Methods Assessed for Detecting Asymptomatic Malaria

Sensitive methods for detecting asymptomatic malaria infections are essential for identifying potential transmission reservoirs and obtaining an accurate assessment of malaria epidemiology in low-endemicity areas aiming to eliminate malaria. Polymerase chain reaction (PCR) techniques to detect parasite nucleic acids (DNA or RNA) are among the most commonly used molecular methods.

Scientists at China Medical University and their colleagues recruited 1,005 healthy individuals (344 males and 661 females, ages 1–82 years) living in Kachin Sate, Myanmar between May and November, 2015. Two fingerprick samples were taken, one was processed in a nearby field laboratory, and the other on filter paper, dried and stored. The study compared three molecular detection methods side-by-side, namely nested PCR targeting the ribosomal ribonucleic acid (rRNA) genes, nested RT-PCR to detect parasite rRNA, and capture and ligation probe-PCR (CLIP-PCR) to detect parasite RNA.

Thick and thin blood films stained with Giemsa were prepared and read. For positive slides, parasite density was quantified in 500 white blood cells (WBCs) on thick blood films assuming that 1 µL of blood contains 8,000 WBCs. Total RNA and genomic DNA were extracted from peripheral blood samples. Modified nested PCR (nD-PCR) was performed based on the 18S rRNA gene. For the CLIP-PCR 3-mm circle of dried blood spot on 3 M Whatman filter paper was punched out and lysed with 100-µL lysis mixture. Two RT-PCR methods were used to detect Plasmodium vivax gametocytes in samples. For detection of P. vivax gametocytes in all 1,005 samples, the 645 bp full-length Pvs25 gene was amplified using Pvs25-specific primers. Amplification and detection were performed on an ABI 7500 apparatus.

Light microscopy detected Plasmodium infections in only 1.19% of the residents harboring the parasites. CLIP-PCR had slightly better performance and detected Plasmodium infections in 1.89% of the population. Further improvement was achieved by nested PCR to detect parasite DNA, which detected P. vivax and P. falciparum infections in 2.39% of the residents. The nested RT-PCR targeting rRNA, however, detected as many as 187 (18.61%) individuals having Plasmodium infections with P. vivax being the predominant species (176 P. vivax, five P. falciparum and six P. falciparum/P. vivax mixed infections). Of the 210 Plasmodium-positive samples detected by all molecular methods, 115 were Pvs25-positive by quantitative polymerase chain reaction (qRT-PCR), indicating that a large proportion of asymptomatic individuals were gametocyte carriers.

The authors concluded that Nested RT-PCR based on the detection of asexual-stage parasite rRNA was the most sensitive, with a more than six-fold higher sensitivity than the other two molecular methods of parasite detection. CLIP-PCR has an increased throughput, but its sensitivity in this study was much lower than those of other molecular methods. The study was published on April 20, 2017, in the Malaria Journal.