Diagnostics in Tuberculosis

1. RECENT ADVANCES IN TUBERCULOSIS DIAGNOSIS
-by Dr. Nishant & Dr. Mukesh

2.  In 2017, 10.4 million incident cases globally while the estimate of incidence was 2.8
million cases in India.
 The diagnosis of tuberculosis in children is challenging, especially in infants and
young children (<5 years), who are at particular risk for disease and adverse
outcomes from tuberculosis.

3. INTRODUCTION
 The diagnostic tests currently available in most public health systems in high-burden
countries (active cases) today largely rely on age old microscopy.
 In 2015, there has been a paradigm shift in the WHO from stop TB to end TB
strategy. To achieve this, newer and faster techniques in diagnosis as well as detection
of MDR with the availability of these facilities at the peripheral centres.

4. Steps taken to eliminate TB
 First objective of NSP (2017-2025) is to find all Drug Sensitive TB and Drug Resistant
TB cases with an emphasis on reaching TB patients seeking care from private providers
and undiagnosed TB in high-risk populations.
 In 2018, the requirements for moving towards TB elimination have been integrated into
the four strategic pillars of “Detect – Treat – Prevent – Build” (DTPB).
 In 2019, the World Health Organization, the Stop TB Partnership, and The Global Fund
to Fight AIDS, Tuberculosis and Malaria have launched a joint initiative titled “FIND.
TREAT. ALL. #ENDTB”, to scale up the End TB response

5.  It is universally acknowledged that improving the diagnosis of tuberculosis in
children both through the development of novel diagnostics and by optimizing the
use of current tools are major research priorities.

6. METHODS
Currently available diagnostics can be classified as those that:
 Directly detect the actively growing bacilli
 Indirectly detect the immune response against the bacilli
 Other newer and experimental methods

7. Direct detection
 Microscopy
 Culture
 Antigen detection and
 Nucleic acid detection
Indirect detection
 Tuberculin skin testing (TST) and
 Interferon-gamma release assays (IGRAs).

8. Year of WHO
endorsement
 2007
 2008
 2010
 2010
 2010
 2013
 2016
 2016
 2016
Techniques
 Commercial liquid culture systems for culture & DST
 Line probe assay for INH & rifampicin
 LED microscopy
 Selected novel culture & DST methods (MODS, CRI, NRA)
 Xpert MTB/RIF for sputum
 Xpert MTB/RIF for CSF & lymph node tissue
 Urine Lipoarbinomannan (LAM) rapid test
 Loop mediated amplification test (LAMP)
 Line probe assay for 2nd line drugs

9. Direct Detection Methods
 1. Microscopy
 Sputum smear microscopy still remains the basis for diagnosis of TB in developing
countries. Higher load needs to be present (10,000 bacilli/ml)
 Sensitivity varies from 20% - 60%.
It is relatively fast, inexpensive and specific for TB in high incidence areas. Although
highly specific, smear microscopy is insensitive – it detects roughly 50% of all the
active cases of TB.

10.  Disadvantages :
Relying on sputum microscopy alone may be dangerous from the public health point
of view as
• 17% of smear-negative cases are known to transmit the disease.
• cannot differentiate between live and dead bacilli and hence cannot be used as a
follow-up diagnostic test.
• Cannot be used to predict MDR.
Despite the multitude of disadvantages, in the absence of better alternatives, it is a useful
tool in the basic laboratories common in developing countries.

11.  Fluorescent microscopy with light-emitting diodes
Light-emitting diodes (LEDs) are more robust, sustainable and user-friendly, thus
allowing advantages of FM at peripheral health-care systems.
FM increases the sensitivity of smear microscopy as it allows a much larger area of the
smear to be seen, resulting in more rapid examination of the specimen (up to four times
faster).

