Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use

Abstract

Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.KEYWORDS 10-color assay, three-phase PCR, XDR-TB, point-of-care test

Evaluation of a Rapid Molecular Drug-Susceptibility Test for Tuberculosis

“…For each probe targeting a resistance-associated genetic region, the melting temperature or temperatures were determined and interpreted as wild-type, mutant (if the measured temperature was different from the known wild-type melting temperature), or heteroresistant (if both wild-type and mutant melting temperatures were present). 11 A specimen was considered to be resistant to a drug if a mutant melting temperature was detected in any of the gene targets associated in high frequency with resistance — namely, katG and the inhA promoter for isoniazid, gyrA for the fluoroquinolones ofloxacin and moxifloxacin, and rrs for kanamycin and amikacin. 3,4,8,11,13–18 A specimen with a wild-type or uninterpretable melting temperature in a high-frequency gene target was considered to be susceptible or of indeterminate susceptibility, respectively, to the corresponding drug, unless there was a mutant melting temperature in the low-frequency gene target — namely, gyrB for fluoroquinolones and the eis promoter for kanamycin.…”

“…11 A specimen was considered to be resistant to a drug if a mutant melting temperature was detected in any of the gene targets associated in high frequency with resistance — namely, katG and the inhA promoter for isoniazid, gyrA for the fluoroquinolones ofloxacin and moxifloxacin, and rrs for kanamycin and amikacin. 3,4,8,11,13–18 A specimen with a wild-type or uninterpretable melting temperature in a high-frequency gene target was considered to be susceptible or of indeterminate susceptibility, respectively, to the corresponding drug, unless there was a mutant melting temperature in the low-frequency gene target — namely, gyrB for fluoroquinolones and the eis promoter for kanamycin. 3,4,8,11,13,15,19,20 The personnel performing the investigational assay were unaware of the other test results.…”

“…3–10 We recently described a new cartridge, for use with the GeneXpert platform, for the rapid molecular detection of these mutations. 11 This assay can provide results from unprocessed sputum samples in just over 2 hours, with minimal hands-on technical time. Here, we describe a clinical study to assess the diagnostic accuracy of this investigational assay for the detection of resistance to fluoroquinolones, aminoglycosides, and isoniazid.…”

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…A potential implementation of the assay targeting relevant regions in gyrA , gyrB , katG and rrs genes and the promoters of inhA and eis genes was described in a pilot study .…”

Some Synonymous and Nonsynonymous gyrA Mutations in Mycobacterium tuberculosis Lead to Systematic False-Positive Fluoroquinolone Resistance Results with the Hain GenoType MTBDR sl Assays

“…Our findings therefore underline the need for diagnostic companies, including Cepheid, which is currently adapting its GeneXpert system for fluoroquinolone testing, to consider the genetic diversity within the MTC at the development stage and to monitor test performance after uptake in clinical settings (19, 33, 34). Importantly, this also applies to software tools designed to automate the analysis of whole-genome sequencing data.…”