Diagnostic Accuracy of FluoroCycler® XT MTBDR Assay for Detection of Rifampicin and Isoniazid ResistantMycobacteria tuberculosisin Clinical Isolates from Kenya

Abstract

AbstractBackgroundDrug-resistant TB (DR-TB) poses a major global challenge to public health and therapeutics. It is an emerging global concern associated with increased morbidity and mortality mostly seen in the low- and middle-income countries. Lack of adequate diagnostic equipment for detection and monitoring of DR-TB leads to delayed diagnosis and subsequent inappropriate treatment. TB drug resistance testing has relied on phenotypic presentations in drug sensitivity testing (DST). The cost of setting up a TB phenotypic testing facility is prohibitive for most healthcare facilities due to its intensive investment in infrastructure, equipment, laboratory consumables, and personnel.Molecular techniques are highly sensitive and offer timely and accurate results for TB drug resistance testing, thereby positively influencing patient management plan. The commonly used assay for detection of rifampicin (RIF) and isoniazid (INH) resistance inMycobacterium tuberculosis(M.tb) is GenoType MTBDRplus. Although the GenoType MTBDRplusis more inexpensive and accurate than DST, when compared to other molecular techniques, it requires more specialized expertise, more hands-on time, substantial laboratory infrastructure and result interpretation is subjective to user. The FluoroCycler® MTBDR is a real-time polymerase chain reaction assay that detects M.tb and at the same time identifies mutations inrpoB, katG and inhAgenes that are associated with RIF and INH resistance. It can detect up to 45 mutations in these genes in a single tube, producing results within 2.5 hours and this ability is only comparable to sequencing.MethodsThe study was carried out at the National Tuberculosis Reference Laboratory (NTRL) in Kenya in the period between January to October 2022. A total of 243 M.tb clinical isolates were included in the study. These isolates comprised of 50 isolates with mutations inrpoB, 51 isolates withkatGmutations, 51 isolates with mutations ininhA. and 91 M.tb isolates lacking mutations in these genes based on Genotype MTBDRplus results. DNA from the isolates was extracted using the FluoroLyse extraction kit. Real-time PCR targeting therpoB, InhA, andkatGgenes was performed using the FluoroType MTBDR amplification mix. Isolates with discordant results between Genotype MTBDRplus and FluoroCycler® MTBDR assays underwent targeted sequencing for the respective genes, then sequences were analyzed for mutations using Geneious version 11.0 software.ResultsThe sensitivity of the Fluorocycler XT MTBDR assay for detection of mutations that confer drug resistance was 86% (95% CI 73.0,94.0) forrpoB, 96% (95% CI 87, 100) forkatGand 92% (95% CI 81, 98) forinhA. The assay’s specificity was 97% (95% CI 93, 99) forrpoB, 98% (95% CI 96, 100) forkatGand 97% (95% CI 93, 99) forinhA. Discrepancy between Genotype MTBDRplusand FluoroType MTBDR results were observed in 28 (11.5%) isolates withrpoB, katGandinhAgenes having 26% (13/50), 10% (5/50), and 20% (10/50) isolates with discrepant results respectively. Sequencing results that were in agreement with FluoroType MTBDR results were 77% (10/13) forrpoB, 80% (4/5) forkatG, and 70% (7/10) forinhAcompared to 23% (3/13), 20% (1/5), and 30% (3/10) for Genotype MTBDRplusassayConclusionThe diagnostic accuracy of FluoroType MTBDR for the detection of mutations conferring resistance to RIF and INH was high compared with that of Genotype MTBDRplus, and demonstrates its suitability as a replacement assay for Genotype MTBDRplus.