HIV感染者口腔白色念珠菌和光滑念珠菌的毒力研究
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摘要
艾滋病是获得性免疫缺陷综合征(acquired immunodeficiency syndrome, AIDS)的简称,是由人类免疫缺陷病毒(human immunodeficiency virus, HIV)感染引起的。HIV主要侵犯、破坏CD4+淋巴细胞,使机体细胞免疫功能受损,最终并发各种严重的机会性感染和肿瘤而导致死亡。1981年,美国发现了1例原因不明的咽喉部严重霉菌感染、体重减轻显著的患者,经多方治疗无效后死亡。后证实其为首例AIDS患者。1982年9月美国疾病控制中心(CDC)正式以“获得性免疫缺陷综合征(acquired immunodeficiency syndrome, AIDS)”为该病命名。CD4淋巴细胞是HIV感染的靶细胞。HIV复制的数量越多,破坏的CD4细胞越多,余留的CD4细胞就越少,患者的免疫力就越差。CD4细胞计数与HIV载量两者均可以独立地预测HIV感染者的临床进程和生存期,广泛用于评估HIV感染的进程、监测抗病毒治疗期间病毒血症的变化。
口腔念珠菌病是HIV感染者的常见并发症,多数学者认为口腔念珠菌病的出现是免疫抑制的先兆,预示疾病即将从HIV感染进入到AIDS阶段,是早期发现和诊断AIDS的重要线索。在HIV感染者病情发展过程中口腔念珠菌病发生率高达90%。其中白色念珠菌是最常见的分离株。
然而,由于免疫抑制治疗药物和广谱抗真菌剂的普遍应用,光滑念珠菌引起的黏膜和系统性感染的发生率明显增多,已成为继白色念珠菌之后,首位最常见的导致念珠菌病的菌种。Polishchuk等1994-2005年检测乌克兰HIV感染者分离的酵母样菌,发现非白色念珠菌增加到46.2%,光滑念珠菌(21.3%)为优势菌种。在以往研究中,我们收集了82例HIV感染者的口腔含漱液,有60例培养出念珠菌,其中有42例可分离出白色念珠菌,44例可分离出光滑念珠菌,同时分离出白色念珠菌和光滑念珠菌的有29例。由此可见,在HIV感染者中,除了主要的白色念珠菌外,光滑念珠菌的检出率也在增高,且可以单独存在,也可与白色念珠菌混合存在。
过去多数研究认为1个个体仅有1种菌株感染,随着念珠菌培养技术的提高,可同时快速鉴定不同菌种,证实混合感染的存在。周菊芬等调查80名正常人群和109例口腔念珠菌病患者口腔念珠菌情况,发现正常人群混合带菌率达11.3%,口腔念珠菌病患者为20.8%。Li等认为白色念珠菌和光滑念珠菌共同导致的感染比单独由白色念珠菌引起的感染更不容易治愈。同样Redding等也发现HIV感染者和癌症患者的白色念珠菌混合光滑念珠菌感染的相关口咽念珠菌病比白色念珠菌单独导致的感染更严重和更难治疗。
HIV感染者易感染口腔念珠菌病的机制较复杂,除宿主免疫因素外,念珠菌本身的毒力变化也可能参与致病过程。与念珠菌相关的毒力因素主要有粘附性、侵袭性的酶、溶血性、形态发生和表型转换等。念珠菌通过粘附来识别定植；通过其利于侵入的侵袭性酶而破坏黏膜完整性,增强毒力；通过溶血性获得营养；通过形态发生及酵母相和菌丝相之间的转换而使其利于侵入宿主,并对该微生物的适应性有利。
HIV感染者口腔白色念珠菌和光滑念珠菌混合感染导致的口咽念珠菌病更难治是否与念珠菌的毒力有关值得关注。此外,从细菌在宿主定植的机制上看,宿主的免疫状况和细菌的毒力亦密切相关。而光滑念珠菌的检出率逐渐增加是否与它的毒力增强相关,它是否具有比白色念珠菌更强的致病力,这也是本实验关注的问题。因此本文从是否混合感染和CD4细胞计数这两个因素出发,选取HIV感染者口腔白色念珠菌和光滑念珠菌各40株,研究并比较其对颊黏膜上皮细胞(Human buccal epithelial cells,BECs)的粘附性、体外磷脂酶活性和溶血性,来探讨其毒力的变化规律。
复苏冻存的HIV感染者口腔白色念珠菌和光滑念珠菌临床分离株,从感染类型和CD4细胞计数两因素研究并比较其对BECs的粘附性、体外磷脂酶活性和溶血性,探讨不同菌种、机体免疫力、感染类型与念珠菌毒力之间的关系。
从冻存的菌株中挑选白色念珠菌40株。单一白色念珠菌感染的20株为单一感染组,其中CD4细胞计数>200个/mm3的菌株10株,CD4细胞计数<200个/mm3的菌株10株；与光滑念珠菌混合感染的20株为混合感染组,其中CD4细胞计数>200个/mm3的菌株10株,CD4细胞计数<200个/mm3的菌株10株。从冻存的菌株中挑选光滑念珠菌40株。单一光滑念珠菌感染的20株为单一感染组,其中CD4细胞计数>200个/mm3的菌株10株,CD4细胞计数<200个/mm3的菌株10株；与白色念珠菌混合感染的20株为混合感染组,其中CD4细胞计数>200个/mm3的菌株10株,CD4细胞计数<200个/mm3的菌株10株。
