Made available in DSpace on 2016-04-28T20:16:21Z (GMT). No. of bitstreams: 1 2007- Franziska Huber.pdf: 2677706 bytes, checksum: 65e703599b63ae016e9aa85d1752e357 (MD5) Previous issue date: 2007-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The objectives of the present study was the genetical characterizations of Cryptosporidium spp. from different hosts, realize the sequencing an phylogenetic analysis, including the deposit in GenBank of the first Cryptosporidium sequences of animal origin, from Brazil. There were obtained fecal samples, containing Cryptosporidium oocysts from chiken, ducks, quails and Guinea pigs from a public market localized in Rio de Janeiro city, from dairy calfs maintained at a farm localized in the same city and from dogs and cats maintained at a shelter localized in the city of Nova Igua?u. For the analysis was utilized the Nested-PCR of the extracted DNA from 200μl of fecal suspension. For primary identification of Cryptosporidium species was realized RFLP with enzymes SspI and VspI. DNA samples were sequenced and phylogenetic analysis were conducted. There were diagnosed and sequenced C. baileyi infecting two ducks (DQ855339 and DQ885340) and one quail (DQ885335) and C. melagridis infecting one chicken (DQ885341). The sequences obtained form Cryptosporidium infecting Guinea pigs received accession numbers DQ885337 and DQ885338, both sequences were not identified with known Cryptosporidium species due to the great genetic distance between them and those already available at GenBank, suggesting that it may be a new genotype or species. Parasitizing cats was diagnosed C. felis (DQ885336) and in one dog C. canis (DQ885334). One sample of C. parvum of calf origin was sequenced and received accession number DQ885333. During analysis of RFLP pattern of the nested- PCR product from 18Sr DNA was stated that only C. baileyi has a characteristic digestion pattern. Other Cryptosporidium species should be digested by several other enzymes, for a accurate diagnosis. At phylogenetic analysis was found a greater genetic distance between C. felis and C. canis from Brazil when compared to the reference sequences obtained from GenBank. Based on the phylogenetic groupings, a possible new species of Cryptosporidium from Guinea Pigs calls attention for the existence of new species even in common pet animals. As is the case of the Guinea Pig. The sequences obtained in this study are the first Brazilian sequences of C. baileyi, C. meleagridis, C. felis, C. canis and C. parvum deposited in GenBank. / O presente trabalho teve por objetivo caracterizar geneticamente as esp?cies de Cryptosporidium oriundos de v?rios hospedeiros, realizar o seq?enciamento e an?lises filogen?ticas, incluindo o dep?sito das primeiras seq??ncias brasileiras de Cryptosporidium spp. de origem animal no GenBank. Foram obtidas amostras fecais contendo oocistos de Cryptosporidium de pintos, patos, codornas e porquinhos da ?ndia comercializados num mercado municipal da cidade do Rio de Janeiro, de bezerros de uma propriedade voltada ? produ??o leiteira localizada no mesmo munic?pio e de gatos e c?es de um abrigo para animais localizado no munic?pio de Nova Igua?u. Para as an?lises foi utilizado Nested-PCR do DNA extra?do a partir de 200μl de solu??o fecal. Foi realizada RFLP dos produtos obtidos no Nested-PCR, utilizando-se as enzimas SspI e VspI, para uma identifica??o preliminar das esp?cies de Cryptosporidium presentes. As amostras de DNA foram seq?enciadas e an?lises filogen?ticas foram conduzidas. Foram diagnosticados e sequenciados C. baileyi infectando dois patos (DQ855339 e DQ885340) e uma codorna (DQ885335) e C. melagridis infectando um pinto (DQ885341). As seq??ncias dos Porquinhos da ?ndia receberam os n?meros de acesso DQ885337 e DQ885338, sendo que ambas as seq??ncias n?o puderam ser identificadas como esp?cie conhecida de Cryptosporidium, devido ? grande dist?ncia gen?tica entre elas e aquelas j? depositadas no GenBank, sugerindo que se trate de um gen?ptipo ou esp?cie nova. Parasitando os gatos foi diagnosticado C. felis (DQ885336) e em um c?o C. canis (DQ885334). Uma das amostras de C. parvum de bovinos foi seq?enciada, sendo depositada no GenBank sob n?mero de acesso DQ885333. Durante as an?lises dos s?tios de corte enzim?tico dos produtos da Nested-PCR do gen 18Sr DNA, a ?nica esp?cie que realmente possue padr?o de corte caracter?stico ? C. baileyi. As demais esp?cies de Cryptosporidium deveriam ser submetidas ? a??o de outras enzimas, para um diagn?stico acurado. Nas an?lises filogen?ticas foi observada uma dist?ncia gen?tica maior entre C. felis e C. canis isolados no Brasil quando comparados ?s seq??ncias do GenBank. Com base nos dados apresentados pelo agrupamento filogen?tico, uma poss?vel nova esp?cie chama a aten??o ? presen?a de esp?cies desconhecidas de Cryptosporidium, mesmo em animais comuns de estima??o, como ? o caso do Porquinho da ?ndia. Estas s?o as primeiras seq??ncias de C. baileyi, C. meleagridis, C. felis, C. canis e C. parvum do Brasil depositadas no GenBank.

