Introduction of Polymerase Chain Reaction (PCR) in Microbiology and Parasitology

Overview

The goal is to develop molecular systems to support or replace in microscopic characterization and in vitro tests with molecular biology systems capable of improving performance in parasitology tests. In particular, we will analyze the main pathogens: several Leishmania species (Old and New World Leishmania species), the five Plasmodium species of human interest (falciparum, oval, vivax, malariae and knowlesi) and Pneumocistys jiroveci, using molecular methods based on the speed, specificity and sensitivity necessary for their diagnosis. Furthermore, we want to provide specific elements for the typing of the species. Our aim is to improve diagnostic and specie classification methods by using PCR in microbiology and parasitology, to evaluate its impact on diagnosis. We would evaluate the impact of this method in terms of timing, sensitivity and specificity of diagnosis. We would also explore future possibilities on quantification techniques, in order to support the clinician to evaluate the efficacy of therapy during the follow-up. Furthermore, we would evaluate these methods in terms of cost effectiveness towards classical direct methods, which are now operator-dependent.

Our project is an observational retrospective and prospective cross-sectional diagnostic pilot study, about the comparison between standard routine laboratory tests and new molecular biology assays. In particular, we would detect principal pathogen Leishmania species (Old and New World Leishmania species), the five species of Plasmodium of human interest (falciparum, oval, vivax, malaria and knowlesi) and Pneumocistys jiroveci, 1. Positive samples for Leishmania amastygotes in peripheral blood and/or bone marrow aspirate/biopsy and/or from cutaneous scraping/biopsy previously tested according to routine diagnostic assays will be tested with a specific qualitative PCR analysis by using commercial diagnosis kit. In addition, we would perform a system of DNA quantification using PCR cycle threshold of the single sample and a virtual scale able to convert the results in the DNA load (Piralla A et al, J clin virol 2013). Then we would perform a specific DNA sequencing of p70 gene to determine specie and the strain. 2. Plasmodium species blood positive samples tested for and analyzed by direct microscopy and immunocromatographic assay, will be tested with multiplex PCR commercial diagnostic kit or with a described method (A.Berg et al, Infection 2020) able to determine single or mixed infection and to quantify the parasitemia respectively. In detail, the blood samples will be analyzed with qualitative and quantitative PCR analysis for Plasmodium spp and Plasmodium Falciparum, respectively. The concentration of P. falciparum DNA in blood samples will be measured by quantitative real-time PCR (qPCR) as described elsewhere (Imwong M, J.clin.microb 2014). In Plasmodium falciparum positive samples, the amount of DNA copies obtained from each sample will be compared with the level of parasitemia obtained from direct microscopy analysis. In this way we would like to determine a range of DNA copy values comparable to the different degrees of classification that are currently obtained with percentages of red blood cells positive for the infection (\<5% vs \>5%). In this way, it could be possible to assess a level of parasitemia with quantitative PCR techniques. 3. Finally, detection of Pneumocistis jirovecii in positive BAL or bronchoaspirate (BAS) samples obtained by direct microscopy will be reanalyzed by using PCR commercial diagnostic kit. As previously mentioned, the CT value will be converted in DNA load in comparison with direct microscopy data.