Browsing by Author "Van Helden, Paul D."
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- ItemAdaptation 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.
- ItemAntigen-specific IP-10 release is a sensitive biomarker of mycobacterium bovis infection in cattle(Public Library of Science, 2016) Parsons, Sven D. C.; McGill, Kevina; Doyle, Mairead B.; Goosen, Wynand J.; Van Helden, Paul D.; Gormley, EamonnThe most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%–100%) and a specificity of 97% (95% CI, 85%–100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle.
- ItemBovine tuberculosis in African buffaloes : observations regarding Mycobacterium bovis shedding into water and exposure to environmental mycobacteria(BioMed Central, 2007-09) Michel, Anita L.; De Klerk, Lin-Mari; Gey van Pittius, Nico C.; Warren, Rob; Van Helden, Paul D.Background: African buffaloes are the maintenance host for Mycobacterium bovis in the endemically infected Kruger National Park (KNP). The infection is primarily spread between buffaloes via the respiratory route, but it is not known whether shedding of M. bovis in nasal and oral excretions may lead to contamination of ground and surface water and facilitate the transmission to other animal species. A study to investigate the possibility of water contamination with M. bovis was conducted in association with a BCG vaccination trial in African buffalo. Groups of vaccinated and nonvaccinated buffaloes were kept together with known infected in-contact buffalo cows to allow natural M. bovis transmission under semi-free ranging conditions. In the absence of horizontal transmission vaccinated and control buffaloes were experimentally challenged with M. bovis. Hence, all study buffaloes in the vaccination trial could be considered potential shedders and provided a suitable setting for investigating questions relating to the tenacity of M. bovis shed in water. Results: Serial water samples were collected from the drinking troughs of the buffaloes once per season over an eleven-month period and cultured for presence of mycobacteria. All water samples were found to be negative for M. bovis, but 16 non-tuberculous Mycobacterium spp. isolates were cultured. The non-tuberculous Mycobacterium species were further characterised using 5'-16S rDNA PCR-sequencing, resulting in the identification of M. terrae, M. vaccae (or vanbaalenii), M. engbaekii, M. thermoresistibile as well as at least two species which have not yet been classified. Conclusion: The absence of detectable levels of Mycobacterium bovis in the trough water suggests that diseased buffalo do not commonly shed the organism in high quantities in nasal and oral discharges. Surface water may therefore not be likely to play an important role in the transmission of bovine tuberculosis from buffalo living in free-ranging ecosystems. The study buffalo were, however, frequently exposed to different species of non-tuberculous, environmental mycobacteria, with an unknown effect on the buffaloes' immune response to mycobacteria.
- ItemClinical relevance of nontuberculous mycobacteria isolated from sputum in a gold mining workforce in South Africa : an observational, clinical study(Hindawi, 2015) Van Halsema, Clare L.; Chihota, Violet N.; Gey van Pittius, Nicolaas C.; Fielding, Katherine L.; Lewis, James J.; Van Helden, Paul D.; Churchyard, Gavin J.; Grant, Alison D.Background.The clinical relevance of nontuberculous mycobacteria (NTM), detected by liquid more than solid culture in sputum specimens from a South African mining workforce, is uncertain. We aimed to describe the current spectrum and relevance of NTMin this population. Methods. An observational study including individuals with sputum NTMisolates, recruited at workforce tuberculosis screening and routine clinics. Symptomquestionnaires were administered at the time of sputumcollection and clinical records and chest radiographs reviewed retrospectively. Results.Of 232 individuals included (228 (98%) male,median age 44 years), M. gordonae (60 individuals), M. kansasii (50), and M. aviumcomplex (MAC: 38) were the commonest species.Of 38MAC isolates, only 2 (5.3%) were from smear-positive sputum specimens and 30/38 grew in liquid but not solid culture. MAC was especially prevalent among symptomatic, HIV-positive individuals. HIV prevalence was high: 57/74 (77%) among those tested.No differences were found in probability of death or medical separation by NTM species. Conclusions. M. gordonae, M. kansasii, andMAC were the commonest NTMamong miners with suspected tuberculosis, withmostMAC fromsmear-negative specimens in liquid culture only. HIV testing and identification of key pathogenic NTM in this setting are essential to ensure optimal treatment.
- ItemComparative 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.
