SUNScholar - Browsing by Author "Warren, Robin M."

Browsing by Author "Warren, Robin M."

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Adaptation and diagnostic potential of a commercial cat interferon gamma release assay for the detection of Mycobacterium bovis infection in African lions (Panthera leo)
(MDPI, 2022-07) Gumbo, Rachiel; Sylvester, Tashnica T.; Goosen, Wynand J.; Buss, Peter E.; De Klerk-Lorist, Lin-Mari; Van Schalkwyk, O. Louis; McCall, Alicia; Warren, Robin M.; Van Helden, Paul D.; Miller, Michele A.; Kerr, Tanya J.
Mycobacterium bovis (M. bovis) infection in wildlife, including lions (Panthera leo), has implications for individual and population health. Tools for the detection of infected lions are needed for diagnosis and disease surveillance. This study aimed to evaluate the Mabtech Cat interferon gamma (IFN-γ) ELISABasic kit for detection of native lion IFN-γ in whole blood samples stimulated using the QuantiFERON® TB Gold Plus (QFT) platform as a potential diagnostic assay. The ELISA was able to detect lion IFN-γ in mitogen-stimulated samples, with good parallelism, linearity, and a working range of 15.6–500 pg/mL. Minimal matrix interference was observed in the recovery of domestic cat rIFN-γ in lion plasma. Both intra- and inter-assay reproducibility had a coefficient of variation less than 10%, while the limit of detection and quantification were 7.8 pg/mL and 31.2 pg/mL, respectively. The diagnostic performance of the QFT Mabtech Cat interferon gamma release assay (IGRA) was determined using mycobacterial antigen-stimulated samples from M. bovis culture-confirmed infected (n = 8) and uninfected (n = 4) lions. A lion-specific cut-off value (33 pg/mL) was calculated, and the sensitivity and specificity were determined to be 87.5% and 100%, respectively. Although additional samples should be tested, the QFT Mabtech Cat IGRA could identify M. bovisinfected African lions.
Alcohol, hospital discharge, and socioeconomic risk factors for default from multidrug resistant tuberculosis treatment in rural South Africa : a retrospective cohort study
(PLoS, 2013-12-13) Kendall, Emily A.; Theron, Danie; Franke, Molly F.; Van Helden, Paul; Victor, Thomas C.; Murray, Megan B.; Warren, Robin M.; Jacobson, Karen R.
Background: Default from multidrug-resistant tuberculosis (MDR-TB) treatment remains a major barrier to cure and epidemic control. We sought to identify patient risk factors for default from MDR-TB treatment and high-risk time periods for default in relation to hospitalization and transition to outpatient care. Methods: We retrospectively analyzed a cohort of 225 patients who initiated MDR-TB treatment between 2007 through 2010 at a rural TB hospital in the Western Cape Province, South Africa. Results: Fifty percent of patients were cured or completed treatment, 27% defaulted, 14% died, 4% failed treatment, and 5% transferred out. Recent alcohol use was common (63% of patients). In multivariable proportional hazards regression, older age (hazard ratio [HR]= 0.97 [95% confidence interval 0.94-0.99] per year of greater age), formal housing (HR=0.38 [0.19-0.78]), and steady employment (HR=0.41 [0.19-0.90]) were associated with decreased risk of default, while recent alcohol use (HR=2.1 [1.1-4.0]), recent drug use (HR=2.0 [1.0-3.6]), and Coloured (mixed ancestry) ethnicity (HR=2.3 [1.1-5.0]) were associated with increased risk of default (P<0.05). Defaults occurred throughout the first 18 months of the two-year treatment course but were especially frequent among alcohol users after discharge from the initial four-to-five-month in-hospital phase of treatment, with the highest default rates occurring among alcohol users within two months of discharge. Default rates during the first two months after discharge were also elevated for patients who received care from mobile clinics. Conclusions: Among patients who were not cured or did not complete MDR-TB treatment, the majority defaulted from treatment. Younger, economically-unstable patients and alcohol and drug users were particularly at risk. For alcohol users as well as mobile-clinic patients, the early outpatient treatment phase is a high-risk period for default that could be targeted in efforts to increase treatment completion rates.
Comparative analysis of Mycobacterium tuberculosis pe and ppe genes reveals high sequence variation and apparent absence of selective constraints
(Public Library of Science, 2012-04-04) McEvoy, Christopher R. E.; Cloete, Ruben; Muller, Borna; Schurch, Anita C.; Van Helden, Paul D.; Gagneux, Sebastien; Warren, Robin M.; Gey van Pittius, Nicolaas C.
