Conventional and molecular typing of Salmonella typhi strains from Brazil

Phenotypic and genotypic characteristics of Salmonella Typhi were studied in 30 strains, isolated in different years, from some areas in Brazil. Conventional typing methods were performed by biochemical tests, Vi phage-typing scheme, and antimicrobial susceptibility test. Molecular typing methods were performed by analysis of plasmid DNA and by random amplified polymorphic DNA (RAPD-PCR). For the latter, an optimization step was performed to ensure the reproducibility of the process in genetic characterization of S. Typhi. The predominance of 76.7% of biotype I (xylose +, arabinose -) was noticed in all studied areas. Three phage types were recognized, with prominence for the phage types A (73.3%) and I+IV (23.3%). All the strains were susceptible to the drugs used. However, 36.7% of the strains contained plasmids, with predominance of the 105 Kb plasmid. RAPD was capable of grouping the strains in 8 genotypic patterns using primer 784, in 6, using primer 787 and in 7, using primer 797. Conventional phenotypic typing methods, as well as the DNA plasmid analysis, presented nonsignificant discriminatory power; however, RAPD-PCR analysis showed discriminatory power, reproducibility, easy interpretation and performance, being considered as a promising alternative typing method for S. Typhi.

Analysis of genetic relatedness between populations of Aedes aegypti from different geographic regions of São Paulo state, Brazil

RAPD markers have been used for the analysis of genetic differentiation of Aedes aegypti, because they allow the study of genetic relationships among populations. The aim of this study was to identify populations in different geographic regions of the São Paulo State in order to understand the infestation pattern of A. aegypti. The dendrogram constructed with the combined data set of the RAPD patterns showed that the mosquitoes were segregated into two major clusters. Mosquitoes from the Western region of the São Paulo State constituted one cluster and the other was composed of mosquitoes from a laboratory strain and from a coastal city, where the largest Latin American port is located. These data are in agreement with the report on the infestation in the São Paulo State. The genetic proximity was greater between mosquitoes whose geographic origin was closer. However, mosquitoes from the coastal city were genetically closer to laboratory-reared mosquitoes than to field-collected mosquitoes from the São Paulo State. The origin of the infestation in this place remains unclear, but certainly it is related to mosquitoes of origins different from those that infested the West and North region of the State in the 80's.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal specie. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.

Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban triatominae

Random amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl2, 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 µL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.

Distribution of virulence markers in clinical and environmental Vibrio cholerae non-O1/non-O139 strains isolated in Brazil from 1991 to 2000

One hundred seventy nine Vibrio cholerae non-O1/non-O139 strains from clinical and different environmental sources isolated in Brazil from 1991 to 2000 were serogrouped and screened for the presence of four different virulence factors. The Random Amplification of Polymorphic DNA (RAPD) technique was used to evaluate the genetic relatedness among strains. Fifty-four different serogroups were identified and V. cholerae O26 was the most common (7.8%). PCR analysis for three genes (ctxA, zot, ace) located of the CTX genetic element and one gene (tcpA) located on the VPI pathogenicity island showed that 27 strains harbored one or more of these genes. Eight (4.5%) strains possessed the complete set of CTX element genes and all but one of these belonged to the O26 serogroup suggesting that V. cholerae O26 has the potential to be an epidemic strain. The RAPD profiles revealed a wide variability among strains and no genetic correlation was observed.

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.

Classic and molecular study of Giardia duodenalis in children from a daycare center in the region of Presidente Prudente, São Paulo, Brazil

Epidemiological studies on giardiasis by using molecular techniques such as RAPD (Randomly Amplified Polymorphic DNA) may give information on factors related to the transmission of Giardia duodenalis. The aim of this work was to assess the epidemiology of G. duodenalis in 101 children attended at a daycare center in Presidente Bernardes, SP, Brazil. After parasitological examinations in feces samples, 15 children presented cysts of G. duodenalis. Their respective parents, brothers and pets, besides the daycare center workers, also had their feces submitted to parasitological analysis. Seven mothers and nine brothers also presented G. duodenalis cysts, while fathers, daycare workers and pets (dogs) did not presented the parasite. Besides the 15 cases with G. duodenalis, other 23 children presented other enteroparasites (Entamoeba coli, Endolimax nana, Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura). Samples of G. duodenalis cysts from children and their relatives were submitted to molecular typing by RAPD after genomic DNA extraction and amplification of a fragment of the 18S rDNA region by PCR. After examining 31 isolates of G. duodenalis (children and their respective mothers and brothers), it was concluded that the parasite transmission occurred in children, probably during daily cohabitation at the daycare center, but not at home among their relatives or pets.

