Contribution of Multiplex Real Time PCR for Detection and Differentiation of Enterocytozoon bieneusi and Encephalitozoon intestinalis in Fecal Samples of Immunocompromised Patients

Background: Intestinal microsporidiosis is among the most frequent opportunistic diseases in immunocompromised patients. Routine diagnosis is generally performed by light microscopy of stained fecal samples. While unequivocal non-molecular species identification, important for cases management, is achievable only through electron microscopy. Objective: This study aimed to evaluate the contribution of multiplex real time PCR for simultaneous detection and differentiation of Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens of patients with immunosuppressive conditions. Methodology: Stool samples were obtained from 78 immunocompromised patients suffering from diarrhea. The samples were screened for intestinal microsporidiosis by light microscopy using Weber's modified trichrome stain. The samples were subjected to multiplex real time PCR using Enterocytozoon bieneusi (E. bieneusi) primers and a probe specific on the internal transcribed spacer (ITS) sequence. Encephalitozoon intestinalis (E. intestinalis) primers and probe were specific for the small ribosomal subunit RNA gene sequence. Results: Of 78 samples, 20 (25.6%) were detected positive by multiplex real time PCR. E. intestinalis was identified in 8 cases (40%), E. bieneusi in 7 (35%), and both species in 5 (25%). Light microscopy detected a total of 22 samples (28.2%), 7 of which did not show the belt-like structure characteristic for microsporidial spores (empty-looking spores). Compared to real time PCR, light microscopy had 75% sensitivity, 87.9% specificity, 68.2% PPV, 91.1% NPV and 84.6% accuracy in detection of microsporidia. No significant difference was found regarding the detection of E. intestinalis, E. bieneusi or both species by microscopy. Conclusion: Multiplex real time PCR proved to be more effective than classical trichrome stain for simultaneous identification and differentiation between E. bieneusi and E. intestinalis.

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