Pesquisas sobre Listeria monocytogenes em indústrias de produtos lácteos no Brasil são escassas. Três laticínios (A, B e C) produtores de queijos do Estado de São Paulo foram monitorados para a presença de L. monocytogenes no período de outubro/2008 a setembro/2009. Foram realizadas 12 coletas, correspondentes a 12 lotes de queijo produzidos, sendo quatro de cada laticínio. Em cada laticínio, as visitas foram realizadas com intervalos de aproximadamente 2 meses entre cada uma. Foram analisadas 393 amostras, sendo 201 de superfícies com e sem contato com alimentos e 192 de alimentos (leite cru e pasteurizado e queijo) água e salmoura, para pesquisa de L. monocytogenes. As análises foram realizadas de acordo com o método do Food and Drug Administration (FDA). Os resultados confirmam a presença de Listeria spp nas instalações dos três laticínios. L. monocytogenes, L. innocua, L. seeligeri e L. welshimeri foram as espécies isoladas neste estudo. Especificamente a espécie L. monocytogenes não foi encontrada no laticínio A, entretanto, o microrganismo foi isolado de 12,5% das amostras do laticínio B e de 9,1% do laticínio C. L. monocytogenes não foi isolada do leite cru dos silos, do leite pasteurizado, da água e dos queijos Minas frescal, nos 3 laticínios. Porém, no laticínio C, L. monocytogenes foi isolada de amostras de queijo Prato que foram incluídas apenas na 4ª coleta deste laticínio, além de ter sido isolada de amostras de salmoura. As maiores prevalências de contaminação por L. monocytogenes ocorreram em superfícies sem contato com alimentos, sendo positivas 51,6% das amostras do laticínio B e 21,7% do laticínio C. Em ambos os laticínios a bactéria também foi isolada de superfícies com contato com alimentos. Os resultados fornecem informações detalhadas dos pontos prioritários para o desenvolvimento de estratégias de controle de L. monocytogenes em laticínios e mostram a importância de programas de monitoramento ambiental do patógeno, mesmo em pequenas indústrias. Os 85 isolados identificados como L. monocytogenes revelaram-se de quatro sorotipos: 1/2a, 1/2b, 1/2c e 4b, com predomínio do 4b, em ambos os laticínios, o que é preocupante para a saúde pública. Com base nos resultados de PFGE (perfis combinados ApaI e AscI), 40 perfis (pulsotipos) foram obtidos. Pulsotipos foram isolados repetidamente entre coletas nos laticínios B e C, sugerindo persistência de linhagens nos laticínios. Apesar dos laticínios serem distantes e independentes, um pulsotipo foi compartilhado entre ambos. O laticínio A apresentou contaminação por mais de um pulsotipo de L. seeligeri e houve isolamento repetido de um pulsotipo dessa espécie, entre as coletas, sugerindo adaptação da bactéria e necessidade de controle do gênero Listeria nessa indústria. A ocorrência de um mesmo pulsotipo de L. monocytogenes com sorotipos diferentes (1/2b e 4b) mostra que a sorotipagem deve acompanhar análises mais refinadas como as de natureza genotípica. / Listeria monocytogenes surveys in cheese manufacturing plants in Brazil are rare. Three cheese manufacturing plants (A, B and C) in São Paulo state were monitored for the presence of Listeria monocytogenes during the period of October/2008 - September/2009. Twelve samples surveys were taken corresponding to 12 cheese lots produced, four in each plant. In each cheese plant, the samples were taken at intervals of approximately 2 months. There were 393 samples analyzed, 201 from surfaces with and without contact with food and 192 of food (raw and pasteurized milk and cheese), water and brine, with the objective of searching for L. monocytogenes. The analyses were performed in accordance with Food and Drug Administration (FDA) method. The results confirmed the presence of Listeria spp in the facilities of three plants. L. monocytogenes, L. innocua, L. seeligeri and L. weshimeri were the species isolated in this study. Specifically the L. monocytogenes specie was not isolated from plant A. However, the microorganism was