An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples. (December 2015) - Adams, Matthew
Abstract Background Highly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans. Methods A reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA ofPlasmodium falciparum andPlasmodium vivax . Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR. Results Limits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80 % humidity, the LoD was less than 16 parasites/mL forP. falciparum and 19.7 copies/µL forP. vivax (using a plasmid surrogate), about 10, 000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive forP. falciparum, 108 were positive forP. vivax, and 127 were positive for eitherP. vivax and/orP. falciparum using ultrasensitive RT-PCR. Conclusions This ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomaticP. falciparum andP. vivax parasitaemia.