Evaluation of three PCR-based diagnostic assays for detecting mixed Plasmodium infection

Abstract

Background: One 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.

Findings: Here 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).

Conclusions: The 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.

Figure 1

Limits of detection of the three different methods at detecting the four Plasmodium species. 10-fold serial dilutions of all four species were prepared starting from 40,000p/μl stock. Lane 1 = 40, 000p/μL, 2 = 4,000p/μL, 3 = 400p/μL, 4 = 40p/μL, 5 = 4p/μL 6 = 0.4p/μL and 7 = 0.04p/μL. One microliter of each of these dilutions was amplified as per the different protocols. Representative gels are shown for the nested (Snounou) (A-D), multiplex (Padley) (E-H), and semi-nested (Rubio) (I-L) results. The nested PCR was able to amplify up to 0.4p/μL for each of the species. The multiplex method amplified up to 0.4 p/μL for P. falciparum, 4 p/μL of P. ovale and 40p/μL of both P. malariae and P. vivax while the semi-nested PCR amplified up to 0.04p/μL for P. malariae, 0.4p/μL for both P. falciparum and P. vivax and 4p/μL for P. ovale. L: 100 bp molecular weight marker.

Figure 2

Detection of mock mixed infections using the three different methods. Mock mixed infections were prepared as per Table 1. One microliter of each of the mock mixed infection was amplified according to the different protocols resulting in the final parasite concentrations given by Additional file 1. Representative gels are shown for the nested (Snounou) (A), multiplex (Padley) (B), and semi-nested (Rubio) (C). Lane numbers correspond to the lanes as shown in Additional file 1. F: P. falciparum, M: P. malariae, O: P. ovale, V: P. vivax, L: 100 bp molecular weight marker, SL: Species Ladder created by mixing equal amounts of individually amplified species DNA using Rubio primers.