将-70℃保存的HIV感染者口腔白色念珠菌和光滑念珠菌临床分离株复苏,并接种到沙保琼脂培养基上,37℃培养18-24 h。
0.5 ml菌悬液和0.5 ml BECs液混匀,37℃水浴振荡1 h,12μm的微孔滤膜过滤,PBS冲洗,置于载玻片上晾干,进行革兰氏染色。计算每100个BECs上粘附的念珠菌个数,用H值表示。
将10μl菌悬液滴于蛋黄培养基表面,37℃培养48 h后观察结果。磷脂酶阳性的菌株在蛋黄培养基上的菌落周围会形成一个与菌落同圆心的沉淀圈(环),测定菌落直径和总直径(菌落+沉淀圈)。用Pz值表示磷脂酶的活力,Pz值=菌落直径／总直径。
将10μl菌悬液滴于羊血培养基上,37℃、5% CO2培养48 h。溶血性阳性的菌株,在透射光下观察菌落周围,可观察到半透明的与菌落同圆心的溶血环,测定菌落直径和总直径(菌落+溶血环)并计算溶血指数：溶血指数=菌落直径/总直径。
白色念珠菌粘附性结果：经析因方差分析结果显示是否混合感染和CD4细胞计数两因素间无交互作用(F=1.990,P=0.167),只需分析主效应。单一感染组和混合感染组对BECs的粘附性差异有统计学意义,白色念珠菌混合感染组比单一感染组粘附性强(F=14.939,P=0.000)；不同CD4水平间差异无统计学意义(F=0.336,P=0.566)。
光滑念珠菌粘附性结果：经析因方差分析结果显示是否混合感染和CD4细胞计数两因素间无交互作用(F=0.758,P=0.390),只需分析主效应。单一感染组和混合感染组对BECs的粘附性差异有统计学意义,光滑念珠菌混合感染组比单一感染组粘附性强(F=10.112,P=0.003)；不同CD4水平间差异有统计学意义,CD4细胞计数低组比CD4细胞计数高组粘附性强(F=5.031,P=0.031)。
白色念珠菌和光滑念珠菌对BECs的粘附性比较结果：当感染类型为单一感染时,存在交互效应(F=7.092,P=0.012),需分析单独效应。CD4细胞计数不同水平上,光滑念珠菌的粘附性均高于白色念珠菌(P<0.05)；当感染类型为混合感染时,两因素间无交互效应(F--0.042,P=0.840),光滑念珠菌的粘附性高于白色念株菌(F=5.661,P=0.023)；当CD4细胞计数>200个/mm3时,两因素间无交互效应(F=1.390,P=0.246),光滑念珠菌的粘附性高于白色念株菌(F=7.614,P=0.009)。当CD4细胞计数<200个/mm3时,两因素间无交互效应(F=1.286,P=0.264),光滑念珠菌粘附性高于白色念株菌(F=11.167,P=0.002)。总之无论是单一感染还是混合感染,无论是CD4细胞计数>200个/mm3还是CD4细胞计数<200个/mm3,光滑念珠菌对BECs的粘附性均高于白色念珠菌。
白色念珠菌体外磷脂酶活性：40株白色念珠菌均为磷脂酶阳性。经析因方差分析,结果显示是否混合感染和CD4细胞计数两因素之间无交互作用(F=0.823,P=0.370),只需分析主效应。白色念珠菌单一感染组和混合感染组磷脂酶活性差异有统计学意义(F=6.319,P=0.017),白色念珠菌混合感染组比单一感染组磷脂酶活性高；不同CD4水平间差异无统计学意义(F=0.440,P=0.512)。
光滑念珠菌体外磷脂酶活性：40株光滑念珠菌均为磷脂酶阳性。是否混合感染和CD4细胞计数这两因素之间存在交互效应(F=38.841,P=0.000),需进行单独效应分析。当感染类型为单一感染时,显示CD4不同水平间无差异(t=1.292,P=0.213),感染类型为混合时,CD4细胞计数低组磷脂酶活性显著高于CD4细胞计数高组(t=8.715,P=0.000)；当CD4细胞计数高时,不同感染类型间无差异(t=0.526,P=0.605),当CD4细胞计数低时,混合感染组磷脂酶活性显著高于单纯感染组(t=11.379,P=0.000)。
