Application of a qPCR assay in the investigation of susceptibility to malaria infection of the M and S molecular forms of An. gambiae s.s. in Cameroon

Abstract

Plasmodium falciparum is the causative agent of malaria, a disease that kills almost one million persons each year, mainly in sub-Saharan Africa. P. falciparum is transmitted to the human host by the bite of an Anopheles female mosquito, and Anopheles gambiae sensus stricto is the most tremendous malaria vector in Africa, widespread throughout the afro-tropical belt. An. gambiae s.s. is subdivided into two distinct molecular forms, namely M and S forms. The two molecular forms are morphologically identical but they are distinct genetically, and differ by their distribution and their ecological preferences. The epidemiological importance of the two molecular forms in malaria transmission has been poorly investigated so far and gave distinct results in different areas. We have developed a real-time quantitative PCR (qPCR) assay, and used it to detect P. falciparum at the oocyst stage in wild An. gambiae s.s. mosquitoes experimentally infected with natural isolates of parasites. Mosquitoes were collected at immature stages in sympatric and allopatric breeding sites and further infected at the adult stage. We next measured the infection prevalence and intensity in female mosquitoes using the qPCR assay and correlated the infection success with the mosquito molecular forms. Our results revealed different prevalence of infection between the M and S molecular forms of An. gambiae s.s. in Cameroon, for both sympatric and allopatric populations of mosquitoes. However, no difference in the infection intensity was observed. Thus, the distribution of the molecular forms of An. gambiae s.s. may impact on the malaria epidemiology, and it will be important to monitor the efficiency of malaria control interventions on the two M and S forms.

Figure 1. Standard curve of qPCR using serial dilutions of DNAs from cultured parasites.

A, Standard curve was obtained by linear regression analysis of Ct values versus log₁₀ copy number of cultured parasites in DNA standards (6 to 6×10⁴ genomes/µl). The slope was calculated from 53 independent reactions for each serial dilution. Errors bars show the standard deviation. B, Calibration curve for absolute quantification representing the starting concentrations of DNA standards (N0), expressed as arbitrary fluorescent units, versus log₁₀ input copy numbers of cultured parasites (6 to 6×10⁴ genomes/µl). N0 were calculated for each sample of the serial dilutions using the LinRegPCR software. The curve shows a good correlation of the N0 values with the estimate parasite number in serial dilutions (slope = 0.952, and R²>0.998). The calibration curve was used to convert the starting concentration of parasites in each mosquito midgut into the number of genomes/µl.

Figure 2. Comparison of standard curves between qPCR assays targeting the cox1 and SSU rRNA genes.

Composite plots were obtained by linear regression analysis of Ct values versus log₁₀ copy numbers of cultured parasites in DNA standards (6 to 6×10⁴ genomes/µl). The slopes were generated from 53 standard curves for the cox1 assay (blue diamonds) and 38 for the SSU rRNA assay (red circles), respectively. Error bars indicate the standard deviation. The SSU rRNA qPCR assay was not reproducible in reactions containing 6 genomes and was not taken into account in the plot.

Figure 3. Forest plots for the infection prevalence between the M and S form of An. gambiae s.s.

in allopatric (A) and sympatric (B) conditions. Each lane corresponds to a feeding on a single gametocyte carrier. Odds ratio (OR) and confidence interval (95% CI) were computed for each carrier and values are shown on the right part of the plot. OR values>1 indicate a higher prevalence of infection in the M form. Grey squares represent the OR estimate for each feeding, and the square size is proportional to the feeding’s weight in the meta-analysis. All feedings were combined in the random effects meta-analysis and the summary OR estimate is indicated at the bottom of the plot by a diamond.

Figure 4. Bland-Altman plot.

The plot shows the difference of infection prevalence between the M and S molecular forms in mosquito populations collected in allopatric (blue triangles) and sympatric (red circles) conditions. Each triangle or circle represents an experiment for which M and S mosquitoes were fed on a same blood donor. A positive value indicates a higher infection rate for the M form, and a negative one a higher infection rate for the S form. The M form was more infected than the S one for 76% (19/25) of feedings, and the difference is significant (X² = 11.52, P<0.001).