Malaria diagnosis by a polymerase chain reaction-based assay using a pooling strategy

Abstract

Pooling clinical specimens reduces the number of assays needed when screening for infectious diseases. Polymerase chain reaction (PCR)-based assays are the most sensitive tests to diagnose malaria, but its high cost limits its use. We adapted a pooling platform that could reduce the number of assays needed to detect malaria infection. To evaluate this platform, two sets of 100 serum samples, with 1% and 5% malaria prevalence, were tested. DNA, extracted from pooled samples, was amplified by malaria-specific PCR. Additional validation was performed by determining the level of PCR detection based on 1:10 and 1:100 dilution. The platform correctly detected all malaria samples in the two test matrices. The use of stored serum samples also has important implications for studies investigating malaria prevalence rates retrospectively. Field studies, using serum and whole blood specimens, are needed to validate this technique for the adaptation of these methods for clinical utility.

Figure 1

Example of using a 10 × 10 matrix to screen for malaria infection. Mini-pools (squares) are made up of 10 samples (circles) each. The octagon represents a master-pool made from 10 mini-pools. A negative master-pool would indicate that all 100 samples constituting the pool are negative, precluding further testing. The black squares and circles represent PCR-positive samples. The gray circles represent negative samples that need to be tested individually to rule out infection.

Figure 2

Specimen pooling strategy using malaria-positive samples to represent 1% and 5% malaria prevalence rates. A, Two pools (black squares) tested positive, and the matrix identified the single malaria positive sample (black circle). B, The positive pools (black squares) led to further testing of individual samples (black and gray circles) and identification of five malaria-positive samples (black circles).

Figure 3

PCR amplification products using Plasmodium genus-specific primers after DNA extraction from pooled and individual serum samples from matrix shown in Figure 2B. Lane 1, 100-bp DNA size marker ladder; Lanes 2–10, DNA extracted from malaria-positive vertical and horizontal mini-pools; Lanes 11–15, DNA extracted from malaria-positive individual specimens that constituted the mini-pools; Lane 16, DNA extracted from serum from a malaria-negative control; Lane 17, DNA extracted from P. falciparum (3D7) strain.