A Rapid Drug Resistance Genotyping Workflow for Mycobacterium tuberculosis, Using Targeted Isothermal Amplification and Nanopore Sequencing

Phenotypic drug susceptibility testing (DST) for tuberculosis (TB) requires weeks to yield results. Although molecular tests rapidly detect drug resistance-associated mutations (DRMs), they are not scalable to cover the full genome and the many DRMs that can predict resistance. Whole-genome sequencing (WGS) methods are scalable, but if conducted directly on sputum, typically require a target enrichment step, such as nucleic acid amplification. We developed a targeted isothermal amplification-nanopore sequencing workflow for rapid prediction of drug resistance of TB isolates. We used recombinase polymerase amplification (RPA) to perform targeted isothermal amplification (37°C for 90 min) of three regions within the Mycobacterium tuberculosis genome, followed by nanopore sequencing on the MinION. We tested 29 mycobacterial genomic DNA extracts from patients with drug-resistant (DR) TB and compared our results to those of WGS by Illumina and phenotypic DST to evaluate the accuracy of prediction of resistance to rifampin and isoniazid. Amplification by RPA showed fidelity equivalent to that of high-fidelity PCR (100% concordance). Nanopore sequencing generated DRM predictions identical to those of WGS, with considerably faster sequencing run times of minutes rather than days. The sensitivity and specificity of rifampin resistance prediction for our workflow were 96.3% (95% confidence interval [CI], 81.0 to 99.9%) and 100.0% (95% CI, 15.8 to 100.0%), respectively. For isoniazid resistance prediction, the sensitivity and specificity were 100.0% (95% CI, 86.3 to 100.0%) and 100.0% (95% CI, 39.8 to 100.0%), respectively. The workflow consumable costs per sample are less than £100. Our rapid and low-cost drug resistance genotyping workflow provides accurate prediction of rifampin and isoniazid resistance, making it appropriate for use in resource-limited settings

Comparative genomics of Cluster O mycobacteriophages

Mycobacteriophages - viruses of mycobacterial hosts - are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages - Corndog, Catdawg, Dylan, Firecracker, and YungJamal - designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange

Genomic and functional analyses of mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance.

CAPRISA, 2016.Abstract available in PDF file

Evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of Mycobacterium tuberculosis isolates from KwaZulu-Natal.

CAPRISA, 2015.Abstract available in pdf

Evolution of extensively drug-resistant tuberculosis over four decades revealed by whole genome sequencing of Mycobacterium tuberculosis from KwaZulu-Natal, South Africa

The largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) was identified in Tugela Ferry, KwaZulu-Natal (KZN), South Africa in 2005. The antecedents and timing of the emergence of drug resistance in this fatal epidemic XDR outbreak are unknown, and it is unclear whether drug resistance in this region continues to be driven by clonal spread or by the development of de novo resistance. A whole genome sequencing and drug susceptibility testing (DST) was performed on 337 clinical isolates of Mycobacterium tuberculosis (M.tb) collected in KZN from 2008 to 2013, in addition to three historical isolates, one of which was isolated during the Tugela Ferry outbreak. Using a variety of whole genome comparative approaches, 11 drug-resistant clones of M.tb circulating from 2008 to 2013 were identified, including a 50-member clone of XDR M.tb that was highly related to the Tugela Ferry XDR outbreak strain. It was calculated that the evolutionary trajectory from first-line drug resistance to XDR in this clone spanned more than four decades and began at the start of the antibiotic era. It was also observed that frequent de novo evolution of MDR and XDR was present, with 56 and 9 independent evolutions, respectively. Thus, ongoing amplification of drug-resistance in KwaZulu-Natal is driven by both clonal spread and de novo acquisition of resistance. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics that assess only rifampicin resistance

Demographic characteristics of participants and phenotypic drug susceptibility of strains.

<p>* HIV-positive and HIV-negative individuals were compared using Fisher’s exact test for categorical variables and nonparametric Mann-Whitney test for continuous variables.</p><p>^† Data in this row are from the XDR data, not the total dataset.</p><p>Results having a <i>p</i>-value < 0.05 were considered statistically significant.</p><p>Data are <i>n</i> (%) or mean ± standard deviation (SD).</p

Isoniazid resistance is the first step towards drug resistance.

<p>Acquisition of <i>katG</i> S315 mutations precedes all other resistance mutations, including rifampicin, in all instances in which the order of acquisition can be disambiguated. For the 214 strains with genotypic resistance to two or more MDR or XDR defining drugs, and in which the order of acquisition of these mutations could be disambiguated, we quantified the number of evolutions in which resistance to one drug was gained before resistance to a second drug. Isoniazid resistance was divided into mutations conferred by the <i>katG</i>S315 codon versus “Other INH” mutations (defined as loss-of-function mutations in <i>katG</i> that do not involve codon 315 or mutations in the <i>inhA</i> promoter). Reported numbers represent the number of independent evolutionary events (not the number of strains) in which the drug resistance indicated by the row labeled “first resistance” was acquired before the drug resistance indicated by the column labeled “second resistance.” The background color is shaded to indicate the fraction of unambiguous evolutionary events in which the “first resistance” was acquired before the “second resistance” for that given drug pair.</p