Molecular and serological detection of occult hepatitis B virus among healthy hepatitis B surface antigen-negative blood donors in Malaysia.

Background: Occult hepatitis B infections are becoming a major global threat, but the available data on its prevalence in various parts of the world are often divergent. Objective: This study aimed to detect occult hepatitis B virus in hepatitis B surface antigen-negative serum using anti-HBc as a marker of previous infection. Patient and Methods: A total of 1000 randomly selected hepatitis B surface antigen-negative sera from blood donors were tested for hepatitis B core antibody and hepatitis B surface antibody using an ELISA and nested polymerase chain reaction was done using primers specific to the surface gene (S-gene). Results: Of the 1000 samples 55 (5.5%) were found to be reactive, of which 87.3% (48/55) were positive for hepatitis B surface antibody, indicating immunity as a result of previous infection however, that does not exclude active infection with escaped mutant HBV. Nested PCR results showed the presence of hepatitis B viral DNA in all the 55 samples that were positive for core protein, which is in agreement with the hepatitis B surface antibody result. Conclusion: This study reveals the 5.5% prevalence of occult hepatitis B among Malaysian blood donors as well as the reliability of using hepatitis B core antibody in screening for occult hepatitis B infection in low endemic, low socioeconomic settings.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Complete genome of a dengue virus serotype 4 strain from Amazonas, Brazil

Dengue virus (DENV) infections represent a significant concern for public health worldwide, being considered as the most prevalent arthropod-borne virus regarding the number of reported cases. In this study, we report the complete genome sequencing of a DENV serotype 4 isolate, genotype II, obtained in the city of Manaus, directly from the serum sample, applying Ion Torrent sequencing technology. The use of a massive sequencing technology allowed the detection of two variable sites, one in the coding region for the viral envelope protein and the other in the nonstructural 1 coding region within viral populations.

Detection of all four dengue serotypes in Aedes aegypti female mosquitoes collected in a rural area in Colombia

The Aedes aegypti vector for dengue virus (DENV) has been reported in urban and periurban areas. The information about DENV circulation in mosquitoes in Colombian rural areas is limited, so we aimed to evaluate the presence of DENV in Ae. aegypti females caught in rural locations of two Colombian municipalities, Anapoima and La Mesa. Mosquitoes from 497 rural households in 44 different rural settlements were collected. Pools of about 20 Ae. aegypti females were processed for DENV serotype detection. DENV in mosquitoes was detected in 74% of the analysed settlements with a pool positivity rate of 62%. The estimated individual mosquito infection rate was 4.12% and the minimum infection rate was 33.3/1,000 mosquitoes. All four serotypes were detected; the most frequent being DENV-2 (50%) and DENV-1 (35%). Two-three serotypes were detected simultaneously in separate pools. This is the first report on the co-occurrence of natural DENV infection of mosquitoes in Colombian rural areas. The findings are important for understanding dengue transmission and planning control strategies. A potential latent virus reservoir in rural areas could spill over to urban areas during population movements. Detecting DENV in wild-caught adult mosquitoes should be included in the development of dengue epidemic forecasting models.

Zika virus damages the human placental barrier and presents marked fetal neurotropism

An unusually high incidence of microcephaly in newborns has recently been observed in Brazil. There is a temporal association between the increase in cases of microcephaly and the Zika virus (ZIKV) epidemic. Viral RNA has been detected in amniotic fluid samples, placental tissues and newborn and fetal brain tissues. However, much remains to be determined concerning the association between ZIKV infection and fetal malformations. In this study, we provide evidence of the transplacental transmission of ZIKV through the detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestation. We observed chronic placentitis (TORCH type) with viral protein detection by immunohistochemistry in Hofbauer cells and some histiocytes in the intervillous spaces. We also demonstrated the neurotropism of the virus via the detection of viral proteins in glial cells and in some endothelial cells and the observation of scattered foci of microcalcifications in the brain tissues. Lesions were mainly located in the white matter. ZIKV RNA was also detected in these tissues by real-time-polymerase chain reaction. We believe that these findings will contribute to the body of knowledge of the mechanisms of ZIKV transmission, interactions between the virus and host cells and viral tropism.

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.