Detection of multiple strains of Mycobacterium tuberculosis using MIRU-VNTR in patients with pulmonary tuberculosis in Kampala, Uganda

<p>Abstract</p> <p>Background</p> <p>Many studies using DNA fingerprinting to differentiate <it>Mycobacterium tuberculosis </it>(MTB) strains reveal single strains in cultures, suggesting that most disease is caused by infection with a single strain. However, recent studies using molecular epidemiological tools that amplify multiple targets have demonstrated simultaneous infection with multiple strains of MTB. We aimed to determine the prevalence of MTB multiple strain infections in Kampala, and the impact of these infections on clinical presentation of tuberculosis (TB) and response to treatment.</p> <p>Methods</p> <p>A total of 113 consecutive smear and culture positive patients who previously enrolled in a house-hold contact study were included in this study. To determine whether infection with multiple MTB strains has a clinical impact on the initial presentation of patients, retrospective patient data (baseline clinical, radiological and drug susceptibility profiles) was obtained. To determine presence of infections with multiple MTB strains, MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeats) -PCR was performed on genomic DNA extracted from MTB cultures of smear positive sputum samples at baseline, second and fifth months.</p> <p>Results</p> <p>Of 113 patients, eight (7.1%) had infection with multiple MTB strains, coupled with a high rate of HIV infection (37.5% versus 12.6%, <it>p </it>= 0.049). The remaining patients (105) were infected with single MTB strains. The proportions of patients with MTB smear positive cultures after two and five months of treatment were similar. There was no difference between the two groups for other variables.</p> <p>Conclusion</p> <p>Infection with multiple MTB strains occurs among patients with first episode of pulmonary tuberculosis in Kampala, in a setting with high TB incidence. Infection with multiple MTB strains had little impact on the clinical course for individual patients. This is the first MIRU-VNTR-based study from in an East African country.</p

Enhancing SARS-CoV-2 Surveillance through Regular Genomic Sequencing in Spain: The RELECOV Network

Millions of SARS-CoV-2 whole genome sequences have been generated to date. However, good quality data and adequate surveillance systems are required to contribute to meaningful surveillance in public health. In this context, the network of Spanish laboratories for coronavirus (RELECOV) was created with the main goal of promoting actions to speed up the detection, analyses, and evaluation of SARS-CoV-2 at a national level, partially structured and financed by an ECDC-HERA-Incubator action (ECDC/GRANT/2021/024). A SARS-CoV-2 sequencing quality control assessment (QCA) was developed to evaluate the network’s technical capacity. QCA full panel results showed a lower hit rate for lineage assignment compared to that obtained for variants. Genomic data comprising 48,578 viral genomes were studied and evaluated to monitor SARS-CoV-2. The developed network actions showed a 36% increase in sharing viral sequences. In addition, analysis of lineage/sublineage-defining mutations to track the virus showed characteristic mutation profiles for the Delta and Omicron variants. Further, phylogenetic analyses strongly correlated with different variant clusters, obtaining a robust reference tree. The RELECOV network has made it possible to improve and enhance the genomic surveillance of SARS-CoV-2 in Spain. It has provided and evaluated genomic tools for viral genome monitoring and characterization that make it possible to increase knowledge efficiently and quickly, promoting the genomic surveillance of SARS-CoV-2 in Spain

The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes

Contains fulltext : 124321.pdf (publisher's version ) (Open Access)BACKGROUND: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. METHODS: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. RESULTS: Seven out of the 11 laboratories (63%), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). CONCLUSIONS: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim

This work was supported by the Swiss National Science Foundation (grants 310030_188888, CRSII5_177163, IZRJZ3_164171 and IZLSZ3_170834) and the European Research Council (309540‑EVODRTB and 883582-ECOEVODRTB)Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Institute of Biomedicine of Valencia. Valencia, Spain.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia. Laboratório de Micobactérias. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Programa de Pós-graduação em Pesquisa Clínica e Doenças Infecciosas. Rio de Janeiro, RJ, Brazil.University of Valencia- joint Unit. I2SysBio,Valencia, Spain.University of Cape Town. Wellcome Centre for Infectious Diseases Research in Africa. Institute of Infectious Diseases and Molecular Medicine. Cape Town, South Africa.Makerere University. Department of Medical Microbiology. Kampala, Uganda.National Health Research Institutes. National Institute of Infectious Diseases and Vaccinology. Zhunan, Taiwan.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / University of Bern. Institute for Social and Preventive Medicine. Switzerland.Victorian Infectious Diseases Reference Laboratory. Victoria, Australia.Fudan University. School of Basic Medical Science. Institutes of Biomedical Sciences and Institute of Medical Microbiology. The Key Laboratory of Medical Molecular Virology of Ministries of Education and Health. Shanghai, China.Instituto de Investigación Sanitaria Gregorio Marañón. Hospital General Universitario Gregorio Marañón. Madrid, Spain / CIBER Enfermedades Respiratorias. Spain.Universitat de Barcelona. Hospital Clínic. Barcelona Institute for Global Health. Barcelona, Spain / Centro de Investigação em Saúde de Manhiça. Maputo, Mozambique.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / United Republic of Tanzania. Ifakara Health Institute, Bagamoyo, Bagamoyo District Hospital. Bagamoyo, Tanzania.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / United Republic of Tanzania. Ifakara Health Institute. Bagamoyo District Hospital. Bagamoyo, Tanzania.University of California. School of Medicine. San Francisco, USA.Fudan University. School of Basic Medical Science. Institutes of Biomedical Sciences and Institute of Medical Microbiology. The Key Laboratory of Medical Molecular Virology of Ministries of Education and Health. Shanghai, China.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / Papua New Guinea Institute of Medical Research. Goroka, Papua New Guinea.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Mahidol University. Faculty of Science. Department of Microbiology. Pornchai Matangkasombut Center for Microbial Genomics / National Science and Technology Development Agency. Bangkok, Thailand.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Mahidol University. Faculty of Science. Department of Microbiology. Pornchai Matangkasombut Center for Microbial Genomics / National Science and Technology Development Agency. Bangkok, Thailand.Institut Pasteur de Madagascar. Mycobacteriology Unit. Antananarivo, Madagascar.Institut Pasteur de Madagascar. Mycobacteriology Unit. Antananarivo, Madagascar.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.University of Basel. Basel, Switzerland / Swiss Tropical and Public Health Institute. Department of Medicine. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland / United Republic of Tanzania. Ifakara Health Institute. Bagamoyo District Hospital. Bagamoyo, Tanzania.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia. Laboratório de Micobactérias. Rio de Janeiro, RJ, Brazil.Université Paris-Saclay. Paris, France / Paris Diderot University. Sorbonne Paris Cité. Paris, France.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brazil.Universidade do Estado do Pará. Centro de Ciências Biológicas e da Saúde. Programa de Pós-graduação em Biologia Parasitária na Amazônia. Belém, PA, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.University of Ghana. Noguchi Memorial Institute for Medical Research. Accra, Ghana.ETH Zürich. Department of Biosystems Science and Engineering. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Department of Medical Parasitology and Infection Biology. Basel, Switzerland / University of Basel. Basel, Switzerland.Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC), causing tuberculosis (TB) in humans. L1 and L3 are endemic to the Rim of the Indian Ocean, the region that accounts for most of the world’s new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Here we analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We show that South Asia played a central role in the dispersion of these two lineages to neighboring regions. Moreover, we found that L1 exhibits signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans