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The development of Xpert MTB / RIF assay, aka Gene Xpert as well as CBNAAT, a molecular assay that could be used close to the point of care and a closed system requiring minimal technical expertise as well as use of single use disposable cartridge (preventing cross contamination) with complete information becoming available in two hours, thus appeared to be the answer to all the diagnostic needs of the community [2].

Following endorsement by the World Health Organization in 2010, Gene Xpert was widely implemented for rapid molecular diagnosis in India and has been available free of charge in government set up and at fixed price in the private laboratory set up.

More than 40 million cartridges have been used worldwide for diagnosis of tuberculosis. It soon became obvious that Gene Xpert was the best diagnostic method not only for smear positive sputum samples but also for smear negative but culture positive samples and later on found to be the best available method for extra pulmonary samples too [3].

Subsequently it emerged that while Gene Xpert was excellent for respiratory samples, it performance was suboptimal in paucibacillary tuberculosis such as meningitis, childhood tuberculosis, genital Tb and household contacts of TB patients. The limit of detection (LOD) of Gene Xpert aka CBNAAT was 131 cfu/ml. Consequently, Gene Xpert Ultra with LOD of 16 cfu/ml was developed and this increase in sensitivity made Gene Xpert Ultra better than Gene Xpert and MGIT Culture in the field. Even Gene Xpert XDR has been developed, but recently the definition of XDR has changes and following success of all oral BPal regimen, WHO has discouraged the use of injectable anti TB drugs, making Gene Xpert XDR not suitable for this purpose [4].

One aspect which has troubled introspective scientists is that Gene Xpert detects only Rifampicin susceptibility and this is used as a surrogate for MDR TB as it is presumed that INH susceptibility mirrors that of Rifampicin susceptibility, which means if the isolate is Rifampicin susceptible, the isolate was termed as DS TB without determining susceptibility of INH and if isolate is Rifampicin resistant, then the isolate would be considered to be MDR TB, which has been shown to be factually incorrect. When INH susceptibility was determined, usually by LPA 1, it was found that INH monoresistant (IMR) was not infrequent in India. This would mean that if the isolate is Rifampicin susceptible but IMR, then during the continuation phase, the patient would receive only one effective drug (Rifampicin) for four months and would lead to emergence of MDR TB, on the other hand if Rifampicin was resistant but INH susceptible, and we treat this patient as MDR TB, we would deny the benefit of INH which has excellent anti-bactericidal activity and make the patient take MDR TB treatment, which runs the risk of default and non-compliance.

When Gene Xpert was being evaluated and endorsed by WHO, it was suggested after a modeling experiment that while detection of Rifampicin has far reaching influence on successful treatment, additional detection of INH resistance added minimally to its effectiveness. Subsequent reports from different parts of the world indicated that IMR had a significant effect on treatment and may lead to more deaths than if IMR did not exist [5].

To overcome this drawback in Gene Xpert Ultra, what is required is that the method should not only detect MTBC but also simultaneously report on both INH and Rifampicin susceptibility. A recent report indicated that in India 86% of isolates were both INH and Rifampin susceptible and in these cases only one test would be enough to put the patient of standard four drugs treatment [6].

Recently WHO has validated, approved and recommended for use four moderately complex automated molecular tests for detection of MTBC but also reporting on INH and Rifampicin susceptibility. Application of any one of these tests would enable initiation of appropriate treatment quickly.

Abbott Real-time MTB and MTB RIF/INH Assays

The automated Real-time MTB (Abbott, Chicago, IL, USA) assay can diagnose MTBC in high-throughput mode (96 samples including two assay controls), with positive specimens reflexed to the MTB RIF/ INH assay (24 samples including two assay controls) for full MDRTB diagnosis within 10.5 hours. DNA extraction and PCR preparation is first performed by the Abbott m2000sp instrument, after which the PCR plate is sealed and transferred to the m2000rt instrument for realtime PCR. For the diagnosis of MTBC, the assay targets the insertion element IS6110 as well as the pab genes. As a reflex test the detection of resistance to rifampicin and isoniazid the assay targets the rpoB gene, and the katG gene and inhA promoter region, respectively. The assay can discriminate and report high (katG) and low (inhA) isoniazid resistance. DNA extraction relies on the capturing of bacterial DNA to magnetic micro particles subsequent to cell lysis [7].

Becton Dickinson MAX MDR-TB Assay

The Becton Dickinson MAX MDR-TB assay is a real time PCR assay that can be run on the BD MAX System to detect MTBC through targeting IS6110 and IS1081. Detection of resistance to rifampicin and isoniazid is done through targeting the rpoB gene, and the katG gene and inhA promoter region. The assay can discriminate and report high (katG) and low (inhA) isoniazid resistance. The assay can include up to 24 sputum samples per run and reports results within 4 hours. Both DNA extraction and the BD MAX MDR-TB assay procedures are done by the BD MAX System. Bacterial cell lysis is done chemically and by heat, and the released nucleic acids are then captured by magnetic affinity beads. This methodology thereby may include the capturing of extracellular DNA [8].

