Xpert MTB/XDR: a 10-Color Reflex Assay Suitable for Point-of-Care Settings To Detect Isoniazid, Fluoroquinolone, and Second-Line-Injectable-Drug Resistance Directly from Mycobacterium tuberculosis-Positive Sputum

Abstract

We describe the design, development, analytical performance, and a limited clinical evaluation of the 10-color Xpert MTB/XDR assay (CE-IVD only, not for sale in the United States). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH), and second-line injectable drugs (SLIDs) in unprocessed sputum samples and concentrated sputum sediments which are positive for Mycobacterium tuberculosis The Xpert MTB/XDR assay simultaneously amplifies eight genes and promoter regions in M. tuberculosis and analyzes melting temperatures (T_m s) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH, and SLID resistance. Results can be obtained in under 90 min using 10-color GeneXpert modules. The assay can differentiate low- versus high-level resistance to INH and FLQ as well as cross-resistance versus individual resistance to SLIDs by identifying mutation-specific T_m s or T_m patterns generated by the SMB probes. The assay has a limit of detection comparable to that of the Xpert MTB/RIF assay and successfully detected 16 clinically significant mutations in a challenge set of clinical isolate DNA. In a clinical study performed at two sites with 100 sputum and 214 clinical isolates, the assay showed a sensitivity of 94% to 100% and a specificity of 100% for all drugs except for ETH compared to that of sequencing. The sensitivity and specificity were in the same ranges as those of phenotypic drug-susceptibility testing. Used in combination with a primary tuberculosis diagnostic test, this assay should expand the capacity for detection of drug-resistant tuberculosis near the point of care.

Keywords: 10-color; GeneXpert; Mycobacterium tuberculosis; TB; XDR; drug resistance; rapid diagnostics.

FIG 1

Limits of detection of the Xpert MTB/XDR and the Xpert MTB/RIF assays. The two assays were performed side by side, with a minimum of 20 replicates for each cell concentration. Both assays were tested at 10, 20, 40, 60, 80, 100, and 200 CFU/ml, and probit analysis was performed to calculate the LoD using R studio.

FIG 2

Mutation detection in mixtures of WT and mutant DNA. Melt peak heights of each target in cell mixtures containing different ratios of WT and mutant plasmids, where blue dots indicate a susceptible call and red dots indicate mutant calls based on their T_m and melt peak height. The melt peak height is determined by highest distance between the peak of the first-derivative melt curve and baseline. The presence of blue and red dots for any concentration designates detection of both a WT and a mutant T_m. In such cases, the result obtained was “resistance detected” for the corresponding drug. The QRDR mutation D94G generates a mutant T_m only with the gyrA3 probe. Each mixture was tested in triplicates, except the 10% and 75% mixtures, which had two valid replicates.

FIG 3

Scatterplot showing clustering of WT and mutant T_m values from samples tested in the clinical study. One hundred M. tuberculosis-positive frozen sputum samples and 214 clinical isolates for all Xpert MTB/XDR targets were tested and plotted using ggplot in R studio. To prevent overplotting, a degree of jitter was introduced. All green dots are WT T_m, while brown, purple, blue, and pink are mutant T_m values.