In silico evaluation of WHO-endorsed molecular methods to detect drug resistant tuberculosis

Abstract

Universal drug susceptibility testing (DST) for tuberculosis is a major goal of the END TB strategy. PCR-based molecular diagnostic tests have been instrumental in increasing DST globally and several assays have now been endorsed by the World Health Organization (WHO) for use in the diagnosis of drug resistance. These endorsed assays, however, each interrogate a limited number of mutations associated with resistance, potentially limiting their sensitivity compared to sequencing-based methods. We applied an in silico method to compare the sensitivity and specificity of WHO-endorsed molecular based diagnostics to the mutation set identified by the WHO mutations catalogue using phenotypic DST as the reference. We found that, in silico, the mutation sets used by probe-based molecular diagnostic tests to identify rifampicin, isoniazid, pyrazinamide, levofloxacin, moxifloxacin, amikacin, capreomycin and kanamycin resistance produced similar sensitivities and specificities to the WHO mutation catalogue. PCR-based diagnostic tests were most sensitive for drugs where mechanisms of resistance are well established and localised to small genetic regions or a few prevalent mutations. Approaches using sequencing technologies can provide advantages for drugs where our knowledge of resistance is limited, or where complex resistance signatures exist.

Mycobacterium tuberculosis Lineage Distribution Using Whole-Genome Sequencing and Bedaquiline, Clofazimine, and Linezolid Phenotypic Profiles among Rifampicin-Resistant Isolates from West Java, Indonesia

“…It was previously believed that phenotypic drug susceptibility testing (DST) employing automated methods like the MGIT 960 system could accurately detect M. tuberculosis susceptibility or resistance to first-line anti-TB medications [ 33 ]. However, these techniques require considerable time and effort and produce results in weeks rather than days [ 33 , 34 ]. Methods that address these limitations are thus needed as alternatives or companions to culture-based methods.…”

Rapid and accurate identification and differentiation of Mycobacterium tuberculosis and non-tuberculous mycobacteria using PCR kits available in a high-burden setting

“…Although these reports endorse the use of PCR-based methods as suitable techniques for rapid mycobacterial detection, clinical performance evaluation studies are necessary as a wide range of sensitivity values were obtained, not only depending on the commercial brand but especially when comparing MTBC with NTM. To date, those reports support that, at least for NTM identification, PCR testing should be accompanied by microbiological analysis for a definitive diagnosis ( 29 , 52 , 54 ).…”

“…Therefore, fast and reliable identification and differentiation of infectious mycobacteria has become a critical priority in the field of microbiological diagnosis, and the implementation of PCR-based techniques has allowed the development of a variety of commercially available kits that perform identification assays in a matter of hours ( 52–54 ). For instance, there are several reports addressing the clinical performance of some commercial kits, such as the Anyplex™ MTB/NTM Real-time Detection V2.0 (Seegene, South Korea), with reported sensitivities for MTB detection ranging from 71 to 86% and reported specificities ranging from 94.9 to 99%; while for NTM detection, the reported sensitivities ranged between 44.9 and 100% and the reported specificities ranged from 97.7 to 97% ( 29 , 31 , 32 , 55 ).…”