Abstract
BackgroundPlasmodium 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.MethodsDBS 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.ResultsAssay 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.ConclusionsThe 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.
Highlights
Plasmodium 18S Ribosomal RNA (rRNA) is a sensitive biomarker for detecting Plasmodium infection in human blood
The Plasmodium 18S rRNA biomarker assay can be applied to Dried blood spots (DBS) collected in field studies
Bland–Altman plots indicated that DBS results yielded quantitative results slightly lower than liquid samples (Fig. 1B) with bias of − 0.37 and − 0.39 log10 estimated parasites/ mL blood by the P. falciparum quantitative triplex reverse transcription polymerase chain reaction (qRT-PCR) and Pan qRTPCR, respectively (Table 1)
Summary
Plasmodium 18S rRNA is a sensitive biomarker for detecting Plasmodium infection in human blood. The use of molecular assays has greatly informed the understanding of malaria epidemiology, especially for asymptomatic, sub-microscopic infections [14,15,16], which may improve effectiveness of interventions like vector control, mass drug administration, and vaccination, but the distribution and dynamics of such infections in endemic countries is largely unknown, and additional studies using highly sensitive molecular assays are needed. Molecular assays (i.e., polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) for the Plasmodium 18S rDNA or rRNA, respectively) are considered high complexity tests, and expanding their use to inform larger field studies can be constrained by limited availability of molecular assay platforms, the need for venous blood collection procedures, sample storage and stability, and testing costs
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