Molecular detection of DHFR and DHPS genes from sporozoite infected mosquitoes

Introduction: Detecting and identifying malaria parasites in mosquitoes are crucial in estimating the efficacy of malaria control strategies. Among several methods which have been used include monoclonal antibodies, DNA probes and polymerase chain reaction (PCR). We are using monoclonal antibody ELISA to screen sporozoite positive mosquitoes and nested PCR to amplify dhfr and dhps genes from these positive samples and from some sporozoite negative samples. Methods: We collected 1090 anopheline mosquitoes from Lupilo village in Ulanga district by indoor resting method. The mosquitoes were transferred to insectary and kept there for 10 days to ensure completion of sporogonic cycle. We examined 646 mosquitoes without their abdomen to minimize the interferences from blood and oocyst stages of Plasmodium. Individual mosquito was homogenized in Tris EDTA buffer and aliquoted into two halves one for ELISA and another for DNA extraction. Sandwich ELISA was used to detect circumsporozoite protein by using monoclonal antibody. DNA from positive and negative sporozoite samples were extracted by chelex-based method and subjected to nested PCR to amplify dhfr and dhps genes with 594 bp and 711 bp, respectively. Results: Of 646 samples processed, 19 samples(2.94%) were positive for ELISA. Of these 19 samples,3 were positive for PCR (dhfr). 36 samples (5.57%)which were negative for ELISA were found to be positive for PCR (dhfr). Of 39 samples where dhfr gene was detected, dhps gene was only detected from two samples. Most of these samples produce characteristically weak bands which may suggests low parasitaemia in most of these samples or poor DNA quality. This problem together with the failure to detect dhps in some samples where dhfr was detected, limited us to undertake further analysis such as screening all possible mutations in these markers of SP resistance. PCR inhibitors that are contained in anopheline mosquitoes may contribute to inhibitory effect to PCR. Nothing is known to whether chelex method is efficient enough to remove these inhibitors. However, the optimisation of extraction methods and PCR conditions are still underway to overcome these problems. Interpretation: Our preliminary results support the fact that PCR is highly sensitive in detecting parasite DNA. The failure to detect these genes from many positive sporozoite samples may be due to melanin, inhibitors that are present in exoskeleton of mosquitoes.