Detection of malaria sporozoites expelled during mosquito sugar feeding

Abstract

Malaria is a severe disease of global importance transmitted by mosquitoes of the genus Anopheles. The ability to rapidly detect the presence of infectious mosquitoes able to transmit malaria is of vital importance for surveillance, control and elimination efforts. Current methods principally rely on large-scale mosquito collections followed by labour-intensive salivary gland dissections or enzyme-linked immunosorbent (ELISA) methods to detect sporozoites. Using forced salivation, we demonstrate here that Anopheles mosquitoes infected with Plasmodium expel sporozoites during sugar feeding. Expelled sporozoites can be detected on two sugar-soaked substrates, cotton wool and Whatman FTA cards, and sporozoite DNA is detectable using real-time PCR. These results demonstrate a simple and rapid methodology for detecting the presence of infectious mosquitoes with sporozoites and highlight potential laboratory applications for investigating mosquito-malaria interactions. Our results indicate that FTA cards could be used as a simple, effective and economical tool in enhancing field surveillance activities for malaria.

Figure 1

Collection of mosquito saliva (‘forced salivation’). An An. stephensi female mosquito with legs and wings removed and proboscis inserted into a pipette tip containing 100 μl of 10% glucose solution. Saliva was collected in this manner for two hours per mosquito.

Figure 2

Detection of expelled sporozoites using qPCR. (A) Box and whisker plot of qPCR Ct values for the P. berghei cytb gene in infected adult mosquitoes (blue) and the resulting saliva extracts from forced salivation (red); (B) Fluorescent profiles for detection of the cytb gene in saliva; (C) Dissociation curve analysis indicating correct target amplification.

Figure 3

Heat map showing the relative amounts of P. falciparum malaria sporozoites in saliva. qPCR targeting the P. falciparum cox1 gene was used to determine the relative amounts in gDNA extracts from saliva in cotton wool collected from 6 replicate pot cages containing ~70 mosquitoes per pot. Lower Ct values (red) indicate a higher level of sporozoite DNA detected and white indicates absence of detection.

Figure 4

Heat map showing the relative amounts of mean P. berghei malaria parasites in mosquitoes and sporozoites in saliva eluted from FTA cards and cotton wool. Sugar substrates were placed on pots of mosquito pools of 5 and 10 with two replicate pots for each pool size over two days of collection. qPCR targeting the P. berghei cytb gene was used to determine the relative amounts and lower Ct values (red) indicate a higher level of sporozoite DNA detected and white indicates absence of detection.

Figure 5

Kaplan-Meier survival curves for three groups of mosquitoes provided with glucose on cotton wool, classic FTA cards or indicating FTA cards. The survival distributions were significantly different for both mosquito species. (A) An. stephensi (Log-Rank statistic Χ²_df = 2 = 82.88, p < 0.0001). (B) An. coluzzii (Log-Rank statistic Χ²_df = 2 = 119.4, p < 0.0001).