Mycobacterium tuberculosis complex (MTBC) genomes contain 2 large gene families termed pe and ppe. The function of pe/ppe proteins remains enigmatic but studies suggest that they are secreted or cell surface associated and are involved in bacterial virulence. Previous studies have also shown that some pe/ppe genes are polymorphic, a finding that suggests involvement in antigenic variation. Using comparative sequence analysis of 18 publicly available MTBC whole genome sequences, we have performed alignments of 33 pe (excluding pe_pgrs) and 66 ppe genes in order to detect the frequency and nature of genetic variation. This work has been supplemented by whole gene sequencing of 14 pe/ppe (including 5 pe_pgrs) genes in a cohort of 40 diverse and well defined clinical isolates covering all the main lineages of the M. tuberculosis phylogenetic tree. We show that nsSNP's in pe (excluding pgrs) and ppe genes are 3.0 and 3.3 times higher than in non-pe/ppe genes respectively and that numerous other mutation types are also present at a high frequency. It has previously been shown that non-pe/ppe M. tuberculosis genes display a remarkably low level of purifying selection. Here, we also show that compared to these genes those of the pe/ppe families show a further reduction of selection pressure that suggests neutral evolution. This is inconsistent with the positive selection pressure of “classical” antigenic variation. Finally, by analyzing such a large number of genes we were able to detect large differences in mutation type and frequency between both individual genes and gene sub-families. The high variation rates and absence of selective constraints provides valuable insights into potential pe/ppe function. Since pe/ppe proteins are highly antigenic and have been studied as potential vaccine components these results should also prove informative for aspects of M. tuberculosis vaccine design.
Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis
(American Society for Microbiology, 2021) Whitfield, Michael G.; Engelthaler, David M.; Allender, Christopher; Folkerts, Megan; Heupink, Tim H.; Limberis, Jason; Warren, Robin M.; Van Rie, Annelies; Metcalfe, John Z.
Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampin- resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods and explored the occurrence of pyrazinamide heteroresistance. We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the pncA gene, whole-genome sequencing (WGS), and phenotypic drug susceptibility testing. We calculated the diagnostic accuracy of the different methods and investigated the prevalence and clinical impact of pncA heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Köser classification and expert rules. We observed 100% agreement across genotypic methods for detection of pncA fixed mutations; only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% confidence interval [CI], 93.8 to 99.3%) and specificity of 100% (95% CI, 100 to 100%), both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI, 94.6 to 99.6%) and specificity of 97.8% (95% CI, 95.7 to 99.9%), and TDS had sensitivity of 98.8% (95% CI, 97.2 to 100%) and specificity of 97.8% (95% CI, 95.7 to 99.9%). We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in Mycobacterium tuberculosis isolates was lower than that identified for other first-line drugs.
Cytokine gene expression assay as a diagnostic tool for detection of Mycobacterium bovis infection in warthogs (Phacochoerus africanus)
(Nature Research (part of Springer Nature), 2019) Roos, Eduard O .; Scott, Leere A.; Ndou, Sedzani; Olea-Popelka, Francisco; Buss, Peter E.; De Klerk-Lorist, Lin-Mari; Warren, Robin M.; Van Helden, Paul D.; Sylvester, Tashnica T .; Miller, Michele A.; Parsons, Sven D. C.
ENGLISH ABSTRACT: Mycobacterium bovis infection has been described in many wildlife species across Africa. However, diagnostic tests are lacking for many of these, including warthogs (Phacochoerus africanus). Most literature on suids has focused on using serological tools, with few studies investigating the use of cell-mediated immune response (CMI) assays. A recent study showed that warthogs develop measurable CMI responses, which suggests that cytokine gene expression assays (GEAs) may be valuable for detecting M. bovis-infection, as shown in numerous African wildlife species. Therefore, the aim of the study was to develop GEAs capable of distinguishing between M. bovis-infected and uninfected warthogs. Whole blood was stimulated using the QuantiFERON-TB Gold (In-Tube) system, using ESAT-6 and CFP-10 peptides, before determining the relative gene expression of five reference (B2M, H3F3A, LDHA, PPIA and YWHAZ) and five target (CXCL9, CXCL10, CXCL11, IFNG and TNFA) genes through qPCR. The reference gene H3F3A was the most stably expressed, while all target genes were significantly upregulated in M. bovis-infected warthogs with the greatest upregulation observed for CXCL10. Consequently, the CXCL10 GEA shows promise as an ante-mortem diagnostic tool for the detection of M. bovis-infected warthogs.
