The general objectives of this thesis were: (i) to determine the etiological and molecular profile of clinical mastitis (CM) in 20 dairy herds of Southeast, Brazil; and (ii) to quantify antimicrobial used for treatment of CM in the study population. To achieve this goals, four studies were performed. In the Study 1, we characterized the pathogen frequency and severity of CM in dairy herds. In addition, we determined the incidence rate of clinical mastitis (IRCM) and its association with the following herd-level descriptors: bulk milk somatic cell count (BMSCC), bulk milk total bacterial count (BMTBC), herd size (number of lactating cows), milk yield, housing system and season. The association between herd-level descriptors and IRCM were determined by two groups of mixed regression models: one based on the overall IRCM, and five based on the following specific-pathogen groups: contagious, other Gram-positive, Gram-negative, other (composed of yeast and Prototheca spp), and negative culture. A total of 5,957 quarter-cases of CM were recorded and the most frequently isolated pathogens were Escherichia coli (6.6% of total cultures), Streptococcus uberis (6.1%), and Streptococcus agalactiae (5.9%). The majority of CM cases were mild (60.3%), while 34.1% were moderate and 5.6% severe. Overall, the IRCM was 9.7 quarter-cases per 10,000 quarter-days at risk (QDAR), and the only herd-level parameter associated with overall IRCM was BMSCC, in which the highest IRCM was observed for herds with BMSCC >600.000 × 103 cells/mL. In the models evaluating the specific-pathogen groups, IRCM with isolation of major contagious pathogens was associated with BMSCC, milk yield and housing system. For the evaluation of other Gram-positive pathogens, the IRCM was higher in the rainy season of 2015 in comparison with the other seasonal categories. In addition, for the model evaluating the Gram-negative group, the IRCM was highest in herds with BMTBC >30 × 103 cfu/mL. The Study 2 aimed to characterize the treatment profile and quantify the antimicrobial consumption for treatment of CM in dairy herds; and to determine the association of antimicrobial use (AMU) and the same herd-level descriptors as described in the Study 1. Data on treatment practices and AMU were obtained from 19 dairy herds for a period of 12 months per herd. The AMU for treatment of CM was quantified monthly in units of defined daily dose (DDD) and expressed as antimicrobial treatment incidence (ATI; number of DDD per 1,000 lactating cows-day). The overall monthly mean ATI was 17.7 DDD per 1,000 lactating cow-days (15.4 for intramammary compounds, and 2.2 for systematically administered antimicrobials). Among intramammary drugs, aminoglycosides had the highest ATI (11.7 DDD per 1,000 lactating cow-days), while for systematically administrated antimicrobials, fluoroquinolones (4.2 DDD per 1,000 lactating cow-days) were the most frequently used antimicrobials. Herd size and BMSCC were positively associated with ATI. In addition, herd-level ATI was higher in freestall herds than in compost bedded-pack barns. In the Study 3, we determined the phylogeny of E. coli strains isolated from CM in dairy cows and the association of most frequent phylogroups with antimicrobial susceptibility. A total of 100 E. coli isolates recovered from CM cases described in the Study 1 were categorized according to their phylogenetic group using a quadruplex PCR method; antimicrobial susceptibility pattern was also evaluated. Most isolates were assigned to phylogenetic group A (52%), followed by B1 (38%), B2 (2%), C (4%), D (3%), and E (1%). Resistant isolates were observed for all evaluated antimicrobials. Overall, more than 96% of E. coli isolates were resistant to ampicillin, and more than 23% were resistant to cephalothin, sulphadimethoxine or tetracycline. High levels of resistance (>70%) were also found to erythromycin, oxacillin, penicillin, penicillin associated with novobiocin, and pirlimycin. In contrary, high susceptibility was observed to ceftiofur (96.8%) among E. coli isolates. Difference in the antimicrobial susceptibility among phylogenetic groups was observed only for cephalothin, in which E. coli strains belonging to the phylogroup A were inhibited at lower antimicrobial concentrations than strains assigned to the phylogroup B1. In Study 4, we evaluated the genotypic diversity among Strep. agalactiae and Strep. uberis isolates recovered from CM in dairy cows; in addition, the study evaluated the association of genotypes clustered by genetic similarity with antimicrobial susceptibility pattern. Isolates were subtyped using randomly amplified polymorphic DNA (RAPD) analysis. A great genotypic diversity was found for both Strep. agalactiae (45 subtypes out of 89 isolates) and Strep. uberis (56 subtypes out of 88 isolates). For evaluation of antimicrobial susceptibility, subtypes of Strep. agalactiae were clustered into three groups (Ia, Ib and II), while Strep. uberis subtypes were clustered into two groups (I and II) according to their genetic similarity. Overall, Strep. agalactiae isolates showed high susceptibility to most antimicrobials, except to tetracycline and erythromycin. Differences in the antimicrobial susceptibility among clusters of Strep. agalactiae were observed for ampicillin, ceftiofur, erythromycin, pirlimycin, sulphadimethoxine and tetracycline. In contrary, Strep. uberis isolates were categorized as resistant to most antimicrobials, except to cephalothin and penicillin+novobiocin. No differences were observed among clusters for all antimicrobials in the analysis of Strep. uberis. In conclusion, the results of this thesis indicated a high IRCM in the evaluated herds, and although environmental pathogens were the most common cause of CM in these herds, contagious pathogens such as Strep. agalactiae and Staph. aureus, are still a concern in some dairy herds of Brazil. Furthermore, high frequencies of AMU and off-label protocols were observed among the evaluated herds. The non-judicious use of antimicrobials can become a risk factor for the development of antimicrobial resistance, which was even observed for isolates belonging to the three most prevalent bacterial species identified from CM cases in our study (E. coli, Strep. agalactiae and Strep. uberis). Finally, because there were some herd-level descriptors associated with the IRCM and AMU in our study, there may be opportunity for management strategies aiming to improve the control of CM in dairy herds of southeastern Brazil. / Os objetivos gerais desta tese foram: (i) determinar o perfil etiológico e molecular da mastite clínica (MC) em 20 rebanhos leiteiros do Sudeste do Brasil; e, (ii) quantificar os antimicrobianos usados para tratamento da MC na população estudada. Para alcançar esses objetivos, quatro estudos foram realizados. No Estudo 1, foi caracterizada a frequência de patógenos causadores de MC e a gravidade das infecções nos rebanhos leiteiros. Além disso, foi determinada a taxa de incidência de mastite clínica (TIMC) e sua associação com as seguintes variáveis em nível de rebanho: contagem de células somáticas em leite de tanque (CCSLT), contagem bacteriana total em leite de tanque (CBTLT), tamanho (número de vacas em lactação), produção de leite, sistema de alojamento e estação do ano. A associação entre as variáveis em nível de rebanho e a TIMC foi determinada por dois grupos de modelos de regressão logística multivariada: um baseado na TIMC geral, e cinco baseados nos seguintes grupos específicos de patógenos: contagiosos, outros Gram-positivos, Gram-negativos, outros patógenos (composto de leveduras e Prototheca spp.), e cultura negativa. Um total de 5.957 casos de MC em nível de quarto mamário foi registrado e os patógenos mais prevalentes foram Escherichia coli (6,6% de todas as culturas), Streptococcus uberis (6,1%), e Streptococcus agalactiae (5,9%). A maioria dos casos de MC foi de gravidade leve (60,3%), enquanto 34,1% dos casos foram moderados e 5,6% foram graves. A TIMC geral foi de 9,7 casos por 10.000 quartos-dia em risco (QDR), e o único parâmetro em nível de rebanho associado com a TIMC geral foi a CCSLT, em que a TIMC mais alta foi observada em rebanhos com CCSLT >600.000 × 103 células/mL. Nos modelos que avaliaram os grupos específicos de patógenos, a TIMC de patógenos contagiosos foi associada com a CCSLT, produção de leite e sistema de alojamento. Na avaliação de outros patógenos Gram-positivos, a TIMC foi maior na estação chuvosa de 2015 em comparação com as outras categorias referentes à estação do ano. Adicionalmente, para o modelo avaliando o grupo de patógenos Gram-negativos, a TIMC foi mais alta em rebanhos com CBTLT >30.000 × 103 ufc/mL. O Estudo 2 teve como objetivo caracterizar o perfil de tratamento e o consumo de antimicrobianos em rebanhos leiteiros; e determinar a associação de uso de antimicrobianos (UAM) e as mesmas variáveis em nível de rebanho descritas no Estudo 1. Dados sobre as práticas terapêuticas e UAM foram obtidos de 19 rebanhos leiteiros durante um período de 12 meses por rebanho. A frequência de UAM para tratamento da MC foi quantificada mensalmente em unidades de doses definidas diárias (DDD) e expressa como incidência de tratamento antimicrobiano (ITA: número de DDD por 1.000 vacas em lactação-dia). A média de ITA mensal foi de 17,7 DDD por 1.000 vacas em lactação-dia (15,4 para compostos intramamários, e 2,2 para compostos sistêmicos). Entre os produtos intramamários, os aminoglicosídeos tiveram a ITA mais alta (11,7 DDD por 1.000 vacas em lactação-dia), enquanto que para os compostos administrados pela via sistêmica, as fluoroquinolonas (4,2 DDD por 1.000 vacas em lactação-dia) foram os antimicrobianos mais frequentemente usados. O tamanho do rebanho e CCSLT foram positivamente associados com a ITA. Além disso, a ITA foi mais alta em rebanhos com freestall do que em rebanhos com sistema tipo compost barn. No Estudo 3, determinou-se a filogenia de cepas de E. coli isoladas de casos de MC em vacas leiteiras, e a associação dos filogrupos mais frequentes com a susceptibilidade aos antimicrobianos. Um total de 100 isolados de E. coli identificados nos casos de MC descritos no Estudo 1 foram categorizados de acordo com os grupos filogenéticos por meio de um método de PCR quadruplex; o perfil de susceptibilidade aos antimicrobianos também foi avaliado. A maioria dos isolados pertenceram ao grupo A (52%), seguido dos grupos B1 (38%), B2 (2%), C (4%), D (3%), e E (1%). Foram encontrados isolados resistentes para todos os antimicrobianos avaliados. De forma geral, mais de 96% dos isolados de E. coli foram resistentes a ampicilina, e mais de 23% foram resistentes a cefalotina, sulfadimetoxina ou tetraciclina. Altos níveis de resistência (>70%) foram encontrados também para eritromicina, oxacilina, penicilina e penicilina associada a novobiocina. Ao contrário, foi observado alta susceptibilidade ao ceftiofur (96.8%) entre os isolados de E. coli. Diferenças na susceptibilidade entre os grupos filogenéticos foi observada apenas para a cefalotina, em que os isolados de E. coli pertencentes ao filogrupo A foram inibidos em concentrações de antimicrobianas mais baixas que isolados pertencentes ao filogrupo B1. No Estudo 4, avaliou-se a diversidade genotípica entre isolados de Strep. agalactiae e Strep. uberis identificados em casos de MC em vacas leiteiras; adicionalmente, o estudo avaliou a associação dos genótipos agrupados de acordo com a similaridade genética com o perfil de susceptibilidade aos antimicrobianos. Os isolados foram genotipados por meio do método de amplificação randômica de DNA polimórfico (RAPD). Grande diversidade genotípica foi observada tanto para o Strep. agalactiae (45 subtipos de 89 isolados) quanto para Strep. uberis (56 subtipos de 89 isolados). Para a avaliação de susceptibilidade aos antimicrobianos, os subtipos de Strep. agalactiae foram agrupados em três clusters (Ia, Ib e II), enquanto que os subtipos de Strep. uberis foram agrupados em dois clusters (I e II) de acordo com a similaridade genética. De forma geral, os isolados de Strep. agalactiae apresentaram alta susceptibilidade à maioria dos antimicrobianos, exceto para tetraciclina e eritromicina. Diferenças na susceptibilidade aos antimicrobianos entre os clusters de Strep. agalactiae foram observadas para ampicilina, ceftiofur, eritromicina, pirlimicina, sulfadimetoxina e tetraciclina. Por outro lado, os isolados de Strep. uberis foram resistentes à maioria dos antimicrobianos, exceto para cefalotina e penicilina + novobiocina. Não foram encontradas diferenças entre os clusters para todos os antimicrobianos na análise de Strep. uberis. Em conclusão, os resultados desta tese indicaram alta TIMC nos rebanhos avaliados, e apesar de os patógenos ambientais serem a causa mais comum de MC nestes rebanhos, patógenos contagiosos como Strep. agalactiae e Staph. aureus, ainda são uma preocupação em alguns rebanhos do Brasil. Além disso, observaram-se altas frequências de UAM e de terapias não recomendadas em bula entre os rebanhos avaliados. O uso não judicioso de antimicrobianos pode se tornar um fator de risco para o desenvolvimento da resistência bacteriana aos antimicrobianos, o que foi inclusive observado para isolados pertencentes as três espécies bacterianas mais prevalentes nos casos de MC no nosso estudo (E. coli, Strep. agalactiae e Strep. uberis). Finalmente, pelo fato de algumas variáveis em nível de rebanho terem sido associadas com a TIMC e com o UAM em nosso estudo, é possível que hajam oportunidades para implementação de estratégias de manejo com o objetivo de melhorar o controle da MC em rebanhos leiteiros do sudeste do Brasil.

