Plasmodium falciparum and Plasmodium vivax account for the majority of malaria cases worldwide . Plasmodium falciparum is responsible for complications and death, mainly in non-immune individuals. On the other hand, P. vivax and Plasmodium ovale induce mild malaria symptoms restricted to fever with rare complications, while Plasmodium malariae infections result in mild symptomatic malaria. Furthermore, P. vivax and P. ovale produce dormant liver stages that may result in relapse of infection months to years later, while P. malariae infections can persist for decades, although dormant liver forms are not thought to occur.
It is currently estimated that 50–80 million individuals from industrialized countries visit malaria-endemic areas each year and approximately 10,000–30,000 travellers contract malaria , among which severe malaria accounts for approximately 5% (range 1–38%) , with a mortality rate ranging from 0.6% to 3.8% mainly resulting mostly from late and/or misdiagnosis, and delayed treatment administration [4–8]. Because about 90% of travellers who contract malaria will not become ill until returning home, preventing malaria-associated morbidity and mortality requires improved rapid and accurate laboratory diagnostic tools detecting low parasitaemia and differentiating febrile patients with P. falciparum from the other Plasmodium species. Such diagnostic, performed at the time of patient admission, will allow a prompt and adequate treatment and follow-up .
Light microscopy of thick and thin Giemsa-stained blood smears remains the gold standard for malaria diagnostic . However, even in expert hands (increasingly missed in industrialized countries), microscopy demonstrated limitations, mostly related to low sensitivity (detection limit: 10–50 trophozoites/μl) and misdiagnosis [4, 11–15]. In some case, parasite morphology is damaged due to exposition to prophylactic medication or auto-medication, making malaria biological diagnosis more difficult.
Alternative methods for laboratory diagnostic of malaria have been developed, including fluorescence microscopy of parasite nuclei stained with acridin orange and rapid dipstick immunoassays. The advantages offered by these methods, such as the fact that a result can be obtained within half an hour by non-skilled technicians, are tempered by three limitations reviewed by Moody et al : 1) the dipstick tests do not improve sensitivity over microscopy and the sensitivity decreases as parasitaemia falls below 100 parasites/μl ; 2) false positives are observed, particularly after treatment, as the parasite antigens detected can remain in the circulation following parasite clearance, or in the presence of pneumococcal meningitis infection ; and 3) many dipstick tests are specific to P. falciparum infections.
A variety of PCR-based techniques have been developed for the genus or species-specific diagnosis of malaria parasite infection [11, 13, 19–21]. While demonstrating increased sensitivity and specificity, they remained labour-intensive, time-consuming and prone to carry more DNA contamination during manipulation of post-amplification products. The recent advance of a real-time quantitative PCR technique has proven usefulness in various applications, including parasite detection, species differentiation, gene expression and regulation, and allelic discrimination [22–30]. However, the large majority of developed real-time PCR assays used many set of primers and/or probes to analyse each sample, thus increasing cost and reliability.
To date, one fluorescence resonance energy transfer (FRET) real-time PCR assay using one set of primer and probe , and two others real-time PCR methods using one set of primer and SYBR green dye [26, 32] for Plasmodium sp. identification and species differentiation have been evaluated. The principal limitation of these assays was the lack of sensitivity of parasite detection: two to 30 parasites/μl of blood according to the study. Furthermore, the real-time PCR approach using SYBR green dye did not avoid the quantification of non-specific amplification products . Here, a real-time PCR assay using a single set of primer and FRET hybridization probe for sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of P. falciparum from other human Plasmodium species was developed and evaluated. Results from the real-time PCR assay were compared to conventional PCR methods and microscopy examination of blood smears.