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.

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 (Tm 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 Tm s or Tm 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.

Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm ) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific "Tm signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.