Hexaplex PCR Detection System for Identification of Five Human Plasmodium Species with an Internal Control

Abstract

Malaria remains one of the major killers of humankind and persists to threaten the lives of more than one-third of the world's population. Given that human malaria can now be caused by five species of Plasmodium, i.e., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and the recently included Plasmodium knowlesi, there is a critical need not only to augment global health efforts in malaria control but also, more importantly, to develop a rapid, accurate, species-sensitive/species-specific, and economically effective diagnostic method for malaria caused by these five species. Therefore, in the present study, a straightforward single-step hexaplex PCR system targeting five human Plasmodium 18S small-subunit rRNAs (ssu rRNAs) was designed, and the system successfully detected all five human malaria parasites. In addition, this system enables the differentiation of single infection as well as mixed infections up to the two-species level. This assay was validated with 50 randomly blinded test and 184 clinical samples suspected to indicate malaria. This hexaplex PCR system is not only an ideal alternative for routine malaria diagnosis in laboratories with conventional PCR machines but also adds value to diagnoses when there is a lack of an experienced microscopist or/and when the parasite morphology is confusing. Indeed, this system will definitely enhance the accuracy and accelerate the speed in the diagnosis of malaria, as well as improve the efficacy of malaria treatment and control, in addition to providing reliable data from epidemiological surveillance studies.

Multiplex 5′ Nuclease Quantitative Real-Time PCR for Clinical Diagnosis of Malaria and Species-Level Identification and Epidemiologic Evaluation of Malaria-Causing Parasites, Including Plasmodium knowlesi

“…Clinical specificity is not ensured, since blood samples from patients with P. ovale and P. malariae are difficult to obtain and infrequently tested. Recently, a multiplex PCR system targeting 18S small-subunit rRNA genes and capable of detecting all 5 known species of malaria in humans was reported, using nested PCR as the molecular gold standard (17). However, these results have yet to be corroborated outside Malaysia; further, the group tested only 2 clinical samples from patients with P. malariae and 1 from a patient with P. ovale malaria.…”

Evaluation of new multiplex PCR primers for the identification ofPlasmodium species found in Sabah, Malaysia

“…One advantage of this single-step multiplex PCR is that it has relatively fewer steps and hence uses less PCR reagents and disposable consumables (17,18,43). Overall, the system has shortened the time needed to identify Plasmodium species to approximately 3 h including DNA extraction per sample, which is half the time needed for nested PCR.…”

“…Molecular methods, such as single-step PCR (13), loopmediated isothermal amplification (LAMP) assay (14), nested PCR (15,16), multiplex PCR (17,18), multiplex real-time PCR (19), and real time PCR (20)(21)(22)(23) basically use conventional PCR techniques to detect and identify malaria parasites. Even though PCR assay initially requires expensive equipment (a thermal cycler and gel electrophoresis tools and documentation), it is considered reasonably cheap in the long run for large-scale malaria detection and speciation, and it is highly sensitive in terms of detection (17), especially in regions where malaria is endemic.…”

Recent Advances in Detection of Plasmodium ovale: Implications of Separation into the Two Species Plasmodium ovale wallikeri and Plasmodium ovale curtisi

“…The results of a study using an 18S SSU rRNA-hexaplex PCR suggest that the primers cover both species (27). In addition, the group around Bauffe et al described a real-time TaqMan PCR (TqPCR) using primer pair PoF and PoR and two probes (pPOC and pPOW) (13).…”