Xpert MTB/XDR: A ten-color reflex assay suitable for point of care settings to detect isoniazid-, fluoroquinolone-, and second line injectable drug-resistance directly fromMycobacterium tuberculosispositive 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 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 probes (SMB) to identify mutations associated with INH, FLQ, ETH and SLID resistance. Results can be obtained under 90 minutes and requires 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 expected to expand the capacity for detection of drug-resistant tuberculosis.

New Xpert MTB/XDR: added value and future in the field

“…This is close to WHO targets for diagnostic sensitivity (>95%) and specificity (>98%) of rapid DST [10]. However, the assays' diminished ability to detect mutations (S91P/A90V, D94A) causing low level fluoroquinolone resistance, especially in hetero-resistant strain populations [3], is a concern and highlights the need for further careful monitoring of discrepant geno/phenotypic DST results in different settings.…”

“…The 2020 World Health Organization (WHO) recommendations for the treatment of RR/MDR-TB recognise the key contribution of later generation fluoroquinolones in shorter duration all-oral regimens [6]. In initial evaluations, Xpert MTB/ XDR was able to identify fluoroquinolone resistance with 91.4% sensitivity and 98.5% specificity compared to phenotypic DST [3]. This is close to WHO targets for diagnostic sensitivity (>95%) and specificity (>98%) of rapid DST [10].…”

“…Isoniazid resistance is a key precursor to MDR-TB [12,13], which indicates the need for improved detection and appropriate treatment of non-MDR isoniazid resistant strains that are not detected by Xpert MTB/RIF or Ultra. Few countries have laboratory capacity to assess isoniazid resistance [14], which the new assay has excellent capacity to detect; sensitivity 98.3% and specificity 95% compared to phenotypic DST [3]. This is important given that WHO recommends a modified treatment regimen for these patients to improve treatment outcomes and to limit MDR-TB generation [6].…”

“…Samples such as fine needle aspiration biopsies may deliver excellent yields, as for Xpert MTB/RIF [25], while reduced sensitivity is expected in cerebrospinal fluid, although a positive test will have major clinical significance [26]. The new assay demonstrated comparable sensitivity to Xpert MTB/RIF in pulmonary samples, but its performance using extra-pulmonary samples has not been evaluated [3,27]. The limit of detection for Mycobacterium tuberculosis by Xpert MTB/XDR (71.9 CFU•mL −1 ) is comparable to MTB/RIF (86.9 CFU•mL −1 ), but not as low as Ultra (15.6 CFU•mL −1 ).…”

“…The recent launch of the new Xpert MTB/XDR assay (Cepheid Inc., Sunnyvale, CA, USA) by the Foundation for Innovative New Diagnostics [1] was widely welcomed by the tuberculosis (TB) community, given the critical need for new and better diagnostics to guide the treatment of drug resistant tuberculosis (DR-TB). Xpert MTB/XDR detects resistance to isoniazid (target genes: inhA promoter, katG, fabG1, oxyR-aphC intergenic region), ethionamide (inhA promoter), fluoroquinolones (gyrA and gyrB), and second-line injectables (rrs and eis promoter) and is positioned as an add-on or "reflex test" in patients with rifampicin resistance detected by Xpert MTB/RIF or Ultra [2,3].…”