In Vitro Virulence Of Candida Albicans And Candida Glabrata In The Oral Cavity Of HIV-infected Patients

AIDS is the abbreviation of acquired immunodeficiency syndrome, is caused by human immunodeficiency virus (HIV). HIV chiefly encroach and destroy CD4+ lymphocyte, induce cell immune function impairment of the host, result in death as coincidencing various of severity opportunity infections and cancers in the last. In 1981, one case was found with unknown aetiology severity throat mycetes infection in USA, body weight losing significantly, after ineffective treatment in many ways, induced death. It is the first AIDS patient witch was confirmed then. In 1982 September, the USA CDC formally designated this disease as AIDS. CD4 lymphocyte is the target of HIV. The more duplication of HIV, the more CD4 lymphocyte it destroyed, so the immunity of the host will be more fragility. CD4 cell count and viral load both can lonely predict the clinical process and life span of HIV infections, and be used generally for forecasting procession of HIV-infected, monitoring the diversify of viremia during antiviral therapy.Oral candidiasis is the most common complication in HIV infected individuals, most scholars considered oral candidiasis was the premonition of immune suppression, signified the disease will change from HIV-infected stage to AIDS stage, was important cue for early discovery and early diagnosis AIDS.The incidence rate of oral candidiasis reach up to 90% during pathogenetic condition development of HIV. Candida albicans was the most common isolations. However, due to the widespread use of immunosuppressive agents and broad-spectrum antimycotic therapy, the frequency of mucosal and systemic C.glabrata infections has increased significantly, making it the second or third most common cause of candidiasis after C. albicans. Polishichuk identified yeast isolations from HIV infections in 1994-2005, non-albicans reached to 46.2%, found in 40% detected patients, and Candida glabrata was the dominant strain. In departed investigation, we collected oral flushing from eighty two HIV infections,60 had positive culture for Candida. Candida albicans were found in 42 cases, Candida glabrata 44 cases, both strains 29 cases. From this, we can found that beside Candida albicans, the incidence of Candida glabrata was increasing, and can survival lonely or both with Candida albicans.In the past, most investigetors considered there was only one strain infected in one case, then along with the advancement in Candida culture technique, it has can identify different strains simultaneously, verified the existence of mixed-infection. Zhou found carrier mixed fungi rate reached to 11.3% in healthy population, and incidence of oral candidiasis was 20.8%. Li considered that infections caused by both Candida albicans and Candida glabrata were more difficult to care than these only caused by Candida albicans. Redding also found that oropharynx candidiasis associated with Candida albicans and Candida glabrata mixed infection in HIV-infected and cancer patients was more severe and more difficulty to treatment than caused only by Candida albicans.The mechanism for HIV infections easily infected Oral candidiasis was complicated, beside immunity factors of the host, the varying of Candida toxicity also participated in pathopoiesis. Virulence factors associated with Candida including adherence, enzyme, haemolyticus, morphogenesis and phenotype switching and so on. Candida can use adherence to discriminate the host; use enzyme to destroy mucosa integrity and enhance virulence; use haemolyticus to acquire mutrition; use morphogenesis and switching between yeast phase and hypha phase to invade host.It is worth to pay attention on that is it whether associate with virulence that oropharynx candidiasis associated with Candida albicans and Candida glabrata mixed infection in HIV-infected was more severe and more difficulty to treatment than that caused only by Candida albicans. Beside this, immunity condition was also closely correlated with fungi virulence. Therefore, we selected forty Candida albicans and forty Candida glabrata from HIV-infected patients, investigated their abilities adhere human buccal epithelial cells, phospholipase activities and haemolyticus, to probe there varying regularity.Revival 40 Candida albicans and 40 Candida glabrata from oral cavities of subjects with HIV-infection witch were freezed under -70℃, and then investigated and compare their in vitro virulence including adherence to healthy buccal epithelial cells, phospholipase activities and haemolysin activities.Forty Candida albican isolations were grouped by single infection or mixed infection and CD4 cell count. Single infection group had 20 isolations(CD4> 200/mm3 and CD4<200/mm3 were 10 isolations, respectively); mixed infection group had 20 isolations(CD4>200/mm3 and CD4<200/mm3 were 10 isolations, respectively). Forty Candida glabrata isolations were grouped by single infection or mixed infection and CD4 cell count. Single infection group had 20 isolations(CD4> 200/mm3 and CD4< 200/mm3 were 10 isolations, respectively); mixed infection group had 20 isolations(CD4>200/mm3 and CD4<200/mm3 were 10 isolations, respectively).