Short report: simultaneous detection by polymerase chain reaction of mosquito species and Plasmodium falciparum infection in Anopheles gambiae sensu lato.

Total RNA purified from Anopheles gambiae mosquitoes can be used for detection of both 1) infection by Plasmodium falciparum using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay specific for P. falciparum ribosomal RNA (rRNA) of sporogonic stage parasites, and 2) mosquito species using a PCR assay that distinguishes members of the Anopheles gambiae complex.

Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa.

As chloroquine resistance spreads across Africa, the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and proguanil are being used as alternative first-line drugs for the treatment and prevention of Plasmodium falciparum malaria. Resistance to these drugs is conferred by point mutations in parasite DHFR. These point mutations can be detected by polymerase chain reaction (PCR) assays, but better methods for sample collection, DNA extraction, and a diagnostic PCR are needed to make these assays useful in malaria-endemic areas. Here we report methods for collecting fingerstick blood onto filter paper strips that are air-dried, then stored and transported at room temperature. Cell lysis and DNA extraction are accomplished by boiling in Chelex-100. We also report a nested PCR technique that has improved sensitivity and specificity. These procedures readily detect mixed infections of parasites with both sensitive and resistant genotypes (confirmed by direct sequencing) and are reliable at parasite densities less than 250/mm3 in field surveys.

Mark-release-recapture experiments with Anopheles gambiae s.l. in Banambani Village, Mali, to determine population size and structure.

Mark-release-recapture experiments with Anopheles gambiae s.l. were performed during the wet seasons of 1993 and 1994 in Banambani, Mali. All recaptured mosquitoes were identified to species by PCR analysis and, when possible, by chromosomal analysis to chromosomal form. Two species of the An. gambiae complex were present: An. gambiae s.s. and An. arabiensis; their ratio differed greatly from one year to the next. Three chromosomal forms of An. gambiae s.s. were found--Bamako, Savanna and Mopti. The drier 1993 was characterized by a high frequency of An. arabiensis and of the Mopti chromosomal forms of An. gambiae s.s. These trends were consistent with large-scale geographical patterns of abundance along a precipitation gradient. We observed no difference in dispersal between the two species, nor among the chromosomal forms of An. gambiae s.s. Therefore, in this situation at least, it is reasonable to group such data on the An. gambiae complex as a whole for analysis. Population size of An. gambiae s.l. females in the village was estimated to be 9000-11,000 in 1993 and 28,000 in 1994. The corresponding numbers were somewhat higher when independently-derived values of daily survival were used. These were consistent with estimates of effective population size obtained from patterns of gene frequency change.