BackgroundWe compared the performance of two new commercial tests for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection—an immunochromatographic test allowing rapid detection of the NS1 antigen, Dengue NS1 Ag STRIP (Bio-Rad Laboratories - Marnes La Coquette, France), and a two-step sandwich-format microplate enzyme-linked immunosorbent assay (ELISA), pan-E Dengue Early ELISA (Panbio - Brisbane, Australia)—with a one-step sandwich-format microplate ELISA, the Platelia Dengue NS1 Ag test (Bio-Rad).MethodsWe tested 272 serum samples from patients with dengue disease. Of these, 222 were from patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation. Forty-eight acute-phase serum samples from patients not infected with dengue virus were also included.ResultsThe sensitivity of the Platelia Dengue NS1 Ag test on acute serum samples (n = 222) was 87.4% (95% confidence interval: 82.3% to 91.5%); that of Dengue NS1 Ag STRIP was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%). The pan-E Dengue Early ELISA had a sensitivity of 60.4% (95% CI: 53.4% to 66.8%) and a specificity of 97.9% (95% CI: 88.9% to 99.9%).ConclusionOur findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The immunochromatographic test, Dengue NS1 Ag STRIP—the first rapid diagnostic test for DENV infection—was highly sensitive and specific, and would therefore be a suitable first-line test in the field. The pan-E Dengue Early ELISA was less sensitive than the Platelia test; this two-step ELISA should be combined with DENV IgM antibody detection for the diagnosis of DENV infection.
The Flavivirus genus includes widespread and severe human pathogens like the four serotypes of dengue virus (DENV1 to DENV4), yellow fever virus, Japanese encephalitis virus and West Nile virus. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by virus neutralizing antibodies. Its structural, antigenic and immunogenic properties have been thoroughly studied contrary to its physico-chemical properties. Here, the ED3 domains of the above pathogenic flaviviruses were produced in the periplasm of Escherichia coli. Their thermodynamic stabilities were measured and compared in experiments of unfolding equilibriums, induced with chemicals or heat and monitored through protein fluorescence. A designed ED3 domain, with the consensus sequence of DENV strains from all serotypes, was highly stable. The low stability of the ED3 domain from DENV3 was increased by three changes of residues in the protein core without affecting its reactivity towards DENV-infected human serums. Additional changes showed that the stability of ED3 varied with the DENV3 genotype. The T(m) of ED3 was higher than 69°C for all the tested viruses and reached 86°C for the consensus ED3. The latter, deprived of its disulfide bond by mutations, was predominantly unfolded at 20°C. These results will help better understand and design the properties of ED3 for its use as diagnostic, vaccine or therapeutic tools.
* RT-PCR = reverse transcriptase-polymerase chain reaction. † Laboratory-confirmed dengue cases based on positive NS1 or/and IgM patients reported by the laboratories. DENGUE VIROLOGICAL SURVEILLANCE USING FILTER PAPERfor DENV-1 (336 of 352), 4.3% for DENV-2 (15 of 352), and 0.3% for DENV-4 (1 of 352) ( Table 3 ).The first epidemic reported in Saint Barthélemy began at the end of October 2008 (Week 43), within 3 weeks after the dengue epidemic alert was issued for Saint Martin ( Table 3 and Figure 2 ). The biological surveillance network reported 152 cases, of which 62 of them were analyzed by RT-PCR ( Table 3 ). This epidemic was similar to the one observed in neighboring Saint Martin in two characteristics: i) DENV-1 was the predominant serotype (91.9%; 57 of 62), and ii) the epidemic ended in February 2010 (Week 07). This dengue outbreak was followed by a 39-week long inter-epidemic period of sporadic transmission.In mid-November 2009, another dengue outbreak took place in Saint Barthélemy. Although the two epidemics had similar durations (17 weeks in 2008-2009 versus 16 weeks in 2009-2010), 2-fold more laboratory-confirmed dengue cases were reported for the latter Saint Barthélemy outbreak ( N = 379) than for the previous one ( N = 152). One hundred seventeen NS1-positive blood samples were subtyped by RT-PCR: 114 were DENV-1 (97.4%) and 3 were DENV-2 (2.6%). Although the DENV-1 serotype predominated during this period in Saint Barthélemy, the DENV-2 serotype was found to predominate among specimens provided from the same period in Saint Martin. DISCUSSIONDengue has become a major public health burden in tropical and sub-tropical regions of the world, partly because of the increased number of reported cases, particularly severe clinical forms, and the increased number of deaths attributable to this disease. With no specific treatments available for patient management and no vaccine yet available to protect vulnerable populations, prevention of this arboviral disease plays a crucial role in its control. Among the different approaches implemented, an active surveillance system was advocated in the 1980s notably to detect dengue outbreaks early and to monitor the introduction of new dengue subtypes. 6,[25][26][27] We reported here the epidemiological usefulness of collecting venous blood samples on filter paper to monitor the dengue serotypes in circulation in two French territories in the Caribbean region: the islands of Saint Martin and Saint Barthélemy. This prospective approach to the virological surveillance of dengue serotypes was evaluated during a 27-month period. It allows detecting dengue serotypes using RT-PCR in 90.1% of overall blood samples provided from NS1-positive dengue cases ( Table 1 ).For samples collected during the acute phase of the disease, the percentage in which dengue virus RNA was detected by RT-PCR analysis was 95.5%. First, these results show the relevance of using, as a first line, such highly specific diagnostic tools for acute dengue diagnosis in areas where techniques for den...
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