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Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60-75% and specificity 71-80%; NS1 RDT sensitivity was 38-71% and specificity 76-80%; the IgM anti-DENV RDTs sensitivity was 30-96%, with a specificity of 86-92%, and IgM anti-DENV ELISA sensitivity was 96-98% and specificity 78-91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88-94%.

El Salvador is a Central American country that has been affected by several dengue outbreaks. This study investigated the levels of IgM, IgA, and IgE anti-dengue antibodies in serum samples from children in El Salvador, with a clinical and serological diagnosis of dengue infection during the dengue 4 outbreak in 2002-2003. Seventy one serum samples were tested by ELISA and cases were classified in three groups: 13 primary dengue fever (PDF), 21 secondary dengue fever (SDF), and 37 secondary dengue hemorrhagic fever (SDHF). Also, the specificity of anti-dengue IgM for the different serotypes was tested. No significant differences in the IgM response were found between PDF and SDF, but these were detected between PDF and SDHF (P = 0.0053) and between SDF and SDHF (P = 0.0003). The IgA and IgE values showed a statistically significant difference between primary and secondary groups. The highest positivity percentage of IgA was between 95% (SDF) and 100% (SDHF) towards day 7 of onset of fever. All secondary cases were positive for IgE antibodies. The specificity of IgM was determined for DENV-4 virus in primary and secondary DF groups. This is the first study on dengue cases in Salvadorian children related to the immune response of different immunoglobulins to the type of infection and the clinical picture. Further prospective studies are needed to define if the pattern of immunoglobulins can determine early dengue infection and/or severity., (© 2013 Wiley Periodicals, Inc.)

Objectives: Recognizing the uniqueness of secondary dengue virus (DENV)-1/3 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) cases at an interval of 24 years, we sought to estimate DENV infections as well as the ratios between mild disease and DHF/DSS by DENV infection sequence in Playa District (Havana, Cuba) during the 2001-2002 outbreak of dengue virus type 3 (DENV-3)., Methods: A retrospective seroepidemiological study was conducted in 2003 in Playa District. Blood samples were collected from a 1% random sample of residents and were studied for the prevalence of dengue neutralizing antibodies., Results: DENV-3 was found to have infected 7.2% (95% confidence interval (95% CI) 6.0-8.4%) of susceptible individuals (the entire cohort), the majority of whom experienced silent infections. Virtually every individual who had a secondary infection in the sequence DENV-1 then DENV-3 became ill, with a ratio of severe to mild cases of 1:35 (95% CI 1:67-1:23). Secondary infections in the sequence DENV-2/3 were less pathogenic than DENV-1/3. Mild disease accompanying secondary DENV2/3 occurred at a ratio of 1:4.49 infections (95% CI 1:5.77-1:3.42) secondary infections., Conclusions: The results obtained highlight the role of the infecting serotype and also the sequence of the viral infection in the clinical outcome of a dengue infection., (Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

In this work, the presence of NS1 protein as a possible early marker of dengue infection was studied in serum samples from confirmed adult patients with a primary and secondary dengue 4 infection. A total of 209 serum samples collected from day 2 up to day 7 of fever onset from 71 patients were tested by Platelia NS1 antigen capture ELISA kit (BioRad, Marnes-la-Coquette, France), and the results were compared with those obtained by capture antidengue virus IgM (MAC)-ELISA and ELISA inhibition method tests. The 83.3% of primary cases and 96.4% of secondary cases were NS1 positive. The kinetics of NS1 protein showed the highest values in optical density mean ratio or in percentage of positives between days 2 and 4. The results obtained in this study show the utility of the NS1 protein as a virologic early marker of dengue infection. Prospective studies should be carried out to confirm its utility as a prognostic marker of severe illness.

Using a serological test, different criteria have been established for classifying a case as primary or secondary dengue virus infection. Considering the dengue epidemiological situation in Cuba, IgG antibody response to dengue virus infection in serum samples from children and adults with a dengue 3 infection, in Havana city during the 2001-2002 epidemic was evaluated. Samples were collected on days 5-7 of fever onset and tested by an ELISA inhibition. A total of 713 serum samples positive for IgM antibody, 93 from children and 620 from adult patients were studied. Serum samples collected from healthy blood donors and patients not infected with dengue were included as controls. An IgG primary infection pattern was observed in sera collected from children, with titers of < or =20 in the 89.3% of the patients, while both, a primary and secondary patterns were observed in sera collected from adult patients with titers of < or =20 (13.4%) and > or =1280 (83.9%), respectively. These results permitted the definition of a primary or secondary case of dengue virus infection in serum samples collected during the acute phase of dengue virus infection.

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.

The humoral immune response in Honduran dengue hemorrhagic fever (DHF) hospitalized pediatric cases from the epidemics of 2004 and 2005 was studied in sera collected from 5 to 7 days of fever onset. A total of 145 cases were included in the study: 40 classified as primary with DHF Grade I or II and 86 classified as secondary; from them, 73 were DHF Grade I or II and 13 were dengue shock syndrome (DSS) Grade III or IV. The highest number of primary cases was found in children < 1 year of age. The highest number of secondary cases was observed in children between 5 and 10 years of age. The IgA values showed a statistically significant difference between primary and secondary groups. The relationship between antibody responses and severity grade is discussed. This is the first study related to the humoral immune response and severity grade in DHF cases in Honduran children.

