Orientador: Clarice Queico Fujimura Leite / Banca: Rosilene Fressatti Cardoso / Banca: Henrique Ferreira / Resumo: O estudo da epidemiologia molecular através de diferentes técnicas vigentes revolucionou a compreensão da epidemiologia da tuberculose. A técnica Spoligotyping tem sido extensamente aplicada no estudo da epidemiologia molecular da tuberculose, para abordar estrutura populacional do M. tuberculosis, atentando para identificar principais linhagens e distribuições geográficas. Por outro lado, técnicas moleculares para diferenciar cepas de M. tuberculosis, tais como RFLP e MIRU possibilitam agrupar isolados clínicos de M. tuberculosis provenientes de pacientes que apresentam ligações epidemiológicas, identificando cadeias de transmissão e discriminando os isolados a nível clonal. A presente dissertação objetivou utilizar as técnicas de Spoligotyping e MIRU, para compreender um pouco mais sobre a tuberculose de uma metrópole, avaliando isolados provenientes do Ambulatório do Instituto Clemente Ferreira (referência no tratamento de tuberculose) na cidade de São Paulo. Os isolados clínicos de pacientes atendidos no período de agosto de 2006 a julho de 2008 foram reidentificados pela técnica molecular de PCR, e as cepas identificadas como de M. tuberculosis foram submetidas à genotipagem pelas técnicas de Spoligotyping e MIRU. Foi confirmada a identificação de M. tuberculosis em 93 isolados clínicos pela técnica de PCR-IS6110. No Spoligotyping, do total de 93 isolados, 21 amostras (22,6%) revelaram spoligotipos ainda não descritos na base de dado mundial (SpolDB4, SITVIT e Spotclust) e 51 (54,8%) estavam envolvidos em 12 cluster, 7 diferentes famílias e 36 spoligotipos. A família mais freqüente foi a LAM (26 isolados), seguida de T (24 isolados) e Haarlem (11 isolados), que juntos representaram 65,4% de todos os isolados. Essas 3 famílias representam os genótipos mais freqüentemente encontrados na África, América Central, América do Sul e Europa. Seis SITs... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The molecular epidemiology study using different techniques has improved the epidemiology of tuberculosis understanding. The Spoligotyping technique has been widely applied in the study of molecular epidemiology of tuberculosis, to address population structure of M. tuberculosis, focusing on the identification of the main lines and geographic distributions. Furthermore, molecular techniques used to differentiate strains of M. tuberculosis, such as RFLP and MIRU can be used for clustering clinical isolates of M. tuberculosis from patients with epidemiological links, identifying chains of transmission and discriminating isolates at the clonal level. In this work the techniques of Spoligotyping and MIRU were used to understand a little more about tuberculosis in a metropolis, assessing isolates from the Ambulatory Clemente Ferreira Institute (reference in the treatment of tuberculosis) in São Paulo. Clinical isolates from patients treated between August 2006 and July 2008 were re-identified by the molecular technique of PCR, were strains identified as M. tuberculosis were evaluated through genotyping the use of Spoligotyping and MIRU techniques. By PCR-IS6110 the identification of M. tuberculosis was confirmed in 93 clinical isolates. The total of 93 isolates, 21 samples (22.6%) in Spoligotyping showed spoligotypes have not yet been described in the world's data base (SpolDB4, SITVIT and Spotclust) and 51 (54.8%) were involved in 12 clusters, seven different families and 36 spoligotypes. The most frequent family was the LAM (26 isolates), followed by T (25 isolates) and Haarlem (11 isolates), which together accounted 65,4% of all isolates. These three families represent the most frequently genotypes found in Africa, Central America, South America and Europe. Six SITs accommodated 51.4% (37/72) of all isolates identified in SpolDB4 (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). By technique... (Complete abstract click electronic access below) / Mestre

La dengue et le chikungunya sont des maladies transmises par Aedes aegypti et Aedes albopictus pouvant causer des pathologies sévères et des atteintes incapacitantes chroniques. Ce sont aussi les maladies qui se diffusent le plus rapidement, en partie à cause du changement climatique. Le Vietnam est une zone d’hyperendémicité pour la dengue et à risque pour le chikungunya comme le Cambodge voisin qui est atteint par les deux maladies.et représente une source de diffusion. L’objectif de cette thèse est d’évaluer le statut de ces deux maladies et la présence du chikungunya, d’évaluer l’efficacité du système de surveillance et d’évaluer la diversité des populations de moustiques vecteurs et leurs rôles respectifs dans la transmission. Une première partie de la thèse est dévolue à une revue bibliographique. La seconde partie comprend trois chapitres associés à trois publications. Le premier chapitre est consacré à une étude de la surveillance à l’hôpital général de la province sud de Dong Thap. Une cohorte de 131 patents avec une fièvre aigue a été étudiée pour tester la présence de dengue et de chikungunya. 