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Mixed strain infections in human tuberculosis: their role in biology, epdemiology and clinical outcome

English title Mixed strain infections in human tuberculosis: their role in biology, epdemiology and clinical outcome
Applicant Gagneux Sebastien
Number 164171
Funding scheme Bilateral programmes
Research institution Swiss Tropical and Public Health Institute
Institution of higher education University of Basel - BS
Main discipline Experimental Microbiology
Start/End 01.09.2016 - 31.12.2020
Approved amount 250'000.00
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All Disciplines (6)

Discipline
Experimental Microbiology
Molecular Biology
Immunology, Immunopathology
Ecology
Medical Microbiology
Infectious Diseases

Keywords (9)

virulence; evolution; HIV; mixed infection; drug resistance; microbiology; mycobacteria; patient outcome; molecular epidemiology

Lay Summary (German)

Lead
Die Tuberkulose (TB) ist nach wie vor weltweit ein grosses Gesundheitsproblem, insbesondere auch in Brasilien. Jedes Jahr sterben weltweit 1.5 Millionen Menschen in dieser Infektionskrankheit, die vom Bakterium Mycobacterium tuberculosis (Mtb) verursacht wird. Ob und wie Mischinfektionen mit verschiedenen Mtb Varianten eine Rolle für die Kontrolle der TB eine Rolle spielt, ist unbekannt.
Lay summary

Inhalt und Ziel des Forschungsprojekts

Unsere Brasiliansichen Mitarbeiter haben vor kurzem festgestellt, dass in Rio de Janeiro TB Patienten oft mit mehreren Mtb Staemmen gleichzeitig infiziert sind. Inwiefern Mischinfektionen für die Behandlung der einzelnen Patienten sowie für die Kontrolle der TB im Land relevant sind, ist nicht bekannt. Solche Mischinfektionen sind insbesondere im Zusammenhang mit Antibiotikaresistenzen wichtig, da sie eine korrekte Diagnose dieser Resistenzen erschweren.

Das Ziel dieses Projektes ist es, die Biologie und Epidemiologie von Mischinfektionen in der TB zu erforschen. Wir wenden dafür neueste Methoden an, die auf hochauflösender Ganzgenomsequenzierung von Patienten Isolaten beruhen und einen detaillierten Blick in die bakteriellen Populationen inerhalb einzelner TB Patienten erlauben. Diese Arbeiten werden in Zusammenarbeit mit unseren Brasliansichen Kollegen durchgeführ, die auch einen direkten Zugang zu TB Patienten und deren Bakterien Isolaten und klinischen Daten ermöglichen.

 Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Unsere Arbeit wird wichtige Information generieren, die zur einem besseren Verständnis der TB führen wird. Diese Ergebnisse werden u.a. für die Entwicklung besserer Diagnosemethoden höchst relevant sein. Gleichzeitig werden wir izusammen mit unseren Brasilianischen Kollegen bioinformatische Methoden in Brasilien etablieren und so zum Forschungspotential im Lande beitragen.

