**6. Acquired Drug-Resistance of Mtb**

A cocktail of different drugs is used to treat TB. Each molecule binds to one or more target, thus inhibiting their functions. The continuous drug exposure during lengthy treatments and the noncompliance of patients to drug regimens, pushes Mtb to select for mutations in genes encoding drug targets, responsible for development of the majority of resistances in clinical strains [20]. A list of the major target genes that, in the case of mutation, confer resistance to the drugs of the WHO groups A, B and C, is shown in Table 1 [97–125]. Many excellent reviews report the genetic mechanisms involved in this resistance to RIF, INH, KM, CP and other drugs [18,23,24,98,118,126].

**Table 1.** Drugs of the World Health Organization (WHO) groups A, B and C, and list of the most common drug resistance-related target genes.



**Table 1.** *Cont.*

Phenotypic testing is still considered a gold standard for Mtb DST, which is accurate, but takes at least two weeks for results [98]. However, a pivotal role has been recently played by the more and more rapid molecular methods to diagnose drug-resistant TB by the identification of chromosomal mutations, including line probe assays, the Xpert MTB/RIF system (Cepheid, Sunnyvale, CA, USA), target gene sequencing, whole genome sequencing (WGS), point-of-care nucleic acid amplification devices [9,127,128].

The Treatment Action Group (TAG) recently released the pipeline report 2019 on TB diagnostics [129]. The TAG-stratified DST tests for decentralized and centralized laboratories. Useful information was provided on what it is currently in TB diagnostics, including tests already recommended by the WHO, and on which tests are expected to be available soon. As to the decentralized tests, the Xpert MTB/RIF assay (sensitivity and specificity for RIF resistance of 96% and 98%, respectively) was recommended by the WHO in 2010, and entered in the market in the same year. The sensitivity of this assay increased with the 2017 rollout of the Xpert MTB/RIF Ultra cartridge. In 2020, there is expected the WHO evaluation and market entry of Xpert XDR, which will detect resistance to INH, MFX, OFL, AK and KM [129]. In 2013, another company (Molbio Diagnostics, Goa, India) released its systems Truenat MTB and Truenat MTB-RIF Dx onto the Indian market [129]. In January 2020, a rapid WHO Communication reported that the Truenat systems MTB, MTB Plus and MTB RIF Dx assays showed comparable accuracy with Xpert MTB/RIF and Xpert Ultra for Mtb detection (Truenat MTB and Truenat MTB Plus), and for sequential RIF resistance detection (Truenat MTB RIF Dx) [130]. Furthermore, the data for Truenat MTB RIF Dx showed similar accuracy to the WHO approved commercial line probe assays indicated by the TAG for centralized DST [GenoType MTBDR*plus* Version 2.0 (Hain Lifescience, Nehren, Germany) and Nipro NTM+MDRTB detection kit2 (Nipro, Osaka, Japan)] [129,130]. Other systems marketed in 2015–2019 and on the pathway to the WHO evaluation for the centralized determination of molecular resistance to INH and RIF are: Cobas MTB-RIF/INH (Roche, Basel, Switzerland), BD MAX MDR-TB (Becton Dickinson, Sparks, MA, USA), real-time MTB-RIF/INH Resistance assay (Abbott, Abbott Park, IL, USA) and FluoroType MTBDR version 2.0 (RIF, INH) (Hain Lifescience) [129,131–133].

Finally, the WGS technology is capable of identifying the complete drug-resistance profile of an Mtb strain, ideally enabling clinicians to obtain the best anti-TB treatment [98,128,130,134]. However, more data are still needed to correlate genetic mutations with phenotypic resistance, in order to definitely guide the clinical care.

In this view, the initiative of the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) project aims at understanding the relationship between genotypes and resistance by sequencing 100,000 whole TB genomes from various countries, in parallel with comprehensive DST assays. Overall, at this stage, the WGS still needs more studies, but it is commonly believed that this technology will be the future of rapid, centralized DST [129,135].
