Nanopore Sequencing for Mycobacterium tuberculosis: a Critical Review of the Literature, New Developments, and Future Opportunities.

The next-generation, short-read sequencing technologies that generate comprehensive, whole-genome data with single nucleotide resolution have already advanced tuberculosis diagnosis, treatment, surveillance, and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long-read ONT sequencing data with potential applications for M. tuberculosis. Only eight studies presented results of ONT sequencing of M. tuberculosis, of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data are available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.

Urgent considerations for booster vaccination strategies against Ebola virus disease.

With two endorsed and prophylactic vaccines against Zaire ebolavirus (referred to hereafter as EBOV), the number of individuals vaccinated against EBOV worldwide is estimated to range between 500 000 and 1 000 000 individuals, increasing with every renewed EBOV threat and vaccination campaign. Therefore, re-exposure of previously vaccinated health-care workers, and possibly community members, could become more frequent. In the absence of long-term data on vaccine efficacy and duration of protection, we urgently need to understand revaccination strategies that could maximise the level of protection. In this Personal View, we highlight the scarcity of available evidence to guide revaccination recommendations for the accumulating groups of previously vaccinated communities or front-line health-care workers that could be redeployed or re-exposed in the next EBOV outbreak(s). This evidence base is crucial to identify optimal target populations and the frequency of booster doses, and guide vaccine interchangeability (especially in settings with limited or unpredictable vaccine supplies), while preventing vaccine mistrust, equity concerns, and exclusion of vulnerable populations. We discuss five priority gaps (to whom, when, and how frequently, to provide booster doses; long-term correlates and thresholds of protection; the effect of vector-directed immunity and viral variant protection; comparative research in mix-and-match schedules; and implementation concerns) that should be urgently tackled to adapt the initial EBOV prophylactic vaccination strategies considering potential booster dose vaccinations.

The Detection of Mutations and Genotyping of Drug-Resistant Mycobacterium tuberculosis Strains Isolated from Patients in the Rural Eastern Cape Province.

Drug-resistant tuberculosis (DR-TB) is still a major public health concern in South Africa. Mutations in M. tuberculosis can cause varying levels of phenotypic resistance to anti-TB medications. There have been no prior studies on gene mutations and the genotyping of DR-TB in the rural Eastern Cape Province; hence, we aimed to identify DR-TB mutations, genetic diversity, and allocated lineages among patients in this area. Using Xpert® MTB/RIF, we assessed the rifampin resistance of sputum samples collected from 1157 patients suspected of having tuberculosis. GenoType MTBDR plus VER 2.0 was used for the detection of mutations causing resistance to anti-TB medications. The next step was to spoligotype 441 isolates. The most prevalent rifampin resistance-conferring mutations were in rpoB codon S531L in INH-resistant strains; the katG gene at codon S315TB and the inhA gene at codon C-15TB had the most mutations; 54.5% and 24.7%, respectively. In addition, 24.6% of strains showed mutations in both the rpoB and inhA genes, while 69.9% of strains showed mutations in both the katG and rpoB genes. Heteroresistance was seen in 17.9% of all cases in the study. According to spoligotyping analysis, Beijing families predominated. Investigation of the evolutionary lineages of M. tuberculosis isolates can be carried out using the information provided by the study's diversity of mutations. In locations wherein these mutations have been discovered, decision-making regarding the standardization of treatment regimens or individualized treatment may be aided by the detection frequency of rpoB, katG, and inhA mutations in various study areas.

Spatial Distribution of Drug-Resistant Mycobacterium tuberculosis Infections in Rural Eastern Cape Province of South Africa.

Tuberculosis (TB), an infectious airborne disease caused by Mycobacterium tuberculosis (Mtb), is a serious public health threat reported as the leading cause of morbidity and mortality worldwide. South Africa is a high-TB-burden country with TB being the highest infectious disease killer. This study investigated the distribution of Mtb mutations and spoligotypes in rural Eastern Cape Province. The Mtb isolates included were 1157 from DR-TB patients and analysed by LPA followed by spoligotyping of 441 isolates. The distribution of mutations and spoligotypes was done by spatial analysis. The rpoB gene had the highest number of mutations. The distribution of rpoB and katG mutations was more prevalent in four healthcare facilities, inhA mutations were more prevalent in three healthcare facilities, and heteroresistant isolates were more prevalent in five healthcare facilities. The Mtb was genetically diverse with Beijing more prevalent and largely distributed. Spatial analysis and mapping of gene mutations and spoligotypes revealed a better picture of distribution.

