SUNScholar - Browsing by Author "Gey van Pittius, Nicolaas C."

Browsing by Author "Gey van Pittius, Nicolaas C."

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Clinical 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.
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 genomics and proteomic analysis of four non-tuberculous Mycobacterium species and Mycobacterium tuberculosis complex : occurrence of shared immunogenic proteins
(Frontiers, 2016-06-07) Gcebe, Nomakorinte; Michel, Anita; Gey van Pittius, Nicolaas C.; Rutten, Victor
The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis identified a total of 609 proteins in the six PPDs and 22 of these were identified as shared between PPD of M.bovis and one or more of the NTM PPDs. Previously characterized M tuberculosis/M. bovis homologous immunogenic proteins detected in one or more of the nonpathogenic NTM in this study included CFP-10 (detected in M. malmesburii sp. nov. PPD), GroES (detected in all NTM PPDs but M. malmesburii sp. nov.), DnaK (detected in all NTM PPDs), and GroEL (detected in all NTM PPDs). This study confirms reports that the ESX-1, ESX-3, and ESX-4 regions are ancestral regions and thus found in the genomes of most mycobacteria. Identification of NTM homologs of immunogenic proteins warrants further investigation of their ability to cause cross-reactive immune responses with MTBC antigens.
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.
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.
Insights into the evolutionary history of tubercle bacilli as disclosed by genetic rearrangements within a PE_PGRS duplicated gene pair
(BioMed Central, 2006-12) Karboul, Anis; Gey van Pittius, Nicolaas C.; Namouchi, Amine; Vincent, Veronique; Sola, Christophe; Rastogi, Nalin; Suffys, Philip; Fabre, Michel; Cataldi, Angel; Huard, Richard C.; Kurepina, Natalia; Kreiswirth, Barry; Ho, John L.; Gutierrez, M. Cristina; Mardassi, Helmi
Background: The highly homologous PE_PGRS (Proline-glutamic acid_polymorphic GC-rich repetitive sequence) genes are members of the PE multigene family which is found only in mycobacteria. PE genes are particularly abundant within the genomes of pathogenic mycobacteria where they seem to have expanded as a result of gene duplication events. PE_PGRS genes are characterized by their high GC content and extensive repetitive sequences, making them prone to recombination events and genetic variability. Results: Comparative sequence analysis of Mycobacterium tuberculosis genes PE_PGRS17 (Rv0978c) and PE_PGRS18 (Rv0980c) revealed a striking genetic variation associated with this typical tandem duplicate. In comparison to the M. tuberculosis reference strain H37Rv, the variation (named the 12/40 polymorphism) consists of an in-frame 12-bp insertion invariably accompanied by a set of 40 single nucleotide polymorphisms (SNPs) that occurs either in PE_PGRS17 or in both genes. Sequence analysis of the paralogous genes in a representative set of worldwide distributed tubercle bacilli isolates revealed data which supported previously proposed evolutionary scenarios for the M. tuberculosis complex (MTBC) and confirmed the very ancient origin of "M. canettii" and other smooth tubercle bacilli. Strikingly, the identified polymorphism appears to be coincident with the emergence of the post-bottleneck successful clone from which the MTBC expanded. Furthermore, the findings provide direct and clear evidence for the natural occurrence of gene conversion in mycobacteria, which appears to be restricted to modern M. tuberculosis strains. Conclusion: This study provides a new perspective to explore the molecular events that accompanied the evolution, clonal expansion, and recent diversification of tubercle bacilli.
Molecular epidemiological interpretation of the epidemic of extensively drug-resistant tuberculosis in South Africa
(American Society for Microbiology, 2015) Streicher, Elizabeth M.; Sampson, S. L.; Dheda, K.; Dolby, T.; Simpson, J. A.; Victor, T. C.; Gey van Pittius, Nicolaas C.; Van Helden, Paul D.; Warren, Robin M.
We show that the interpretation of molecular epidemiological data for extensively drug-resistant tuberculosis (XDR-TB) is dependent on the number of different markers used to define transmission. Using spoligotyping, IS6110 DNA fingerprinting, and DNA sequence data, we show that XDR-TB in South Africa (2006 to 2008) was predominantly driven by the acquisition of second-line drug resistance.
The non-clonality of drug resistance in Beijing-genotype isolates of Mycobacterium tuberculosis from the Western Cape of South Africa
(BioMed Central, 2010-11) Loerger, Thomas R.; Feng, Yicheng; Chen, Xiaohua; Dobos, Karen M.; Victor, Thomas C.; Streicher, Elizabeth M.; Warren, Robin M.; Gey van Pittius, Nicolaas C.; Van Helden, Paul D.; Sacchettini, James C.
