Carriage Frequency And In Vitro Virulence Of Candida Spp. In The Oral Cavity Of Chinese HIV-infected Patients

It was estimated that China would have more than 0.7 million people living with HIV/AIDS by 2007.Opportunistic mucosal infections caused by Candida species are common in individuals with human immunodeficiency virus(HIV) infection.Oral candidiasis remains the most commonly encountered oral complication in HIV/AIDS patients.Although episodes of mucosal candidiasis are rarely lethal,they contribute significantly to morbidity and reduced quality of life of patients with AIDS.However,only a limited number of studies have embarked on identifying the colonizing or infecting yeast species in China.The majority of candidal infections are caused by Candida albicans,however, reports of the emergence of other species of Candida have begun to appear.Despite the increased prevalence of Candida species and the increased frequency of candidiasis in HIV-infected patients,little is known about the changes.There are several factors that may contribute to the transition of Candida species from asymptomatic commensal to pathogen,including changes in the host and/or the virulence of the organism.Virulence factors that contribute to pathogenicity include dimorphism,increased adhesion,production of phospholipases, production of hemolysin,drug resistance,and antigenic modulation.Although there are many studies on the adherence of yeasts to mucosal surfaces, little is known of this critical step initiating fungal infection in HIV-infected individuals.While there have been a number of detailed studies on some of these hydrolytic enzymes,such as proteases,lipases,and phospholipases,little is known of the hemolytic activity exhibited by Candida species.Chapter One:Carriage frequency,species distribution of Candida spp.in the oral cavity of Chinese HIV-infected patients1.Objective:The present study was conducted to evaluate the frequency of colonization by Candida species and Candida species distribution in the oral cavity of HIV-infected patients.2.Methods:82 individuals(49 male and 33 female) diagnosed as HIV-infected patients by ELISA and Western-blot were included in the study.Oral rinses were collected and the yeast identification was performed by morphology in cornmeal agar,Gram staining reaction,CHROMagar Candida test and the API 20 C Aux,growth at 45℃.3.Result:Out of 82 HIV-infected patients studied,60(73.17%) had positive culture for Candida.The frequency of isolation of Candida in HIV-infected patients was:C. albicans,70%(42/60);C.glabrata,77.33%(44/60);tropicalis,1.67%(1/60);C. parapsilosis,3.33%(2/60);Candida pelliculosa,1.67%(1/60),others,3.33%(2/60). There were 51.67%(31/60) episodes of colonization by at least 2 different yeast species. Chapter Two:Carriage frequency,species distribution of Candida spp.in the oral cavity of Chinese healthy persons1.Objective:To evaluate the frequency of colonization by Candida species and Candida species distribution in the oral cavity of non HIV-infected persons.2.Methods:Oral rinses were collected in 82 individuals(49 male and 33 female) diagnosed as non HIV-infected persons.The yeast identification was performed by morphology in cornmeal agar,Gram staining reaction,CHROMagar Candida test and the API 20 C Aux,growth at 45℃.3.Result:Out of 82 HIV-free patients studied,17(20.73%) had positive culture for Candida.The frequency of isolation of Candida in HIV-infected patients was:C. albicans,64.71%(11/17);Candida parapsilosis,29.41%(5/17);others,5.88% (1/60).There were 1(5.88%)episodes of colonization by at least 2 different yeast species,Chapter Three:In vitro virulence of Candida spp.in the oral cavity of Chinese HIV-infected patients and healthy persons1.Objective:To examine the in vitro virulence of Candida species isolates from oral cavities of subjects with and without H IV-infection,including adhesion to healthy buccal epithelial cells,phospholipase activity and hemolytic activity.2.Methods:24 isolates were recovered from distilled water stored at -70℃.They were divided into 3 groups.A group:8 Candida albican isolates from oral cavities of subjects with HIV infection;B group:8 Candida glabrata isolates from oral cavities of subjects with HIV-infection;Cgroup:8 Candida albican isolates from oral cavities of subjects without HIV- infection.A loopful of the stock culture was streaked onto Sabouraud dextrose agar and incubated at 37℃for 18-24 h.