Diversity of Mycobacterium tuberculosis genotypes circulating in Ndola, Zambia

<p>Abstract</p> <p>Background</p> <p>Tuberculosis (TB) is one of the major public health problems in Zambia. However, information about lineages of <it>M. tuberculosis </it>complex (MTBC) isolates useful for epidemiology investigations is unknown. In this study, we investigated the diversity of MTBC isolates from Ndola, a typical Zambian urbanized city with a documented high HIV prevalence.</p> <p>Methods</p> <p>This was part of a prospective cohort study in subjects with sputum smear-positive pulmonary TB. Spoligotyping was used to genotype the MTBC isolates and establish the circulating lineages. The 15-locus Mycobacterial Interspersed Repetitive Units - Variable Number Tandem Repeats (MIRU-VNTR) typing was used to study recent transmission.</p> <p>Results</p> <p>A total of 98 different spoligotypes were identified among 273 MTBC isolates. The majority (64.8%) of the isolates belonged to 9 known families, while 96 (35.2%) of the isolates were orphans. While LAM (41.8%) was the largest spoligotype family observed, most of the isolates (87.7%) belonging to the SAF1 family, with a significant portion coming from the T (13.6%), and X (5.9%) families. A few isolates (3.6%) belonged to the CAS, EAI, H, S, X1-LAM9 or U families. MIRU-VNTR typing was highly discriminatory (h = 0.988) among the 156 isolates tested in our sample, and increased the discrimination among 82 SAF1 isolates from 6 to 46 distinct patterns. In addition, 3.2% (5/156) of cases with available MIRU-VNTR results harbored more than one MTBC strain.</p> <p>Conclusions</p> <p>Our findings show a limited diversity of MTBC in Ndola with a high clustering rate (37.7%), which indicates that recent transmission plays an appreciable role in the dynamics of TB disease in this setting. This conclusion emphasizes the importance of early diagnosis and timely treatment. The results also confirm that MIRU-VNTR typing is suitable for studying the molecular epidemiology of TB in Ndola.</p

Phylo-epidemiological and pathogenic diversity of Mycobacterium tuberculosis strains in London with implications for vaccine develpoment

PhDApproximately one-third of the global population is infected with tuberculosis causing approximately 1.7 million deaths. Currently, the BCG vaccine is used to protect against TB, but it cannot prevent primary infection or reactivation of latent infection. Ideally a vaccine should protect against a diverse array of Mycobacterium tuberculosis strains and promote a strong, long-lasting TH1 cell-mediated immune response. Whilst evaluating the efficiency of novel vaccines using laboratory control strains (M. tuberculosis H37Rv, H37Ra and M. bovis-BCG), it is important to test efficacy against a representative panel of wild-type circulating strains. In England 42.2% of TB cases are reported in London and the diversity of nationalities generates a diverse pool of strains consisting of globally representative TB strains. The aim of the study was to construct a representative panel of strains for vaccine evaluation studies and general TB research. Common M. tuberculosis strains were identified by performing molecular MIRUVNTR and spoligotyping on 2363 isolates from TB cases reported in London during a one-year period. Epidemiological analysis demonstrated there were representatives from 13 global regions, including high TB burden countries. An algorithm was designed to select strains for a preliminary panel based on associations between MTBC families in clusters of more common strains, the country of birth and VNTR sub-clusters. The preliminary panel contained 42 MTBC strains belonging to 10 MTBC families from patients born in 17 countries. Results of phylogenetic analysis of all 2363 isolates was used to select a smaller panel of strains from the preliminary panel to represent MTBC lineages to investigate if wild-type strains were phenotypically similar. The final panel included five strains from each of the Baker et al., 2004 M. tuberculosis lineages (M. tuberculosis Beijing, LAM10, two CAS, EAI5 strains representing lineage I, II, III, IV, respectively) and an M. africanum strain. In vitro tissue culture experiments demonstrated significantly higher growth of the Beijing strain compared to the other wild-type and laboratory strains. Higher growth rates of this strain were also observed in a cell-free culture system. Aerosol challenge of guinea pigs with wild-type strains showed a quicker dissemination of the EAI5 strain from the lung to the spleen 16 days post-challenge, but significantly higher c.f.u. count of the Beijing strain in the spleen 56 days post-challenge. Collectively, the data demonstrated that there are phenotypic differences between wild-type circulating MTBC strains

