Igor Adolfo Dexheimer Paploski

Outbreak of Exanthematous Illness Associated with Zika, Chikungunya, and Dengue Viruses, Salvador, Brazil

To the Editor: Zika virus (ZIKV) has been recognized as an emerging mosquito-borne flavivirus since outbreaks were reported from Yap Island in 2007 (1), French Polynesia in 2013 (2), and Cook Island and New Caledonia in 2014 (3). It has joined dengue virus (DENV) and chikungunya virus (CHIKV) as global public health threats (4). ZIKV infection typically causes a self-limited dengue-like illness characterized by exanthema, low-grade fever, conjunctivitis, and arthralgia, and an increase in rates of Guillain-Barre syndrome have been observed during ZIKV outbreaks (5). In Brazil, clusters of cases of acute exanthematous illness have been reported from various regions since late 2014, and in April 2015, ZIKV was identified as the etiologic agent (6). In May 2015, the Brazilian Ministry of Health recognized circulation of ZIKV in Brazil. We report epidemiologic findings for an ongoing outbreak of acute exanthematous illness in the population of Salvador, the third largest city in Brazil. The Salvador Epidemiologic Surveillance Office (ESO) was first alerted to cases of an acute exanthematous illness early in 2015. Reporting of cases increased during March, and in April the ESO established 10 public emergency health centers in Salvador as sentinel units for systematic surveillance of patients with acute exanthematous illness of unknown cause. The units searched retrospectively for suspected cases by review of medical charts of patients treated since February 15, continued with prospective case detection, and submitted weekly reports of identified cases to the ESO. During February 15−June 25, a total of 14,835 cases of an indeterminate acute exanthematous illness were reported from the 12 sanitary districts in Salvador. The overall attack rate was 5.5 cases/1,000 persons (4.6 cases/1,000 men and 6.3 cases/1,000 women, 8.2 cases/1,000 children 40 years of age). The epidemic curve peaked in the first week of May, which was 1 week after molecular diagnosis of ZIKV in 8 patients residing ≈50 km from Salvador and during a period of intense media coverage of the outbreak (Figure) (6). Reporting of suspected dengue cases in Salvador did not vary substantially from that in other years and was >5 times lower: 2,630 cases, of which 165/366 (45.1%) were positive for dengue IgM, 20/590 (3.4%) positive for dengue virus nonstructural protein 1, and 1/11 (9.1%) positive for dengue virus by reverse transcription PCR (Figure). During the same period, 58 cases of suspected chikungunya were reported and 24 patients with suspected Guillain-Barre syndrome were hospitalized. Figure Reported cases of indeterminate acute exanthematous illness and suspected dengue fever in Salvador, Brazil, by date of medical care, February 15−June 25, 2015. Letters indicate specific events. A) February 15: systematic reporting of cases of ... The median age of case-patients was 26 years (interquartile range 11–39 years), but all age groups were affected, which is a pattern typical of spread of new microorganisms (or subtypes) in a susceptible population. Median duration of symptoms at time of medical attention was 1 day (interquartile range 0–3 days). All patients had exanthema and most (12,711/14,093 [90.2%]) had pruritus. Fever (4,841/13,786, 35.1%), arthralgia (278/1,048 [26.5%]), headache (3,446/13,503 [25.6%]), and myalgia (223/1,033 [21.6%]) were less common. Serum samples from some patients were examined for rubella IgM (2/200, 1.0% positive), rubella IgG (15/18, 83.3% positive), measles IgM (0/11, 0% positive), dengue nonstructural protein 1 (3/185, 1.6% positive), dengue IgM (17/80, 21.3% positive), parvovirus B19 IgM (0/1, 0% positive), and parvovirus B19 IgG (1/1, 100% positive). Reverse transcription PCR was performed on 58 serum samples stored at −20°C and confirmed ZIKV in 3 (5.2%) samples, CHIKV in 3 (5.2%) samples, DENV type 3 in 1 (1.7%) sample, and DENV type 4 in 1 (1.7%) sample. Identification of ZIKV, CHIKV and DENV as etiologic agents of acute exanthematous illness suggests that these 3 Aedes spp. mosquito−transmitted viruses were co-circulating in Salvador and highlights the challenge in clinically differentiating these infections during outbreaks. Although we were not able to determine the specific incidence of each virus, the low frequency of fever and arthralgia, which are indicators of dengue and chikungunya, point to ZIKV as the probable cause of several of the reported cases. Furthermore, laboratory-confirmed cases of infection with ZIKV were simultaneously identified in other cities within metropolitan Salvador (6,7) and in other states in Brazil (8). Low diagnosis of ZIKV infection is likely because viremia levels among infected patients appear to be low (9). The spread of ZIKV represents an additional challenge for public health systems, particularly because of the risk for concurrent transmission of DENV and CHIKV by the same vectors, Ae. aegypti and Ae. albopictus mosquitoes, which are abundant throughout tropical and subtropical regions. To date, the largest outbreak of chikungunya in Brazil occurred in 2014 in Feira de Santana, Bahia, ≈100 km from Salvador, where dengue is also prevalent (10). This report illustrates the potential for explosive simultaneous outbreaks of ZIKV, CHIKV, and DENV in the Western Hemisphere and the increasing public health effects of Aedes spp. mosquitoes as vectors. The apparent increase in reports of Guillain-Barre syndrome during the outbreak deserves further investigation to elucidate whether this syndrome is associated with ZIKV infection. Public health authorities in Brazil and neighboring countries should plan accordingly.