12.  Being WHO recommended in 2009, the RNTCP has adopted LED microscopy to
replace ZN method in its designated microscopy centres (DMCs) across India.
 Disadvantages:
Instability of fluorescent stains.
Proper training.
Unlike Ziehl Neelsen, international guidance on quality assurance for FM does not
currently exist and is under development.
The sensitivity, specificity, cost-effectiveness and cost-benefit of this approach have not
yet been adequately established.
Front-loaded microscopy (2009)

13.  Vital fluorescent staining
 Simple and instant method for TB treatment monitoring (fluorescein diacetate (FDA)
marker, in combination with smear microscopy). Unlike most fluorescent stains, FDA
stains only living, cultivable bacteria thus guiding antimicrobial therapy before culture
reports come in.

14. Newer microscopic technologies
1. Automated microscopic technology by TBDx (Signature Mapping Medical Sciences,
USA) integrates robotic loading of stained slides and automated high-resolution
digital image analysis to provide a result in minutes.
2. Capture-XT (Quantum Dx, UK) which concentrate low number of Mycobacterium
tuberculosis (MTB) cells into a capture chamber.
3. A second innovation to assist a microscopist is CellScope, a portable digital FM that
provides enlarged digitalised images for review.

15.  2. Culture
Culture still remains the gold standard for diagnosis of TB, and it also permits the
diagnosis of drug resistance, including the emerging mutations.
 Traditional egg-based (Lowenstein-Jensen) and agar-based (Middlebrook 7H10/11)
methods are widely used. The limit of detection is 10-100 CFU/ml, thus increasing the
sensitivity compared to smear.

16.  Disadvantages: Long TAT: from 4 to 8 weeks with an additional 4 weeks for drug
sensitivity (total 70 days) by the conventional proportion method.
 Other limitations: requirement of biosafety facilities ( expensive and specially trained
technicians). Hence, TB cultures are performed only at national reference laboratories or
in-hospital laboratories in large cities, distant from rural areas.

17.  The current recommendation from WHO is to use automated liquid culture platform
because of its better sensitivity and faster results (average 10–15 d for culture and
additional 10–14 d for DST) than solid cultures.
 The liquid culture: Bactec 960 MGIT (Becton Dickinson), BacT/ALERT (bioMerieux),
Myco-ESP culture system (Trek Diagnostics) etc.
 The sensitivity of culture, which is considered as the present gold standard is only 30–
40% in clinically diagnosed cases of pediatric TB.
 There are studies that recommend stool culture in pulmonary tuberculosis, but evidence
is poor.

18. Table 1: Culture methods for Mycobacterium tuberculosis

19.  3. Antigen detection
Lipoarabinomannan (LAM) is a glycolipid found in the outer cell wall of
mycobacterial species.
 An ELISA or dipstick method with TAT of 4–6 h or 20 min depending on the test kit
used. The WHO recommends that it should not be used for the diagnosis of TB, except
for HIV-positive in-patients (good sensitivity).

20.  4. Molecular detection: Nucleic acid amplification tests
Currently, three methods exist; the first two being WHO recommended:
 Cartridge-based NAAT (CB-NAAT)
 Line probe assay (LPA)
 Loop-mediated amplification (LAMP).

21.  Cartridge-based nucleic acid amplification test: Xpert MTB/RIF
 The CB-NAAT is a semi-quantitative nested real-time PCR which detects both MTB
(DNA) and RIF resistance (mutations in the rpoB gene) directly from clinical
specimens. 2010
 Recommended method under the current RNTCP guidelines.

22.  The analytical limit is 131 CFU/ml and the TAT is 2–3 h. Because the cartridges are self-
contained, the problem of cross-contamination between samples is eliminated.
 Procedure: Sputum is liquefied and inactivated with a sample reagent which kills over 99.9% of
TB bacilli in the specimen, and 2 ml of the material is transferred into a cartridge and this is
inserted in the MTB-RIF test platform. Inside the cartridge, the sample is automatically filled,
washed, filtered, by ultrasonic lysis of the filter captured organisms to release the DNA. It uses
three specific primers and five unique molecular probes to ensure high degree of specificity. The
primers amplify a portion of the rpoB gene 81 bp RIF resistance determining region.