Molecular characterization of mycobacterium tuberculosis complex isolates in Mozambique

Mozambique is one of the high burden tuberculosis (TB) and human immunodeficiency virus (HIV) countries with a prevalence of HIV infection in adults of 11.5% and an estimated TB prevalence of 559 per 100 000 population. Fifty six percent of the TB patients in Mozambique are estimated to be HIV positive. TB control strategies might significantly be affected by differences in virulence, epidemiologic characteristics and epidemiology of particular strains of the Mycobacterium tuberculosis complex. Molecular epidemiology studies allow the identification of circulating strain types, understanding of transmission dynamics, as well as investigations of the evolution of the M. tuberculosis complex. The studies included in this thesis described the molecular epidemiology of M. tuberculosis complex in Mozambique, identified predominant genotypes responsible for TB transmission and prevalence and investigated the association between predominant spoligotypes and HIV sero-status. The prevalence and transmission of the Beijing genotype in Mozambique was also evaluated. With the aim to explore the public health risk for bovine TB, isolates from two sites were investigated, Maputo (tuberculous lymphadenitis or TBLN cases) and Govuro district (TBLN and pulmonary cases), the last site, Govuro, with known high prevalence of bovine TB in cattle (39.6%). Furthermore, a phylogenetic phylogeographic snapshot of worldwide M. tuberculosis complex diversity was created based on the classification of the Multiple-locus variable-number tandem repeat analysis (MLVA). For the first time, the genetic diversity of circulating M. tuberculosis complex strains in Mozambique was described. It was found that the TB epidemic in Mozambique was caused by a wide diversity of spoligotypes with predominance of the Latin-American Mediterranean (LAM, n=165 or 37%); East African-Indian (EAI, n=132 or 29.7%); the evolutionary recent T clade (n=52 or 11.6%) and the globally-emerging Beijing clone (n=31 or 7%). The predominant lineages were also common in neighboring countries, indicating TB transmission by migration from one country to another. The Beijing lineage, distributed worldwide and responsible for large epidemics was found to be particularly common in the Southern region of Mozambique, especially in Maputo City (17%) and associated with HIV infection (p=0.023). By combined use of region of difference (RD) analysis and spacer oligonucleotide typing (spoligotyping), a distinct group of four isolates had deletion of RD150, a signature of the “sublineage 7” recently emerging in South Africa. The same group was very similar to the South African “sublineage 7” by Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetitive Units–Variable-Number Tandem Repeat (MIRU-VNTR), suggesting that this sublineage could have been recently introduced in Mozambique from South Africa. No M. bovis was found in TBLN cases from Maputo. It was demonstrated that TBLN in Maputo was caused by a variety of M. tuberculosis genotypes, similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. For the first time, evidence of the occurrence of M. bovis in humans in Mozambique was revealed. In a study presently being conducted in the district of Govuro, among six M. tuberculosis complex isolates, one was M. bovis. Nevertheless, further research is needed on cases of abdominal TB and other forms of extrapulmonary TB, in Govuro and in other pastoral areas, where the prevalence of bovine TB in cattle is known to be high, in order to have a better answer about the public health importance of this zoonotic disease in Mozambique

Determination of circulating Mycobacterium tuberculosis strains and transmission patterns among pulmonary TB patients in Kawempe municipality, Uganda, using MIRU-VNTR

<p>Abstract</p> <p>Background</p> <p>Mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR) genotyping is a powerful tool for unraveling clonally complex <it>Mycobacterium tuberculosis </it>(MTB) strains and detection of transmission patterns. Using MIRU-VNTR, MTB genotypes and their transmission patterns among patients with new and active pulmonary tuberculosis (PTB) in Kawempe municipality in Kampala, Uganda was determined.</p> <p>Results</p> <p>MIRU-VNTR genotyping was performed by PCR-amplification of 15 MTB-MIRU loci from 113 cultured specimens from 113 PTB patients (one culture sample per patient). To determine lineages, the genotypes were entered into the MIRU-VNTR<it>plus </it>database [<url>http://www.miru-vntrplus.org/</url>] as numerical codes corresponding to the number of alleles at each locus. Ten different lineages were obtained: Uganda II (40% of specimens), Uganda I (14%), LAM (6%), Delhi/CAS (3%), Haarlem (3%), Beijing (3%), Cameroon (3%), EAI (2%), TUR (2%) and S (1%). Uganda I and Uganda II were the most predominant genotypes. Genotypes for 29 isolates (26%) did not match any strain in the database and were considered unique. There was high diversity of MIRU-VNTR genotypes, with a total of 94 distinct patterns. Thirty four isolates grouped into 15 distinct clusters each with two to four isolates. Eight households had similar MTB strains for both index and contact cases, indicating possible transmission.</p> <p>Conclusion</p> <p>MIRU-VNTR genotyping revealed high MTB strain diversity with low clustering in Kawempe municipality. The technique has a high discriminatory power for genotyping MTB strains in Uganda.</p

