Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs

A importância do cão como reservatório de L. infantum chagasi no meio urbano tem estimulado a realização de inúmeros trabalhos de avaliação de técnicas de diagnóstico, uma vez que este procedimento, quando realizado corretamente, torna-se um importante passo na prevenção da doença em humanos. Dentre os métodos de diagnóstico, as técnicas moleculares têm adquirido destaque. Objetivou-se neste trabalho verificar o desempenho da Reação em Cadeia da Polimerase (PCR) e da PCR em tempo real (qPCR) para diagnóstico da Leishmaniose Visceral Canina (LVC) utilizando diferentes amostras biológicas. Para tanto foram utilizados 35 cães provenientes de uma área endêmica para LVC, onde foram utilizados para o diagnóstico molecular, aspirado de medula óssea, fragmentos de linfonodo e baço. Neste estudo a qPCR foi capaz de detectar um maior número de animais positivos quando comparada com a PCR. Já entre as diferentes amostras biológicas utilizadas não foi observada diferença significativa na detecção de DNA de L. infantumchagasi por meio da PCR e qPCR. Mesmo assim, considerando a facilidade de obtenção, o linfonodo pode ser considerada como a melhor amostra para diagnóstico molecular da infecção por L. infantum chagasi.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Co-infecção HIV/HCV em pacientes de Botucatu e região

Devido à similaridade nas rotas de transmissão, a co-infecção HIV/HCV é freqüente, afetando em média 30 a 50% dos portadores de HIV. O presente estudo visou avaliar uma possível associação entre os subtipos do HIV e genótipos do HCV em pacientes co-infectados, com base na análise das freqüências em pacientes mono e co-infectados. Para determinação da freqüência dos subtipos HIV e genótipos HCV, foram analisados respectivamente 124 e 496 pacientes mono-infectados. O estudo da co-infecção foi realizado num grupo de 150 pacientes HIV positivos e esteve presente em 22 (14,7%) dos pacientes. A freqüência dos subtipos do HIV-1 em mono-infectados foi: subtipo B (85,5%), subtipo F (12,9%) e recombinante B/F (1,6%), enquanto nos genótipos HCV foi: 1a (25%), 1b (29,4%), 1a/1b (3,6%), 3a (35%), 2 (1,8%) e 5 (0,4%). Nos co-infectados o padrão de distribuição dos subtipos HIV-1 é semelhante aos mono-infectados, ou seja, subtipo B (85,0%), seguido do subtipo F (15,0%). A distribuição de freqüência de genótipos HCV nos co-infectados foi: 1a (36,3%), 1b (27,3%), 1a/1b (9,1%) e 3a (27,3%) mostrando um aumento de 10% na freqüência do genótipo 1, queda de 7,7% no genótipo 3 e ausência de outros genótipos. A análise estatística de associação entre os subtipos HIV e genótipos HCV (Goodman) mostrou que no genótipo 1 (HCV) ocorreu predominância do subtipo B, enquanto no genótipo 3 (HCV) a distribuição dos subtipos B e F (HIV-1) foi casual. Isto aponta para a necessidade de mais estudos desse grupo e um maior valor amostral.

In vitro detection of hepatitis C virus in platelets from uninfected individuals exposed to the virus

Introduction ,,,,,Despite hepatocytes being the target cells of hepatitis C virus (HCV), viral ribonucleic acid RNA has been detected in other cells, including platelets, which have been described as carriers of the virus in the circulation of infected patients. Platelets do not express cluster differentiation 81 CD81, the main receptor for the virus in hepatocytes, although this receptor protein has been found in megakaryocytes. Still, it is not clear if HCV interacts with platelets directly or if this interaction is a consequence of its association with megakaryocytes. The aim of this study was to evaluate the interaction of HCV with platelets from non-infected individuals, after in vitro exposure to the virus. ,,,, ,,,, ,,,,,Methods ,,,,,Platelets obtained from 50 blood donors not infected by HCV were incubated in vitro at 37°C for 48h with serum containing 100,000IU∕mL of genotype 1 HCV. After incubation, RNA extracted from the platelets was assayed for the presence of HCV by reverse transcription – polymerase chain reaction RT-PCR. ,,,, ,,,, ,,,,,Results ,,,,,After incubation in the presence of virus, all samples of platelets showed HCV RNA. ,,,, ,,,, ,,,,,Conclusions ,,,,,The results demonstrate that, in vitro, the virus interacts with platelets despite the absence of the receptor CD81, suggesting that other molecules could be involved in this association.

