Game changing revolutionary newer lab techniques in TB

My co-bacterium TB ! TB poses a significant occupational hazard for healthcare workers in India, which carries the world’s highest TB burden (28 per cent globally), surpassing all other infectious diseases in mortality [1,2] . Annually, India sees approximately 2.8 million new TB cases and 480,000 deaths [1]. MDR-TB and XDR-TB are critical challenges, with India accounting for 27 per cent of the global MDRTB burden (~124,000 cases in 2019)[3-4]. Approximately 9.5 per cent of MDR-TB cases exhibit XDR-TB, rising to 38 per cent in some studies [3-5]. This crisis leads to substantial economic costs too. Treatment success rates for MDR-TB in India are 46 per cent, with a 20 per cent mortality rate [6], and MDR-TB patients have a significantly higher transmission rate (48-99 per cent)[7].

Current TB Diagnosis And Treatment: Scope For Improvement

Conventional TB diagnosis takes 4-6 weeks [8], causing unacceptable delays. Moreover, DOTS requires patients to collect medication thrice weekly, incurring costs due to lost earnings and transportation. Despite current guidelines, MDRTB is often only considered after 4 months of standard antiTB drug treatment (AKT), likely due to the significantly higher cost: Rs 4,000-9,000 for regular TB treatment versus Rs 4-7 Lakh for MDR-TB, with an outlier study stating MDRTB drug costs being 135-fold higher [9], excluding indirect costs [10].

Isn’t it time we change? Why should an average poor patient have to wait for 3-4 months to know whether his TB is MDR or not? In fact, he is expecting a cure by then, or at least being half way through. One should not forget the heavy toll the side effects of AKT takes. Ask me, I’ve been there ! Hence, many simply stop believing in the doctor and discontinue treatment. Thus contributing to the wider spreading of MDR-TB [7]. The incessant rise of MDR and extensively drug-resistant (XDR) TB also poses immense other challenges, including a huge financial burden on the exchequer [5].

Do we just sit by and watch? Definitely not ! Recent advances in TB diagnosis have the potential to be a game changer; especially revolutionary PCR based technologies ( that amplify DNA segments ) which diagnose TB within 90 mins, including knowledge of MDR-TB. As Rifampicin is a surrogate marker for INH, if the patient is Rifampicin resistant on these tests, he can be diagnosed as an MDR-TB case, right up-front and treated thus; instead of commencing initial treatment with regular anti-TB drugs. It’s as simple as that !

Gene-Xpert MTB/RIF, is a 2010 year WHO recommended primary test. It is a cartridge based nucleic acid amplification technique [CBNAAT] to increase TB detection and improve diagnosis of rifampicin (RIF) resistance in pulmonary and extra-pulmonary TB specimens [11] and has already reduced TB mortality by 35 per cent [12]. It requires no special technicians/rooms, can literally be run by anyone; and barely occupies the space of a computer printer. Line Probe Assay (LPA) systems on the other hand are very elaborate, large systems that diagnose TB within 2 days. However they give evidence of resistance to 1st and 2nd line of AKT directly [13]

We should roll out many more GeneXpert/ similar machines throughout the country for quick diagnosis, screening high-risk groups etc. The initial investment is totally worth it, just considering reduction in the long term costs incurred. The Math is very simple. Although, India has performed over 35 lakh CBNAAT tests in 2020 [14], much more needs be done, especially utilising its role to diagnose MDR-TB upfront. A newer GeneXpert, MTB/RIF Ultra has shown a ten-fold improvement in the lower limit of TB detection as well as improved differentiation of [a] certain silent mutations, [b] RIF resistance; and reduced falsepositive results in detecting RIF resistance in paucibacillary specimens [15]. GenXpert MTB/XDR detects resistance to isoniazid (INH), fluoroquinolones (FQ), second-line injectable drug (SLI) (amikacin, kanamycin, capreomycin) and ethionamide (Eto), in a single test [16]. Truenat MTB and Truenat MTB-Rif Dx are recent indigenous chip-based, micro real-time PCR-based NAAT systems for TB detection and rifampicin resistance detection respectively. Results are comparable to similar GenXpert systems; and being portable and battery-operated, are ideal for point-of-care testing [4]

