ORCID Profile
0000-0002-2511-7534
Current Organisations
Institute of Tropical Medicine Antwerp
,
University of Antwerp Antwerp
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Publisher: Springer Science and Business Media LLC
Date: 21-02-2020
DOI: 10.1186/S12916-019-1487-2
Abstract: Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide ( ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin ( gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to lification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to lification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.
Publisher: Cold Spring Harbor Laboratory
Date: 21-06-2021
DOI: 10.1101/2021.06.14.21258812
Abstract: Rif icin mono-resistant TB (RMR-TB) constitutes 38% of all rif icin-resistant TB (RR-TB) in South Africa and is increasing. We aimed to compare RMR-TB with multidrug-resistant TB (MDR-TB) within a high TB, RR-TB and HIV burden setting. Patient-level clinical data and stored RR-TB isolates from 2008-2017 with available whole genome sequencing (WGS) data were used to describe risk factors associated with RMR-TB and to compare rif icin-resistance (RR) conferring mutations between RMR-TB and MDR-TB. A subset of isolates with particular RR-conferring mutations were subjected to semi-quantitative rif icin phenotypic drug susceptibility testing. Among 2,041 routinely diagnosed RR-TB patients, 463 (22.7%) had RMR-TB. HIV-positive in iduals (adjusted Odds Ratio 1.4, 95% CI 1.1-1.9) and diagnosis between 2013-2017 versus 2008-2012 (aOR 1.3, 1.1-1.7) were associated with RMR-TB. Among 1,119 (54.8%) patients with available WGS data showing RR-TB, significant differences in the distribution of rpoB RR-conferring mutations between RMR-TB and MDR-TB isolates were observed. Mutations associated with high-level RR were more commonly found among MDR-TB isolates (811/889, 90.2% versus 162/230, 70.4% among RMR-TB, p .01). In particular, the rpoB L430P mutation, conferring low-level RR, was identified in 32/230 (13.9%) RMR-TB versus 10/889 (1.1%) in MDR-TB (p .01). Among 10 isolates with an rpoB L430P mutation, 7 were phenotypically susceptible using the critical concentration of 0.5 µg/ml (range 0.125-1 µg/ml). The majority (215/230, 93.5%) of RMR-TB isolates showed susceptibility to all other TB drugs, highlighting the potential benefits of WGS for simplified treatment. These data suggest that the evolution of RMR-TB differs from MDR-TB with a potential contribution from HIV infection.
Publisher: Elsevier BV
Date: 02-2023
Publisher: Elsevier BV
Date: 11-2021
Publisher: Cold Spring Harbor Laboratory
Date: 08-03-2022
DOI: 10.1101/2022.03.04.22271870
Abstract: Mycobacterium tuberculosis whole-genome sequencing (WGS) using Illumina technology has been widely adopted for genotypic drug susceptibility testing (DST) and outbreak investigation. Oxford Nanopore Technologies is reported to have higher error rates but has not been thoroughly evaluated for these applications. We analyse 151 isolates from Madagascar, South Africa and England with phenotypic DST and matched Illumina and Nanopore data. Using PacBio assemblies, we select Nanopore filters for BCFtools (software) detection of single nucleotide polymorphisms (SNPs). We compare transmission clusters identified by Nanopore and the United Kingdom Health Security Agency Illumina pipeline (COMPASS). We compare Illumina and Nanopore WGS-based DST predictions using Mykrobe (software). Nanopore/BCFtools identifies SNPs with median precision/recall of 99·5/90·2% compared with 99·6/91·9% for Illumina/COMPASS. Using a threshold of 12 SNPs for putative transmission clusters, Illumina identifies 98 isolates as unrelated and 53 as belonging to 19 distinct clusters (size range 2-7). Nanopore reproduces this distribution with addition of 5 singleton isolates to distinct clusters and merging of two cluster pairs. Illumina-based clusters are also replicated using a 5 SNP threshold. Clustering accuracy is maintained using mixed Illumina/Nanopore datasets. Genotyping resistance variants is highly concordant, with 0(4) discordant SNPs (indels) across 151 isolates genotyped at (60,000) SNPs (indels). Illumina and Nanopore sequence data provide comparable cluster-identification and DST results. Academy for Medical Sciences (SGL018\\110), Oxford Wellcome Institutional Strategic Support Fund (ISSF TT17 4). Swiss South Africa Joint Research Award (Swiss national science Foundation and South African national research foundation). Two key types of information can be obtained from laboratory testing of M. tuberculosis isolates to help directly guide public health interventions: drug susceptibility testing (DST) to guide