- ItemThe complete genome sequence of the African buffalo (Syncerus caffer)(BioMed Central, 2016-12-07) Glanzmann, Brigitte; Moller, Marlo; Le Roex, Nikki; Tromp, Gerard; Hoal, Eileen G.; Van Helden, Paul D.Background: The African buffalo (Syncerus caffer) is an important role player in the savannah ecosystem. It has become a species of relevance because of its role as a wildlife maintenance host for an array of infectious and zoonotic diseases some of which include corridor disease, foot-and-mouth disease and bovine tuberculosis. To date, no complete genome sequence for S. caffer had been available for study and the genomes of other species such as the domestic cow (Bos taurus) had been used as a proxy for any genetics analysis conducted on this species. Here, the high coverage genome sequence of the African buffalo (S. caffer) is presented. Results: A total of 19,765 genes were predicted and 19,296 genes could be successfully annotated to S. caffer while 469 genes remained unannotated. Moreover, in order to extend a detailed annotation of S. caffer, gene clusters were constructed using twelve additional mammalian genomes. The S. caffer genome contains 10,988 gene clusters, of which 62 are shared exclusively between B. taurus and S. caffer. Conclusions: This study provides a unique genomic perspective for the S. caffer, allowing for the identification of novel variants that may play a role in the natural history and physiological adaptations.
- ItemContext‐dependent costs and benefits of tuberculosis resistance traits in a wild mammalian host(Wiley Open Access, 2018) Tavalire, Hannah F.; Beechler, Brianna R.; Buss, Peter E.; Gorsich, Erin E.; Hoal, Eileen G.; Le Roex, Nikki; Spaan, Johannie M.; Spaan, Robert S.; Van Helden, Paul D.; Ezenwa, Vanessa O.; Jolles, Anna E.Disease acts as a powerful driver of evolution in natural host populations, yet individuals in a population often vary in their susceptibility to infection. Energetic trade‐offs between immune and reproductive investment lead to the evolution of distinct life history strategies, driven by the relative fitness costs and benefits of resisting infection. However, examples quantifying the cost of resistance outside of the laboratory are rare. Here, we observe two distinct forms of resistance to bovine tuberculosis (bTB), an important zoonotic pathogen, in a free‐ranging African buffalo (Syncerus caffer) population. We characterize these phenotypes as “infection resistance,” in which hosts delay or prevent infection, and “proliferation resistance,” in which the host limits the spread of lesions caused by the pathogen after infection has occurred. We found weak evidence that infection resistance to bTB may be heritable in this buffalo population (h2 = 0.10) and comes at the cost of reduced body condition and marginally reduced survival once infected, but also associates with an overall higher reproductive rate. Infection‐resistant animals thus appear to follow a “fast” pace‐of‐life syndrome, in that they reproduce more quickly but die upon infection. In contrast, proliferation resistance had no apparent costs and was associated with measures of positive host health—such as having a higher body condition and reproductive rate. This study quantifies striking phenotypic variation in pathogen resistance and provides evidence for a link between life history variation and a disease resistance trait in a wild mammalian host population.
- ItemCytokine 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.
- ItemDifferences in primary sites of infection between zoonotic and human tuberculosis : results from a worldwide systematic review(Public Library of Science, 2013-08-29) Durr, Salome; Muller, Borna; Alonso, Silvia; Hattendorf, Jan; Laisse, Claudio J. M.; Van Helden, Paul D.; Zinsstag, JakobTuberculosis (TB) is one of the most devastating infectious diseases worldwide. Whilst global burden estimates for M. tuberculosis infection (MtTB) are well established, accurate data on the contribution of zoonotic TB (zTB) caused by M. bovis or M. caprae to human TB are scarce. The association of M. bovis infection with extrapulmonary tuberculosis has been suggested repeatedly, though there is little scientific evidence available to support this relationship. The present study aimed to determine globally the occurrence of extrapulmonary TB and the primary site (i.e. primary body location affected) of zTB in comparison with MtTB, based on previously published reports. A systematic literature review was conducted in 32 different bibliographic databases, selecting reports on zTB written in English, French, German, Spanish or Portuguese. Data from 27 reports from Africa, America, Europe and the Western Pacific Region were extracted for analyses. Low income countries, in Africa and South-East Asia, were highly underrepresented in the dataset. The median proportion of extrapulmonary TB cases was significantly increased among zTB in comparison with data from registries of Europe and USA, reporting mainly MtTB cases (47% versus 22% in Europe, 73% versus 30% in the USA). These findings were confirmed by analyses of eight studies reporting on the proportions of extrapulmonary TB in comparable populations of zTB and MtTB cases (median 63% versus 22%). Also, disparities of primary sites of extrapulmonary TB between zTB and MtTB were detected. Our findings, based on global data, confirm the widely suggested association between zTB and extrapulmonary disease. Different disability weights for zTB and MtTB should be considered and we recommend separate burden estimates for the two diseases.
