Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

Abstract

Background: This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for Plasmodium species-specific real-time PCR.

Methods: First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag Plasmodium falciparum/Pan test) were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four Plasmodium species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples.

Results: Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of P. falciparum (n = 60), Plasmodium vivax (n = 10), Plasmodium ovale (n = 10) and Plasmodium malariae (n = 10). Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20) gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests.

Conclusions: RDTs are a reliable source of DNA for Plasmodium real-time PCR. This study demonstrates the best method of RDT fragment sampling for a wide range of RDT brands in combination with a simple and low cost extraction method, allowing RDT quality control.

Figure 1

Different components of a two-band RDT. Blood is applied on the sample pad at the proximal part of the RDT and buffer is dropped on the buffer pad. The absorption pad that consists of filter paper sucks the solution to the distal part of the RDT. Detecting antibodies conjugated with colloid gold are present on the conjugate pad coloring it pink. On the nitrocellulose membrane, test lines with capture antibodies for HRP-2, pLDH or aldolase and a control line with control antibodies are present. Depending on the RDT brand, the sample pad, buffer pad and conjugate pad are either separate or combined in one component. Those three pads consist of fiberglass-like paper strips and partly overlap the nitrocellulose membrane. All components are glued onto a plastic backing. Some RDTs contain a plastic cover on top of the nitrocellulose membrane (not illustrated). Most RDTs are packed in a plastic cassette housing or available as cardboard or hybrid format. The arrow indicates the direction of the lateral flow from proximal to distal RDT components.

Figure 2

Fragment sampling of OptiMAL (A) and SDFK60 (B). NC = nitrocellulose strip (nitrocellulose membrane inclusive plastic backing), F = filter paper fragment of absorption pad, S = sample pad, C = conjugate pad.

Figure 3

Boxplot illustration of Ct-values obtained by PCR on whole blood, PCR on OptiMAL and PCR on SDFK60. The median Ct-value (indicated by the horizontal bar in the box) of PCR on whole blood is significantly (p < 0.01) lower than the median Ct-value of PCR on RDTs. * indicated minimum (min) and maximum (max) outlier.