 
Direct link to Lay Summary Last update: 30.06.2016

Responsible applicant and co-applicants

Employees

Name Institute

Project partner

Publications

Publication
Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history
Coscolla Mireia, Gagneux Sebastien, Menardo Fabrizio, Loiseau Chloé, Ruiz-Rodriguez Paula, Borrell Sonia, Otchere Isaac Darko, Asante-Poku Adwoa, Asare Prince, Sánchez-Busó Leonor, Gehre Florian, Sanoussi C. N’Dira, Antonio Martin, Affolabi Dissou, Fyfe Janet, Beckert Patrick, Niemann Stefan, Alabi Abraham S., Grobusch Martin P., Kobbe Robin, Parkhill Julian, Beisel Christian, Fenner Lukas, Böttger Erik C., et al. (2021), Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history, in Microbial Genomics, 1.
Multiple Merger Genealogies in Outbreaks of Mycobacterium tuberculosis
Menardo Fabrizio, Gagneux Sébastien, Freund Fabian (2021), Multiple Merger Genealogies in Outbreaks of Mycobacterium tuberculosis, in Molecular Biology and Evolution, 38(1), 290-306.
The within-host evolution of antimicrobial resistance in Mycobacterium tuberculosis
Castro Rhastin A D, Borrell Sonia, Gagneux Sebastien (2020), The within-host evolution of antimicrobial resistance in Mycobacterium tuberculosis, in FEMS Microbiology Reviews, fuaa071.
A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region
Ngabonziza Jean Claude Semuto, Loiseau Chloé, Marceau Michael, Jouet Agathe, Menardo Fabrizio, Tzfadia Oren, Antoine Rudy, Niyigena Esdras Belamo, Mulders Wim, Fissette Kristina, Diels Maren, Gaudin Cyril, Duthoy Stéphanie, Ssengooba Willy, André Emmanuel, Kaswa Michel K., Habimana Yves Mucyo, Brites Daniela, Affolabi Dissou, Mazarati Jean Baptiste, de Jong Bouke Catherine, Rigouts Leen, Gagneux Sebastien, Meehan Conor Joseph, et al. (2020), A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region, in Nature Communications, 11(1), 2917-2917.
Natural Polymorphisms in Mycobacterium tuberculosis Conferring Resistance to Delamanid in Drug-Naive Patients
Reichmuth Martina L., Hömke Rico, Zürcher Kathrin, Sander Peter, Avihingsanon Anchalee, Collantes Jimena, Loiseau Chloé, Borrell Sonia, Reinhard Miriam, Wilkinson Robert J., Yotebieng Marcel, Fenner Lukas, Böttger Erik C., Gagneux Sebastien, Egger Matthias, Keller Peter M. (2020), Natural Polymorphisms in Mycobacterium tuberculosis Conferring Resistance to Delamanid in Drug-Naive Patients, in Antimicrobial Agents and Chemotherapy, 64(11), e00513-20.
HIV Coinfection Is Associated with Low-Fitness rpoB Variants in Rifampicin-Resistant Mycobacterium tuberculosis
Loiseau Chloé, Brites Daniela, Reinhard Miriam, Zürcher Kathrin, Borrell Sonia, Ballif Marie, Fenner Lukas, Cox Helen, Rutaihwa Liliana K., Wilkinson Robert J., Yotebieng Marcel, Carter E. Jane, Abimiku Alash'le, Marcy Olivier, Gotuzzo Eduardo, Avihingsanon Anchalee, Zetola Nicola, Doulla Basra, Böttger Erik C., Egger Matthias, Gagneux Sebastien (2020), HIV Coinfection Is Associated with Low-Fitness rpoB Variants in Rifampicin-Resistant Mycobacterium tuberculosis, in Antimicrobial Agents and Chemotherapy, 64(10), e00782-20.
Model-based integration of genomics and metabolomics reveals SNP functionality in Mycobacterium tuberculosis
Øyås Ove, Borrell Sonia, Trauner Andrej, Zimmermann Michael, Feldmann Julia, Liphardt Thomas, Gagneux Sebastien, Stelling Jörg, Sauer Uwe, Zampieri Mattia (2020), Model-based integration of genomics and metabolomics reveals SNP functionality in Mycobacterium tuberculosis, in Proceedings of the National Academy of Sciences, 117(15), 8494-8502.
An African origin for Mycobacterium bovis