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp, at bovine artificial insemination centers. (C) 2000 Elsevier B.V. B.V. All rights reserved.

Use of PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphism) in the gene of the enzyme Stearoyl-CoA-Desaturase in Bubalus bubalis

The milk is an important food because it contents Conjugated Linoleic Acids (CIA). These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD) and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD's gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z (sic) (sic) D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren't genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

A new polymerase chain reaction/restriction fragment length polymorphism protocol for Plasmodium vivax circumsporozoite protein genotype (VK210, VK247, and P-vivax-like) determination

For the molecular diagnosis of Plasmodium vivax variants (VK210, VK247, and P. vivax-like) using DNA amplification procedures in the laboratory, the choice of rapid and inexpensive identification products of the 3 different genotypes is an important prerequisite. We report here the standardization of a new polymerase chain reaction/restriction fragment length polymorphism technique to identify the 3 described P. vivax circumsporozoite protein (CSP) variants using amplification of the central immunodominant region of the CSP gene of this protozoan. The simplicity, specificity, and sensitivity of the system described here is important to determine the prevalence and the distribution of infection with these P. vivax genotypes in endemic and nonendemic malaria areas, enabling a better understanding of their phylogeny. (c) 2007 Published by Elsevier B.V.

Periodontal status in smokers and never-smokers: Clinical findings and real-time polymerase chain reaction quantification of putative periodontal pathogens

Background: Smoking is a well-known risk factor for destructive periodontal disease, but its relationship with periodontal status and subgingival microbiota remains unclear. Inherent limitations of microbiological methods previously used may partly explain these mixed results, and real-time polymerase chain reaction (PCR) has been presented as a valid alternative. The aim of the present study was to investigate the clinical condition and microbiological profile of patients with chronic periodontitis as related to the habit of smoking.Methods: Fifty patients (33 to 59 years old), 25 smokers and 25 never-smokers, constituted the sample. The visible plaque index (VPI), gingival bleeding index (GBI), bleeding on probing (BOP), periodontal probing depth (PD), clinical attachment loss (CAL), and gingival crevicular fluid (GCF) volume were recorded. Real-time PCR quantified Porphyromonas gingivalis, Micromonas micros, Dialister pneumosintes, Actinobacillus actinomycetemcomitans and total bacteria in subgingival samples.Results: Smokers and never-smokers showed similar values for VPI, GBI, and BOP. Smokers had deeper PD in buccal/lingual sites and higher CAL independently of the tooth surface. The GCF volume was smaller in smokers, independent of the PD. Similar amounts of total bacteria and P. gingivalis were observed for both groups. Significantly higher numbers of D. pneumosintes and M. micros were present in smokers and associated with moderate and deep pockets. When heavy smokers were considered, higher counts of total bacteria, M. micros, and D. pneumosintes were observed.Conclusions: Smoking seems to have a detrimental impact on the periodontal status and microbiological profile of patients with periodontitis. Compared to never-smokers, smokers had deeper pockets, greater periodontal destruction, and higher counts of some putative periodontal pathogens.

Detection of cross infections by Leishmania spp. and Trypanosoma spp. in dogs using indirect immunoenzyme assay, indirect fluorescent antibody test and polymerase chain reaction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein

In this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L-1 of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and continued by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1: 10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1: 1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application. (c) 2007 Elsevier B.V. All rights reserved.

Detection of leptospires in bovine semen by polymerase chain reaction

Objective: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. Design: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. Result: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. Conclusion: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.