Annals of Tropical Medicine & Parasitology, Vol. 97, No. 1, 000–000 (2003) A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax and P. falciparum E. PATSOULA*,† , G. SPANAKOS*,† , D. SOFIANATOU‡ , M. PARARA‡ and N. C. VAKALIS* *Department of Parasitology, Entomology and Tropical Diseases, National School of Public Health, 196 Alexandras Avenue, 11521, Athens, Greece † Hellenic Centre for the Control of Infectious Diseases, Laboratory of Opportunistic Infections, National School of Public Health, 196 Alexandras Avenue, 11521, Athens, Greece ‡ Blood Bank, NIMTS Veterans’ Army Hospital, 10 Monis Petraki Street, 11521, Athens, Greece Received 4 September 2002, Accepted 3 October 2002 The ability to detect and differentiate between Plasmodium falciparum and P. vivax is of great importance for the routine laboratory diagnosis of malaria, donor-blood screening and epidemiological studies. Most PCR-based methods for the discrimination of these two species require nested protocols or an additional hybridization reaction, leading to high labour costs and long turn-around times. A simple, time-effective and yet sensitive and specific technique, based on a multiplex PCR, has now been developed for the simultaneous detection and differentiation of P. falciparum and P. vivax in blood samples. Compared with the ‘gold standard’ of microscopy, this method had a sensitivity and specificity of 100%, with a detection limit of just one P. falciparum or three P. vivax parasites/ml blood. Over the last few years the flow of immi- limit of 10–20 parasites/ml blood (Gilles grants from the tropics to many European and Warrell, 1993). Serological tests are countries has increased. In Europe, this available but, because antimalarial anti- has led to increases both in the annual bodies may persist after cure, they cannot numbers of malaria cases who present at always be used to distinguish between past medical institutions and in the incidence and present infections. The limitations of of autochthonous malaria. In Greece, for microscopy and serology have encouraged example, eight autochthonous cases were researchers to develop alternative diagnostic recorded between 1991 and 2000, compared procedures based on molecular biology. PCR with only three in the previous decade (Prifti offers a sensitive method for the detection et al., 1993; Kampen et al., 2002). of infectious agents in blood, and in recent The detection and identification of malarial years methods based on this reaction have infections can be difficult, particularly in been used successfully for the detection of non-endemic regions where clinicians may Plasmodium spp. (Tirasophon et al., 1991; rarely see the disease. The classic diag- Barker et al., 1992; Brown et al., 1992). nostic procedure remains the examination Initially, the target was the parasite’s repetitive of Giemsa-stained bloodsmears. Although DNA (Franzen et al., 1984). However, once specific, this method is subjective, requires the ribosomal RNA (rRNA) genes of the a skilled microscopist, and has detection malarial parasites infecting humans had been characterized, species-specific regions of these genes were exploited in developing Reprint requests to: E. Patsoula. E-mail: epatsoul@hotmail.com; fax: +30 210 6458578. sensitive diagnostic tests (Lal et al., 1989; © 2003 The Liverpool School of Tropical Medicine DOI: 10.1179/000349803125002535 atm0000018 28-11-02 13:18:09 Rev 14.05 The Charlesworth Group, Huddersfield 01484 517077