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

Abstract

(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 ≥10 3 cfU/ml (c tmin <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.

Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis Isolates from Moroccan Patients: Systematic Review

“…In this review, 49 isolates with confirmed phenotypic RIF-resistance do not harbor any known mutation in the rpoB gene, which may be explained by the fact that RIF resistance-conferring mutations are present elsewhere in the rpoB gene (such as a V146F and I572F) [ 42 , 43 ], suggesting that the nature and frequency of mutations in the rpoB gene vary considerably, between different geographical regions [ 44 ], by the fact that not all the mutations are targeted by the probes used [ 45 ], or as it has been reported that molecular assays still have some drawbacks, such as product cross contamination which is a major problem leading to false positive results [ 20 ]. The reason for this cross contamination has not been elucidated properly [ 46 ], but it may be due to laboratory procedures (protocol for pretreatment, DNA extraction, and detection of the amplification product) [ 47 ]. Therefore, failure to detect RIF-resistance with rapid molecular tests (about 10% of cases in this study), added to the risk factors for unsuccessful TB treatment and MDR-TB development, could risk gains made in the fight against TB.…”

Rapid Tuberculosis Diagnostics Including Molecular First- and Second-Line Resistance Testing Based on a Novel Microfluidic DNA Extraction Cartridge

“…However, the cartridges had to be punctured with a syringe to extract DNA. 22,23 A strength of this TB-Diskebased workflow is that, besides providing DNA for TB testing, a separate external reaction tube containing DNA for additional tests provides maximal flexibility; and no puncturing of the cartridge is required. After diagnosis of TB, Inh, Rif, and SLD resistance markers can be immediately investigated by PCR, LPA, or potentially targeted next-generation sequencing in a stepwise algorithm.…”

Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS

“…In the past, workarounds using the Xpert MTB/Rif Ultra cartridges were considered for further use of the extracted DNA for downstream testing. 48,49 This involves puncturing of the cartridges with a syringe to recover the amplification product. In addition to the risk of injury from syringe handling, the DNA used from Xpert cartridges is already amplified, posing a serious risk of bias and high error rates in downstream analyses.…”

“…In the past, workarounds using the Xpert MTB/Rif Ultra cartridges were considered for further use of the extracted DNA for downstream testing. 48,49…”