Laboratory and field evaluation of polymerase chain reaction-based forensic DNA profiling for use in identification of human blood meal sources of Aedes aegypti (Diptera: Culicidae).

ABSTRACT

We modified polymerase chain reaction (PCR)-based forensic DNA profiling for field studies on the feeding behavior of Aedes aegypti, the principal mosquito vector of dengue virus. Human DNA was extracted from oral swabs of human subjects and from blood-engorged mosquitoes, DNA was quantified by slot blot, and alleles at variable number tandem repeats and three short tandem repeats loci were amplified by PCR. Alleles were separated electrophoretically and then visualized by silver staining. A custom software program was written to match DNA fingerprints of potential human hosts to allelic profiles detected in engorged mosquitoes, and to calculate error rates for identification of human hosts of single and multiple-host blood meals. At 29 degrees C in the laboratory, human DNA recovered from mosquito blood meals declined an average of 67% 8 h after feeding and 90% after 24 h. We obtained complete allelic profiles from seven of 10 mosquitoes collected after 24 h. In a field trial, complete DNA profiles were obtained successfully for 43 people living in a rural village in south central Thailand and for 20 of 100 Ae. aegypti that contained blood and were collected in those peoples' homes. Blood imbibed from more than one person was detected in 45% (9 of 20) of the meals. Sixty-five percent of the meals contained blood from nonresidents of the house in which the mosquito was collected or from people who were not profiled; data consistent with the hypothesis that human movement is important for the spread of dengue virus within and among communities. When using alleles at four loci, all of the Thais and nine members spanning three generations of a Chinese-American family had unique allelic profiles. Error rates from classifying possible multiple-host meals as single-host meals were low (1-8%), with the highest error associated with closely related people. Results from our laboratory and field studies indicated that DNA profiling can be used to study the details and epidemiological implications of Ae. aegypti blood-feeding behavior.