Reliable Classifier to Differentiate Primary and Secondary Acute Dengue Infection Based on IgG ELISA

Dengue virus infection causes a wide spectrum of illness, ranging from sub-clinical to severe disease. Severe dengue is associated with sequential viral infections. A strict definition of primary versus secondary dengue infections requires a combination of several tests performed at different stages of the disease, which is not practical.We developed a simple method to classify dengue infections as primary or secondary based on the levels of dengue-specific IgG. A group of 109 dengue infection patients were classified as having primary or secondary dengue infection on the basis of a strict combination of results from assays of antigen-specific IgM and IgG, isolation of virus and detection of the viral genome by PCR tests performed on multiple samples, collected from each patient over a period of 30 days. The dengue-specific IgG levels of all samples from 59 of the patients were analyzed by linear discriminant analysis (LDA), and one- and two-dimensional classifiers were designed. The one-dimensional classifier was estimated by bolstered resubstitution error estimation to have 75.1% sensitivity and 92.5% specificity. The two-dimensional classifier was designed by taking also into consideration the number of days after the onset of symptoms, with an estimated sensitivity and specificity of 91.64% and 92.46%. The performance of the two-dimensional classifier was validated using an independent test set of standard samples from the remaining 50 patients. The classifications of the independent set of samples determined by the two-dimensional classifiers were further validated by comparing with two other dengue classification methods: hemagglutination inhibition (HI) assay and an in-house anti-dengue IgG-capture ELISA method. The decisions made with the two-dimensional classifier were in 100% accordance with the HI assay and 96% with the in-house ELISA.Once acute dengue infection has been determined, a 2-D classifier based on common dengue virus IgG kits can reliably distinguish primary and secondary dengue infections. Software for calculation and validation of the 2-D classifier is made available for download

Acute norovirus gastroenteritis in children in a highly rotavirus-vaccinated population in Northeast Brazil.

Background: Gastroenteritis is one of the most important causes of morbidity and mortality in children and an important etiological agent is norovirus. Objective: We describe the occurrence and characteristics of norovirus diarrhoea in children from Sergipe, Northeast-Brazil, over two consecutive periods of three years following rotavirus vaccine introduction. Study design: A cross sectional hospital-based survey conducted from October-2006 to September-2009 and from July-2011 to January-2013. Acute diarrhoea cases had a stool sample collected and tested for norovirus by RT-PCR and positive samples were sequenced. Results: In total 280 (19.6%) of 1432 samples were norovirus positive, including 204 (18.3%) of 1,113 samples collected during the first period and 76 (23.9%) of 318 collected during the second period. The proportion of children with norovirus infection increased significantly through the second study period (χ2 for trend = 6.7; p = 0.009), was more frequent in rotavirus vaccinated and in younger children (p < 0.001). Of 280 norovirus-positive specimens, 188 (67.1%) were sequenced. Of these, 12 were genogroup I and 176 genogroup II. The main genotype was GII.4 (149/188, 79.3%), followed by GII.2 (6, 3.2%) and GII.6 (5, 2.6%). Conclusion: Norovirus annual detection rates increased over the study period. The detection of norovirus was higher among young children

Characterization of Dengue Virus Type 2: New Insights on the 2010 Brazilian Epidemic

Dengue viruses (DENV) serotypes 1, 2, and 3 have been causing yearly outbreaks in Brazil. In this study, we report the re-introduction of DENV2 in the coast of São Paulo State. Partial envelope viral genes were sequenced from eighteen patients with dengue fever during the 2010 epidemic. Phylogenetic analysis showed this strain belongs to the American/Asian genotype and was closely related to the virus that circulated in Rio de Janeiro in 2007 and 2008. The phylogeny also showed no clustering by clinical presentation, suggesting that the disease severity could not be explained by distinct variants or genotypes. The time of the most recent common ancestor of American/Asian genotype and the São Paulo and Rio de Janeiro (SP/RJ) monophyletic cluster was estimated to be around 40 and 10 years, respectively. Since this virus was first identified in Brazil in 2007, we suggest that it was already circulating in the country before causing the first documented outbreak. This is the first description of the 2010 outbreak in the State of São Paulo, Brazil, and should contribute to efforts to control and monitor the spread of DENVs in endemic areas

Comparison of Three Commercially Available Dengue NS1 Antigen Capture Assays for Acute Diagnosis of Dengue in Brazil

Dengue is the one of the most prevalent arthropod-borne viral diseases in tropical regions of the world. Manifestations may vary from asymptomatic to potentially fatal complications. Laboratorial diagnosis is essential to diagnose dengue and differentiate it from other diseases. Dengue virus non-structural protein 1 (NS1) may be used as a marker of acute dengue virus infection. Our results, based in the comparison of three NS1 antigen capture assays available, have shown that this approach is reliable for the early diagnosis of dengue infections, especially in the first four days after the onset of the symptoms. A lower sensitivity was observed in DENV-3 cases. Serum positive by virus isolation were more often detected than those positive by RT-PCR by all three assays. Only the Platelia™ NS1 test showed a higher sensitivity in confirming primary infections than secondary ones. In conclusion, NS1 antigen capture commercial kits are useful for diagnosis of acute primary and secondary dengue infections and, in endemic countries where secondary infections are expected to occur, may be used in combination with MAC-ELISA to increase the overall sensitivity of both tests

Identification of Continuous Human B-Cell Epitopes in the Envelope Glycoprotein of Dengue Virus Type 3 (DENV-3)

BACKGROUND:Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. METHODOLOGY/PRINCIPAL FINDINGS:Synthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51-65, 71-90, 131-170, 196-210 and 246-260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51-65), 27 and 28 (aa131-150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51-65, 131-170, 196-210 and 246-260 elicited the highest antibody response and epitopes131-170, 196-210 and 246-260, elicited IFN-gamma production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. CONCLUSIONS/SIGNIFICANCE:Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine

Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection

ABSTRACT: Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV) replication. These cells are activated after infection, producing proinflammatory mediators, including tumour-necrosis factor-α, which has also been detected in vivo. Nitric oxide (NO), usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities. METHODS: The expression of DENV antigens and inducible nitric oxide synthase (iNOS) in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using N(G)-methyl L-Arginine (N(G)MLA) as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP), a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis. RESULTS: INOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days), significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with N(G)MLA. In the mosquito cell line C6/36, virus detection was significantly reduced in the presence of SNP, when compared to that of untreated cells. CONCLUSION: This study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production