BACKGROUND Dengue virus type 4 (DENV-4) was first reported in Brazil in 1982 and since then no more cases were detected again in Brazil until 2010, when the virus was reintroduced. Over the following years, the virus spread to several Brazilian states and resulted in about 1,400,000 dengue cases, in 2013. The largest number of cases were documented in the Southeast macro-region. OBJECTIVES To determine the phylogeography of DENV-4 Genotype IIB strains isolated during the epidemics in 2012-2013 in São Paulo, Brazil, we aimed to contextualise the contribution of viruses sampled in different localities across the overall movement of DENV-4 in Brazil. METHODS Based on the envelope gene sequences retrieved from GenBank, we employed a Bayesian phylogeographic approach to assess the spatiotemporal dynamics of DENV-4 Genotype IIB in São Paulo, Brazil. FINDINGS The dispersal dynamics of DENV-4 Genotype IIB in Brazil indicated Rio de Janeiro and Mato Grosso states as the most likely routes toward São Paulo before the 2012-2013 outbreak. Likewise, Guarujá and São José do Rio Preto facilitated viral spread and transmission to other localities in the South and Southeast macro-regions in Brazil. CONCLUSIONS The spread pattern of DENV-4 Genotype IIB strains across the country supports two independent introductions of the virus in São Paulo in a short period of time. Furthermore, São Paulo appears to have played a pivotal role in the dissemination of DENV-4 to other locations in Brazil.

ABSTRACT Many countries in the Americas have detected local transmission of multiple arboviruses that cause febrile illnesses. Therefore, laboratory testing has become an important tool for confirming the etiology of these diseases. The present study aimed to compare the sensitivity and specificity of three different Zika virus detection assays. One hundred serum samples from patients presenting with acute febrile symptoms were tested using a previously reported TaqMan® RT-qPCR assay. We used a SYBR® Green RT-qPCR and a conventional PCR methodologies to compare the results. Of the samples that were determined to be negative by the TaqMan® RT-qPCR assay, 100% (Kappa = 0.670) were also found to be negative by SYBR® Green RT-qPCR based on Tm comparison; however, 14% (Kappa = 0.035) were found to be positive by conventional PCR followed by agarose gel electrophoresis. The differences between the ZIKV strains circulating worldwide and the low viremia period can compromise diagnostic accuracy and thereby the accuracy of outbreak data. Therefore, improved assays are required to improve the diagnosis and surveillance of arbovirus.

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.

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.

Abstract Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1–4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population.

RESUMO A co-infecção por dengue vírus pode ocorrer em áreas com circulação endêmica, nas quais diferentes sorotipos vêm circulando durante muitos anos. Neste trabalho relatamos um caso de co-infecção por DENV-1/DENV-4 em soro humano, detectado por testes moleculares. Análises filogenéticas das sequências obtidas indicaram a presença do genótipo V e II de DENV-1 e DENV-4, respectivamente.

SUMMARY The natural co-infection with dengue virus can occur in highly endemic areas where different serotypes have been observed for many years. We report one case of DENV-1/DENV-4 co-infection in human serum detected by molecular tests. Phylogenetic analysis of the sequences obtained indicated the presence of genotype V and II for DENV-1 and DENV-4, respectively.

OBJETIVO: Verificar se existe contaminação por fungos antes e após limpeza e desinfecção terminal de colchões hospitalares utilizados por portadores de candidemia. MÉTODOS: Estudo transversal que investigou 25 colchões de diferentes unidades hospitalares e utilizados por pacientes com candidemia, confirmados por hemocultura. Utilizou-se amostragem por conveniência. Após crescimento em Ágar Sabouraud Dextrose as leveduras isoladas foram identificadas pelas características macroscópicas, microscópicas e fisiológicas. RESULTADOS: Totalizou-se 15 (60%) colchões contaminados com Candida spp. Desse total, 10 (66,7%) e cinco (33,3%) corresponderam respectivamente à coleta antes e após a desinfecção dos colchões, sendo que a espécie mais frequentemente isolada foi Candida parapsilosis. CONCLUSÃO: Considerando que a metade dos colchões permaneceram contaminados após o processo de limpeza e desinfecção, pode-se inferir sobre o risco destes atuarem como reservatórios secundários na cadeia de infecção.

OBJECTIVE: To verify the existence of fungal contamination prior to and following the cleaning and disinfection process of hospital mattresses used by patients with Candidemia. METHODS: Cross-sectional study analyzing 25 mattresses used by patients with Candidemia confirmed by blood culture from different hospital wards. The study made use of convenience samples. After growing the samples in an Agar Sabouraud Dextrose environment, isolated yeasts were identified by macroscopic, microscopic and physiologic characteristics. RESULTS: Analyses showed 15 (60%) mattresses contaminated by Candida spp. From these, 10 (66.7%) and five (33.3%) mattresses corresponded respectively to the collection prior to and following disinfection, with Candida parapsilosis being the isolated species with the highest frequency. CONCLUSION: Considering that half of the mattresses remained contaminated after cleaning and disinfection, there is a risk that these mattresses may act as potential secondary reservoirs in the infection chain.

OBJECTIVE: To verify the existence of fungal contamination prior to and following the cleaning and disinfection process of hospital mattresses used by patients with Candidemia. METHODS: Cross-sectional study analyzing 25 mattresses used by patients with Candidemia confirmed by blood culture from different hospital wards. The study made use of convenience samples. After growing the samples in an Agar Sabouraud Dextrose environment, isolated yeasts were identified by macroscopic, microscopic and physiologic characteristics. RESULTS: Analyses showed 15 (60%) mattresses contaminated by Candida spp. From these, 10 (66.7%) and five (33.3%) mattresses corresponded respectively to the collection prior to and following disinfection, with Candida parapsilosis being the isolated species with the highest frequency. CONCLUSION: Considering that half of the mattresses remained contaminated after cleaning and disinfection, there is a risk that these mattresses may act as potential secondary reservoirs in the infection chain.

OBJETIVO: Verificar se existe contaminação por fungos antes e após limpeza e desinfecção terminal de colchões hospitalares utilizados por portadores de candidemia. MÉTODOS: Estudo transversal que investigou 25 colchões de diferentes unidades hospitalares e utilizados por pacientes com candidemia, confirmados por hemocultura. Utilizou-se amostragem por conveniência. Após crescimento em Ágar Sabouraud Dextrose as leveduras isoladas foram identificadas pelas características macroscópicas, microscópicas e fisiológicas. RESULTADOS: Totalizou-se 15 (60%) colchões contaminados com Candida spp. Desse total, 10 (66,7%) e cinco (33,3%) corresponderam respectivamente à coleta antes e após a desinfecção dos colchões, sendo que a espécie mais frequentemente isolada foi Candida parapsilosis. CONCLUSÃO: Considerando que a metade dos colchões permaneceram contaminados após o processo de limpeza e desinfecção, pode-se inferir sobre o risco destes atuarem como reservatórios secundários na cadeia de infecção.