Mosquito feeding assays to determine the infectiousness of naturally infected Plasmodium falciparum gametocyte carriers

Abstract

Introduction: In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes.

Methods: WE COMPARED TWO COMMONLY USED MOSQUITO FEEDING ASSAY PROCEDURES: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum.

Results: 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94-2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68-2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52-0.70).

Conclusions: Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.

Figure 1. Mosquito feeding assays.

In direct skin feeding assays, mosquitoes are placed directly on the skin of a parasitaemic host. In direct membrane feeding assays, a venous or finger prick blood sample from a naturally infected individual is offered to mosquitoes. Water-jacketed glass feeders are kept at approximately 37°C and mosquitoes feed through a membrane. In a whole blood assay, the blood sample is offered to mosquitoes directly. In a serum replacement experiment, the blood cells are offered to mosquitoes in a paired experiment in the presence of own plasma or of control serum.

Figure 2. A forest plot showing the overall result of each study together with the overall estimates of the meta-analysis.

All data were combined to generate the summary odds ratio and 95% confidence interval estimates denoted by the black diamond. Odds ratios are calculated with the latter feeding condition as reference; e.g. for Figure 2A the odds ratio for mosquito infection in skin feeding experiments is calculated with whole blood membrane feeding as reference. Points are weighted according to the number of paired experiments in the study (indicated by the size of the data point).

Figure 3. Skin feeding versus membrane feeding.

Results are from 161 paired experiments plotted in a Bland-Altman plot. Points above the line had higher infectivity in the skin feeding assay than the whole blood SMFA. Point colour denotes the study using the same key as in Figure 2. The shape of the point denotes the country the experiment was carried out in, be it Cameroon (circle), Mali (triangle) or Senegal (diamond). The size of the points indicates the relative average number of mosquitoes dissected in the experiment, with points>50 mosquitoes dissected having the same size). In 117 experiments (72.7%), the proportion of infected mosquitoes was higher in skin feeding assays compared to the paired whole blood membrane feeding assay; in 43 experiments (26.7%) the opposite was observed.

Figure 4. The association between mosquito infection rates in skin feeding experiments and whole blood membrane feeding assays.

The proportion of infected mosquitoes in whole blood membrane feeding assays (X-axis) is strongly associated with the proportion of infected mosquitoes in skin feeding assays (Spearman's rho 0.36, p<0.0001). Point size, shape and colour are as described in Figure 3. The shape of the point denotes the country the experiment was carried out in, be it Cameroon (circle), Mali (triangle) or Senegal (diamond).

Figure 5. Membrane feeding with whole blood and with own plasma.

The results are from 159 paired experiments. In 116 experiments (73.0%), the proportion of infected mosquitoes was higher in experiments with whole blood samples (i.e., without sample modulation) compared to serum replacement experiments where the blood pellet went through additional manipulation before being re-suspended with the gametocyte donor's own plasma. In 38 experiments (23.9%) the opposite was observed. Point size, shape and colour are as described in Figure 3. All experiments were performed in Cameroon.

Figure 6. Membrane feeding with and without serum replacement.

The results are from 325 paired experiments. In 221 experiments (68.0%), the proportion of infected mosquitoes was higher in experiments with malaria naïve control serum compared to the gametocyte donor's own plasma; in 92 experiments (28.3%) the opposite was observed. The shape of the point denotes the country the experiment was carried out in, be it Cameroon (circle) or The Gambia (square). Point size, shape and colour are as described in Figure 3.

Figure 7. The association between gametocyte density and mosquito infection rates for different feeding conditions.

Plotted is the association between gametocyte density and mosquito infection rates (oocyst prevalence) for skin feeding assays (red squares), whole blood membrane feeding assays (blue circles) and serum replacement experiments with own plasma (green triangles) and control serum (yellow diamonds). Data were grouped according to gametocyte density estimates into 8 equally sized bins (on a log scale). Panel A shows all the data grouped together whilst B to E show the data grouped by country. The size of the data points is relative to the number of mosquitoes dissected in each bin.