Nous avons exploré la diversité génétique du complexe d'espèces Ralstonia solanacearum (ceRs) pour caractériser et comprendre les graves épidémies de flétrissement bactérien qui sévissent à Madagascar. Une large collection de souches (n=1224; 75 sites) est constituée. Les souches sont assignées aux phylotypes I, III, et la grande majorité associée à l'épidémie appartiennent au groupe IIB-1 (‘Brown rot’ des anglo-saxons) signalé pour la première fois à Madagascar. L'approche MLVA (RS2-MLVA9) a révélé leur apparenté aux souches IIB-1 distribuées mondialement, suggérant ainsi une introduction malheureuse à Madagascar. Trois populations clonales se propagent par des tubercules infectées. Le génotypage des souches du phylotype III, avec le schéma hautement résolutif RS3-MLVA16 que nous avons développé, a révélé une forte diversité génétique structurée géographiquement en onze populations clonales bien différenciées. Cette structure reflète un caractère endémique des populations suggérant l'absence de transmission par tubercules. Les souches malgaches sont différentes de celles d'Afrique continentale. Les souches IIB-1 et III présentent deux modèles épidémiologiques différents. Les variétés de pomme de terre cultivées à Madagascar n'ont montré aucune résistance génétique vis-à-vis du panel de souches malgaches. Cependant, dans nos conditions expérimentales les accessions 720118 et 800934 sont résistantes aux souches I-31 non détectées pour l'instant à Madagascar, mais prévalentes dans l'océan Indien. Nous disposons ainsi d'un outil robuste pouvant être appliqué à l'étude du phylotype III, d'une vue globale de la structure des populations et d'épidémiologie du ceRs. / This thesis is exploring genetic diversity, population structure and molecular epidemiology of the Ralstonia solanacearum species complex (Rssc) causing potato bacterial wilt outbreaks in Madagascar. We characterized a large collection of strains (n=1224; 75 sites) collected from potato production areas. Surprisingly, the large outbreaks were associated with IIB-1 strains (Brown rot) while a few were associated with phylotypes I and III. This is the first report of phylotype IIB-1 in Madagascar. The IIB-1 strains were genotyped based on MLVA (RS2-MLVA9). And Malagasy phylotype IIB-1 clustered with worldwide distributed strains. Fine scale genetic investigation suggested three clonal populations that were introduced and spread through latently infected tuber-seeds. Phylotype III strains were genotyped with the highly discriminatory RS3-MLVA16 scheme we developed. Genetic population analyses revealed a high genetic diversity within phylotype III strains that geographically structured into 11 clonal populations. This support the endemic character of the phylotype III population in Madagascar and suggests no transmission with potato tubers. Malagasy strains were distinct from continental African strains. A clear-cut epidemiological profile is shown between IIB-1 and III strains. Genetically, no bacterial wilt resistance properties were shown for the most popular Malagasy potato cultivars, except two cultivars: 720118 and 800934 that showed strong resistance to phylotype I-31 strain that are predominantly distributed in the Indian Ocean. This study offered tool to genotype phylotype III strains and gives an insights into population structure and epidemiology of the Rssc.

O Herpesvírus 8 humano (HHV-8) é endêmico em populações africanas e indígenas da região Amazônica. A infecção nestas populações acontece durante a infância e, na África, envolve o contato íntimo no ambiente intrafamiliar. Diversos estudos confirmam a distribuição geográfica dos diferentes subtipos de HHV-8, sendo que o subtipo E é típico das populações indígenas. Objetivos: 1. Caracterizar o(s) subtipo(s) de HHV-8 que circula(m) em população indígena da Amazônia brasileira baseado na análise da região ORF K1 do vírus; 2. Construir a árvore filogenética dos subtipos virais encontrados; 3. Comparar filogeneticamente os subtipos encontrados com os subtipos prevalentes em outras populações indígenas do Brasil e de outros países da América do Sul; 4. Calcular a taxa de substituição para a região VR1 do HHV-8 para as amostras estudadas; 5. Estimar a data de entrada do vírus na população do estudo; 6. Investigar a dinâmica de transmissão do vírus no ambiente intrafamiliar; 7. Averiguar se há correlação entre os alelos de HLA classe I (A e B) e II (DQB1 e DRB1) e suscetibilidade à infecção por HHV-8. Casuística e métodos: Estudo de soroprevalência da infecção por HHV-8 em amostra de população indígena da Amazônia brasileira utilizando IFI para detecção de antígenos da fase latente (LANA) e lítica (Lítico) do vírus. Análise filogenética da amostras encontradas utilizando-se o DNA/HHV-8 extraído de amostras de saliva, submetidas à reação de nested PCR para amplificar as regiões hipervariáveis VR1 e VR2. Cálculo da taxa de substituição do HHV-8, utilizando-se os métodos de distância e técnica bayesiana. Estimar a data do ancestral comum mais recente para as amostras em estudo, utilizando-se o programa BEAST. Tipagem de HLA de indivíduos positivos e negativos para a infecção por HHV-8, utilizando-se a técnica de PCR-SSO. Resultados: A soroprevalência geral da infecção por HHV-8 na população em estudo foi de 75,3% (399/530). Observou-se que a soropositividade dos filhos está correlecionada com a soropositividade materna. O único subtipo viral encontrado foi o subtipo E. A taxa de substituição de nucleotídeos do HHV-8 utilizando a região VR1 foi da ordem de 6x10-4 substituições por sítio por ano (s/s/a). Ao analisar todas as seqüências estudadas o ancestral comum mais recente está em torno de 138 anos. Não houve correlação entre a susceptibilidade à infecção por HHV-8 e alelos de HLA classe I ou II. Conclusões: A população estudada é endêmica para a infecção por HHV-8. A infecção ocorre principalmente na infância, por via horizontal não-sexual, e a transmissão se dá provavelmente pela saliva. Assim como em outras populações endêmicas da África, a soropositvidade dos filhos está correlacionada com a soropositividade das mães. Confirmando achados anteriores, o único subtipo do HHV-8 circulante na população estudada, foi o subtipo E. Nossos dados sugerem que a região do gene VR1 do HHV-8 evolui com uma taxa de 6x10-4 substituições por sítio por ano (s/s/a), e que o ancestral comum mais recente do vírus, a partir das amostras analisadas está em torno de 138 anos. Os dados sugerem, também, que não há correlação entre a susceptibilidade à infecção por HHV-8 e os alelos de HLA classe I ou II. / The human herpesvirus 8 (HHV-8) is endemic in Africa and Amerindian populations from Amazon region. The infection in those populations occurs during childhood and, in Africa, involves a close contact in intrafamilial environment. Several studies confirm the geographical distribution of different subtypes of HHV-8, and the subtype E is typical of the Amerindian population. Objectives: 1. To characterize the HHV-8 subtypes circulating in Amerindian population from Brazilian Amazon, based on the analysis of ORF K1 region of the virus. 