A real-time PCR for the detection of Plasmodium ovale variant strains.

Objectives: Malaria is the most frequent imported parasitic infection in Italy, mainly from Africa. Molecular assays based on 18S-rDNA developed by several researchers, including us, revealed that recently the prevalence of P. ovale (Po) infections was higher than previously thought and allowed to demonstrate that most prevalent malaria cases in our area as well as in Italy were due to P. falciparum (Pf), followed by Po and P. vivax (Pv). Sequence variations in 18S-rDNA lead to describe a classic and a variant type of Po (vtPo); therefore primers and probe specific for classic type of Po (ctPo) can fail in identification of vtPo. In this study, a new primer and probe for the identification of vtPo were described in order to accurately and promptly diagnose cases of imported malaria. Methods: A new Rt-PCR assay to identify vtPo was developed using a new (OVA-Fv) and a previously described (OVA-R) primer with a new probe specific for vtPo (OVA-v) in a variant Rt-PCR (Rtv-PCR) assay. This assay was comparatively evaluated with the classic Rt-PCR assay (Rtc-PCR) to identify Po testing 24 selected blood samples from 24 patients with malaria. Some samples were also pre-amplified by a genus-specific conventional PCR (primers rPLU1-rPLU5) before testing with Rt-PCR assays. Results: Among the 24 samples resulted positive for Po 15 were ctPo and 9 vtPo. Fourteen samples were Po positive by Rtc-PCR and 6 samples were Po positive by Rtv-PCR. Two samples resulting indeterminate by Rtc-PCR and Rtv-PCR, respectively, were positive by the same assays after pre-amplification. Two samples negative by both Rt-PCR assays resulted positive by Rtv-PCR after pre-amplification. No signal was detected by Rtv-PCR assay testing DNA from 4 Pf, 2 Pv and 5 Pm positive samples. Conclusion: The present study reports that some cases of malaria by vtPo (9 of 24 in our experience) could not be diagnosed by both microscopy and Rtc-PCR based on 18S-rDNA. The use of Rtcand Rtv-PCR to accurately identify Po strains can improve sensitivity of diagnostic assays, especially in case of malaria by Po, usually occurring with low parasitaemia as was particularly in 4 of 24 samples which needed a pre-amplification. The association of microscopic and molecular assays demonstrated to be essential for a rapid and accurate diagnosis of imported malaria in our area and allowed to administer a targeted therapy