Although GenXpert was initially standardized for only sputum and pleural effusions, knowing that DNA is present even in avascular structures such as hair, nails etc; and having amongst the first few GenXperts, we were the first in the world ( 2012 ) to successfully use the same on a lymph node sample to diagnose TB (and Rifampicin sensitivity ) in 90 mins. How did this innovation come about? I was fortunate that while motivating some teenagers at Georgia Tech University, Atlanta, USA, I had learnt about the discovery of PCR there, by a tipsy student. Despite the extremely odd late night timing he was able to work in the lab there and make this remarkable discovery. I have mentioned this repeatedly since then; no, not to encourage tipsiness, but to remind everyone that permitting students to use the lab 24 hours a day is probably what enabled this remarkable discovery. Maybe our universities can do the same. Who knows what brilliant young minds can come up with?

A few years later a senior journalist friend, who normally was a very strong and composed person (having fought many a hardy battle, including his wife’s chronic illness) broke down while talking to me, stating that his wife was now suspected to have cancer. I told him not to worry and get her for an Ultrasound examination, during which I assured him that it wasn’t cancer but TB (based on an unpublished sign of mine). Despite the same his wife was operated upon to get a better sample of the lymph node in question. I requested for a tiny piece from there and knowing fully well that PCR technology required just a bit of DNA I got the tiny tissue sample processed on our new GenXpert machine. Voila, within 2 hours we had a TB positive diagnosis (+ rifamipicin sensitive TB); which offered immense relief to my senior journalist friend. An Indian’s jugaad ( based on scientific knowledge, used innovatively) had paid extremely rich dividends; setting off a worldwide phenomenon in the bargain ! This is now widely used worldwide for all kinds of pulmonary and extra-pulmonary samples and offers humongous potential to revolutionize TB diagnosis & treatment, including MDR-TB [17,18,19,20].

Virtually any sample including – Pleural, Ascitic and other sterile/nonsterile fluids, tissue specimens from FNACs, biopsies, abscess aspirates, stool specimens and any material collected during PHC/OPD/ ward/imaging/related procedures, including from extra-pulmonary sites, should be routinely sent for GenXpert/ similar testing too. What took weeks earlier via cultures etc, can now be diagnosed within 2 hours.

Recent Advances : CRISPER has captivated the imagination of researchers worldwide. TBQUICK is a novel rapid and ultrasensitive MTB detection platform which combines loopmediated isothermal amplification (LAMP) and CRISPRCas12b reaction for TB detection [21]. It is highly sensitive (with a near single-copy sensitivity), demands less sample input and offers shorter turnaround time than GenXpert. The CRISPR-MTB test exhibited an overall improved sensitivity over both culture (79 per cent vs 33 per cent) and GenXpert (79 per cent vs 66 per cent), without comprise in specificity (62/63, 98 per cent), thus offering great potential as a new diagnostic technique for both pulmonary and extra-pulmonary tuberculosis.

The initial time loss in diagnosis of MDR-TB can be minimised with the use of GeneXpert/Truenat; greatly reducing the frequency and impact of unnecessary empiric treatment, contact investigation [22], also providing substantial patient and programmatic benefits if used in management decisions [15]. It could also be used for developing treatment strategies to end TB [23].

It is also time to endorse the indigenous Truenat MTB and Truenat MTB-Rif Dx chipbased, micro real-time PCRbased NAAT for TB detection and rifampicin resistance detection respectively. They have results comparable to similar GenXpert systems and are much cheaper. Being extremely portable and battery-operated, they are advantageous for remote areas where [a] electricity could be a problem and [b] sending samples to GenXpert Labs is a herculean task [24].