2. To construct a phylogenetic tree of viral subtypes found among Amerindians 3. To compare by phylogenetic methods the subtypes found in Mapuera Amerindians with the subtypes prevalent in others Amerindians populations of Brazil and South America 4. To determine the substitution rate of VR1 region of HHV-8 for the sequences obtained in the present study 5. To estimate the date of entry of the viruses in the Mapuera population 6. To investigate the dynamic of transmission of the virus in the intrafamilial environment 7. To investigate if there is a correlation between susceptibility to HHV-8-infection and HLA class I (A and B) and II (DQB1 and DRB1) alleles. Patients and methods: The seroprevalence of HHV-8 infection in a sample of the indigenous population of the Brazilian Amazon was carried out using IFA to detect antibodies to latent (LANA) and lytic phase antigens of HHV-8. Phylogenetic analysis of the sequences was performed by using the DNA extracted from samples of saliva, using a nested PCR to amplify the hypervariable regions VR1/ VR2 of HHV-8. Estimation of the substitution rate of HHV-8 nucleotides was performed by using the method of distance and the Bayesian technique. Estimates of the time of the most recent common ancestor (TMRCA) for all samples studied were done by using the BEAST program. HLA typing of positive and negative subjects for HHV-8 infection was performed by using the PCR-SSO technique. Results: The overall HHV-8 seroprevalence was 75.3% (399/530). There was a positive correlation between soropositivity of children and maternal seropositivity. The only viral subtype found was subtype E. The substitution rate of HHV-8 using the VR1 region was estimated around 6x10-4 substitutions per site per year (s / s / y). By using this rate of substitution, the TMRCA of the Mapuera viruses sequences was estimated to be around 138 years. There was no correlation between susceptibility to HHV-8-infection and HLA class I or II alleles. Conclusions: The population studied is endemic for HHV-8 infection. The infection occurs mainly in childhood, by horizontal, nonsexual transmission, probably by saliva. As in endemic populations of Africa, the soropositvity of children is positively correlated with the seropositivity of the mothers. In agreement with previous reports, the subtype E was the only HHV-8 subtype found in Mapuera Amerindians. Our data suggest that the VR1 gene region of HHV-8 evolves with a rate of 6x10-4 substitutions per site per year (s / s / y), which results in a time of the most recent common ancestor for Mapuera HHV-8 sequences of 138 years. There was no correlation between susceptibility to HHV-8-infection and HLA class I or II alleles.

Le protozoaire Blastocystis et la bactérie Campylobacter sont des microorganismes zoonotiques ayant un impact majeur en santé publique. Même si son pouvoir pathogène reste controversé, Blastocystis est l’eucaryote unicellulaire le plus fréquemment retrouvé dans les selles humaines alors que Campylobacter est considérée comme la cause la plus courante de gastroentérite humaine. Ces microorganismes sont fréquemment présents chez les animaux destinés à l’alimentation comme les volailles ou les bovins et leur transmission peut être liée à la consommation ou à un contact répété avec les manipulateurs de ces animaux. Malgré ce risque potentiel majeur pour l’Homme, très peu de données étaient disponibles concernant la prévalence et les espèces de Campylobacter ou sous-types de Blastocystis présents dans les filières avicole et bovine au Liban. De plus, le risque de transmission zoonotique par contact direct avec le personnel des abattoirs ou les éleveurs n’avait jamais été évalué. Pour ce qui est des bovins, Blastocystis a été identifié par PCR quantitative en temps réel dans plus de 60% des échantillons de selle de vaches laitières analysés au Nord-Liban. Une prédominance du sous-type (ST) 10 et du ST14 a été observée confirmant que les bovins sont les hôtes naturels de ces deux STs. Les ST2, ST1, ST5, ST3 et ST7 ont aussi été identifiés avec de faibles prévalences. Chez les éleveurs comme dans un groupe de patients sans contact avec ces animaux, une prévalence importante dépassant les 50% a été rapportée avec une prédominance du ST3. Cependant, la comparaison des isolats identifiés dans ces cohortes humaine et animale suggère que les bovins joueraient un rôle négligeable en tant que réservoirs zoonotiques de Blastocystis. Dans la filière avicole, la prévalence de ce même parasite dépassait les 30% dans les caeca de poulets de chair analysés dans des abattoirs du Nord-Liban. Tous les isolats aviaires caractérisés appartenaient aux ST6 et ST7 confirmant que les oiseaux sont des hôtes naturels de ces deux STs. Chez le personnel d’abattoir comme dans un groupe de patients sans contact avec les volailles, la prévalence de Blastocystis dépassait les 50% avec une prédominance du ST3. De plus, l’identification du ST6 aviaire chez le personnel d’un abattoir confirmait le potentiel zoonotique de ce ST. Toujours dans cette filière avicole et en analysant à la fois les même caeca et des carcasses de poulets en fin de chaîne d’abatage, la prévalence de Campylobacter était respectivement de 67% et 17,2% dans ces prélèvements. Les deux principales espèces identifiées étaient C. jejuni et C. coli. Des niveaux élevés de diversité génétique ont été observés parmi les 51 isolats de C. jejuni identifiés chez les poulets et répartis en 25 profils distincts. Une prédominance des profils 1, 13, 30 et 38 a été observée alors qu’ils sont aussi fréquemment retrouvés parmi les cas cliniques humains français suggérant que ces animaux représentent un réservoir potentiel de campylobactériose humaine. Une prévalence significativement plus élevée de Campylobacter a été rapportée chez le personnel de l'abattoir par rapport à celle observée dans la cohorte de patients sans contact avec la volaille montrant que les poulets de chair contaminés à l'abattoir représentaient une source non négligeable de transmission zoonotique de la bactérie. Les mêmes caeca de poulets ont pu être comparés pour la présence de Campylobacter et de Blastocystis. Cette comparaison révélait une association statistiquement significative de ces deux microorganismes suggérant que la présence de Campylobacter serait favorisée par celle de Blastocystis et vice versa [...] / The protozoan Blastocystis and