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 US). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH) and second-line injectable drugs (SLID) on 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 (Tms) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH and SLID resistance. Results can be obtained in under 90 minutes 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 Tms or Tm patterns generated by the SMB probes. The assay has a limit of detection comparable to the Xpert MTB/RIF assay and succesfully 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 94-100% and a specificity of 100% for all drugs except for ETH when compared to sequencing. The sensitivity and specificity when compared to phenotypic drug-susceptibility testing were in the same range. Used in combination with a primary tuberculosis diagnostic test, this assay is should expand the capacity for detection of drug-resistant tuberculosis near the point of care.

Clinical evaluation of the Xpert MTB/XDR assay for rapid detection of isoniazid, fluoroquinolone, ethionamide and second-line drug resistance: A cross-sectional multicentre diagnostic accuracy study

“…As the study was designed to assess the Xpert MTB/XDR assay as a reflex test to any TB positive result, 7 only Xpert MTB/RIF and Ultra M. tuberculosis -positive patients were enrolled in our study, and so no data were available to confirm Xpert MTB/XDR specificity for TB detection. Although previous studies have demonstrated the limit of detection of the Xpert MTB/XDR assay to be equivalent to Xpert MTB/RIF, 10,18 additional prospective, clinical data would be helpful to support this finding and define best use cases. Furthermore, the study was designed to evaluate a high number of drug-resistant specimens to provide accurate sensitivity estimates, and this was achieved by testing mostly RIF resistant samples, but also meant that sample size was relatively low to assess Xpert MTB/XDR specificity for INH resistance detection.…”

“…Upon closer examination, it was determined that 11 of the 14 false-negative results in New Delhi were from specimens that were phenotypically SLI-susceptible but heteroresistant by WGS, with rrs c1402a and g1484t mutations present in only a fraction of WGS reads at levels likely undetectable by the Xpert MTB/XDR assay (the rrs c1402a mutation was detected in <50% of reads for nine of the 11 samples). 10,18 Ultimately, the clinical relevance of these minor resistant populations is unclear given that these samples were phenotypically SLI-susceptible. If only pDST was considered as a reference standard in this study, the sensitivity of the Xpert MTB/XDR assay would improve to 67–75% for the detection of SLI resistance in New Delhi and 75–92% overall.…”

“…In a retrospective study of 314 clinical sputum samples and sediments in two laboratories, the final assay demonstrated high performance compared with sequencing (99·7%, 97·5%, 100%, 96·5%, 94·1% and 88·5% for detection of INH, FLQ, AMK, KAN, CAP and ETH resistance respectively, with a specificity of 100% for all the drugs except for ETH, for which the assay demonstrated a specificity of 97·3%). 10…”

Controlling the drug-resistant tuberculosis epidemic in India: challenges and implications

“…As a result, despite the enormous success of scaling up CBNAATs, just 44% of estimated MDR cases were diagnosed in India in 2019 [ 11 ]. In July 2020, the Foundation for Innovative New Diagnostics and Cepheid, Inc., announced the launch of the new Xpert MTB/XDR cartridges, which enable expanded DR profiling against multiple drugs such as isoniazid, ethionamide, fluoroquinolones, amikacin, kanamycin, and capreomycin with a turnaround time of less than 90 minutes [ 20 ]. While such tests can greatly overcome the diagnostic limitations of existing CBNAAT systems, they are still being evaluated by the WHO, and it may take considerable time to scale them up (with consideration of a cost-benefit analysis and infrastructure) after being introduced into the Indian market.…”

Personalised Medicine for Tuberculosis and Non-Tuberculous Mycobacterial Pulmonary Disease

“…Xpert ® MTB/RIF Ultra and Xpert ® MTB/RIF have a similar sensitivity (94.9% vs. 95.3%, respectively) and specificity (99.1% vs. 98.8%) for detecting rifampicin resistance [61]. The recently developed Xpert ® MTB/XDR (Cepheid, Sunnyvale, CA, USA), capable of identifying 16 resistance-conferring mutations in M. tuberculosis (Table 1) [62], has been shown to have a high sensitivity (94.1-100%) and specificity (100%) for detecting resistance to isoniazid, fluoroquinolones and second-line injectable drugs; the sensitivity and specificity for detecting ethionamide resistance was slightly lower (88.5% and 97.3%, respectively) [63]. Another study has shown that the assay is capable of detecting 100% of the tested mutations for resistance to these drugs but has more variable outcomes for detecting heteroresistance [64].…”