Loiseau Chloé, Menardo Fabrizio, Aseffa Abraham, Hailu Elena, Gumi Balako, Ameni Gobena, Berg Stefan, Rigouts Leen, Robbe-Austerman Suelee, Zinsstag Jakob, Gagneux Sebastien, Brites Daniela (2020), An African origin for Mycobacterium bovis, in Evolution, Medicine, and Public Health, 2020(1), 49-59.
The Genetic Background Modulates the Evolution of Fluoroquinolone-Resistance in Mycobacterium tuberculosis
Castro Rhastin A D, Ross Amanda, Kamwela Lujeko, Reinhard Miriam, Loiseau Chloé, Feldmann Julia, Borrell Sonia, Trauner Andrej, Gagneux Sebastien (2020), The Genetic Background Modulates the Evolution of Fluoroquinolone-Resistance in Mycobacterium tuberculosis, in Molecular Biology and Evolution, 37(1), 195-207.
Classifying recurrent Mycobacterium tuberculosis cases in Georgia using MIRU-VNTR typing
Maghradze Nino, Jugheli Levan, Borrell Sonia, Tukvadze Nestani, Aspindzelashvili Rusudan, Avaliani Zaza, Reither Klaus, Gagneux Sebastien (2019), Classifying recurrent Mycobacterium tuberculosis cases in Georgia using MIRU-VNTR typing, in PLOS ONE, 14(10), e0223610-e0223610.
The Genetic Background Modulates the Evolution of Fluoroquinolone-Resistance in Mycobacterium tuberculosis
Castro Rhastin A D, Ross Amanda, Kamwela Lujeko, Reinhard Miriam, Loiseau Chloé, Feldmann Julia, Borrell Sonia, Trauner Andrej, Gagneux Sebastien (2019), The Genetic Background Modulates the Evolution of Fluoroquinolone-Resistance in Mycobacterium tuberculosis, in Molecular Biology and Evolution, msz214.
The molecular clock of Mycobacterium tuberculosis
Menardo Fabrizio, Duchêne Sebastian, Brites Daniela, Gagneux Sebastien (2019), The molecular clock of Mycobacterium tuberculosis, in PLOS Pathogens, 15(9), e1008067-e1008067.
Revised Interpretation of the Hain Lifescience GenoType MTBC To Differentiate Mycobacterium canettii and Members of the Mycobacterium tuberculosis Complex
Loiseau Chloé, Brites Daniela, Moser Irmgard, Coll Francesc, Pourcel Christine, Robbe-Austerman Suelee, Escuyer Vincent, Musser Kimberlee A., Peacock Sharon J., Feuerriegel Silke, Kohl Thomas A., Niemann Stefan, Gagneux Sebastien, Köser Claudio U. (2019), Revised Interpretation of the Hain Lifescience GenoType MTBC To Differentiate Mycobacterium canettii and Members of the Mycobacterium tuberculosis Complex, in Antimicrobial Agents and Chemotherapy, 63(6), e00159.
Transition bias influences the evolution of antibiotic resistance in Mycobacterium tuberculosis
Payne Joshua L., Menardo Fabrizio, Trauner Andrej, Borrell Sonia, Gygli Sebastian M., Loiseau Chloe, Gagneux Sebastien, Hall Alex R. (2019), Transition bias influences the evolution of antibiotic resistance in Mycobacterium tuberculosis, in PLOS Biology, 17(5), e3000265-e3000265.
Whole-Genome Sequencing for Drug Resistance Profile Prediction in Mycobacterium tuberculosis
Gygli Sebastian M., Keller Peter M., Ballif Marie, Blöchliger Nicolas, Hömke Rico, Reinhard Miriam, Loiseau Chloé, Ritter Claudia, Sander Peter, Borrell Sonia, Collantes Loo Jimena, Avihingsanon Anchalee, Gnokoro Joachim, Yotebieng Marcel, Egger Matthias, Gagneux Sebastien, Böttger Erik C. (2019), Whole-Genome Sequencing for Drug Resistance Profile Prediction in Mycobacterium tuberculosis, in Antimicrobial Agents and Chemotherapy, 63(4), e02175.
Insights into the genetic diversity of Mycobacterium tuberculosis in Tanzania
Rutaihwa Liliana K., Sasamalo Mohamed, Jaleco Aladino, Hella Jerry, Kingazi Ally, Kamwela Lujeko, Kingalu Amri, Malewo Bryceson, Shirima Raymond, Doetsch Anna, Feldmann Julia, Reinhard Miriam, Borrell Sonia, Brites Daniela, Reither Klaus, Doulla Basra, Fenner Lukas, Gagneux Sebastien (2019), Insights into the genetic diversity of Mycobacterium tuberculosis in Tanzania, in PLOS ONE, 14(4), e0206334-e0206334.
