CHO-CH5: Diagnostic Technologies

Testing for TB diagnosis

Content

National Tuberculosis Elimination Programme (NTEP) strives for all presumptive TB patients to be microbiologically confirmed. Under NTEP, the acceptable methods for microbiological diagnosis of TB are:

Sputum Smear Microscopy (for Acid Fast Bacilli - AFB): Sputum Smear microscopy is the primary tool which is reliable, inexpensive, easily accessible and rapid method of diagnosing PTB, where in the bacilli are demonstrated in the sputum. Two types:

Ziehl-Neelsen Staining
Fluorescence staining

Rapid diagnostic molecular test: Rapid molecular tests that use techniques like NAAT are very specific. They amplify the genomic material in the patient sample and hence enhances detection

Nucleic Acid Amplification Test (NAAT) e.g., GeneXpert, TrueNat

Figure: Genxpert Machine for CBNAAT

Figure: Truenat Machine
Line Probe Assay

Culture and DST: A culture test involves studying bacteria by growing the bacteria on different substances. This is to find out if particular bacteria are present. In the case of the TB culture test, the test is to see if the TB bacteria Mycobacterium tuberculosis, are present.

Two types:

Solid (Lowenstein Jensen) media
Liquid media (Middlebrook) e.g., Bactec MGIT etc.

Cartridge Based Nucleic Acid Amplification Test [CBNAAT]

Content

Cartridge Based Nucleic Acid Amplification Test (CBNAAT) is a rapid molecular diagnostic test. It is used for diagnosis of Tuberculosis (TB) and Rif-resistant Tuberculosis (RR-TB) in NTEP. Results are obtained from unprocessed sputum samples in about 2hours which helps in early detection and treatment of TB patients.

India has vast number of CBNAAT laboratories which are utilized for TB/RR-TB detection and Universal Drug Susceptibility Testing (UDST) under the National TB Elimination Program (NTEP).

Figure: CBNAAT Cartridge and Machine in Use (Image courtesy: USAID supported Challenge TB Project)

The CB-NAAT system detects DNA sequences specific for Mycobacterium tuberculosis complex and rifampicin resistance by Polymerase Chain Reaction (PCR). It concentrates Mycobacterium tuberculosis bacilli from sputum samples, isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR. The process identifies clinically relevant rifampicin resistance-inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real-time format using fluorescent probes called molecular beacons.

Video: Cartridge-Based Nucleic Acid Amplification Test [CBNAAT] - GeneXpert Technology

Resources

Assessment Questions

Question

Answer 1

Answer 2

Answer3

Answer 4

Correct Answer

Correct explanation

Part of pre-test

Part of post-test

Under NTEP, CBNAAT is offered upfront for which of these categories?

PLHIV

Paediatric presumptive TB

Presumptive DR-TB

All of the above

4

Under NTEP, CBNAAT is recommended upfront for People living with HIV, Paediatric Presumptive TB patients, Presumptive DR-TB patients and patients notified from the Private sector.

Yes

CBNAAT requires the processing of sputum samples before testing

True

False

2

Results are obtained from unprocessed sputum samples in about 2hours from a CBNAAT machine

Yes

Truenat

Content

Truenat is an indigenous rapid molecular test platform that is currently under use in NTEP for diagnosis of TB and Rif Resistance. It is a platform utilising real-time Polymerase Chain Reaction (PCR) technology built into micro-PCR chips.

Testing on Truenat involves three components:

Workstation (consisting of 2 devices)
- Trueprep AUTO Universal Cartridge-based Sample Prep Device for the automated extraction and purification of DNA
- Truelab Real-time micro PCR Analyzer for performing real-time PCR. It is available as 1 (Uno), 2 (Duo) or 4 (Quattro) chip ports.
Cartridge and Chip
Reagent kits (Sample Pre-treatment and Prep kits)

Figure: Truenat Source: MolBio Products.

Test results for MTB detection and Rif Resistance has a turn around time of 1-2 hours. Depending on the micro-PCR chips used various tests can be performed using Truenat. Truenat MTB micro-PCR chips detect Mycobacterium tuberculosis bacteria for TB diagnosis. Truenat MTB RIF micro-PCR chip is used as a reflex test to detect resistance to Rifampicin (RIF), the first-line drug for TB treatment

Truenat has many advantages. Truenat is designed to be mobile and is battery operated (~8 hours on full charge). It can be deployed in peripheral laboratories and microscopy centres with minimal or no added facilities and hence it is more point-of-care. Biosafety requirements are similar to smear microscopy. However, it is multi staged and partially automated, requiring the presence of a Lab Technician through out the test.