101 patients sur 131 ont été positifs pour la dengue et les quatre sérotypes ont été détectés avec une prédominance de DENV1 et DENV4. Aucun cas de chikungunya n’a été détecté. Une variation d’efficacité dans la détection sérologique de la dengue a été observée avec un passage de 29% lors de l’admission à 53% sept jours après admission. Il y a donc clairement un risque de sous-estimation de la dengue alors que le chikungunya, n’est pas du tout testé. Des changements dans la procédure de détection et de surveillance sont proposés pour améliorer l’efficacité de la surveillance et surveiller l’émergence du chikungunya. Le deuxième chapitre est consacré à l’étude du rôle respectif d’A. aegypti et A. albopictus dans l’épidémie de Dengue de 2011 à Hanoi. Seuls DENV1 et DENV2 ont été détectés chez les 140 patients étudiés. Une corrélation positive a été observée entre la densité de population d’A. aegypti et le nombre de cas humains et la durée des épidémies. Ceci n’a pas été observé chez A. albopictus. Trois lots d’A. aegypti se sont révélés positifs pour la dengue, deux pour DENV1 et un pour DENV2. Cette étude montre clairement le rôle d’A. aegypti dans l’épidémie de 2011 à Hanoi. Le dernier chapitre est consacré à une analyse transversale sur 5 provinces frontalières du Laos et du Cambodge. 558 sérums collectés chez des patients admis pour fièvre aigue et de symptômes compatibles avec la dengue et chikungunya entre 2012 et 2014 dans des centres de médecine préventive. Les quatre sérotypes ont été détectés mais pas tous dans la même province. Seulement deux sérotypes ont été détectés au maximum dans une même province. Le chikungunya n’a pas été détecté. Un total de 1104 moustiques adultes a été prélevé dans les mêmes zones à l’intérieur et à l’extérieur des habitations. La densité de population de moustiques et les indices entomologiques ont été évalués suite à la capture de larves. Des densités très différentes ont été observées et la densité la plus importante a été obtenue dans la province de Long An, voisine du Cambodge. Le virus de la dengue a été détecté principalement chez A. albopictus. Le virus du chikungunya a également été détecté chez A. albopictus. L’analyse phylogénétique des moustiques collectés a montré une grande diversité génétique avec des génotypes décrits sur d’autres continents. Cette partie de la thèse met en évidence le rôle différentiel d’A. aegypti et A. albopictus, le rôle croissant de ce dernier et le transport anthropique des moustiques sur de grandes distances. Ce travail souligne le besoin de nouvelles approches de surveillance, au niveau clinique et au niveau entomologique, pour s’attaquer de façon efficace au risque épidémique de dengue et de chikungunya. / Dengue and chikungunya are both transmitted by Aedes aegypti and Aedes albopictus and can cause potentially severe and or debilitating chronic disease. They are the fastest spreading diseases, in part because of the climate change. Vietnam is a hyperendemicity country for dengue and is at risk to be like neighboring Cambodia affected both by dengue and chikungunya and be an overlapping area of distribution for both viruses. The aim of this PhD work was therefore to assess the status of single and dual infections all over the country, investigate the presence of chikungunya, assess the efficiency of the surveillance procedures routinely established and assess the diversity of mosquito populations and their potential respective role. A first part of the PhD dissertation is devoted to a bibliographic review. The second part comprises three chapters associated to three different publications. The first chapter is devoted to a surveillance study in the general hospital if the Southern Province of Dong Thap. A cohort of 131 patients with acute fever symptoms was investigated for the presence of dengue and chikungunya. 101 patients out of 131 were confirmed with dengue. All four dengue serotypes were detected with a predominance of DENV2 and DENV4. No chikungunya infection was detected although reported in neighboring Cambodia. A differential efficiency of serological dengue detection was observed. Efficiency was 29% upon admission and 53% after seven days on the same patients. There is thus a clear risk of dengue being underestimated while chikungunya is not systematically detected. Changes in detection and surveillance procedures are therefore proposed to increase the efficiency of dengue detection and continue the monitoring the emergence of CHIKV. The second Chapter is dedicated to the respective role of A. aegypti and A. albopictus in the 2011 outbreak in the Northern capital city of Hanoi. Only DENV-1 and DENV-2 serotypes were detected from the 140 patients hospitalized. A positive correlation was found between the population density of A. aegypti and the number of human cases and duration of outbreaks. This was not observed for A. albopictus. Three pools of A. aegypti were positive with dengue virus, two with DENV-1 and one with DENV-2. This work indicate clearly the role of A. aegypti in the 2011 Hanoi epidemics. The last chapter of the PhD is devoted to a crosscutting country wide survey in five provinces border with Lao PDR and Cambodia. In this work, a total of 558 serum samples were collected from patients admitted in the 2012-2014 period in five provincial preventive medicine centers with acute fever and symptoms compatible to DENV-CHIKV infection. All four dengue serotypes were found altogether but not in the same province. Only two serotypes were found at the maximum in a single province. No CHIKV was detected. A total of 1104 adult mosquitoes were collected inside and outside houses at the same place. Mosquito population density and vector indexes were assessed following capture of larvae. Differing densities of mosquito populations were found with the highest one being in the Long An province border with Cambodia. Dengue viruses were detected mostly in A. albopictus. CHIKV was also detected in A. albopictus mosquitoes. The phylogenetic analysis of the collected mosquitoes showed a large diversity of genotypes, all of them having been described in other parts of the world. This part of the PhD work underline the dual role of A. aegypti and A. albopictus, the increasing role of the latter and the high level of man-related very long distance mobility of mosquitoes. This work underlines the need of novel approaches for surveillance both at the clinical and at the entomological level to efficiently tackle the risk of dengue and chikungunya outbreaks.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O vÃrus da hepatite C à considerado como o principal agente causador de hepatite nÃo-A, nÃo-B e, desde sua descoberta em 1989, tem sido reconhecido como a principal causa de doenÃa crÃnica do fÃgado em todo o mundo. O VHC possui um genoma de RNA de sentido positivo e à classificado como um flavivÃrus. O vÃrus apresenta considerÃvel variabilidade em sua seqÃÃncia e as variantes do VHC podem ser divididas em seis genÃtipos principais (numerados de 1 a 6) e diversos subtipos. A distribuiÃÃo dos genÃtipos varia tanto geograficamente, quanto entre os grupos de risco. Este estudo teve como objetivo analisar a distribuiÃÃo dos genÃtipos do VHC entre pacientes atendidos em um hospital de referÃncia em Fortaleza, CearÃ. Cento e vinte pacientes com sorologia positiva para anti-VHC ou histÃria clÃnica sugestiva de infecÃÃo pelo vÃrus foram estudados. O RNA viral foi extraÃdo a partir do soro dos pacientes e a detecÃÃo molecular do vÃrus foi feita por PCR, utilizando iniciadores especÃficos para a regiÃo 5 (linha) nÃo-codificadora do genoma viral. A genotipagem foi baseada em tÃcnica de RFLP utilizando as enzimas de restriÃÃo HaeIII, RsaI, MvaI e HinfI. Noventa e seis pacientes (80%) foram positivos no teste qualitativo para VHC. A mÃdia de idade desses pacientes foi de 44 anos. HistÃria clÃnica de cirurgia foi o fator de risco mais presente, seguido por transfusÃo sangÃÃnea, DST, uso de drogas, tatuagem, diÃlise e exposiÃÃo ocupacional. A relaÃÃo entre o resultado do teste qualitativo e o uso de drogas apresentou significÃncia estatÃstica, com 96% dos usuÃrios de drogas positivos para VHC. NÃo houve diferenÃa significativa no resultado do teste qualitativo quando transfusÃes sangÃÃneas feitas antes ou depois de 1993 foram analisadas. ManifestaÃÃes clÃnicas ou Ãndices de ALT alterados tambÃm nÃo foram preditivos de positividade para VHC. O genÃtipo 1 foi o mais prevalente na populaÃÃo estudada (46,9%), seguido pelos genÃtipos 3 (34,4%) e 2 (8,3%). O genÃtipo 4 viral foi detectado em um paciente. Em nove amostras nÃo foi possÃvel a genotipagem do VHC. Esses casos foram denominados indeterminados. CaracterÃsticas epidemiolÃgicas como idade, sexo, fatores de risco, Ãndices de ALT e manifestaÃÃes clÃnicas foram relacionadas com os diversos genÃtipos virais. A distribuiÃÃo dos genÃtipos virais entre as categorias estudadas ocorreu de forma homogÃnea na maioria dos casos. Foi observada significÃncia estatÃstica apenas na relaÃÃo entre genÃtipo e exposiÃÃo ocupacional ao VHC, com predominÃncia do genÃtipo 1 neste grupo de risco. NÃo foram observadas co-infecÃÃes com mais de um genÃtipo viral. / Hepatitis C virus is considered as the main causative agent of non-A non-B hepatitis and has been recognised as the major cause of chronic liver disease worldwide, since its discovery in 1989. It has a positive-sense RNA and belongs to the flavivirus family. The HCV shows considerable sequence variability and its variants can be divided into six main genotypes (numbered from 1 to 6) and several subtypes. The genotypes distribution varies from the geographic area and among risk groups. This study had the main aim to study HCV genotypes distribution in patients from a reference hospital in Fortaleza, Ceara. It was investigated a hundred and twenty patients with anti-HCV positive or clinical history suggesting virus infection. Viral RNA was extracted from patients serum and the virus molecular detection was made by PCR, using specific primers to the 5â non-coding region of viral genome. Genotyping was based in a RFLP technique, using the restriction enzymes HaeIII, RsaI, MvaI and HinfI. Ninety six patients (80%) were positive in the qualitative test for HCV. The mean age of these patients was 44 years old. Surgery clinical history was the most frequent risk factor, followed by blood transfusion, sexual transmitted disease, drugs use, tattoos, dialysis, and occupacional exposure. The relation between qualitative test result and drug users showed statistical significance, with 96% of drugs users being positive for HCV. There was no significant difference in qualitative test result when we analysed blood transfusions done after or before the year 1993. Clinical symptoms and ALT levels also were not predictive of HCV positive result. Genotype 1 was the most prevalent in the studied population (46,9%), followed by genotype 3 (34,4%) and 2 (8,3%). There was also a molecular characterization of one patient with genotype 4 whereas nine samples were not HCV genotyped and were called as undetermined. The epidemiological characteristics such as age, gender, risk factors, ALT levels and clinical manifestations were related with viral genotypes. The HCV genotypes had a homogeneous distribution, in the majority of cases, among the different categories. A statistical significance was observed only when genotype 1 was related to HCV occupational exposure. Co-infections with more than one viral genotype were not observed.

