microscopy identification

M. tuberculosis is a acidfast bacterium, rod-shaped bacterium measuring 2-4 x 0.2-0.5 m. 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 None per 100 oil immersion fields 1-9 per 100 oil immersion fields 10-99 per 100 oil immersion fields 1-10 per oil immersion field > 10 per oil immersion field Result reported Negative Scanty, report exact number 1+ 2+ 3+

AFB MICROSCOPY

Advantages
- High specificity (AFB in sputum = TB)

-Rapid

- Accurate diagnoses

All mycobacterium are acid fast, no exception ; > 98% for AFB in high burden countries - 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% 100% 93% 81%

Cumulative Positivity

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,

patient may persons/year

Smear-positive

smear-positive infect 10-15

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.

24 h pooled ?

Urine -3 or more consecutive samples

Volume
Pleural, peritoneal fluids Cerebrospinal fluid

E p i d e m i o l o g y 3

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 palmaticacid, producing radioactively labeled 14CO2.
Middle Brook 7H12 containing palmatic acid PANTA (Polymyxin B , Amphotericin B , Nalidic acid , trimethoprim ,Azlocillin ) OADC enrichment

*.Containing

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

Can be done anywhere

Unclear correlation with development of TB

Blood must be sent quickly to lab Reliable Does not cross-react with BCG, most NTM

Reliability fair
Cross-reacts with BCG, NTM

TST vs. Blood Tests

TST
Sensitivity
(in pts with active TB)

Blood Tests
67-96% 95-100%

60-80% As low as 35%

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%.
Identification of the target sequence of DNA doesn't imply organism viability. Contamination of samples by product from previous PCR experiments

Disadvantages:

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