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 Table of Contents  
EDITORIAL
Year : 2021  |  Volume : 1  |  Issue : 2  |  Page : 38-40

Newer molecular diagnostic tests for tuberculosis: How good are they? Where can we use them?


1 Department of Pulmonary Medicine, Government Medical College, Thrissur, Kerala, India
2 Intermediate Reference Laboratory, State TB Cell, Thiruvananthapuram, Kerala, India

Date of Submission07-Feb-2021
Date of Acceptance11-Feb-2021
Date of Web Publication21-Jun-2021

Correspondence Address:
Dr. Sanjeev Nair
5A, Omega Paradise, Thekkemadom Road, Thrissur - 680 001, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jalh.jalh_3_21

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How to cite this article:
Nair S, Murthy N. Newer molecular diagnostic tests for tuberculosis: How good are they? Where can we use them?. J Adv Lung Health 2021;1:38-40

How to cite this URL:
Nair S, Murthy N. Newer molecular diagnostic tests for tuberculosis: How good are they? Where can we use them?. J Adv Lung Health [serial online] 2021 [cited 2021 Oct 21];1:38-40. Available from: http://www.jalh.com/text.asp?2021/1/2/38/318910



Tuberculosis (TB) remains a major cause of mortality in the world and in India with an estimated global incidence of 10 million and mortality of 1.4 million in 2019. India is the highest TB burden country in the world with an estimated incidence of 28 lakhs and mortality of 4.5 lakhs in 2019.[1] The world is moving toward TB elimination, with strategies based on the END TB strategy. The targets set are ambitious and would require widespread use of existing and new tools for case detection, new drugs and regimes, new vaccines, and other innovative strategies for success.[2]

Molecular tests for TB have made a huge impact on the diagnosis of TB as well as drug-resistant TB (DR-TB) worldwide. Diagnostic tests such as line probe assays (LPAs), nucleic acid amplification tests (NAATs) have revolutionized the diagnosis of TB and DR TB. NAATs include “Xpert MTB/RIF,” Xpert® MTB/RIF Ultra' which are cartridge based nucleic acid amplification tests (CBNAATs) and the Truenat Mycobacterium tuberculosis (MTB), MTB Plus and MTB RIF Dx which are chip based real time polymerase chain reaction tests. The newer tests offer higher sensitivity, specificity, as well as faster results. These tests have been endorsed by the World Health Organization (WHO) and the latest among these tests to be endorsed is the Truenat.[3]

Truenat is an indigenously developed diagnostic test from Molbio Diagnostics, Goa. The test was endorsed by the WHO based on the preliminary results available from a multicentric study being conducted in four countries (India, Ethiopia, Peru, and Papua New Guinea) coordinated by the Foundation for Innovative New Diagnostics. The available interim results show high sensitivity and specificity of the Truenat assays for the diagnosis of disease caused by MTB as well as for the sequential rifampicin resistance determination. Based on this evidence, the rapid communication from the WHO endorsed the test saying, “The performance of Truenat MTB, MTB Plus and MTB-RIF Dx assays show comparable accuracy with Xpert MTB/RIF and Xpert Ultra for TB detection (Truenat MTB and Truenat MTB Plus) and for sequential rifampicin resistance detection (Truenat MTB-Rif Dx).”[3]

While the newer diagnostic technologies are extremely good and have made a huge difference in both clinical practice and program management, there is still lack of clarity on how congruous these tests are in the local setting and in relation to patient outcome improvement.

The first among the commonly used molecular tests to be endorsed by the WHO was LPA. This family of DNA strip based tests can be applied on acid fast bacilli smear microscopy positive pulmonary specimens (direct testing) and on culture isolates (indirect testing). However, LPA bears no endorsement for smear-negative and extrapulmonary specimens, leading to significant delay in the results, particularly for extrapulmonary TB (EPTB). LPA is less efficient than conventional culture-based drug susceptibility testing (DST) in diagnosing resistance in samples harboring both drug-susceptible and -resistant MTB isolates (heteroresistance), synonymous mutations, and phylogenetic mutations.

The test which got endorsement for use in EPTB by the WHO was the CBNAAT (Xpert MTB/Rif). The initial documents from the WHO mentioned a sensitivity of 72% and specificity of 99% in the diagnosis of sputum smear-negative TB.[4] A later document in 2014 placed the sensitivity and specificity of CBNAAT as a replacement of smear microscopy in pulmonary TB at 88% and 99% and the sensitivity and specificity in smear-negative TB at 68% and 99%. The sensitivity and specificity for the detection of rifampicin resistance were found to be 95% and 98%.[5] The sensitivity and specificity of CBNAAT in EPTB varied for each form of EPTB. The sensitivity was estimated to be 84.9% for lymph node TB, 79.5% for CSF, 43.7% for pleural fluid, and 83.8% for gastric lavage, when culture was used as the comparator. However, when a composite reference standard (CRS) was used as the comparator, the sensitivity came down to 83.7% for lymph node TB, 55.5% for CSF, and 17% for pleural fluid. Hence, while the test was recommended for most forms of EPTB, it was not recommended for testing pleural fluid in presumptive TB pleural effusion due to the very low yield.[5] This analysis also highlights the problem with choosing the gold standard for comparison in such analysis.

Various articles have discussed the concept of 'reference standard misclassification' associated with imperfect gold standards resulting in variation of validity measures of index tests with varying disease prevalence.[6] A common solution in this regard is to use CRS. The advantages and disadvantages of using CRS as a gold standard have been discussed very clearly by Naaktgeboren et al.[7] While pointing out the advantages in terms of CRS being able to deal with the imperfect reference standard and also being transparent and reproducible, the authors also point out that a combination of imperfect tests to make a CRS is unlikely to produce a CRS of perfect sensitivity and specificity.