the bacterium Campylobacter are zoonotic microorganisms with a major impact on public health. Although its pathogenicity remains controversial, Blastocystis is the most common single-celled eukaryote found in human stool, while Campylobacter is considered the most common cause of human gastroenteritis. These microorganisms are frequently present in food-producing animals such as poultry or cattle and their transmission may be related to consumption or repeated contact with the handlers of these animals. Despite this major potential risk to humans, very little data were available on the prevalence and species of Campylobacter or Blastocystis subtypes present in the poultry and bovid sectors in Lebanon. In addition, the risk of zoonotic transmission through direct contact with slaughterhouse staff or farmers had never been assessed. Regarding bovids, Blastocystis was identified by PCR in more than 60% of the dairy cattle stool samples analysed in North Lebanon. A predominance of ST10 and ST14 has been observed confirming that cattle are the natural hosts of these two STs. ST2, ST1, ST5, ST3 and ST7 were also identified with lower prevalences. In breeders as well as in a group of patients without contact with these animals, a significant prevalence exceeding 50% has been reported with a predominance of ST3. However, a comparison of the isolates identified in these human and animal cohorts suggests that cattle would play a negligible role as zoonotic reservoirs of Blastocystis. In the poultry sector, the prevalence of the same parasite exceeded 30% in broiler caeca analysed in slaughterhouses in North Lebanon. All characterized avian isolates belonged to ST6 and ST7 confirming that birds are natural hosts of these two STs. Among slaughterhouse staff as well as in a group of patients without contact with poultry, the prevalence of Blastocystis exceeded 50% with a prevalence of ST3. In addition, the identification of avian ST6 in slaughterhouse staff confirmed the zoonotic potential of this ST. Still in this poultry sector and by analysing both the same caeca samples and chicken carcasses, the prevalence of Campylobacter was 67% and 17.2%, respectively, in these specimens. The two main species identified were C. jejuni and C. coli. High level of genetic diversity was observed among the 51 C. jejuni isolates identified in chickens and distributed in 25 distinct profiles. A predominance of profiles 1, 13, 30 and 38 has been observed, although they are also frequently found among French human clinical cases suggesting that these animals represent a potential reservoir of human campylobacteriosis. A significantly higher prevalence of Campylobacter was reported among slaughterhouse staff compared to that observed in the cohort of patients without contact with poultry, showing that contaminated broilers at the slaughterhouse were a significant source of zoonotic transmission of the bacteria. The same chicken caeca samples were compared for the presence of Campylobacter and Blastocystis. This comparison revealed a statistically significant association of these two microorganisms suggesting that the presence of Campylobacter would be enhanced by that of Blastocystis and vice versa. This first large-scale survey conducted in Lebanon provided major data on the prevalence and circulation of Blastocystis and Campylobacter in this ocuntry and assessed the need to put in place measures to prevent and control these microorganisms in poultry and cattle breeding or slaughter facilities in order to limit their transmission.

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UNIFSJ / A colonização por Staphylococcus aureus representa o principal risco para infecções, principalmente em crianças, que apresentam elevada morbimortalidade relacionada a este patógeno. Tal microrganismo apresenta grande variabilidade molecular que confere mudanças epidemiológicas constantes. Linhagens com características variadas de resistência e virulência emergem e sucumbem, constituindo-se em um desafio para a saúde pública mundial. Nos últimos anos, alguns estudos têm sugerido uma mudança epidemiológica nas linhagens de S. aureus resistentes a meticilina (MRSA) no Brasil. O objetivo do presente estudo é atualizar o conhecimento acerca da epidemiologia molecular de MRSA colonizando crianças e adolescentes em Niterói – Rio de Janeiro. Trata-se de estudo de corte transversal, realizado com crianças e adolescentes, de zero a 16 anos, em creches, ambulatórios e hospitais na cidade de Niterói-RJ, no período de agosto de 2011 a junho de 2013. Um total de 1500 participantes (500 de creches, 500 de ambulatório e 500 de hospitais) foi submetido à coleta de secreção nasal por meio de swabs para pesquisa de MRSA. Destes, 749 (49,9%) das 1500 amostras foram caracterizados como S. aureus, sendo 288 (57,6%) das 500 amostras de ambulatório, 239 (47,8%) das 500 de hospitais e 222 (44,4%) das 500 das creches desta região. Do total, 144 (9,6%) das 1500 amostras foram caracterizadas como MRSA, sendo 31 (6,2%) das 500 das creches, 45(9%) das 500 de ambulatório e 68 (13,6%) das 500 dos hospitais. As 144 amostras de MRSA foram submetidas às técnicas de genotipificação por PCR dos genes mecA, lukS/lukFPV e SA442 para a identificação do gene de resistência a meticilina, da leucocidina Panton-Valentine (PVL) e confirmação da espécie S. aureus, respectivamente. Foram realizados testes de susceptibilidade antimicrobiana com 14 antimicrobianos selecionados de acordo com o CLSI (2012). Também foram realizados PCR-multiplex para identificação do tipo de cassete mec (SCCmec) e sequenciamento dos genes da proteína A (spa-Typing) e genes constitutivos (MLST), para a caracterização molecular das linhagens de MRSA. Observou-se a prevalência de diferentes clones de MRSA, incluindo os mais frequentes, ST5-MRSA-IV (CC5) e ST30-MRSA-IV (CC30), cujas características, corroboram com a literatura de linhagens de grande relevância, disseminadas em nível pandêmico, “Clone pediátrico” e “Clone SWP”, respectivamente. Nas creches e nos hospitais, foram identificados seis de complexos clonais (CC) diferentes em cada cenário. Já o ambulatório apresentou nove CCs diferentes. O PVL foi observado em 30 (54,5%) das 55 CC30. Das amostras classificadas como CC5 34 (57,6%) de 59 apresentaram resistência ou resistência intermediária à eritromicina. Evidenciou-se variação sazonal de colonização por MRSA, com maior frequência no verão. Nossos resultados confirmam a mudança epidemiológica do até então conhecido Clone Epidêmico Brasileiro para linhagens amplamente descritas pela literatura e disseminadas em nível pandêmico, com grande relevância, conhecidas como “Clone Pediátrico” e “Clone SWP” em crianças e adolescentes no Brasil, com variação sazonal na frequência