Study Design
LSS based on defined event for symptomatic infection of arbovirus transmitted by Aedes sp. was performed in a health unit ofslum area, this healthfacility being the first destination of local individuals who seek medical support: all individuals who sought the health unit duringthe morning period (between 8:00 am and 12:00 pm) from Monday to Friday, complaining of febrile and/or exanthematicacute syndrome - the defined event - were invited to participate. The following inclusion criteria were considered for this prospective cohort: being over 18; being residents (sleeping at least four nights a week, for at least three months); and have no contraindication for venipuncture. For exclusion criteria, were considered homeless and not literate (according to sign the Informed Consent Form for research). Verifying the seasonality ofthe mosquito-borne diseases,for this researcha period of 1 year of observation was established, at least (from June 2019 to June 2020).
Following the definition of probable case from the Manual for Adult and Child Diagnosis and Clinical Management for Dengue Fever [22], febrile and/or exanthematic acute syndromes were considered the defined event based on the symptomatology of three arbovirus infection: for DENV, the first manifestations are high fever (39ºC to 40ºC), associated with cephalea, myalgia, arthralgia and retroorbital pain, with exanthem presents in 50% of the symptomatic infections, stillaccompanied by anorexia, nausea, vomiting and diarrhea; for CHIKV, the disease manifestation is similar, with emphasis on the intensity of arthralgias; for ZIKV, fever may present with less intensity or absent, while exanthem occurs more frequently even in the first days of symptomatic infection. Cough and/or coryza, which are not associated with arbovirus infection, was considered for the define event here investigated:therefore,we opted for a wider spectrum than the definition of suspectcase (individualshowing suggestive signs and symptoms of a group of diseases that share the same symptoms) and probable case (clinically compatible case, without evidence of epidemiological link or/and laboratory confirmation)for DENV, CHIKV and ZIKV, thus reducing possible losses.
The primary identification of patients presenting the defined event at health unit was conducted by the family health teams (FHT), in accordance with the Family Health Strategy [23]. Each FHT is composed by a group of health professionals (medical doctor; nurse; community health agent), andthis group are responsible for a limited geographical area from respective region served by the health unit. When a patient seeks the health unit for medical support, the respective FHT makes the first appointment. The health unitwhere LSS was developed has 12 FHS, these being oriented to refer for the LSSthe patients with suggestedsymptoms of defined event.
The methodology recommended by Ministry of Health [22] was followed by laboratory confirmation: RT-PCR was performed for DENV (by serotype), CHIKV and ZIKV on acute samples; ELISA-IgM serology was performed for DENV and CHIKV on both acute and convalescent. Considering the serological assay on both moments, two parameters were established according to the positivity: acute infection when seroconversion occurs; and recent infection when serology was positive at the acute moment [14]. Due to the possibility of cross-reaction between dengue and Zika (bothflavivirus), ELISA-IgM was only performed for DENV.Patients who reported cough and/or coryza were not contacted for convalescent collection.
Study Area
LSS was performed at the Health Unit N.º 4 (15 ° 47 '00.89 "S 47 ° 49' 49.96" O) in the Administrative Region of “CidadeEstrutural”(AR: political-administrative unit that configures the territory of Federal District). CidadeEstrutural is less than 10 km from the political center of Brazil, and the respectiveExecutive, Legislative and Judiciary federal powers (Figure 1). Established as a dumping ground in the early 1960s, CidadeEstrutural is one of the least consolidated ARs in the Federal District, consequently with a vulnerable population facing poverty and thecorresponding absence of urban planning, infrastructure and environmental sanitation [24]. Currently, as seen in Figure 2, the study area is geographically delimited by Estrutural Highway in the south; by the farm sector in the west; by the industrial AR “SCIA” in the east; andby the Brasilia National Park in the north/northeast.CidadeEstrutural presented approximately 35,730 habitants in 2018, corresponding 1.23% of Federal District population (2,894,953 in same year) [24]. According to epidemiological data of the State Health Secretariat of Federal District, CidadeEstruturalhas an important contribution with Federal District numbers: considering probable cases reported by 100,000 inhabitants in 2016, 2017, 2018 and 2019, the indicators of the CidadeEstrutural were, respectively: 1,132; 475; 92.5; 1613, higher than the general incidence at same period, respectively: 583; 141; 79; 1,428.
