Characterization of Mycobacterium tuberculosis Central Asian Strain1 using mycobacterial interspersed repetitive unit genotyping

<p>Abstract</p> <p>Background</p> <p>The Central Asian Strain1 (CAS1) genogroup of <it>Mycobacterium tuberculosis </it>(MTB) is the most prevalent in Pakistan, India and Bangladesh. Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing is a reliable and reproducible method for differentiation of MTB isolates. However, information of its utility in determining the diversity of CAS1 strain is limited. We performed standard 12 loci based MIRU-VNTR typing on previously spoligotyped CAS1 strains and 'unique' strains in order to evaluate its discriminatory power for these isolates.</p> <p>Methods</p> <p>Twelve loci based MIRU- VNTR typing was used to type178 CAS1 and 189 'unique' MTB strains. The discriminatory index for each of the loci was calculated using the Hunter Gaston Discriminatory Index (HGDI). A subset of these strains (n = 78) were typed using IS<it>6110 </it>restriction fragment length polymorphism (RFLP). MIRU-VNTR profiles were studied together with their drug susceptibility patterns.</p> <p>Results</p> <p>A total of 349 MIRU patterns were obtained for the 367 strains tested. The CAS1 strains were subdivided into 160 distinct patterns; 15 clusters of 2 strains each, 1 cluster of four strains and 144 unique patterns. Using HGDI, seven MIRU loci, (numbers 26, 31, 27, 16, 10, 39, and 40) were found to be "highly discriminatory" (DI: ≥0.6), four MIRU loci (numbers 20, 24, 23, and 4) were "moderately discriminatory" (DI: 0.3–0.59), and one locus (number 2) was "poorly discriminatory" (DI< 0.3). Loci 26 and 31 were the most discriminatory for the CAS1 isolates. Amongst 'unique' strains in addition to loci 26, 31, 27, 16, 10, 39, and 40, locus 23 was highly discriminatory, while no locus was poorly discriminating. DI values for loci 4, 10 and 26 were significantly lower (P-value < .01) in CAS1 strains than in 'unique' strains. The association between CAS1 strains and MDR was not found to be significant (p value = 0.21).</p> <p>Conclusion</p> <p>We propose that MIRU typing could be used to estimate the phylogenetic relatedness amongst prevalent CAS1 strains, for which MIRU loci 26, 31, 16, 10, 27, 39 and 40 were found to be the most discriminatory.</p

Human Leukocyte Antigen Class 1 Phenotype Distribution and Analysis in Persons from Central Uganda with Active Tuberculosis and Latent Mycobacterium tuberculosis Infection

Background: The Ugandan population is heavily affected by infectious diseases and Human leukocyte antigen (HLA) diversity plays a crucial role in the host-pathogen interaction and affects the rates of disease acquisition and outcome. The identification of HLA class 1 alleles and determining which alleles are associated with tuberculosis (TB) outcomes would help in screening individuals in TB endemic areas for susceptibility to TB and to predict resistance or progression to TB which would inevitably lead to better clinical management of TB. Aims: To be able to determine the HLA class 1 phenotype distribution in a Ugandan TB cohort and to establish the relationship between these phenotypes and active and latent TB. Methods: Blood samples were drawn from 32 HIV negative individuals with active TB and 45 HIV negative individuals with latent MTB infection. DNA was extracted from the blood samples and the DNA samples HLA typed by the polymerase chain reaction-sequence specific primer method. The allelic frequencies were determined by direct count. Results: HLA-A*02, A*01, A*74, A*30, B*15, B*58, C*07, C*03 and C*04 were the dominant phenotypes in this Ugandan cohort. There were differences in the distribution of HLA types between the individuals with active TB and the individuals with LTBI with only HLA-A*03 allele showing a statistically significant difference (p=0.0136). However, after FDR computation the corresponding q-value is above the expected proportion of false discoveries (q-value 0.2176). Key findings: We identified a number of HLA class I alleles in a population from Central Uganda which will enable us to carry out a functional characterization of CD8+ T-cell mediated immune responses to MTB. Our results also suggest that there may be a positive association between the HLA-A*03 allele and TB implying that individuals with the HLA-A*03 allele are at a higher risk of developing active TB

DNA restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from HIV-seropositive and HIV-seronegative patients in Kampala, Uganda

<p>Abstract</p> <p>Background</p> <p>The identification and differentiation of strains of <it>Mycobacterium tuberculosis </it>by DNA fingerprinting has provided a better understanding of the epidemiology and tracing the transmission of tuberculosis. We set out to determine if there was a relationship between the risk of belonging to a group of tuberculosis patients with identical mycobacterial DNA fingerprint patterns and the HIV sero-status of the individuals in a high TB incidence peri-urban setting of Kampala, Uganda.</p> <p>Methods</p> <p>One hundred eighty three isolates of <it>Mycobacterium tuberculosis </it>from 80 HIV seropositive and 103 HIV seronegative patients were fingerprinted by standard IS<it>6110</it>-RFLP. Using the BioNumerics software, strains were considered to be clustered if at least one other patient had an isolate with identical RFLP pattern.</p> <p>Results</p> <p>One hundred and eighteen different fingerprint patterns were obtained from the 183 isolates. There were 34 clusters containing 54% (99/183) of the patients (average cluster size of 2.9), and a majority (96.2%) of the strains possessed a high copy number (≥ 5 copies) of the IS<it>6110 </it>element. When strains with <5 bands were excluded from the analysis, 50.3% (92/183) were clustered, and there was no difference in the level of diversity of DNA fingerprints observed in the two sero-groups (adjusted odds ratio [aOR] 0.85, 95%CI 0.46–1.56, <it>P </it>= 0.615), patients aged <40 years (aOR 0.53, 95%CI 0.25–1.12, <it>P </it>= 0.100), and sex (aOR 1.12, 95%CI 0.60–2.06, <it>P </it>= 0.715).</p> <p>Conclusion</p> <p>The sample showed evidence of a high prevalence of recent transmission with a high average cluster size, but infection with an isolate with a fingerprint found to be part of a cluster was not associated with any demographic or clinical characteristics, including HIV status.</p

