Laboratory diagnosis of Tubercular Infections

1. DIAGNOSIS OF TUBERCULAR INFECTIONS
MAJ (DR) ROHIT VIKAS
MS (ORTHO)

2. DIAGNOSIS
CLINICAL
– PULM/ EXTRA PULM
RADIOLOGICAL
ROUTINE LAB
IDENTIFICATION OF MTB/ MDR
TB
HISTOPATHOLOGICAL

3. Routine Lab
Anaemia
Lymphocytosis/ Leukopenia
Raised ESR
Detection of adenosine deaminase (ADA)
An enzyme present in almost all mammalian cells, principally in the lymphocytes,
being directly related to lymphocyte activation.
In diseases presenting greater lymphocyte activation and participation, elevated
levels of ADA are usually detectable.
Other disease conditions that cause pleural effusion with predominance of
lymphocytes such as SLE, lymphoma etc, high levels of ADA may also be seen.
Pleural TB - an ADA cut off value of 35 U/L

4. Chest Radiographs

5. Collection of Right Clinical Sample
Pulm TB
Sputum (Min requirement - At least 02 samples 01 hr apart)
Induced Sputum
Laryngeal swabs
Transtracheal aspiration
Gastric aspiration
BAL
Transbronchial lung biopsy
Blood samples are of no value
Extra pulm TB – from site of disease
Pleural effusion – Pleural tap/ biopsy
TB Lymphadenitis - FNA/ LN Biopsy
TB Meningitis – CSF tap
Urine
Body fluids
Tissue biopsy
Smear microscopy for AFB
Nucleic acid amplification (NAAT)
Culture

6. Sputum Microscopy
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.
Auramine Rhodamine stain
Fluorescence Microscopy - More accurate: 10%
more sensitive than light microscopy, with
specificity comparable to ZN staining.
Sputum processing - Concentration +/- Chemical
pretreatment  Higher sensitivity (15-20%
increase) and higher smear positive rate
Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (9):570-81
Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (10):664-74

7. Sputum Microscopy
> 10 bacilli/ field +++
1 – 10 / field ++
10 – 99 / 100 field +
1 – 9 / 100 field Write the no
No bacilli seen Negative
Oil immersion Field
5000 – 10000 bacilli per ml sputum

11. Culture
Higher sensitivity than smear microscopy - If TB
suspected and sputum smears are negative, culture
may provide diagnosis
Allows for identification of mycobacterial species.
Allows for drug susceptibility testing.
Tuberculosis culture room of the Lala Ram Swawrup (LRS)
Institute of Tuberculosis and Respiratory Diseases in New Delhi
10 – 100 bacilli per ml sputum

12. Culture
Solid media
Lowenstein-Jensen: egg-based
Middlebrook 7H 10 or 7H11: agar-based
Liquid media
Middlebrook 7H 12
Liquid media
More sophisticated equipment
Faster detection of growth
Higher sensitivity than solid media
Can also be used for drug-susceptibility testing
BACTEC 460 TB
MGIT
MGIT Incubator
Conventional Automated

13. Smooth, buff-colored colonies
suggestive of Mycobacterium
avium complex
Rough, buff-colored colonies suggestive of
Mycobacterium tuberculosis
Visual assessment of colony morphology:
Culture – Identification of MTb
Nitrate reduction and niacin production are definitive for M.tb

14. Mycobacteriophage Based Assay
Rapid - Results available within 24 hr of
sample preparation
From clinical samples
Detects patient positive samples missed by
smear
No instrumentation required
Safe - no culturing of pathogen
Sensitive - assay sensitivity 100 - 300
cells/ml
Only detects live bacilli - reduces the
possibility of false positives
Technology can be extended for antibiotic
susceptibility testing
FastPlaque ® TB

15. Molecular Methods of Diagnosis
Direct detention of MTB
from Specimen
Identification of MTB
from Cultures
Genotypic
PCR
LAMP
NAATs
Ligase chain reaction
Phenotypic - FAST Plaque TB
PCR based sequencing
1 – 10 bacilli per ml sputum
Rapid – 3 – 4 hr
Cannot differentiate between dead and living MTB
Expensive

16. NAATs Nucleic acid amplification tests (NAAT)
Not recommended to be used on blood samples
NAA assays amplify M. tuberculosis-specific nucleic acid sequences using a
nucleic acid probe.
Sensitivity - 80% in most studies - Require as few as 10 bacilli in a given sample.
Specificity - 98% to 99%.
Official statement of ATS and CDC, July 1999

17. LAMP (Loop Mediated Isothermal Amplification) for TB
A manual simplified NAAT for TB
Particularly applicable to resource-poor
settings.
Amplifies target DNA with high
specificity, efficiency and rapidity under
isothermal conditions
DNA can be amplified 10 9 -10 10 times
in 15-60 min
Visually detect DNA directly from
clinical samples, in less than two hours
and with minimal instrumentation.