In the context of molecular tests in the diagnosis of TB and DRTB, one of the major problems has been the discordance between phenotypic (DST) as determined by culture as opposed to the genotypic DST as determined by the molecular tests. This issue has been highlighted in the Kerala scenario by Sanker et al., when it was shown that keeping culture and DST as the gold standard, the NAATs lead to “false identification” of rifampicin resistance to the tune of 20%.[8] However, the same issue has been addressed by Armand Van Deun in his article with the interesting title “Rifampin drug resistance tests for tuberculosis: challenging the gold standard.” The author makes a suggestion after following up patients with discordant results on genotypic and phenotypic DST and observing their treatment response that when the clinical suspicion is high, the susceptive results on a phenotypic DST may not always be correct.[9] Current National TB Elimination program (NTEP) guidelines do not recommend “confirming” discordance rifampicin results with phenotypic DST.

EPTB is often difficult to diagnose. This is more so for TB pleural effusion, where the yield of even mycobacterial culture on pleural fluid is low. Currently, the best way of diagnosing pleural TB with certainty is thoracoscopy-directed pleural biopsy, with specimen being sent for both histopathology and CBNAAT. However, there are often issues with access to CBNAAT. With the Truenat machines becoming widely available, there is likely to be more interest in using Truenat to test EPTB samples, despite the fact that the machine is endorsed only for testing for sputum in presumptive pulmonary TB.[3] Thomas et al.[10] in their interesting article in this issue of the journal studied the utility of Truenat in the diagnosis of pleural TB, both when testing pleural fluid and when testing the thoracoscopic pleural biopsy specimen. The results are very exciting and should lead to further studies in this regard, so that the findings can be confirmed in multiple centers.

However, the study also brings forth the issues related to such studies. The ideal comparison as gold standard for EPTB specimen would be liquid culture. However, often, liquid culture is not available and investigators are forced to fall back on solid culture which has lower yield in EPTB specimen. Culture results vary from laboratory to laboratory, particularly with respect to false negatives due to harsh decontamination, focusing on the need for culture laboratories to undergo strict quality control and enroll in the quality assurance mechanisms already in place. The issue related to CRS is also to be considered as discussed above.

The NTEP guidelines recommend in the diagnostic algorithm for EPTB that whenever a sample is available for testing in EPTB, such a sample should be sent for CBNAAT.[11] With the broader availability of Truenat, whether such samples can be sent for Truenat would be determined by the results of more studies on the performance of Truenat in such samples. In view of the increasing gap between the demand for testing with NAAT and the access to these tests, in view of limited number of labs and machines, such studies need to be taken up urgently to guide practice and for incorporation into guidelines.



 
  References Top

1.
Global Tuberculosis Report 2020. Available from: https://www.who.int/publications-detail-redirect/9789240013131. [Last accessed on 2020 Dec 03].  Back to cited text no. 1
    
2.
WHO | The End TB Strategy. WHO. World Health Organization; Available from: http://www.who.int/tb/strategy/en/. [Last accessed on 2020 Nov 07].  Back to cited text no. 2
    
3.
WHO Announces Updates on New Molecular Assays for the Diagnosis of Tuberculosis and Drug Resistance. Available from: https://www.who.int/news/item/13-01-2020-who-announces-updates-on-new-molecular-assays-for-the-diagnosis-of-tuberculosis-and-drug-resistance. [Last accessed on 2021 Jan 24].  Back to cited text no. 3
    
4.
WHO | Automated Real-time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifampicin Resistance: Xpert MTB/RIF System. WHO. World Health Organization; Available from: https://www.who.int/tb/publications/tb-amplificationtechnology-statement/en/. [Last accessed 2021 Jan 24].  Back to cited text no. 4
    
5.
WHO | Xpert MTB/RIF Assay for the Diagnosis of Pulmonary and Extrapulmonary TB in Adults and Children. WHO. World Health Organization; Available from: http://www.who.int/entity/tb/publications/xpert_policyupdate/en/index.html. [Last accessed on 2021 Jan 24].  Back to cited text no. 5
    
6.
Valenstein PN. Evaluating diagnostic tests with imperfect standards. Am J Clin Pathol 1990;93:252-8.  Back to cited text no. 6
    
7.
Naaktgeboren CA, Bertens LC, van Smeden M, de Groot JA, Moons KG, Reitsma JB. Value of composite reference standards in diagnostic research. BMJ. 2013 Oct 25;347:f5605. doi: 10.1136/bmj.f5605. PMID: 24162938.  Back to cited text no. 7
    
8.
Sanker P, Ambika AP, Santhosh VT, Kottuthodi RP, Balakrishnan R, Mrithunjayan SK, et al. Are WHO approved nucleic acid amplification tests causing large-scale “false identification” of rifampicin-resistant tuberculosis?: Programmatic experience from south India. Int J Mycobacteriol 2017;6:21.  Back to cited text no. 8
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9.
Van Deun A, Aung KJ, Bola V, Lebeke R, Hossain MA, de Rijk WB, et al. Rifampin drug resistance tests for tuberculosis: Challenging the gold standard. J Clin Microbiol 2013;51:2633-40.  Back to cited text no. 9
    
10.
Thomas Kurian, A R Paramez, Rohita S. C, Nimmy Jose. The Yield of Thoracoscopic Biopsy Truenat in the Diagnosis of Tuberculous Pleural Effusion. Jalh 2021;1:50-4.  Back to cited text no. 10
    
11.
NTEPTrainingModules1to4.pdf. Available from: https://tbcindia.gov.in/WriteReadData/NTEPTrainingModules1to4.pdf. [Last acessed on 2021 Jan 24].  Back to cited text no. 11
    




 

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