Made available in DSpace on 2017-07-10T17:37:22Z (GMT). No. of bitstreams: 1 Ana_Paula_G_S_Wengrat.pdf: 2096466 bytes, checksum: eab7f1fa4bfc4b23b72f4b2bcd8697d8 (MD5) Previous issue date: 2016-08-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Species of Vatiga (Hemiptera: Tingidae) are pests of Cassava, Manihot esculenta Crantz (Euphorbiaceae), an important food source for more than 800 million people. Vatiga infestations in Cassava can cause serious production losses. Although importance economic, there are few research work about morphological description this species, relevant item in pest identification at the beginning of infestation and for use in phylogenetic analysis. Furthermore, not many records occur of Vatiga species and resistant genotypes to prague. That way, has been described and illustred the external morphology all the phases of V. illudens life, evaluated at resistance of five genotypes of cassava at this species and raising the occurence of insects of the genus Vatiga in Cassava comercial crops. About morphology, the eggs´s whitish, oblong-shaped and deposited inside of leaf. Nymphs, the dorsal surface of the head and body side margins have spikes; from the second to fifth instar the cephalic and lateral spikes become more visible, but in least amount that V. manihotae. Genotypic resistance showed that insects supplied Mecu 72 genotype had affected their development, thereby increasing nymphal and eggs-adults period, reducing significantly the fertility. The occurrence of the species recorded four species of Vatiga / As espécies de Vatiga (Hemiptera: Tingidae) são pragas da cultura da mandioca, Manihot esculenta Crantz (Euphorbiaceae), uma importante fonte de alimento para mais de 800 milhões de pessoas. Infestações de Vatiga em mandioca podem causar graves perdas de produtividade. Embora apresente importância econômica, são raros os trabalhos de descrição morfológica desta espécie, ítem relevante na identificação da praga já no inicio das infestações e para utilização em análises filogenéticas. Além disso, são escassos os registros de ocorrência das espécies de Vatiga e de genótipos resistentes à praga. Assim, foi descrita e ilustrada a morfologia externa de todas as fases de vida de V. illudens, avaliada a resistência de cinco genótipos de mandioca a esta espécie e levantada a ocorrência das espécies do gênero Vatiga em cultivos comerciais de mandioca. Acerca da morfologia, os ovos são esbranquiçados, oblongos e depositados no interior do tecido vegetal. Nas ninfas, a superfície dorsal da cabeça e margens laterais do corpo apresentam espinhos; a partir do segundo ao quinto ínstar os espinhos cefálicos e laterais tornam-se mais visíveis porém, em menor quantidade em relação à V. manihotae. Verificou-se que os insetos alimentados com o genótipo Mecu 72 tiveram seu desenvolvimento afetado, aumentando o período ninfal e de ovo-adulto e reduzindo significativamente a fecundidade. A ocorrência das espécies registrou quatro espécies de Vatiga

Orientador: André Rodrigues dos Reis / Resumo: O selênio (Se) é um nutriente para humanos e animais e um elemento benéfico para as plantas, sua baixa concentração nos solos do Brasil pode gerar deficiência nos animais e humanos. O fitato é a principal forma de reserva de fósforo (P) encontrado nas sementes de plantas, sendo considerado um “anti-nutriente” por formar complexos não digeríveis com nutrientes como Fe, Ca e Zn. Desta forma, existe a necessidade de se buscar alternativas para aumentar os teores de Se e reduzir o teor de fitato nas partes comestíveis de cultivares modernos. O objetivo do trabalho foi avaliar doses e fonte ótimas de Se a serem aplicadas em condições brasileiras, bem como a influência do Se na produção e qualidade nutricional em 29 genótipos de feijão-caupi. Para isso, foram desenvolvidos dois experimentos a seguir: Experimento 1: Foi avaliada a eficiência da biofortificação agronômica utilizando 2 fontes de Se (selenato e selenito) e 7 doses de Se (0; 2,5; 5,0; 10,0; 20,0; 40,0 e 60 g ha-1) aplicados via solo, foi realizada analise de fitatos nos grãos. Experimento 2: Foi realizado um experimento para caracterizar a absorção e acúmulo de Se, teor de fitatos, açucares, proteínas de reversa e amino ácidos nos grãos de diferentes genótipos de feijão-caupi, nesse experimento, 29 genótipos foram avaliados na presença e ausência de Se (0 e 25 kg ha-1), cultivados até o final do ciclo para a obtenção dos grãos. No experimento 1, observou-se que a aplicação de selenato proporciona maiores concentrações d... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Reptiles exhibit marked diversity in sex-determining mechanisms. Many species exhibit genotypic sex determination (GSD) with male heterogamety (XX females/XY males), others have GSD with female heterogamety (ZW females/ZZ males), and still others exhibit temperature-dependent sex determination (TSD). The distribution of these mechanisms throughout the reptile phylogeny implies evolutionary lability in sex determination, and in some lineages there has been a number of transitions between GSD and TSD. Despite this diversity, GSD and TSD have traditionally been viewed as mutually-exclusive mechanisms of sex determination in reptiles, since there is little evidence for their co-occurrence. Considerable empirical and theoretical effort has been directed towards understanding the adaptive significance of TSD in reptiles. In comparison, there has been little focus on understanding how evolutionary transitions between GSD and TSD occur at a genetic and mechanistic level. I addressed this question by applying both empirical and theoretical approaches to investigate interaction of genotypic and temperature influences in the sex determination of two endemic species of Australian lizards. The three-lined skink, Bassiana duperreyi, has XX/XY chromosomal sex determination, yet a previous investigation reported a significant male bias in the sex ratio of eggs incubated at low temperatures. To enable an explicit test for temperature induced sex reversal in this species, a 185 bp Y chromosome marker was isolated by Amplified Fragment Length Polymorphism (AFLP) analysis. The marker was subsequently converted into a duplex PCR assay that co-amplified a 185 bp (or 92 bp) Y chromosome fragment and a 356 bp fragment of the single-copy nuclear gene C-mos (from both sexes) as a positive control. The accuracy of the PCR sex assay was tested on 78 individuals for which sex reversal was not expected. PCR genotype and sex phenotype were concordant for 96% of the animals. This is one of the very few sex tests developed for a reptile, and the first report of Y chromosome sequence from a reptile. The PCR assay was subsequently applied to genotype hatchlings from both cool (16-7.5C) and warm (22-7.5C) cyclical incubation temperature treatments, and identified sex reversal in 15% of genotypically female (XX) embryos (n=26) from the cool treatment, but no sex reversal in eggs from the warmer treatment (n=35). Thus, low incubation temperatures can over-ride genotypic sex determination in B. duperreyi, indicating that GSD and TSD co-occur in this species. The Central bearded dragon, Pogona vitticeps (Agamidae), has ZZ/ZW chromosomal sex determination, and is a member of a lizard family in which GSD and TSD are both widespread, indicating evolutionary lability in sex determination. AFLP analysis was applied to isolate homologous Z and W chromosome-linked markers (71 bp and 72 bp, respectively) from this species. The AFLP sequences were subsequently extended into larger genomic fragments by a reiterated genome walking procedure, producing three non-overlapping contigs of 1.7 kb, 2.2 kb and 4.5 kb. The latter two fragments were verified as distinct, homologous Z/W chromosome fragments by PCR analyses. An amplified 3 kb fragment of the 4.5 kb contig was physically mapped to metaphase spreads, identifying the W microchromosome, and for the first time in this species, the Z microchromosome. PCR analyses indicated the presence of homologous sequences in other Australian agamid species, including both GSD and TSD species. The isolated sequences should therefore prove useful as a comparative genomic tool for investigating the genomic changes that have occurred in evolutionary transitions between sexdetermining mechanisms in agamids, by enabling the identification of chromosomes in TSD species that are homologous to the sex chromosomes of P. vitticeps. The isolated sequences were further converted into a duplex DNA sex assay that co-amplified a 224 bp W chromosome fragment and a 963 bp positive control fragment in both sexes. This PCR assay diagnosed chromosomal sex in three Pogona species, but was not effective outside the genus. Incubation treatment of P. vitticeps eggs revealed a strong and increasing female bias at high constant temperatures (34-36C), but an unbiased sex ratio between 22-32C. Hatchlings from three clutches split between 28C and 34 or 36C incubation treatments were genotyped with the W chromosome AFLP marker. At 28C, the sex ratio was 1:1 but the high temperature treatments produced 2 males and 33 females. All but one of the 30 lizards (97%) incubated at 28C had concordant sex phenotype and genotype, but only 18 of 35 animals (51%) from the high temperature treatment were concordant. All discordant animals were genotypic males (ZZ) that developed as females. Thus, temperature and genotypic influences can interact to determine sex in P. vitticeps. These empirical findings for B. duperreyi and P. vitticeps were extended into