Time Lags between Exanthematous Illness Attributed to Zika Virus, Guillain-Barré Syndrome, and Microcephaly, Salvador, Brazil.

There is strong evidence of a temporal relationship between virus infection in pregnant women and birth outcome.

Variation in aedes aegypti mosquito competence for zika virus transmission

To test whether Zika virus has adapted for more efficient transmission by Aedes aegypti mosquitoes, leading to recent urban outbreaks, we fed mosquitoes from Brazil, the Dominican Republic, and the United States artificial blood meals containing 1 of 3 Zika virus strains (Senegal, Cambodia, Mexico) and monitored infection, dissemination, and virus in saliva. Contrary to our hypothesis, Cambodia and Mexica strains were less infectious than the Senegal strain. Only mosquitoes from the Dominican Republic transmitted the Cambodia and Mexica strains. However, blood meals from viremic mice were more infectious than artificial blood meals of comparable doses; the Cambodia strain was not transmitted by mosquitoes from Brazil after artificial blood meals, whereas 61% transmission occurred after a murine blood meal (saliva titers up to 4 log10 infectious units/collection). Although regional origins of vector populations and virus strain influence transmission efficiency, Ae. aegypti mosquitoes appear to be competent vectors of Zika virus in several regions of the Americas.

Accuracy of Dengue Reporting by National Surveillance System, Brazil.