isolated in 12.5% of the samples from plant B and 9.1% from plant C. Listeria monocytogenes was not isolated from raw milk in storage tanks, pasteurized milk, water or Minas frescal cheese samples from the three plants. Nevertheless, in plant C, L. monocytogenes was isolated in Prato cheese that was included only in the 4th sampling survey and also from the brine samples. The major prevalence of contamination by L. monocytogenes occurred on surfaces without contact with food, with 51.6% of the samples positive from plant B and 21.7% from plant C. In both plants, the microorganism was also isolated from food contact surfaces. The results provide detailed information about the critical points for the development of L. monocytogenes control strategies in cheese processing plants and, moreover, show the relevance of sampling programs of the pathogen, even in small cheese processing plants. The 85 isolates identified as L. monocytogenes were classified in four serotypes: 1/2a, 1/2b, 1/2c and 4b, with 4b dominating in both cheese plants, which is of concern to human health. On the basis of PFGE results (combined profiles ApaI and AscI), 40 profiles (pulsotypes) were found. Pulsotypes were isolated repeatedly among sampling surveys in plants B and C, suggesting persistence of lineages in the plants. Despite these plants being distant and independent, one pulsotype was shared between them. Plant A presented contamination by more than one pulsotype of L. seeligeri and there was a repetitive isolation of one pulsotype of this specie among samplings, suggesting adaptation of the bacterium and the need for control of the Listeria genus in this plant. The occurrence of one single pulsotype of L. monocytogenes with different serotypes (1/2b and 4b) show that serotyping should follow more refined analyses as the ones of genotypic nature.

The increasing infection of Cryptococcus species and Tricosporon asahii emerged in clinical patients who were immunocompromised. They usually induce lung, skin, brain and systemic infection. Morbidity and mortality of immunocompromised patients are higher than normal healthy people. Cryptococcus neoformans var. grubii ¡]serotype A¡^ infections were reported in clinical cases predominantly and they were isolated from birds¡¦ droppings in large amount. Cryptococcus neoformans var. gattii ¡]serotype B, C¡^ had a natural life in plants, especially Eucalypticus trees. Isolations from other trees were reported increasingly in the tropical and subtropical areas. Comparing to Cryptococcus species, Tricosporon asahii is the normal mycoses of soil. In this study, we performed an environmental investigation concerning Cryptococcus species and Tricosporon asahii in Southern Taiwan. 120 droppings of racing pigeons and 114 samples from Eucalypticus trees were obtained. The results revealed that 30 Cryptococcus neoformans were isolated from racing pigeons¡¦ droppings ¡]25%¡^, as well as 4 Cryptococcus laurentii ¡]3.3%¡^ and 2 Cryptococcus albidus ¡]1.7%¡^. In addition, 25 Tricosporon asahii ( 20.8% ) were isolated from droppings of racing pigeons. But, none of Cryptococcus species or Tricosporon asahii is isolated from Eucalypticus trees ¡]0%¡^. All of Cryptococcus neoformans isolated from pigeons¡¦ droppings were var. grubii ¡]serotype A¡^ and their drug susceptibility tests showed sensitive to Amphotericin B ¡]minimal inhibitory concentration ¡Ø0.25£gg/ml¡^ and Fluconazole ¡]minimal inhibitory concentration 2£gg/ml¡^ and Flucytosine ¡]minimal inhibitory concentration ¡Ø1£gg/ml¡^. To sum up, both Cryptococcus species and Tricosporon asahii were isolated from droppings of racing pigeons in our study, especially Tricosporon asahii in large amount. Opportunistic infection caused by these species should be given more attention to racing pigeons which have close contact with human . Intensive investigation and surveillance should be carried out in the future to provide an information for the control and prevention of diseases.