therapy, and bacterial typing to enrich understanding of the epidemiology and guide interventions to mitigate transmission. DST is typically performed by the “gold standard” culture-based phenotyping method or nucleic acid lification assays targeting specific resistance-conferring mutations. Studies over the last 7 years have shown that prediction of susceptibility profile using Illumina-technology genome sequence data is possible, and can be automated. In a key publication, the CRyPTIC consortium and UK 100,000 Genomes project evaluated the method on over 10,000 genomes including prospectively s led isolates and showed that for first-line tuberculosis (TB) drugs (isoniazid, rif icin, ethambutol, pyrazinamide) a pan-susceptibility profile is accurate enough to be used clinically. The genetic basis of resistance remains imperfectly understood for second-line TB drugs, in particular for new and repurposed drugs (bedaquiline, clofazimine, delamanid, linezolid). Prior work in the field of genotypic DST was heavily based on Illumina technology, which provides short (70-300 base pair) sequence reads of very high quality. Many different softwares (e.g. TBProfiler, Mykrobe, MTBseq, kvarq) have been designed for sequence analysis and genotypic DST. However, the increasingly used Nanopore sequencing platforms yield very different data with much longer sequence reads (frequently over 1kb) and higher error rates including systematic biases. To date, very limited evaluation of Nanopore-based drug susceptibility prediction has been performed using the only two compatible tools (Mykrobe (n=5 independent s les), TBProfiler (n=3 independent s les)). Molecular typing of M. tuberculosis allows lineage identification and detection of putative transmission clusters. In the last decade, multiple M. tuberculosis molecular epidemiology studies have shown how genomic information can complement traditional epidemiology in identifying person-to-person transmission clusters with a high level of resolution. Typically, the number of single nucleotide polymorphism (SNP) disagreements between genomes, or SNP distance, is calculated and single-linkage clustering is performed for genomes falling within retrospectively established transmission thresholds of either 5 or 12 SNPs. Just as with DST, these thresholds were established with Illumina sequencing data. The increased error rate in Nanopore sequencing is believed to lead to inflated SNP distances if standard genome analysis tools are used. Prior to this study it was unknown what impact on isolate-clustering this would incur. Full-scale adoption of genomic sequencing in tuberculosis reference laboratories has so far taken place in a limited number of settings - England, the Netherlands, and New York State - all using Illumina-based sequencing data. Building on current evidence, specific WHO technical guidance and ersification and democratisation of technology, sequencing is expected to be increasingly used in tuberculosis control globally. For the first time, our study offers 4 key deliverables intended to inform adoption of Nanopore technology as an alternative, or a complement, to Illumina. First: a systematic head-to-head comparison of Nanopore and Illumina data for M. tuberculosis drug susceptibility profiling and isolate clustering, including quantitative metrics for cluster precision and recall. Second: an assessment of the impact of mixed Illumina and Nanopore data on clustering which represents an increasingly common challenge. Third: an open-source software pipeline allowing research and reference laboratories to replicate our analytical approach. Fourth: a publicly available curated test set of 151 isolates, including matched Illumina and Nanopore sequence data, and (for a subset of seven isolates) high-quality PacBio assemblies, for method development and validation. Catalogues of drug resistance conferring mutations will keep improving, especially for new and repurposed drugs. Our data confirms that Illumina and Nanopore sequencing technologies can be used to identify those mutations equally accurately in M. tuberculosis . Bacterial molecular typing is constantly shown to support the understanding of disease transmission and tuberculosis control in new settings. The bioinformatics tools and filters we have developed, assessed, and made publicly available allow the use of Nanopore or mixed-technology data to appropriately cluster genetically related isolates. We provide a measure of the expected level of over-clustering associated with Nanopore technology. This study confirms that Illumina and Nanopore sequence data provide comparable DST results and isolate cluster-identification.