Interview and Blood sampling
With the patient referred by the FHT, andagreeing to participate, was followed an explanation of the project's objectives, asking him or her to sign the Informed Consent Form. Blood collection was performed using a serum-separating tube (5 mL), and interview was conducted which addressed: demographic and socio-economic characteristics (age, gender, address, enviromment sanitation conditions of the domicile); day of onset of symptoms and clinical characteristics of the event; daily routine in the last 15 days before the onset of symptoms (school, university, work, travel); possible exposures and epidemiologic link by some close individual with positive diagnosis (by medical doctor) for dengue fever (family, neighbors, co-workers, schoolmates). After 15-21 days from the onset of symptoms, the participants who not reported cough and/or coryzawere approached again to a new blood sample collection (5 mL), in addition performing a second part of the questionnaire focusing on the outcome of the symptoms reported at the acute moment (cure or persistence).
Standardized questionnaire was applied using REDCap electronic platform version 7.5.0 (www.project-redcap.org/).
Laboratory Preparation
Tubes with collectedblood were kept at room temperature in the health unit laboratory, followed by centrifugation of 1500 x g/10 min, separating 0.5 mL of the supernatant serum in threemicrotubes(1.5 mL). Properly labeled with the respective patient code, the samples were first kept in the freezer of the health unit laboratory (-20° C), to be transportedby thermal boxesto the biorepository of Center for Tropical Medicine, UnB (-80° C), where were keep until molecular and serological analysis.
Viral RNA extraction
Extraction of viral RNA from serum collected at the acute moment was performed using the QIAamp Mini kit, as recommended by the manufacturers (www.qiagen.com/us/resources), from 140 μL of serum resulting 60 μL of eluted.
Reverse transcription and Arboviral RNA detection
RT-PCR was performed using a ZDC multiplex kit from the Institute of Molecular Biology of Paraná from 38 μL of eluted, following the manufacturers' recommendations (www.ibmp.org.br/en-us/). In this protocol, a standardized 96-well microplate is subdivided into four isometric subgroups containing 24 wells, applying the respective PCR mixture according to the primers and probes for the targetarbovirus (DENV 1/4; DENV 2/3; CHIKV; ZIKV), with 1 Positive Control and 23 samples to be tested. RT-PCR was performed using QuantStudio 5 (http://thermofisher.com/), with the results being analyzed by company's software.
Serological testing for anti-DENV IgM
Both samples collected (acute and convalescent) were tested for anti-DENV using Panbio Dengue IgM Capture ELISA kit, as recommended by the manufacturers (www.globalpointofcare.abbott). The reading was performed on a Kasuaki absorbance reader (450 nm), using Panbio units for cut-off values, following the criteria: <9 non-reactive; between 9 and 11 inconclusive; > 11 reactive for anti-DENV IgM.
Serological testing for anti-CHIKV IgM
Both samples collected (acute and convalescent) were tested for anti-CHIK using EuroimunChikungunyaIgM ELISA kit, as recommended by the manufacturers (www.euroimmun.com). The reading was performed on a Kasuaki absorbance reader (450 nm), using relative units (RU/mL) for cut-off values, following the criteria: <0.8 RU/mL negative; between 0.8 and 1.1 RU/mL inconclusive; >1.1 RU/mL positive for anti-CHIKV IgM.
Statistical Analyses
Frequencies of defined event andlab-confirmed caseswere organized by epidemiological week (EW)concerningthe day of onset of symptoms, according to the period covered by the research.Clinical-epidemiological characteristics were crossed with lab-confirmed cases, by frequency tables. Information obtained from the interviews was used to estimate the likely local of infection. Traditional epidemiologic surveillance data from CidadeEstrutural was arranged by EW and compared with LSS results, using probable cases for DENV (clinically compatible case, without evidence of epidemiological link or/and laboratory confirmation). Statistical difference between probable cases from traditional epidemiology surveillance and LSS results was verified using Wilcoxon matched pairs signed rank test, according to the subject of analysis is paired (by EW), the use of discrete count variables, and respective no-parametric distribution (overdispersion).
Microsoft Excel was used to management data sets, and todesign tables and graphs.Stata 14.0 Software was used for statistical analysis. Google Earth was used for satellite images. GPS points from residences were collected using a Garmin Etrex 10 device.