Avalia����o da ocorr��ncia de Giardia spp. por diferentes m��todos coprol��gicos

O g��nero Giardia inclui esp��cies com potencial zoon��tico e com uma distribui����o mundial, e em que alguns gen��tipos de Giardia duodenalis s��o respons��veis anualmente por milhares de novos casos em humanos. Existem v��rios ciclos de transmiss��o, sendo a ��gua de consumo, por contamina����o fecal de origem animal, uma das principais fontes de infec����o humana. Neste estudo foram colhidas 162 amostras fecais de animais de quatro origens diferentes (Zool��gico = 55; Produ����o = 25; Dom��stico = 5; Canil = 77) que foram testadas por duas t��cnicas coprol��gicas diferentes, a t��cnica de flutua����o com sacarose e a t��cnica de sedimenta����o com formol-acetato. Para a implementa����o de Nested PCR foram testados v��rios genes, ��-giardina, Glutamato desidrogenase e 18SrRNA, ocorrendo apenas amplifica����o das amostras com o gene 18SrRNA. Com esta t��cnica foram analisadas 26 amostras, que inclu��a a treze positivas �� microscopia e as restantes escolhidas aleatoriamente. Este trabalho permitiu determinar a ocorr��ncia de Giardia spp. atrav��s das t��cnicas coprol��gicas em treze animais de diferentes origens e verificar que o n��mero de animais de canil positivos n��o foi o esperadas de acordo com o descrito na literatura que refere ser este o grupo com maior preval��ncia. Este estudo tamb��m permitiu uma compara����o entre os dois m��todos de concentra����o de quistos de Giardia spp., com maior recupera����o utilizando a t��cnica de flutua����o com sacarose. Atrav��s da t��cnica molecular confirmaram-se dez dos positivos encontrados por microscopia e ainda se detectaram dois novos positivos.

In vitro effects of synbiotic fermentation on the canine faecal microbiota.

Stirred, pH-controlled anaerobic batch cultures were used to investigate the in vitro effects of galacto-oligosaccharides (GOS) alone or combined with the probiotic Bifidobacterium bifidum 02 450B on the canine faecal microbiota of three different donors. GOS supported the growth of B. bifidum 02 450B throughout the fermentation. Quantitative analysis of bacterial populations by FISH revealed significant increases in Bifidobacterium spp. counts (Bif164) and a concomitant decrease in Clostridium histolyticum counts (Chis150) in the synbiotic-containing vessels compared with the controls and GOS vessels. Vessels containing probiotic alone displayed a transient increase in Bifidobacterium spp. and a transient decrease in Bacteroides spp. Denaturing gradient gel electrophoresis analysis showed that GOS elicited similar alterations in the microbial profiles of the three in vitro runs. However, the synbiotic did not alter the microbial diversity of the three runs to the same extent as GOS alone. Nested PCR using universal primers, followed by bifidobacterial-specific primers illustrated low bifidobacterial diversity in dogs, which did not change drastically during the in vitro fermentation. This study illustrates that the canine faecal microbiota can be modulated in vitro by GOS supplementation and that GOS can sustain the growth of B. bifidum 02 450B in a synbiotic combination.

Avalia����o de duas t��cnicas de rea����o em cadeia da polimerase na detec����o do Mycobacterium tuberculosis em diferentes amostras biol��gicas

