A Comprehensive Evaluation of GeneLEAD VIII DNA Platform Combined to Deeplex Myc-TB® Assay to Detect in 8 Days Drug Resistance to 13 Antituberculous Drugs and Transmission of Mycobacterium tuberculosis Complex Directly From Clinical Samples

Abstract

The GeneLEAD VIII (Diagenode, Belgium) is a new, fully automated, sample-to-result precision instrument for the extraction of DNA and PCR detection of Mycobacterium tuberculosis complex (MTBC) directly from clinical samples. The Deeplex Myc-TB® assay (Genoscreen, France) is a diagnostic kit based on the deep sequencing of a 24-plexed amplicon mix allowing simultaneously the detection of resistance to 13 antituberculous (antiTB) drugs and the determination of spoligotype. We evaluated the performance of a strategy combining the both mentioned tools to detect directly from clinical samples, in 8 days, MTBC and its resistance to 13 antiTB drugs, and identify potential transmission of strains from patient-to-patient. Using this approach, we screened 112 clinical samples (65 smear-negative) and 94 MTBC cultured strains. The sensitivity and the specificity of the GeneLEAD/Deeplex Myc-TB approach for MTBC detection were 79.3% and 100%, respectively. One hundred forty successful Deeplex Myc-TB results were obtained for 46 clinical samples and 94 strains, a total of 85.4% of which had a Deeplex Myc-TB susceptibility and resistance prediction consistent with phenotypic drug susceptibility testing (DST). Importantly, the Deeplex Myc-TB assay was able to detect 100% of the multidrug-resistant (MDR) MTBC tested. The lowest concordance rates were for pyrazinamide, ethambutol, streptomycin, and ethionamide (84.5%, 81.5%, 73%, and 55%, respectively) for which the determination of susceptibility or resistance is generally difficult with current tools. One of the main difficulties of Deeplex Myc-TB is to interpret the non-synonymous uncharacterized variants that can represent up to 30% of the detected single nucleotide variants. We observed a good level of concordance between Deeplex Myc-TB-spoligotyping and MIRU-VNTR despite a lower discriminatory power for spoligotyping. The median time to obtain complete results from clinical samples was 8 days (IQR 7–13) provided a high-throughput NGS sequencing platform was available. Our results highlight that the GeneLEAD/Deeplex Myc-TB approach could be a breakthrough in rapid diagnosis of MDR TB in routine practice.

Targeted sequencing from cerebrospinal fluid for rapid identification of drug-resistant tuberculous meningitis

“…Our results are consistent with previous findings that both culture and Xpert MTB/RIF are more sensitive than Deeplex for samples with low bacillary burdens ( 16 ). Deeplex failed to detect Mtb in any of the 17 samples that were Xpert negative but culture positive.…”

“…tNGS from direct samples has potential to greatly accelerate the time to results. As demonstrated elsewhere, it takes 32–71 days to get results by pDST or 22–58 days for WGS (including 20–56 days of culture positive plus 2 days of sequencing), while tNGS takes just 2–3 days ( 16 ). Another potential advantage of tNGS is the opportunity to detect hetero-resistance from direct clinical samples.…”

“…The number of CSF samples required for the study was calculated following the formula for diagnostic studies with binary test outcome (here resistance or susceptible) ( 15 ). Deeplex can detect Mtb in 80.0% of culture-positive sputum samples, with high sensitivity (95.0%) and specificity (97.0%) for detecting Mtb drug resistance ( 11 , 12 , 16 , 17 ). Therefore, around 43 CSF samples, including at least 29 resistant to any first-line drug or streptomycin and 14 fully susceptible samples, would be required to detect a sensitivity and specificity of 95.0% with the marginal errors for sensitivity and specificity of 8.0% and 11.0%, respectively.…”

Tuberkulose im Erwachsenenalter

“…Mit der zurzeit modernsten Technologie, der "targeted next generation Sequenzierung" (targeted NGS), kann parallel eine Vielzahl von resistenzassoziierten Bereichen des Erregergenoms bereits im Untersuchungsmaterial untersucht sowie eine vorläufige Erregertypisierung vorgenommen werden [30,31]. Der Erfolg der Untersuchung korreliert mit der Erregerlast im Ausgangsmaterial und ist bei mikroskopisch positiven Proben größer als bei negativer Mikroskopie.…”

Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory

“…For proximity laboratories unable/unwilling to provide high inoculum MTBC cultures (requiring BSL3 facilities and costly transportation), it was necessary to develop an in silico spoligotyping technique compatible with inactivated samples containing low DNA amounts. A new commercial in vitro diagnostic-targeted NGS test, the Deeplex Myc-TB (GenoScreen, Lille, France), allows for the targeting of 18 MTBC drug resistance-associated genes, combined with genomic targets for mycobacterial species identification and MTBC spoligotyping from samples containing low DNA amounts [ 11 , 12 ]. However, this test remains expensive and no comparison of the outputs of these different methods is available.…”