Induction of phagocytic activity and nitric-oxide production in natural populations of Trypanosoma Cruzi I and II from the state of Paraná, Brazil

Twelve strains of Trypanosoma cruzi isolated from wild reservoirs, triatomines, and chronic chagasic patients in the state of Paraná, southern Brazil, and classified as T. cruzi I and II, were used to test the correlation between genetic and biological diversity. The Phagocytic Index (PI) and nitric-oxide (NO) production in vitro were used as biological parameters. The PI of the T. cruzi I and II strains did not differ significantly, nor did the PI of the T. cruzi strains isolated from humans, triatomines, or wild reservoirs. There was a statistical difference in the inhibition of NO production between T. cruzi I and II and between parasites isolated from humans and the strains isolated from triatomines and wild reservoirs, but there was no correlation between genetics and biology when the strains were analyzed independently of the lineages or hosts from which the strains were isolated. There were significant correlations for Randomly Amplified Polymorphic Deoxyribonucleic acid (RAPD) and biological parameters for T. cruzi I and II, and for humans or wild reservoirs when the lineages or hosts were considered individually.

Molecular characterization of Aeromonas spp. and Vibrio cholerae O1 isolated during a diarrhea outbreak

This work aimed to assess pathogenic potential and clonal relatedness of Aeromonas sp. and Vibrio cholerae isolates recovered during a diarrhea outbreak in Brazil. Clinical and environmental isolates were investigated for the presence of known pathogenic genes and clonal relatedness was assessed by intergenic spacer region (ISR) 16S-23S amplification. Four Aeromonas genes (lip, exu, gcat, flaA/B) were found at high overall frequency in both clinical and environmental isolates although the lip gene was specifically absent from selected species. A fifth gene, aerA, was rarely found in A. caviae, the most abundant species. The ISR profile revealed high heterogeneity among the Aeromonas isolates and no correlation with species identification. In contrast, in all the V. cholerae isolates the four genes investigated (ctxA, tcpA, zot and ace) were amplified and revealed homogeneous ISR and RAPD profiles. Although Aeromonas isolates were the major enteric pathogen recovered, their ISR profiles are not compatible with a unique cause for the diarrhea events, while the clonal relationship clearly implicates V. cholerae in those cases from which it was isolated. These results reinforce the need for a better definition of the role of aeromonads in diarrhea and whether they benefit from co-infection with V. cholerae.

MOLECULAR TYPING OF Candida albicans ISOLATES FROM HOSPITALIZED PATIENTS

SUMMARYIntroduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals.Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD) and microsatellite, two different primers for each technique were used.Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91.Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions) and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.

FREQUENCY OF Candida SPECIES IN A TERTIARY CARE HOSPITAL IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL

Infections by Candida species are a high-impact problem in public health due to their wide incidence in hospitalized patients. The goal of this study was to evaluate frequency, susceptibility to antifungals, and genetic polymorphism of Candida species isolated from clinical specimens of hospitalized patients. The Candida isolates included in this study were obtained from blood cultures, abdominal fluids, and central venous catheters (CVC) of hospitalized patients at the Clinical Hospital of the Federal University of Uberlândia during the period of July 2010 - June 2011. Susceptibility tests were conducted by the broth microdilution method. The RAPD-PCR tests used employed initiator oligonucleotides OPA09, OPB11, and OPE06. Of the 63 Candida isolates, 18 (28.5%) were C. albicans, 20 (31.7%) were C. parapsilosis complex species, 14 (22.2%) C. tropicalis, four (6.4%) C. glabrata, four (6.4%) C. krusei, two (3.3%) C. kefyr, and one (1.6%) C. lusitaniae. In vitro resistance to amphotericin B was observed in 12.7% of isolates. In vitroresistance to azoles was not detected, except for C. krusei. The two primers, OPA09 and OPB11, were able to distinguish different species. Isolates of C. albicans and C. parapsilosis complex species presented six and five clusters, respectively, with the OPA09 marker by RAPD-PCR, showing the genetic variability of the isolates of those species. It was concluded that members of the C. parapsilosis complex were the most frequent species found, and most isolates were susceptible to the antifungals amphotericin B, flucozanole, and itraconazole. High genetic polymorphisms were observed for isolates of C. albicans and C. parapsilosis complex species, mainly with the OPA09 marker.

Estudo radiológico longitudinal do esôfago, em área endêmica de doença de Chagas, em um período de seis anos

Foi realizado estudo longitudinal de seis anos através do esofagograma, em 494 pessoas do projeto Mambaí, das quais 212 (43%) eram soropositivas. O estudo realizado em 1975176 e 1980/82 constou em ambas as ocasiões, de duas abreugrafias do esôfago: a primeira, imediatamente após a ingestão de 75ml de solução baritada e outra 60 segundos após. Entre as 201 pessoas soropositivas normais no primeiro estudo, foram encontrados 4 (2%) casos novos de megaesôfago do Grupo I, e entre as 11 com megaesôfago, 2 dos Grupos I e 11 evoluíràm, respectivamente, para os Grupos II e IV, indicando progressão da esofagopatia em 2,8% (6/212) dos soropositivos. Quatro pessoas com megaesôfago do Grupo I apresentaram esofagograma normalno segundo exame radio lógico, com aparente "regressão "da esofagopatia. Chama a atenção a existência de 10 pessoas com esofagogramas duvidosos no primeiro exame e, outras seis no segundo, sendo 75 % delas soropositivas. Este achado pode estar indicando o esofagograma duvidoso como um marcador precoce da esofagopatia.