23.  The MTB/RIF test correctly detected RIF resistance with a sensitivity of 99.1% and
100% specificity. Thus, the test detects TB in essentially all smear-positive samples
and the majority of smear-negative samples.
 In future, the same technology may be also used for HIV viral load detection.
 A next generation catridge called Xpert Ultra has been developed by Cepheid with
improved sensitivity, to detect low number of bacilli (16 CFU/ml of sputum) .

24.  Disadvantages:
 the presence of mono-resistance to INH which is not detected in this test. INH mono-
resistance is documented to be 7%–11% in the first-line treatment failures.
 False-positive RIF resistance results; hence, samples found to be resistant must be
confirmed by a second Xpert MTB/RIF test or an LPA and phenotypic culture testing.
 The test requires a trained and computer-literate operator, a stable supply of electricity
and air-conditioned settings.

27.  Line probe assay
This strip test detects TB DNA and genetic mutations (inhA promoter & katG regions
for INH and rpoB gene for Rifampicin) associated with drug resistance from smear-
positive sputum specimens or culture isolates after DNA extraction and PCR
amplification.
 This is a hybridisation assay that allows differentiation
between Mycobacterium species.

28.  In a meta analysis, the pooled sensitivity of LPA for detection of INH and Rif
resistance was estimated as 84% & 98% respectively and the pooled specificity was
99% in both the cases.
 The time- to-result may be reduced to as low as 8 h in comparison to 60 d using
conventional culture and sensitivity.

29.  It has a good sensitivity and specificity for the detection of MDR compared to
conventional results, thus confirming their value in rapid screening of patients suspected
of MDR-TB. WHO has endorsed LPA for MDR-TB in 2009.
 Another LPA, GenoTypeMTBDRsl is recently endorsed by WHO (May 2016) for
detection of drug resistance to 2nd line drugs, fluoroquinolones and aminoglycosides
which shorten the TAT of second-line DST testing to 1–2 days. (the 'gold standard' DST
method with TAT between 10 days and 6 weeks )

30.  Disadvantages: LPAs are as complex to perform as conventional culture and DST and
require skilled and well-trained laboratory personnel as well as adequate laboratory
space and design (BSL-2/3 level laboratory with Class II Biological Safety Cabinet) to
reduce the risk of false-positive results.
 LPAs do not eliminate the need for conventional culture and DST capability as culture
remains necessary for definitive diagnosis of TB in smear-negative patients while DST
is required to confirm if not diagnose MDR/XDR TB.

32.  Loop-mediated amplification
LAMP is a simple, rapid, specific and cost-effective nucleic acid amplification method solely
developed by Eiken Chemical Co., Ltd, Japan.
 It is characterised by the use of four different primers specifically designed to recognise six
distinct regions on the target gene and the reaction process proceeds at a constant temperature
using auto-cycling strand displacement reaction targeting the six regions of the gyrB and 16S
rRNA genes. Amplification and detection of gene can be completed in a single step, by
incubating the mixture of samples, primers, DNA polymerase with strand displacement activity
and substrates at a constant temperature (about 65°C).

33.  It provides high amplification efficiency, with DNA being amplified 109–1010 times
in 15–60 min.
 LAMP is a simple isothermal DNA amplification method. It detects M.
tuberculosis complex but does not detect resistance.

34.  Various other isothermal NAAT includes:
• Recombinase polymerase amplification (RRA, Twist Dx, UK)
• Cross-priming amplification (CPA-China)
• Helicase-dependent amplification (HAD-USA)
• Nicking enzyme amplification reaction (NEAR-USA)
• Epistem (UK) and Xceleris (India) are working on providing a rapid TB diagnostic
test using gene drive technology. This is a hand-held device which incorporates a rapid
nucleic acid amplification specific to M. tuberculosis complex as well as RIF
resistance.