Genotypic and epidemiological characterization of Mycobacterium tuberculosis complex in Ghana

Tuberculosis (TB) remains a public health challenge. In 2013, TB was estimated to have caused 9 million incident cases of which 1.1 million were co infected with HIV and 1.5 million deaths worldwide. For the effective control of TB, the use of simplified diagnostic tools for case detection diagnosis of drug resistant TB and understanding the effects of comorbidities such as HIV on the prevalence of TB is paramount. Ghana, housing six of the seven phylogenetic lineages of Mycobacterium tuberculosis complex (MTBC) with high TB/HIV prevalence provides a unique opportunity to study and better understand the dynamics of TB. In the context of TB control, we studied the level of drug resistance using phenotypic drug susceptibility testing (DST) and correlated the DST results with patient treatment outcome (Chapter 3). We found a low rate of multidrug-resistant (MDR)-TB rate (1.9%), high isoniazid (INH) mono resistance (15%) and the dependence of treatment outcome on the susceptibility to rifampicin (RIF). For the rapid diagnosis of MDR cases, we further evaluated the accuracy of a molecular base diagnostic tool (Genotype MTBDRplus) and compared it with the gold standard phenotypic DST method (Chapter4). We found 100% correlation for detection of both MDR and RIF mono resistance and 83% for INH mono resistance. The remaining 17% INH resistance detected by standard phenotypic DST but not Genotype MTBDRplus are likely due to molecular mechanisms whose targets are not interrogated by Genotype MTBDRplus. The high overall sensitivity and the relative short turn- around time of Genotype MTBDRplus makes it a valuable addition to diagnostic algorithm in Ghana. The control of TB also depends on understanding the patterns and dynamics of TB transmission to reduce source of infection. Existing tools for studying transmission such as MIRU-15 used for routine molecular epidemiological studies have been shown to exhibit varying discriminatory power among the different human-associated MTBC lineages. We established a robust and cost-effective PCR based reduced but lineage-specific set of MIRU-VNTR loci with high discrimination power in the main MTBC circulating in Ghana (Chapter 5). This assay will help identify risk factors that enhance transmission and patient groups at increased risk of developing TB. In addition, this assay can be used to differentiate between exogenous re-infection from true relapse cases SNP- based genotyping and spoligotyping established that M. africanum (MAF) still causes 20% of all TB cases in Ghana (Chapter 6 and 7). Reasons for the restriction of MAF to West Africa have eluded researchers for many years. Using retrospective isolates, we provide for the first time plausible reason why MAF is restricted to parts of West Africa. We showed a significant association between MAF and the Ewe ethnic group. This association was confirmed using prospective isolates and supports possible host pathogen co-evolution inn TB. In addition, we observed a strong association between MAF2 and HIV co-infection supporting the notion that MAF might have a lower virulence compared to other MTBC in human

Genetic Diversity of Mycobacterium tuberculosis Isolates from Tibetans in Tibet, China

BACKGROUND: Tuberculosis (TB) is a serious health problem in Tibet where Tibetans are the major ethnic group. Although genotyping of Mycobacterium tuberculosis (M. tuberculosis) isolates is a valuable tool for TB control, our knowledge of population structure of M. tuberculosis circulating in Tibet is limited. METHODOLOGY/PRINCIPAL FINDINGS: In our study, a total of 576 M. tuberculosis isolates from Tibetans in Tibet, China, were analyzed via spoligotyping and 24-locus MIRU-VNTR. The Beijing genotype was the most prevalent family (90.63%, n = 522). Shared-type (ST) 1 was the most dominant genotype (88.89%, n = 512). We found that there was no association between the Beijing genotype and sex, age and treatment status. In this sample collection, 7 of the 24 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. An informative set of 12 loci had similar discriminatory power with 24 loci set. CONCLUSIONS/SIGNIFICANCE: The population structure of M. tuberculosis isolates in Tibetans is homogeneous and dominated by Beijing genotype. The analysis of 24-locus MIRU-VNTR data might be useful to select appropriate VNTR loci for the genotyping of M. tuberculosis

