Extract from used Xpert MTB/RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk

Rouxjeane Venter,Margaretha De Vos,Grant Theron,Brigitta Derendinger,Stephanie Minnies,Ashley Ruiters,Robin M Warren,Happy Tshivhula,Tania Dolby

doi:10.1038/s41598-020-59164-3

Abstract

Xpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra’s predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥103 CFU/ml (CTmin <25, >“low semi-quantitation”) detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10−3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.

Highlights

Drug-resistant tuberculosis (TB) remains a global threat[1]
As Chamber 2 (C2), Chamber 4 (C4) and diamond-shaped chamber of the cartridge (dCE) gave positive quantitative PCR (qPCR) results on cartridges done on some dilutions, and Chamber 1 (C1) and Chamber 3 (C3) gave none, we only explored the utility of the former for downstream testing using MTBDRplus, MTBDRsl, and FluoroType MTBDR (FT)
We show: (1) MTBDRsl on Ultra dCE when CTmin

Summary

Introduction

Drug-resistant tuberculosis (TB) remains a global threat[1]. Of 10 million estimated incidence cases reported in 2017, 588 000 were rifampicin-resistant[2]. Previous work showed that mycobacterial genomic DNA can be recovered from the rear diamond-shaped chamber of used Xpert MTB/RIF (Xpert) cartridges after the test is complete This diamond cartridge extract (dCE) is useful for downstream testing with the MTBDRsl line probe assay (LPA) (Hain Lifescience, Germany), the only World Health Organization (WHO)-endorsed molecular test for second-line drug resistance, and spoligotyping[6], a method useful for monitoring the molecular epidemiology of TB outbreaks. Showing that the extracted cartridge approach in Ultra is compatible with MTBDRsl and represents minimal rpoB amplicon cross-contamination risk would increase the likelihood of implementation, especially as Xpert is in the process of being phased out in lieu of Ultra This could reduce both sputum collection requirements for complete DST and time-to-effective-treatment initiation

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Results

Conclusion