High-throughput pooling and real-time PCR-based strategy for malaria detection

Abstract

Molecular assays can provide critical information for malaria diagnosis, speciation, and drug resistance, but their cost and resource requirements limit their application to clinical malaria studies. This study describes the application of a resource-conserving testing algorithm employing sample pooling for real-time PCR assays for malaria in a cohort of 182 pregnant women in Kinshasa. A total of 1,268 peripheral blood samples were collected during the study. Using a real-time PCR assay that detects all Plasmodium species, microscopy-positive samples were amplified individually; the microscopy-negative samples were amplified after pooling the genomic DNA (gDNA) of four samples prior to testing. Of 176 microscopy-positive samples, 74 were positive by the real-time PCR assay; the 1,092 microscopy-negative samples were initially amplified in 293 pools, and subsequently, 35 samples were real-time PCR positive (3%). With the real-time PCR result as the referent standard, microscopy was 67.9% sensitive (95% confidence interval [CI], 58.3% to 76.5%) and 91.2% specific (95% CI, 89.4% to 92.8%) for malaria. In total, we detected 109 parasitemias by real-time PCR and, by pooling samples, obviated over 50% of reactions and halved the cost of testing. Our study highlights both substantial discordance between malaria diagnostics and the utility and parsimony of employing a sample pooling strategy for molecular diagnostics in clinical and epidemiologic malaria studies.

FIG. 1.

Sample processing and assay work flow schematic. Microscopy-positive samples were amplified directly in the pan-species assay, and positive samples were subsequently tested in the speciation assay. Microscopy-negative samples were first grouped into pools of four and then amplified in the pan-species assay; the individual constituents of positive pools were then retested in the pan-species assay, and positive samples were subsequently tested in the speciation assay.

FIG. 2.

Real-time PCR output from masked validation of pan-species and speciation assays using artificial test samples. Panel A shows the output of real-time PCR testing with 5 artificial test pools (pools A to E) in the pan-species assay. Each pool contained 2 μl of DNA from 10 samples. Pool A contained a total of 1.111 ng/μl, divided among 4 samples, and pool B contained 1 ng/μl of DNA in a single sample. Pools C, D, and E contained DNA at total concentrations of 0.1, 0.01, and 0.001 ng/μl, respectively. Panel B shows the output observed for the speciation real-time PCR assay when tested on a sample with 0.01 ng/μl of P. falciparum plasmid (Pf), 0.000001 ng/μl of P. ovale plasmid (Po), and 2.5 ng/μl of human DNA (Hu). Delta Rn, fluorescent signal generated relative to background fluorescence.