(1) Adhesion to buccal epithelial cells.Human buccal epithelial cells(BECs) were used for the adhesion assay.The cells were collected by gently rubbing the cheek mucosa of healthy adult volunteers with sterile swabs and then resuspending the cells by rotating the swabs in 10 ml PBS.Equal volumes of BECs(1×10~5/ml) and yeast cell suspension(1×10~7/ml) were mixed and incubated at 37℃for 1 h on a shaking incubator at 1500 r.p.m.The cells were then filtered with a manifold filter apparatus through 12um pore polycarbonate filters.The filters were washed with 70 ml of PBS to get rid of unattached yeasts,removed carefully and then pressed gently onto glass slides cleaned with chromic acid.Afterwards the filters were carefully peeled off,thus leaving most of the BECs and the attached yeasts on the glass slide.The cells were air-dried and then stained by Gram's method.The number of yeasts attached to 100 epithelial cells was counted microscopically at a magnification of×400.Counting was undertaken randomly without prior knowledge of the source of the sample,and only uniform,unfolded epithelial cells were included.(2) Phospholipase detectionExtracellular phospholipase activity was detected using the egg yolk agar plate method.5 ul aliquots of the yeast suspension(approximately 10~8/ml) were inoculated onto the surface of the egg-yolk medium in quadruplicated samples,left to dry at room temperature and after incubation at 37℃for 48 h the diameter of the precipitation zone around the colony was determined.Each experiment was carried out on two separate occasions.The plates were read using a rule.Phospholipase activity(so called Pzvalue) was determined by the ratio of the diameter of the colony to the total diameter of the colony plus the precipitation zone.Thus,a Pz value of 1 indicates no activity,and less than one indicates the degree of phospholipase positivity.(3) Haemolysin production.Hemolysin production was evaluated using a modification of the plate assay described by Manns et al.The resultant cultures were harvested and washed with sterile saline,and a yeast suspension with an inoculum size of 10~8 cells/ml was prepared using hemocytometric counts.Ten microliters of this suspension was spot inoculated on a sugar-enriched sheep blood agar medium so as to yield a circular inoculation site of about 5 mm in diameter.The final pH of the medium so prepared was 5.6±0.2.The plates were incubated at 37℃in 5%CO2 for 48 h.The presence of a distinct translucent halo around the inoculum site,viewed with transmitted light, indicated positive hemolytic activity.The diameters of the zones of lysis and the colony were measured with a rule,and this ratio was used as a hemolytic index to represent the intensity of the hemolysin production by different Candida species.The assay was conducted in quadruplicate on two separate occasions for each yeast isolate tested.3.Results:H value of three groups were statistically significant(F=6.182,P=0.008).H value between A and C group were not statistically significant(P=0.682).H value between A and B group were statistically significant(P＝0.010).H value between B and C group were statistically significant P=0.004).Enzymatic activity was more pronounced in Candida albicans with 100%PL activity.In contrast,C.glabrata demonstrated only 25%PL.Pz value(The phospholipase activity) of three groups were statistically significant(F=25.562,P =0.000).Pz value between A and C group were not statistically significant(P= 0.660).Pz value between A and B group were statistically significant(P=0.000). Pz value between B and C group were statistically significant P=0.000).Hemolytic index of three groups were not statistically significant(F=0.284, P=0.756).Conclusion:1.Our results showed that the species isolated most frequently was Candida albicans and Candida glabrata in the oral cavity of HIV-infected patients.2.The species isolated most frequently was Candida albicans and Candida parapsilosis in the oral cavity of healthy persons.Asymptomatic oral Candida spp. carriage has been demonstrated in HIV-infected patients,and an increased incidence of asymptomatic oral Candida spp.carriage in HIV-infected patients compared to that in other at-risk groups has also been noted.Thus,a higher prevalence of oral Candida spp.colonization may be a predisposing factor for the subsequent development of clinical thrush.3.The pathogenetic isolates of Candida glabrata from HIV-infected patients were significantly lower than that of Candida albicans from HIV- infected patients and healthy persons in adhesion to buccal epithelial cells and phospholipase activities.There was no difference between Candida albicans isolates from oral cavities of HIV-infected patients and healthy persons in adhesion to buccal epithelial cells and phospholipase activities.And there was no significant difference in hemolytic index of Candida albicans isolates from oral cavities of subjects with and without HIV infection and Candida glabrata isolates from oral cavities of subjects with H IV infection.These results indicate that oral candidiasis was not correlated with some p redominant strains of Candida albicans with higher virulence, but was correlated with strains of Candida glabrata with lower virulence in H TV-infected patients.