First insights into the genetic diversity of Mycobacterium tuberculosis isolates from HIV-infected Mexican patients and mutations causing multidrug resistance

<p>Abstract</p> <p>Background</p> <p>The prevalence of infections with <it>Mycobacterium tuberculosis </it>(MTb) and nontuberculous mycobacteria (NTM) species in HIV-infected patients in Mexico is unknown. The aims of this study were to determine the frequency of MTb and NTM species in HIV-infected patients from Mexico City, to evaluate the genotypic diversity of the <it>Mycobacterium tuberculosis </it>complex strains, to determine their drug resistance profiles by colorimetric microplate Alamar Blue assay (MABA), and finally, to detect mutations present in <it>kat</it>G, <it>rpo</it>B and <it>inh</it>A genes, resulting in isoniazid (INH) and rifampin (RIF) resistance.</p> <p>Results</p> <p>Of the 67 mycobacterial strains isolated, 48 were identified as MTb, 9 as <it>M. bovis</it>, 9 as <it>M. avium </it>and 1 as <it>M. intracellulare</it>. IS<it>6110</it>-RFLP of 48 MTb strains showed 27 profiles. Spoligotyping of the 48 MTb strains yielded 21 patterns, and 9 <it>M. bovis </it>strains produced 7 patterns. Eleven new spoligotypes patterns were found. A total of 40 patterns were produced from the 48 MTb strains when MIRU-VNTR was performed. Nineteen (39.6%) MTb strains were resistant to one or more drugs. One (2.1%) multidrug-resistant (MDR) strain was identified. A novel mutation was identified in a RIF-resistant strain, GAG → TCG (Glu → Ser) at codon 469 of <it>rpo</it>B gene.</p> <p>Conclusions</p> <p>This is the first molecular analysis of mycobacteria isolated from HIV-infected patients in Mexico, which describe the prevalence of different mycobacterial species in this population. A high genetic diversity of MTb strains was identified. New spoligotypes and MIRU-VNTR patterns as well as a novel mutation associated to RIF-resistance were found. This information will facilitate the tracking of different mycobacterial species in HIV-infected individuals, and monitoring the spread of these microorganisms, leading to more appropriate measures for tuberculosis control.</p

Developing customized stepwise MIRU-VNTR typing for tuberculosis surveillance in Georgia

INTRODUCTION: Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) typing has been widely used for molecular epidemiological studies of tuberculosis (TB). However, genotyping tools for Mycobacterium tuberculosis (Mtb) may be limiting in some settings due to high cost and workload. In this study developed a customized stepwise MIRU-VNTR typing that prioritizes high discriminatory loci and validated this method using penitentiary system cohort in the country of Georgia. METHODS: We used a previously generated MIRU-VNTR dataset from recurrent TB cases (32 cases) in Georgia and a new dataset of TB cases from the penitentiary system (102 cases) recruited from 2014 to 2015. A Hunter-Gaston Discriminatory Index (HGDI) was calculated utilizing a 24 standard loci panel, to select high discriminatory power loci, subsequently defined as the customized Georgia-specific set of loci for initial typing. The remaining loci were scored and hierarchically grouped for second and third step typing of the cohort. We then compared the processing time and costs of the customized stepwise method to the standard 24-loci method. RESULTS: For the customized Georgia-specific set that was used for initial typing, 10 loci were selected with a minimum value of 0.32 to the highest HGDI score locus. Customized 10 loci (step 1) typing of 102 Mtb patient isolates revealed 35.7% clustered cases. This proportion was reduced to 19.5% after hierarchical application of 2nd and 3rd step typing with the corresponding groups of loci. Our customized stepwise MIRU-VNTR genotyping approach reduced the quantity of samples to be typed and therefore overall processing time and costs by 42.6% each. CONCLUSION: Our study shows that our customized stepwise MIRU-VNTR typing approach is a valid alternative of standard MIRI-VNTR typing panels for molecular epidemiological investigation in Georgia that saves time, workload and costs. Similar approaches could be developed for other settings