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No. of bitstreams: 2 Tese - Maria Edileuza Soares Moura - 2014.pdf: 2617829 bytes, checksum: 8fe2eea4d03e82310f1d867e6bfbcf37 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In a vast country, like Brazil surveillance of transmitted drug resistance to antiretroviral drugs/TDR should be continuous and extended to different locations and exposure groups. In the last decade significant geographic differences have been reported in the Brazilian AIDS epidemic: reduction in incidence of AIDS cases and related mortality in the southeast region (epicenter of the epidemic) contrasting with significant rise in both parameters in the northeast region. This study describes TDR and HIV-1 subtypes in protease-PR and reverse transcriptase-RT regions among antiretroviral (ARV) naïve patients from Piauí State (n=89) and Maranhão State (n=106), northeast, Brazil recruited between August 2011 and June 2012. HIV-1 pol gene (protease/PR and 2/3 of reverse transcriptase/RT) was sequenced from RNA. TDR was evaluated using the Calibrated Population Resistance (CPR) tool/Stanford HIV-1 Database, HIV-1 subtypes were defined by REGA software and phylogenetic analyses. Among patients from Piauí State (44 females, 45 males, 22 of them were men who have sex with men-MSM), overall TDR rate was 13.5%. TDR rate among MSM was 27.3% while among heterosexual men TDR rate was 10% and 9.1% among females. Single-class mutations to ARV (RT nucleoside and non nucleoside inhibitors NRTI/NNRTI or PR inhibitors/PI) predominated (10/12): M46L/V82F/L90M (PI), M41L/D67N (NRTI); K103NS/E138A (NNRTI). Two patients had dual class mutations: T215L (NRTI) and E138G/Y188L (NNRTI); T215N (NRTI) and F227L (NNRTI). Subtype B predominated (86.6%), non-subtype B isolates were rare: subtype F1 (n=1), subtype C (n=1). B/F1, F1/B and B/C intersubtype recombinants were observed in 11.2%. In Maranhão State females predominated (54.7%), median age was 31 years (range: 18-72); 78.3% reported heterosexual unprotected sex, 17.9% were MSM, two reported intravenous drug use/IDU. SDRM TDR rate was 3.8% (4/106). TDR was identified in adults (21-45 years), mostly males (3/4), 2 MSM, one IDU. Single-class mutations associated with NRTI (M184V; T215S) or NNRTI (K103S/N) were detected. No major PI mutation was identified; four isolates presented accessory mutations to PI (L10I/F, A71T/V). Subtype B represented 81.1% (86/106), subtype F1 1.9% (2/106), subtype C 2.8% (3/106) and 14.2% (15/106) were recombinants. The moderate frequency of BF recombinants in PI and MA where subtype F1 is rare suggests that recombinants generated in southeast/central-western were introduced and are circulating in northeast Brazil. Among patients from PI, high TDR rate observed among MSM ccontrasts with moderate rate among heterosexual patients. This result indicates that genotyping tests to detect drug resistance and TDR, mainly among MSM can contribute to define surveillance policies and to delineate prevention strategies to help control AIDS epidemic in northeast, Brazil. / Em um país extenso como o Brasil, a vigilância da resistência transmitida a antirretrovirais/TDR deve ser contínua e estendida a diferentes grupos populacionais e regiões geográficas. Na última década, diferenças regionais significativas foram relatadas na epidemia de HIV/aids no Brasil: redução na incidência de casos de aids e mortalidade na região sudeste (epicentro da epidemia) contrastando com aumento significativo de ambos parametros na região nordeste. O objetivo deste estudo foi descrever a prevalência de TDR e subtipos de HIV-1 na protease-PR e parte da transcriptase reversa-TR em isolados de pacientes virgens de tratamento que vivem nos estados do Piauí e do Maranhão, nordeste, Brasil. Pacientes virgens de tratamento antirretroviral/ARV recrutados entre agosto 2011- junho 2012 (Maranhão, n=106; Piauí, n=89) tiveram o gene pol (protease/PR e 2/3 da transcriptasecreversa/TR) sequenciado a partir do RNA. TDR foi avaliada mediante uso da Calibrated Population Resistance (CPR) tool/Stanford HIV-1 Database, subtipos de HIV-1 foram definidos pelo software REGA e por análise