Resources

Assessment

Question	Answer 1	Answer 2	Answer 3	Answer 4	Correct answer	Correct explanation	Page id	Part of Pre-test	Part of Post-test
Truenat is used in NTEP for:	MTB detection	Rif Resistance Detection	INH resistance Detection	MTB and Rif Resistance Detection	4	Truenat is used for MTB and Rif Resistance detection in NTEP		Yes	Yes
The Truelab Analyzer is available in how many chip ports?	2 (Duo)	1 (Uno), 2 (Duo) and 4 (Quattro)	1 (Uno)	4 (Quattro)	2	The Truelab Analyzer is available as 1 (Uno), 2 (Duo) and 4 (Quattro) chip ports.		Yes	Yes

Line Probe Assay [LPA]

Content

Line Probe Assay (LPA) is a rapid molecular test available at centralised laboratories.

The assay is based on Polymerase Chain Reaction (PCR) that can simultaneously detect Mycobacterium tuberculosis complex as well as drug sensitivity to anti-TB drugs.

Figure 1: The GenoType MTBDRplus Molecular LPA Procedure; Source: Molecular Detection of Drug-resistant Tuberculosis by Line Probe Assay.

Advantages of LPA

Rapid molecular test. (Turnaround time: 3-5 days)
Highly sensitive and specific.
Performed directly from sputum smear-positive specimens and on isolates of M. tuberculosis complex grown from smear-negative and smear-positive specimens.
Detects multiple gene mutations in anti-TB drugs.
- First-line LPA detects mutations to rifampicin and isoniazid
- Second-line LPA detects mutations to fluoroquinolones and aminoglycosides.
Suitable for low and high-throughput labs.

Disadvantages of LPA

Cannot be used as a point-of-care test.
Requires appropriate laboratory infrastructure, equipment and biosafety precautions.
Different rooms (DNA extraction, pre-amplification, amplification, post-amplification/ hybridization) are required to perform different steps (Figure 2).
Requires trained manpower to perform tests and interpret test results.
Stringent internal quality control is required to prevent contamination.

Figure 2: Amplification (A) and Post-amplification Laboratory (B) for LPA; Source: Molecular Detection of Drug-resistant Tuberculosis by Line Probe Assay.

Resources

Assessment

Question	Answer 1	Answer 2	Answer 3	Answer 4	Correct answer	Correct explanation	Page id	Part of Pre-test	Part of Post-test
LPA can be used as a point-of-care test.	True	False			2	LPA cannot be used as a point-of-care test.		Yes	Yes

Solid and Liquid Culture in TB

Content

Culturing TB Bacilli is well known and historic method for detection/ confirmation of Tuberculosis. It is a highly sensitive and specific phenotypic test; it can detect even a few viable bacilli in the sample (Upto 10 Colony Forming Units- CFUs). TB bacilli multiply in the culture and form colonies of TB bacilli which can are easily be identified.

Based on the growth media used Culture is divided in to two types, Solid and Liquid Culture methods. Types Culture:

Solid Culture on Lowenstein Jensen media : Historic gold standard culture test. Results take usually upto 2 months (60 days).
Modern Liquid culture systems: (e.g. BACTEC MGIT 960, BacT Alert or Versatrek etc.) Results take usually up to 42 days.

Uses

Solid culture is the gold standard diagnostic test for TB. But it is not used for the purpose of TB diagnosis due to the long turn around time of 2 months. It is largely used for research purposes where it is used as the baseline test on which the sensitivity and specificity of other tests are calculated.
Liquid Culture is being used for follow-up monitoring of patients on drug resistant TB treatment to detect treatment failure. Liquid culture is also used for long term follow up patients who have successfully completed treatment to detect recurrence.
Liquid culture is used as a previous step to grow bacilli and obtain isolates prior to Drug Susceptibility Testing.
Liquid cultures are also used in TB prevalence surveys for its high sensitivity and specificity

Resources

Guidelines for Programmatic Management of Drug Resistant Tuberculosis in India 2021

Kindly provide your valuable feedback on the page to the link provided HERE

Culture Drug Susceptibility Testing [CDST]

Content

Culture Drug Susceptibility Testing (CDST) is a growth-based phenotypic method used to check the susceptibility of Mycobacterium tuberculosis strains to various first and second line anti-TB drugs. Mycobacterial resistance to a particular drug is identified if there is growth observed in culture in presence of that drug.

In NTEP CDST is the standard method to detect resistance in samples of patients who have tested positive on followup. While CDST is possible on both Solid and Liquid culture, currently, the NTEP utilizes only liquid culture as a method for DST, due to faster Turn around times.

CDST testing services are available under NTEP in designated, specialized laboratories called CDST Labs both in public and private sector. Currently there are 80 such laboratories (60 certified for First Line and 49 for Second line drugs). Such designated laboratories are subject to regular external quality assessment, often by the National Reference Laboratory at that region.

Quality assured DST to R, H, Z, Mfx, Lfx, Lzd, Am, Km and Cm are available across the country.

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