Pesquisas sobre Listeria monocytogenes em indústrias de produtos lácteos no Brasil são escassas. Três laticínios (A, B e C) produtores de queijos do Estado de São Paulo foram monitorados para a presença de L. monocytogenes no período de outubro/2008 a setembro/2009. Foram realizadas 12 coletas, correspondentes a 12 lotes de queijo produzidos, sendo quatro de cada laticínio. Em cada laticínio, as visitas foram realizadas com intervalos de aproximadamente 2 meses entre cada uma. Foram analisadas 393 amostras, sendo 201 de superfícies com e sem contato com alimentos e 192 de alimentos (leite cru e pasteurizado e queijo) água e salmoura, para pesquisa de L. monocytogenes. As análises foram realizadas de acordo com o método do Food and Drug Administration (FDA). Os resultados confirmam a presença de Listeria spp nas instalações dos três laticínios. L. monocytogenes, L. innocua, L. seeligeri e L. welshimeri foram as espécies isoladas neste estudo. Especificamente a espécie L. monocytogenes não foi encontrada no laticínio A, entretanto, o microrganismo foi isolado de 12,5% das amostras do laticínio B e de 9,1% do laticínio C. L. monocytogenes não foi isolada do leite cru dos silos, do leite pasteurizado, da água e dos queijos Minas frescal, nos 3 laticínios. Porém, no laticínio C, L. monocytogenes foi isolada de amostras de queijo Prato que foram incluídas apenas na 4ª coleta deste laticínio, além de ter sido isolada de amostras de salmoura. As maiores prevalências de contaminação por L. monocytogenes ocorreram em superfícies sem contato com alimentos, sendo positivas 51,6% das amostras do laticínio B e 21,7% do laticínio C. Em ambos os laticínios a bactéria também foi isolada de superfícies com contato com alimentos. Os resultados fornecem informações detalhadas dos pontos prioritários para o desenvolvimento de estratégias de controle de L. monocytogenes em laticínios e mostram a importância de programas de monitoramento ambiental do patógeno, mesmo em pequenas indústrias. Os 85 isolados identificados como L. monocytogenes revelaram-se de quatro sorotipos: 1/2a, 1/2b, 1/2c e 4b, com predomínio do 4b, em ambos os laticínios, o que é preocupante para a saúde pública. Com base nos resultados de PFGE (perfis combinados ApaI e AscI), 40 perfis (pulsotipos) foram obtidos. Pulsotipos foram isolados repetidamente entre coletas nos laticínios B e C, sugerindo persistência de linhagens nos laticínios. Apesar dos laticínios serem distantes e independentes, um pulsotipo foi compartilhado entre ambos. O laticínio A apresentou contaminação por mais de um pulsotipo de L. seeligeri e houve isolamento repetido de um pulsotipo dessa espécie, entre as coletas, sugerindo adaptação da bactéria e necessidade de controle do gênero Listeria nessa indústria. A ocorrência de um mesmo pulsotipo de L. monocytogenes com sorotipos diferentes (1/2b e 4b) mostra que a sorotipagem deve acompanhar análises mais refinadas como as de natureza genotípica. / Listeria monocytogenes surveys in cheese manufacturing plants in Brazil are rare. Three cheese manufacturing plants (A, B and C) in São Paulo state were monitored for the presence of Listeria monocytogenes during the period of October/2008 - September/2009. Twelve samples surveys were taken corresponding to 12 cheese lots produced, four in each plant. In each cheese plant, the samples were taken at intervals of approximately 2 months. There were 393 samples analyzed, 201 from surfaces with and without contact with food and 192 of food (raw and pasteurized milk and cheese), water and brine, with the objective of searching for L. monocytogenes. The analyses were performed in accordance with Food and Drug Administration (FDA) method. The results confirmed the presence of Listeria spp in the facilities of three plants. L. monocytogenes, L. innocua, L. seeligeri and L. weshimeri were the species isolated in this study. Specifically the L. monocytogenes specie was not isolated from plant A. However, the microorganism was isolated in 