filogenética. Entre os pacientes do Piauí (44 mulheres, 45 homens, 22 deles declararam-se homens que fazem sexo com homens/HSH), a taxa de TDR foi 13,5%. TDR entre HSH foi 27,3%. Entre os homens heterossexuais, a taxa de TDR foi de 10% e entre as mulheres 9,1%. Mutações de resistência a uma classe de ARV (inibidores nucleosidicos ou não-nucleosidicos da TR/ITRN/ITRNN ou inibidores da PR/IP) predominaram (10/12): M46L/V82F/L90M (IP); M41L/D67N (ITRN); K103NS/E138A (ITRNN). Dois pacientes tiveram mutações de resistência a duas classes de ARV: T215L (ITRN) e E138G/Y188L (ITRNN); T215N (ITRN) e F227L (ITRNN). O subtipo B representou 86,6%, subtipos não B foram raros: subtipo F1 (n=1) e o subtipo C (n=1). Recombinantes intersubtipos B/F1, F1/B e B/C foram observados em 11,2 % dos isolados. No Maranhão predominou mulheres (54,7%), com mediana de idade de 31 anos (variação: 18-72); 78,3% relatou relação heterossexual desprotegida, 17,9% dos homens declararam-se HSH, dois referiram uso de drogas injetáveis. Entre os pacientes do MA 3,8% apresentou TDR (4/106). TDR foi identificada em adultos (21-45 anos), a maioria do sexo masculino (3/4), 2 HSH, um usuário de droga injetável. Mutações associadas a resistência aos ITRN (M184V; T215S) e aos ITRNN (K103S/N) foram detectadas. Mutações principais aos IP não foram identificadas, quatro isolados apresentaram mutações acessórias aos IP (L10I/F, A71T/V). O subtipo B representou 81,1% (86/106) dos isolados, o subtipo F1 1,9% (2/106), o subtipo C 2,8% (3/106) e 14,2% (15/106) eram recombinantes. A moderada prevalência de recombinantes BF detectada no PI e MA onde a frequência de subtipo F1 é rara, sugere que recombinantes gerados no sudeste/centro-oeste tenham sido introduzidos e estão circulando no nordeste. Nos pacientes do PI, alta taxa de TDR observada entre HSH contrastou com taxa moderada entre heterossexuais. Este resultado indica que a realização de testes de genotipagem para resistência para avaliar TDR, principalmente entre HSH pode contribuir para definir diretrizes de vigilância e delinear estratégias de prevenção para auxiliar o controle da epidemia de aids na região nordeste, Brasil.

Ce travail vise à estimer l’impact de co-infections sur le terrain et à mieux comprendre le synergisme possible entre agents pathogènes en conditions expérimentales. Nous nous sommes intéressés à E. coli (O78) et au virus influenza faiblement pathogène (LPAIV, H6N1) dans le modèle dinde. Les oiseaux ont été infectés par voie aérosole pour reproduire l’infection respiratoire. L’excrétion virale ainsi que les lésions ont été plus importantes lors de la co-infection, ce qui suggère une patho-génicité accrue. Ces résultats montrent que E. coli et LPAIV ont un effet additif sur la maladie respiratoire lors qu’ils ont été inoculés soit simultanément soit en différé (à 3 jours d’intervalle) à des dindes naïves. En parallèle, nous avons étudié les virus respiratoires en circulation dans les élevages pakistanais. Des co-infections avec le LPAIV H9 (lignage G1) et le virus de la maladie de Newcastle (génotype VII) ont été fréquemment observées. / The purpose of this study was to assess the burden of co-infections in the field and to better understand the possible synergism between pathogens in a laboratory setting. We focussed on E. coli (O78) and low pathogenic avian influenza virus (LPAIV, H6N1) in turkey model and infected the birds via the aerosol route to reproduce respiratory disease. Viral shedding and lesions were more severe and persisted longer during coinfection indicating possible enhancement of pathogenesis for LPAIV by E. coli and vice versa. These findings all endorse our conclusions that E. coli and LPAIV exercise an additive pathogenic effect in the reproduction of respiratory disease if given simultaneously or spaced by three days between the viral and the bacterial challenges to susceptible turkeys. In parallel, we studied avian respiratory agents circulating in the field in Pakistani farms. There, we focussed on co-infections as well, targeting viruses only as a first study. We observed frequent LPAIV H9 (G1 lineage) and Newcastle disease virus (genotype VII) coinfections in the field.