Hain Lifescience FluoroType MTBDR Assay

The Hain Lifescience (Hain) FluoroType MTBDR assay (Hain Lifescience, Nehren, Germany) uses LATE-PCR amplification and lightson/lights-off chemistry to detect MTBC by targeting the rpoB gene. Detection of resistance to rifampicin and isoniazid is done through targeting the rpoB gene, and the katG gene and inhA promoter region. The high throughput platform can include up to 96 samples (including assay controls) per run and reports results within 4 hours. Not only can the assay differentiate between high- and low-level isoniazid resistance, the run report also includes the specific mutations identified for the three gene targets. DNA extraction and PCR preparation are done by the Geno Xtract 96 (GXT96) instrument, after which the PCR plate is transferred to the FluoroCycler XT instrument for the FluoroType MTBDR assay. For the DNA extraction by the GXT96, the methodology includes the capturing of intact cells to magnetic beads, from where the cells are washed and then lysed. The binding of extracellular DNA to the magnetic beads are very low and not competitive to the bacteria binding but dependent on the salt concentration and pH which may vary between raw specimens, decontaminated specimens and cultured isolates [9].

The Roche cobas MTB assay (Roche, Basel, Switzerland) uses realtime PCR to detect MTBC by targeting 16S rRNA and 5 esx genes. It can generate results for 96 tests (including assay controls) in one 3.5 hour run. MTBC positive specimens are reflexed to the RIF/INH assay (96 tests including assay controls per run) for MDR-TB diagnosis in an additional 3.5 hours.

Similar to the Abbott Real-time platform, the assay targets the rpoB gene, and the katG gene and inhA promoter region for detection of resistance to rifampicin and isoniazid. DNA extraction, PCR preparation, and the cobas MTB and MTB-RIF/INH assays are done in cobas 6800/8800 systems. The sample preparation procedure requires sonication and centrifugation, for which additional instrumentation is needed. Bacterial cell lysis is done chemically (lysis reagent), enzymatically (proteinase), and physically (sonication). Subsequently, the released bacterial DNA is captured by magnetic glass particles. This methodology implies that extracellular DNA may also be captured [8].

The comparative analytical evaluation showed that the platforms from BD, Abbott, and Roche had a similar or lower Limit of Detection (LoD) for MTBC compared to Xpert MTB/RIF. The platform from Hain Lifescience showed an increased LoD compared to Xpert MTB/ RIF, but the clinical significance of this difference is uncertain. All assays were comparable (or slightly more sensitive) to Hain Lifescience GenoType MTBDRplus for the detection of rifampicin and isoniazid resistance.

Similarly, the systematic review and meta-analyses showed overall similar performance of the centralized platforms to the WHO-endorsed assays, although the amount and quality of available clinical data were very limited with available head-to-head studies. Available direct headto- head studies showed comparable performance of Abbott Realtime MTB, BD MAX MDR-TB, and Roche cobas MTB for MTBC detection to Xpert MTB/RIF [9,10].

The assays from Roche, Abbott, and BD showed lower or similar LoD for MTBC compared to Xpert MTB/RIF, while the assay from Hain Lifescience showed 5-6 fold increase in the limit to detect MTBC compared to Xpert MTB/RIF. The higher analytical sensitivity of Abbott Real-time MTB, BD MAX MDR-TB, and Roche cobas MTB compared to Xpert MTB/RIF is most likely explained by these assays targeting multiple genetic regions for MTBC DNA amplification. These three assays also have the capability of capturing extracellular DNA, which may increase the sensitivity of these assays in analytical studies. All centralized assays assessed with the resistant strain panel showed similar or increased accuracy for the detection of rifampicin and isoniazid resistance compared to the comparator test Genotype MTBDRplus. Sensitivity for detection of tested mutations conferring rifampicin and isoniazid resistance was ≥95% of tested strains for all assays when accounting for the global frequency of tested mutations [11].

While all these four tests are high throughput and best suitable for a centralized model of laboratory diagnosis, recently SD Biosensor based in South Korea but with a branch in Manesar has developed a singleuse cartridge-based POCT, which is able to do the same, and its small size makes it suitable for decentralized use. It is under evaluation of ICMR and WHO.

In a meta-analysis of newer molecular methods, Kohli et al. [12] included 21 studies, which contributed a total of 26 datasets. Data limitations meant that they could only meta-analyze data for three out of the five identified assays. For TB detection, the included assays had a sensitivity of 91% or higher and a specificity ranging from 97% to 100%. All included assays for rifampicin resistance detection had a sensitivity of over 92% and a specificity of 99-100%. Sensitivity for isoniazid resistance detection varied from 70% to 91%, but specificity remained high at 99-100% across all index tests. Studies that compared these assays with Xpert MTB/RIF for detection of TB and rifampicin resistance indicated comparable diagnostic accuracy. Centralized molecular assays showed similar diagnostic accuracy to the WHOrecommended Xpert MTB/RIF assay for detecting TB, rifampicin resistance, and isoniazid resistance in people with symptoms of pulmonary TB.

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