Rastreamento sorológico para hepatite B em profissionais de saúde na cidade de Goiânia - Goiás

Pesquisou-se marcadores para o vírus da hepatite B em profissionais de saúde, ligados a três instituições na cidade de Goiânia-Goiás. A prevalência da infecção virai encontrada foi de 23,4% em relação aos diferentes marcadores (AgHBs, anti-HBs e anti-HBc). Entre os indivíduos positivos, 2,3% eram portadores do vírus e 21,1% demonstraram infecção prévia. Destes, 9,8% apresentaram como marcador, apenas o anti- HBs e 32,6% o anti-HBc. Dezenove indivíduos relataram vacinação ao vírus, sendo que 8 soroconverteram para o anti-HBs e 6 apresentaram também o anti-HBc, e os demais não apresentaram qualquer marcador virai. A maior prevalência foi a partir dos 30 anos de idade (p< 0,05). O sexo masculino mostrou percentual superior ao feminino, 29,5% e 21,1% respectivamente (p< 0,05). Quando considerada a prevalência virai em relação a contatos com sangue e/ou pacientes, o percentual de positividade para contactantesfoi 25,9% (p < 0,05), e a maior prevalência do vírus ocorreu a partir de 10 anos de trabalho (p < 0,05). A hemodiálise mostrou-se como setor de maior risco cujo percentual foi de 77,0% (p<0,05).

Prevalência dos marcadores sorológicos dos vírus da hepatite B e D em crianças das tribos Caiabietxucarramãe do parque indígena do Xingu, Brasil central

O Parque Indígena do Xingu (PIX) está localizado no estado do Mato Grosso, na região de transição de cenado ao sul e da floresta Amazônica ao norte. Dados de literatura mostram que a população adulta apresenta elevada prevalência de marcadores do vírus da hepatite B (HBV). O presente estudo visa determinar a prevalência dos marcadores do HBV e do HDV na população indígena do PIX de zero a 14 anos, e investigar a forma de transmissão do HBV na região. Entre as 17 tribos existentes no PIX escolheu-se os Caiabi e os Txucairamãe que diferem em seus hábitos de vida e habitam a região Norte do parque com características de clima, vegetação e fauna semelhantes ás da região Amazônica. Avaliaram-se 222 crianças (116 Txucarramãe e 106 Caiabi) e 33 mulheres em idade fértil. A pesquisa de marcadores sorológicos para HBV e HDV foi feita por técnica imunoenzimãtica. A prevalência global dos marcadores sorológicos nas crianças foi: HBsAg 4,5%; anti-HBs 39,6%; anti- HBc 44,1%; presença de algum marcador do HBV 47,3% e anti-HDV 0,0%, enquanto que nas mulheres em idade fértil foi: HBsAg 12%, todas anti-HBe positivas. A infecção pelo HBV ocoireu mais precocemente entre os Txucarramãe, quando se considerou algum marcador do HBV (p < 0,001). No PIX, área de alta endemicidadepara infecção pelo HBV, a transmissão do vírus ocorre provavelmente de maneira horizontal e não vertical como seria de se esperar, características culturais, condições de habitação e presença de insetos hematófagos são importantes determinantes na sua transmissão. Apesar de não ter sido detectado nenhum caso com hepatite Delta, o PIX é uma área propícia para a sua disseminação.

Imunogenicidade da cepa avirulenta RV194-2 do vírus rábico em camundongos

O vírus rábico RV194-2, uma variante avirulenta da cepa CVS (Challenge Vírus Standard), produz uma infecção inaparente quando inoculado intracerebralmente em camundongos adultos. Sugerindo que a resposta imunológica do hospedeiro permite a eliminação do vírus do sistema nervoso central. Por esta razão foram estudadas a indução de interferon e a resposta imune humoral em camundongos BALB/c inoculados com vírus RV194-2. Durante a infecção, estes camundongos apresentaram elevados níveis de interferon no plasma e no cérebro com altos títulos de anticorpos neutralizantes anti-rábicos. A 2-5A sintetase. um marcador da ação dos interferons,foi também analisada no cérebro destes animais. Sua atividade, aumentou, paralelamente, á produção de interferon, demonstrando que este interferon é bioquímicamente ativo. O vírus RV194-2 também induziu, 45 dias após sua inoculação, proteção aos animais quando desafiados com a cepa virulenta CVS. Estes resultados demonstram que a cepa RV194-2possui um alto nível imunogênico.