In Summary: India faces a significant TB burden, exacerbated by rising MDR-TB rates. Traditional diagnostic methods are slow, delaying appropriate treatment. Rapid PCRbased diagnostics, like GeneXpert/ Truenat offer a game-changing solution for upfront MDR-TB detection, leading to faster treatment initiation, reduced transmission, and improved patient outcomes. By investing in widespread implementation of these tools and embracing indigenous innovations, India can significantly curb the TB epidemic and move closer to achieving TB elimination.

The Future: Next-generation molecular imaging offers unparalleled holistic 3D characterization and non-invasive monitoring of TB disease dynamics within individual patients, surpassing current diagnostic tools, allowing detailed insights into the dynamics and spatiotemporal disease heterogeneity noted with TB. These readily available technologies hold the potential to advance fundamental TB biology research, accelerate therapeutic development, and serve as precision tools for diagnosis, monitoring, and prognostication in clinical management [25].

Bibliography: 

1. Global Tuberculosis Report 2024. World Health Organization; 2024. [Accessed on Feb 14, 2025]. ISBN 978-92-4-010153-1 https://iris.who.int/bitstream/h andle/10665/379339/ 9789240101531-eng.pdf?sequence=1 
2. National Tb elimination Program. India TB report 2024. GOI,MOHFA, Central T division. [Accessed on Feb 14, 2025]. https://tbcindia.mohfw.gov.in/w p-content/uploads/2024/10/TBReport_for-Web_08_10-2024- 1.pdf
3. Global Tuberculosis Report 20217. World Health Organization; 2017. [Accessed on Feb 14, 2025]. https://www.who.int/publications/i/item/9789241565516
4. Mansi Gupta, et al. Recent updates in diagnosis and management of drug-resistant tuberculosis in India: A paradigm shift and the way ahead during the COVID-19 crisis, Indian Journal of Tuberculosis, Volume 69:3, 264-267, 2022. https://doi.org/10.1016/j.ijtb.2021 .08.013. https://www.sciencedirect.com/science/article/pii/S001 9570721001554 
5. Prasad R, et al. Extensively drug-resistant tuberculosis in India: Current evidence on diagnosis & management. Indian J Med Res. 2017 Mar;145(3):271-293. doi: 10.4103/ijmr. IJMR_177_16. PMID: 28749390; PMCID: PMC5555056. 
6. Report of the first anti-TB drug resistance survey 2104- 2016. MOHFA, GOI. [Accessed on Feb 14, 2025]. https://tbcindia.mohfw.gov.in/wp-content/uploads/2023/05/4187947827National-Anti-TB-Drug-Resistance-Survey.pdf 
7. Kendall EA, et al. Burden of transmitted multidrug resistance in epidemics of tuberculosis: a transmission modelling analysis. The Lancet Respiratory Medicine 2015; 3: 963-972. DOI: https://doi.org/10.1016/ S2213- 2600(15)00458-0. 
8. Zaporojan N, et al. Evolution of Laboratory Diagnosis of Tuberculosis. Clin Pract. 2024 Feb 23;14(2):388-416. doi: 10.3390/clinpract14020030. PMID: 38525709; PMCID: PMC10961697. 9. Kundu D, et al. Innovative social protection mechanism for alleviating catastrophic expenses on multidrug-resistant tuberculosis patients in Chhattisgarh, India. WHO South East Asia J Public Health. 2015 JanJun;4(1):69-77. doi: 

     10.4103/2224- 3151.206624. PMID: 28607277. 