Optimizing liquefaction and decontamination of sputum for DNA extraction from Mycobacterium tuberculosis.

Whole genome sequencing (WGS) can investigate the entire Mycobacterium tuberculosis (Mtb) genome but currently requires large amounts of mycobacterial DNA, necessitating culture. Culture-free Mtb WGS could revolutionize the clinical use of WGS but is hampered by the high viscosity, low mycobacterial load, and high contamination with bacterial and human DNA in sputum samples. To improve the sputum liquefaction and decontamination step prior to DNA extraction, we assessed the efficiency of Myco-TB, MycoPrep, and Sputolysin with/without TiKa-Kic in liquefying and decontaminating sputum and aimed to evaluate the effect of these approaches on mycobacterial viability, and Mtb DNA quality and quantity. Experiments using spiked sputum samples showed that Myco-TB and BD MycoPrep with standard (15 min) or increased (30 min) incubation time, but not reduced (7,5 min) incubation time performed well in liquefying and decontaminating sputum. No difference in DNA quality or quantity, contamination, or the amount of human DNA present was observed. In comparison, Sputolysin with/without TiKa-Kic was less effective for liquefaction and decontamination of sputum. PCR amplification of the human GAPDH gene after sputum treatment, showed the presence of human DNA in all samples, regardless of sputum treatment. Focused efforts are needed to deplete contaminating DNA for culture-free Mtb WGS.

Transmission, distribution and drug resistance-conferring mutations of extensively drug-resistant tuberculosis in the Western Cape Province, South Africa.

Extensively drug-resistant tuberculosis (XDR-TB), defined as resistance to at least isoniazid (INH), rifampicin (RIF), a fluoroquinolone (FQ) and a second-line injectable drug (SLID), is difficult to treat and poses a major threat to TB control. The transmission dynamics and distribution of XDR Mycobacterium tuberculosis (Mtb) strains have not been thoroughly investigated. Using whole genome sequencing data on 461 XDR-Mtb strains, we aimed to investigate the geographical distribution of XDR-Mtb strains in the Western Cape Province of South Africa over a 10 year period (2006-2017) and assess the association between Mtb sub-lineage, age, gender, geographical patient location and membership or size of XDR-TB clusters. First, we identified transmission clusters by excluding drug resistance-conferring mutations and using the 5 SNP cutoff, followed by merging clusters based on their most recent common ancestor. We then consecutively included variants conferring resistance to INH, RIF, ethambutol (EMB), pyrazinamide (PZA), SLIDs and FQs in the cluster definition. Cluster sizes were classified as small (2-4 isolates), medium (5-20 isolates), large (21-100 isolates) or very large (>100 isolates) to reflect the success of individual strains. We found that most XDR-TB strains were clustered and that including variants conferring resistance to INH, RIF, EMB, PZA and SLIDs in the cluster definition did not significantly reduce the proportion of clustered isolates (85.5-82.2 %) but increased the number of patients belonging to small clusters (4.3-12.4 %, P=0.56). Inclusion of FQ resistance-conferring variants had the greatest effect, with 11 clustered isolates reclassified as unique while the number of clusters increased from 17 to 37. Lineage 2 strains (lineage 2.2.1 typical Beijing or lineage 2.2.2 atypical Beijing) showed the large clusters which were spread across all health districts of the Western Cape Province. We identified a significant association between residence in the Cape Town metropole and cluster membership (P=0.016) but no association between gender, age and cluster membership or cluster size (P=0.39). Our data suggest that the XDR-TB epidemic in South Africa probably has its origin in the endemic spread of MDR Mtb and pre-XDR Mtb strains followed by acquisition of FQ resistance, with more limited transmission of XDR Mtb strains. This only became apparent with the inclusion of drug resistance-conferring variants in the definition of a cluster. In addition to the prevention of amplification of resistance, rapid diagnosis of MDR, pre-XDR and XDR-TB and timely initiation of appropriate treatment is needed to reduce transmission of difficult-to-treat TB.