Ultrasensitive Assay Detects Asymptomatic Low-Density Malaria

Highly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions and 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.

An improved assay has been developed to detect both ribosomal ribonucleic acid (RNA) and DNA, thus taking advantage of the high copy numbers of RNA in small blood volumes, using a stabilizing reagent that allows preservation of nucleic acids from small volumes of blood suitable for collection by finger or ear stick, with minimal sample processing, and no cold chain.

Scientists at the University of Maryland School of Medicine (Baltimore, MD, USA) and their colleagues collected blood samples across three townships in Southeastern Myanmar. Blood collected by finger stick was used to perform Malaria Ag P.f/Pv, RDTs (Standard Diagnostics; Yongin-si, Republic of Korea) on site, and 0.3 mL of blood was placed into collection tubes. Total nucleic acid was extracted from 200 μL of blood/stabilizer mixture using the QIAamp 96 DNA Blood Kit (Qiagen; Valencia, CA, USA) but with an elution volume of 50 µL of buffer.

A reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1,750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR, which was performed on a Roche Lightcycler 96 or 480 real time PCR machine (Roche Diagnostics; Indianapolis, IN, USA).

The limits of detection (LoD) were determined under simulated field condition and was found to be was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1,739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR.

The authors concluded that the 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 asymptomatic P. falciparum and P. vivax parasitemia. The study was published on December 23, 2015, in the Malaria Journal.

Related Links:
University of Maryland School of Medicine
Standard Diagnostics 
Qiagen