Diagnostic Accuracy of FluoroCycler® XT MTBDR Assay for Detection of Rifampicin and Isoniazid Resistant <i>Mycobacteria tuberculosis</i> in Clinical Isolates from Kenya

Abstract Background Drug-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 in Mycobacterium tuberculosis (M.tb) is GenoType MTBDR plus . Although the GenoType MTBDR plus is 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 in rpoB, katG and inhA genes 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. Methods The 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 in rpoB , 51 isolates with katG mutations, 51 isolates with mutations in inhA . and 91 M.tb isolates lacking mutations in these genes based on Genotype MTBDR plu s results. DNA from the isolates was extracted using the FluoroLyse extraction kit. Real-time PCR targeting the rpoB, InhA , and katG genes was performed using the FluoroType MTBDR amplification mix. Isolates with discordant results between Genotype MTBDR plu s and FluoroCycler® MTBDR assays underwent targeted sequencing for the respective genes, then sequences were analyzed for mutations using Geneious version 11.0 software. Results The sensitivity of the Fluorocycler XT MTBDR assay for detection of mutations that confer drug resistance was 86% (95% CI 73.0,94.0) for rpoB , 96% (95% CI 87, 100) for katG and 92% (95% CI 81, 98) for inhA . The assay’s specificity was 97% (95% CI 93, 99) for rpoB , 98% (95% CI 96, 100) for katG and 97% (95% CI 93, 99) for inhA . Discrepancy between Genotype MTBDR plus and FluoroType MTBDR results were observed in 28 (11.5%) isolates with rpoB, katG and inhA genes 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) for rpoB , 80% (4/5) for katG , and 70% (7/10) for inhA compared to 23% (3/13), 20% (1/5), and 30% (3/10) for Genotype MTBDR plus assay Conclusion The diagnostic accuracy of FluoroType MTBDR for the detection of mutations conferring resistance to RIF and INH was high compared with that of Genotype MTBDR plus , and demonstrates its suitability as a replacement assay for Genotype MTBDR plus .