a novel theory for the evolution of sex-determining mechanisms in reptiles, working within the framework that species with temperature-induced reversal of chromosomal sex determination are a window to transitional stages of evolution between GSD and TSD. A model was derived from the observation that in both lizards, an extreme of incubation temperature causes sex reversal of the homogametic genotype. In this model, the strength of a genetic regulatory signal for sex determination must exceed a threshold for development of the homogametic sex to occur (male in Pogona, female in Bassiana). The strength of this signal is also temperature-sensitive, so diminishes at extremes of temperature. Simulation modelling demonstrated that increasing the relative magnitude of the threshold for sexual development can cause evolutionary transitions between GSD and TSD. Even more remarkably, decreasing the relative magnitude of the threshold value causes an evolutionary transition between female and male heterogametic GSD. Quantitative adjustment of a single model parameter (the threshold value) thus charts a continuous evolutionary pathway between the three principal mechanisms of sex determination in reptiles (XX/XY-ZZ/ZW-TSD), which were previously considered to be qualitatively distinct mechanisms. The experimental demonstration of temperature-induced reversal of chromosomal sex determination in both B. duperreyi and P. vitticeps presents a challenge to the traditional view that reptilian sex determination is strictly dichotomous (GSD or TSD), and suggests instead that sex determination in reptiles consists of a continuum of systems of interaction between genotypic and temperature influences. Simulation modelling provided solid theoretical support for this proposition, demonstrating that transitions along this continuum are effected simply through shifts in the mean population value for the sex-determining threshold, without requiring substantial genotypic innovation. An important implication of this theory is that transitions between XX/XY and ZZ/ZW modes of GSD may retain the same sex chromosome pair, and the same primary sexdetermining gene, in contrast to previous models for heterogametic transitions. A more immediate implication of these findings is that many reptile species believed to have strict TSD (in particular, lizards and crocodilians), may in fact have a sex-determining system of GSD-TSD interaction, where there is an equilibrium between GSD and TSD individuals within the population.

Για περισσότερο από έναν αιώνα, οι εντερόκοκκοι προβληματίζουν τους ερευνητές ως προς την ταξινόμηση, τη λοιμογόνο ικανότητα, την επιδημιολογία και την ανθεκτικότητα τους στα αντιβιοτικά, αλλά και ως προς τη διασπορά τους στο περιβάλλον, αφού αυτή συνδέεται άμεσα με τη Δημόσια Υγεία. Οι εντερόκοκκοι, αποτελούν μέρος της φυσιολογικής εντερικής χλωρίδας ανθρώπων και ζώων και μπορούν να επιβιώσουν στο υδάτινο περιβάλλον για μεγάλο χρονικό διάστημα, κάτω από αντίξοες περιβαλλοντικές συνθήκες. Στον άνθρωπο προκαλούν βακτηριαιμία, ενδοκαρδίτιδα, λοιμώξεις του ουροποιητικού και άλλων συστημάτων. Μέσο διασποράς τους μπορεί να είναι και το περιβάλλον, γεγονός που καθιστά τη μελέτη της οικολογίας τους και την επιδημιολογική τους επιτήρηση ζωτικής σημασίας. Κατά τη χρονική περίοδο 10/2009-7/2010 στο νομό Αχαΐας, από 2115 δείγματα θαλάσσιου, επιφανειακού και πόσιμου ύδατος, 168 δείγματα βρέθηκαν θετικά για παρουσία εντερόκοκκων μετά από καλλιέργεια σε SB agar, δοκιμασία υδρόλυσης εσκουλίνης, αρνητική αντίδραση καταλάσης, ανάπτυξη σε 6,5% NaCl και βιοτυπία με Vitek. Επιπλέον, προσδορίστηκε η αντοχή στα αντιβιοτικά Penicilin, Erythromycin, Vancomycin, Teicoplanin, Chloramphenicol, Ciprofloxacin και Quinupristin/Dalfopristin. Ακολούθησε Pulsed-Field Gel Electrophoresis για την τυποποίηση των στελεχών σε κλώνους. Τέλος, πραγματοποιήθηκε συσχέτιση με αντίστοιχα κλινικά στελέχη ασθενών από το ΠΓΝΠ. Τα 121 από τα 168 δείγματα ανήκουν στο γένος των εντεροκόκκων, σύμφωνα με τον βιοχημικό φαινότυπο (50 E. faecalis, 43 E. faecium, 10 E. villorum, 9 E. gallinarum, 5 E. casseliflavus και 4 E.durans). To 85% των εντεροκόκκων ήταν ευαίσθητα στην Ρenicillin, το 17% στην Erythromycin, το 95% στην Vancomycin, το 100% στην Teicoplanin, το 70% στην Chloramphenicol και το 1% στην Ciprofloxacin. Συνολικά ταυτοποίηθηκαν σε κλώνους 76 από τα 105 στελέχη. Τα E. faecalis σε 2 ομάδες, τα E. faecium σε 5, τα E. gallinarum σε 3, τα E. villorum σε 1, τα E. casseliflavus σε 1 και τα E.durans σε 1. / For more than a century, there is great concern about enterococci, regarding the classification, pathogenicity, epidemiology and resistance to antibiotics, but also the distribution into the environment, since this fact is directly linked to Public Health. Enterococci are part of the