First malaria infections in a cohort of infants in Benin: Biological, environmental and genetic determinants. Description of the study site, population methods and preliminary results

Objectives: Malaria infection of the placenta during pregnancy was found to be associated with infant susceptibility to malaria. Other factors such as the intensity of malaria transmission and the nutritional status of the child might also play a role, which has not been adequately taken into account in previous studies. The aim of this study was to assess precisely the parts played by environmental, nutritional and biological determinants in first malaria infections, with a special interest in the role of placental infection. The objective of this paper is not to present final results but to outline the rationale of the study, to describe the methods used and to report baseline data. Design: A cohort of infants followed with a parasitological (symptomatic and asymptomatic parasitaemia) and nutritional follow-up from birth to 18 months. Ecological, entomological and behavioural data were collected along the duration of the study. Setting: A rural area in Benin with two seasonal peaks in malaria transmission. Participants: 656 infants of women willing to participate in the study, giving birth in one of the three maternity clinics and living in one of the nine villages of the study area. Primary Outcome Measures: The time and frequency of first malaria parasitaemias in infants, according to Plasmodium falciparum infection of the placenta. Results: 11% of mothers had a malaria-infected placenta at delivery. Mosquito catches made every 6 weeks in the area showed an average annual P falciparum entomological inoculation rate of 15.5, with important time and space variations depending on villages. Similarly, the distribution of rainfalls, maximal during the two rainy seasons, was heterogeneous over the area. Conclusions: Considering the multidisciplinary approach of all factors potentially influencing the malaria status of newborn babies, this study should bring evidence on the implication of placental malaria in the occurrence of first malaria infections in infants. The study was conducted in the district of Tori Bossito located on the coastal plain of southern Benin (6°25–6°37 N and 2°1–2°17 E), 40 km north-east of Cotonou, the economic capital (figure 1A).15 The area, where the vegetation is composed of a mosaic of wooded savannah and food-living agriculture, is located on a clayey plateau with a central marshy depression. Small-scale agriculture (maize, pineapple and cassava, market gardening) and fishing are the primary sources of food and income for most residents of the study area. Southern Benin is characterised by a subtropical climate, with two rainy seasons (April–July and October–November) and two dry seasons (August–September and December–March). The annual rainfall is over 1300 mm. The main mosquito vectors of malaria in this region of Benin are Anopheles gambiae ss and Anopheles funestus.16 Previous entomological studies in Benin were made in Cotonou or in a village on the Nokoué Lake, two sites ecologically different from the region of Tori Bossito but probably with similar vectorial species.17 18 There is a lack of entomological studies in this particular area19 but climatic data suggest that malaria transmission is probably important. The study area included nine villages (figure 1B) and three health centres providing birth attendance and primary healthcare. The nearest well-equipped hospital is located in the town of Ouidah, 17 km south of Tori Bossito. (A and B). Geographical location of Tori Bossito and the nine villages of the study area, Benin. A birth cohort with a close follow-up of children was set up in June 2007, with an enrolment lasting 13 months, until July 2008. It was designed to detect biological and clinical signs of malaria infection from birth to the age of 18 months and was planned to last 30 months, until January 2010. All epidemiological, behavioural and family data were collected at recruitment and a nutritional, environmental and malaria follow-up was carried over, starting at delivery. Three field supervisors (all state nurses) were recruited by the programme and were responsible for three villages each. In each village, two community health workers worked under the responsibility of a supervisor. In the study carried out in Cameroon by Le Hesran and collaborators,8 65% of the placental malaria-infected infants had a parasitaemia during the first year of life versus 50% in placental malaria-negative infants. Transmission being lower in the south of Benin and in the absence of pre-existing data concerning malaria transmission in the area, it was difficult to predict the relative proportions of parasitaemic children in the two groups during the 12-month follow-up. However, assuming 30% of the children would present with a first peripheral infection in the placental malaria-infected group and 15% in the placental malaria-negative group, with a ratio of 5, a 80% power and a 5% α