La cryptosporidiose est une infection du tube digestif, causée par un protozoaire du genre Cryptosporidium, à l'origine de diarrhées néonatales chez les jeunes ruminants. Cette affection revêt une importance en santé publique et en santé animale, ces deux aspects étant liés par l’existence d'espèces zoonotiques, la principale étant C. parvum. Les données sur l'épidémiologie moléculaire de Cryptosporidium spp chez les ruminants abondent au niveau international mais sont limitées pour la France. Ce travail a confirmé la forte prévalence et les forts niveaux d'excrétion en oocystes de Cryptosporidium chez les jeunes ruminants. L'espèce C. parvum a été identifiée aussi bien chez les veaux, les chevreaux que chez les agneaux. A l'inverse, les espèces C. xiaoi (chez le jeune) et C. ubiquitum (adultes en gestation) n'ont été retrouvées que chez les caprins et les espèces C. bovis et C. ryanae uniquement chez les veaux.Deux espèces zoonotiques ont été identifiées, C. ubiquitum et C. parvum. Tous les sous-types de C. parvum identifiés appartiennent à la famille zoonotique IIa. Le sous-type IIaA15G2R1 a été majoritairement retrouvé quelle que soit l'espèce hôte. Cette observation confirme ce qui a été observé dans d'autres pays à savoir le rôle de réservoir des jeunes ruminants dans la transmission d'isolats de C. parvum à l'homme. Enfin, ce travail a mis en évidence la complexité de l'épidémiologie de l'infection par Cryptosporidium spp avec une évolution de la prévalence et du niveau d'excrétion ainsi que de la distribution des espèces et sous-types de C. parvum d'une année sur l'autre au sein d'un même élevage. / Cryptosporidiosis is an infection of the digestive tract due to protozoa of the genus Cryptosporidium, which cause neonatal diarrhoea in young ruminants. This infection has a real significance in public and animal health; in fact, both of these aspects are bound by the existence of zoonotic species including C. parvum, the predominant species. Molecular epidemiology data on Cryptosporidium spp are very numerous over the world; however, little information has been published in France.This work confirmed the high prevalence and high level of excretion of Cryptosporidium oocysts in young ruminants. C. parvum species was identified in calves, goat kids and also in lambs. By contrast, the two species, C. xiaoi (goat kids) and C. ubiquitum (pregnant goats) were only found in goats whereas C. bovis and C. ryanae were observed only in calves.Two zoonotic species have been identified: C. ubiquitum and C. parvum. All C. parvum subtypes belongs to the IIa zoonotic family. IIaA15G2R1 subtype was mainly found in all ruminants' species. This observation confirms the potential role of young ruminants in transmission of zoonotic isolates of C. parvum to humans which has already been observed in previous studies over the world. Finally, this work highlighted the complexity of the epidemiology of Cryptosporidium spp infection, in particular the evolution in the prevalence and level of excretion and in the distribution of Cryptosporidium spp species or C. parvum subtypes according to the year of sampling in the same livestock.

Rabies, one of the oldest recognized viral zoonotic diseases, is a fatal encephalomyelitis transmitted to man via contact with infected animals. Evan today, rabies still is a disease of public health concern with many potentially preventable deaths occurring mainly in Asia, Africa and Latin America. Rabies and rabies-related viruses are members of the Lyssavirus genus, which comprises the rabies virus (genotype 1), Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), European bat lyssaviruses 1 and 2 (genotypes 5 and 6) and the Australian bat lyssavirus (genotype 7). Antigenic and genetic studies have shown that rabies virus strains circulating in particular host species tend to undergo genetic adaptation and evolve into distinct biotypes that differ in antigenicity and pathogenicity. Two biotypes of rabies virus are recognized in southern Africa. The first called the canid viruses, infect carnivores of the family Canidae (dogs, jackals and bat-eared foxes) and the second, the viverrid viruses, infect carnivores of the family Herpestidae (the yellow mongoose Cynictis penicil!ata and the slender mongoose Galerella sanguinea). In an endeavour to better understand the molecular epidemiology of lyssaviruses in Zimbabwe and South Africa, we analysed nucleotide sequences of the glycoprotein and the G-L intergenic region (rabies viruses) and the nucleoprotein gene (Mokola viruses). The main aim of the studies described in this thesis was to characterise lyssaviruses (genotypes I and 3) from Zimbabwe and compare them to those present in South Africa. In addition, we wanted to establish the role of the various rabies variants in rabies epizootics in the southern African subcontinent. It could be shown from this study that all the southern African canid viruses were closely related, with no general distinction between viruses from any of the canid species. Despite the general overall similarity between the canid viruses, certain phylogenetic groupings were apparent and by association with host species, geography and year of isolation, certain groups could be identified as particular epidemiological cycles. A high genetic diversity was evident amongst viverrid rabies viruses, the opposite of our observation for canid viruses. The viverrid virus groups corresponded to geographical pockets that were independent of host species. Mokola viruses from Zimbabwe were shown to be different from those from South Africa and phylogenetic relationships of these viruses were related to their geographical location of origin. This study has demonstrated the value of multinational surveillance and investigation in understanding the epidemiology of lyssaviruses in southern Africa and elsewhere in Africa. The results presented here will serve as basis for future studies on lyssaviruses in Africa and will contribute to the improved surveillance and control programs of rabies and Mokola viruses in the region. / Thesis (PhD (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

With over 35 million people currently infected, the World Health Organization considers HIV a global pandemic. HIV is characterized by a high mutation rate, which allows it to evade the host immune system and develop resistance to drugs. However, this extraordinary adaptive ability may also be the key to HIV’s demise. Through the field of phylodynamics, the evolutionary behavior of the virus is being studied in an attempt to control the epidemic. In this thesis, three papers are presented in which we analyze sequences generated through the Canadian HIV Strain and Drug Resistance Surveillance program. In chapter 2 we validate a classifier which distinguishes between recent and established infections based on the proportion of mixed bases observed in population-based pol sequences. Our results will help identify recent infections and improve incidence calculations. In chapter 3, we investigate immune-induced patterns in HIV that are shared by patients of the same ethnicity. An understanding of the forces shaping HIV evolution is instrumental to the development of a vaccine relevant to the Canadian epidemic. In chapter 4, we present preliminary results of a historical reconstruction of HIV across the provinces of Canada. This analysis will highlight strategies that have succeeded or failed in controlling the epidemic. Furthermore, our work will establish whether non-B subtypes of HIV are an increasing threat to Canadian public health. Overall, this thesis provides the first country-wide evolutionary and phylogenetic analysis of the HIV epidemic.