Detection of natural infection with Mycobacterium intracellulare in healthy wild-caught Chacma baboons (Papio ursinus)by ESAT-6 and CFP-10 IFN- ELISPOT tests following a tuberculosis outbreak
(BioMed Central, 2008-02) Chege, Gerald K.; Warren, Robin M.; Gey van Pittius, Nico C.; Burgers, Wendy A.; Wilkinson, Robert J.; Shephard, Enid G.; Williamson, Anna-Lise
Background: Both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates (NHP), indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. Screening of mycobacterial infections in NHP is traditionally done by tuberculin skin test (TST), which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. In this study, we investigated the use of ESAT-6 and CFP-10 for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis (TB). Methods: Peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay (IFN-γ ELISPOT) assay were obtained from TST positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture. Results: Both ESAT-6 and CFP-10 IFN-γ ELISPOT assays were able to detect early M. tuberculosis but also M. intracellulare infection. Although this indicates potential cross-reactivity with M. intracellulare antigens, the method was able to distinguish M. bovis BCG vaccination from M. tuberculosis infection. This assay performed better than the TST, which failed to detect one M. tuberculosis and two early M. intracellulare infections. Conclusion: These results suggest that the IFN-γ ELISPOT assay could improve the detection of M tuberculosis infections when screening NHP. There is some doubt, however, concerning specificity, as the assay scored positive three animals infected with M. intracellulare.
Detection of second line drug resistance among drug resistant Mycobacterium tuberculosis isolates in Botswana
(MDPI, 2019-10-28) Mogashoa, Tuelo; Melamu, Pinkie; Derendinger, Brigitta; Ley, Serej D.; Streicher, Elizabeth M.; Iketleng, Thato; Mupfumi, Lucy; Mokomane, Margaret; Kgwaadira, Botshelo; Rankgoane-Pono, Goabaone; Tsholofelo, Thusoyaone T.; Kasvosve, Ishmael; Moyo, Sikhulile; Warren, Robin M.; Gaseitsiwe, Simani
ENGLISH ABSTRACT: The emergence and transmission of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (M.tb) strains is a threat to global tuberculosis (TB) control. The early detection of drug resistance is critical for patient management. The aim of this study was to determine the proportion of isolates with additional second-line resistance among rifampicin and isoniazid resistant and MDR-TB isolates. A total of 66 M.tb isolates received at the National Tuberculosis Reference Laboratory between March 2012 and October 2013 with resistance to isoniazid, rifampicin or both were analyzed in this study. The genotypes of the M.tb isolates were determined by spoligotyping and second-line drug susceptibility testing was done using the Hain Genotype MTBDRsl line probe assay version 2.0. The treatment outcomes were defined according to the Botswana national and World Health Organization (WHO) guidelines. Of the 57 isolates analyzed, 33 (58%) were MDR-TB, 4 (7%) were additionally resistant to flouroquinolones and 3 (5%) were resistant to both fluoroquinolones and second-line injectable drugs. The most common fluoroquinolone resistance-conferring mutation detected was gyrA A90V. All XDR-TB cases remained smear or culture positive throughout the treatment. Our study findings indicate the importance of monitoring drug resistant TB cases to ensure rapid detection of second-line drug resistance.