A tuberculose (TB) �� uma doen��a infecto-contagiosa causada pelo bacilo Mycobacterium tuberculosis e que permanece como um importante problema de sa��de p��blica mundial, sendo a TB pulmonar a forma mais comum de apresenta����o da doen��a. O diagn��stico precoce e tratamento adequado s��o essenciais para a efic��cia dos programas p��blicos de controle da TB. Novos metodologias mais r��pidas, sens��veis e espec��ficas, como a rea����o em cadeia da polimerase (PCR), vem sendo propostas no diagn��stico da doen��a. O objetivo desse estudo foi avaliar o desempenho de duas PCR, a PCR em tempo real (qPCR) e a Nested PCR em ��nico tubo (STNPCR), em diferentes amostras biol��gicas, no diagn��stico da tuberculose pulmonar, al��m de compar��-las com as metodologias convencionais (baciloscopia e cultura) e entre si. Para isso foram analisados 125 pacientes que tiveram amostras de sangue (125 amostras de plasma e 116 amostras de PBMC), urina (n=125) e escarro (n=125) coletadas, totalizando a an��lise de 491 amostras biol��gicas. Amostras de escarro e urina foram descontaminadas pelo m��todo de Petroff NAOH 4 por cento modificado e semeadas em meio de cultura L��wenstein-Jensen (LJ), enquanto as amostras de sangue eram separadas em plasma e PBMC. Ap��s processamento, deu-se a extra����o de DNA atrav��s do kit comercial da Qiagen seguida de amplifica����o pelas duas metodologias de PCR. Para an��lise estat��stica calculou-se a sensibilidade, especificidade, valores preditivos positivo e negativo e ��ndice kappa das t��cnicas. A STNPCR apresentou, em amostras de sangue, sensibilidade de 26,3 por cento e especificidade de 97,7 por cento. Em amostras de urina observou-se uma S = 7,9 por cento e E = 98,9 por cento e em escarro S = 21,1 por cento e E = 98,9 por cento. Quando analisadas as asmotras em paralelo, a sensibilidade da STNPCR foi igual a 44,7 por cento enquanto sua especificidade foi 97,7 por cento. J�� a qPCR, em amostras de sangue, obteve sensibilidade igual a 26,3 por cento e especificidade de 95,4 por cento. Em amostras de urina a sensibilidade obtida foi 47,4 por cento e a especificidade 79,3 por cento e, em escarro, S = 36,8 por cento e E = 95,4 por cento. Quando analisada em paralelo, a sensibilidade da qPCR foi 65,8 por cento e a especificidade foi 79,3 por cento. A baciloscopia de escarro apresentou sensibilidade de 41,7 por cento e especificidade de 100 por cento, enquanto as culturas em urina e escarro apresentaram sensibilidade e especificidade, respectivamente, de 10,5 por cento e 100 por cento e 60,5 por cento e 96,6 por cento. Pode-se concluir que a qPCR apresentou melhor desempenho quando comparada �� STNPCR e tamb��m bom desempenho quando comparada ��s metodologias convencionais, e que quando analisa-se mais de um tipo de amostras biol��gica, a efic��cia das t��cnicas �� aumentada. Espera-se que com a utiliza����o dessa t��cnica molecular, seja poss��vel a melhor elucida����o dos casos de TB pulmonar, promovendo maior taxa de tratamento dos pacientes e menor risco de transmiss��o da doen��a

Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota

With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intenstity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans.

Detection of assemblages A and B of Giardia duodenalis in water and sewage from Sao Paulo state, Brazil

Giardia duodenalis is a protozoan that parasitizes humans and other mammals and causes giardiasis. Although its isolates have been divided into seven assemblages, named A to G, only A and B have been detected in human faeces. Assemblage A isolates are commonly divided into two genotypes, AI and AII. Even though information about the presence of this protozoan in water and sewage is available in Brazil, it is important to verify the distribution of different assemblages that might be present, which can only be done by genotyping techniques. A total of 24 raw and treated sewage, surface and spring water samples were collected, concentrated and purified. DNA was extracted, and a nested PCR was used to amplify an 890 bp fragment of the gdh gene of G. duodenalis, which codes for glutamate dehydrogenase. Positive samples were cloned and sequenced. Ten out of 24 (41.6%) samples were confirmed to be positive for G. duodenalis by sequencing. Phylogenetic analysis grouped most sequences with G. duodenalis genotype AII from GenBank. Only two raw sewage samples presented sequences assigned to assemblage B. In one of these samples genotype AII was also detected. As these assemblages/genotypes are commonly associated to human giardiasis, the contact with these matrices represents risk for public health.

One-step reverse transcriptase polymerase chain reaction for the diagnosis of respiratory syncytial virus in children

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory tract infections in infants and children. Rapid diagnosis is required to permit appropriate care and treatment and to avoid unnecessary antibiotic use. Reverse transcriptase (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been considered important tools for virus detection due to their high sensitivity and specificity. In order to maximize use-simplicity and minimize the risk of sample cross-contamination inherent in two-step techniques, a RT-PCR method using only a single tube to detect HRSV in clinical samples was developed. Nasopharyngeal aspirates from 226 patients with acute respiratory illness, ranging from infants to 5 years old, were collected at the University Hospital of the University of Sao Paulo (HU-USP), and tested using IFA, one-step RT-PCR, and semi-nested RT-PCR. One hundred and two (45.1%) samples were positive by at least one of the three methods, and 75 (33.2%) were positive by all methods: 92 (40.7%) were positive by one-step RT-PCR, 84 (37.2%) by IFA, and 96 (42.5%) by the semi-nested RT-PCR technique. One-step RT-PCR was shown to be fast, sensitive, and specific for RSV diagnosis, without the added inconvenience and risk of false positive results associated with semi-nested PCR. The combined use of these two methods enhances HRSV detection. (C) 2007 Elsevier B.V. All rights reserved.

