Yes / Background: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. Methods: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005–2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. Findings: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. Interpretation: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting. / ERC grant [INTERRUPTB; no. 311725] to BdJ, FG and CJM; an ERC grant to TS [PhyPD; no. 335529]; an FWO PhD fellowship to PM [grant number 1141217N]; the Leibniz Science Campus EvolLUNG for MM and SN; the German Centre for Infection Research (DZIF) for TAK, MM, CU, PB and SN; a SNF SystemsX grant (TBX) to JP and TS and a Marie Heim-Vögtlin fellowship granted to DK by the Swiss National Science Foundation. The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation - Flanders (FWO) and the Flemish Government – department EWI.

Made available in DSpace on 2016-01-26T12:51:36Z (GMT). No. of bitstreams: 1 juarezeliassantosjunior_dissert.pdf: 1690383 bytes, checksum: 9dd87174c2d25f09841337ec79d6968f (MD5) Previous issue date: 2010-11-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The parasite Giardia duodenalis, responsible for giardiasis, is commonly found in intestines of mammals, including man, currently considered to be important public health. Molecular research showed that G. duodenalis presents seven genotypes: A, B, C, D, E, F and G. Only genotypes A, which owns the subgenotypes A1 and A2 and B were detected in humans but also in other mammalian hosts. Objective: Evaluate the genotypic frequency of this parasite in humans in the Northwest region and to correlate the presence of diarrhea and their genotypes. Material and Method: Fecal samples were collected in the city of São José do Rio Preto (n = 150) and Araçatuba (n = 154), Northwest of São Paulo, Brazil. The parasitologic diagnosis was done by light microscopy and genotyping of β-Giardin gene by PCR-RFLP. Results: The subgenotype A1 was the most prevalent, however, the subgenotype A2 more frequent in Araçatuba. Genotype B was not found. No significant result was observed between significant stool consistency and subgenotype detected. Conclusion: The distribution of these genotypes may be related to host-parasite interactions in several areas and may influence the clinical and epidemiologic each region. Keywords: 1. Molecular epidemiology; 2. Gene β-giardin; 3. Giardiasis; 4. Northwest of São Paulo. / O parasito Giardia duodenalis, responsável pela giardíase, é comumente encontrado no intestino de mamíferos, inclusive o homem, sendo de importância em saúde pública. Pesquisas moleculares evidenciaram que Giardia duodenalis apresenta sete genótipos: A, B, C, D, E, F e G. Apenas o genótipo A, que possui os subgenótipos A1 e A2, e B foram detectados em humanos, mas também em outros hospedeiros mamíferos. Objetivo: Avaliar a freqüência genotípica deste parasito em humanos no Noroeste Paulista e correlacionar a presença deste enteropatógeno e seus genótipos à diarréia. Material e Método: Amostras fecais foram coletadas nos municípios de São José do Rio Preto (n=150 ) e Araçatuba (n=154), Noroeste do Estado de São Paulo, Brasil. O diagnóstico parasitológico foi feito por meio de microscopia ótica e a genotipagem do gene β-giardina pela técnica de PCR-RFLP. Resultados: O subgenótipo A1 foi o mais prevalente, no entanto, o subgenótipo A2 mais freqüente em Araçatuba. O genótipo B não foi encontrado e nenhum resultado significativo foi observado entre a consistência das fezes e o subgenótipo detectado. Conclusão: A distribuição destes genótipos pode ser relacionada as interações parasito-hospedeiro em diversas áreas, podendo influenciar as características clínico-epidemiológicas de cada região.