Diagnostic accuracy of the FluoroType MTB and MTBDR VER 2.0 assays for the centralized high-throughput detection of Mycobacterium tuberculosis complex DNA and isoniazid and rifampicin resistance
(Elsevier Ltd, 2021-09) Dippenaar, Anzaan; Derendinger, Brigitta; Dolby, Tania; Beylis, Natalie; Van Helden, Paul D.; Theron, Grant; Warren, Robin M.; De Vos, Margaretha
Objectives To evaluate the accuracy of two new molecular diagnostic tests for the detection of drug-resistant tuberculosis, the FluoroType MTB and MTBDR VER 2.0 assays, in combination with manual and automated DNA extraction methods. Methods Sputa from 360 Xpert Ultra Mycobacterium tuberculosis complex (MTBC)-positive patients and 250 Xpert Ultra MTBC-negative patients were tested. GenoType MTBDRplus served as reference for MTBC and drug resistance detection. Sanger sequencing was used to resolve discrepancies. Results FluoroType MTB VER 2.0 showed similar MTBC sensitivity compared with FluoroType MTBDR VER 2.0 (manual DNA extraction: 91.6% (294/321) versus 89.8% (291/324); p 0.4); automated DNA extraction: 92.1% (305/331) versus 87.7% (291/332); p 0.05)). FluoroType MTBDR VER2.0 showed comparable diagnostic accuracy to FluoroType MTBDR VER1.0 as previously reported for the detection of MTBC and rifampicin and isoniazid resistance. Conclusions The FluoroType MTB and MTBDR VER 2.0 assays together with an automated DNA extraction and PCR set-up platform may improve laboratory operational efficiency for the diagnosis of MTBC and resistance to rifampicin and isoniazid and show promise for the implementation in a centralized molecular drug susceptibility testing model.
Drug-associated adverse events and their relationship with outcomes in patients receiving treatment for extensively drug-resistant tuberculosis in South Africa
(Public Library of Science, 2013-05-07) Shean, Karen; Streicher, Elizabeth M.; Pieterson, Elize; Symons, Greg; Van Zyl Smit, Richard; Theron, Grant; Lehloenya, Rannakoe; Padanilam, Xavier; Wilcox, Paul; Victor, Tommie C.; Van Helden, Paul D.; Groubusch, Martin; Warren, Robin M.; Badri, Motasim; Dheda, Keertan
Background: Treatment-related outcomes in patients with extensively drug-resistant tuberculosis (XDR-TB) are poor. However, data about the type, frequency and severity of presumed drug-associated adverse events (AEs) and their association with treatment-related outcomes in patients with XDR-TB are scarce. Methods: Case records of 115 South-African XDR-TB patients were retrospectively reviewed by a trained researcher. AEs were estimated and graded according to severity [grade 0 = none; grade 1–2 = mild to moderate; and grade 3–5 = severe (drug stopped, life-threatening or death)]. Findings: 161 AEs were experienced by 67/115(58%) patients: 23/67(34%) required modification of treatment, the offending drug was discontinued in 19/67(28%), reactions were life-threatening in 2/67(3.0%), and 6/67(9.0%) died. ∼50% of the patients were still on treatment at the time of data capture. Sputum culture-conversion was less likely in those with severe (grade 3–5) vs. grade 0–2 AEs [2/27(7%) vs. 24/88(27%); p = 0.02]. The type, frequency and severity of AEs was similar in HIV-infected and uninfected patients. Capreomycin, which was empirically administered in most cases, was withdrawn in 14/104(14%) patients, implicated in (14/34) 41% of the total drug withdrawals, and was associated with all 6 deaths in the severe AE group (renal failure in five patients and hypokalemia in one patient). Conclusion: Drug-associated AEs occur commonly with XDR-TB treatment, are often severe, frequently interrupt therapy, and negatively impact on culture conversion outcomes. These preliminary data inform on the need for standardised strategies (including pre-treatment counselling, early detection, monitoring, and follow-up) and less toxic drugs to optimally manage patients with XDR-TB.
Evidence for a rapid rate of molecular evolution at the hypervariable and immunogenic Mycobacterium tuberculosis PPE38 gene region
(BioMed Central, 2009-09) McEvoy, Christopher R. E.; Van Helden, Paul D.; Warren, Robin M.; Gey van Pittius, Nicolaas C.
