Relationship between Antibody Levels, IgG Binding to Plasmodium falciparum-Infected Erythrocytes, and Disease Outcome in Hospitalized Urban Malaria Patients from Dakar, Sénégal

Abstract

Background. Management of clinical malaria requires the development of reliable diagnostic methods and efficient biomarkers for follow-up of patients. Protection is partly based on IgG responses to parasite antigens exposed at the surface of infected erythrocytes (iRBCs). These IgG responses appeared low during clinical infection, particularly in severe disease. Methods. We analyzed the IgG binding capacity to the surface of live erythrocytes infected by knob positive FCR3 strain. Sera from 69 cerebral malaria (CM) and 72 mild malaria (MM) cases were analyzed by ELISA for IgG responses to five antigens from iRBC and by flow cytometry for IgG binding as expressed in labeling index ratio (LIR). The relationship between IgG levels, LIR, parasitemia, age, and the clinical outcomes was evaluated. Results. We found a significant decrease of LIR in adult CM fatal cases compared to surviving patients (p = 0.019). In MM, LIRs were correlated to IgG anti-iRBC and anti-PfEMP3/5 levels. In CM, no correlation was found between LIR, IgG levels, and parasitemia. Conclusion. The IgG binding assay was able to discriminate outcome of cerebral malaria cases and it deserves further development as a potential functional-associated assay for symptomatic malaria analysis.

Figure 1

Examples of histograms of flow cytometry data acquisition. The two histograms show results from the acquisition of 5,000 events of a 3% P. falciparum culture after gating on FL₁ > 10¹ (a) and on FL₁ > 10² (b). The gating on high FL₁ values (>10²) selected the mature population of the cultured parasites. The binding of human IgG to IRBC was revealed by an anti-IgG conjugated to thiazole orange (TO). The fluorescence of TO-positive iRBCs was measured in the FL₂ channel. The reference negative control (blue curve histogram, Pool NEG), the positive control (green curve, Pool HIS), and a strong responder from Ndiop (red curve, positive sample) are shown. Analysis was done by Flow Jo® Software.

Figure 2

IgG antibodies levels against whole parasite extract antigens (a) and recombinants proteins (b) in survivors and fatal CM patients. Scatter plot of IgG responses to whole parasite extracts antigens (a) and recombinant proteins (b). Comparison between surviving cerebral malaria patients (SCM; n = 51; open circles) and fatal cerebral malaria patients (FCM; n = 18; black circles) for each antigen. Horizontal blue bar indicates medians of OD ratio value. The Mann-Whitney rank test was employed for between-groups comparison for each antigen. IgG levels were highest in surviving CM group for P. falciparum schizont extract, iRBC membrane, PfEB200, and PfEMP3/5. Significant p values are included (p < 0.05).

Figure 3

Variations of labeling index ratio according to disease severity (a), CM outcome (b), and age of individuals in CM patients (c). Box plot of labeling index ratio (LIR). The horizontal line within each box represents the median value of LIR. Comparison were done (a) between mild malaria patients (MM, n = 35; white boxes) and cerebral malaria group (CM, n = 69; dark boxes), (b) between fatal cerebral malaria group (FCM; n = 18; hacked boxes) and surviving cerebral malaria patients (SCM; n = 51; grey boxes), and (c) in CM patients according to both outcome and age of children (left) and adults (right). The Mann-Whitney rank test was employed for comparison between groups. Sera from survivors showed significant high LIR compared to fatal CM patients (p < 0.05) in adults. Significant p values are included.

Figure 4

Relationship between labeled index of recognition and IgG responses in MM (a) and CM (b–d) groups. The correlations between labeled index of recognition (LIR) and IgG anti-iRBCm levels (OD ratio) are plotted in MM patients (a) (dark circles) and in CM group (b) with a dichotomization in fatal CM (dark squares) and surviving CM (open circles). Relationships between LIR and the levels of IgG anti-PfEMP3/5 (OD ratio) or IgG anti-PfEB200 levels (OD ratio) are, respectively, plotted in (c) and (d), only in CM surviving patients (open circles). Results from statistical analyses done by nonparametric Spearman rank test are indicated.