Reference set of Mycobacterium tuberculosis clinical strains: A tool for research and product development
Borrell Sònia, Trauner Andrej, Brites Daniela, Rigouts Leen, Loiseau Chloe, Coscolla Mireia, Niemann Stefan, De Jong Bouke, Yeboah-Manu Dorothy, Kato-Maeda Midori, Feldmann Julia, Reinhard Miriam, Beisel Christian, Gagneux Sebastien (2019), Reference set of Mycobacterium tuberculosis clinical strains: A tool for research and product development, in PLOS ONE, 14(3), e0214088-e0214088.
Comparative genomics of Mycobacterium africanum Lineage 5 and Lineage 6 from Ghana suggests distinct ecological niches
Otchere Isaac Darko, Coscollá Mireia, Sánchez-Busó Leonor, Asante-Poku Adwoa, Brites Daniela, Loiseau Chloe, Meehan Conor, Osei-Wusu Stephen, Forson Audrey, Laryea Clement, Yahayah Abdallah Iddrisu, Baddoo Akosua, Ansa Gloria Akosua, Aboagye Samuel Yaw, Asare Prince, Borrell Sonia, Gehre Florian, Beckert Patrick, Kohl Thomas A., N’dira Sanoussi, Beisel Christian, Antonio Martin, Niemann Stefan, de Jong Bouke C., et al. (2018), Comparative genomics of Mycobacterium africanum Lineage 5 and Lineage 6 from Ghana suggests distinct ecological niches, in Scientific Reports, 8(1), 11269-11269.
Treemmer: a tool to reduce large phylogenetic datasets with minimal loss of diversity
Menardo Fabrizio, Loiseau Chloé, Brites Daniela, Coscolla Mireia, Gygli Sebastian M., Rutaihwa Liliana K., Trauner Andrej, Beisel Christian, Borrell Sonia, Gagneux Sebastien (2018), Treemmer: a tool to reduce large phylogenetic datasets with minimal loss of diversity, in BMC Bioinformatics, 19(1), 164-164.
Analysis of potential household transmission events of tuberculosis in the city of Belem, Brazil
Conceição Emilyn Costa, Guimarães Arthur Emil dos Santos, Lopes Maria Luíza, Furlaneto Ismari Perini, Rodrigues Yan Corrêa, da Conceição Marília Lima, Barros Wandyra Araújo, Cardoso Ninarosa Calzavara, Sharma Abhinav, Lima Luana Nepomuceno Gondim Costa, Gomes Harrison Magdinier, Duarte Rafael Silva, Frota Cristiane, Rutaihwa Liliana K., Gagneux Sebastien, Suffys Philip Noel, Lima Karla Valéria Batista (2018), Analysis of potential household transmission events of tuberculosis in the city of Belem, Brazil, in Tuberculosis, 113, 125-129.
A New Phylogenetic Framework for the Animal-Adapted Mycobacterium tuberculosis Complex
Brites Daniela, Loiseau Chloé, Menardo Fabrizio, Borrell Sonia, Boniotti Maria Beatrice, Warren Robin, Dippenaar Anzaan, Parsons Sven David Charles, Beisel Christian, Behr Marcel A., Fyfe Janet A., Coscolla Mireia, Gagneux Sebastien (2018), A New Phylogenetic Framework for the Animal-Adapted Mycobacterium tuberculosis Complex, in Frontiers in Microbiology, 9, 2820.
Tuberculosis outbreak investigation using phylodynamic analysis
Kühnert Denise, Coscolla Mireia, Brites Daniela, Stucki David, Metcalfe John, Fenner Lukas, Gagneux Sebastien, Stadler Tanja (2018), Tuberculosis outbreak investigation using phylodynamic analysis, in Epidemics.
Ecology and evolution of Mycobacterium tuberculosis
Gagneux Sebastien (2018), Ecology and evolution of Mycobacterium tuberculosis, in Nature Reviews Microbiology, 16(4), 202-213.
Tuberculosis in Swiss captive Asian elephants: microevolution of Mycobacterium tuberculosis characterized by multilocus variable-number tandem-repeat analysis and whole-genome sequencing
Ghielmetti Giovanni, Coscolla Mireia, Ruetten Maja, Friedel Ute, Loiseau Chloé, Feldmann Julia, Steinmetz Hanspeter W., Stucki David, Gagneux Sebastien (2017), Tuberculosis in Swiss captive Asian elephants: microevolution of Mycobacterium tuberculosis characterized by multilocus variable-number tandem-repeat analysis and whole-genome sequencing, in Scientific Reports, 7(1), 14647-14647.