Publisher: American Society for Microbiology
Date: 18-10-2021
DOI: 10.1128/AAC.00364-21
Abstract: Rif in monoresistance (RMR rif in resistance and isoniazid susceptibility) accounts for 38% of all rif in-resistant tuberculosis (RR-TB) in South Africa and is increasing. We aimed to compare RMR-TB with multidrug-resistant TB (MDR-TB) in a setting with high TB, RR-TB, and HIV burdens.
Publisher: Microbiology Society
Date: 26-04-2022
Abstract: Extensively drug-resistant tuberculosis (XDR-TB), defined as resistance to at least isoniazid (INH), rif icin (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 ( isolates) to reflect the success of in idual 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 lification 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.
Publisher: American Society for Microbiology
Date: 19-07-2022
DOI: 10.1128/AAC.00322-22
Abstract: Studies have shown that variants in bedaquiline-resistance genes can occur in isolates from bedaquiline-naive patients. We assessed the prevalence of variants in all bedaquiline-candidate-resistance genes in bedaquiline-naive patients, investigated the association between these variants and lineage, and the effect on phenotype.
Publisher: Elsevier BV
Date: 2022
DOI: 10.1016/J.TUBE.2021.102159
Abstract: 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 h ered by the high viscosity, low mycobacterial load, and high contamination with bacterial and human DNA in sputum s les. 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 s les 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 lification of the human GAPDH gene after sputum treatment, showed the presence of human DNA in all s les, regardless of sputum treatment. Focused efforts are needed to deplete contaminating DNA for culture-free Mtb WGS.
Publisher: American Society for Microbiology
Date: 16-03-2022
DOI: 10.1128/JCM.02362-21
Abstract: Treatment of multidrug-resistant or rif icin-resistant tuberculosis (MDR/RR-TB), although improved in recent years with shorter, more tolerable regimens, remains largely standardized and based on limited drug susceptibility testing (DST). More in idualized treatment with expanded DST access is likely to improve patient outcomes.
Publisher: Oxford University Press (OUP)
Date: 03-10-2020
DOI: 10.1093/BIB/BBAA246
Abstract: Whole genome sequencing (WGS) is increasingly used for Mycobacterium tuberculosis (Mtb) research. Countries with the highest tuberculosis (TB) burden face important challenges to integrate WGS into surveillance and research. We assessed the global status of Mtb WGS and developed a 3-week training course coupled with long-term mentoring and WGS infrastructure building. Training focused on genome sequencing, bioinformatics and development of a locally relevant WGS research project. The aim of the long-term mentoring was to support trainees in project implementation and funding acquisition. The focus of WGS infrastructure building was on the DNA extraction process and bioinformatics. Compared to their TB burden, Asia and Africa are grossly underrepresented in Mtb WGS research. Challenges faced resulted in adaptations to the training, mentoring and infrastructure building. Out-of-date laptop hardware and operating systems were overcome by using online tools and a Galaxy WGS analysis pipeline. A case studies approach created a safe atmosphere for students to formulate and defend opinions. Because quality DNA extraction is paramount for WGS, a biosafety level 3 and general laboratory skill training session were added, use of commercial DNA extraction kits was introduced and a 2-week training in a highly equipped laboratory was combined with a 1-week training in the local setting. By developing and sharing the components of and experiences with a sequencing and bioinformatics training program, we hope to stimulate capacity building programs for Mtb WGS and empower high-burden countries to play an important role in WGS-based TB surveillance and research.
Location: South Africa
No related grants have been discovered for Anzaan Dippenaar.