Cacipacore virus as an emergent mosquito-borne Flavivirus

Abstract INTRODUCTION: Cacipacore virus (CPCV), a possible bird-associated flavivirus, has yet to be detected in mosquitoes. Our purpose is examining CPCV in mosquitoes from the Amazon region of Brazil. METHODS: Approximately 3,253 Culicidae (grouped into 264 pools) were collected from the Amazon region during 2002-2006 and analyzed using a Flavivirus genus-specific reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assay and by nucleotide sequencing of amplicons. RESULTS: Nucleotide sequences from five mosquito samples showed high similarity to the those of CPCV originally isolated in the Amazon region. CONCLUSIONS: This is the first report of CPCV-infected mosquitoes which has implications on the arbovirus maintenance in nature and transmission to man.

Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Molecular and clinical evaluation of the acute human parvovirus B19 infection: comparison of two cases in children with sickle cell disease and discussion of the literature

Human parvovirus B19 is a well-known cause of severe conditions in patients with sickle cell disease, but the molecular mechanisms of the infection are insufficiently understood. The different clinical outcome of the acute parvovirus B19 infection in two pediatric patients with sickle cell disease has been examined. One of them developed life-threatening condition requiring emergency transfusions, while the other had asymptomatic infection, diagnosed occasionally. Both cases had high viral load and identical subgenotype, indicating that the viral molecular characteristics play a minimal role in the infection outcome.

Padronização e uso de um método imunoenzimático que utiliza células infectadas como antígeno no diagnóstico rotineiro do dengue / Standardization and use of an immunoenzymatic method using infected cells as antigens in routine diagnosing of dengue

INTRODUÇÃO: Este trabalho mostra a padronização e o uso do método imunoenzimático utilizando células infectadas como antígeno (EIA-ICC) no diagnóstico sorológico rotineiro do dengue. MÉTODOS: Na otimização do teste, com a dose de 1.000 TCID50 de vírus do dengue tipo 3 (DENV-3), foram utilizadas 100.000 células C636 infectadas 1000 TCID50 (DENV-3). RESULTADOS: Os resultados obtidos com EIA-ICC foram comparados com o kit comercial de dengue HUMAN. Os resultados foram altamente coincidentes; o EIA-ICC mostrou-se moderadamente sensível e com alta especificidade. O teste foi usado no diagnóstico sorológico de 1.797 amostras sorológicas de casos suspeitos de dengue durante a epidemia de Ribeirão Preto, em 2006. Na avaliação sorológica, 228 amostras foram positivas para IgM contra DENV-3, e 235 amostras foram positivas para IgG contra DEV-3, e em 35 amostras detectou-se positividade para IgM e IgG. CONCLUSÕES: O EIA-ICC mostrou-se confiável e simples sendo adequado ao diagnóstico sorológico do dengue.

INTRODUCTION: This paper show the standardization and use of the immunoenzymatic method using infected cells as antigens (EIA-ICC) for routine serological diagnosing of dengue. METHODS: In optimizing the test, a dose of 1,000 TCID50 of dengue type 3 virus (DENV-3) was used, and 100,000 C636 cells infected with 1,000 TCID50 (DENV-3) were used. RESULTS: The results obtained with EIA-ICC were compared with the HUMAN commercial dengue kit. The results were highly concordant. The EIA-ICC showed moderate sensitivity and high specificity. The test was used for serologically diagnosing 1,797 blood samples from suspected dengue cases during the 2006 epidemic in Ribeirao Preto. From the serological evaluation, 228 samples were positive for IgM against DENV-3; 235 samples were positive for IgG against DENV-3; and 35 samples were positive for both IgG and IgM. CONCLUSIONS: EIA-ICC was shown to be reliable and simple, and suitable for serologically diagnosing dengue.

The use of qualitative and quantitative polymerase chain reactions for diagnosis of cytomegalovirus infections in bone marrow and kidney transplant recipients

O objetivo deste trabalho foi testar uma PCR qualitativa e uma PCR semiquantitativa para CMV para determinar a carga de CMV nos leucócitos de pacientes transplantados de medula óssea e transplantados de rim. Trinta e três pacientes TMO e 35 TR participaram deste estudo. O DNA foi testado pela PCR qualitativa utilizando primers que amplificam parte do gene gB de CMV. As cargas de CMV das amostras positivas foram determinadas pela PCR semi-quantitativa utilizando como controle plasmídios quantificáveis inseridos com parte do gene gB de CMV. A sensibilidade do teste foi de 867 plamídios/µg DNA. Cargas de CMV entre 2.118 e 72.443 copias/µg DNA foram observadas em 12,1 por cento dos TMO entre 1,246 e 58,613 cópias/µg DNA foram observadas em 22,9 por cento dos TR. Futuros estudos, com maiores casuísticas são necessários para confirmar a utilidade desta PCR semiquantitativa para CMV em pacientes transplantados.

Diagnosis of cytomegalovirus infections by qualitative and quantitative PCR in HIV infected patients

A high incidence of cytomegalovirus (CMV) infections is observed in Brazil. These viruses are causatives of significant morbidity and mortality among patients with advanced human immunodeficiency virus (HIV) infection. This work, shows the application of a PCR on determination of CMV load in the buffy coat and plasma. We analyzed the samples of 247 HIV infected patients in order to diagnose CMV infection and disease. We developed a semi-quantitative PCR that amplifies part of the glycoprotein B (gB) gene of CMV. The semi-quantitative PCR was carried out only in positive clinical samples in a qualitative PCR confirmed by a nested-PCR. CD4 lymphocyte count, HIV viral load and CMV disease symptom were correlated with CMV load. CMV genome was detected in the buffy coat of 82 of 237 (34.6 percent) patients, in 10 of these the CMV load was determined varying between 928 and 332 880 viral copies/mug DNA. None of these 237 patients developed any suggestive manifestation of CMV disease. For the other 10 HIV infected patients selected based on the suspicion of CMV disease, CMV genome was detected in only one case. This patient presented a high CMV load, 8 000 000 copies/mug DNA, and developed a disseminated form of CMV disease including hepatitis and retinitis. Our results were greatly influenced by the impact of the highly active antiretroviral therapy that reduced incidence of CMV viremia and occurrence of CMV disease in the HIV infected patients