Amazonia ICEMR Brazil Cohort

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Amazonia ICEMR Peru Cohort

Background: Malaria remains a major public health concern in Brazil, particularly in areas of recent colonization in the densely forested Amazon Basin. Official and informal colonization projects have originated in a number of new frontier settlements, attracting migrant farmers from malaria-free regions. The creation of these settlements can have a major impact on vector biology by creating or expanding mosquito breeding habits through slash-and-burn agriculture and extensive logging, and by changing the vector species composition. Furthermore, the immigration of non-immune settlers can favor malaria transmission, creating hotspots until communities become more stable. This population-based longitudinal study addresses the epidemiology of malaria during the early stages of colonization of frontier settlements in Remansinho area, rural Amazonia.

Objectives:

Estimate the prevalence and incidence of asymptomatic malaria parasite carriage, by combining microscopic and molecular diagnosis, and to characterize risk factors for clinical disease among parasite carriers in rural Amazonia
Estimate the prevalence, incidence and risk factors for gametocyte carriage in symptomatic and asymptomatic infections
Estimate the average duration of gametocytemia in asymptomatic infections
Estimate prospectively the risk of subsequent clinical malaria among asymptomatic parasite carriers and non-infected controls living in the same communities
Determine whether consecutive malaria episodes diagnosed in the population-based cohorts are due to parasite lineages that persist in human populations or to new, genetically unrelated parasites introduced by migration of both symptomatic and asymptomatic carriers

Methodology:

Geographic Location/Study Sites: Remansinho area, Brazilian Amazonia

Dates of Data Collection: March 2010 - October 2014

Study Design: Longitudinal cohort study

Eligibility Criteria: The study population is the entire community of Remansinho, which was chosen based on geographic location and community malaria rates. All residents 3 months old and greater were invited to participate in the baseline survey and cross-sectional screenings.

Data Collection: The study design is a population-based open cohort study and consists of various methods of evaluating malaria in the study site:

Baseline surveys: Each household in Remansinho was visited to give a briefing to eligible subjects on study objectives (in Portuguese) by a field team member. Interested subjects then met with a field clinician to review the consent form. After the study had been fully explained and informed consent obtained from each subject or their parents/guardians, a unique subject identification number (SIN) was assigned to each participant. A standardized questionnaire was applied to all study participants to collect demographic, health, and socioeconomic data. Information on selected household assets, land ownership, and house characteristics was collected to derive a wealth index, from which socioeconomic status will be estimated. A blood draw of 10-12 mL was taken via venipuncture to make blood smears, evaluate for malaria by nested PCR and antibodies to malaria, and if P. vivax was diagnosed, for genotyping. For small children 3 months of age up to 2 years, only 5 mL of blood was taken; when venipuncture was not possible, especially in very young children, blood samples for malaria diagnosis (both microscopy and PCR) was obtained by heel or finger prick.

Cross-sectional surveys (AACD): A door-to-door cross-sectional survey of the entire population of Remansinho was carried out every four months (first year) or six months (years 2-5), with a total of 10 surveys. From each study participant we, regardless of the presence of symptoms, drew 5 mL of blood for slide and PCR evaluation to measure parasite prevalence and immunological parameters, to capture seasonal and year-to-year variation in these estimates. Because study subjects may have left the study site (and therefore be lost for follow-up) and newcomers were enrolled, this is an open cohort study in which the participation of each individual was measured as person-months of follow-up.

Passive and active case detection: The whole population of Remansinho was monitored using passive surveillance for acute febrile illnesses for five years. Local malaria control personnel (two MOH officials who live in the area), under the supervision of a study clinician or nurse, surveyed the cohort for symptoms Monday through Friday every week. The study clinician or nurse also checked at the local health posts if any study participants had been evaluated for symptoms that could be attributed to malaria. Subjects with concern or symptoms suggestive of malaria were evaluated by blood smear and PCR.

Laboratory Methods:

Blood Smears: Thick blood smears were stained with Giemsa and examined for malaria parasites, under 700x magnification (at least 100 fields), by local microscopists with >20 years of experience. Malaria infection (either symptomatic or asymptomatic) diagnosed by onsite microscopy during either ACD, PCD or AACD were treated according to the latest malaria therapy guidelines of the Brazilian MOH. Official MOH case notification forms(known in Brazil by the acronym SIVEP) were filled for every slide-confirmed infection.

PCR: Quantitative real-time PCR amplification of a species-specific segment of the 18S rRNA gene of human malaria parasites was used to confirm blood-stage infection. According to the therapy guidelines of the Ministry of Health of Brazil at the time of study, asymptomatic carriage of parasites detected by PCR only were left untreated unless they became slide-positive over the next days or weeks.

Plasma separation: EDTA or heparin-containing vacutainer tubes were transported to the field-site laboratory on ice and were centrifuged at 200 g for 10 minutes to obtain plasma. Following separation, plasma samples were transferred to cryotubes and stored at -20C. Red blood cell pellets were used for DNA extraction.

DNA and RNA extraction: DNA was extracted from 200 µL of red cell pellets using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) according to the blood and body fluid protocol. DNA samples were stored at -20C. For RNA isolation, venous blood aliquots were stored in liquid nitrogen.

Study Documentation:

Household data dictionary
Participant data dictionary

ClinEpiDB Data Integration: Data files were provided to ClinEpiDB as Stata files. Data were rearranged to be longer with one row for each observation per participant or household, and each set of variables collecting the same information at each survey were collapsed into one variable each. These datasets were merged by unique ID and redundant or administrative columns were dropped from presentation on ClinEpiDB.org. All dates were obfuscated per participant through the application of a random number algorithm that shifted dates no more than seven days to comply with the ethical conduct of human subjects research.

Acknowledgements: We thank all inhabitants in Remansinho for their enthusiastic participation in this study; Cleide F. Nunes and Eusueli Arraes da Silva for onsite microscopic diagnosis of malaria; and Márcio C. Santana, Andrecresa N. Duarte, and Francisco Naildo C. Leitão for overall support during fieldwork.

Financial Support: ICEMR program, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), United States, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.

Ethics Statement: Approved by University of California San Diego (UCSD), Instituto de Ciências Biomédicas da Universidade de São Paulo - ICB/USP, and the National Committee on Ethics in Research of the Ministry of Health of Brazil.

Last updated: December 23, 2021A longitudinal cohort study of malaria was conducted in the Brazilian Amazon. All residents >3 months of age were recruited to the open cohort with visits every 4-6 months. Household data was collected twice, but participants may have changed households at any study visit. Note that data subsets based on participant or sample variables will only include the first household a participant belonged to.