de colonização por MRSA. Tais achados são relevantes para uma melhor compreensão do comportamento epidemiológico da colonização por MRSA em nível local, com informações que auxiliem nas estratégias de vigilância epidemiológica para o controle desse patógeno. / The colonization by Staphylococcus aureus (S. aureus) is the main risk factor for infections, especially in children who have high morbidity and mortality related to this pathogen. This microorganism has large molecular variability that provides constant epidemiological changes. Lineages with varied characteristics of resistance and virulence emerge and succumb, constituting a challenge to global public health. In recent years, some epidemiologic studies have suggested a change in strains of methicillin-resistant S. aureus (MRSA) in Brazil. The aim of this study is to update the knowledge of the molecular epidemiology of MRSA colonizing children and adolescents in Niterói - Rio de Janeiro. This is a cross-sectional study, conducted with children and adolescents in community day care centers, outpatient clinics and hospitals in Niterói, RJ, from August 2011 to June 2013. A total of 1500 participants (500 of day care centers, 500 of outpatient clinic and 500 of hospitals) was submitted to nasal secretion collection using swabs for MRSA research. Of these, 749/1500 (49.9%) were characterized as S. aureus, being that 288/500 (57.6%) were of outpatient clinic, 239/500 (47.8%) of hospitals and 222/500 (44.4 %) of the day cares centers. Among the samples of S. aureus 144/1500 (9.6%) were characterized as MRSA, with 31/500 (6.2%) from day care centers, 45/500 (9%) from outpatient clinics and 68/500 (13 6%) of hospitals. The 144 samples of MRSA were submitted to genotyping by PCR from the mecA, luks/lukFPV and SA442 genes to identifi of methicillin resistance gene, the Panton-Valentine leukocidin (PVL) toxin and species, respectively. Antimicrobial susceptibility tests were performed with 14 antimicrobials selected according to the CLSI (2012). It was also performed multiplex-PCR to identify the type of cassette mec (SCCmec) and sequencing of the genes of protein A (spa-Typing) and constitutive genes (MLST), for molecular characterization of MRSA lineages. We observed the prevalence of different MRSA, including the most commons, ST5-MRSA-IV (CC5) and ST30-MRSA-IV (CC30), whose characteristics corroborate with the literature of relevant lineages, disseminated on pandemic level, "Pediatric Clone" and "SWP Clone", respectively. In day care centers and hospitals six types of different clonal complexes (CC) have been identified in each environment, and the outpatient clinics had nine different CCs. The CC30 showed a frequency of 54.5% (30/55) for positivity of PVL genes. Samples classified as CC5 showed 57.6% (34/59) of resistance or intermediate resistance to erythromycin. This finding corroborates with literature, which describes about the epidemiological distribution of these strains. Our results confirm the epidemiological change of the Epidemic Brazilian Clone to strains widely described in the literature with pandemic level of disseminated and great relevance, known as "Pediatric Clone" and "SWP Clone" in children and adolescents in Brazil with seasonal variation in the frequency of MRSA colonization. These findings are relevant for a better understanding of the epidemiological changes of MRSA colonization locally, with information that assists in epidemiological surveillance strategies for the control of this pathogen.

O Herpesvírus 8 humano (HHV-8) é endêmico em populações africanas e indígenas da região Amazônica. A infecção nestas populações acontece durante a infância e, na África, envolve o contato íntimo no ambiente intrafamiliar. Diversos estudos confirmam a distribuição geográfica dos diferentes subtipos de HHV-8, sendo que o subtipo E é típico das populações indígenas. Objetivos: 1. Caracterizar o(s) subtipo(s) de HHV-8 que circula(m) em população indígena da Amazônia brasileira baseado na análise da região ORF K1 do vírus; 2. Construir a árvore filogenética dos subtipos virais encontrados; 3. Comparar filogeneticamente os subtipos encontrados com os subtipos prevalentes em outras populações indígenas do Brasil e de outros países da América do Sul; 4. Calcular a taxa de substituição para a região VR1 do HHV-8 para as amostras estudadas; 5. Estimar a data de entrada do vírus na população do estudo; 6. Investigar a dinâmica de transmissão do vírus no ambiente intrafamiliar; 7. Averiguar se há correlação entre os alelos de HLA classe I (A e B) e II (DQB1 e DRB1) e suscetibilidade à infecção por HHV-8. Casuística e métodos: Estudo de soroprevalência da infecção por HHV-8 em amostra de população indígena da Amazônia brasileira utilizando IFI para detecção de antígenos da fase latente (LANA) e lítica (Lítico) do vírus. Análise filogenética da amostras encontradas utilizando-se o DNA/HHV-8 extraído de amostras de saliva, submetidas à reação de nested PCR para amplificar as regiões hipervariáveis VR1 e VR2. Cálculo da taxa de substituição do HHV-8, utilizando-se os métodos de distância e técnica bayesiana. Estimar a data do ancestral comum mais recente para as amostras em estudo, utilizando-se o programa BEAST. Tipagem de HLA de indivíduos positivos e negativos para a infecção por HHV-8, utilizando-se a técnica de PCR-SSO. Resultados: A soroprevalência geral da infecção por HHV-8 na população em estudo foi de 75,3% (399/530). Observou-se que a soropositividade dos filhos está correlecionada com a soropositividade materna. O único subtipo viral encontrado foi o subtipo E. A taxa de substituição de nucleotídeos do HHV-8 utilizando a região VR1 foi da ordem de 6x10-4 substituições por sítio por ano (s/s/a). Ao analisar todas as seqüências estudadas o ancestral comum mais recente está em torno de 138 anos. Não houve correlação entre a susceptibilidade à infecção por HHV-8 e alelos de HLA classe I ou II. Conclusões: A população estudada é endêmica para a infecção por HHV-8. A infecção ocorre principalmente na infância, por via horizontal não-sexual, e a transmissão se dá provavelmente pela saliva. Assim como em outras populações endêmicas da África, a soropositvidade dos filhos está correlacionada com a soropositividade das mães. Confirmando achados anteriores, o único subtipo do HHV-8 circulante na população estudada, foi o subtipo E. Nossos dados sugerem que a região do gene VR1 do HHV-8 evolui com uma taxa de 6x10-4 substituições por sítio por ano (s/s/a), e que o ancestral comum mais recente do vírus, a partir das amostras analisadas está em torno de 138 anos. Os dados sugerem, também, que não há correlação entre a susceptibilidade à infecção por HHV-8 e os alelos de HLA classe I ou II. / The human herpesvirus 8 (HHV-8) is endemic in Africa and Amerindian populations from Amazon region. The infection in those populations occurs during childhood and, in Africa, involves a close contact in intrafamilial environment. Several studies confirm the geographical distribution of different subtypes of HHV-8, and the subtype E is typical of the Amerindian population. Objectives: 1. To characterize the HHV-8 subtypes circulating in Amerindian population from Brazilian Amazon, based on the analysis of ORF K1 region of the virus. 