The authors have declared that no competing interests exist.
Conceived and designed the experiments: VRFS ACC RQG RLCS. Performed the experiments: VRFS ACC BTDN EVPS. Analyzed the data: VRFS ACC RQG BTDN EVPS SSD RLCS. Contributed reagents/materials/analysis tools: ACC EVPS SSD RLCS. Wrote the paper: VRFS ACC RQG SSD RLCS.
We have identified fifty-eight samples that were positive for Dengue-4 among 119 samples with negative diagnoses for dengue via the Platelia™ dengue NS1 Ag in Aracaju, State of Sergipe, Brazil. We determined that the low sensitivity of the NS1 Ag test could be related to secondary dengue infections in the studied population. Therefore, we concluded that the sensitivity and specificity of the Platelia™ dengue NS1 Ag test as a screening method for monitoring circulating dengue serotypes must be reevaluated. In addition, regional endo-epidemic profiles should also be considered due to the prevalence of secondary responses.
Dengue is considered the most important of the arboviruses affecting humans. It is transmitted mainly by the mosquito
The first dengue epidemic that was documented in both the clinic and laboratory in Brazil occurred in 1981 in Boa Vista, State of Roraima, and DENV-1 and DENV-4 were identified
Since 2009, the NS1 antigen-capture ELISA test has been implemented by the Health Ministry in the Public Health laboratories network. Patients arriving for care within five days of the onset of disease symptoms are screened for viral isolation in health care sentinel units and, thus, provide early detection of circulating serotypes in a given area
Lines 1–4: positive samples; 5–8: negative samples; 9–12: positive control for DENV-1, DENV-2, DENV-3 and DENV-4; NC: negative control; MW: molecular weight marker.
In Sergipe state, routine NS1 Ag test detection protocol is applied to patients in the sentinel units according to Health Ministry determinations. Blood is collected and send to the Central Laboratory (Laboratório Central - LACEN/SE) for the Platelia™ test. Positive biological samples are then forwarded to the reference laboratory in Rio de Janeiro for viral isolation. Negative samples are stored in a −80°C and for this study were provided to our group in order to evaluate the presence of other arbovirus.
This study followed the ethical principles according to the Helsinki Declaration and was approved by the Research Ethics Committee of the Federal University of Sergipe (02872312.7.0000.0058). The samples analyzed were derived from routine diagnosis for dengue fever conducted by the Central Laboratory of Public Health (LACEN). Patients were asked and provided their oral consent to enter in the Ministry of Health protocol of diagnosis and isolation of serotype dengue virus. They were all informed that the serum was kept for further investigations of the circulation of other arboviruses and they were asked to return after 14 days for serological diagnosis. As that step was part of the routine of the dengue control program, there was not written term of consent available. Ethics committee dispensed patient’s written informed consent after we presented signed agreement by the authority responsible for the custody of the samples. Confidentiality of the identity and results was maintained.
To investigate the co-circulation of arboviruses in the State of Sergipe, blood samples negative for the NS1 antigen test from suspected dengue patients were requested from the Central Laboratory of Public Health. Platelia Dengue NS1 Ag test is a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen. Tests were carried out on samples of serum or blood from patients and controls according to the manufacturer’s recommendations. Before being included in the protocol for the detection of other arboviruses these negative blood samples were initially evaluated by semi-nested RT-PCR for dengue
In total, 119 samples of serum or blood from symptomatic patients collected between September 2011 and February 2012 were obtained, and sample RNA was extracted using TRIzol® Plus reagent (Invitrogen™, California, US) according to the manufacturers’ instructions. Target viral RNA was converted to a DNA copy (cDNA) prior to enzymatic DNA amplification by use of reverse transcriptase (RT - MMLV/Invitrogen™) and the dengue virus downstream consensus primer (D2), homologous to the genomic RNA of the four serotypes. Subsequent
When processing the samples, 58 (48.7%) samples were unexpectedly found to be positive for DENV-4 (Fig). No other serotype was detected. Of the 58 positive samples, 33 (56.9%) were from the capital city, Aracaju, while two (3.4%) were from the municipality of Itabaiana, and 23(39.7%) samples were from the municipality of Simão Dias; these two municipalities are 51 and 101 km from the capital, respectively. Positive samples were subjected to viral isolation in
The DENV-4 serotype was detected for the first time in Brazil in 1982 during a dengue outbreak in the north of the country, and since then, no new cases of DENV-4 were detected until 2010
The low sensitivity of the NS1 Ag test should be considered in relation to secondary dengue infections in the studied population
Although the implementation of the NS1 Ag test for viremic sample screening has efficiently increased the viral isolation percentage and permitted the identification of the circulating serotypes in Brazil
Most likely due to the low sensitivity of the Platelia™ NS1 Ag test for DENV-4, there was a delay in detection and an underreporting of DENV-4 dengue cases in Brazil. As this serotype has been increasingly detected in many regions
The authors would like to thank Dr. Pedro Vasconcelos for the critical evaluation of the manuscript, Danuza Duarte Costa and Sandra Maria Araujo Menezes Cavalcante for support with storage and provision of samples.