12.5% of the samples from plant B and 9.1% from plant C. Listeria monocytogenes was not isolated from raw milk in storage tanks, pasteurized milk, water or Minas frescal cheese samples from the three plants. Nevertheless, in plant C, L. monocytogenes was isolated in Prato cheese that was included only in the 4th sampling survey and also from the brine samples. The major prevalence of contamination by L. monocytogenes occurred on surfaces without contact with food, with 51.6% of the samples positive from plant B and 21.7% from plant C. In both plants, the microorganism was also isolated from food contact surfaces. The results provide detailed information about the critical points for the development of L. monocytogenes control strategies in cheese processing plants and, moreover, show the relevance of sampling programs of the pathogen, even in small cheese processing plants. The 85 isolates identified as L. monocytogenes were classified in four serotypes: 1/2a, 1/2b, 1/2c and 4b, with 4b dominating in both cheese plants, which is of concern to human health. On the basis of PFGE results (combined profiles ApaI and AscI), 40 profiles (pulsotypes) were found. Pulsotypes were isolated repeatedly among sampling surveys in plants B and C, suggesting persistence of lineages in the plants. Despite these plants being distant and independent, one pulsotype was shared between them. Plant A presented contamination by more than one pulsotype of L. seeligeri and there was a repetitive isolation of one pulsotype of this specie among samplings, suggesting adaptation of the bacterium and the need for control of the Listeria genus in this plant. The occurrence of one single pulsotype of L. monocytogenes with different serotypes (1/2b and 4b) show that serotyping should follow more refined analyses as the ones of genotypic nature.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Na região Norte, Roraima destaca-se como um dos Estados hiper-endêmicos para o dengue, com circulação dos quatro sorotipos nos últimos três anos, e com uma elevada incidência da doença na última década. Por outro lado, sua localização geográfica tem um papel importante na entrada de novos genótipos/sorotipos do dengue ao Brasil. O DENV-1, depois de uma breve incursão no estado nos anos de 1981-1982, foi reintroduzido no ano 2000, e tem sido um dos sorotipos mais isolados até o ano de 2011, porém, existem poucos dados que mostram a ocorrência de variabilidade genética ou de alguma evolução na composição genética deste sorotipo durante as infecções ocorridas nos diferentes anos. O objetivo deste trabalho foi realizar a caracterização molecular do gene do envelope de isolados de DENV-1 durante epidemias ocorridas no período de 2008 a 2010 no estado de Roraima. As amostras foram inoculadas em células da linhagem C6/36 de Aedes albopictus, e identificadas por imunoflorescência indireta e pela técnica de RT-Hemi-Nested-PCR. Para a obtenção de um amplicom da região do envelope, uma RT-PCR foi realizada com o uso de iniciadores específicos, gerando um produto com 1724 pb. Os amplicons foram sequenciados e as sequências consenso foram obtidas usando o programa Geneious v.5.5.4. Para realização da análise molecular, as sequências foram comparadas com sequências referência dos quatro sorotipos do vírus dengue, e dos cinco genótipos do DENV-1 de diferentes partes do mundo, disponíveis no GenBank. A reconstrução filogenética foi realizada por dois métodos, o de Máxima Verossimilhança e o Bayesiano. Todos os isolados foram agrupados no genótipo V do DENV-1. As sequências utilizadas neste estudo apresentaram de 99-100% de similaridade com cepas de DENV-1 de Países e Estado vizinhos, como República Bolivariana da Venezuela, Guiana Inglesa, Colômbia e Amazonas, e se agruparam na árvore filogenética formando um subclado com cepas destes países e com outros da região do Caribe, indicando, mais uma vez, Roraima, como possível via de entrada de sorotipos/genótipos de dengue no país, fato totalmente comprovado