risk, 450 newborns had to be included (75 in the placental malaria-infected group and 375 in the placental malaria-negative group) (sampsi command, Stata V.8). At study initiation, having no better estimation for the final proportions, to be conservative and to account for loss to follow-up, we decided to include 600 newborn babies. Recruitment was performed in three health centres providing deliveries and postnatal care (Tori Avame, Tori Cada and Tori Gare). The study objectives and study protocol were explained by midwives to all women attending the health centre for antenatal care from the seventh month of pregnancy. Inclusion criteria were to live in one of the nine villages of the study area, to have no intention to move in the next month and to plan to deliver in the health centre. At enrolment, ie, before delivery, midwives again gave information about the study and collected signed informed consents. The approval of husbands was systematically looked for by pregnant women themselves. Consequently, husbands were invited to come and sign the informed consent form. After delivery, newborns were listed and received an identification card, giving them an access to free treatment in health centres during the 18 months of the follow-up. The general follow-up included a weekly home visit by health workers to detect fever and the general health status of infants, and a monthly home visit by supervisors and field workers to search for malaria and to collect nutritional data. In addition, women were told to come with their offspring to the health centre for blood sampling once a trimester. At least three attempts were made before a scheduled visit was considered missed and infants were considered lost to follow-up after having missed more than four consecutive monthly visits. According to a recent entomological survey, P falciparum is the commonest species in the study area (95%), Plasmodium malariae and Plasmodium ovale representing 3% and 2%, respectively.20 To investigate the relation between malaria infection of the placenta and infant parasitaemias and to be consistent with previous studies, we considered only P falciparum infections for the data analysis. At delivery, maternal blood and umbilical cord blood samples were drawn to search for malaria infection and anaemia. Thick and thin placental smears were made by midwives to look for placental malaria infection. During weekly home visits, the axillary temperature was measured by health workers with a digital thermometer to detect P falciparum symptomatic infections. In the case of temperature higher than 37.5°C, mothers were told to bring their children to the health centre. On arrival at the health centre, a questionnaire was filled in and a Parascreen rapid diagnostic test (RDT) was made, to obtain an immediate diagnosis of symptomatic malaria infection (ie, the presence of parasites and temperature higher than 37.5°C). A thick blood smear (TBS) was made to provide a later confirmation of the RDT result. When RDT was positive, infants were treated by an artemisinin-based combination (arthemeter and lumefantrin) as recommended by the National Malaria Control Program of Benin. If the mother did not bring her child to the health centre the following day, a field supervisor visited the family to get information on the infant’s clinical status and to invite the mother to consult. Mothers were also invited to bring their infants to the health centre at any time, for free attendance in the case of fever (suspected by the mother) or any clinical signs, related to malaria or not, and the same protocol (ie, questionnaire and RDT) was applied. Moreover, a TBS for confirmation of malaria infection was made and a drop of blood was deposited on filter paper for parasite genotyping. Once a month, a TBS and a sample of blood were systematically collected to assess asymptomatic P falciparum infection (figure 2). The presence of insecticide-treated nets in the house was checked during the visit. Infant follow-up, Tori Bossito, Benin. Anthropometric measures were performed once a month during the first 6 months and every 3 months afterwards. ITN, insecticide-treated net; RDT, rapid diagnostic test; TBS, thick blood smear. At 3, 6, 9, 12, 15 and 18 months, women were asked to attend the health centre with their offspring. In addition, venous blood was sampled (3 ml with EDTA). The level of haemoglobin was determined, blood was centrifuged and both plasma and buffy coat were kept frozen for further immunological and genetic studies. At birth anthropometric measurements were performed by midwives (weight, length, head circumference and mid-upper arm circumference; MUAC), according to WHO recommendations.21 Estimation of the gestational age of the newborn was carried out by field supervisors within 72 h after delivery by the neurological and morphological Ballard score.22 On monthly home visits a nutrition questionnaire was administered by supervisors to collect information about breastfeeding and to assess the quality of feeding practices through a qualitative