HCV genotype 6 is restricted in its distribution in South East Asia and it has been circulating for a long period of time. Phylogenetic and phylodynamic analysis on epidemic history of HCV focusing genotype 6 in South East Asia was explored, taking references from the pattern delineated in Liuzhou. Our results show that the date of most recent common ancestor (MRCA) of the whole HCV genotype 6 was estimated to be 100 years ago or more. There was an obvious increase of effective number of HCV genotype 6 infections in the part 20 years. Epidemic history of Subtype 6a and 6e/6d also showed the similar time course as that of the genotype 6. Interestingly, there was an increase of effective number of infections around 15-20 years ago which was maintained in the following decade for subtype 6a as well as 6e/6d. This specific pattern was consistent with the history of needle sharing in South East Asia, where the number of IDUs increased in the 1980s. The epidemic then spread to Southern China as evident by the increasing trend in Liuzhou. There was an exponential growth around 5 years ago involving subtype 6a predominantly, which might remain prevalent in Southern China in the coming decades. In conclusion, the study has shed new light on the transmission history of HCV, providing an explanation on the emergence of HCV genotype 6 in South East Asia. / HCV infection is an important public health problem associated with blood transfusion and needle-sharing in injection drug users (IDU) in Southern China. An understanding of the epidemiological pattern of the HCV infection, in conjunction with the transmission dynamics, would be beneficial for supporting effective prevention and control. This is accessed using a combination of molecular and public health approaches. / Through the Liuzhou Methadone Clinic, a total of 117 IDUs were recruited from Guangxi, Southern China. A majority of the IDUs (96%) were HCV antibody positive, of which 21% were HIV infected. Unlike HCV monoinfection, there was spatial heterogeneity in the distribution of HIV/HCV coinfection. The latter was also characterised by a higher prevalence of needle-sharing. Phylogenetic analysis of HCV revealed that genotype 6a was predominant in the study population. There were shorter genetic distances among the 6a samples compared to 3 other HCV genotypes/subtypes, 1a, 3a, and 3b. Our results suggested that HIV and HCV were both introduced at around the same time to the IDU populations in Southern China, followed by their differential spread as determined by the biologie characteristics of the virus and the intensity of behavioural risk. This pattern might be different from that in other South East Asian countries where HCV infections have probably predated HIV. / Tan, Yi. / Adviser: Kung Hsiang-fu. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3404. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 164-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

The Human immunodeficiency virus (HIV) pandemic has resulted in a heavy global disease burden, and clinically causes Acquired Immuno-Deficiency Syndrome (AIDS). The development of highly active antiretroviral therapy (HAART) has achieved remarkable control of the rapidly evolving HIV. However, HIV remains neither curable nor preventable by vaccine, and in the developing regions worst affected by HIV, HAART remains inaccessible to most patients. Furthermore, the change in both immunology and viral evolution during chronic HIV infection and its relation to AIDS pathogenesis remains unknown. Following the failure of recent HIV vaccines, it is believed that a better understanding of host-pathogen interaction is vital to advance therapeutic (vaccine and drug) design. In this thesis, I have performed an investigation of viral adaptation in response to different selection forces during advanced HIV infection and AIDS. The thesis first examined a case study that reveals the potential role of B cell-mediated neutralising antibody (NAb) in chronic HIV infection through the unexpected effect of B cell depletion agent, anti-CD20 (Rituximab). Here, longitudinal results have shown that viral load (VL), env gene diversity, and NAb sensitive strains increased during B cell and NAb depletion as a result of Rituximab administration, and reversed as B cells recovered. The study provides preliminary evidence to support the idea that NAb may be effective at suppressing HIV. The rest of the thesis focused on the cross-sectional cohort at Bloemfontein, South Africa (n=1491), a resource-limited region affected by the pandemic. Here, we used methods that include molecular and pretherapy drug resistance epidemiology, mathematical modelling, phylogenetically adjusted bioinformatics analysis and in vitro viral replication capacity (VRC) assay to study materials including cohort demography, plasma samples, CD4 cell count, VL, viral genetic sequences and host human leukocyte antigen (HLA) tissue types. Our analysis was further augmented by the additional data kindly contributed by our neighbouring Durban cohort collaborators (n=775), which also includes an IFN! ELISPOT assay that measures cytotoxic T lymphocyte (CTL) responses. Using the HIV pol sequencing data and phylogenetic analysis we confirmed that the local molecular epidemiology is similar to the circulating strains documented in the regional database. However, the pretherapy drug resistance mutation screening results have revealed an unexpected high incidence of drug-induced viral mutants in the AIDS patients with CD4 counts <100 cells/μl. According to mathematical modelling, this finding is attributable to additional sources of antiretroviral therapy exposure, which warrants public health caution. The investigation then focused on studying the changes in HLA class I mediated CTL selection and viral evolution as CD4 counts are reduced in AIDS. Interestingly we have noted evidence that suggest weakening CTL immune selection against gag during AIDS is associated with increased viral fitness (measured by VRC) and reversion of previous immune-escape mutations which conferred high fitness costs. In conclusion, this thesis compared different sources of host and drug mediated HIV selection and its implication for viral evolution. The identification of more bottleneck sites conferring high fitness costs to the selection of escape mutants is expected to be helpful in the design of future therapeutics (via vaccine, drug, immune therapy, or public health strategy). As we have learnt from the principle of combinational ARV, it would be desirable for a vaccine to select HIV at multiple sites of high escape-mutation fitness cost, hence offering protective effect.