quando houve a entrada e dispersão do genótipo II do vírus DENV-4 no ano de 2010. As cepas brasileiras do genótipo V do DENV-1 formaram três linhagens distintas. Os isolados deste estudo (2008-2010) se agruparam na linhagem III, diferente da cepa que circulou no Estado no de 2001, a qual era pertencente à linhagem II. Ao comparar estas sequências, foram encontradas quatro substituições de aminoácidos, duas (E428 e E436) provavelmente neutras que não modificaram a conformação tridimensional do envelope, pois trocaram aminoácidos de características físico-químicas similares (Leucina x Valina; Isoleucina x Valina) e duas substituições (E227 e E338) com alta probabilidade de modificar o envelope (Prolina x Serina; Leucina x Serina). Estes resultados provavelmente indicam uma evolução adaptativa no DENV-1, ou entrada de uma nova linhagem ao Estado, enfatizando a importância do monitoramento molecular de cepas de DENV circulantes em determinada região, permitindo um melhor entendimento do vírus. / In northern Brazil, Roraima is highlighted as one of the dengue virus hyperendemic states, where the four serotypes has been circulating for the last three years and with a high incidence of this disease in the last decade. On the other hand, its geographic localization has an important part on the entry of new genotypes/serotypes of dengue in Brazil. DENV-1, after a short incursion in the state in years 1981 and 1982, was reintroduced in 2000 and it was one of most isolated serotypes until 2011. Nevertheless, there is little data that shows the occurrence of genetic variability or any evolution in genetic composition from this serotype on infections occurred in different years. The objective of this project was to perform a molecular characterization of the envelope gene isolated from DENV-1 on infections occurred between 2008 and 2010 in Roraima. The samples were inoculated in C6/36 cells from Aedes albopictus, and identified by indirect immunofluorescence and RT-Hemi-Nested-PCR techniques. To obtain an amplicom from the envelope region, it was made an RT-PCR test with specific primers, generating a product with 1724 bp. The amplicons were sequenced and the consensus sequences were obtained using the program Geneious V.5.5.4. For molecular analysis, the current sequences were compared to sequences from the four serotypes of dengue virus and also compared to five genotypes of DENV-1 from different parts of the world available on GenBank. The phylogenetic reconstruction was made by two methods, Maximum-likelihood and Bayesian. All of the isolates were grouped in genotype V of DENV-1. The sequences showed 99-100% of similarity with strains of DENV-1 from neighbor countries and states as the Bolivarian Republic of Venezuela, Guyana, Colombia and Amazonas, they were grouped on a phylogenetic tree forming a subclade with strains of these countries and Caribbean region, and besides indicating, once more, Roraima as a possible entry route of serotypes/genotypes of dengue in Brazil, fact totally proven in 2010 with the entry and dispersion of genotypes II of DENV-4. The Brazilian strains of genotype V of DENV-1 formed three distinct lineages. The isolates of this study (2008-2010) were grouped in lineage III, different from the strain that circulated in the state in 2001, which belonged to lineage II. When these sequences were compared, it was found four aminoacid substitutions, two of them (E428 e E436) probably neutral which did not change tridimensional conformation of the envelope, since amino acids with similar physicochemical characteristics were changed (Leucine x Valine; Isoleucine x Valine), and two substitutions (E227 e E338) with high probability of changing the envelope (Proline x Serine; Leucine x Serine). These results may indicate an adaptive evolution of DENV-1 or the entry of a new lineage in the State, emphasizing the importance of molecular monitoring strains of DENV in circulation in certain regions, allowing better understanding of the virus.