Genetic diversity and potential routes of transmission of Mycobacterium bovis in Mozambique

CITATION: Machado, A., et al. 2018. Genetic diversity and potential routes of transmission of Mycobacterium bovis in Mozambique. PLOS Neglected Tropical Diseases, 12(1):e0006147, doi:10.1371/journal.pntd.0006147.The original publication is available at https://journals.plos.org/plosntdsBovine tuberculosis is a zoonotic disease with largely unknown impact in Africa, with risk factors such as HIV and direct contact with animals or consumption of Mycobacterium bovis infected animal products. In order to understand and quantify this risk and design intervention strategies, good epidemiological studies are needed. Such studies can include molecular typing of M. bovis isolates. The aim of this study was to apply these tools to provide novel information concerning the distribution of bovine tuberculosis in cattle in Mozambique and thereby provide relevant information to guide policy development and strategies to contain the disease in livestock, and reduce the risk associated with transmission to humans. A collection of 178 M. bovis isolates was obtained from cattle in Mozambique. Using spoligotyping and regions of difference analysis, we classified the isolates into clonal complexes, thus reporting the first characterisation of M. bovis strains in this region. Data from MIRU-VNTR typing was used to compare isolates from a number of African countries, revealing a deeply geographically structured diversity of M. bovis. Eastern Africa appears to show high diversity, suggesting deep evolution in that region. The diversity of M. bovis in Africa does not seem to be a function of recent importation of animals, but is probably maintained within each particular region by constant reinfection from reservoir animals. Understanding the transmission routes of M. bovis in Mozambique and elsewhere is essential in order to focus public health and veterinary resources to contain bovine tuberculosis.https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0006147Publisher's versio

The Guinea-Bissau Family of Mycobacterium tuberculosis Complex Revisited

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048)

Predominance of Uganda genotype of Mycobacterium tuberculosis isolated from Ugandan patients with tuberculous lymphadenitis.

International audienceIn Uganda, the emerging Uganda genotype of Mycobacterium tuberculosis is the most common cause of pulmonary tuberculosis (PTB), and accounts for up to 70% of isolates. Extrapulmonary TB (EPTB) is less studied in Uganda. Molecular characterization using deletion analysis and spoligotyping was performed on 121 M. tuberculosis isolates from lymph node fine needle biopsy aspirates of Ugandan patients with tuberculous lymphadenitis. The evolutionary relationships and worldwide distribution of the spoligotypes were analyzed. Mycobacterium tuberculosis was the only cause of EPTB in this study. The T2 sublineage was the most predominant lineage and the Uganda genotype was the dominant genotype. There were 54 spoligotype patterns among the 121 study isolates. The dominant spoligotypes were shared international types (SIT) SIT420, SIT53, SIT 135, SIT 128 and SIT590 in descending order. All but SIT420 were previously reported in pulmonary TB in this setting. The phylogenetic analysis showed a long descendant branch of spoligotypes belonging to the T2-Uganda sublineage containing specifically SITs 135, 128 and 420. In most cases, the spoligotypes were similar to those causing PTB, but the Uganda genotype was found to be less common in EPTB than previously reported for PTB in Uganda. The phylogenetic analysis and the study of the worldwide distribution of clustered spoligotypes indicate an ongoing evolution of the Uganda genotype, with the country of Uganda at the center of this evolution

A sensitive urinary lipoarabinomannan test for tuberculosis

CITATION: Hamasur, B., et al. 2015. A sensitive urinary lipoarabinomannan test for tuberculosis. PLoS ONE, 10(4):1-11, doi:10.1371/journal.pone.0123457.The original publication is available at http://journals.plos.org/plosoneWe have previously developed a diagnostic test for tuberculosis based on detection of mycobacterial lipoarabinomannan (LAM) in urine. The method depended on a laborious concentration step. We have now developed an easy to perform test based on a magnetic immunoassay platform, utilizing high avidity monoclonal antibodies for the detection of LAM in urine. With this method the analytical sensitivity of the assay was increased 50-100-fold compared to conventional ELISA. In a pilot study of HIV-negative patients with microbiologically verified TB (n=17) and healthy controls (n=22) the sensitivity of the test was 82% and the specificity 100%. This is in stark positive contrast to a range of studies using available commercial tests with polyclonal anti-LAM Abs where the sensitivity of the tests in HIVnegative TB patients was very low.http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0123457Publisher's versio