High-Throughput Pooling and Real-Time PCR-Based Strategy for Malaria Detection

Author:

Taylor Steve M.12,Juliano Jonathan J.3,Trottman Paul A.1,Griffin Jennifer B.1,Landis Sarah H.1,Kitsa Paluku4,Tshefu Antoinette K.4,Meshnick Steven R.1

Affiliation:

1. Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina

2. Division of Infectious Diseases and International Health, Duke University Medical Center, Durham, North Carolina

3. Division of Infectious Diseases, School of Medicine, University of North Carolina, Chapel Hill, North Carolina

4. Ecole de Santé Publique, Faculté de Medecine, Université de Kinshasa, Kinshasa, Democratic Republic of the Congo

Abstract

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.

Publisher

American Society for Microbiology

Subject

Microbiology (medical)

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