- ItemDrug-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, KeertanBackground: 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.
- ItemThe effect of diagnostic delays on the drop-out rate and the total delay to diagnosis of tuberculosis(Public Library of Science, 2008-04-09) Millen, Stephen J.; Uys, Pieter W.; Hargrove, John; Van Helden, Paul D.; Williams, Brian G.Background: Numerous patient and healthcare system-related delays contribute to the overall delay experienced by patients from onset of TB symptoms to diagnosis and treatment. Such delays are critical as infected individuals remain untreated in the community, providing more opportunities for transmission of the disease and adversely affecting the epidemic. Methodology/Principal Findings: We present an analysis of the factors that contribute to the overall delay in TB diagnosis and treatment, in a resource-poor setting. Impact on the distribution of diagnostic delay times was assessed for various factors, the sensitivity of the diagnostic method being found to be the most significant. A linear relationship was found between the sensitivity of the test and the predicted mean delay time, with an increase in test sensitivity resulting in a reduced mean delay time and a reduction in the drop-out rate. Conclusions/Significance: The results show that in a developing country a number of delay factors, particularly the low sensitivity of the initial sputum smear microscopy test, potentially increase total diagnostic delay times experienced by TB patients significantly. The results reinforce the urgent need for novel diagnostic methods, both for smear positive and negative TB, that are highly sensitive, accessible and point of care, in order to reduce mean delay times. © 2008 Millen et al.
- ItemErgothioneine is a secreted antioxidant in mycobacterium smegmatis(American Society for Microbiology, 2013-07) Emani, Carine Sao; Williams, Monique J.; Wiid, Ian J.; Hiten, Nicholas F.; Viljoen, Albertus J.; Pietersen, Ray-Dean D.; Van Helden, Paul D.; Baker, BienyameenErgothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH.
- ItemEvidence 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.
- ItemEvidence 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.
- ItemEvolution 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.
- ItemExome sequencing identifies a novel TTC37 mutation in the first reported case of Trichohepatoenteric syndrome (THE-S) in South Africa(BioMed Central, 2017-03-14) Kinnear, Craig; Glanzmann, Brigitte; Banda, Eric; Schlechter, Nikola; Durrheim, Glenda; Neethling, Annika; Nel, Etienne; Schoeman, Mardelle; Johnson, Glynis; Van Helden, Paul D.; Hoal, Eileen G; Esser, Monika; Urban, Michael; Moller, MarloBackground Trichohepatoenteric syndrome (THE-S) or phenotypic diarrhoea of infancy is a rare autosomal recessive disorder characterised by severe infantile diarrhoea, facial dysmorphism, immunodeficiency and woolly hair. It was first described in 1982 in two infants with intractable diarrhoea, liver cirrhosis and abnormal hair structure on microscopy. We report on two siblings from a consanguineous family of Somali descent who, despite extensive clinical investigation, remained undiagnosed until their demise. The index patient died of fulminant cytomegalovirus pneumonitis at 3 months of age. Methods Whole exome sequencing (WES) was performed on a premortem DNA sample from the index case. Variants in a homozygous recessive state or compound heterozygous state were prioritized as potential candidate variants using TAPER™. Sanger sequencing was done to genotype the parents, unaffected sibling and a deceased sibling for the variant of interest. Results Exome sequencing identified a novel homozygous mutation (c.4507C > T, rs200067423) in TTC37 which was confirmed by Sanger sequencing in the index case. The identification of this mutation led to the diagnosis of THE-S in the proband and the same homozygous variant was confirmed in a male sibling who died 4 years earlier with severe chronic diarrhoea of infancy. The unaffected parents and sister were heterozygous for the identified variant. Conclusions WES permitted definitive genetic diagnosis despite an atypical presentation in the index case and suggests that severe infection, likely secondary to immunodeficiency, may be a presenting feature. In addition definitive molecular diagnosis allows for genetic counseling and future prenatal diagnosis, and demonstrates the value of WES for post-mortem diagnosis of disorders with a non-specific clinical presentation in which a Mendelian cause is suspected.
