PCR detection of malaria parasites in desiccated Anopheles mosquitoes is uninhibited by storage time and temperature.
BACKGROUND
Reliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR).


METHODS
Laboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein.


RESULTS
Varying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification.


CONCLUSIONS
Cold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely ensure accurate assessment of malaria parasite presence without diminishing PCR-detection of parasites in mosquitoes stored for at least six months.
Department of Tropical Medicine, Tulane University, New Orleans, LA 70112, USA. mrider@tulane.edu
Mark A
MA
Rider
Brian D
BD
Byrd
Joseph
J
Keating
Dawn M
DM
Wesson
Kevin A
KA
Caillouet
D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D003890:Desiccation; D004772:Entomology / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D010961:Plasmodium / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors
D016054:DNA, Protozoan