The introduction of DENV-4 in the state of Roraima, North Brazil in 2010 warned for the possibility of a new dengue epidemics in Brazil [16], as the main vector, Aedes aegypti is widespread in the country. In the disease surveillance, the identification of the vectors has important implications for the disease outbreak control, especially with the rapid disease spread when a new serotype is introduced.
In 2011, the first isolation of DENV- 4 in RJ from cases occurred in the city of Niteroi resulted in the emergence of this serotype in the state in 2012, characterized by isolation of this serotype in 48.7% of confirmed cases. That same year showed for, the first time, the co-circulation of the four DENV serotypes in the state. However, in 2013, DENV-4 was responsible for the largest number of cases in the state.
The Metropolitan region of the state was responsible for most cases occurred during the studied period, mainly in the municipalities of RJ and Niterói. This region has a high population density and high-traffic, which partly explains the concentration of the cases studied [12]. Furthermore, as the Flavivirus Laboratory, FIOCRUZ acts as a Regional Reference Laboratory for dengue diagnosis in Brazil, samples from other municipalities were also received and analyzed.
Although viral isolation is considered the “gold standard” method for dengue viral detection, the sensitivity of the molecular methods in detecting the viral genome offers a great advantage during an epidemic period, as shown by the results presented here. However, it is noteworthy that the high percentage of positive results obtained in RT-PCR may be the result of the selection criteria for the analysis in this study, as known DENV-4 cases were selected. Despite this, previous studies have demonstrated the usefulness of RT-PCR to confirm the cases and identification of the infecting serotype, when the virus isolation was not possible [25, 26].
Serological methods are still the most useful tool for the diagnosis of the disease during epidemics, as observed since its establishment in 1986 in Rio de Janeiro [27]. In this study, the low detection rate of anti-DENV IgM by ELISA (39.4%) was probably due to the acute nature of the cases selected for analysis, as previously described. When it has been described that patients with a primary infection have evidently undetectable IgM antibody titers and there is an increase in detection from a paired sample in the later disease [9]. Usually, higher anti-DENV IgM detection rates are observed after the sixth day after the onset of symptoms. Therefore, considering cases with ≥4 days of symptoms, the detection rate significantly increases.
The usefulness of NS1 Ag capture tests for the early diagnosis of dengue, even in limited resourced setting has been shown [28] and evaluated to confirm primary and secondary acute dengue infections [28,29,30,31]. In this study, initially, the NS1 capture ELISA detection rate was 41.5% on previously confirmed DENV-4 cases. However, after using a heat immune complex dissociation step, the NS1 detection rate significantly increased to 87.6%, as previously described.
In this study, hemorrhage, shock, increased transaminases levels and central nervous system involvement were considered for severe cases classification. In the study, 3.2% of DENV-4 confirmed cases were classified as severe dengue and 12.8% as dengue with warning signs. We observed that abdominal pain is an important warning sign regarding the evolution of the disease and which can help in the correct patient management and thus, avoiding increased severity and fatal outcome. However, due to the small number of severe cases available in this study, no statistical analysis on the disease severity was performed. Furthermore, during the study period, no increase in the disease severity was observed and this is in agreement with previous observations in the same period in the state of RJ [32]. The lower severity on DENV-4 cases is known [33] and here, 14 deaths due to DENV-4 were confirmed in the period studied.
No significant differences between the viremia on mild dengue and severe cases were observed, which does not corroborate with previous studies analyzing other serotypes [34,35,36]. However, a limitation of the present study was the small sample size of severe cases. Moreover, the sampling collection represents the patient’s first visit to the health unit and the information on their progression to a more severe disease is not available.
DENV-4 strains isolated from 2011 to 2013, following this serotype introduction and spread in RJ were partially sequenced and the phylogenetic analysis demonstrated that those belong to genotype II. Moreover, based on previous studies, the sampling size sequenced here is reliable for this analysis considering the epidemic duration, site and the low-mutation characteristic of DENV [16, 37]. The strains of genotype II first isolated during the outbreak of 1982 in Roraima, North Brazil [38] did not spread to other states at that time. However, this genotype was once again detected in Roraima in 2010, being circulated through Central America, Northeastern South America and the Caribbean [39]. According to Temporão et al. (2011) [16], these strains of genotype II were genetically different from those isolated in the 1980s in Roraima and demonstrated a probable origin from Venezuela and Colombia. However, Nunes et al. [40] complete and partial DENV-4 sequences isolated in Brazil identified the circulation of two genotypes (I and II). Genotype II is the most commonly one observed circulating in South America and the Caribbean. Genotype I, one the other hand is represented by a strain isolated in the Northeast of Brazil (Bahia), which may have some evolutionary relationship with Asian strains, according to phylogenetic analysis performed by Campos et al. [41].