Evaluation of a multiplex Real-Time PCR assay for the diagnosis of human intestinal protozoa in Azienda Ospedaliero Universitaria Pisana

INTRODUCTION.The gold standard for diagnosis of human infection with intestinal protozoa is microscopic examination of stool specimens following concentration. However, this method has limited sensitivity at low parasite densities, requiring observation of multiple specimens and specific staining. Furthermore, morphologically identical taxa with different pathogenicity such as species of the Entamoeba histolytica complex cannot be identified. This study aims to evaluate a new multiplex Real-Time PCR assay (Allplex™ GI-Parasite Assay, Seegene) for the detection of DNA of Giardia duodenalis, E. histolytica sensu stricto, Cryptosporidium spp., Cyclospora cayetanensis, Diantamoeba fragilis and Blastocystis hominis. MATERIALS AND METHODS. Multiplex Real-Time PCR was performed on: i) positive controls stool samples (N=9) to evaluate performance in alternative storage conditions (RT, 4°C, -20 °C, EtOH, formalin and Ecofix) from the validated one; ii) stool samples from patients (N=100) previously tested with an immunochromatographic test (ImmunoCard STAT! CGE, Meridian Bioscience) for antigen detection of G. duodenalis, E. histolytica complex and Cryptosporidium spp. to compare results of the two methods; iii) stool samples from patients (N=54) following introduction of the new method in the diagnostic routine to assess prevalence of infection. RESULTS AND CONCLUSIONS. Preservation in EtOH showed 100% sensitivity andwas the best alternative to freezing, allowing storage at RT and thereby avoidance of the cold chain. Compared to ICT, multiplex Real-Time PCR was equally sensitive but allowed to discriminate between E. dispar/moskovskii and E. histolytica ss., as well as detection of B. hominis e D. fragilis. Prevalence of infection was: 0% for E. histolytica ss. and C. cayetanensis, 2% for G. duodenalis and Cryptosporidium spp., 13% for D. fragilis and 20% for B. hominis. The observed prevalence of B. hominis and D. fragilis is in line with data from Italy and other European countries reported in the literature. Detection of these protozoa is fundamental to gain new insights into their uncertain pathogenetic role, making multiplex Real-Time PCR a useful diagnostic and investigation tool.