dietary 24-h recall. Dietary diversity was assessed through the means of a list of 24 food groups that were further collapsed into 14 groups to create an individual dietary diversity score according to Food and Agriculture Organization recommendations.23 Other indicators of infant and young child practices were constructed as recommended by WHO.24 Anthropometric measurements (weight, length and MUAC) were performed every month from birth to 6 months, then at 9, 12, 15 and 18 months. Weight was recorded to the nearest 10 g using mechanical baby scales (SECA type 745, PHI Medical S.A., France). Length was measured to the nearest millimetre with a locally made wooden board equipped with two metal measuring tapes. MUAC was measured to the nearest millimetre using non-stretchable tapes (SECA 200). To ensure good quality anthropometric data, all measurements were performed twice, by two different operators. Entomological measures were made during 2 years in 36 sampling houses (simultaneously indoor and outdoor in four houses per village). Human landing catches were performed every 6 weeks, during three nights, simultaneously in three villages. Collections were made inside and outside the houses simultaneously, from 22:00 to 06:00 hours the following morning. At daybreak, morphological identification was made and Anopheles gambiae and Anopheles funestus were sent to Cotonou laboratory where they were kept and frozen at −20°C. Thereafter, P falciparum infected anopheles were identified by ELISA to determine sporozoite rates.25 The entomological inoculation rate (EIR), expressed in the number of infected anopheles per night and per man, was calculated for each village. To account for environmental factors that might influence transmission, climatic measures (temperature, humidity rate) were recorded every hour by electronic microchips placed in each of the nine villages. Rain levels were collected twice a day by means of pluviometers. At the end of the study, these data will be entered into a geographical information system, based on high resolution imagery (Spot 5, 10m colour, 2003), as well as data from mosquito catches, climate data, data related to the soil occupation of the area, vegetation favourable to breeding sites, water collections, information concerning human behaviour (use of bed nets, etc) and way of life (housing characteristics, habitat conditions, water supply, use of pesticides, number of persons sleeping in the house, etc) to characterise the spatial and temporal variability of malaria transmission and to build up an individual indicator of the risk of infection. All blood samples were processed in the Tori Bossito laboratory. Haemoglobin rates were measured at birth and quarterly on blood samples with a Hemocue analyser (Hemocue@AB, Sweden). TBS obtained monthly or during consultations were stained with Giemsa. Leucocytes and parasites were counted simultaneously until leucocyte or parasite numbers reached 500. A TBS was declared negative if no parasite was found after counting 500 leucocytes. Blood samples (maternal peripheral blood, cord blood or infant 3-monthly peripheral blood) were centrifuged at 1500 rpm for 10 min. Plasma and buffy coat were sent to Cotonou to be frozen (at −80°C and −20°C, respectively) for subsequent immunological and genetic analyses. Collections of whole blood or plasma were deposited on filter papers for further parasite genotyping and immunological analyses. Questionnaires were transferred to the Cotonou laboratory to be entered using Epi Data software V.3.1. In case of discordance after double entry control, data were sent back to the field to be corrected. The first phase of the analysis, which is ongoing, consists of a semiparametric model to study the association between the first occurrence of parasitaemia and the existence of an infected placenta at delivery.26 A Cox proportional hazards model was used to identify potential risk factors from fixed covariates, collected at the beginning of the study, and time-dependent covariates collected during the follow-up. Entomological and nutritional factors have been integrated in the model as time-dependent covariates.27 As there were few missing data, and as they were randomly distributed, no specific procedure was used to deal with them. In particular, we checked that there was no problem of informative censure (malaria infections not related to loss to follow-up). A second phase of the analysis will focus on the succession of recurrent malaria events in the same individual. The first parasitaemia (or the first malaria attack) and the following ones will be analysed with appropriate models based on an extension of the Cox model.28 This analysis will allow us to assess if the risk factors identified for the first malaria episodes persist in the following episodes. Data are analysed using Stata V.8.0 and SAS V 9.0 software. The protocol was approved by both the Ethical Committee of the Faculté des Sciences de la Santé (FSS) in Benin and the IRD Consultative Committee on Professional Conduct and Ethics (CCDE).