Abstract Chlamydia trachomatis epidemic continues at a slowly, albeit steadily increasing rate in the Western world despite health education, easy/user-friendly diagnostic measures, and effective treatment. In Finland, 8,031 and 13,227 C.trachomatis infections were reported in 1995 and 2012, respectively. Over half of the Chlamydia cases were diagnosed among young women, who suffer from the Chlamydia-related complications such as infertility many years after initial infection. The rates of all but first of the following major Chlamydia-related complications: cervical intraepithelial neoplasia, ectopic pregnancy, hospitalized pelvic inflammatory disease, tubal factor infertility have, however, decreased since the 1990s. The aim of this study was to clarify the discordance between the apparently increasing incidence of C. trachomatis and decreasing C.trachomatis IgG antibody rates (seroprevalence). The study material consisted of a random subsample of first trimester serum samples of 7,999 women from the population-based Finnish Maternity Cohort (FMC) registry from 1983 to 2005, and 147,148 women and men with a total of 177,138 C. trachomatis genital infections reported to the Finnish National Infectious Diseases Registry (NIDR) during 1995–2009. Both registries are maintained by the National Institute for Health and Welfare (THL). Serum IgG antibodies were measured by a C.trachomatis major outer membrane protein-specific peptide enzyme immunoassay (EIA) and the standard micro-immunofluorescence (MIF) method. We found that while C. trachomatis seroprevalences decreased >50% among fertile-aged women the seroconversion rates (seroincidences) were comparable to the NIDR reported rates. The numbers of annual repeated C. trachomatis infection in the NIDR increased until 2009 by 49% in women and 39% in men. In 2009, about 25% of the females and 20% of the males had had an earlier C.trachomatis infection. During the whole follow-up time, 34% of all the repeat diagnoses occurred within 12 months. Most of the first infections were observed among females and males under 25 years of age, but the numbers of repeated chlamydial infections increased up to the age of 30 years. The C. trachomatis serotype distribution changed between the 1980s and 1990s, but the leading 1980 serotypes bounced back by 2005. The numbers of women with multiple serotype infections peaked in the 1990s, and serotypes G and J were temporarily replaced by serotypes E and D. In conclusion, the serological observations fit the polymerase chain reaction (PCR) -based data on C. trachomatis epidemiology. The observed increases in the repeated chlamydial infections among young women and men comply with increasing sexual risk-taking behaviour in Finland. Our observations help to understand the discrepancy between C. trachomatis occurrence and sequelae rates as the overall C. trachomatis infection burden in the population may be decreasing despite the increasing incidence trend. / Tiivistelmä Chlamydia trachomatis -epidemia jatkuu yhä länsimaissakin terveyskasvatuksesta, helposta diagnostiikasta sekä tehokkaasta hoidosta huolimatta. Vuonna 1995 maassamme raportoitujen klamydiainfektioiden määrä oli 8 031 ja viime vuonna 13 227. Näistä yli puolet todettiin nuorilla naisilla, joilla klamydian pitkäaikaishaitat, kuten hedelmättömyysongelmat, tulevat esille usein vasta usean vuoden kuluttua. Klamydiainfektioon liittyvät komplikaatiot, eli kohdunkaulan syövän esiasteet, kohdunulkoinen raskaus, vaikea sisäsynnytintulehdus ja munanjohdinperäinen hedelmättömyys ovat ensimmäistä lukuun ottamatta olleet maassamme laskussa 1990-luvulta lähtien. Tämän väitöskirjatyön tarkoituksena oli selvittää klamydian ilmaantuvuuden nousun ja C. trachomatis IgG vasta-ainetasojen laskun välistä ristiriitaa. Väestötasoisen tutkimuksen aineisto koostui äitiseerumipankista (Finnish Maternity Cohort, FMC) vuosilta 1983–2005 satunnaistetusti poimittujen 7 999 naisen verinäytteistä ja kansalliseen tartuntatautirekisteriin vuosina 1995–2009 raportoiduista klamydiatapauksista, joita havaittiin 147 148 naisella ja miehellä yhteensä 177 138 tapausta. Molemmat rekisterit ovat Terveyden ja hyvinvoinnin laitoksen (THL) ylläpitämiä. Klamydia IgG vasta-aineet määritettiin entsyymi-immunologisesti (EIA) sekä mikroimmunofluoresenssimenetelmällä (MIF) verinäytteistä. Keskeisinä tuloksina havaittiin klamydian seroprevalenssin