Greetings from Chandigarh!

Newer laboratory diagnosis for

tuberculosis

CHETANA VAISHNAVI
Professor & Chief
Division of Clinical Microbiology
Department of Gastroenterology Postgraduate
Institute of Medical Education and Research,
Chandigarh, 160012
India.

Introduction

Tuberculosis

major

public

health

problem

in

developing countries
Mycobacterium tuberculosis (human)
M.bovis (bovine)
M. avium and M. intracellulare (atypical mycobacteria)
In 2006 Global Plan to Stop TB 2006-2015 launched
Epidemic growing by about 1% annually
Each year 9 million new cases; 2 million deaths
85% of cases occur in Africa and Asia (30%; 55%)
India and China together represent 35% of the burden

 In India 3-4 million new cases annually

Increasing AIDS incidence diagnosis is becoming a
challenge.
Tuberculosis involve any organ system
Pulmonary tuberculosis most common presentation
Extrapulmonary tuberculosis
Major clinical problem
Accounts for 15-25% of reported cases
More

frequent

in

children

and

people

with

HIV

infection.
More difficult to diagnose
Require invasive procedures & sophisticated lab.
techniques
Diagnosed on the basis of clinical experience

 Gastrointestinal tract sixth most frequent

site of extrapulmonary involvement for
tuberculosis

Autopsies

on

patients

with

pulmonary

tuberculosis before the use of effective antitubercular drugs

55-90% cases

intestinal involvement in

Diagnostic aids
Microbiological techniques
Smear/Light Microscopy

Acid fast staining

followed by smear microscopy most

freq. used test

Sensitivity threshold of detection 5000-10,000 bacilli/ml

of specimen.

Smear AFB negative does not eliminate diagnosis active

tuberculosis

Concentration

centrifugation/sedimentation/chemical

Fluorescence microscopy

Fluorescence microscopy using an acid-fast fluorochrome

dye (auramine O/auramine-rhodamine)

sensitive, specific

and cost effective.

Mycobacteria brightly fluorescing rods against dark

background.
Possibility of false-positive inorganic objects incorp. dyes
Culture
Conventional method
Egg-based (Lowenstein-Jensen/Ogawa media)
Agar-based (Middlebrook 7H9, 7H10 and 7H11).
Culture most specific of currently available tests
Provides
sensitivity

starting

point

for

species

identification/drug

Automated or semi-automated method

Faster culture of mycobacterial isolates manual culture
systems (Septi-Chek AFB/manual mycobact. growth
indicator tube MGIT).
Commercially available automated/semi-automated
liquid culture systems reduced recovery time to 2-3
weeks.
Automated culture systems (MB/BacT system, BACTEC
9000MB, BACTEC MGIT 960 and ESP Myco and
AccuMed/Difco ESPII BACTEC 12B, MB/BacT Mycobacterial
detection system and the ESP culture system II).
Radiometric liquid culture
radiolabelled carbon
measure changes in gas pressure, carbon dioxide
production
or
oxygen
consumption
fluorimetrically/colorimetrically
Allow continuous monitoring of cultures

ascitic fluid due to stimulation of T-cells by mycobacterial

antigens.

High interferon levels in tubercular ascites useful for lab.

diagnosis.

Quantiferon-tuberculosis Gold (in vitro test)detects release

of IFN- after stimulation of WBCs in
M.tuberculosis

antigen

ESAT-6

(early

blood sample by
secretory

antigenic

target 6) and CFP-10 (culture filtrate protein 10).

Corresponding burst in cytokines examined by EIA.

Test may have a possible role in follow-up of patients on ATT

and in the diagnostic dilemma of Crohns disease versus
tuberculosis

Immunodiagnostic tests

Mantoux test delayed hypersensitivity response to the

tuberculin antigen.

Cannot differentiate between active tuberculosis and previous

sensitization with the tuberculin antigen due to:

Past exposure or BCG vaccination in patients exposed to nontuberculous Mycobacteria

Subjectivity in interpretation of results

Need for 2 clinical encounters to administer and interpret the

results.

Laparoscopic findings
Excellent but sparingly used diagnostic technique

Visual appearance (85-95% accurate)

Endoscopic examination for GI tuberculosis

Colonoscopy
Double balloon enteroscopy
Capsule endoscopy
Very useful in diagnosis of abdominal tuberculosis

Colonoscopic findings
On colonoscopy, up to 8-10 biopsies are generally taken for

histopathology and culture.

