Antibodies to full-length and the DBL5 domain of VAR2CSA in pregnant women after long-term implementation of intermittent preventive treatment in Etoudi, Cameroon

In high malaria transmission settings, the use of sulfadoxine-pyrimethamine-based intermittent preventive treatment during pregnancy (IPTp-SP) has resulted in decreased antibody (Ab) levels to VAR2CSA. However, information of Ab levels in areas of low or intermediate malaria transmission after long-term implementation of IPTp-SP is still lacking. The present study sought to evaluate antibody prevalence and levels in women at delivery in Etoudi, a peri-urban area in the capital of Yaoundé, Cameroon, that is a relatively low-malaria transmission area. Peripheral plasma samples from 130 pregnant women were collected at delivery and tested for IgG to the full-length recombinant VAR2CSA (FV2) and its most immunogenic subdomain, DBL5. The study was conducted between 2013 and 2015, approximately ten years after implementation of IPTp-SP in Cameroon. About 8.6% of the women attending the clinic had placental malaria (PM). One, two or 3 doses of SP did not impact significantly on either the percentage of women with Ab to FV2 and DBL5 or Ab levels in Ab-positive women compared to women not taking SP. The prevalence of Ab to FV2 and DBL5 was only 36.9% and 36.1%, respectively. Surprisingly, among women who had PM at delivery, only 61.5% and 57.7% had Ab to FV2 and DBL5, respectively, with only 52.9% and 47.1% in PM-positive paucigravidae and 77.7% of multigravidae having Ab to both antigens. These results suggest that long-term implementation of IPTp-SP in a low-malaria transmission area results in few women having Ab to VAR2CSA. The study protocol was reviewed and approved by the National Ethics Committee of Cameroon (Ethical Clearance 2013/02/ N° 029/L/CNERSH/SP). Administrative Authorizations were obtained from the Ministry of the Public Health of Cameroon (No D30-392 AAR/MINSANTE/SG/DROS/ CRC/ CEA1) and from Health Centre. Participation in the study was voluntary with written informed consent from each woman. Upon collection, all samples and clinical information about the mother and baby were de-identified. The study was performed following the guidelines and regulations of human clinical research as recommended by the Ministry of the Public Health of Cameroon. Malaria rapid diagnostic test (RDT) was performed for each woman at the time of enrollment and positive results were reported to the physician for prescription of treatment according to the national policy. This cross-sectional study was carried out between 2013 and 2015 at the Marie Reine Health Center in Etoudi, situated in a peri-urban area of Yaoundé, Cameroon. Malaria transmission in this area is relatively low and perennial, peaking in May (during the long-wet season from March to June) and October (during short wet season from September to November). A total of 130 HIV-negative women aged 16 to 39 years were recruited. Information on the mother’s health, estimated length of pregnancy, parity, age, use of anti-malarial drugs, IPTp-SP usage, HIV status, and baby birth weight were recorded. Peripheral blood samples were collected in EDTA tubes from women immediately following delivery. A portion of the blood was used to prepare thick and thin smears for microscopy and to measure hemoglobin levels. The remainder was centrifuged and plasma was collected and stored at -80º C for antibody studies. Placental tissues were also collected and a section excised to prepare impression smears and for histology. Thick and thin blood smears, were prepared using Giemsa-Wright stain and read by two skilled microscopists to determine the presence of malaria parasites. In addition, thin film of peripheral blood was used to determine parasite species. Results from blood smears were compared with RDT results obtained at enrollment using the CarestartTMHRP2 (Pf) (Access Bio Inc. NJ, USA). Placental sections were fixed in buffered formalin, embedded, stained with hematoxylin-eosin, and examined for IE and malaria pigments. Women were considered PM+ if IE were detected in impression smears of villous tissue and/or in histological sections. Hemoglobin levels in maternal blood were determined using a Coulter Counter (URIT-3300, Europe). Plasma IgG Ab levels to VAR2CSA recombinant proteins (FV2 and DBL5) of the FCR3 strain, expressed in Baculovirus-transfected insect cells, were measured using a multi-analyte platform (MAP). The coupling of the recombinant proteins to MagPix microspheres and basic protocol have been described previously [21, 24, 25]. Briefly, 50 μl of antigen-coupled microspheres (2000 microspheres/antigen) were incubated in a well of a microtiter plate (U-bottom microplate) with 50 μl of plasma diluted to 1:100 in phosphate buffered saline containing 1% bovine serum albumin (PBS 1% BSA) for 1 h at room temperature on a rotating shaker at 500 rpm (Microplate Shaker, Lab-line, Melrose Park, IL, USA). After washing twice with PBS-0.05% Tween 20 and once with PBS-1% BSA, 100 μl of secondary Ab (R-phycoerythrin-conjugated, Affini Pure F(ab′)2 fragment, Goat anti-human IgG Fc fragment specific, Jackson Immunoresearch, West Grove, PA, USA) diluted to 2 μg/ml in PBS-1% BSA was added to each well and incubated on a shaker in the dark for 1 h. Wells were washed and microspheres re-suspended in 100 μl PBS-1% BSA. Plates were analysed immediately using MAGpix Analyser (MAGpix Technology, USA). The reader was programmed to read a minimum of 100 beads per spectral address, DD Gate 7500–15,000 and 35s timeout. The results were expressed as median fluorescence intensity (MFI). Positive and negative controls were included on each plate to control for plate-to-plate variation. Positive control was a pool of eight Cameroonian multigravidae with high Ab levels to VAR2CSA. Negative controls were plasma from 12 Cameroonian men, age 23 to 30 years of age, living in the study area. The cut-off value for seropositivity was determined based on the mean MFI + 2SD of the male samples. Graph Pad Prism 6.0.1 was used for the statistical analyses. Continuous variables are reported as means +/- standard deviations (SD) or medians with interquartile range (IQR). Differences between groups were compared using unpaired t-test for normally distributed continuous data or Mann-Whitney Rank Sum test for non-normal distributed continuous data, while categorical variables were reported as percentages and were compared using Fisher’s exact test. P values <0.05 were considered statistically significant.