A multiplex assay for the simultaneous detection of antibodies against 15 Plasmodium falciparum and Anopheles gambiae saliva antigens

Abstract

Background: Assessment exposure and immunity to malaria is an important step in the fight against the disease. Increased malaria infection in non-immune travellers under anti-malarial chemoprophylaxis, as well as the implementation of malaria elimination programmes in endemic countries, raises new issues that pertain to these processes. Notably, monitoring malaria immunity has become more difficult in individuals showing low antibody (Ab) responses or taking medications against the Plasmodium falciparum blood stages. Commonly available techniques in malaria seroepidemiology have limited sensitivity, both against pre-erythrocytic, as against blood stages of the parasite. Thus, the aim of this study was to develop a sensitive tool to assess the exposure to malaria or to bites from the vector Anopheles gambiae, despite anti-malarial prophylactic treatment.

Methods: Ab responses to 13 pre-erythrocytic P. falciparum-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the Anopheles gambiae saliva protein gSG6 were tested. In this study, 253 individuals from three Senegalese areas with different transmission intensities and 124 European travellers exposed to malaria during a short period of time were included.

Results: The multiplex assay was optimized for most but not all of the antigens. It was rapid, reproducible and required a small volume of serum. Proportions of Ab-positive individuals, Ab levels and the mean number of antigens (Ags) recognized by each individual increased significantly with increases in the level of malaria exposure.

Conclusion: The multiplex assay developed here provides a useful tool to evaluate immune responses to multiple Ags in large populations, even when only small amounts of serum are available, or Ab titres are low, as in case of travellers. Finally, the relationship of Ab responses with malaria endemicity levels provides a way to monitor exposure in differentially exposed autochthonous individuals from various endemicity areas, as well as in travellers who are not immune, thus indirectly assessing the parasite transmission and malaria risk in the new eradication era.

Figure 1

Determining the optimal Ag concentration to be used for bead coating and the optimal serum dilution. The figure shows MFI values obtained from the serum of an exposed adult from Dielmo village at seven different dilutions (from 1:50 to 1:3200) with beads coated with three concentrations (0.075, 0.3 and 1.2 nmol) of synthetic peptides. All peptides were detected by the tested serum and their signals were much higher than the background signal against BSA-coated beads (upper left graph).

Figure 2

Comparison between exposed and unexposed adults: monoplex and multiplex assays showing coated bead-stability over time. In the upper panel (a), the same serum from the exposed individual from Dielmo village (black bars) tested in figure 1 and serum from an unexposed adult (white bars) were compared at a dilution of 1:100 by the multiplex assay. The assay allows a clear separation of peptide-specific MFI between the two samples, whereas the backgrounds (MFI values with BSA-coated beads) were equivalent. The serum from the exposed individual from Dielmo village was also tested for the same Ab responses by the monoplex assay with single peptide-coated beads (gray bars). Results with monoplex and multiplex assays were almost identical (correlation coefficient R² = 0.9896). The lower panel (b) shows results obtained using peptide-coupled beads after 1 (white bars), 2 (light gray bars) and 3 (dark gray bars) months of storage at 4°C against the 1:100 diluted serum from the exposed adult from Dielmo village and the 1:100 diluted serum from the unexposed adult (black bars, superimposed to the corresponding time delay). MFIs were consistent for all Ags, except StarpR (the correlation coefficient comparing values using coated beads after 1 and 3 months for each peptide, except StarpR, was R² = 0.9874).

Figure 3

The proportion of seropositive individuals increases with malaria exposure level. Among the adults (124 travelers, 45 from Dielmo, 40 from Ndiop and 38 from Diama), the proportion of seropositives for Lsa1-41 (p < 0.001), Lsa1J (p < 0.001), Lsa3NR2 (p < 0.001), Glurp (p < 0.001), GlurpP3 (p < 0.001), Salsa1 (p < 0.001), Salsa2 (p < 0.001), StarpR (p < 0.001), CSP (p < 0.001), SR11.1 (p < 0.001) and Saliv1 (p = 0.001) peptides differed significantly between groups (Fischer's exact test). In travellers, no Abs against Saliv1 Ag were detected; Abs were detected in exposed adults (three far left bars). 95% confidence intervals are shown in table 2.

Figure 4

MFI response in seropositive individuals increased with exposure to malaria. The distribution of corrected MFI (median fluorescent intensity) values (MFI_Ag/MFI_BSA) within groups is shown for the peptides Lsa1-41, Lsa1J, Lsa3NR2, Glurp, GlurpP3, Salsa1, Salsa2, StarpR, CSP, SR11.1 and Saliv1. The difference between groups was significant for Lsa1-41 (p = 0.02), Glurp (p = 0.0014) and CSP (p = 0.002), Kruskal-Wallis test. The number of seropositive individuals per group is presented in table 2.

Figure 5

The number of P. falciparum peptides that produced seropositive results increased with malaria exposure. The distribution of the total number of P. falciparum peptides for which each individual was seropositive is shown. The number of recognized peptides increased significantly with the malaria exposure level (p < 0.0001). The middle lines of the boxes represent the median distribution; the upper lines indicate the upper quartiles and the lower lines show the lower quartile. The upper and the lower lines outside the boxes represent the upper and lower non-outsider observations, respectively, while the dots are the outliers.