To the Editor: Dengue is an underreported disease globally. In 2010, the World Health Organization recorded 2.2 million dengue cases (1), but models projected that the number of symptomatic dengue cases might have been as high as 96 million (2). Brazil reports more cases of dengue than any other country (1); however, the degree of dengue underreporting in Brazil is unknown. We conducted a study to evaluate dengue underreporting by Brazil’s Notifiable Diseases Information System (Sistema de Informacao de Agravos de Notificacao [SINAN]). From January 1, 2009, through December 31, 2011, we performed enhanced surveillance for acute febrile illness (AFI) in a public emergency unit in Salvador, Brazil. The surveillance team enrolled outpatients >5 years of age with measured (>37.8°C) or reported fever. Patients or their legal guardians provided written consent. The study was approved by the Oswaldo Cruz Foundation Ethics Committee, Brazil’s National Council for Ethics in Research, and the Yale Institutional Review Board. We collected participants’ blood samples at study enrollment and >15 days later. Acute-phase serum samples were tested by dengue nonstructural protein 1 ELISA and IgM ELISA (Panbio Diagnostics, East Brisbane, Queensland, Australia). Convalescent-phase serum samples were tested by IgM ELISA. In concordance with case-reporting guidelines in Brazil (3), we defined dengue cases by a positive nonstructural protein 1 ELISA result or a positive acute-phase or convalescent-phase IgM ELISA result. All others were classified as nondengue AFI. We then identified which study patients were officially reported to SINAN as having a suspected case of dengue. In Brazil, notification of suspected dengue cases is mandatory. A suspected case is defined as illness in a person from an area of dengue transmission or Aedes aegypti mosquito infestation who has symptoms of dengue (fever of 2 of the following symptoms: nausea/vomiting, exanthema, myalgia, arthralgia, headache, retro-orbital pain, petechiae/positive tourniquet test, or leukopenia). We used Link Plus software (CDC-Link Plus Production 2.0; Centers for Disease Control and Prevention, Atlanta, GA, USA) to perform probabilistic record linkage from our database with official reports in the SINAN database. The records were matched based on the patients’ first names, last names, and dates of birth. We then manually reviewed the matches to confirm the pairs. On the basis of the results, we calculated the sensitivity, specificity, positive predictive value (PPV), and negative predictive value of the national surveillance system. We calculated accuracy measurements with 95% CIs for the overall study period and for each study year, age group (5–14 vs. >15 years), and seasonal prevalence of dengue (months of low vs. high dengue transmission, defined by dengue detection in 20% of the AFI patients, respectively). We estimated multiplication factors by dividing the number of dengue cases in our study by the number of study patients who were reported to SINAN as having dengue. Of the 3,864 AFI patients identified during the 3-year study period, 997 (25.8%) had laboratory evidence of dengue infection, and 2,867 (74.2%) were classified as having nondengue AFI. Of the 997 dengue cases, 57 were reported to SINAN (sensitivity 5.7%) (Table). Of the 2,867 nondengue AFI cases, 26 were reported to SINAN as dengue cases (false-positive ratio 0.9%, specificity 99.1%). None of these 26 cases had laboratory confirmation in the SINAN database. The PPV for reporting to SINAN was 68.7%, and the negative predictive value was 75.1% (Table). PPV was higher among patients >15 years of age, which might be attributable to atypical presentations of dengue in children (4,5). Table Accuracy of a national surveillance system for recording cases of suspected dengue among patients with acute febrile illness who visited an emergency health unit of Salvador, Brazil, January 1, 2009–December 31, 2011* We found that 1 in 4 patients with AFI had laboratory evidence of dengue infection. However, for every 20 dengue patients that we identified, only about 1 had been reported to SINAN as having dengue. During periods of low dengue transmission, only about 1 in 40 dengue cases identified was reported. Conversely, among the patients who were reported as having dengue, 31.2% did not have the disease; this percentage reached 61.5% in low-transmission periods. We estimated that overall, there were 12 dengue cases per reported case in the community, but in months of low dengue transmission, this ratio was >17:1 (Table). Comparable results have been observed in Nicaragua, Thailand, and Cambodia (6–8). By applying the estimated multiplication factor to the study period’s mean annual incidence of 303.8 reported dengue cases/100,000 Salvador residents (9), we estimated that the actual mean annual dengue incidence for Salvador was 3,645.7 cases/100,000 residents. We showed that dengue surveillance substantially underestimated disease burden in Brazil, especially in what are considered low-transmission periods. Dengue underreporting has been attributed to passive case detection, which fails to identify persons with dengue who do not seek health care (1). We also showed that surveillance failed to detect dengue cases among symptomatic patients seeking health care. Novel surveillance tools, such as active syndromic surveillance and point-of-care testing, should be applied to improve estimates of dengue incidence. Furthermore, given the recent emergence of chikungunya and Zika viruses in Brazil (10), improved surveillance and laboratory diagnostics are needed to avert misclassification and mismanagement of cases.

Spatial Distribution of Dengue in a Brazilian Urban Slum Setting: Role of Socioeconomic Gradient in Disease Risk

Background Few studies of dengue have shown group-level associations between demographic, socioeconomic, or geographic characteristics and the spatial distribution of dengue within small urban areas. This study aimed to examine whether specific characteristics of an urban slum community were associated with the risk of dengue disease. Methodology/Principal Findings From 01/2009 to 12/2010, we conducted enhanced, community-based surveillance in the only public emergency unit in a slum in Salvador, Brazil to identify acute febrile illness (AFI) patients with laboratory evidence of dengue infection. Patient households were geocoded within census tracts (CTs). Demographic, socioeconomic, and geographical data were obtained from the 2010 national census. Associations between CTs characteristics and the spatial risk of both dengue and non-dengue AFI were assessed by Poisson log-normal and conditional auto-regressive models (CAR). We identified 651 (22.0%) dengue cases among 2,962 AFI patients. Estimated risk of symptomatic dengue was 21.3 and 70.2 cases per 10,000 inhabitants in 2009 and 2010, respectively. All the four dengue serotypes were identified, but DENV2 predominated (DENV1: 8.1%; DENV2: 90.7%; DENV3: 0.4%; DENV4: 0.8%). Multivariable CAR regression analysis showed increased dengue risk in CTs with poorer inhabitants (RR: 1.02 for each percent increase in the frequency of families earning ≤1 times the minimum wage; 95% CI: 1.01-1.04), and decreased risk in CTs located farther from the health unit (RR: 0.87 for each 100 meter increase; 95% CI: 0.80-0.94). The same CTs characteristics were also associated with non-dengue AFI risk. Conclusions/Significance This study highlights the large burden of symptomatic dengue on individuals living in urban slums in Brazil. Lower neighborhood socioeconomic status was independently associated with increased risk of dengue, indicating that within slum communities with high levels of absolute poverty, factors associated with the social gradient influence dengue transmission. In addition, poor geographic access to health services may be a barrier to identifying both dengue and non-dengue AFI cases. Therefore, further spatial studies should account for this potential source of bias.