DGGE with genomic DNA: Suitable for detection of numerically important organisms but not for identification of the most abundant organisms

Identification of all important community members as well as of the numerically dominant members of a community are key aspects of microbial community analysis of bioreactor samples. A systematic study was conducted with artificial consortia to test whether denaturing gradient gel electrophoresis (DGCE) is a reliable technique to obtain such community data under conditions where results would not be affected by differences in DNA extraction efficiency from cells. A total of 27 consortia were established by mixing DNA extracted from Escherichia coli K12, Burkholderia cepacia and Stenotrophomonas maltophilia in different proportions. Concentrations of DNA of single organisms in the consortia were either 0.04, 0.4 or 4 ng/mu l. DGGE-PCR of genomic DNA with primer sets targeted at the V3 and V6-V8 regions of the 16S rDNA failed to detect the three community members in only 7% of consortia, but provided incorrect information about dominance or co-dominance for 85% and 89% of consortia with the primer sets for the V6-V8 and V3 regions, respectively. The high failure rate in detection of dominant B. cepacia with the primers for the V6-V8 region was attributable to a single nucleoticle primer mismatch in the target sequences of both, the forward and reverse primer. Amplification bias in PCR of E. coli and S. maltophilia for the V6-V8 region and for all three organisms for the V3 region occurred due to interference of genomic DNA in PCR-DGGE, since a nested PCR approach, where PCR-DGGE was started from mixtures of 16S rRNA genes of the organisms, provided correct information about the relative abundance of original DNA in the sample. Multiple bands were not observed in pure culture amplicons produced with the V6-V8 primer pair, but pure culture V3 DGGE profiles of E. coli, S. maltophilia and B. cepacia contained 5, 3 and 3 bands, respectively. These results demonstrate DGGE was suitable for identification of all important community members in the three-membered artificial consortium, but not for identification of the dominant organisms in this small community. Multiple DGGE bands obtained for single organisms with the V3 primer pair could greatly confound interpretation of DGGE profiles. (C) 2008 Elsevier Ltd. All rights reserved.

Development and application of polymerase chain reaction-based assays for Rathayibacter Toxicus and a Bacteriophage associated with annual ryegrass (Lolium Rigidum) toxicity

Annual ryegrass toxicity (ARGT) is responsible for significant stock losses in South Australia and Western Australia. The toxicity is caused by corynetoxins produced by the bacterium <i>Rathayibacter toxicus</i> (with the possible involvement of a bacteriophage), which infects annual ryegrass (<i>Lolium</i> <i>rigidum</i>). Polymerase chain reaction (PCR)-based assays, compatible with an existing enzyme-linked immunosorbent assay for the corynetoxins, have been developed and used to screen <i>L. rigidum</i> for both the presence of <i>R</i>. <i>toxicus</i> and for the bacteriophage isolate NCPPB 3778. The results from analysing bacterially infected galls from toxic grain screenings showed a positive correlation between the presence of the bacterium and corynetoxins but not with the bacteriophage. Analysis of pasture-derived samples of annual ryegrass showed about a 50% correlation of corynetoxins with bacterial presence and about a 5% correlation of phage with the presence of the bacterium. These observations support the potential application of the PCR-based assays in providing a useful, complementary tool in the assessment of the likelihood of pasture and feed to cause ARGT and to enable a better understanding of the complex aetiology of ARGT.<br />

Associa����o do papilomav��rus humano com o carcinoma colorretal

OBJETIVO: Investigar a presen��a do papiloma v��rus humano no adenocarcinoma de c��lon e reto. M��TODOS: Setenta e dois pacientes com adenocarcinoma de c��lon e reto foram analisados. Foram coletadas duas amostras de cada paciente: uma amostra do tumor e outra de mucosa n��o neopl��sica distante 15 cm do tumor. Como grupo de controle, tamb��m foram estudadas amostras de mucosa de quinze pacientes sem c��ncer colorretal. Os tecidos foram analisados por ���auto nested��� PCR usando o primer consensus GP5+/GP6+. Dois primers espec��ficos para regi��o E6 dos HPV 16 e HPV 18 tamb��m foram utilizados. RESULTADOS: O DNA do HPV foi detectado em tecidos de c��lon e reto de 60 pacientes com c��ncer ( 83 por cento), enquanto que este n��o foi encontrado em nenhum dos pacientes controles sem tumor ( p<0,001). Em vinte e tr��s pacientes, o DNA do HPV estava presente tanto no tecido tumoral como na mucosa n��o neopl��sica adjacente. Em vinte e tr��s pacientes o DNA do HPV foi encontrado apenas no tumor, enquanto que em quatorze, s�� foram encontrados nos tecidos normais coletados pr��ximos ao tumor de c��lon e reto. Em setenta e cinco por cento dos casos positivos foram identificados os HPV tipo 16 ou 18 por PCR com primers E6 espec��ficos. Os achados positivos obtidos por PCR foram confirmados por seq��enciamento viral. CONCLUS��O: O HPV est�� presente no c��lon e reto da maioria dos pacientes com carcinoma de c��lon e reto estudados, sugerindo que este v��rus pode estar relacionado �� patog��nese do c��ncer colorretal. Ser��o necess��rios mais estudos para determinar o papel do HPV na carcinog��nese colorretal.

