Molecular methods for accurate diagnosis and epidemiological picture of imported malaria.

Objectives: Malaria is the most frequent imported infection in Italy, related to the increasing number of travellers and migratory flows from endemic countries. Microscopic examination has poor sensitivity and may give problems in the differentiation of Plasmodium falciparum (Pf), P. malariae (Pm), P. ovale (Po), and P. vivax (Pv), especially in cases of low parasitaemia or mixed infections: to circumvent these limitations molecular assays based on 18S-rDNA were developed by several research groups, including us. Our study aimed to accurately and promptly diagnose cases of malaria and to describe their occurrence in our area comparing the results of microscopy and molecular assays, in order to assess the usefulness of these assays in the diagnostic practice. Methods: Blood samples from 701 patients presenting to the University Hospital of Parma from 2000 to 2007 with clinical suspicion of malaria were subjected to microscopic examination and to 6 different PCR protocols targeting plasmodial 18S-rDNA alternatively used during 2000−07, including nested- and Real-time PCR assays. Results: By microscopy 153 cases of malaria were diagnosed [129 Pf (84.3%), 7 Po (4.6%), 10 Pv (6.5%), 5 P. spp. (3.3%), 1 Pf/P. spp. (0.65%), 1 mixed infection (0.65%)], whilst 159 were diagnosed by PCRs [129 Pf (81.1%), 14 Po (8.8%), 6 Pv (3.8%), 3 Pm (1.9%), 7 mixed infections (4.4%)]. Conclusion: Despite microscopy remains the reference diagnostic method (rapid and inexpensive), in some cases molecular assays are the only ones allowing a correct diagnosis of malaria, particularly to detect infections by species other than Pf and mixed infections. However, in our study only one PCR assay developed by us showed the higher accuracy in Po detection due to specific primer design done to recognise all the variants in Po 18S-rRNA gene. PCR proved to be more sensitive and specific than microscopy and changed the picture of malaria epidemiology in our area detecting 5 single and 1 mixed infections missed by microscopy, revealing 5 single and 2 mixed infections incorrectly diagnosed by microscopy and giving speciation in 6 cases in which microscopy had limited the result to genus identification. The most prevalent malaria cases in our area as well as in Italy were imported from Africa and due to Pf, followed by Po and Pv. In our experience a rapid and accurate diagnosis of malaria allowed to administer a prompt and targeted therapy with positive impact on the clinical management of the patients.