Differential vector competency of aedes albopictus populations from the Americas for Zika Virus

To evaluate the potential role of Aedes albopictus (Skuse) as a vector of Zika virus (ZIKV), colonized mosquitoes of low generation number (≤ F5) from Brazil, Houston, and the Rio Grande Valley of Texas engorged on viremic mice infected with ZIKV strains originating from Senegal, Cambodia, Mexico, Brazil, or Puerto Rico. Vector competence was established by monitoring infection, dissemination, and transmission potential after 3, 7, and 14 days of extrinsic incubation. Positive saliva samples were assayed for infectious titer. Although all three mosquito populations were susceptible to all ZIKV strains, rates of infection, dissemination, and transmission differed among mosquito and virus strains. Aedes albopictus from Salvador, Brazil, were the least efficient vectors, demonstrating susceptibility to infection to two American strains of ZIKV but failing to shed virus in saliva. Mosquitoes from the Rio Grande Valley were the most efficient vectors and were capable of shedding all three tested ZIKV strains into saliva after 14 days of extrinsic incubation. In particular, ZIKV strain DakAR 41525 (Senegal 1954) was significantly more efficient at dissemination and saliva deposition than the others tested in Rio Grande mosquitoes. Overall, our data indicate that, while Ae. albopictus is capable of transmitting ZIKV, its competence is potentially dependent on geographic origin of both the mosquito population and the viral strain.

Unrecognized Emergence of Chikungunya Virus during a Zika Virus Outbreak in Salvador, Brazil

Background Chikungunya virus (CHIKV) entered Brazil in 2014, causing a large outbreak in Feira de Santana, state of Bahia. Although cases have been recorded in Salvador, the capital of Bahia, located ~100 km of Feira de Santana, CHIKV transmission has not been perceived to occur epidemically, largely contrasting with the Zika virus (ZIKV) outbreak and ensuing complications reaching the city in 2015. Methodology/Principal Findings This study aimed to determine the intensity of CHIKV transmission in Salvador between November 2014 and April 2016. Results of all the CHIKV laboratory tests performed in the public sector were obtained and the frequency of positivity was analyzed by epidemiological week. Of the 2,736 tests analyzed, 456 (16.7%) were positive. An increasing in the positivity rate was observed, starting in January/2015, and peaking at 68% in August, shortly after the exanthematous illness outbreak attributed to ZIKV. Conclusions/Significance Public health authorities and health professionals did not immediately detect the increase in CHIKV cases, likely because all the attention was directed to the ZIKV outbreak and ensuing complications. It is important that regions in the world that harbor arbovirus vectors and did not experience intense ZIKV and CHIKV transmission be prepared for the potential co-emergence of these two viruses.

Epizootic Outbreak of Yellow Fever Virus and Risk for Human Disease in Salvador, Brazil.