Class of 2006 Abstract / Background: The processes’ underlying a recent rise in the rate of Clostridium difficile associated diarrhea (CDAD) at the Southern Arizona Veterans Administration Health Care System (SAVAHS) is unclear. Past changes to formulary in workhorse oral flouroquinolone from levofloxacin to gatifloxacin are under scrutiny. An infection-control component was also possible. Methods: 142 patients suspected of having CDAD had stool specimens submitted for toxin assay from late July to late Oct of 2004. A retrospective chart review was performed using the Veterans Administration Computerized Patient Record System (CPRS) to examine total antibiotic use in the three months prior to having specimens submitted for laboratory toxin analysis. A subset-analysis was performed on 100 specimens submitted for toxin analysis. Parallel culture was performed and 9 isolates of C. difficile were obtained for molecular analysis and fingerprinting. Results: Of the 142 patients sampled, 20 tested positive for C. difficile toxin with the remaining 122 patients testing negative. Antibiotic usage was categorized by total antibiotic use and gatifloxacin use. 98 patients received at least 1 antibiotic within the preceding 3 months with 44 patients receiving no antibiotic therapy of any kind. Of the 98 patients that received antibiotic therapy, 44 received gatifloxacin, however, all of these patients also received at least one other antibiotic. Of the nine isolates fingerprinted, two distinct genetic clusters were identified.

Blastocystis est un protozoaire anaérobie trouvé dans le tube digestif de l’homme et de nombreux animaux. Il est à ce jour le parasite intestinal le plus fréquemment retrouvé dans les selles humaines. Dix-sept sous-types (ST1 à ST17) ont été décrits en se basant sur la comparaison des séquences du gène de l’ARNr 18S. L’infection à Blastocystis est associée à une variété de troubles gastro-intestinaux et plusieurs études suggèrent une corrélation entre la pathogénicité et le ST du parasite. Trois différents axes de recherche ont été développés. Le premier s’est focalisé sur la prévalence et la biodiversité génétique de ce parasite dans les populations humaines. Des études épidémiologiques ont été menées en France et au Liban mais aussi en Afrique en réalisant la première enquête au Sénégal. Le sous-typage des isolats a été réalisé par PCR en temps réel en ciblant un domaine du gène de l’ARNr 18S suivi d’un séquençage direct du produit de PCR. Au Liban, la prévalence de Blastocystis était de 20% dans la population globale avec une corrélation entre le ST1 et le développement de symptômes gastro-intestinaux. Dans le même pays, cette prévalence dépassait les 60% chez des patients symptomatiques et des écoliers. Au Sénégal, la prévalence observée est la plus importante jamais décrite pour ce parasite puisqu’elle atteignait 100% dans une population d’une centaine d’enfants vivant en milieu rural. Ces données soulignent l’impact socioéconomique de la blastocystose dans les pays en développement où les conditions sanitaires sont souvent précaires. En France, une prévalence importante de 18% a pourtant été observée dans une large étude épidémiologique englobant des patients présentant ou non des symptômes et suivis dans 11 hôpitaux répartis sur tout le territoire français. Le ST3 est prédominant suivi des STs 1, 2 et 4 comme dans une majorité de pays à travers le monde. Le deuxième axe s’est concentré sur l’identification des facteurs de risque de transmission de Blastocystis à l’homme. Le parasite a été recherché dans les selles de vaches et de patients ainsi que dans des échantillons d’eau consommée par l’homme et les animaux dans une région géographique limitée du Nord Liban. 30% des échantillons humains, 69% des échantillons d'eau et 80% des échantillons de bovins étaient positifs pour le parasite. Le ST3 était prédominant dans les échantillons humains et d’eau suivi des ST1, ST2 et ST4. Par contre, ST10 et ST14 étaient prédominants chez les bovins mais ces deux STs n’ont pas été retrouvés dans les autres types d’échantillons. Pour expliquer l'absence des ST10 et ST14 dans ces échantillons, une transmission de ces STs par contact direct entre les bovins et/ou l'absence de formes kystiques transmissibles pour ces STs ont été proposées. Ce parasite a aussi été recherché dans les selles de nombreux groupes d’animaux du zoo de La Palmyre en France. Nous avons montré que près de 40% des selles analysés étaient positives pour Blastocystis et identifié de nouveaux réservoirs d'infections pour l’homme chez les carnivores. La prévalence du parasite atteignait 60% chez les primates chez lesquels les ST1 à ST5 identifiés sont identiques à ceux observés chez l'homme confirmant la