الفهرس 
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Abstract
Vulvovaginal candidiasis (VVC) is common among women of different age groups, irrespective of their sexual activity. It occurs in 75% of all women at least once in their lifetimes, most often during childbearing years. VVC is treatable and symptoms are usually mild; however, when left untreated, these lesions cause significant morbidity in women. The occurrence of VVC appears to have increased in recent decades; and many predisposing factors have been proposed to explain this trend. Yet, few studies have linked use of intrauterine contraceptive devices (IUDs) with Candida colonization or infection among women with VVC. However, very little is known about the formation of candidal biofilm on genital devices such as the IUD, which can be of importance in the development of VVC.
Candida biofilm formation is of particular interest because Candida involved in biofilm exhibits a poor response to antifungal agents. The evolution of antifungal drug resistance poses a growing public health problem because of the sharp increase in the incidence of opportunistic fungal infections in recent years. Thus, this study aimed at:
1. Determination of Candida spp. in clinicaly suscepted VVC cases.
2. Studying of virulence factors of the isolated Candida spp. (i.e: phospholipase and proteinase activity)
3. Determination of the biofilm formation ability of Candida spp isolated in IUD users and non-users.
The present study extended from December 2015 until August 2016. Two hundred and ten clinically suspected female patients were microbiologically tested for VVC. Patients were attending the outpatient clinic of EL Shatby Obstetrics and Gynecology Hospital in Alexandria aged from 18 to 59 years old.
All were subjected to history taking in a questionnaire sheet and two vaginal swabs were used for microscopic examination using 10% potassium hydroxide (KOH) preparation to detect the presence of budding yeast cells and pseudohyphae of Candida species and the other swab was inoculated on SDA supplemented with 0.05g/L chloramphenicol and incubated at 37°C for 48 hours. Typical Candida colonies were creamy and smooth white yeast colonies with yeast like odour, then positive culture results were subjected to germ tube test by incubating 0.5 ml of human serum with a young Candida colony at 37oC for 2-3 hours then microscopic examination recorded tube like out growths in positive cases. Corn meal agar test was done by inoculating yeast colony by scraping of the agar then a cover slip placed over the cut were examined for terminal double walled spheres chlamydospore production after 2 to 7 days of aerobic incubation at 25 ± 2.
CHROMagar Candida plates were inoculated then incubated aerobically at 35± 2°C for 24 to 48 hours and positive plates were inspected for different colors of colonies as compared to the guiding table of the manufacturer. Twenty API Candida tests were inoculated then incubated at 35± 2°C for 24 to 48 hours and compared to the guiding table of the manufacturer.
Extracellular proteinase activity of Candida isolates was determined by inoculation of bovine serum albumin (BSA) and measuring the zone of proteolysis. The Candida phospholipase activity was detected by measuring the size of the precipitation zone inoculating the egg yolk agar. Biofilm formation tissue culture plate method (TCP) was done by filling 96 well-flat bottom tissue culture plates wells with 0.2 ml aliquots of the diluted cultures and incubating for 24 hours at 37°C then washing four times then fixing for one hour at 56oC and staining with 0.1 ml crystal violet for 15 min., formed biofilm on all side wells were uniformly stained with crystal violet. Optical densities (OD) of stained adherent yeasts were determined with a micro ELISA auto reader at wavelength of 570 nm.
Antifungal susceptibility by disc diffusion was done on Mueller Hinton-glucose agar plate that was streaked by a homogeneous 0.5 McFarland Candida suspension to cover the entire surface and left to dry then discs of the antifungal agents; fluconazole, voriconazole, and nystatin were applied onto the surface. The plates were incubated at 35°C for 24 then diameters of zones of inhibition were measured and interpreted.
Out of the 210 studied cases, only 132 were positive for Candida by culture on SDA (63.2%). The commonest complaint was vaginal burning sensation, dyspareunia and vaginal discharge. The majority of patients (91.4%) had no specific signs on examination. There was a clear assosciation between positive microscopic examination (sensitivity was 68.18) and positive SDA culture results. API Candida biotypic diagnosis of Candida spesies is mandatory and complementary to phenotypic chrOMagar Candida mixed infection diagnosis. Identification of Candida species showed that the most common isolated Candida was C. albicans followed by C. glabrata, C. tropicalis, C. krusie, C. famata then C. parapsilosis.
C. albicans was significantly higher in the production of phospholipase and proteinase enzyme in comparison to CNA isolates. In the current study, the biofilm formation abilities in different Candida spp. were arranged in a descending manner as follow: C. krusie, C. tropicalis, C. albicans, and then C. glabrata.
C. albicans was more sensitive to voriconazole, fluconazole than nystatin while CNA was more sensitive to nystatin, voriconazole than fluconazole. Mixed Candida infection had the same sensitivity pattern as CNA (significantly sensitive to nystatin).
The present study showed that the use of IUD made CNA isolates more resistant to fluconazole and less sensitive to voriconazole and nystatin and had significant higher production of proteinase enzyme in IUD users than non-users. However it did not support the clinical impression that the IUD predisposes to colonization and infection by yeast cells and development of VVC. VVC cases distribution was 61% in users 63% in non-users of IUD. The current study results had not found significant association between biofilm formation among C. albicans or CNA/ with or without IUD use/ or with any other clinical characteristics.
from the present study it was concluded that:
• Culture of vaginal discharge was more sensitive than direct smear.
• The use of chrOMagar Candida for the vaginal swab had several advantages over SDA regarding recognition of mixed cultures; but was still deficient in recognition of some Candida species which were identified by API Candida test.
• C. albicans followed by C. glabrata were the most leading pathogens causing VVC followed by C. tropicalis, C. krusie, C. famata then C. parapsilosis.
• Candida virulence factors (phospholipase and proteinase) were more secreted by C. albicans than CNA.
• Copper IUD did not predispose to VVC, however, when the infection occurred by a CNA isolates, the IUD played a role in aggravating antifungal resistance of CNA to fluconazole and nystatin.
• There was a difference in the response of C. albicans/CNA to the azole (fluconazole) antifungals and nystatin where C. albicans was more sensitive to fluconazole while CNA isolates were more sensitive to nystatin.