Study Design
Event-based LSS defined by acute arbovirus infection transmitted by Aedes sp. was conducted in a health unit, the only facility in the slum, and the first destination of individuals seeking medical care. Based on the definition of a probable case in the Manual for Adult and Child Diagnosis and Clinical Management for Dengue Fever [26], acute febrile and/or exanthematous illness was considered the defined event for LSS, thereby opting for broad spectrum symptomatology that addressed the symptomatic infections of the three arboviruses investigated here: for DENV, probable case symptomatology is defined as a sharp temperature increase (39-40ºC) accompanied by two or more of the following symptoms: headache, myalgia, arthralgia and retro-orbital pain, with exanthem present in 50% of symptomatic infections, as well as with anorexia, nausea, vomitting and diarrhea; for CHIKV, symptoms are similar, underscoring the intensity of arthralgia; for ZIKV, symptoms are characterized by absent or low to moderate-intensity fever, while exanthem occurs more frequently in the first days of the infection.
The sample population consisted of residents in the study area who visited the health unit in the morning (from 8:00 am to 12:00 pm) between Monday and Friday, complaining of acute fever and/or exanthemathous illness (the defined event). The primary identification of patients was conducted by family health teams (FHT) [27]. Each FHT is composed of a group of health professionals (doctor, nurse, community health agent) responsible for a geographic area within the respective region served by the health unit, which provides primary care. The health unit where LSS was carried out has 12 FHTs, which are instructed to refer patients presenting with the defined event. After the patient was referred, the following inclusion criteria were established: being older than 18 years of age; being a resident of the study area (at least four days a week for at least three months before the visit to the health unit); and not exhibiting any contraindication for venipuncture. The exclusion criteria were being a homeless person and illiterate (given the need to provide written informed consent).
After the patients were referred by the FHT and had agreed to take part in the study, they signed the informed consent form and were given a brief explanation of the study objectives. Blood was collected – characterized as the “acute moment” - and 15-21 days after symptom onset, participants were contacted again for a second blood collection – characterized as the “convalescent moment” - provided they did not exhibit cough and/or coryza (flu-like upper respiratory tract syndrome) [28]. The laboratory methodology recommended by the Ministry of Health [26] was applied for molecular and serological confirmation: RT-PCR was used for the DENV (for serotypes), CHIKV and ZIKV in acute samples; and ELISA-IgM for DENV and CHIKV in both acute and convalescent samples. For the ELISA-IgM serological assay, two parameters were established: acute infection when seroconversion occurs, and recent infection when serology is positive in the acute sample [16]. To confirm acute infection, positive RT-PCR and/or seroconversion are considered laboratory evidence. ELISA-Igm was only tested for DENV due to the possible cross-reaction between dengue and Zika (both flaviviruses).
Based on the seasonal pattern of mosquito-transmitted diseases, an initial 1-year observation period was established for the present study, between June 2019 and June 2020. However, given the lack of knowledge regarding SARS-CoV-2 infection at the time, as well as the lack of biosecurity for conducting research at the health unit, it was decided to halt the study in March 2020, after 9 months of observation.
Study Area
LSS was conducted at Health Unit No. 4 (15 ° 47 '00.89 "S 47 ° 49' 49.96" W) in the “Cidade Estrutural” administrative region (AR) of the Federal District (Figure 1-A). Cidade Estrutural is less than 10 km from the political center of Brazil and the executive, legislative and judiciary branches of government (Figure 1-B). Established as a landfill site in the early 1960s, the area is one of the least developed ARs in the Federal District, with a vulnerable population that suffer from poverty and a lack of urban planning, infrastructure and environmental sanitation [29]. Albeit isolated, it is near the central region of the District: the Pilot Plan (plano piloto in Portuguese) (Figure 1-B). Cidade Estrutural is bordered by the Structural Highway to the south; the farm sector to the west; the “SCIA” industrial AR to the east; and the National Park of Brasilia to the north/northeast (Figure 1-C). Aproximately 35,730 inhabitants were estimated for 2018, corresponding to 1.23% of the Federal District population of 2,894,953 [29]. According to the Federal District Health Department (Secretária de Saúde do Distrito Federal – SES-DF in Portuguese), the number of probable cases per 100,000 notified in the Cidade Estrutural in 2016, 2017, 2018 and 2019 was 1,132; 475; 92.5; and 1,613, respectively, and for the same period in the Federal District 583; 141; 79; and 1,428.
Figure 1. A: Location of the Federal District in Brazil; Federal District, highlighting Cidade Estrutural/SCIA in red, and Praça dos Três Poderes in yellow; B: Satellite image of the Pilot Project (central region of the Federal District) and surrounding area, highlighting Cidade Estrutural/SCIA in red and Praça dos Três Poderes in yellow; C: Satellite image of Cidade Estrutural, highlighting the key locations and homes with positive laboratory confirmations for the arboviruses investigated: HU: health unit; DD: deactivated dumping ground; NP: National Park of Brasilia; SCIA: AR bordering Cidade Estrutural, commercial and industrial area with a low number of houses. The homes with positive laboratory confirmations detected by LSS are represented by the following colors: Green: DENV-1; Red: DENV-2; Purple: CHIKV; Orange: DENV-IgM (seroconversion); Blue: recent DENV infection.