2. To construct a phylogenetic tree of viral subtypes found among Amerindians 3. To compare by phylogenetic methods the subtypes found in Mapuera Amerindians with the subtypes prevalent in others Amerindians populations of Brazil and South America 4. To determine the substitution rate of VR1 region of HHV-8 for the sequences obtained in the present study 5. To estimate the date of entry of the viruses in the Mapuera population 6. To investigate the dynamic of transmission of the virus in the intrafamilial environment 7. To investigate if there is a correlation between susceptibility to HHV-8-infection and HLA class I (A and B) and II (DQB1 and DRB1) alleles. Patients and methods: The seroprevalence of HHV-8 infection in a sample of the indigenous population of the Brazilian Amazon was carried out using IFA to detect antibodies to latent (LANA) and lytic phase antigens of HHV-8. Phylogenetic analysis of the sequences was performed by using the DNA extracted from samples of saliva, using a nested PCR to amplify the hypervariable regions VR1/ VR2 of HHV-8. Estimation of the substitution rate of HHV-8 nucleotides was performed by using the method of distance and the Bayesian technique. Estimates of the time of the most recent common ancestor (TMRCA) for all samples studied were done by using the BEAST program. HLA typing of positive and negative subjects for HHV-8 infection was performed by using the PCR-SSO technique. Results: The overall HHV-8 seroprevalence was 75.3% (399/530). There was a positive correlation between soropositivity of children and maternal seropositivity. The only viral subtype found was subtype E. The substitution rate of HHV-8 using the VR1 region was estimated around 6x10-4 substitutions per site per year (s / s / y). By using this rate of substitution, the TMRCA of the Mapuera viruses sequences was estimated to be around 138 years. There was no correlation between susceptibility to HHV-8-infection and HLA class I or II alleles. Conclusions: The population studied is endemic for HHV-8 infection. The infection occurs mainly in childhood, by horizontal, nonsexual transmission, probably by saliva. As in endemic populations of Africa, the soropositvity of children is positively correlated with the seropositivity of the mothers. In agreement with previous reports, the subtype E was the only HHV-8 subtype found in Mapuera Amerindians. Our data suggest that the VR1 gene region of HHV-8 evolves with a rate of 6x10-4 substitutions per site per year (s / s / y), which results in a time of the most recent common ancestor for Mapuera HHV-8 sequences of 138 years. There was no correlation between susceptibility to HHV-8-infection and HLA class I or II alleles.

Parmi les parasites, les échinocoques revêtent une importance majeure en santé publique de par leur distribution mondiale et les maladies potentiellement très graves, qu’ils occasionnent. En Europe, les espèces présentes sont E. multilocularis, qui a un cycle sylvatique et E. granulosus spp., dont les hôtes sont essentiellement domestiques.En France, peu de données étaient disponibles quant à la distribution de ces espèces parasitaires lors de la création du LNR Echinococccus spp. en 2006. Une forte réduction supposée des zones et du niveau d’enzootie pour E. granulosus laissait envisager un risque zoonotique moindre voire désormais absent. S’agissant d’E. multilocularis, l’expansion constatée en Europe était également suspectée en France. La modification des techniques de parasitologie classique ainsi que le développement d’outils moléculaires par le LNR ont contribué à l’actualisation et à une meilleure connaissance de la distribution des espèces parasitaires en France.Une large étude de surveillance menée chez le renard avec la méthode SSCT validée par le LNR a révélé une expansion de la zone d’enzootie d’E. multilocularis jusqu’en Ille-et-Vilaine. L’analyse par microsatellite EmsB a permis d’estimer à plusieurs décennies la dispersion du parasite vers l’ouest et le nord à partir du foyer historique de l’est. Cette expansion a été confirmée à l’ouest chez des rongeurs aquatiques et rétrospectivement au sud chez le renard.Des études de surveillance d’E. granulosus en abattoir ont confirmé sa présence dans le sud de la France et en Corse. Les caractérisations moléculaires ont permis d’identifier E. granulosus sensu stricto dans le sud et E. canadensis G6-7 en Corse. Un plan de surveillance national en abattoir a ensuite montré la présence d’E. granulosus s.s. à travers l’ensemble du territoire continental. Les niveaux de prévalence d’infestation par E. granulosus s.s. estimés chez les ovins à 15,3 et chez les bovins à 8,3 cas pour 1 million de têtes abattues sont très inférieurs à ceux décrits il y a vingt ans. Deux foyers majeurs constitués par les Alpes pour E. granulosus s.s. et la Corse pour E. canadensis G6-7 demeurent, alors que l’identification d’E. ortleppi confirme le maintien de cette espèce zoonotique pourtant désormais rare en Europe.Les niveaux de prévalence d’infestation par E. multilocularis chez le chien et le chat en France confirment leurs importances mineures dans le maintien du cycle parasitaire. Le rôle zoonotique du chat semble négligeable d’après les observations d’infestations naturelles, corroborées par les données obtenues lors d’infestations expérimentales. La vermifugation régulière et adaptée des chiens, en particulier des chiens de chasse, apparait nécessaire. Cette recommandation de vermifugation canine s’applique également pour E. canadensis G6-7 en Corse, où l’accès aux viscères des porcs et des sangliers implique un volet sylvatique dans le cycle.La reconnaissance de l’expertise du LNR a permis d’initier des collaborations internationales qui ont en retour enrichi la diversité des situations épidémiologiques étudiées. Ainsi, l’étude de la diversité génétique d’E. multilocularis (microsatellite EmsB) a contribué à une meilleure compréhension de la dynamique d’expansion du parasite en Europe (Pologne, Suède, Danemark). L’étude des foyers d’hyper enzootie d’E. granulosus (Moldavie, Maroc, Algérie) et la caractérisation des espèces présentes et de leurs niveaux d’infestation chez les hôtes intermédiaires a permis d’envisager des actions de lutte adaptées. Le