Detection of Ehrlichia chaffeensis in Brazilian marsh deer (Blastocerus dichotomus)

Ehrlichia chaffeensis was detected for the first time in blood samples from Brazilian marsh deers (Blastocerus dichotomus) captured in the marshes of Parana River in Southeast Brazil in 1998. Seven EDTA-blood samples from deers were analyzed by PCR and nested PCR for presence of Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Neoriickettsia risticii, Anaplasma phagocytophilum and Anaplasma marginale. Three samples showed positive reactions for E. chaffeensis and Anaplasma marginale. None contained detectable A. phagocytophilum, E. ewingii, E. canis or Neorickettsia risticii DNA. In Brazil, the wild marsh deer may be a natural reservoir of the agents that cause human monocytotropic ehrlichiosis and ruminant erythrocytic anaplasmosis. (c) 2006 Elsevier B.V. All rights reserved.

Chlamydophila psittaci in free-living Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil

Chlamydophila psittaci (C. psittaci) infection was evaluated in 77 free-living nestlings of Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil. Tracheal and cloacal swab samples from 32 wild parrot and 45 macaw nestlings were submitted to semi-nested PCR, while serum samples were submitted to complement fixation test (CFT). Although all 32 Amazon parrot serum samples were negative by CFT, cloacal swabs from two birds were positive for Chlamydophila DNA by semi-nested PCR (6.3%); these positive birds were 32 and 45 days old. In macaws, tracheal and cloacal swabs were positive in 8.9% and 26.7% of the samples, respectively. Complement-fixing antibodies were detected in 4.8% of the macaw nestlings; macaw nestlings with positive findings were between 33 and 88 days old. These results indicate widespread dissemination of this pathogen in the two evaluated psittacine populations. No birds had clinical signs suggestive of chlamydiosis. To the best of our knowledge, this is the first report on C. psittaci in free-living Blue-fronted Amazon parrots and Hyacinth macaws in Brazil. (c) 2006 Elsevier B.V. All rights reserved.

Molecular diagnosis of Anaplasmataceae organisms in dogs with clinical and microscopical signs of ehrlichiosis

Coordena����o de Aperfei��oamento de Pessoal de N��vel Superior (CAPES)

Canine ehrlichiosis: clinical, hematological, serological and molecular aspects

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Serologic evidence of equine granulocytic anaplasmosis in horses from central West Brazil

A Erliquiose �� uma doen��a zoon��tica causada por bact��rias gram-negativas e intracelulares obrigat��rias. A Anaplasmose Granuloc��tica Equina - AGE (anteriormente denominada Erliquiose Granuloc��tica Equina, EGE) �� uma enfermidade sazonal, normalmente auto-limitante em equinos. No Brasil, existem poucos relatos deste agente erliquial, bem como de seus vetores naturais. Atualmente, veterin��rios t��m levantado a suspeita de casos de AGE em equinos com sinais cl��nicos sugestivos de erliquiose e n��o responsivos ao tratamento para a piroplasmose equina. O objetivo do presente estudo foi identificar equinos expostos a A. phagocytophilum por meio de t��cnicas sorol��gicas e moleculares. Vinte amostras de sangue e soro de equinos da regi��o Centro-oeste do Brasil foram avaliados por meio do exame microsc��pico de capa leucocit��ria, ensaio imunoenzim��tico indireto (ELISA), rea����o de imunofluoresc��ncia indireta (RIFI) e rea����o em cadeia da polimerase (nested PCR). Adicionalmente, o diagn��stico sorol��gico de Theileria equi pela RIFI e ELISA foram realizados, assim como o diagn��stico molecular pelo nPCR. Treze (65%) amostras de soro foram positivas para A. phagocytophilum pelo teste de ELISA, entretanto nenhum equino foi positivo pelo exame microsc��pico da capa leucocit��ria ou nPCR. Anticorpos IgG anti-T. equi foram detectados em 18 (90%) e 17 (85%) equinos pela RIFI e ELISA, respectivamente e o agente foi detectado em 9 (45%) animais pelo nPCR. Estes dados sugerem importante informa����o para o entendimento da ocorr��ncia da AGE e piroplasmose equina no Centro-oeste do Brasil.

Molecular and serological detection of Theileria equi and Babesia caballi in donkeys (Equus asinus) in Brazil

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)