Background: PPE38 (Rv2352c) is a member of the large PPE gene family of Mycobacterium tuberculosis and related mycobacteria. The function of PPE proteins is unknown but evidence suggests that many are cell-surface associated and recognised by the host immune system. Previous studies targeting other PPE gene members suggest that some display high levels of polymorphism and it is thought that this might represent a means of providing antigenic variation. We have analysed the genetic variability of the PPE38 genomic region on a cohort of M. tuberculosis clinical isolates representing all of the major phylogenetic lineages, along with the ancestral M. tuberculosis complex (MTBC) member M. canettii, and supplemented this with analysis of publicly available whole genome sequences representing additional M. tuberculosis clinical isolates, other MTBC members and non tuberculous mycobacteria (NTM). Where possible we have extended this analysis to include the adjacent plcABC and PPE39/40 genomic regions. Results: We show that the ancestral MTBC PPE38 region comprises 2 homologous PPE genes (PPE38 and PPE71), separated by 2 esat-6 (esx)-like genes and that this structure derives from an esx/esx/PPE duplication in the common ancestor of M. tuberculosis and M. marinum. We also demonstrate that this region of the genome is hypervariable due to frequent IS6110 integration, IS6110-associated recombination, and homologous recombination and gene conversion events between PPE38 and PPE71. These mutations result in combinations of gene deletion, gene truncation and gene disruption in the majority of clinical isolates. These mutations were generally found to be IS6110 strain lineage-specific, although examples of additional within-lineage and even within-cluster mutations were observed. Furthermore, we provide evidence that the published M. tuberculosis H37Rv whole genome sequence is inaccurate regarding this region. Conclusion: Our results show that this antigen-encoding region of the M. tuberculosis genome is hypervariable. The observation that numerous different mutations have become fixed within specific lineages demonstrates that this genomic region is undergoing rapid molecular evolution and that further lineage-specific evolutionary expansion and diversification has occurred subsequent to the lineage-defining mutational events. We predict that functional loss of these genes could aid immune evasion. Finally, we also show that the PPE38 region of the published M. tuberculosis H37Rv whole genome sequence is not representative of the ATCC H37Rv reference strain.
Evidence that the spread of Mycobacterium tuberculosis strains with the Beijing genotype is human population dependent
(American Society for Microbiology, 2007-07) Hanekom, M.; Van der Spuy, G. D.; Gey van Pittius, N. C.; McEvoy, C. R. E.; Ndabambi, S. L.; Victor, T. C.; Hoal, E. G.; Van Helden, Paul D.; Warren, Robin M.
This study describes a comparative analysis of the Beijing mycobacterial interspersed repetitive unit types of Mycobacterium tuberculosis isolates from Cape Town, South Africa, and East Asia. The results show a significant association between the frequency of occurrence of strains from defined Beijing sublineages and the human population from whom they were cultured (P < 0.0001). Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions
(BioMed Central, 2006-11) Gey van Pittius, Nicolaas C.; Sampson, Samantha L.; Lee, Hyeyoung; Kim, Yeun; Van Helden, Paul D.; Warren, Robin M.
Background: The PE and PPE multigene families of Mycobacterium tuberculosis comprise about 10% of the coding potential of the genome. The function of the proteins encoded by these large gene families remains unknown, although they have been proposed to be involved in antigenic variation and disease pathogenesis. Interestingly, some members of the PE and PPE families are associated with the ESAT-6 (esx) gene cluster regions, which are regions of immunopathogenic importance, and encode a system dedicated to the secretion of members of the potent T-cell antigen ESAT-6 family. This study investigates the duplication characteristics of the PE and PPE gene families and their association with the ESAT-6 gene clusters, using a combination of phylogenetic analyses, DNA hybridization, and comparative genomics, in order to gain insight into their evolutionary history and distribution in the genus Mycobacterium. Results: The results showed that the expansion of the PE and PPE gene families is linked to the duplications of the ESAT-6 gene clusters, and that members situated in and associated with the clusters represent the most ancestral copies of the two gene families. Furthermore, the emergence of the repeat protein PGRS and MPTR subfamilies is a recent evolutionary event, occurring at defined branching points in the evolution of the genus Mycobacterium. These gene subfamilies are thus present in multiple copies only in the members of the M. tuberculosis complex and close relatives. The study provides a complete analysis of all the PE and PPE genes found in the sequenced genomes of members of the genus Mycobacterium such as M. smegmatis, M. avium paratuberculosis, M. leprae, M. ulcerans, and M. tuberculosis. Conclusion: This work provides insight into the evolutionary history for the PE and PPE gene families of the mycobacteria, linking the expansion of these families to the duplications of the ESAT-6 (esx) gene cluster regions, and showing that they are composed of subgroups with distinct evolutionary (and possibly functional) differences.
Extract from used Xpert MTB/ RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk
(Nature Research (part of Springer Nature), 2020) Venter, Rouxjeane; Minnies, Stephanie; Derendinger, Brigitta; Tshivhula, Happy; De Vos, Margaretha; Dolby, Tania; Ruiters, Ashley; Warren, Robin M.; Theron, Grant
Xpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra’s predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥103 CFU/ml (CTmin <25, >“low semi-quantitation”) detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10−3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.