Collaboration

Group / person Country
Types of collaboration
Federal University of Rio de Janeiro Brazil (South America)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Seminar at University of Oxford Individual talk TB Treatment and Resistance 02.07.2019 Oxford, Great Britain and Northern Ireland Gagneux Sebastien;
European Society of Mycobacteriology Talk given at a conference Middelbrook-Award Lecture 30.06.2019 Valencia, Spain Gagneux Sebastien;
Fogarty Symposium Talk given at a conference Ecology and Evolution of MDR M. tuberculosis 05.06.2019 Lopota, Georgia Gagneux Sebastien;
9. Union Conference on TB and Lung Health, North-American Region Talk given at a conference Population Genomics of the M. tuberculosis Complex 21.02.2019 Vancouver, Canada Gagneux Sebastien;
Seminar at the Ifakara Health Institute Talk given at a conference The Ecology and Evolution of Mycobacterium tuberculosis 30.01.2019 Bagamoyo, Tanzania Gagneux Sebastien;
A History of the Microbe: Co-evolution of Humans/Animals and TB Talk given at a conference World Conference on TB and Lung Health 26.10.2018 The Hague, Netherlands Gagneux Sebastien;
TB Science 2018 Conference Talk given at a conference WGS To Quantify MDR-TB Transmission 23.10.2018 The Hague, Netherlands Gagneux Sebastien;
International Conference on Clinical Metagenomics Talk given at a conference WGS To Quantify MDR-TB Transmission 18.10.2018 Geneva, Switzerland Gagneux Sebastien;
Seminar at the Insitut efor Evolutionary Medicine, University of Zurich Individual talk Ecology and Evolution of Mycobacterium tuberculosis 26.09.2018 Zurich, Switzerland Gagneux Sebastien;
Seminar at Biozentrum, University of Basel Individual talk Ecology and Evolution of Multidrug-resistant Mycobacterium tuberculosis 24.09.2018 Basel, Switzerland Gagneux Sebastien;
Bilbo TB Conference Talk given at a conference Ecology and Evolution of (MDR) Mycobacterium tuberculosis 17.09.2018 Bilbao, Spain Gagneux Sebastien;
International Symposium on Antibiotic Resistance Talk given at a conference Epistasis and Compensatory Evolution in Multidrug-resistant Mycobacterium tuberculosis 30.05.2018 Stockholm, Sweden Gagneux Sebastien;
Seminar at Institute for Infectious Diseases, University of Bern Individual talk Ecology and Evolution of Mycobacterium tuberculosis 25.05.2018 Bern, Switzerland Gagneux Sebastien;
Webinar - Collaboration for TB Vaccine Discovery – Bill & Melinda Gates Foundation Individual talk Population genomics of M. tuberculosis and implications for product development 24.05.2018 Seattle, United States of America Gagneux Sebastien;
Symposium at University of Cape Town Talk given at a conference Ecology and Evolution of the M. tuberculosis Complex 12.05.2018 Cape Town, South Africa Gagneux Sebastien;
28th European Congress of Clinical Microbiology and Infectious Diseases Talk given at a conference Clinical Relevance of Genetic Diversity in M. tuberculosis 24.04.2018 Madrid, Spain Gagneux Sebastien;
