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.

Keywords: Deeplex Myc-TB; GeneLEAD VIII; Mycobacterium tuberculosis; diagnostic; resistance; spoligotype.

Figure 1

Three examples of Deeplex Myc-TB results. (A) Successful Deeplex Myc-TB result. (B) Successful Deeplex Myc-TB result with deletion in ethA. (C) Failed Deeplex Myc-TB result. hsp65-based identification best match, spoligotype, and SNP-based lineage identification are shown in the center of the map. Targeted loci are displayed in groups, depending on which antibiotic(s) they impact: rpoB for rifampicin (RIF); katG-ahpC for isoniazid (INH); inhA-fabG1 for INH and ethionamide (ETH); pncA for pyrazinamide (PZA); embB for ethambutol (EMB); rpsL-gidB for streptomycin (SM); gyrA-gyrB for fluoroquinolones (FQ); eis for kanamycin (KAN); rrs for amikacin, capreomycin, and kanamycin (AMI-CAP-KAN); tlyA for CAP, ethA for ETH, rplC-rrl for linezolid (LNZ); and rv0678 for bedaquiline-clofazimine (BDQ-CFZ). Wild-type alleles are shown in green, mutant alleles with mutations linked to resistance in red, and uncharacterized variants in blue (dark blue if the SNV is fixed, light blue if the SNV is unfixed). Gray color indicates that more than 5% of the locus is missing (e.g., in ethA in B). Sequencing depths are indicated above the circle representing the amplified genes, colored in gray if the read depth is >100× on average, and in orange if the read depth is <100× on average. Filled lines shown above the loci names are colored according to the amplification efficiency of each gene: green if >95% of the locus has been sequenced, and black if <95% of the locus has been sequenced. Each variant is noted with the reference codon, codon number, and the alternate codon, followed by the reference amino acid, amino acid position, and the mutated amino acid.