35.  Microarray, Gene sequencing & Next generation sequencing are other
tools under study.

36. Indirect Method of Detection
Exposure to MTB may result in latent TB infection. A person with latent TB infection
usually leads a healthy life without developing active TB disease. Two billion people
have latent TB infection, but only a fraction (<10 million a year) fall sick with active TB
disease.
 TST and IGRA cannot differentiate between latent TB and active TB.

37.  Tuberculin skin testing
Popularly known as Mantoux test involves injecting the purified protein derivative
(PPD) of MTB intradermally in the forearm and the resulting reaction is read after
48–72 h.

38.  Interferon-gamma release assay (IGRA)
IGRAs were developed based on the principle that the T-cells of individuals who had
prior TB infection, respond to re-stimulation with MTB specific antigens (early
secretory antigenic target 6 [ESAT-6]) by secreting IFN-ɣ.
 Advantages of this test are that the antigen used is recognised by T-cells of TB patients
and not by BCG-vaccinated or healthy unvaccinated individuals.
 It has a very high specificity (which reflects only recent exposure) and much less likely
than the TST to be confounded by exposure to environmental mycobacteria or by prior
BCG vaccination.

39.  It does not boost responses that will be measured by subsequent tests as happens with
TST.
 IGRAs do not require a second visit to the clinic to evaluate the test result, thus
potentially reducing costs to the patient. Results can be available within 24 h.
 Disadvantages: the sample drawn should be incubated within 16 h for older IGRAs and
8 h for the T-SPOT of collection which may require the use of portable incubators or
establishment of systems enabling transportation to properly equipped laboratories
for testing.
 Commercially available tests are QuantiFERON-TB Gold (QFT-G) and
QuantiFERON-TB Gold in Tube (QFT-GIT) (Cellestis, Australia) and T-SPOT TB
(Immunotec, UK).

40.  There are insufficient data and low-quality evidence on the performance of IGRAs in
low- and middle-income countries, typically those with a high TB and/or HIV burden.
In 2011, WHO has recommended against the use of IGRAs in these scenarios.
 Neither IGRAs nor the TST should be used for the diagnosis of active TB disease.

41. Test based on Biomarkers
 The current requirement of sensitive and specific diagnostic tests which can be
performed in peripheral laboratory settings using easily accessible samples such as
blood, urine or breath led us to biomarkers as a diagnostic option.
 Biomarkers may be classified as:
A. Those related to pathogen
B. Those related to host

42. Those Related to Pathogen
 Lipoarabinomannan (low
sensisitivity. High HIV prevalent
population)
 Transdermal MPB64Ag: low
sensitivity (latent TB)
 Volatile organic compounds;
Sputum, urine, breath
Those Related to Host
 ADA in body fluids (produced by
mycobacterial anti- gens
stimulating T cell lymphocytes),
poor sensitivity
 Other markers under evaluation:
erum Micro RNAs, IFN ɣ, IFN
αβ
 MTB specific antibody detection

45. • Case definition: There are significant changes in the definition of cases as per New
Guidelines:
 Microbiologically confirmed TB case refers to a presumptive TB patient with
biological specimen positive for acid-fast bacilli, or positive for mycobacterium TB
on culture, or positive for TB through Quality Assured Rapid Diagnostic molecular
test.
 Clinically diagnosed TB case refers to a presumptive TB patient who is not
microbiologically confirmed, but has been diagnosed with active TB by a clinician on
the basis of X-ray abnormalities, histopathology or clinical signs with a decision to
treat the patient with a full course of anti-TB treatment.

46. Current recommendations.
 At DMCs, LED microscopy to detect MTB has been adopted by the RNTCP is going
to replace conventional ZN staining soon.
 In IRLs, commercial automated liquid culture-MGIT is the current gold standard for
detecting MDR-TB and XDR-TB, including in smear-negative cases.
 The LPA is suitable to detect MDR-TB only in smear-positive cases.
 Genotypic automated NAATs have considerable advantage in programme management
and surveillance of MDR-TB.
 The RNTCP discourages the use of the indirect test IGRA in India.