PCR-based pooling of dried blood spots for detection of malaria parasites: optimization and application to a cohort of Ugandan children

Abstract

Sensitive, high-throughput methods to detect malaria parasites in low-transmission settings are needed. PCR-based pooling strategies may offer a solution. We first used laboratory-prepared samples to compare 2 DNA extraction and 4 PCR detection methods across a range of pool sizes and parasite densities. Pooled Chelex extraction of DNA, followed by nested PCR of cytochrome b, was the optimal strategy, allowing reliable detection of a single low-parasitemic sample (100 parasites/μl) in pool sizes up to 50. This PCR-based pooling strategy was then compared with microscopy using 891 dried blood spots from a cohort of 77 Ugandan children followed for 2 years in an urban setting of low endemicity. Among 419 febrile episodes, 35 cases of malaria were detected using the PCR-based pooling strategy and 40 cases using microscopy. All five cases of malaria not detected by PCR were from samples stored for >2 years with parasitemia of <6,000/μl, highlighting the issue of possible DNA degradation with long-term storage of samples. Among 472 samples collected from asymptomatic children as part of routine surveillance, 15 (3.2%) were positive by PCR-based pooling compared to 4 (0.8%) by microscopy (P = 0.01). Thus, this PCR-based pooling strategy for detection of malaria parasites using dried blood spots offers a sensitive and efficient approach for malaria surveillance in low-transmission settings, enabling improved detection of asymptomatic submicroscopic infections and dramatic savings in labor and costs.

FIG. 1.

Schematic of pooling strategy applied to cohort samples. The white circles represent Plasmodium-negative dried blood spots (DBS); the solid black circles represent Plasmodium-positive dried blood spots. At each stage, the dried blood spots were extracted together. The DNA extraction product was then used as a template for cytochrome b nested PCR.

FIG. 2.

AluI digestion for species determination. P. ovale digestion patterns may vary depending on the strain type. Our samples showed a pattern that correlates with a published sequence (GenBank accession number AB182497). However, another sequence (GenBank accession number AB182496) would produce a different AluI restriction pattern with expected band sizes of 205 and 610 bp. Pf, P. falciparum; Pm, P. malariae; Po, P. ovale; Pv, P. vivax.