A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector

• Published in: International journal for parasitology 2004-06, Vol.34 (7), p.795-802
• Main Authors: Bell, A.S, Ranford-Cartwright, L.C
• Format: Article
• Language: English
• Subjects: animal parasites and pests; Animals; Anopheles - parasitology; Anopheles stephensi; assays; Biological and medical sciences; carrier state; Culicidae; developmental stages; disease transmission; disease vectors; Female; Freezing; Fundamental and applied biological sciences. Psychology; genes; hematophagous insects; Human protozoal diseases; Infectious diseases; insect vectors; Insect Vectors - parasitology; Malaria; Malaria, Falciparum - transmission; Medical sciences; Medically important nuisances and vectors, pests of stored products and materials: population survey and control; midgut; monoclonal antibodies; oocysts; Oocysts - chemistry; Parasitic diseases; Plasmodium falciparum; Plasmodium falciparum - chemistry; polymerase chain reaction; Polymerase Chain Reaction - methods; Protozoal diseases; Real-time quantitative PCR; Reproducibility of Results; ribosomal RNA; RNA, Protozoan - analysis; RNA, Ribosomal - analysis; sporozoites; standard membrane feeding assay; SYBR Green I; transmission blocking activity; Transmission-blocking vaccine; transmission-blocking vaccines; vaccines; vector competence; Vectors. Intermediate hosts
• ISSN: 0020-7519; 1879-0135
• DOI: 10.1016/j.ijpara.2004.03.008

Transmission-blocking vaccines prevent the development of Plasmodium parasite within the mosquito vector, thereby thwarting the spread of malaria through a community. The gold standard for determining the efficacy of a transmission-blocking vaccine is the standard membrane feeding assay. This assay requires the dissection of mosquitoes and microscopic counting of oocysts present on the mosquito mid-gut, typically at 7–10 days p.i. Here we describe a real-time quantitative PCR assay that is rapid, target-specific and robust, with a sensitive detection threshold and which may be employed earlier p.i. than the standard membrane feeding assay and is applicable to preserved material. The real-time PCR assay utilises the LightCycler platform and SYBR Green I detection system to amplify 180 bp of the asexual form of the Plasmodium falciparum rRNA gene. It has a quantitative range of greater than four orders of magnitude and a detection threshold of 10 parasites. Validation experiments using a monoclonal antibody of known blocking activity revealed the real-time PCR assay to give equivalent results to the standard membrane feeding assay. In addition, the PCR assay can establish the effect of such a monoclonal antibody on the parasites' development within the oocyst and on the sporozoite (the transmissible stage) yield, providing a more pertinent assessment of transmission blocking activity than is possible by the standard membrane feeding assay. This assay may also be employed to monitor the sporogonic development of P. falciparum parasites within the mosquito vector.