Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings

Abstract

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.

Keywords: Plasmodium falciparum; Plasmodium vivax; low-transmission setting; malaria; nested PCR; pooling strategy.

Fig. 1.

Experimental design for optimal number of the pooling strategy. Lane 1, 100 bp DNA ladder; lane 2, negative blood only (200 μl); lane 3, parasitized blood only (200 μl of 200 p/μl); lane 4, parasitized blood only (200 μl of 20 p/μl); lane 5, 67 μl of 200 p/μl with 133 μl of negative blood; lane 6, 40 μl of 200 p/μl with 160 μl of negative blood; lane 7, 20 μl of 200 p/μl with 180 μl of negative blood; lane 8, 67 μl of 20 p/μl with 133 μl of negative blood; lane 9, 40 μl of 20 p/μl with 160 μl of negative blood; lane 10, 20 μl of 20 p/μl+180 μl of negative blood; lane 11, negative control for PCR reaction.

Fig. 2.

Experimental design for lowest detection limit of pooling strategy. Falciparum and vivax were done independently. (A) P. falciparum. (B) P. vivax. Lane 1, ladder (100 bp); lane 2-5, 20 μl each of 200 p/μl, 20 p/μl, 2 p/μl, and 0.2 p/μl of parasite blood with 180 μl of negative blood, respectively; lane 6, falciparum control (3D7) or known vivax sample; lane 7, negative control for PCR reaction.

Fig. 3.

Proposed pooling strategy. Twenty microliters (20 μl) from each sample and 10 samples in 1 pool were prepared, and DNA extractions were done for each pooled sample. Using these genomic DNA from each pool, genus-specific PCR was done. Only genus positive pools were selected for individual DNA extraction followed by genus-specific PCR to get individual positive samples. Species identification was done on these individual genus positive samples.