Evaluation &validation of a highly multiplexed LATE-PCR single-tube assay for M(X)DR-TB

Abstract

Background: In 2006, the World Health Organization (WHO) and the Stop TB partnership called for strengthening of diagnostic services and highlighted the need for the development of rapid diagnostics to fight the tuberculosis pandemic. In 2011, WHO estimated that approximately 630,000 (5.3%) of the 12 million TB cases had multiple drug resistant (MDR)-TB, while more than 80 countries have reported cases of extremely drug-resistant (XDR)TB. Only a small fraction of reported cases (<20%) were correctly diagnosed and even fewer were treated according to WHO standards. In response the WHO endorsed the Genotype® MTBDRplus (version 1.0) line probe assay (LPA) in 2008 and theXpert®MTB/RIF assay in 2010. But these tests only provide evidence for resistance to isoniazid and/or rifampicin. There continues to be a critical need for a more comprehensive convenient diagnostic technology. The highly multiplexed LATE-PCR assay for M(X)DR-TB described here was designed to meet that need. Our goal was to firmly establish that a highly multiplexed Linear-After-the-Exponential (LATE) PCR single closed-tube assay can simultaneously detect and distinguish multiple mutations in multiple gene targets that are known to confer resistance to isoniazid, rifampicin, ethambutol, ofloxacin, amikacin, kanamycin and capreomycin. Methods & Materials: In this initial study, DNA from clinical isolates with different rpoB, katG, embB, inhA promoter, gyrB, gyrA and rrs genotypes were selected from a DNA bank housed at Stellenbosch University. Each DNA samples was amplified and the singled-strandedDNAproductswere scanned formutations at endpoint using the same mixture of Lights-On/Lights-Off Probes. The resulting fluorescent signatures were compared to that of H37Rv, a pan-susceptible “wildtype” strain. Results: Each clinical isolate harbouring a uniquemutation had its own, highly reproducible fluorescent signature distinct from that of H37Rv, as well as all other isolates with differentmutations. Conclusion: This study achieved the intended transfer of the Brandeis University technology to Stellenbosch University. This study also demonstrates that this single tube multiplexed assay can simultaneously distinguish the different mutations that confer resistance to rifampicin, isoniazid, ethambutol, fluoroquinolones, aminoglycosides and ethionamide in less than three hours. (Supported by NIH Grant R01 A1099532)