Background: Serological assays are widely used to confirm dengue virus infections and to differentiate between a primary and a secondary infection., Objective: Two commercial dengue diagnostic kits, Panbio Dengue IgM Capture and Dengue IgG Capture ELISA (Brisbane, Australia) were evaluated., Study Design: Three hundred and seventy-three serum samples were tested. Panel sera included samples from dengue confirmed cases (representing both primary and secondary infections), from non-dengue infectious diseases, and from healthy individuals. The MAC-ELISA/Dengue IPK was used for the detection of anti-dengue virus IgM antibody in the sera and the ELISA inhibition method (EIM/Dengue IPK) was used to differentiate between primary and secondary infections. Both these reference assays, which were previously developed in the Arbovirus Laboratory at the "Pedro Kouri" Tropical Medicine Institute, were employed as the gold standard., Results: High sensitivity (96.8%) and specificity (99.4%) were found with the commercial diagnostics when compared to the reference methods. Furthermore, high concordance 95.5% in classifying dengue infection types (primary or secondary infections) was observed., Conclusions: The Panbio Dengue IgM and IgG assays offer a good alternative for dengue diagnosis. They are easy to perform and results can be obtained in less than 3h.

Severity of disease is markedly increased when infection with dengue virus type 2 (DENV-2) follows infection with DENV-1 at an interval of 20 years. Studies have shown that heterologous neutralizing antibody titers are inversely correlated with severity of a second infection. If this mechanism controlled disease severity in Cuba, heterotypic antibody titers should have declined over time. To determine whether phenotypic changes in dengue antibodies occur over time, we analyzed serum samples collected 4-8 and 20-22 years after DENV-1 infection. We found a significant increase in mean titer of homologous DENV-1 neutralizing antibodies and a significant decrease in heterologous antibodies to 1 of 2 genotypes of DENV-2 virus (the American genotype). Asian DENV-2 viruses were not neutralized during either interval; however, the American genotype underwent phenotypic changes in heterotypic viral neutralizing antibodies in the predicted direction. This finding may be related to the time-dependent changes in severity of disease found with secondary dengue infection.

Chimeric yellow fever (YF)-dengue type 2 (Den 2) viruses were constructed by replacing the premembrane (prM) and envelope (E) genes of YF 17D virus with those from Den 2 virus strains of south-east Asian genotype. Whereas viable chimeric viruses were successfully recovered when the YF 17D C gene and the Den 2 prM gene were fused at the signalase cleavage site, no virus could be rescued from the constructions fused at the viral protease cleavage site. Unlike YF virus that replicated in all the cell lines tested and similar to the Den 2 virus, the recombinant viruses did not replicate in vaccine-production certified CEF and MRC5 cells. Besides, chimeric 17D/Den 2 viruses and their parental viruses reached similar growth titers in Vero and C6/36 cell cultures. Analysis of mouse neurovirulence, performed by intracerebral inoculation, demonstrated that the 17D/Den 2 chimera is more attenuated in this system than the YF 17DD virus. Immunization of mice with this chimera induced a neutralizing antibody response associated with a partial protection against an otherwise lethal dose of mouse neurovirulent Den 2 NGC virus. Overall, these results provide further support for the use of chimeric viruses as an attractive methodology for the development of new live flavivirus vaccines.

During the 1981 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) Cuban epidemic, bronchial asthma (BA) was frequently found as a personal or family antecedent in dengue hemorrhagic fever patients. Considering that antibody dependent enhancement (ADE) plays an important role in the etiopathogenic mechanism of DHF/DSS, we decide to study the Dengue 2 virus (D2V) capability of replication in peripheral blood leukocytes (PBL) from asthmatic patients and healthy persons. In 90% of asthmatic patients and 53.8% of control group it was obtained PBL with a significant D2V enhancing activity (X2 p < 0.01). Power enhancement was higher in asthmatic group. This is the first in vitro study relating BA and the dengue 2 virus immuno enhancement. The results obtained support the role of BA as a risk factor for DHF/DSS as already described on epidemiological data.

In Cuba, 2 epidemics of dengue virus occurred: 1 caused by DEN-1 in 1977 and 1 caused by DEN-2 in 1981. The latter was associated with cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). To study viral risk factors for DHF/DSS, a retrospective seroepidemiological survey was conducted in Cerro, a densely populated district in Havana City. The prevalence of plaque reduction neutralizing antibodies to DEN-1 and DEN-2 viruses was measured in 1,295 individuals (children and adults). Of these, 43.7% were immune to DEN-1 virus and 23.6% to DEN-2 virus. Of those individuals who were immune, 26.1% were immune to DEN-1 virus only, 6% to DEN-2 virus only, and 17.6% to both viruses. The DEN-2 virus infection rate in DEN-1 immune individuals was 3.8 times higher than in non-immune individuals. The 5 DHF/DSS cases in the sample had evidence of DEN-1 virus plus DEN-2 virus infections. Three were children and 2 were young adults. No cases were found in individuals infected with DEN-1 virus or DEN-2 virus only. Children infected by DEN-1 virus followed by DEN-2 virus had a high risk of acquiring DHF/DSS. Blacks and whites were equally infected with DEN-1 and DEN-2 viruses.