Researching Frequency of Microsporidia on diarrheic patients with Calcofluor white and Uvitex 2B Chemiluminescence painting methods and molecular typing of Microsporidia

Microsporidia, imperative intracellular parasite, was first defined by Nageli in 1857. In microsporidia phylum; there are 170 genuses and 1.400 species. There is a wide range of host scope such as insects, fishes and mammals. It is shown that it can infect both animals and humans and it can be symptomatically and asymptomatically found in humans. There are 8 genuses which infect humans. These genuses are; Anncaliia ( Brachhiola Nosema), Encephalitozoon, Entrocytozoon, Microsporidium, Nosema, Pleistophora, Trachipleistophora and Vittaforma. In the studies, the research group is individuals who are HIV positive, immune suppressive, homosexuals; people who travel and healthy individuals. The species that develop infection in humans are Encephalitozoon intestinalis and Enterocytozoon bieneusi. In the study, it was aimed to identify Microporidia frequency and type it as molecular by means of Uvitex 2B and Calcofluor, two different chemiluminescence strain. In this study; 200 patients? diarrhea stool samples, sent to Gazi University Medical Faculty Hospital Microbiology Laboratory and Ankara Numune Education and Research Hospital Microbiology Laboratory between the years 2011-2012, were studied. Stool samples were strained by means of Uvitex 2B and Calcofluor methods which are chemiluminescence strain and microsporidia frequency was confirmed as %38.5 (77/200). It was understood that in terms of Microsporidia characterization, there is a statistically perfect compatibility between Uvitex 2B and Calcofluor which are chemiluminescence strain. (Kappa=0,881) With regard to Microsporidia spore?s quantitative concentration( low, high) and spore brightness (colourless, bright) on microscope field, Uvitex 2B and Calcofluor compatibilities were statistically pointed out. While there is a medium level compatibility between tow dyes in terms of quantitative concentration (Kappa=0,354), there is no compatibility in spore brightness (Kappa=0,001). In the study; it was determined that microsporidia frequency has no meaningful difference with regard to the age groups and clinics (p > 0,05). It was confirmed that microsporidia frequency is %50 for women and %30,8 for men (p < 0,05). In our study; 77 samples, confirmed as positive by means of Uvitex 2B and Calcofluor, were applied to multiplex nested PCR and they were classified as E.bieneusi and Encephalitozoon spp. in order to distinguish genuses in the first stage and it was confirmed as 7(%91) E.bieneusi and 70(%90,9) Encephalitozoon spp. When microporidia genuses are considered, there is no statistically meaningful difference in Micropodia with regard to age, gender, clinics (p >0,05). To distinguish species, 70 Encephalitozoon spp. genus, confirmed in our study, were typed as E. intestinalis, E.cuniculi and E. hellem by means of second stage multiplex nested PCR. According to this, it was confirmed that there are 44 (62,9) E. intestinalis, 22 (%31,4) E. cuniculi, 4(%5,7) E. hellem in 70 Encephalitozoon spp. . Encephalitozoon species statistically has no meaningful microsporidia frequency difference with regard to age, gender and clinic. It was resulted that Uvitex 2B and Calcofluor straining methods, chemiluminescence strains, are beneficial to be used as first scanning test and it was concluded that multiplex nested PCR method is beneficial for genus and species characterization. There are few studies addressing to Microsporidia in our country. Microscopic methods were used in the studies. This is the first study that microsporidia diagnose and species characterization have ever been done. It is required that molecular method applications must become widespread to determine treatment options and to epidemiologically determine Microsporidia species. Key Words: Microsporidia, chemiluminescence strain, multiplex nested PCR