Genetic diversity of Mycobacterium tuberculosis strains circulating in Botswana
(PLoS, 2019-05-07) Mogashoa, Tuelo; Melamu, Pinkie; Ley, Serej D.; Streicher, Elizabeth M.; Iketleng, Thato; Kelentse, Nametso; Mupfumi, Lucy; Mokomane, Margaret; Kgwaadira, Botshelo; Novitsky, Vladimir; Kasvosve, Ishmael; Moyo, Sikhulile; Warren, Robin M.; Gaseitsiwe, Simani
Background: Molecular typing of Mycobacterium tuberculosis (M.tb) isolates can inform Tuberculosis (TB) control programs on the relative proportion of transmission driving the TB epidemic. There is limited data on the M. tb genotypes that are circulating in Botswana. The aim of this study was to generate baseline data on the genetic diversity of M.tb isolates circulating in the country. Methods: A total of 461 M.tb isolates received at the Botswana National Tuberculosis Reference Laboratory between March 2012 and October 2013 were included in this study. Drug susceptibility testing was conducted using the BD BACTEC MGIT 960 System. M.tb strains were genotyped using spoligotyping and spoligotype patterns were compared with existing patterns in the SITVIT Web database. A subset of drug resistant isolates which formed spoligo clusters (n = 65) was additionally genotyped with 12-loci MIRU. Factors associated with drug resistance and clustering were evaluated using logistic regression. Results: Of the 461 isolates genotyped, 458 showed 108 distinct spoligotype patterns. The predominant M.tb lineages were Lineage 4 (81.9%), Lineage 2 (9%) and Lineage 1 (7.2%). The predominant spoligotype families within Lineage 4 were LAM (33%), S (14%), T (16%), X (16%). Three hundred and ninety-two (86%) isolates could be grouped into 44 clusters (2– 46 isolates per cluster); giving a clustering rate of 76%. We identified 173 (37.8%) drug resistant isolates, 48 (10.5%) of these were multi-drug resistant. MIRU typing of the drug resistant isolates allowed grouping of 46 isolates into 14 clusters, giving a clustering rate of 49.2%. There was no association between age, sex, treatment category, region and clustering. Conclusions: This study highlights the complexity of the TB epidemic in Botswana with multiple strains contributing to disease and provides baseline data on the population structure of M.tb strains in Botswana.
Geospatial distribution of Mycobacterium tuberculosis genotypes in Africa
(Public Library of Science, 2018-08-01) Chihota, Violet N.; Niehaus, Antoinette; Streicher, Elizabeth M.; Wang, Xia; Sampson, Samantha L.; Mason, Peter; Kallenius, Gunilla; Mfinanga, Sayoki G.; Pillay, Marnomorney; Klopper, Marisa; Kasongo, Webster; Behr, Marcel A.; Van Pittius, Nicolaas C. Gey; Van Helden, Paul D.; Couvin, David; Rastogi, Nalin; Warren, Robin M.
Objective: To investigate the distribution of Mycobacterium tuberculosis genotypes across Africa. Methods: The SITVIT2 global repository and PUBMED were searched for spoligotype and published genotype data respectively, of M. tuberculosis from Africa. M. tuberculosis lineages in Africa were described and compared across regions and with those from 7 European and 6 South-Asian countries. Further analysis of the major lineages and sub-lineages using Principal Component analysis (PCA) and hierarchical cluster analysis were done to describe clustering by geographical regions. Evolutionary relationships were assessed using phylogenetic tree analysis. Results: The SITVIT2 global repository and PUBMED were searched for spoligotype and published genotype data respectively, of M. tuberculosis from Africa. M. tuberculosis lineages in Africa were described and compared across regions and with those from 7 European and 6 South-Asian countries. Further analysis of the major lineages and sub-lineages using Principal Component analysis (PCA) and hierarchical cluster analysis were done to describe clustering by geographical regions. Evolutionary relationships were assessed using phylogenetic tree analysis. Results: A total of 14727 isolates from 35 African countries were included in the analysis and of these 13607 were assigned to one of 10 major lineages, whilst 1120 were unknown. There were differences in geographical distribution of major lineages and their sub-lineages with regional clustering. Southern African countries were grouped based on high prevalence of LAM11-ZWE strains; strains which have an origin in Portugal. The grouping of North African countries was due to the high percentage of LAM9 strains, which have an origin in the Eastern Mediterranean region. East African countries were grouped based on Central Asian (CAS) and East-African Indian (EAI) strain lineage possibly reflecting historic sea trade with Asia, while West African Countries were grouped based on Cameroon lineage of unknown origin. A high percentage of the Haarlem lineage isolates were observed in the Central African Republic, Guinea, Gambia and Tunisia, however, a mixed distribution prevented close clustering. Conclusions: This study highlighted that the TB epidemic in Africa is driven by regional epidemics characterized by genetically distinct lineages of M. tuberculosis. M. tuberculosis in these regions may have been introduced from either Europe or Asia and has spread through pastoralism, mining and war. The vast array of genotypes and their associated phenotypes should be considered when designing future vaccines, diagnostics and anti-TB drugs.
