V. Evaluation of Cross-Immunity against Type 1 Dengue Fever in Human Subjects Convalescent from Subclinical Natural Japanese Encephalitis Virus Infection and Vaccinated with 17D Strain Yellow Fever Vaccine
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department d'Epidemiologie des Affectations Parasitaires, Ecole Nationale de Medecine et de Pharmacie, Bethesda, Maryland, Mali
We used sequences specific to the small subunit ribosomal RNA (SSU rRNA) of the sporogonic stages of Plasmodium falciparum to design a reverse transcriptase-polymerase chain reaction (RT-PCR) assay that can detect 0.1 sporozoites in total RNA purified from potentially infected mosquitoes. We made a synthetic RNA that is amplified in the RT-PCR by the same primers as the parasite SSU rRNA and that serves as an internal control and competitive quantitation standard. We calibrated the assay for quantitation of sporozoites by making a standard curve with RNA from purified and counted sporozoites. The assay accurately measured sporozoite number with a linear range of at least three orders of magnitude in a single reaction. Some applications and limitations of the assay are discussed.