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 10ml PBS.Equal volumes of BECs(1x105/ml) and yeast cell suspension (1x107/ml) were mixed and incubated at 37℃for 1 h on a shaking incubator an 1500 r.p.m. The cells were then filtered with a manifold filter apparatus through 12μm pore polycarbonate filters. The filters were washed with 70 ml 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 stain.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.Extracellular phospholipase activity was detected using the egg yolk agar plate method. 10μl aliquots of the yeast suspension (approximately 108/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 48h 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 Pz value) 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 phosphholipase positivity.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 108cells/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 5mm 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 48h. 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 testedAdherence of Candida albicans:After analysis of variance for 40 Candida albicans isolations, the results indicated that the adherence of single infection and mixed infection groups was statistically different, Candida albicans mixed infection group was higher than single infection group(F=14.939, P=0.000); The adherence between different CD4 cell counts has not statistically significance (F=0.336, P= 0.566). there was not interaction between infection type and CD4 cell counts.Adherence of Candida glabrata:After analysis of variance for 40 Candida glabrata isolations, the results indicated that the adherence of single infection and mixed infection groups was ststistically signifinance, Candida glabrata mixed infection group was higher than shingle infection group (F=10.112, P=0.003); The adherence during different CD4 cell counts was also statistically significance, the CD4>200/mm3 group was higher than CD4<200/mm3 (F=5.031, P=0.031). there was not interaction between infection type and CD4 cell counts(F=0.758,P=0.390).Comparison of adherence between Candida albicans and Candida glabrata: When it was single infection, there was interaction between strains and CD4 cell counts(F=7.092,P=0.012), so we must analyze the solitude effect. At distinct level of CD4 cell counts, the adherence of Candida glabrata both were higher than Candida albicans (P< 0.05); When it was mixed infection, there was no interaction (F=0.042,P=0.840), the adherence of Candida glabrata was higher than Candida albicans(F=5.661,P=0.023); When the level of CD4 cell counts was high, there was no interaction (F=1.390,P=0.246), the adherence of Candida glabrata was higher than Candida albicans(F=7.614,P=0.009); When the level of CD4 cell counts was low, there was no interaction (F=1.286,P=0.264), the adherence of Candida glabrata was higher than Candida albicans(F=11.167,P=0.002). In conclusion, whether single infection or mixed infection, whether CD4 cell counts high or low, the adherence to BECs of Candida glabrate was higher than Candida albicans.Phospholipase activities of Candida albicans:All of 40 Candida albicans were phosopholipase positive. After analysis variance, the results indicated that the in vitro phospholipase activity of single infection and mixed infection groups was statistically different, Candida albicans mixed infection group was higher than single infection group(F=6.319, P=0.017); The phospholipase activity between different CD4 cell counts has not statistically significance (F=0.440, P=0.512). there was not interaction between infection type and CD4 cell counts (F=0.823, P=0.370)Phospholipase activities of Candida glabrata:All of 40 Candida glabrata isolations were phosopholipase positive. After analysis variance, there was an interaction effect (F=38.841,P=0.000), we must analyze the solitude effect. When it was single infection, the phosopholipase activities between distinct levels of CD4 cell counts were not statistically significance (t=1.292,P=0.213); When mixed infection, the phosopholipase activities with high CD4 cell counts were obviously higher than that with low CD4 cell counts (t=8.715,P=0.000); When the level of CD4 