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Microsporidiosis is caused by infection with microsporidia, which are obligatory intracellular, spore-forming parasites. Microsporidia are recognized as parasitic infectious agents worldwide in both developed and developing countries.
About 1,000 species have now been identified. Five genera: Enterocytozoon, Encephalitozoon, Pleistophora, Nosema and unclassified microsporidia referred to as Microsporidium have been associated with human disease, which appears to manifest primarily in immunocompromised persons. Cases of microsporidiosis in both immune compromised and immune competent patients were reported.
The present work aimed to investigate the occurrence of intestinal microsporidia and to clarify their genotype patterns in symptomatic and asymptomatic immunocompromised patients to detect their role and the genotypes responsible for their pathogenicity.
Stool samples were collected from patients admitted to radiotherapy, hematology, nephrology, geriatric departments and patients of the Pediatric hospital, Faculty of Medicine, Ain Shams University. In addition, stool samples were collected and examined from immune competent individuals attending Parasitology Research Unit, Medical Parasitology Department, Faculty of Medicine, Ain Shams University.
Patients were divided into two groups according to immune status:
Group I: immune suppressed patients (n=173).
Group II: immune competent individuals (n=150).
Staining techniques and molecular studies were carried out in Parasitology Research Unit, Medical Parasitology Department and Professor Dr. Ali Khalifa Cancer Detection Unit, Biochemistry department, Faculty of Medicine, Ain Shams University, respectively.
The mean age for microsporidia positive patients was 27.8 years. where 48.9% of microsporidia positive patients were males and 51.1% were females. There was no statistical significant difference as regards age or gender and microsporidial infection.
One hundred seventy three faecal samples from patients with different causes of immune suppression were collected such as organ transplant recipient, patients with autoimmune diseases or on corticosteroids therapy, patients with malignancies, patients with renal failure and the elderly. In addition to 150 faecal samples from individuals without underlying cause of immune suppression who were considered immune competent. Faecal samples were examined microscopically by modified trichrome, modified Ziehl-Neelsen stains and confirmed genetically by nested and RFLP-PCR and by direct sequencing.
A total of 45 faecal samples were found positive for microsporidial spores by both modified trichrome and modified Ziehl-Neelsen stains where staining was considered the gold standard test in this study. Out of which, 25 samples belonged to group I and 20 belonged to group II. Out of the 45 microsporidia positive samples by microscopic examination 44 samples were found positive by nested and RFLP-PCR, where 25 samples belonged to group I and 19 samples belonged to group II.
In relation to staining and light microscopic examination nested and RFLP-PCR had 97.8% sensitivity, 100% specificity, positive predictive value 100% and negative predictive value 99.6%.
Nested PCR could detect all the 25 (100%) positive cases for microsporidia (previously detected by staining and light microscopic examination) in the immune suppressed patients (group I). Out of these 20% had infection with Ent. bieneusi only, 20% had infection with Encephalitozoon species only and 60% patients had mixed infection with Ent. bieneusi and Encephalitozoon species together.
Nested PCR could detect 19 (95%) out of the microsporidia positive samples detected by staining and light microscopic examination in the immune competent individuals (group II). Infection with Ent. bieneusi only was reported in 31.6%, Encephalitozoon species only in 15.8% and mixed infection with Ent. bieneusi and Encephalitozoon species in 52.6%.
RFLP-PCR gave the same results as nested PCR. Besides, it could detect a single case of Enc. cuniculi infection in a 21 year old male patient with a history of occupational animal contact with birds and rabbits.
It was noted that 4% and 40% of microsporidia positive patients in group I and group II, respectively had animal contact. Out of the 44 microspridia positive cases detected by RFLP-PCR, 36.4% had Ent. bieneusi infection only, 0% had Enc. intestinalis infection only, 100% had Enc. cuniculi infection only and 20% had mixed infection by both Ent. bieneusi and Enc. intestinalis had animal contact.
It was noted that 60% and 50% of microsporidia positive patients in group I and group II, respectively suffered from different abdominal symptoms. Out of the 45 microsporidia positive patients, 51.1% had diarrhea, 33.3% had abdominal pain, 31.1% had distension, 22.2% showed maldigestion and 22.2% showed weight loss.
As for infection by Ent.bieneusi only, 45.5% of patients were symptomatic, 45.5% had diarrhea, 45.5% had abdominal pain, 36.4% had distension, 9.1% had maldigestion and 27.3% had weight loss.
In infection by Enc. intestinalis only, 85.7% of patients were symptomatic, 85.7% had diarrhea, 42.9% had distension and maldigestion, 28.6% had weight loss and 28.6% had abdominal pain.
As for positive cases with mixed infection by Ent. bieneusi and Enc. intestinalis, 56% were symptomatic, where 48% had diarrhea, 32% had abdominal pain, 28% had distension, 24% had maldigestion, 20% had weight loss.
In the present work 55.6% and 44.4% of microsporidia positive patients belonged to the immune suppressed group (group I) and the immune competent group (group II), respectively showing no statistical significant difference between infection with microsporidia and the immune status.
There was no statistical significant difference between microsporidial infection and the etiology of immune suppression among patients of group I. There was no relation between etiology of immune suppression and infection with certain species of microsporidia except for Enc. intestinalis with renal transplant recipient where there was high statistical difference.
Five samples were processed and sequenced in the present work and compared to the genotypes reported by Prof. Dr. Karim Aoun in Tunisia who kindly provided the microsporidia positive control DNA extract. The reported Ent. bieneusi genotypes in Tunisia were B, D and Peru 8.
Three genotypes for Ent. bieneusi were determined in the present work. Genotype B was detected in an immune competent patient, genotype D in a renal transplant recipient immune suppressed patient and genotype K which was detected in two patients, an elderly immune suppressed patient and the other case was an immune competent patient. The sequence of Enc. intestinalis belonged to an immune suppressed patient on corticosteroid therapy.