LNR a participé à l’expérimentation de méthodes de lutte contre E. multilocularis, démontrant la complexité des conditions requises pour leur efficacité tant par la vermifugation des renards que par la régulation de leur population. Au bilan, les nouvelles données épidémiologiques obtenues au cours des dix années de travaux ont permis d’aboutir à une meilleure appréhension du risque zoonotique actuel lié aux échinocoques en France / Among parasites, Echinococcus species are of major public health importance due to their worldwide distribution and the potential severity of the diseases they cause. In Europe, the endemic species are E. multilocularis, which has a sylvatic lifestyle, and E. granulosus spp., which the hosts are mainly domestic species. When the Echinococcus spp. NRL was created in France in 2006, few data were available on the distribution of these parasitic species. The implementation of health measures made it possible to consider a marked reduction of the endemic level and geographical distribution of E. granulosus and consequently a reduced or inexistent zoonotic risk. With regard to E. multilocularis, the parasite spread already observed in Europe was also suspected in France. The development of classical parasitology and molecular techniques for diagnosis and epidemiology has helped improve understanding of the distribution of these parasitic species in France.A large-scale surveillance study in foxes, using the SSCT method validated by the NRL, led to the description of significant westward and northward expansion from E. multilocularis’s historical endemic focus, which was estimated, using a spatio-temporal scenario deduced by EmsB microsatellite analysis, to have begun several decades ago. Molecular analyses of various types of animal samples confirmed its westward expansion in aquatic rodents. Its extension southward was confirmed thanks to fecal samples of foxes.Surveillance studies of E. granulosus at the slaughterhouse confirmed its presence in southern France and in Corsica. The first molecular characterization of this parasite in France resulted in the identification of E. granulosus sensu stricto in southern France and E. canadensis G6-7 in Corsica. The presence of E. granulosus s.s. throughout continental France was then observed on the basis of a national surveillance study at the slaughterhouse. The prevalence level of infection by E. granulosus s.s. estimated per million was 15.3 cases in sheep and 8.3 cases in cattle which was much lower than that described 20 years ago. The two main endemic foci – the Alps for E. granulosus s.s. and Corsica for E. canadensis G6-7 – still exist, while the identification of E. ortleppi confirmed maintenance of this species despite its current rarity in Europe.In France, the low prevalence of E. multilocularis infection in dogs and cats was ascertained, confirming the minor contribution of these hosts to the lifecycle. The zoonotic role of the cat appears to be negligible based on observations of natural infection cases and on data obtained from experimental infection. The regular and adapted deworming of dogs, especially hunting dogs, appears to be necessary. This deworming recommendation is also relevant against E. canadensis G6-7 in Corsica where access to viscera of pigs and wild boar adds a sylvatic component at this lifecycle.An acknowledgment of the NRL’s expertise led to several international collaborations which in turn contributed to the diversity of the epidemiological situations studied. Thus, the study of the genetic diversity of E. multilocularis by EmsB microsatellite led to a better understanding of the parasite’s expansion dynamics throughout Europe. The study of foreign foci of E. granulosus led to characterization of the endemic parasitic species and of their prevalence levels in intermediate hosts, which made it possible to plan appropriate control measures. The NRL participated in the testing of control methods against E. multilocularis, including deworming of foxes and population regulation, which demonstrated the complexity of the conditions required for these methods to be effective.The epidemiological data obtained over ten years of studies has led to a better understanding of the current zoonotic risk associated with the Echinococcus species in France

Title from PDF of title page. / Document formatted into pages; contains 86 pages. / Thesis (M.S.P.H.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: The Enterovirus genus within the family Picornaviridae contains over 100 serotypes, of which sixty-four are known to be human pathogens. Infection with this group of RNA viruses produces a myriad of clinical conditions including poliomyelitis, meningitis, encephalitis, respiratory illnesses, and hand-foot-and-mouth disease. Outbreaks have been documented worldwide; significant morbidity and mortality exist to warrant laboratory surveillance. Traditionally, enteroviruses have been identified to the level of serotype by the serum neutralization assay. However, numerous problems are associated with this assay. The serum neutralization assay is labor intensive, results are often ambiguous, and reagents are becoming difficult to obtain. Recently, molecular-based typing protocols have been described that are cost effective and produce results that are more reliable. / ABSTRACT: The overall objective of this thesis was to implement a molecular-based typing protocol to replace the serum neutralization method currently used. Three specific aims were identified to reach this objective. First, a database cataloging all enteroviruses isolated at the Florida Department of Health - Tampa Branch Laboratory from 1981 through 2002 was created. Serotype prevalence, specimen submission rates, and temporal trends were analyzed to demonstrate the public health importance of enterovirus surveillance. Next, five oligonucleotide primer sets were compared with respect to sensitivity, specificity, and overall utility in molecular typing protocols developed to accurately determine enterovirus type. Finally, the most effective molecular assay was used to conduct two basic molecular epidemiological analyses of intratypic variation of Coxsackievirus B5 isolates, and of intratypic variation of successive Echovirus 9 passages. / ABSTRACT: The results from this study show that implementation of a molecular-based typing system for enteroviruses would be an improvement over current enterovirus serotyping methods. Results are obtained more rapidly and are more reliable. The implementation of such a system would improve the surveillance capabilities of the State of Florida Department of Health. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.

HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.