cell counts were high, between distinct infection types the phosopholipase of Candida glabrata were not different (t=0.526,P=0.605); When the level of CD4 cell counts were low, the phosopholipase activities of mixed infection isolations were significantly higher than that of single infection (t=11.379,P=0.000).Comparison of in vitro phosopholipase activities between Candida albicans and Candida glabrata:When it was single infection, there was no interaction between strains and CD4 cell counts (F=1.251, P=0.271), the in vitro phosopholipase activities of Candida glabrata were higher than Candida albicans (F=6.268,P=0.017); When it was mixed infection, there was interaction (F=13.708,P=0.001,P<0.05), we must analyze the solitude effect. only at low level of CD4 cell counts, the phosopholipase activities of Candida glabrata were higher than Candida albicans (t=4.308,P=0.001); When the level of CD4 cell counts were high, there was interaction (F=4.759,P=0.036), we must analyze the solitude effect. At distinct level of CD4 cell counts, the phosopholipase activities of Candida albicans and Candida glabrata were not statistically different (P>0.05); When the level of CD4 cell counts were low, there was interaction (F=10.703,P=0.002), we must analyze the solitude effect. Only in mixed infection group, the phosopholipase activities of Candida glabrata were higher than Candida albicans (t=4.308, P=0.001).Haemolysin activities of Candida albicans:All of the 40 Candida albicans isolations were haemolysin positive, had both alpha and beta haemolysin. After analysis variance, the results indicated that the haemolysin activities of single infection and mixed infection groups of Candida albicans isolations were not statistically significance (F=0.021,P=0.886); The haemolysin activities between different CD4 cell counts has not statistically significance (F=0.408,P=0.527). there was not interaction between infection type and CD4 cell counts (F=0.408,P=0.527).Haemolysin activities of Candida glabrata:All of the 40 Candida glabrata isolations were haemolysin positive, had both alpha and beta haemolysin. After analysis variance, the results indicated that the haemolysin activities of single infection and mixed infection groups were not statistically significance (F=0.007,P= 0.934); The haemolysin activities between different CD4 cell counts has not statistically significance (F=0.194,P=0.662). there was not interaction between infection type and CD4 cell counts (F=0.124,P=0.727)Comparison of haemolysin activities between Candida albicans and Candida glabrata:When it was single infection, there was no interaction between strains and CD4 cell counts (F=0.095, P=0.759), the haemolysin activities of Candida glabrata were higher than Candida albicans (F=11.921,P=0.001); When it was mixed infection, there was no interaction (F=1.816,P=0.186), the haemolysin activities of Candida glabrata were higher than Candida albicans (F=11.994,P=0.001); When the level of CD4 cell counts was high, there was no interaction (F=0.532,P=0.471), the haemolysin activities of Candida glabrata were higher than Candida albicans (F=8.520, P=0.006); When the level of CD4 cell counts was low, there was no interaction (F=0.920,P=0.344), the haemolysin activities of Candida glabrata were higher than Candida albicans (F=15.925,P=0.000). In conclusion, whether single infection or mixed infection, whether CD4 cell counts high or low, the haemolysin activities of Candida glabrate were obviously higher than Candida albicans (P<0.01).1.When Candida albicans and Candida glabrata mixed infected in oral cavity of HIV-infected patients, the phospholipase activities and adherence to BECs of these isolations were higher than single infections, it indicated that virulence of mixed infected isolations were stronger than single infected isolations, which may be leading to incurable oral candidiasis with mixed infection.2.Compare with Candida albicans, the adherence, phospholipase activities and haemolyticus of Candida glabrata in oral cabity of HIV-infected patients were higher. This may be the reason why oropharynx candidiasis caused by Candida albicans and Candida glabrata were more difficult to care than those caused only by Candida albicans.3.The results of this three virulence factors of Candida albicans and Candida glabrata were different each respectively, the results of virulence comparision of the two strains were also different, which suggested that adherence, phospholipase activities and haemolyticus were independent each other. And the results hint during Monilia adherence, invasion and reach to deep part of the tissues, the competition between strain interior and other strains was incline to relax gradually.