normal intestinal flora of humans and animals and can survive in the aquatic environment for long periods under adverse environmental conditions. In humans, enterococci are leading causes of bacteraemia, endocarditis and urinary tract infections.. The environment is involved in their distribution, which makes the study of their ecology and the epidemiological surveillance of vital importance. During the period 10/2009-7/2010, in the geographic area of the prefecture of Achaia, 2115 samples from marine, surface and drinking water were collected and 168 of them were positive for the presence of enterococci according to inoculation in SB agar, bile-aesculin agar, negative catalase reaction, growth in 6,5% NaCl and biotyping with Vitek. Moreover, resistance to Penicillin, Erythromycin, Vancomycin, Teicoplanin, Chloramphenicol, Ciprofloxacin and Quinupristin / Dalfopristin was determined. Pulsed-Field Gel Electrophoresis was applied for clonal identification. Finally, a correlation with corresponding clonal types isolated from patients hospitalised at the University Hospital of Patras was performed. One hundrend and twenty-one out of 168 isolates were identified as enterococci, according to their biotypes (50 E. faecalis, 43 E. faecium, 10 E. villorum, 9 E. gallinarum, 5 E. casseliflavus and 4 E.durans). 85% of enterococci were sensitive to Penicillin, 17% to Erythromycin, 95% to Vancomycin, 100% to Teicoplanin, 70% to Chloramphenicol, and 1% to Ciprofloxacin. Overall 76 of the 105 strains were grouped. E. faecalis strains were classified into two PFGE types, E. faecium into five, E. gallinarum into three, E. villorum into one, E. casseliflavus into one and E.durans into one.

Amebas de vida livre (AVL) do gênero Acanthamoeba estão amplamente distribuídas no ambiente e podem tornar-se amebas patogênicas ao homem. O objetivo deste trabalho foi isolar em de água de torneira amebas de vida livre do gênero Acanthamoeba, identificá-las e classificá-las. Um total de 132 amostras de água de torneira foi coletado de escolas estaduais e municipais entre os meses de março a novembro de 2009. As amostras passaram pelo processo de filtração e as membranas foram semeadas em ágar não-nutriente 1,5% coberto por uma suspensão de E. coli inativadas pelo calor. Todas as amostras positivas para AVL foram submetidas à clonagem celular e identificadas como pertencentes ao gênero Acanthamoeba, através da morfologia dos cistos e trofozoitos e pela PCR utilizando oligonucleotídeos gênero-específicos que amplificam a região ASA.S1 do gene 18S rDNA. Ensaios fisiológicos de termo e osmotolerância foram utilizados para avaliar a patogenicidade dos isolados. Vinte sete isolados foram positivos para AVL e 10 foram identificados como pertencentes ao gênero Acanthamoeba tanto pelas características morfológicas quanto pela análise molecular. Destes, nove isolados apresentaram características do grupo II e um do grupo III, segundo Pussard e Pons (1977). A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição no grupo genotípicos T2 (40%), T2/T6 (40%), T6 (10%) e T4 (10%). Nos ensaios de termotolerância e osmotolerância 50% dos isolados obtiveram um baixo potencial patogênico. Os resultados indicaram a presença do gênero Acanthamoeba em água tratada no estado do RS, revelando sua importância epidemiológica e a necessidade de mais estudos para determinar sua distribuição no ambiente e seu potencial patogênico. / Free-living amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can become human pathogenic amoebae. The aim of this study was to isolate from tap water in free-living amoebae of Acanthamoeba, identify them and then classify them. A total of 132 samples of tap water was collected from state and municipal schools between march and november 2009. The samples passed through the filtration process and the membranes were seeded in non-nutrient 1.5% covered by a suspension of E. coli heatinactivated. All samples of AVL were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites by PCR using primers and genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Tests of physiological thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. Twenty seven isolates of AVL and 10 were identified as belonging to the genus Acanthamoeba through the morphological and molecular analysis. Nine of the isolates showed characteristics of group II and one isolate showed characteristics of group III, according Pussard and Pons (1977). The sequencing analysis by comparing the sequences submitted to GenBank, showed that genotype distribution in group T2 (40%), T2/T6 (40%), T6 (10%) and T4 (10%). In tests of thermotolerance and osmotolerance 50% of isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in the RS, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential.

