الفهرس 
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Abstract
Microsporidiosis is an emerging infectious disease, with extensive host range including most invertebrates and all classes of vertebrates. Out of 100 genera and almost 1000 identified Microsporidia species, seven genera have been documented in humans. The most common are genus Enterocytozoon and genus Encephalitozoon. Studies showed that microsporidiosis is not only confined to patients with AIDS, but also, it has recently been detected as a cause of self-limiting diarrhea in immunocompetent travellers, as well as in non-HIV immunosuppressed patients.
Each species differs in its site of infection, clinical picture and treatment, thus identification of the Microsporidia to the species level is mandatory. Several studies were previously held aiming at determining a diagnostic technique with good sensitivity and ability of identification of different species of Microsporidia.
In the current study we aimed at simultaneous diagnosis and identification of Microsporidia species (Enterocytozoon bieneusi and Encephalitozoon intestinalis, cuniculi and hellem) in stool samples of 100 immunosuppressed patients by application of SYBR Green real-time PCR, and comparing its results with Weber`s modified trichrome stain in relation to age, sex and different causes of immunosuppression of the patients.
To fulfill this objective, the stool samples were collected in sterile screw capped, and properly labelled plastic bottles. Each sample was divided into two parts, the first part was preserved at 4˚C for routine parasitological examination and staining with MTS and MZN. The spores’ sizes were measured in the Microsporidia positive modified trichrome stained samples using the micrometre in order to identify the species, based on the reference spore’s size of each species. The second part was freshly frozen at -70˚C for the molecular study. The molecular study included DNA extraction using ISOLATE faecal DNA extraction kit, followed by DNA amplification using SensiFAST TM SYBR NO-ROX PCR kit in Step one real-time PCR using MsRTf1 and MsRTr1 primers. Identification of different species of Microsporidia was achieved by melting curve analysis which was a core step in the SYBR Green real-time PCR.
Of 100 stool samples stained by MTS and examined microscopically, 49 were positive for Microsporidia. By measuring the spore size, determination of the species of the positive cases was as follows, 17 E. intestinalis, 15 E. bieneusi, 11 E. hellem and six E. cuniculi. On the other hand, by SYBR Green real-time PCR, 55 stool samples were positive for microsporidial DNA and by determination of their specific melting curves, they were identified as follows: 16 E. cuniculi, 14 E. hellem, 13 E. intestinalis, 10 E. bieneusi and two unspecified species.
When microscopy was used as the reference standard technique in diagnosis of Microsporidia, the sensitivity, specificity, negative and positive predictive values of SYBR Green real-time PCR were 89.8 %, 78.43 %, 80 % and 88.89 % respectively. Microscopic examination and SYBR Green real-time PCR showed no statistically significant difference in the number of the positive cases for Microsporidia in stool samples with a degree of agreement reaching 84 %