Comparison of Three Commercial Molecular Assays for Detection of Rifampin and Isoniazid Resistance among Mycobacterium tuberculosis Isolates in a High-HIV-Prevalence Setting

Abstract

cIn a head-to-head comparison of the MTBDRplus version 2.0 (Hain Lifescience), the Xpert MTB/RIF (Cepheid), and the Anyplex MTB/NTM (Seegene) assays, we demonstrated equal sensitivity (59/61; 96.7%) and specificity (53/54; 98.1%) for detecting rifampin resistance with further analysis of discordances. The Xpert assay does not detect isoniazid resistance while the Anyplex assay showed high false positivity. There are a limited number of commercial molecular assays available for the rapid detection of drug-resistant tuberculosis (DR-TB), and currently only two are endorsed by the WHO for this purpose: the GenoType MTBDRplus version 1.0 (Hain Lifescience GmbH, Germany) and the Xpert MTB/RIF (Cepheid, USA) (1-3). The MTBDRplus has been further optimized, and this new version (2.0) can now be performed on both smearpositive and smear-negative clinical specimens as well as cultured isolates, according to the manufacturer (4, 5).The Anyplex MTB/NTM (Seegene, South Korea) assay has not undergone WHO review for use in detecting DR-TB but is widely used. It is a multiplex real-time PCR assay capable of distinguishing between Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) while allowing for the amplification of drug resistance-related gene (rpoB, katG, and inhA) sequences simultaneously (6, 7).In this study, we evaluated the performance characteristics of these three molecular assays for the detection of DR-TB by performing a head-to-head comparison of these technologies.This was a retrospective laboratory-based evaluation study. Cultured isolates were collected from the TB laboratory, Diagnostic Division of the Department of Medical Microbiology, Tshwane Academic Division, National Health Laboratory Services (NHLS). The results from the MTBDRplus version 1.0 assay, performed as part of the routine laboratory testing formed the basis of stratification into 50 multidrug-resistant (MDR) isolates, 30 fully susceptible isolates, 20 monoresistant (10 rifampin and 10 isoniazid resistant) isolates, and 20 isolates with undefined mutations. The 20 isolates with undefined mutations, having a wild-type missing with no corresponding mutation band on the MTBDRplus version 1.0 assay, were further characterized by means of Sanger sequencing.These isolates were collected consecutively to make up a total of 120, which was calculated to provide the required sample size for the comparative evaluation. Four isolates from this sample subset were excluded as they were duplicates and another had poor banding repeatedly on the MTBDRplus version 2.0 assay. A total of 115/120 (96%) cultured isolates were analyzable on all three systems.The MTBDRplus version 2.0, the Xpert MTB/RIF (GXP) (G3 cartridge), and the Anyplex MTB/NTM (Anyplex) assays were performed according to the manufacturer's instructions. For the GXP, 1 ml of cultured isolate was used, and the sample reagent buffer was added in a 2:1 ratio. The extracted DNA templates of isolates with discordant results and isolates with undefined mutations based on MTBDRplus versio...

MTBDRplus for the rapid diagnosis of ocular tuberculosis and screening of drug resistance

“…On one and a half year of follow-up, this particular patient improved clinically without receiving MDR treatment. Similar observation has been made by prior researchers who have documented at least one case of false rifampicin resistance by MTBDRplus assay in comparison with rpoB sequencing, 24 DST, 20,21 or CRS. 18 Such cases of false rifampicin resistance could have arisen due to cross-contamination at any step of the assay thus emphasizing the importance of separate working areas for each step.…”

“…Further, unlike our study, none of these studies have confirmed their results by carrying out gene sequencing for the offending drug. Two other studies, 15,24 comparing results of MTBDRplus assay version 2.0 with sequencing have reported a sensitivity of 96.7 and 100%, respectively, for detecting rifampicin resistance and 100% each for detecting isoniazid resistance. The present study differs from these two by the fact that while the former was carried out on 115 culture-positive undefined (PTB or EPTB) samples, the latter was conducted on mere eleven VF samples from cases of IOTB.…”

“…Interestingly, another commercially available molecular platform, the GeneXpert MTB/RIF assay, is also known to report 'false' rifampicin resistance 26,27 despite being a semi-automated closed system with minimal chances of cross contamination. Prior studies have also documented cases of false rifampicin-susceptibility by MTBDRplus assay, i.e., these cases were wrongly labeled rifampicin susceptible by MTBDRplus assay whereas they were resistant either by sequencing (n = 2) 24 or DST (n = 1 each). 20,22 Discordant results for isoniazid resistance, as reported earlier, [20][21][22] could arise due to loss of DNA during manual processing (unlike GeneXpert), presence of inhibitors, or the phenomenon of 'heteroresistance'.…”