International Symposium on “Inflammation at Interfaces” Talk given at a conference Genome Evolution of Mycobacterium tuberculosis 26.03.2018 Hamburg, Germany Gagneux Sebastien;
Seminar at Nanyang Technological University Individual talk The Ecology and Evolution of Mycobacterium tuberculosis 16.01.2018 Singapore, Singapore Gagneux Sebastien;
Workshop on “TB ONE Health” Talk given at a conference Ecology and Evolution of the Animal-adapted MTBC Revisited 23.11.2017 Dublin, Ireland Gagneux Sebastien;
Conference of the French Society of Microbiology Talk given at a conference MTBC: un compagnon fidèle de l’hommme 09.10.2017 Paris, France Gagneux Sebastien;
Symposium on Microbiological Diagnostics Talk given at a conference GENOMIQUE: Typisation des Mycobactéries 03.10.2017 Lausanne, Switzerland Gagneux Sebastien;
Conference of the Swiss Society of Microbiology Talk given at a conference Ecology and Evolution of Human Tuberculosis 01.09.2017 Basel, Switzerland Gagneux Sebastien;
Symposium on Biosafety Talk given at a conference Multidrug-Resistant Tuberculosis: A Threat to Global Public Health 25.08.2017 Basel, Switzerland Gagneux Sebastien;
Symposium on Pathogen Evolution Talk given at a conference Ecology and Evolution of Human Tuberculosis 20.07.2017 Kiel, Germany Gagneux Sebastien;
Yearly Conference of the European Society of Mycobacteriology Talk given at a conference Population Structure and Transmission of the MTBC 26.06.2017 Sibenik, Croatia Gagneux Sebastien;
Seminar at VetSuisse Individual talk Evolution and Epidemiology of MDR-TB 20.06.2017 Bern, Switzerland Gagneux Sebastien;
Seminar at Institut Pasteur Individual talk Evolution of the MTBC 16.06.2017 Paris, France Gagneux Sebastien;
Meeting of Microbiologists of South-Western France Talk given at a conference The Cause and Consequence of Genetic Diversity in Mycobacterium tuberculosis 24.04.2017 Toulouse, France Gagneux Sebastien;
10th National Conference on Laboratory Aspects of Tuberculosis Talk given at a conference Evolution of MDR-TB 19.04.2017 Atlanta, United States of America Gagneux Sebastien;
Symposium on "Future of Health" Talk given at a conference Evolution of Human TB: Implications for Global Control 03.04.2017 Institute for Systems Biology, Seattle, United States of America Gagneux Sebastien;
TB Symposium Talk given at a conference A Joint History of TB and Humankind 23.03.2017 Magglingen, Switzerland Gagneux Sebastien;
Lecture series on antibiotic development Individual talk Evolution of Multidrug-resistant Tuberculosis 21.11.2016 Biozentrum, Basel, Switzerland Gagneux Sebastien;
Latin-American Society of Tuberculosis Talk given at a conference Evolution and Epidemiology of MDR-TB 29.09.2016 Rosario, Argentina Gagneux Sebastien;
Argentine Association of Microbiology Talk given at a conference Coevolution of M. tuberculosis and H. sapiens 27.09.2016 Rosario, Argentina Gagneux Sebastien;
EMBO Conference on Tuberculosis Talk given at a conference Generalists and Specialists in M. tuberculosis 20.09.2016 Pasteur Institute, Paris, France Gagneux Sebastien;