A global perspective on pyrazinamide resistance: systematic review and meta-analysis
(Public Library of Science, 2015) Whitfield, Michael G.; Soeters, Heidi M.; Warren, Robin M.; York, Talita; Sampson, Samantha L.; Streicher, Elizabeth M.; Van Helden, Paul D.; Van Rie, Annelies
Background: Pyrazinamide (PZA) is crucial for tuberculosis (TB) treatment, given its unique ability to eradicate persister bacilli. The worldwide burden of PZA resistance remains poorly described. Methods Systematic PubMed, Science Direct and Scopus searches for articles reporting phenotypic (liquid culture drug susceptibility testing or pyrazinamidase activity assays) and/or genotypic (polymerase chain reaction or DNA sequencing) PZA resistance. Global and regional summary estimates were obtained from random-effects meta-analysis, stratified by presence or risk of multidrug resistant TB (MDR-TB). Regional summary estimates were combined with regional WHO TB incidence estimates to determine the annual burden of PZA resistance. Information on single nucleotide polymorphisms (SNPs) in the pncA gene was aggregated to obtain a global summary. Results: Pooled PZA resistance prevalence estimate was 16.2% (95% CI 11.2-21.2) among all TB cases, 41.3% (29.0-53.7) among patients at high MDR-TB risk, and 60.5% (52.3-68.6) among MDR-TB cases. The estimated global burden is 1.4 million new PZA resistant TB cases annually, about 270,000 in MDR-TB patients. Among 1,815 phenotypically resistant isolates, 608 unique SNPs occurred at 397 distinct positions throughout the pncA gene. Interpretation: PZA resistance is ubiquitous, with an estimated one in six incident TB cases and more than half of all MDR-TB cases resistant to PZA globally. The diversity of SNPs across the pncA gene complicates the development of rapid molecular diagnostics. These findings caution against relying on PZA in current and future TB drug regimens, especially in MDR-TB patients.
High frequency of resistance, lack of clinical benefit, and poor outcomes in capreomycin treated South African patients with extensively drug-resistant tuberculosis
(Public Library of Science, 2015) Pietersen, Elize; Peter, Jonny; Streicher, Elizabeth M.; Sirgel, Frik; Rockwood, Neesha; Mastrapa, Barbara; Te Riele, Julian; Davids, Malika; Van Helden, Paul; Warren, Robin M.; Dheda, Keertan
Background: There are limited data about the epidemiology and treatment-related outcomes associated with capreomycin resistance in patients with XDR-TB. Capreomycin achieves high serum concentrations relative to MIC but whether capreomycin has therapeutic benefit despite microbiological resistance remains unclear. Methods We reviewed the susceptibility profiles and outcomes associated with capreomycin usage in patients diagnosed with XDR-TB between August 2002 and October 2012 in two provinces of South Africa. Patients whose isolates were genotypically tested for capreomycin resistance were included in the analysis. Results Of 178 XDR-TB patients 41% were HIV-infected. 87% (154/178) isolates contained a capreomycin resistance-conferring mutation [80% (143/178) rrs A1401G and 6% (11/178) were heteroresistant (containing both the rrs A1401G mutation and wild-type sequences)]. Previous MDR-TB treatment, prior usage of kanamycin, or strain type was not associated with capreomycin resistance. 92% (163/178) of XDR-TB patients were empirically treated with capreomycin. Capreomycin resistance decreased the odds of sputum culture conversion. In capreomycin sensitive and resistant persons combined weight at diagnosis was the only independent predictor for survival (p=<0.001). By contrast, HIV status and use of co-amoxicillin/clavulanic acid were independent predictors of mortality (p=<0.05). Capreomycin usage was not associated with survival or culture conversion when the analysis was restricted to those whose isolates were resistant to capreomycin. Conclusion: In South Africa the frequency of capreomycin conferring mutations was extremely high in XDR-TB isolates. In those with capreomycin resistance there appeared to be no therapeutic benefit of using capreomycin. These data inform susceptibility testing and the design of treatment regimens for XDR-TB in TB endemic settings.