lasku > 50 % hedelmällisessä iässä olevilla naisilla, vaikka infektion ilmaantuvuus serologisessa aineistossa oli samaan aikaan samankaltainen kuin tartuntatautirekisterissä. Lisäksi havaitsimme, että vuositasolla toistuvien klamydiainfektioiden määrä on tartuntatautirekisterin perusteella lisääntynyt tutkimusaikana naisilla 49 % ja miehillä 39 %. Vuonna 2009 neljännes naisten ja viidennes miesten klamydiainfektioista oli uusintainfektioita. Koko seuranta-aikana 34 % toistuvista tapauksista ilmaantui 12 kuukauden sisällä. Suurin osa klamydiainfektioista todettiin alle 25-vuotiailla naisilla ja miehillä, mutta toistuvia infektioita havaittiin vielä runsaasti 30 ikävuoteen asti. Klamydiaserotyyppien jakaumassa tapahtui muutoksia 1980- ja 1990-lukujen välissä, mutta jakauma palautui vuoteen 2005 mennessä samankaltaiseksi kuin 1980-luvulla. Useamman serotyypin infektioiden lukumäärä oli korkeimmillaan 1990-luvulla, jolloin serotyypit G ja J korvautuivat väliaikaisesti serotyypeillä E ja D. Yhteenvetona voimme todeta, että serologiset havaintomme ovat yhteneväisiä polymeraasiketjureaktiolla (PCR) tutkittujen klamydiaepidemiologisten tulosten kanssa. Toistuvien klamydiainfektioiden määrän nousu aineistossamme sopii havaintoon nuorten naisten ja miesten lisääntyneestä seksuaalisesta riskikäyttäytymisestä maassamme. Tutkimustuloksemme auttavat ymmärtämään ristiriitaa klamydian ilmaantuvuuden nousun ja klamydiaperäisten komplikaatioiden laskun välillä, koska kaiken kaikkiaan klamydiaan liittyvä pitkäaikaissairastavuus väestötasolla voi olla vähenemässä.

La cryptococcose neuroméningée (CNM) est la seconde infection opportuniste chez les patients infectés par le VIH. Il s’agit de la 4ème cause de décès dus aux maladies infectieuses en Afrique avec une mortalité annuelle de 600.000 cas. Les levures responsables appartiennent au complexe d’espèces Cryptococcus neoformans / C. gattii. Notre étude décrit, l’épidémiologie et la résistance aux antifongiques de souches environnementales et cliniques de cryptocoques en Côte d’Ivoire. Les isolats sont issus d’une file active de 1750 PVVIH et de 667 prélèvements réalisés dans l’environnement de vie des patients. Nous démontrons une grande diversité génotypique au sein de notre cohorte, la présence de plusieurs espèces de cryptocoques dans un seul prélèvement chez un même patient ainsi que dans des prélèvements issus de suivi de patients, ce qui n’avait jamais été démontré en Afrique de l’Ouest. Nous avons constaté que la récurrence de la CNM est due à des infections multiples par des souches différentes au cours du temps. Nos résultats décrivent également pour la première fois, l’isolement de cryptocoques à partir de fientes de pigeons à Abidjan. Et nous constatons que les génotypes des isolats environnementaux et cliniques sont très différents, ce qui exclut les fientes de pigeons comme source de contamination des patients dans notre échantillon. Enfin, la majorité des isolats est sensible aux antifongiques de référence mais un patient peut être contaminé par des isolats de sensibilité différente. / Cryptococcal meningitis (CM) is the second opportunistic infection in HIV infected patients. It is the fourth cause of death due to infectious diseases in Africa with an annual mortality of 600,000. The yeasts responsible belong to the C. neoformans / Cryptococcus gattii species complex. Our study describes epidemiology and resistance to antifungal of environmental and clinical strains of Cryptococcus in Ivory Coast. The isolates are from an active list of 1,750 patients VIH positive and 667 samples taken in the living environment of patients. We demonstrate a high genotypic diversity within our cohort and the presence of several species of Cryptococcus in one sample from the same patient as well as in samples from patients follow up, which had never been shown in West Africa. We found that the recurrent cryptococcosis is caused by multiple infections by different strains over time. Our results describe also, for the first time, the isolation of Cryptococcus from pigeon droppings from Abidjan. And we notice that, as the genotypes of environmental and clinical isolates are very different, that excludes contamination of patients by pigeon droppings. Finally, most of the isolates were susceptible to reference antifungal but a patient might be contaminated by isolates with different susceptibility.