Biopsies should be taken from the edge of the ulcers to obtain
tissue for bacterial culture or to identify AFB by histology.
Caseating granulomas 85-90% of biopsies

Radioimaging methods

Chest radiography

Barium studies
Ultrasonography
Computed tomographic scan
Very useful in diagnosis of abdominal tuberculosis.

Pathological /Histopathological evidence

Pathological features (tuberculous granulomas) imp. for
lab.diagnosis
Most reliable method for the diagnosis is demonstration of AFB
through a combination of histology and culture of the biopsy material.
Establish diagnosis in over 60% of cases.
Histopathology is gold standard

Molecular methods

(a) DNA probes

Rapid and specific identification of M.tuberculosis

Useful along with newer methods of detection of early growth

(viz. BACTEC, Septi-Chek, MGIT) rapidly confirming diagnosis
(within 1-2 days)
For direct confirmation from clinical specimens need >10000
organisms in the specimen for positivity.
(b) Ribosomal RNA based probes
Ribosomal RNA probes target rRNA, ribosomal DNA, spacer and

flanking sequences.
rRNA targeting probes are 10-100 fold more sensitive than DNA
targeting
Confirm diagnosis directly in clinical specimens
Lowest detection limit is around 100 organisms

(c) Gene amplification methods

For the diagnosis of tuberculosis gene amplification, several
techniques based on PCR and isothermal amplification assay have
been developed.

(d) Loop mediated isothermal amplification

In this technique, different enzymes other than taq polymerase
are used and various steps of amplification are completed at one
temperature only.

(e) PCR technology

Gene targets are MPB 64, repetitive sequences, GC repeats, dev
R, 38kD, TRC 4, and IS-1081.
Tuberculosis PCR assay based on augmenting highly specific
oligonucleotides found in chromosomes of M.tuberculosis
Sensitivity modest
No correlation between PCR positivity and histological lesions
(caseation or granuloma).
Multiplex PCR (from India) promising results in diagnosis and
detection of drug resistance, including extensively drug-resistant
tuberculosis.

(f) Line-probe assays

Line probe assay technology newer NAATs available for
diagnosis of tuberculosis as well as MDR tuberculosis.
PCR amplification of resistance-determining region of gene
performed using biotinylated primers.
Labeled PCR products are hybridized with specific oligonucleotide
probes immobilized on a strip and detected by colorimetric
development.
Commercially available
INNO-LiPA Rif. tuberculosis kit (Innogenetics, Belgium)
GenoType MTBDRplus assay (Hain Lifescience, Germany)

(g) Xpert MTB/RIF test

GeneXpert microfluidic and molecular testing platform

Attempts to overcome known limitations
Combines 3 separate steps for specimen processing and
nucleic acid extraction, nucleic acid amplification, and detection
of amplified products into a single, automated process
Xpert MTB/RIF test being developed uses molecular beacons
and 6-color fluorescence detection for real time identification of
both M. tuberculosis and rifampin resistance in <120 minutes.

Some of the newer options (under development

or evaluation) include:
Options for case detection and/or drug sensitivity (growthbased)
The microscopic-observation drug-susceptibility assay
Thin-layer culture
Phage-based detection
Options for direct visualization
Florescent microscopy with molecular probes (Florescent
In situ hybridization)
Computer-assisted microscopy

Options for species identification

Luminescent probe of culture isolate
Line probe from culture isolates
Dipstick detection of tuberculosis antigens in positive
cultures
Species-specific amplification or sequencing (Research
use)

Options for latent tuberculosis infection

detection
M. tuberculosis protein-64 (MTP-64) skin patch

Options for volatile organic compound

detection
Electronic nose analysis of headspace gas
Gas chromatographic (GC)/Mass spectrometric
analysis of exhaled air
Handheld surface acoustic wave-GC
Use of giant African pouched rats to detect
Mycobacteria

Some limitations that neglect imp. aspects of

tuberculosis

epidemic
Biggest concern lack of a rapid, simple, inexpensive, pointof-care test
Major step forward an easy to use, inexpensive diagnostic
test (better than smear microscopy) required to deliver results
within minutes without sophisticated equipment or highly-trained
laboratory personnel
Could have

tremendous impact on global tuberculosis

control
Adoption and implementation new tools

THANK YOU