Introdução: Resistência aos antirretrovirais pode limitar opções de tratamento, principalmente em pacientes com acúmulo de falhas terapêuticas, o que pode comprometer resultados clínicos. Objetivos: caracterizar o perfil de mutações na transcriptase reversa e protease do HIV-1 de pacientes em falha ao tratamento. Secundariamente, avaliar associação entre mutações e número de falhas terapêuticas, associação entre mutações e subtipos do HIV-1 e apresentar a evolução temporal da prevalência dos subtipos do HIV-1, no estado do Pará, ao Norte do Brasil. Método: Estudo transversal, no qual se avaliam genotipagens, entre janeiro de 2004 a dezembro de 2013, com dados obtidos de formulário de solicitação do exame padronizado pela RENAGENO e de impressos dos resultados, ambos arquivados em quatro serviços de atendimento especializados. Foram incluídos os testes realizados por laboratório da RENAGENO, em maiores de 18 anos, e o primeiro exame daqueles que o realizaram em mais de um momento, totalizando 377 amostras. As mutações são descritas de acordo com o banco de dados de resistência do HIV da Universidade de Stanford (http://hivdb.stanford.edu), estimam-se suas prevalências e avaliam-se mutações de resistência de acordo com o número de falhas no momento da genotipagem, bem como diferenças de mutações entre subtipos B e não-B do HIV-1. Resultados: A mutação M184V foi a mais prevalente (80,1%), seguida da K130N (40,6%) e TAM. Em pacientes multiexperimentados previamente à genotipagem, resistência a ZDV, d4T e TDF foi associada às mutações M41L, D67N, V118I, L210W, K219Q e T69D; bem como resistência a todos os IP/r associou-se às mutações principais M46I, V82A, L90M, I54V, I84V, M46L e L76V. O subtipo B é o predominante no Pará (90,7%) e diferenças de prevalência de mutações entre subtipos ocorreram entre as mutações L63P e A71T versus subtipo B, enquanto as mutações L76V, M36I, K20R, L10V, L89M e F53L associaram-se ao subtipo não-B. Conclusão: A seleção de mutações de resistência do HIV-1 relacionada aos antirretrovirais é similar ao descrito em literatura. O acúmulo de falhas ao tratamento favorece a emergência de mutações, o que reforça o monitoramento de falha virológica, seguida de genotipagem para minimizar o impacto de resistência. Estudos adicionais de epidemiologia molecular são necessários para avaliar melhor a questão da prevalência de subtipos de HIV-1 no estado e possíveis associações com mutações de resistência do HIV-1. / Introduction: Resistance to antiretroviral treatment can limit treatment options, especially in patients with accumulation of therapeutic failures, which may compromise clinical outcomes. Objectives: characterizing the profile of mutations in the protease and reverse transcriptase of HIV-1 patients in the treatment failure. Secondarily to evaluate the association between mutations and the number of treatment failures, association between mutations and subtypes of HIV-1 and present the temporal evolution of the prevalence of subtypes of HIV-1 in the state of Pará in northern Brazil. Method: cross-sectional study in which genotyping is evaluated between January, 2004 and December, 2013 with data obtained from the standardized application form for the examination RENAGENO and printed the results, both filed in four specialty care services. We included those by laboratory RENAGENO in 18 years and the first examination in those who underwent more than one time, totaling 377 samples. Mutations are described according to the database of HIV resistance at Stanford University (http://hivdb.stanford.edu), estimated their prevalence and resistance is evaluated according to the number of failures at the time of genotyping as well as differences between mutations and subtype B and non-B HIV-1. Results: The M184V mutation was the most prevalent (80.1%), followed by K130N (40.6%) and TAM. In patients who received at least