The value of local malaria strains for serological studies: local strains versus Palo Alto reference strain

Since 1990, the Dielmo project was established between the Institut Pasteur of Dakar, the Institute for Research and Development, the Senegal Ministry of Health and the University of Dakar. This project was initially approved by the Ministry of Health of Senegal and the assembled village population. The project aim was to study the natural history of malaria and more specifically the determinants of protection and biological markers associated to protection/exposure through longitudinal follow up of the inhabitants of the villages of Dielmo and Ndiop [14]. For this purpose clinical, entomological and parasitical studies were performed and blood samples were collected for immunological studies and stored in a biobank.

Written informed consent from individuals enrolled in the project and parents or guardians of children who provided blood samples was obtained. The consent to participate to the project was regularly renewed. Approval was obtained from the National Ethics Committee for Health Research of Senegal and ad-hoc committees of the Ministry of Health, the Pasteur Institutes (Dakar and Paris), and Institute for Research and Development did audits regularly.

This study is based on the samples stored in the biobank and is in line with the objectives of the Dielmo project [14].

This comparative analysis study was conducted using sera collected in 2000 from inhabitants of the villages of Ndiop and Dielmo. A total of 220 sera from individuals less than 20 years old were used: 76 from Ndiop and 141 from Dielmo. In 2000, Ndiop and Dielmo had differences in malaria endemicity and contrasting contexts of transmission: in Ndiop, transmission was seasonal with an entomological inoculation rate of 80 infectious bites/person/year, whereas in Dielmo the transmission was continuous with 482 infectious bites/person/year [15]. Blood samples were collected before the end of the rainy season in 2000 when transmission was high in both Ndiop and Dielmo [16]. The sera were separated from blood cells and stored at −20 °C until use for immunological studies.

Four parasite strains (0703, F15, F16 and PA) were used in this study and continuously cultured on O⁺ erythrocytes in RPMI containing 0.5 % Albumax using the candle jar method [17]. Three field strains of P. falciparum isolated from clinical or asymptomatic malaria-infected Senegalese patients and adapted for in vitro culture as described elsewhere [13, 18]. The 0703 strain was isolated from an inhabitant of Dielmo on November 1991 and successfully adapted to continuous in vitro culture thereafter [19]. F15 and F16 strains were isolated in Dakar on February 2010 and March 2010, respectively, from patients of the medical analysis laboratory and were also successfully adapted to continuous in vitro culture. The Palo Alto Uganda reference strain FCR3 (PA, Uganda) had been isolated from a Ugandan patient [20] and had been routinely cultured for years in the laboratory. Strains were cultured separately at different time periods to avoid cross contamination between strains. Extracts of schizonts of P. falciparum were prepared as previously described [12, 18]. Briefly, for schizont extracts, after centrifugation, one volume of the pellet was lysed in three volumes of sterile distilled water and vortexed for homogenization; the extracted schizonts were frozen without further centrifugation in liquid nitrogen in working aliquots. The merozoite extract was prepared from synchronous parasites as described by others [12, 21]. In brief, merozoites were collected by stepwise centrifugation of culture supernatants at 2,000 and 4,000 g, washed three times in sterile phosphate buffered saline (PBS), counted, and frozen in liquid nitrogen.

Genomic DNA was extracted from wild field Senegalese isolates of P. falciparum adapted in continuous culture (0703, F15, F16) and from the PA reference strain using a QiAmp ® DNA Blood MiniKit (Qiagen Catalogue # 51106) according to the manufacturer’s recommendations. Nested PCR genotyping was performed for the variable block 3 region of msp-2, considered to be the most informative genetic marker for assessment of P. falciparum genetic diversity [22]. The initial PCR amplification with primers pairs flanking the msp-2 (block 3) region was followed by individual nested PCR reactions using family-specific primers for msp-2 (FC27 and 3D7/IC), using previously described standard protocols [23]. The PCR conditions and primers sequences were as described by Snounou et al. [24, 25]. The PCR-amplified msp-2 products were separated by electrophoresis on 1.5 % agarose gel and the fragments were visualized under UV transillumination with ethidium-bromide. The sizes were estimated by comparison with a 100 bp (Hyperladder, Bioline®) molecular weight marker.

ELISA Maxisorp plates (Nunc, Roskilde, Denmark) and a dose effect assay using positive control sera were used to determine the optimal dilution of the extracts to use in the assay. The optimal dilution found for each extract was used for coating the plates. Plates were coated with 100 μl of water-soluble extract for all crude extracts. Non-infected red blood cell extract was prepared in the same conditions, incubated overnight at 4 °C and washed. Then, 200 μl of a blocking buffer (2 % BSA in PBS with 0.05 % Tween) was added to each well and the plates were incubated for 1 h at 37 °C. Plasma samples were diluted at 1/200 in a dilution buffer (1 % BSA in PBS with 0.05 % Tween) and 100 μl of diluted serum was distributed into each well. Negative and positive controls were included on each plate: positive controls were from Dielmo/Ndiop hyperimmune individuals and negative controls were from European individuals. Polyclonal goat anti-human IgG conjugated to peroxidase at a dilution of 1/6,000 in the dilution buffer (1 % BSA in PBS-Tween 0.05 %) was then added. Bound peroxidase was detected with ortho-toluidine /H₂O₂ (100 μl) and the reaction was stopped by addition of 4 N H₂SO₄ (50 μl/well). Between each incubation phase, the ELISA plates were washed extensively with PBS-0.05 % Tween. For each assay, plasma samples were tested in duplicates on the same ELISA plate. The optical density (OD) at 450 nm was read in a BIO-RAD Microplate Reader (iMark) [12]. The final OD value of each serum tested was obtained by calculating the mean OD of the duplicates. Sera from negative controls were used to define the cutting-off value for positivity based on an optical density of 2 fold the mean optical density (OD) of these negative controls. Results was expressed as OD ratio calculated as mean OD test/mean OD neg. and a ratio OD test/mean OD neg >2 was defined as cutting-off value for positive response. Inter-assay variations for positive controls did not exceed 20 %. The reproducibility of this technique has been found satisfactory with coefficients of variation of 4 % by using two positive controls (two different pools of hyperimmune serums), 10 negative controls and the same crude extract preparation on each plate [12, 26].

R software was used for statistical analysis. Non parametric tests (Kruskal Wallis, Wilcoxon) and the Spearman test were used to study non-parametric quantitative variables. Fisher’s exact test was used for qualitative variables. Differences were considered statistically significant for p < 0.05 [27].