10. Sinha P, et al. How much do Indians pay for tuberculosis treatment? A cost analysis. Public Health Action. 2020 Sep 21;10(3):110-117. doi: 10.5588/pha.20.0017. PMID: 33134125; PMCID: PMC7577002. 
11. Xpert MTB/RIF implementation manual: technical and operational ‘how-to’; practical considerations. World Health Organization. [Accessed on Feb 14, 2025]. https://www.who.int/publications/i/item/9789241506700 
12. Stevens WS. Et al.2017.Impact of the GeneXpert MTB/RIF Technology on Tuberculosis Control. Microbiol Spectr5:10.1128/microbiolspec.tb tb2-0040-2016. https://doi.org/10.1128/microbiolspec.tbtb2-0040-2016 
13. Line Probe Assay [LPA]. Knowledge base for National TB elimination Program. [Accessed on Feb 14, 2025]. https://ntep.in/node/478/CPline-probe-assay-lpa 
14. National Tb elimination Program. India TB report 2024. GOI,MOHFA, Central T division. [Accessed on Feb 14, 2025]. https://tbcindia.mohfw.gov.in/w p-content/uploads/2023/05/India-TB-Report-2020.pdf 
15. Osei Sekyere, J., et al. A Comparative Evaluation of the New Genexpert MTB/RIF Ultra and other Rapid Diagnostic Assays for Detecting Tuberculosis in Pulmonary and Extra Pulmonary Specimens. Sci Rep 9, 16587 (2019). https://doi.org/10.1038/s41598- 019-53086-5 
16. Pillay S, et al Xpert MTB/XDR for detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin. Cochrane Database Syst Rev. 2022 May 18;5(5):CD014841. doi: 10.1002/14651858. CD014841.pub2. PMID: 35583175; PMCID: PMC9115865. 
17. Vankayala VR, et al. Efficacy of cartridge-based nucleic acid amplification test (CBNAAT) in comparison with line probe assay and liquid culture in the evaluation of tubercular lymph node abscess. J Family Med Prim Care. 2024 Jun;13(6):2260-2265. doi: 10.4103/jfmpc.jfmpc_1284_23. Epub 2024 Jun 14. PMID: 39027873; PMCID: PMC11254079. 18. Sachdeva K, et al. CBNAAT: A Boon for Early Diagnosis of Tuberculosis-Head and Neck. Indian J Otolaryngol Head Neck Surg. 2018 Dec;70(4):572-577. doi: 10.1007/s12070-018-1364-x. Epub 2018 Apr 16. PMID: 30464918; PMCID: PMC6224834. 
18. World Health Organization. Xpert MTB/RIF implementation manual: technical and operational ‘how-to’: practical considerations. 2014. ISBN: 978-92-4- 150670-0 [Accessed on Feb 14, 2025]. https://www.who.int/publications/i/item/9789241506700. 
19. Perez-Risco D, et al. Evaluation of the Xpert MTB/RIF Ultra Assay for Direct Detection of Mycobacterium tuberculosis Complex in Smear-Negative Extrapulmonary Samples. J Clin Microbiol. 2018 Aug 27;56(9):e00659-18. doi: 10.1128/JCM.00659-18. PMID: 29950333; PMCID: PMC6113499.
20. Sam IK, et al. TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis. J Infect. 2021 Jul;83(1):54-60. doi: 10.1016/j.jinf.2021.04.032. Epub 2021 May 2. PMID: 33951419. 
21. WHO Global TB report 2021. https://www.who.int/publications/digital/global-tuberculosisreport-2021/tb-diagnosis-treatment/diagnostic-testing#fig–3-2 -8 
22. Chauhan A, et al. The prevalence of tuberculosis infection in India: A systematic review and meta-analysis. Indian J Med Res. 2023 Feb-Mar;157(2&3):135- 151. doi: 10.4103/ijmr.ijmr_382_23. PMID: 37202933; PMCID: PMC10319385.
23. Gomathi NS, et al. Validation of an indigenous assay for rapid molecular detection of rifampicin resistance in presumptive multidrug-resistant pulmonary tuberculosis patients. Indian J Med Res. 2020 Nov;152(5):482-489. doi: 10.4103/ijmr.IJMR_2557_19. PMID: 33707390; PMCID: PMC8157890. 
24. Ordonez AA, et al. Visualizing the dynamics of tuberculosis pathology using molecular imaging. J Clin Invest. 2021 Mar 1;131(5):e145107. doi: 10.1172/JCI145107. PMID: 33645551; PMCID: PMC7919721.