three treatments prior to genotyping, resistance to ZDV, d4T and TDF was associated with mutations M41L, D67N, V118I, L210W, K219Q and T69D; well as resistance to all PI / r was associated with the major mutations M46I, V82A, L90M, I54V, I84V M46L and L76V. HIV-1 subtype B was the most prevalent (90.7%) and there were differences between subtypes B versus mutations: L63P and A71T were more frequent in the subtype B, whereas mutations L76V, K20R, L10V, L89M and F53L were in non-B subtypes. Conclusion: The selection of resistance mutations in HIV-1 related to antiretroviral is similar to that described in the literature. The accumulation of failures to treatment favors the emergence of mutations, reinforcing the monitoring and evaluation of virologic failure by genotyping to minimize the impact resistance. Additional molecular epidemiological studies are needed to better assess the issue of prevalence of subtypes of HIV-1 in the state and possible associations with resistance mutations in HIV-1.

Amebas de vida livre (AVL) são distribuídas mundialmente no solo e na água. Um número pequeno delas é considerado importante para a saúde dos seres humanos: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris e Sappinia diploidea. Algumas das infecções são oportunistas, ocorrendo em indivíduos imunocomprometidos, enquanto outras são não oportunistas. Amostras de água foram coletadas de banheira de hidromassagens e piscinas térmicas na cidade de Porto Alegre, RS, Brasil, com o objetivo de determinar a presença de Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e na amplificação por PCR com oligonucleotídeos gênero-específico. De 72 amostras analisadas, 20 (27,77%) foram positivas para amebas de vida livre, e identificadas morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 11 possuíam características compatíveis com o grupo morfológico II e 9 com o grupo III. Entre os isolados, 11(55%) foram considerados potencialmente patogênicos a partir de testes de osmotolerância e termotolerância. Somente 9 isolados quando submetidos à Reação da PCR, confirmaram pertencer ao gênero Acanthamoeba. A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição nos grupos genotípicos T3 (11,1%), T5 (11,1%), T4 (33,3%) e T15 (44,4%).Os resultados obtidos com este confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana nos ambientes de banheiras de hidromassagem e piscinas térmicas. / Free-living amoebae (FLA) are widely distributed in soil and water. A few number of them are implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Some of the infections were opportunistic, occurring mainly in immunocompromised hosts, while others are non opportunistic. Water samples were collecyed from both hot tubs and thermal swimming pools in the city of Porto Alegre, RS, Brazil, to determine the presence of Acanthamoeba in the water as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Eschererichia coli. The identification of the isolates was based on the cysts morphology and PCR amplification using genus-specific oligonucleotides. From 72 samples analyzed, 20 (27,77%) were positive for free-living amoebae, and the isolates were morphologically identified as belonging to the genus Acanthamoeba. Out of these, 11 presented morphological characteristics compatible with group II, and 9 with group III. Among the isolates, 11 (55%) were considered potentially pathogenic according to osmotolerance and temperature assays. The isolates when submitted to PCR reaction only 9 were confirmed as belonging to the genus Acanthamoeba. The sequences analysis when compare to the sequences in the GenBank, showed that genotype distribution in group T3 (11,1%), T5 (11,1%), T4 (33,3%) and T15 (44,4%). The results of this study confirmed the presence of potentially pathogenic isolates of free living amoebae in hot swimming pool and spas which can present risks to human health.