Orientador: Carlos Magno Castelo Branco Fortaleza / Resumo: Staphylococcus aureus resistente à meticilina (Methicillin-resistant S. aureus, MRSA) é cada vez mais reconhecido como uma ameaça para pessoas vivendo com HIV/AIDS (PVHA). No entanto, a magnitude da colonização por MRSA varia entre diferentes países e regiões geográficas. Nós realizamos um estudo que teve por objetivo identificar a prevalência e os fatores de risco para colonização por S. aureus como um todo e MRSA em PVHA residindo em cidades de pequeno porte do interior do Estado de São Paulo. Isolados de MRSA foram caracterizados por Eletroforese em Gel de Campo Pulsado (Pulsed-Field Gel Electrophoresis, PFGE) e tiveram o Cassete Cromossômico Estafilocóccico (Staphylococcal Chromosome Cassete, SCC) mec tipado. Análise espacial foi realizada para identificar agregados geográficos e correlação com indicadores socioeconômicos. No primeiro momento, realizamos um estudo de prevalência pontual coletando swab nasal e de orofaringe de 368 PVHA atendidas em ambulatório de referência em Botucatu, SP. Sessenta e sete sujeitos residentes na cidade sede foram seguidos com coletas em dois outros momentos, e tiveram seus contactantes domiciliares também investigados para colonização. As taxas de prevalência de S. aureus e MRSA no primeiro levantamento foram 25,8% e 2,7%. A colonização por S. aureus foi negativamente associada com o uso de antibióticos beta-lactâmicos e drogas ilícitas. Por outro lado, fatores de risco para MRSA incluíam uso de crack e internação hospitalar recente. Inqué... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is increasingly recognized as a threat for people living with HIV/AIDS (PLWHA). However, the magnitude of asymptomatic MRSA colonization in that group varies among different countries and geographic regions. We conducted a study that aimed at identifying the prevalence and risk factors for both overall S. aureus and MRSA colonization among PLWHA attending in small cities from inner São Paulo State, Brazil. MRSA isolates were characterized using Pulsed-Field Gel Electrophoresis (PFGE), and submitted to typing of the Staphylococcal Chromosome Cassete (SCC)mec. Spatial analysis was performed to search for geographical clusters and correlation with socioeconomic indicators. In a first point prevalence survey, nasal and oropharyngeal swabs of 368 people were collected. Sixty-seven subjects from the main city (Botucatu) were surveyed for colonization in two other occasions, and had swabs collected from household members. The prevalence rates for S. aureus and MRSA in the first survey were 25.8% and 2.7%. The overall S. aureus colonization was negatively associated with the use of beta-lactams and of illicit drugs. On the other hand, MRSA colonized subjects were more likely to use crack and to have been admitted to a hospital during the past year. Repeated surveys found additional cases of MRSA colonization, but all subjects were positive in only one occasion. Four PFGE clusters were characterized, grouping subjects in household, ... (Complete abstract click electronic access below) / Mestre

A Febre Maculosa Brasileira causada por riquétsias do Grupo Febre Maculosa (GFM) e transmitida por carrapatos ocorre principalmente na Região Sudeste, onde óbitos humanos são registrados. No estado do Rio de Janeiro, a letalidade devido à riquetsiose é alta, mas só recentemente investigações epidemiológicas foram realizadas, e indicaram a participação de novas espécies de ectoparasitas na circulação das riquétsias. O objetivo geral do projeto é avaliar riquétsias em ectoparasitos coletados em áreas de casos humanos de Febre Maculosa, suspeitos, compatíveis ou confirmados, em municípios do estado com focos recentemente comunicados. A