faible spécificité d’hôte de ces STs. Dans une autre étude, la prévalence de Blastocystis était de seulement 3,5% dans une population de chiens en France suggérant que cet animal n'est pas un hôte naturel de Blastocystis. Enfin, pour clarifier la pathogénicité de ce parasite, le troisième axe de mes travaux a souligné le caractère invasif de Blastocystis dans un cas de péritonite appendiculaire chez une fillette de 9 ans de retour du Maroc. Seul Blastocystis a été détecté dans les selles, l’appendice, le liquide péritonéal et le sac de Douglas de cette patiente. Une gastro-entérite s’est de plus déclarée simultanément chez 26 membres de la famille de l'enfant suggérant une épidémie qui pourrait trouver son origine dans la consommation commune d’une eau contaminée. / Blastocystis sp. is an anaerobic parasitic protozoa found in the digestive tract of humans and numerous animals. To date, it is the most common intestinal parasite found in human feces with worldwide distribution. Seventeen subtypes (ST1-ST17) have been described based on the comparison of SSU rRNA gene sequences. Blastocystis infection is associated with various gastrointestinal disorders and many studies suggest a correlation between Blastocystis STs and pathogenicity. My work was developed on three different topics. The first concerned the prevalence and the genetic biodiversity of the parasite in human populations. Epidemiological studies were conducted in France and Lebanon but also in Africa by performing the first survey of this parasite in Senegal. Subtyping of the isolates was performed by real-time PCR targeting a domain of the SSU rRNA gene followed by direct sequencing of the PCR product. In Lebanon, the prevalence of Blastocystis reached 20% in the general population and we demonstrated a correlation between ST1 infection and the presence of symptoms. In the same country, this prevalence was 60% in schoolchildren and patients presenting gastrointestinal symptoms. Strikingly, the prevalence of Blastocystis in a population of one hundred children living in a rural area reached 100% in Senegal and more than half of the infected children by the parasite presented gastrointestinal disorders. These latter studies highlighted the socioeconomic impact of blastocystosis in developing countries with poor hygiene sanitation. In France, a large-scale molecular epidemiological study was performed including patients presenting or not gastrointestinal symptoms. Stool samples were collected during winter and summer in 11 hospitals spread all over the French territory. We observed a high prevalence of Blastocystis in the french population with an average of 18.2% and the predominance of ST3 followed by ST1, ST4 and ST2 as in numerous countries. We also identified seasonal variations since the average prevalence of the parasite is 13.6% in winter and 23.1% in summer. The second topic focused on the identification of the risk factors of Blastocystis transmission to humans. We searched this parasite in bovid and human stools as well as in drinking water samples consumed by bovids and breeders in a limited geographic area of North-Lebanon. 30% of human samples, 69% of water samples and 80% of bovid samples were positive for the parasite. Interestingly ST3 is predominant in human and water samples followed by ST1, ST2 and ST4. ST10 and ST14 were predominant in bovid but both STs are lacking in human and water samples. To explain the lack of ST10 and ST14 in human and water samples, we suggested a transmission of these STs occurring through direct contact between bovid and / or the absence of transmissible cystic forms of these STs. Furthermore, this parasite was searched in the stools of numerous animal groups in the zoo of La Palmyre in France. We showed that nearly 40% of the analyzed stools were positive for Blastocystis and identified new reservoirs of human infections in carnivores. The prevalence of the parasite reached 60% in primates in which the identified ST1 to ST5 are identical to those observed in humans confirming the limited host specificity of these STs. In another study, we showed that the prevalence of Blastocystis was of only 3.5% in a population of one hundred dogs in France suggesting that this pet is not a natural host of Blastocystis. Finally, to clarify the pathogenicity of this parasite, the third topic highlighted the invasive character of Blastocystis observed in a case of appendicular peritonitis in a 9-year old girl returning from Morocco. Only Blastocystis was detected in stools, appendix, peritoneal liquid and Douglas pouch of the patient. Interestingly, simultaneous gastroenteritis occurred in 26 members of the child’s family suggested an outbreak with contaminated water as probable origin.