Abstract
BackgroundA rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas.MethodsA semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene.ResultsThis study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix.ConclusionsThe development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples.
Highlights
A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas
Diagnosis of malaria is based on four different approaches: microscopy, antigen detection using immunochromatographic rapid diagnostic tests (RDTs), malaria antibody detection, and nucleic acid-based assays
The amplicons generated were detected by agarose gel electrophoresis and lateral flow up-converting phosphor (UCP) detection (Figure 2B)
Summary
A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. To alleviate some of the difficulties of microscopy-based diagnosis of malaria, RDTs that detect parasite-specific antigens were developed [2]. RDTs offer ease of operation, a timely diagnosis, and do not require trained personnel or special equipment [2,7]. They are relatively expensive and prone to false-positive responses due to persistence of pfHRP2 antigen in blood for up to two weeks after the parasite is cleared [2,8]. The relatively low RDT sensitivity is a constraint for endemic regions attempting malaria pre-elimination, where detection and treatment of low-grade reservoir infections is required for effective elimination of infection [9]
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