LE METODICHE DI BIOLOGIA MOLECOLARE NELLA DIAGNOSI E NEL MONITORAGGIO CLINICO DELLA LEISHMANIOSI.

My PhD work was entirely devoted to the application of molecular biology to diagnosis and follow-up of leishmaniasis. Leishmaniasis is an infectious disease with a prevalence of 12 million cases and an estimated 1.5-2 million cases per year incidence [1 million cutaneous leishmaniasis (CL), 500.000 leishmaniasis visceral (VL)]. A first approach to the problem was carried out to evaluate the use of molecular biology in the identification, quantification and typing of Leishmania species at the time of diagnosis of cutaneous and visceral leishmaniasis; we also evaluated their follow-up, with particular attention to Real Time polymerase chain reaction (PCR), using specific Taqman probes, compared with traditional methods like semiquantitative PCR. Were tested a total of 174 biological samples: 147 peripheral blood, 17 bone marrows and 10 scraping or skin biopsy. All samples assayed came from patients with a confirmed diagnosis of either VL or CL: 22 VL [7 immunocompetent human immunodeficiency virus (HIV)-negative children, and 15 adults (9 HIV-positive and 6 HIV-negative)] and 5 CL. The methods used to detect Leishmania spp. had several targets. For the diagnosis and quantification of parasites were used PCRs which had as target a ribosomial RNA, 18S rRNA, repeated 150 times for parasite, a region of minieson genes kinetoplast DNA (kDNA) which comes out in a number of about 10.000, and a Real Time PCR, which we have developed, directed against the α -polymerase of Leishmania, repeated in single copy. Molecular Old World Leishmania identification was carried out by PCR-restriction fragment lengh polymorphism (RFLP); molecular typing of the New World Leishmania was carried out by amplification and sequencing of a large fragment of the SSU rRNA. Control samples resulted all negative with all methods used, this means a 100% specificity. PCR kDNA showed a higher sensitivity of conventional PCR for Leishmania spp. Our single copy Real Time PCR missed one diagnosis of VL as compared to the conventional PCR, whereas both PCR methods were able to detect Leishmania parasites in CL. Under anti-leishmania treatment the kinetics of parasitemia obtained with PCR SSU rRNA and Real Time PCR were comparable. Despite Real Time PCR is able to make a more accurate quantification of parasites, the method doesn’t help the decision-making compared with a semiquantitative PCR; furthermore it is comparatively expensive.