Amebas de vida livre (AVL) do gênero Acanthamoeba estão amplamente distribuídas no ambiente e podem tornar-se amebas patogênicas ao homem. O objetivo deste trabalho foi isolar em de água de torneira amebas de vida livre do gênero Acanthamoeba, identificá-las e classificá-las. Um total de 132 amostras de água de torneira foi coletado de escolas estaduais e municipais entre os meses de março a novembro de 2009. As amostras passaram pelo processo de filtração e as membranas foram semeadas em ágar não-nutriente 1,5% coberto por uma suspensão de E. coli inativadas pelo calor. Todas as amostras positivas para AVL foram submetidas à clonagem celular e identificadas como pertencentes ao gênero Acanthamoeba, através da morfologia dos cistos e trofozoitos e pela PCR utilizando oligonucleotídeos gênero-específicos que amplificam a região ASA.S1 do gene 18S rDNA. Ensaios fisiológicos de termo e osmotolerância foram utilizados para avaliar a patogenicidade dos isolados. Vinte sete isolados foram positivos para AVL e 10 foram identificados como pertencentes ao gênero Acanthamoeba tanto pelas características morfológicas quanto pela análise molecular. Destes, nove isolados apresentaram características do grupo II e um do grupo III, segundo Pussard e Pons (1977). A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição no grupo genotípicos T2 (40%), T2/T6 (40%), T6 (10%) e T4 (10%). Nos ensaios de termotolerância e osmotolerância 50% dos isolados obtiveram um baixo potencial patogênico. Os resultados indicaram a presença do gênero Acanthamoeba em água tratada no estado do RS, revelando sua importância epidemiológica e a necessidade de mais estudos para determinar sua distribuição no ambiente e seu potencial patogênico. / Free-living amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can become human pathogenic amoebae. The aim of this study was to isolate from tap water in free-living amoebae of Acanthamoeba, identify them and then classify them. A total of 132 samples of tap water was collected from state and municipal schools between march and november 2009. The samples passed through the filtration process and the membranes were seeded in non-nutrient 1.5% covered by a suspension of E. coli heatinactivated. All samples of AVL were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites by PCR using primers and genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Tests of physiological thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. Twenty seven isolates of AVL and 10 were identified as belonging to the genus Acanthamoeba through the morphological and molecular analysis. Nine of the isolates showed characteristics of group II and one isolate showed characteristics of group III, according Pussard and Pons (1977). The sequencing analysis by comparing the sequences submitted to GenBank, showed that genotype distribution in group T2 (40%), T2/T6 (40%), T6 (10%) and T4 (10%). In tests of thermotolerance and osmotolerance 50% of isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in the RS, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential.

Amebas de vida livre (AVL) são distribuídas mundialmente no solo e na água. Um número pequeno delas é considerado importante para a saúde dos seres humanos: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris e Sappinia diploidea. Algumas das infecções são oportunistas, ocorrendo em indivíduos imunocomprometidos, enquanto outras são não oportunistas. Amostras de água foram coletadas de banheira de hidromassagens e piscinas térmicas na cidade de Porto Alegre, RS, Brasil, com o objetivo de determinar a presença de Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e na amplificação por PCR com oligonucleotídeos gênero-específico. De 72 amostras analisadas, 20 (27,77%) foram positivas para amebas de vida livre, e identificadas morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 11 possuíam características compatíveis com o grupo morfológico II e 9 com o grupo III. Entre os isolados, 11(55%) foram considerados potencialmente patogênicos a partir de testes de osmotolerância e termotolerância. Somente 9 isolados quando submetidos à Reação da PCR, confirmaram pertencer ao gênero Acanthamoeba. A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição nos grupos genotípicos T3 (11,1%), T5 (11,1%), T4 (33,3%) e T15 (44,4%).Os resultados obtidos com este confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana nos ambientes de banheiras de hidromassagem e piscinas térmicas. / Free-living amoebae (FLA) are widely distributed in soil and water. A few number of them are implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Some of the infections were opportunistic, occurring mainly in immunocompromised hosts, while others are non opportunistic. Water samples were collecyed from both hot tubs and thermal swimming pools in the city of Porto Alegre, RS, Brazil, to determine the presence of Acanthamoeba in the water as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Eschererichia coli. The identification of the isolates was based on the cysts morphology and PCR amplification using genus-specific oligonucleotides. From 72 samples analyzed, 20 (27,77%) were positive for free-living amoebae, and the isolates were morphologically identified as belonging to the genus Acanthamoeba. Out of these, 11 presented morphological characteristics compatible with group II, and 9 with group III. Among the isolates, 11 (55%) were considered potentially pathogenic according to osmotolerance and temperature assays. The isolates when submitted to PCR reaction only 9 were confirmed as belonging to the genus Acanthamoeba. The sequences analysis when compare to the sequences in the GenBank, showed that genotype distribution in group T3 (11,1%), T5 (11,1%), T4 (33,3%) and T15 (44,4%). The results of this study confirmed the presence of potentially pathogenic isolates of free living amoebae in hot swimming pool and spas which can present risks to human health.