Human whole genome sequencing in South Africa
(Nature, 2021-01) Glanzmann, Brigitte; Jooste, Tracey; Ghoor, Samira; Gordon, Richard; Mia, Rizwana; Mao, Jun; Li, Hao; Charls, Patrick; Douman, Craig; Kotze, Maritha J.; Peeters, Armand V.; Loots, Glaudina; Esser, Monika; Tiemessen, Caroline T.; Wilkinson, Robert J.; Louw, Johan; Gray, Glenda; Warren, Robin M.; Moller, Marlo; Kinnear, Craig
The advent and evolution of next generation sequencing has considerably impacted genomic research. Until recently, South African researchers were unable to access affordable platforms capable of human whole genome sequencing locally and DNA samples had to be exported. Here we report the whole genome sequences of the first six human DNA samples sequenced and analysed at the South African Medical Research Council’s Genomics Centre. We demonstrate that the data obtained is of high quality, with an average sequencing depth of 36.41, and that the output is comparable to data generated internationally on a similar platform. The Genomics Centre creates an environment where African researchers are able to access world class facilities, increasing local capacity to sequence whole genomes as well as store and analyse the data.
Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics
(BioMed Central, 2020-03-23) Kriel, Nastassja L.; Heunis, Tiaan; Sampson, Samantha L.; Gey Van Pittius, Nico C.; Williams, Monique J.; Warren, Robin M.
Background: Transcriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, is in part mediated by several nucleic-acid associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acidassociated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results: AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification. Conclusions: Effective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA- and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase β- subunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress.
Impact of alcohol consumption on tuberculosis treatment outcomes : a prospective longitudinal cohort study protocol
(BioMed Central, 2018-09-29) Myers, Bronwyn; Bouton, Tara C.; Ragan, Elizabeth J.; White, Laura F.; McIlleron, Helen; Theron, Danie; Parry, Charles D. H.; Horsburgh, C. R.; Warren, Robin M.; Jacobson, Karen R.
Background: An estimated 10% of tuberculosis (TB) deaths are attributable to problematic alcohol use globally, however the causal pathways through which problem alcohol use has an impact on TB treatment outcome is not clear. This study aims to improve understanding of these mechanisms. Specifically, we aim to 1) assess whether poor TB treatment outcomes, measured as delayed time-to-culture conversion, are associated with problem alcohol use after controlling for non-adherence to TB pharmacotherapy; and 2) to determine whether pharmacokinetic (PK) changes in those with problem alcohol use are associated with delayed culture conversion, higher treatment failure/relapse rates or with increased toxicity. Methods: Our longitudinal, repeated measures, prospective cohort study aims to examine the associations between problem alcohol use and TB treatment outcomes and to evaluate the effect of alcohol on the PK and pharmacodynamics (PD) of TB drugs. We will recruit 438 microbiologically confirmed, pulmonary TB patients with evidence of rifampicin susceptibility in Worcester, South Africa with 200 HIV uninfected patients co-enrolled in the PK aim. Participants are followed for the six months of TB treatment and an additional 12 months thereafter, with sputum collected weekly for the first 12 weeks of treatment, alcohol consumption measures repeated monthly in concert with an alcohol biomarker (phosphatidylethanol) measurement at baseline, and in person directly observed therapy (DOT) using real-time mobile phone-based adherence monitoring. The primary outcome is based on time to culture conversion with the second objective to compare PK of first line TB therapy in those with and without problem alcohol use. Discussion: Globally, an urgent need exists to identify modifiable drivers of poor TB treatment outcomes. There is a critical need for more effective TB treatment strategies for patients with a history of problem alcohol use. However, it is not known whether poor treatment outcomes in alcohol using patients are solely attributable to noncompliance. This study will attempt to answer this question and provide guidance for future TB intervention trials. Trial registration: Clinicaltrials.gov, Registration Number: NCT02840877. Registered on 19 July 2016.

Browsing by Author "Warren, Robin M."

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