Background: Yellow fever virus (YFV) is an RNA virus maintained in an enzootic, sylvatic cycle involving nonhuman primates (NHPs) and sylvatic mosquito vectors primarily of the genus Haemagogus and Sabethes. Transmission occasionally spills over to humans entering forested regions. In the Americas, urban transmission of YFV to humans has not occurred since the mid-1900s because of vaccination and near-elimination of the anthropophilic Aedes aegypti, the urban vector (1). However, concerns about reemergence of urban YFV have recently increased because of the reappearance and rapid spread of A aegypti in the urban environment. Furthermore, immunization coverage for YFV is insufficient because vaccination is generally indicated only for higher-risk populations, such as those living in or travelling to areas with sylvatic transmission. Objective: To investigate the 2017 epizootic outbreak of YFV and the risk for human disease in Salvador, Brazil. Methods and Findings: Since November 2016, deaths of NHPs due to YFV in Brazil have been reported in the state of So Paulo. Beginning in December 2016, human cases were also reported in the states of So Paulo and Minas Gerais. By the end of May 2017, the YFV outbreak in humans had spread to 9 Brazilian states, with more than 130 municipalities reporting confirmed cases (Appendix Figure 1), all deemed of sylvatic origin (rather than via urban A aegypti transmission). Of 3240 suspected reported human cases, 792 were laboratory-confirmed. Among all reported patients, 435 died and 274 of these had laboratory-confirmed infection with YFV (casefatality ratio for laboratory-confirmed cases, 34.6%) (2). Appendix Figure 1. Brazilian states where human cases of YFV were reported between December 2016 and May 2017, according to laboratory confirmation status. YFV = yellow fever virus. Reports of deaths of NHPs due to YFV also simultaneously increased. By the end of May 2017, a total of 3850 NHPs was suspected to have died of YFV infection, 642 of which were confirmed from 12 states (2). Since the beginning of the outbreak, the Brazilian Ministry of Health has distributed more than 25 million YFV vaccine doses to persons in areas with confirmed human or NHP cases, likely reducing incidence (2). On 14 January 2017, deaths of NHPs suspected of having YFV were first reported in Salvador, the fourth largest Brazilian city, which had not been considered a risk area for transmission (Appendix Figure 1). The number of NHPs found dead throughout the city rapidly increased, and by 24 August, 205 NHPs of the genus Callithrix suspected of having infection with YFV had been collected (Appendix Figure 2). Reverse transcriptase polymerase chain reaction testing for the virus was done on tissue samples of 21 dead animals at the Oswaldo Cruz Foundation in Rio de Janeiro; samples from 13 animals (61.9%) tested positive. Animals suspected of being infected were found throughout Salvador, with a cluster (3 of the 13 YFV-positive animals) around a city park, a 0.66-km2 recreational area of residual Atlantic forest with trees less than 10 m high (Figure). Appendix Figure 2. Temporal distribution of NHPs collected in Salvador, Brazil, by epidemiologic week in 2017. NHP = nonhuman primate. Figure. Spatial distribution of locations of NHP collections according to YFV status and of sites of mosquito collections in Salvador, Brazil. Distribution of 7 of the 8 YFV-negative NHPs, all 13 of the YFV-positive NHPs, and 173 of the 184 NHPs that were not tested is shown. NHP = nonhuman primate; YFV = yellow fever virus. To investigate potential vectors involved in YFV transmission among NHPs in Salvador, we initiated ground-level mosquito collections within and around the city park and the naval base, another preserved Atlantic forest area where dead, YFV-positive NHPs were found. During 10 days of fieldwork in 26 sites between 10 April and 7 June 2017, a total of 435 adult mosquitoes (307 females) was captured using human landing catches (Table). Most females captured were Wyeomyia species and A albopictus. Although we did not capture Haemagogus or Sabethes species, Haemagogus mosquitoes were identified in Salvador in January 2017 in a suburban, forested area. Pools of captured female mosquitoes from each species were tested for YFV by reverse transcriptase polymerase chain reaction (primers CAG and YF7) (3) and cell culture (C6/36, A albopictus), and none was positive. Table. Number of Mosquitos Collected at Ground Level in Salvador, Brazil* Discussion: To date, urban Aedes mosquitoes have not been associated with YFV transmission to humans in Brazil. However, cases of YFV in NHPs in densely urbanized areas pose a considerable risk for resurgence of A aegyptimediated YFV transmission to humans. Salvador has long been an epicenter of dengue transmission and more recently of Zika (4) and chikungunya (5) viruses, all with A aegypti as the main vector. In addition, A albopictus, which is susceptible to YFV infection in laboratory settings, is commonly found here, particularly in peridomestic and green areas. Although never implicated in natural YFV transmission, this species was only introduced into the Americas in the 1980s. Continuous entomologic and veterinary surveillance of mosquitoes and NHP deaths, accompanied by laboratory testing for YFV, is the cornerstone of assessing the risk that this virus will establish an urban transmission cycle. In parallel, scaling up YFV vaccination coverage is critical to prevent additional human cases of this disease.

Lack of evidence for Zika virus transmission by Culex mosquitoes

Emerging Microbes & Infections (2017) 6, e90; doi:10.1038/emi.2017.85; published online 18 October 2017

Does immunity after Zika virus infection cross-protect against dengue?

Brazilian National Council for Scientific and Technological Development (grant 550160/2010-8 to MGR, grants 400830/2013-2 and 440891/2016-7