Introduction 1Introduction 1

Laboratory DiagnosisLaboratory DiagnosisTuberculosisTuberculosis

Dr. Kiarash Ghazvini

Department for bacteriology and virology,

Mashhad University of medical Sciences

long, slender, straight or curved, acid fast bacilli

Mycobacterium tuberculosis

slow growers, obligate aerobes, intracellular bacterium

structure composed of high molecular weight acidic waxes,

mycolic acid, cord factor

Diagnosis

EpidemiologEpidemiology 2y 2

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.

Direct Microscopy identification

M. tuberculosis appearing as bright red bacilli (rods) in a sputum smear stained with the Ziehl-Neelsen stain

Reporting on AFB MicroscopyReporting on AFB Microscopy

Number of bacilli seen Result reported

None per 100 oil immersion fields Negative

1-9 per 100 oil immersion fields Scanty, reportexact 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.

81%

93%100%

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50%

100%

First Second Third

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Three sputum smears are optimal

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 smear-positive patient may infect 10-15 persons/year

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 techniquesAppropriate 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

CultureCulture

EpidemioEpidemiology 3logy 3

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 buff-coloured colonies are visible on the medium.

Typical small, buff coloured colonies of M. tuberculosis on Lowenstein Jensen medium

CultureCulture 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 Other Methods for Laboratory Diagnosis of Laboratory Diagnosis of

Tuberculosis

BACTEC –TB SystemBACTEC –TB System

based on the principal that the organisms multiply in the broth and metabolize C 14-containing 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 Mycobacterium Growth Indicator Tubes (MGIT)(MGIT)

A fluorescent compound is embedded in

silicone on the bottom of 16 x 100 mm

round-bottom tubes.

Tests based on Immune responceTests based on Immune responce

PPD test

Gama interferone response

Invitro Blood Test (ELISPOT)

Serodiagnostic Test (ELISA)

Blood Tests for TBBlood 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 TBBlood 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)

Meier T et al, Eur J Clin Microbiol Infect Dis 24:529 (2005)

T-SPOT (ELISPOT) test

TSTTST Blood testsBlood tests

Test itself cheap

Clearly correlated with development of TB

Can be done anywhere

Reliability fair

Cross-reacts with BCG, NTM

More expensive

Unclear correlation with development of TB

Blood must be sent quickly to lab

Reliable

Does not cross-react with BCG, most NTM

TST vs. Blood TestsTST vs. Blood Tests

TST Blood TestsSensitivity

(in pts with active TB)60-80% 67-96%

Specificity

(in low-risk pts)As low as 35% 95-100%

SummarySummary

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) 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 TBImmune 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 methodMolecular method

Sensitivity and Specificity of PCRSensitivity 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 cost-effectiveness

Real time PCRReal 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