detecção e análise de genes riquetsiais indicam a presença de Rickettsia bellii, Rickettsia felis e Rickettsia rickettsii, nos vetores Amblyomma cajennense, Amblyomma dubitatum, Boophilus microplus, Ctenocephalides felis e Ctenocephalides canis, sugerindo ampla distribuição geográfica de riquétsias GFM, nas regiões Serrana, Noroeste Fluminense e Médio Paraíba do estado. / Brazilian spotted fever caused by spotted fever group (SFG) rickettsiae and mainly transmitted by ticks occurs in the southeast, where human deaths are recorded. In the state of Rio de Janeiro, lethality due to rickettsial infection is high, but only recently epidemiological investigations were conducted, and indicated the participation of new species of ectoparasites in rickettsiae circulation. The project\'s overall objective is to evaluate rickettsiae in ectoparasites collected in areas of human suspected, confirmed or compatible cases of spotted fever, in cities of the State with the recently reported outbreaks. The detection and analysis of rickettsial genes indicate the presence of Rickettsia bellii, Rickettsia felis and Rickettsia rickettsii in the vectors Amblyomma cajennense, Amblyomma dubitatum, Boophilus microplus, Ctenocephalides felis and Ctenocephalides canis, suggesting broad geographic distribution of SFG rickettsiae in the regions Serrana, Noroeste Fluminense and Médio Paraíba of the State.

Orientador: Ida Maria Foschiani Dias Batista / Resumo: As bactérias Mycobacterium leprae e mais recentemente Mycobacterium lepromatosis são os agentes etiológicos da hanseníase que causam sérios danos neuromotores e podem evoluir para incapacidades irreversíveis. A incidência de casos novos de hanseníase em todo o mundo foi de 2.77/100 mil habitantes. No Brasil, em 2016, foram 2.665 casos somente do Estado de Mato Grosso na Amazônia Meridional, esses valores representam 88,9/100 mil habitantes no índice geral de detecção para a hanseníase. Foram capturados animais silvestres naturalmente infectados por Mycobacterium leprae e Mycobacterium lepromatosis das Ordens Cingulata, Didelphimorphia, e Rodentia, todos estavam em fragmentos florestais próximos a grupos humanos. Um total 327 amostras de biópsias foram avaliados, dos quais recuperou-se 254, sendo 187 amostras de orelhas, 77 baço, e 63 fígado de 187 animais silvestres das Ordens Cingulata, Rodentia e Didelphimorphia. Após extraídos DNA e RNA de baço, fígado, e orelha, foram avaliados por qPCR para os genes RLEP (enumerador) e 16S rRNA (viabilidade). Três gêneros apresentaram positividade nas orelhas para ambos os genes RLEP e 16S rRNA, sendo 18% para Dasypus (Cingulata), 60% para Proechimys (Rodentia) e 64% para Marmosa (Didelphimorphia). Enquanto que nos testes utilizando PCR multiplex obteve-se 12 amostras positivas para o gene henM referente a Mycobacterium lepromatosis, dos 13 gêneros avaliados apenas Proechimys e Marmosa apresentaram presença para o bacilo. A presença freq... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bacteria Mycobacterium leprae and more recently Mycobacterium lepromatosis are the etiological agents of leprosy that cause serious neuromotor damage and can progress to irreversible impairments. The incidence of new cases of leprosy worldwide was 2.77 / 100 thousand inhabitants. In Brazil, in 2016, there were 2,665 cases of the State of Mato Grosso alone in the Southern Amazon, which represent 88.9 / 100 thousand inhabitants in the general detection index for leprosy. Wild animals naturally infected with Mycobacterium leprae and Mycobacterium lepromatosis from the Cingulata, Didelphimorphia, and Rodentia orders were all captured in forest fragments close to human groups. A total of 327 biopsy specimens were evaluated, of which 254 were