Background. The Beijing genotype of M. tuberculosis is a virulent strain that is disseminating worldwide and has a strong association with drug resistance. In the Western Cape of South Africa, epidemiological studies have identified the R220 cluster of the Beijing genotype as a major contributor to a recent outbreak of drug-resistant tuberculosis. Although the outbreak is considered to be due to clonal transmission, the relationship among drug resistant isolates has not yet been established. Results. To better understand the evolution of drug resistance among these strains, 14 drug-resistant clinical isolates of the Beijing genotype were sequenced by whole-genome sequencing, including eight from R220 and six from a more ancestral Beijing cluster, R86, for comparison. While each cluster shares a distinct resistance mutation for isoniazid, mapping of other drug-resistance mutations onto a phylogenetic tree constructed from single nucleotide polymorphisms shows that resistance mutations to many drugs have arisen multiple times independently within each cluster of isolates. Thus, drug resistance among these isolates appears to be acquired, not clonally derived. This observation suggests that, although the Beijing genotype as a whole might have selective advantages enabling its rapid dissemination, the XDR isolates are relatively less fit and do not propagate well. Although it has been hypothesized that the increased frequency of drug resistance in some Beijing lineages might be caused by a mutator phenotype, no significant shift in synonymous substitution patterns is observed in the genomes. Conclusion. While MDR-TB is spreading by transmission in the Western Cape, our data suggests that further drug resistance (i.e. XDR-TB) at this stage is acquired.
Novel cause of tuberculosis in meerkats, South Africa
(Centers for Disease Control and Prevention, 2013-12) Parsons, Sven D. C.; Drewe, Julian A.; Gey van Pittius, Nicolaas C.; Warren, Robin M.; Van Helden, Paul D.
ENGLISH ABSTRACT: The organism that causes tuberculosis in meerkats (Suricata suricatta) has been poorly characterized. Our genetic analysis showed it to be a novel member of the Mycobacterium tuberculosis complex and closely related to the dassie bacillus. We have named this epidemiologically and genetically unique strain M. suricattae.
Population structure of mixed Mycobacterium tuberculosis infection is strain genotype and culture medium dependent
(Public Library of Science, 2013-07-30) Hanekom, Madeleine; Streicher, Elizabeth M.; Van de Berg, Doreen; Cox, Helen; McDermid, Cheryl; Bosman, Marlein; Gey van Pittius, Nicolaas C.; Victor, Tommie C.; Kidd, Martin; Van Soolingen, Dick; Van Helden, Paul D.; Warren, Robin M.
Background Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection. Aim This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein–Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes. Method A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes. Results The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with “other genotype” strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype. Conclusion The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.
Programmatically selected multidrug-resistant strains drive the emergence of extensively drug-resistant tuberculosis in South Africa
(Public Library of Science, 2013-08-23) Muller, Borna; Chihota, Violet N.; Pillay, Manormoney; Klopper, Marisa; Streicher, Elizabeth M.; Coetzee, Gerrit; Trollip, Andre; Hayes, Cindy; Bosman, Marlein E.; Gey van Pittius, Nicolaas C.; Victor, Thomas C.; Gagneux, Sebastien; Van Helden, Paul D.; Warren, Robin M.
Background: South Africa shows one of the highest global burdens of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). Since 2002, MDR-TB in South Africa has been treated by a standardized combination therapy, which until 2010 included ofloxacin, kanamycin, ethionamide, ethambutol and pyrazinamide. Since 2010, ethambutol has been replaced by cycloserine or terizidone. The effect of standardized treatment on the acquisition of XDR-TB is not currently known. Methods: We genetically characterized a random sample of 4,667 patient isolates of drug-sensitive, MDR and XDR-TB cases collected from three South African provinces, namely, the Western Cape, Eastern Cape and KwaZulu-Natal. Drug resistance patterns of a subset of isolates were analyzed for the presence of commonly observed resistance mutations. Results: Our analyses revealed a strong association between distinct strain genotypes and the emergence of XDR-TB in three neighbouring provinces of South Africa. Strains predominant in XDR-TB increased in proportion by more than 20-fold from drug-sensitive to XDR-TB and accounted for up to 95% of the XDR-TB cases. A high degree of clustering for drug resistance mutation patterns was detected. For example, the largest cluster of XDR-TB associated strains in the Eastern Cape, affecting more than 40% of all MDR patients in this province, harboured identical mutations concurrently conferring resistance to isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, ethionamide, kanamycin, amikacin and capreomycin. Conclusions: XDR-TB associated genotypes in South Africa probably were programmatically selected as a result of the standard treatment regimen being ineffective in preventing their transmission. Our findings call for an immediate adaptation of standard treatment regimens for M/XDR-TB in South Africa.

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