- ItemGeneration and characterization of thiol-deficient Mycobacterium tuberculosis mutants(Nature Research, 2018) Emani, C. Sao; Williams, M. J.; Van Helden, Paul D.; Taylor, M. J. C.; Carolis, C.; Wiid, I. J.; Baker, B.Mycothiol (MSH) and ergothioneine (ERG) are thiols able to compensate for each other to protect mycobacteria against oxidative stress. Gamma-glutamylcysteine (GGC), another thiol and an intermediate in ERG biosynthesis has detoxification abilities. Five enzymes are involved in ERG biosynthesis, namely EgtA, EgtB, EgtC, EgtD and EgtE. The role of these enzymes in the production of ERG had been unclear. On the other hand, the enzyme MshA is known to be essential for MSH biosynthesis. In this manuscript, we describe the raw data of the generation and characterization of Mycobacterium tuberculosis (M.tb) mutants harbouring a deletion of the gene coding for each of these enzymes, and the raw data of the phenotypic characterization of the obtained thiol-deficient M.tb mutants. High throughput screening (HTS) of off-patent drugs and natural compounds revealed few compounds that displayed a higher activity against the thiol-deficient mutants relative to the wild-type strain. The mode of action of these drugs was further investigated. Raw data displaying these results are described here.
- ItemGenetic diversity of Mycobacterium tuberculosis complex strains isolated from livestock workers and cattle in Nigeria(PLoS, 2019-02-20) Adesokan, Hezekiah K.; Streicher, Elizabeth M.; Van Helden, Paul D.; Warren, Rob M.; Cadmus, Simeon I. B.ENGLISH ABSTRACT: Molecular typing techniques are useful in understanding tuberculosis epidemiology; yet, they have been under-utilised at the human-animal interface in Nigeria. Sixty-four Mycobacterium tuberculosis complex (MTBC) isolates including 42 M. tuberculosis, 13 M. bovis and nine M. africanum obtained from livestock workers (LW, n = 47) and their cattle (n = 17) in three geographical zones of Nigeria were genotyped to identify and evaluate the genetic diversity of the circulating MTBC using spoligotyping. Distribution into clades of M. tuberculosis revealed; 45.3% Uganda I- [SIT46- cattle: 1; LW: 28], 14.1% Latin American Mediterranean- [SIT61, cattle: 1; LW: 8], and 1.6% T- [SIT53—LW: 1]. The M. bovis strains were 6.3% SB0944 [cattle: 4] and 1.6% each of SB0300, SB1026, SB1027 and SB1439 [cattle: 4]. Seventeen MTBC isolates [cattle: 7; LW: 10] yielded 14 new spoligotype patterns including three M. tuberculosis strains (three isolates), five M. bovis strains (five isolates) and six M. africanum strains (nine isolates), two of which belonged to MAF1. Only few families namely, the not previously described Uganda I-, LAM and SB0944 are predominant among the LW and cattle, with other types in lower prevalences. The strain population structure indicates an intriguing diversity and possible zoonotic linkage with consequences for TB control in the country. The need to employ newer molecular techniques such as Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats and whole genome sequence to decipher circulating MTBC strains in Nigeria is advocated.
- ItemGenetic resistance to mycobacterium tuberculosis infection and disease(Frontiers Media, 2017) Moller, Marlo; Kinnear, Craig J.; Orlova, Marianna; Kroon, Elouise E.; Van Helden, Paul D.; Schurr, Erwin; Hoal, Eileen G.Natural history studies of tuberculosis (TB) have revealed a spectrum of clinical outcomes after exposure to Mycobacterium tuberculosis, the cause of TB. Not all individuals exposed to the bacterium will become diseased and depending on the infection pressure, many will remain infection-free. Intriguingly, complete resistance to infection is observed in some individuals (termed resisters) after intense, continuing M. tuberculosis exposure. After successful infection, the majority of individuals will develop latent TB infection (LTBI). This infection state is currently (and perhaps imperfectly) defined by the presence of a positive tuberculin skin test (TST) and/or interferon gamma release assay (IGRA), but no detectable clinical disease symptoms. The majority of healthy individuals with LTBI are resistant to clinical TB, indicating that infection is remarkably well-contained in these non-progressors. The remaining 5–15% of LTBI positive individuals will progress to active TB. Epidemiological investigations have indicated that the host genetic component contributes to these infection and disease phenotypes, influencing both susceptibility and resistance. Elucidating these genetic correlates is therefore a priority as it may translate to new interventions to prevent, diagnose or treat TB. The most successful approaches in resistance/susceptibility investigation have focused on specific infection and disease phenotypes and the resister phenotype may hold the key to the discovery of actionable genetic variants in TB infection and disease. This review will not only discuss lessons from epidemiological studies, but will also focus on the contribution of epidemiology and functional genetics to human genetic resistance to M. tuberculosis infection and disease.
- ItemGeospatial 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.