There is a rise in prevalence of antibiotic resistance in Streptococcus pneumoniae, and its FDA-approved vaccines often do not mount effective immune responses in children, the elderly, or the immunocompromised. One reason these vaccines are generally less effective is because they do not utilize T-cell help. T-cell help can be accessed when di-, tri-, or tetra-saccharides positioned inside major histocompatibility complex (MHC) II are presented to T-cell receptors as a target antigen. Pairing MHC II-antigen complexes with T-cell receptors enables development of B and T lymphocytes that are highly specific to these antigens, granting an increase in antibody affinity and cell memory. One problem with today's vaccines against S. pneumoniae, in contrast, is that extracted, polymeric sugars cannot be presented to T-cells by MHC because they do not fit inside the MHC II complex due to their large molecular size. Thus, FDA-approved vaccines generate antibodies which have inadequate affinities and are largely nonspecific in their targets. This thesis covers the synthesis of a functionalized S. pneumoniae serotype 6A disaccharide and trisaccharide, which are core components of the repeating unit of natural capsular polysaccharides, and can be used to obtain necessary T-cell help in working vaccines and good monoclonal antibodies.

The rise in antibiotic resistant strains of bacteria has led to the need for new methods of combatting bacterial infection. Since the surfaces of bacteria are covered in uniquely patterned capsular polysaccharides, vaccines targeting these polysaccharides have become a popular field of research for protection against pathogenic bacteria. Though licensed polysaccharide vaccines are commercially available, they lack the efficiency to protect patients that are immunocompromised or at high risk because they elicit T cell independent immune responses, resulting in low-affinity antibodies. To elicit a T cell dependent response and thereby recruit B cells that produce high-affinity antibodies, a vaccine was developed consisting of a short target oligosaccharide antigen of no more than four carbohydrate units, a virus-like particle that the antigen is conjugated to, and an NKT cell adjuvant that recruits T cell help. Previously the vaccine utilized in this work successfully elicited the T cell dependent adaptive immune response desired, and B cells were isolated producing high-affinity antibodies to our specific targets. The previous results were from using tetrasaccharide antigens for serotypes 14 and 3 of Streptococcus pneumonia (Sp). To further optimize this vaccine, disaccharides were tested as minimal epitopes for high-affinity antibody production to specific serotypes of Sp. Four disaccharides were synthesized for serotypes 3, 4, 7F, and 9v, one for each serotype. The synthesis of each disaccharide is described. These disaccharides were tested using the vaccine platform we developed. Reactive B cells were isolated producing high-affinity antibodies to the serotype 3 disaccharide antigen. For the other disaccharides only weak antibody responses were observed.

Botulinum Neurotoxin (BoNT) is one of the most potent toxins known to human kind. The Atomic Force Microscope (AFM) was employed to investigate the conditions under which BoNT type A heavy chain would bind and/or insert into mica supported dipalmitoylphosphatidylcholine (DPPC) lipid bilayers. As an alternate technique, DPPC/GT1b or total ganglioside extract (80:20) monolayers of a Langmuir Blodgett (LB) Trough were adapted to be artificial membrane models for toxin insertion studies. We conclude that LB monolayer studies are a promising candidate for BoNT/A membrane insertion investigation. Botulinum neurotoxin serotype A insertions into the LB monolayers in the presence of BoNT/A low affinity ganglioside receptor alone, independent of pH. This thermodynamic evidence indicates that BoNT/A may begin its heavy chain insertion into the cholinergic nerve ending before endocytosis and acidification.

Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.