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

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.