Application of dried blood spot sample pooling strategies for Plasmodium 18S rRNA biomarker testing to facilitate identification of infected persons in large-scale epidemiological studies.

BACKGROUND: Plasmodium 18S rRNA is a sensitive biomarker for detecting Plasmodium infection in human blood. Dried blood spots (DBS) are a practical sample type for malaria field studies to collect, store, and transport large quantities of blood samples for diagnostic testing. Pooled testing is a common way to reduce reagent costs and labour. This study examined performance of the Plasmodium 18S rRNA biomarker assay for DBS, improved assay sensitivity for pooled samples, and created graphical user interface (GUI) programmes for facilitating optimal pooling.

METHODS: DBS samples of varied parasite densities from clinical specimens, Plasmodium falciparum in vitro culture, and P. falciparum Armored RNA® were tested using the Plasmodium 18S rRNA quantitative triplex reverse transcription polymerase chain reaction (qRT-PCR) assay and a simplified duplex assay. DBS sample precision, linearity, limit of detection (LoD) and stability at varied storage temperatures were evaluated. Novel GUIs were created to model two-stage hierarchy, square matrix, and three-stage hierarchy pooling strategies with samples of varying positivity rates and estimated test counts. Seventy-eight DBS samples from persons residing in endemic regions with sub-patent infections were tested in pools and deconvoluted to identify positive cases.

RESULTS: Assay performance showed linearity for DBS from 4 × 107 to 5 × 102 parasites/mL with strong correlation to liquid blood samples (r2 > 0.96). There was a minor quantitative reduction in DBS rRNA copies/mL compared to liquid blood samples. Analytical sensitivity for DBS was estimated 5.3 log copies 18S rRNA/mL blood (28 estimated parasites/mL). Properly preserved DBS demonstrated minimal degradation of 18S rRNA when stored at ambient temperatures for one month. A simplified duplex qRT-PCR assay omitting the human mRNA target showed improved analytical sensitivity, 1 parasite/mL blood, and was optimized for pooling. Optimal pooling sizes varied depending on prevalence. A pilot DBS study of the two-stage hierarchy pooling scheme corroborated results previously determined by testing individual DBS.

CONCLUSIONS: The Plasmodium 18S rRNA biomarker assay can be applied to DBS collected in field studies. The simplified Plasmodium qRT-PCR assay and GUIs have been established to provide efficient means to test large quantities of DBS samples.