18. Xpert MTB/RIF
Endorsed by WHO (2010).
Updated WHO
recommendations for diagnosis
of pulmonary TB, paediatric TB,
extrapulmonary TB and
rifampicin resistance (2013).
Implementation in endemic
countries is ongoing.
Cost ~ Rs 3500/-
A recent Cochrane review has shown that the Xpert MTB/RIF test has 88%
sensitivity and 98% specificity when compared to culture
Xpert MTB/RIF can detect rifampicin resistance with a sensitivity of 94% and
specificity of 98%.

19. Line Probe Assay
DNA strip test - rapid, manual
NAAT
Use PCR and reverse
hybridization methods
Can diagnose MDR-TB directly
from smear-positive sputum
samples, providing results in
just 05 hrs.
2008 - WHO issued a
recommendation for the use of
molecular LPA for the rapid
diagnosis of MDR-TB in high
TB-burden, low-income
settings.
Highly accurate in detecting
MDR-TB and cost-effective
when compared with TB culture
and DST
GenoType MTBDRplus®
by Hain Lifescience, Germany

20. Liquid culture significantly faster: the
average time-to-growth detection with
liquid culture is 10 to 14 days, as opposed
to four to six weeks with traditional solid
culture.
Up to 20% more sensitive than solid
culture.
Considered the gold standard for TB
diagnosis and the only technology that can
detect resistance to all major TB drugs.
Liquid cultures are also very useful for
smear-negative TB and extrapulmonary TB.
Liquid Cultures
MGIT (Mycobacterium Growth Indicator Tube method) by BD, USA
BacT/Alert by BioMerieux, France

21. Liquid Cultures
Rs 35,00,000 + Rs 120 per MGIT vial.
BACTEC™ MGIT™ 960 Mycobacterial Detection System

22. Role of Blood Tests in TB
ELISA to detect IgG, IgM, IgA Abs
Rapid strip or card tests
‘Mycodot’
TB IgG/IgM
Serological Tests

23. June 2012 - Govt of India, acting on the 2011 WHO policy against serological tests,
banned the use, import, sale, and manufacture of Ab - based blood tests for TB, and
discouraged the use of tests like "TB Gold" for active TB
Role of Blood Tests in TB

24. Interferon-γ Release AssaysIGRAs
Based on the detection of IFN-γ - Released by sensitized
T cells on stimulation with very specific Ags –
Early secretory antigen target-6 (ESAT 6)
Culture filtrate protein-10 (CFP 10)
Both derived from a very specific region of MTb, the
region of difference 1 (RD1).
This segment (RD1) is deleted from all strains of BCG and
the majority of environmental mycobacteria (except M.
kansasii, M. szulgai, M. marinum, M. flavescens, and M.
gastrii).
Advantage - Discriminate between MTB infection and
previous use of BCG vaccine.
T-Spot TB test - Directly count the number of IFN-γ-
secreting T cells
Quantiferon TB Gold In-Tube test - Measures the
concentration of IFN-γ secretion.

25. Role in Indian ScenarioIGRAs
Like Mantoux test - Do not differentiate between active pulmonary TB disease and
latent TB infection.
High False positive results – unnecessary treatment with ATT
Cross-reactivity - L-ESAT, a M. leprae Ag homolog to the T-ESAT-6 seen in M. tuberculosis,
may induce the production of IFN-γ, therefore making the test less useful in populations
in which leprosy is endemic.
Expensive.
Meant for detection of Latent MTB Infection (Important in non endemic countries)
LTBI – 6-9 months single agent Isoniazid
Active TB – 6 months short course
High negative predictive value - A negative test in a healthy patient excludes tuberculosis
infection.
(40% Indian population latently infected)

27. Role of Tuberculin (Montaux) test in Indian Scenario
Indicates previous exposure and
carriage of T.B.
Tuberculin positive persons may
develop reactivation type of T.B.
False positive reactions are mainly due
to Infection with nontuberculous
mycobacteria/ BCG vaccination
Children below 5 years of age with no
exposure history - Positive test must
be regarded suspicious
Even the induration of 5 mm to be
considered positive when tested on
HIV patients.
(40% Indian population latently infected)
Excludes infection in suspected
persons
Tuberculin negative persons are at risk
of gaining new infection
False negative reactions may be due to
Severe tuberculosis infection
(Miliary T.B.)
Hodgkin’s disease
Corticosteroid therapy
Malnutrition
AIDS
Positive test Negative test

28. Histopathological Examination
Central Ceseation Necrosis
Epitheloid Cell Granuloma
Multinucleated Langhans
Giant Cells

29. Aptamers
Aptamers (Oligonucleotides) recognise a target with high affinity and
specificity, but can also discriminate between very subtle structural
differences.
The main competitive advantage of aptamers over conventional
approaches includes their high specificity, high sensitivity, relatively low
production costs, convenience and simplicity, which allows for rapid
point-of-care diagnosis.
MODS (Microscopic Observation Drug Susceptibility)
ROAD AHEAD