Abstract
BackgroundOne of the most commonly used molecular test for malaria diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA (rDNA) gene. Published diagnostic assays based on the 18S gene include the "gold standard" nested assay, semi-nested multiplex assay, and one tube multiplex assay. To our knowledge, no one has reported whether the two multiplex methods are better at detecting mixed Plasmodium infections compared to the nested assay using known quantities of DNA in experimentally mixed cocktails.FindingsHere we evaluated three PCR assays (nested, semi-nested multiplex, and one-tube multiplex) for the simultaneous detection of human malaria parasites using experimentally mixed cocktails of known quantities of laboratory derived DNA. All three assays detected individual species with high sensitivity and specificity when DNA was from any one single species; however, experimentally mixed DNA cocktails with all four species present were correctly identified most consistently with the nested method. The other two methods failed to consistently identify all four species correctly, especially at lower concentrations of DNA -subclinical levels of malaria (DNA equivalent to or less than 10 parasites per microliter).ConclusionsThe nested PCR method remains the method of choice for the detection of mixed malaria infections and especially of sub-clinical infections. Further optimization and/or new molecular gene targets may improve the success rate of detecting multiple parasite species simultaneously using traditional PCR assays.
Highlights
One of the most commonly used molecular test for malaria diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA gene
The nested PCR method remains the method of choice for the detection of mixed malaria infections and especially of sub-clinical infections
The semi-nested multiplex assay was as sensitive as the nested assay at detecting P. falciparum and P. vivax, more sensitive at detecting P. malariae (0.04p/μL), and was only able to detect down to 4 p/μL for P. ovale (Figure 1I-L)
Summary
The nested PCR method remains the method of choice for the detection of mixed malaria infections and especially of sub-clinical infections. The nested PCR method developed by Snounou et al [20] has been widely used in laboratory studies and in clinical diagnosis, including in a reference diagnostic laboratory in the United States [13,15,17,22] This method is time consuming, expensive, and labor intensive as it requires five separate PCR reactions to detect P. falciparum, P. vivax, P. ovale, and P. malariae. The three previously described PCR-based methods (nested, semi-nested, and single-tube multiplex) are all good alternatives to microscopy and RDTs. to our knowledge, no one has evaluated which of these traditional PCR methods is truly better at detecting mixed Plasmodium infections; we compared the two multiplex methods to the nested method using known quantities of laboratory derived Plasmodium DNA alone and in experimentally mixed cocktails containing all four species of DNA
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