Evaluation of a novel real-time PCR assay for the detection, identification and quantification of Plasmodium species causing malaria in humans

Abstract

Background The entry of PCR-based techniques into malaria diagnostics has improved the sensitivity and specificity of the detection of Plasmodium infections. It has been shown that humans are regularly infected by at least six different Plasmodium species. The MC004 real-time PCR assay for malaria diagnosis is a novel single-tube assay that has been developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium knowlesi (including differentiation of three strains) and Plasmodium cynomolgi (including differentiation of three strains). Detection and identification of Plasmodium species relies on molecular beacon probe-based melting curve analysis. In addition, this assay might be used to quantify the parasitaemia of at least P. falciparum by calculating the level of parasitaemia directly from the Cq-value. Methods The samples used in this study comprised reference samples, patient samples, and synthetic controls. The following analytical performance characteristics of the MC004 assay were determined: analytical specificity, limit of detection, the ability to detect mixed infections, and the potential to determine the level of parasitaemia of P. falciparum, including assessment of within-run and between-run precisions. Results No false positive or false negative results were observed. The limit of detection of P. falciparum was 1 × 10–3 IU/mL (WHO standard). Mixed infections with P. falciparum and non-falciparum species were correctly identified. A calibration curve could be established to quantify the parasitaemia of at least P. falciparum. The within-run and between-run precisions were less than 20% CV at the tested parasitaemia levels of 0.09%, 0.16%, 2.15% and 27.27%. Conclusion Based upon the analytical performance characteristics that were determined, the MC004 assay showed performance suitable for use in clinical settings, as well as epidemiological studies.

Assessing the diagnostic performance of a novel RT-PCR fluorescence method for the detection of human plasmodium species

“…The MC004 assay specifically targets Plasmodium mitochondrial DNA for amplification, and includes post-PCR melting curve analysis based on three different molecular beacon probes. More details on the methodology can be found in our previous study [ 17 ]. The MC004 RT-PCR assay for malarial parasites were in almost perfect agreement with microscopy (k = 0.958, specificity = 96.7% and a sensitivity of 100%.…”

“…It is designed to detect mitochondrial DNA encompassing the cyclo-oxygenase 3 (COX-3), cyclo-oxygenase 1 (COX-1) and cytochrome b (CYTB) genes having different melting temperatures. The detailed methodology has been described in detail elsewhere [ 17 ].…”

“…The MC004 RT-PCR technique is a single-tube assay that has been developed for the simultaneous detection and identification of human plasmodia including P. falciparum, P. vivax, P. malariae, and P. knowlesi. The initial evaluation study using reference samples, patient samples and synthetic controls has been recently published [17]. We build upon this to further confirm the assay's performance in a different population setup.…”

“…knowlesi . The initial evaluation study using reference samples, patient samples and synthetic controls has been recently published [ 17 ]. We build upon this to further confirm the assay’s performance in a different population setup.…”

Diagnostic methods for Plasmodium knowlesi: performance, limitations and recommendations

“…In 2021, van Bergen et al [50] presented a study on the analytical performance of MC004 real-time PCR assay (MRC Holland, Amsterdam, Netherlands) for malaria diagnosis. MC004 is a single-tube multiplex assay which can detect all human Plasmodium species including P. knowlesi and Plasmodium cynomolgi.…”