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
The Evolution of Human Tuberculosis Talk 16.11.2016 Murten, Switzerland Gagneux Sebastien;


Awards

Title Year
Gardner Middlebrook Lifetime Achievement Award 2019

Associated projects

Number Title Start Funding scheme
166687 Integrating population and functional genomics with molecular epidemiology to explore the interaction between host and pathogen diversity in human tuberculosis 01.04.2016 Project funding (Div. I-III)

Abstract

Background: Tuberculosis (TB) is a major public health problem globally, and particularly in Brazil, which remains one of the 22 TB high-burden countries defined by the World Health Organization. Pulmonary TB is primarily caused by Mycobacterium tuberculosis (Mtb), but other nontuberculous mycobacteria (NTMs) are increasingly being isolated from such patients. The global TB epidemic is partially driven by co-infection with HIV and worsening due to the emergence of multidrug-resistant and extensively drug-resistant Mtb strains. One of the striking facts about TB is that patients can be re-infected following successful treatment. Hence, prior TB infection does not protect against reinfection. Similarly, and against common believes, TB patients can be infected with multiple Mtb genotypes at the same time. Indeed, studies have been reporting mixed infection in up to 60% of patients depending on TB incidence and study setting, but mathematical models predict that in most cases these figures are underestimates, because of the inherent limitations of the methods used to detect mixed infections. Moreover, little is known on the co-occurrence of Mtb and NTMs in TB patients. Recent advances in sample preparation and DNA sequencing offer an opportunity to explore mixed-strain infections in TB. Because mixed infections in TB have been highly neglected, little empirical data exist with respect to their impact on treatment outcome. Another neglected field in TB research is hetero-resistance, which refers to the co-occurrence of drug-susceptible and drug-resistant Mtb strains in the same individual. Hetero-resistance can emerge de novo during the development of drug resistance in a patient failing anti-TB drug therapy, or through initial co-infection or sequential superinfection of different Mtb strains with varying drug susceptibility profiles. The relative importance of these different phenomena for the evolution of drug-resistant Mtb and the outcome of patient treatment has not been determined. A few studies have reported that HIV co-infected TB patients were more likely to carry multiple Mtb genotypes, but it is unknown if and how this relates to the comparably poor treatment outcome of TB/HIV co-infected patients. More generally, little is known on the impact of HIV co-infection on the evolution of drug-susceptible and drug-resistant Mtb. To address these knowledge gaps, we will study TB patients in Rio de Janeiro and apply state-of-the art genotyping and whole genome sequencing (WGS) of Mtb isolates directly from sputum samples.Goal, Hypotheses and Objectives: The overall goal of this project is to study the biology and epidemiology of mixed infections with different strains of Mtb and/or NTMs and hetero-resistance in patients who start anti-TB treatment and the association with treatment outcome. We hypothesise that i) mixed infections (as defined above) and hetero-resistance occur more often in TB patients suffering from treatment failure, ii) mixed infection and hetero-resistance occur more often in patients co-infected with HIV, and iii) mixed infection and hetero-resistance occur more often in patients with previous TB treatment and/or drug-resistant TB. We will test these hypotheses by addressing the following Objectives:1.To study the nature and frequency of mixed infections and hetero-resistance in TB patients from Rio de Janeiro;2.To analyse the association of mixed infections and hetero-resistance with epidemiological and laboratory variables;3.To link mixed infections and/or hetero-resistance to treatment failure in TB patients;4.To investigate the nature and frequency of mixed infections and hetero-resistance as a function of HIV co-infection and anti-TB drug resistance.Study Setting, General Approach, and Methods: This project is embedded in a larger study funded by the National Institutes of Health and the Departamento Ciência Tecnologia - Secretaria de Ciência Tecnologia do Ministério da Saúde, on which the Brazilian Principal Investigator of this proposal (Prof. Kritski) is a Co-Principal Investigator. This larger study focuses on the immunology of progression from latent to active TB. The proposed project will take advantage of the available infrastructure and resources to recruit TB patients at four TB clinics in Rio de Janeiro during a period of 18 months. A total of 780 culture-positive TB patients will be recruited and their sputum samples subjected to standard genotyping to detect signals of mixed infection by different Mtb and/or NTM strains. Based on our preliminary data, we anticipate that a minimum of 10-20% of patients will harbour more than one mycobacterial strain, depending on HIV-status and drug resistance profile. All patient samples showing evidence of mixed infection will be subjected to WGS performed directly on sputum to avoid any potential bias introduced during the culturing step. As a back-up, WGS will be performed on multiple colonies obtained from bacterial cultures. Because TB patients with HIV co-infection and/or drug resistance will be comparably few, these will all be subjected to WGS analysis, irrespective of whether they show evidence of mixed infection based on standard Mtb genotyping. Moreover, to study hetero-resistance in detail and differentiate intra-host evolution from mixed or superinfection, we will clone individual PCR products obtained from sputum samples and directly sequence. This will allow us to reconstruct the specific haplotypes in mixed bacterial populations, which currently cannot easily be inferred from Illumine sequencing data alone. We will combine these Mtb genetic data to detailed clinical and epidemiological data obtained through our ongoing cohort study and look for associations between mixed strain infections and patient variables, including culture conversion at 2 months and treatment outcome at 6 months post treatment initiation. We will use a nested case-control study to compare TB patients with and without mixed infections and identify risk factors and patient characteristics associated with mixed infections. Moreover, we will use the WGS data to study the intra-host evolution of Mtb as a function of HIV co-infection and anti-TB drug resistance.Significance: This project will generate for the first time detailed data on the nature and frequency of mixed-strain infections in TB patients from Brazil as well as its impact on patient treatment outcome. Moreover, we will define risk factors and other patient variables associated with mixed infections. Because we will be able to combine the data generated in this project with data from the larger immunological study, this project will also shed new light on the impact of mixed-strain infections on the immune response to TB infection, and inform the development of new TB vaccines and biomarkers. Finally, our project will explore the diversity of Mtb in individual patients during treatment and generate new knowledge on the evolution of Mtb in the context of HIV co-infection and drug-resistant TB, as well as contribute to a better understanding of the role of hetero-resistance in the emergence of anti-TB drug resistance and its impact on treatment outcome.
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