recovered, being 187 samples of ears, 77 spleen, and 63 liver of 187 wild animals of the Orders Cingulata, Rodentia and Didelphimorphia. After extracting DNA and RNA from spleen, liver, and ear, they were assessed by qPCR for the RLEP (enumerator) and 16S rRNA (viability) genes. Three genera presented positivity in the ears for both RLEP and 16S rRNA genes, 18% for Dasypus (Cingulata), 60% for Proechimys (Rodentia) and 64% for Marmosa (Didelphimorphia). While in the tests using multiplex PCR, 12 samples were positive for the henM gene for Mycobacterium lepromatosis, of the 13 genera evaluated only Proechimys and Marmosa showed presence for the bacillus. The frequent presence of man in the forest fragments where M. leprae or Mycobacterium lepro... (Complete abstract click electronic access below) / Doutor

Orientador: Márcio Garcia Ribeiro / Resumo: As infecções mamárias por Prototheca têm sido registradas, de modo crescente, em todo o mundo, como um dos agentes mais patogênicos de origem ambiental na mastite bovina. Estas algas provocam lesões graves no tecido mamário e, até o momento, não existe protocolo efetivo de tratamento. O objetivo do presente estudo foi investigar o efeito algicida “in vitro” da guanidina em 75 isolados de Prototheca zopfii identificados de 60 casos de mastite clínica bovina (80,0%), 14 (18,7%) casos de mastite subclínica e um (1,3%) caso sem o diagnóstico de mastite clínica ou subclínica. Os 75 isolados foram submetidas a testes fenotípicos convencionais e caracterização genotípica por PCR multiplex, permitindo a identificação de todas as estirpes como P. zopfii genótipo 2. O efeito “in vitro” da guanidina revelou que todas os isolados mostraram variações na concentração algicida mínima que variaram de 0,001% a 0,035%. A guanidina tem alto efeito microbicida e é considerado um antisséptico/desinfetante da nova geração de microbicidas. O composto não é tóxico para as membranas mucosas e conjuntivas de humanos em baixas concentrações. É utilizado como desinfetante de piscinas e na desinfecção de superfícies, bem como antisséptico em feridas humanas. A ação algicida da guanidina em baixas concentrações indica que poderia ser usada na higienização de ambiente, utensílios e equipamentos de ordenha, no pré e pós-dipping em propriedades com casos de prototecose mamária, assim como na ablação química ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Prototheca species have increasingly been reported as the most common opportunistic pathogens causing mastitis worldwide. The protothecal mastitis poses a major health and economic problem in dairy herds. To date, there is any effective therapy against protothecal mastitis. The aim of the present study was to investigate in vitro algaecide effect of guanidine on 75 Prototheca zopfii genotype 2 strains isolated from 75 cases of clinical and subclinical bovine mastitis cases. In vitro effect of guanidine revealed that all strains were susceptible to the compound with minimal algaecide concentration ranging from 0.001% to 0.035%. Guanidine has high microbicidal effect and is considered a new-generation microbicidal compound. It is non-toxic to human mucous membranes and conjunctivas at low concentrations; it has been used as a disinfectant of surfaces and in swimming pools, as well as antiseptic on human wounds. The algaecide action of guanidine at low concentrations indicates that it could be an alternative disinfectant or antiseptic to clean the environment and milking dairy equipment, in pre- and post-dipping solutions, in the chemical dry therapy of bovine teats, and even in intramammary therapy of P. zopfii infections. This is the first report of the in vitro algaecide effect of guanidine on P. zopfii strains from animal origin. / Doutor