Interview and Blood sampling
Blood collection at the acute moment was performed using a tube with separator gel (5 mL), followed by the interview to obtain demographic and socioeconomic characteristics (age, sex, ethnicity, address, domestic sanitary conditions); first day of symptoms; clinical characteristics of the event; daily routine in the last 15 days before symptom onset (school, university, work or travel); the epidemiological link to an individual (family, neighborhood, work or school mate) with a positive medical diagnosis for DENV. Between 15 and 21 days after symptom onset, participants who did not report cough and/or coryza were contacted for convalescent blood collection at the conclusion of the second part of the questionnaire (focusing on symptoms reported at the acute moment: cure or persistence).
A standardized questionnaire from the REDCap website, version 7.5.0 (www.project-redcap.org/) was applied using mobile devices (tablets).
Laboratory Preparation
Tubes containing the collected blood were kept at ambient temperature in the health unit laboratory, followed by centrifugation of 1500 x g/10 min, aliquoting the supernatant serum in triplicate microtubes (usually 0.5 mL in each microtube). Once duly labeled with the respective patient code, the samples were initially kept in the laboratory freezer of the health unit (-20° C), then taken in thermal containers to the biorepository of the Center for Tropical Medicine, University of Brasilia (UnB) (-80° C), where they were stored until molecular and serological analyses.
Viral RNA extraction
Viral RNA extraction of 140 μL of serum from the acute sample was conducted using the QIAamp Mini kit, according to the manufacturer’s recommendation (www.qiagen.com/us/resources), resulting in 60 μL of eluate.
Reverse transcription and arboviral RNA detection
RT-PCR was performed using the “multiplex ZDC” kit from the Institute of Molecular Biology of Paraná with 38 μL of eluate, in accordance with the manufacturer’s recommendation (www.ibmp.org.br/en-us/). In this protocol [30], a standardized 96-well microplate is subdivided into four isometric subgroups of 24 wells for the respective target arboviruses: DENV 1/4; DENV 2/3; CHIKV; and ZIKV. Each of these four subgroups on the plate is composed of 23 wells to test the samples (38 uL/4 subgroups= 9.5 uL of eluate for each subgroup), and 1 positive control from the kit itself. RT-PCR was amplified using the QuantStudio 5 real-time system (http://thermofisher.com/), and the results analyzed by the company’s software.
Serological testing for anti-DENV IgM
Both acute and convalescent samples were tested for anti-DENV using the Panbio Dengue IgM Capture ELISA kit, according to the manufacturer’s recommendation (www.globalpointofcare.abbott). Reading was done on a Kasuaki absorbance reader (450 nm), using Panbio units for cutoff values, following the criterion: <9 non-reactive; between 9 and 11 inconclusive; > 11 reactive for anti-DENV IgM.
Serological testing for anti-CHIKV IgM
Both acute and convalescent samples were tested for anti-CHIKV using the Euroimun Chikungunya IgM ELISA kit, in line with the manufacturer’s recommendation (www.euroimmun.com). Reading was done on a Kasuaki absorbance reader (450 nm), using relative units (RU/mL) for cutoff values, based on the following criterion: <0.8 RU/mL negative; between 0.8 and 1.1 RU/mL inconclusive; >1.1 RU/mL positive for anti-CHIKV IgM.
Data Analyses
The number of defined events and laboratory confirmations was illustrated on epidemic curves, organized by epidemiological week (EW) starting with symptom onset. Clinical-epidemiological characteristics were cross-referenced with laboratory confirmations, using frequency tables. Information on routines for the 15 days prior to symptom onset was used to estimate the probable infection site.
In order to compare LSS results with traditional epidemiological surveillance data, probable arbovirus cases notified by the Cidade Estrutural and the Federal District were extracted from the Ministry of Health (datasus.gov.br) and Federal District Health Department websites (info.saude.df.gov.br): for the former, the interface allows extraction per period (year, month, EW) and final classification of the probable case (confirmed, discarded, not investigated), but not separation by the ARs of the Federal District; for the latter, the interface makes it possble to extract by period and site (including AR), but only dengue data are made available. Epidemiological bulletins from the Federal District Health Department were consulted to complement this information [31]. The final classification criteria of the probable case (clinical-epidemiological or laboratory) for Cidade Estrutural was obtained; the laboratory methods used (rapid NS1 test; IgM serology; PCR; viral isolation); and the dates of the first symptoms and blood collections for the respective laboratory tests established. Epidemic curves of probable dengue and Chikunguya cases from traditional surveillance for Cidade Estrutural were organized by EW starting on the day of symptom onset for the study period, discriminating the respective final classification (dengue, Chikunguya, discarded and non-investigated). Probable Zika cases and the respective final classification were not arranged by EW for the study area due to the non-availability of these notifications at the AR level for the Federal District. In order to observe the statistical differences between traditional surveillance and LSS results, the Wilcoxon test for paired data was conducted. To contextualize the COVID-19 situation in the first quarter of 2020, Federal District Health Department epidemiological bulletins from the Center of Emergency Operations for Covid-19, established in February of that year, were consulted [32].
Microsoft Excel was used to manage the datasets and create tables and graphs, Stata 14.0 software for statistical analyses, and Google Earth for satellite images. The GPS points of the dwellings were collected using a Garmin Etrex 10 handheld navigator.