白色念珠菌与光滑念珠菌磷脂酶活性比较结果：当感染类型为单一感染时,菌种和CD4细胞计数水平之间不存在交互效应(F=1.251,P=0.271),光滑念珠菌的磷脂酶活性高于白色念珠菌(F=6.268,P=0.017)；当为混合感染时,存在交互效应(F--13.708,P=0.001),需分析单独效应,不同CD4细胞计数水平上,高时,白色念珠菌和光滑念珠菌体外磷脂酶活性无差异(t=-1.182,P=0.261)低时,光滑念珠菌的磷脂酶活性高于白色念珠菌(t=4.308,P=0.001)；当CD4细胞计数>200个/mm3时,存在交互效应(F=4.759,P=0.036),需分析单独效应。不同感染类型,白色念珠菌和光滑念珠菌的磷脂酶活性均无差异(P>0.05)；CD4细胞计数<200个/mm3时,存在交互效应(F=10.703,P=0.002),需分析单独效应。不同感染类型,单一感染时,光滑念珠菌和白色念珠菌的磷脂酶活性无统计学意义(t=1.372,P=0.187),混合感染时,光滑念珠菌磷脂酶活性高于白色念珠菌(t=4.308,P=0.001)。
白色念珠菌溶血性结果：40株白色念珠菌均为溶血性阳性,且均有alpha溶血和beta溶血。经析因方差分析,结果显示是否混合感染和CD4细胞计数这两因素之间无交互效应(F=2.435,P=0.127),只需分析主效应。不同感染类型间白色念珠菌的溶血活性无统计学意义(F=0.021,P=0.886)；不同CD4细胞计数水平间无统计学意义(F=0.408,P=0.527)。
光滑念珠菌溶血性结果：40株光滑念珠菌均为溶血性阳性,且均有alpha溶血和beta溶血。经析因方差分析,结果显示是否混合感染和CD4细胞计数这两因素之间无交互效应(F=0.124,P=0.727),只需分析主效应。不同感染类型间光滑念珠菌的溶血活性无统计学意义(F=0.007,P=0.934)；不同CD4水平间无统计学意义(F=0.194,P=0.662)。
白色念珠菌和光滑念珠菌的溶血性比较结果：当单一感染时,无交互效应(F--0.095,P=0.759),光滑念珠菌的溶血性高于白色念珠菌(F=l 1.921,P=0.001)；当混合感染时,无交互效应(F=1.816,P=0.186),光滑念珠菌的溶血性高于白色念珠菌(F=11.994,P=0.001)；CD4细胞计数>200个/mm3时,无交互效应(F=0.532,P=0.471),光滑念珠菌的溶血性高于白色念珠菌(F=8.520,P=0.006)；CD4细胞计数<200个/mm3时,无交互效应(F=0.920,P=0.344),光滑念珠菌的溶血性高于白色念珠菌(F=15.925,P=0.000)。总之,无论是单一感染还是混合感染,无论是CD4细胞计数>200个/mm3还是CD4细胞计数<200个/mm3,光滑念珠菌的溶血性均高于白色念珠菌。
1.在HIV感染者口腔白色念珠菌和光滑念珠菌混合感染时,菌株的磷脂酶和粘附性比在单一感染时要高,说明混合感染时的菌株毒力比单一感染时高,这可能是混合感染时念珠菌病更难治疗的原因之一。
2.与白色念珠菌相比,HIV感染者口腔光滑念珠菌分离株的粘附性、磷脂酶活性和溶血性均较高,说明光滑念珠菌的毒力比白色念珠菌高,这可能也是白色念珠菌与光滑念珠菌混合感染所致的口咽念珠菌病比单一白色念珠菌感染更难治疗的另一原因。
3.白色念珠菌和光滑念珠菌三种毒力因素的结果分别不同,两菌种毒力比较的结果也有所不同,这说明粘附性、磷脂酶活性和溶血性是相互独立的。且从结果提示,在念珠菌粘附定植、侵入并进入组织深部过程中,菌株内部和菌种之间的竞争可能逐渐趋于缓和。
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 tested
Adherence 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.
口腔念珠菌病是HIV感染者的常见并发症,多数学者认为口腔念珠菌病的出现是免疫抑制的先兆,预示疾病即将从HIV感染进入到AIDS阶段,是早期发现和诊断AIDS的重要线索。在HIV感染者病情发展过程中口腔念珠菌病发生率高达90%。其中白色念珠菌是最常见的分离株。
然而,由于免疫抑制治疗药物和广谱抗真菌剂的普遍应用,光滑念珠菌引起的黏膜和系统性感染的发生率明显增多,已成为继白色念珠菌之后,首位最常见的导致念珠菌病的菌种。Polishchuk等1994-2005年检测乌克兰HIV感染者分离的酵母样菌,发现非白色念珠菌增加到46.2%,光滑念珠菌(21.3%)为优势菌种。在以往研究中,我们收集了82例HIV感染者的口腔含漱液,有60例培养出念珠菌,其中有42例可分离出白色念珠菌,44例可分离出光滑念珠菌,同时分离出白色念珠菌和光滑念珠菌的有29例。由此可见,在HIV感染者中,除了主要的白色念珠菌外,光滑念珠菌的检出率也在增高,且可以单独存在,也可与白色念珠菌混合存在。
过去多数研究认为1个个体仅有1种菌株感染,随着念珠菌培养技术的提高,可同时快速鉴定不同菌种,证实混合感染的存在。周菊芬等调查80名正常人群和109例口腔念珠菌病患者口腔念珠菌情况,发现正常人群混合带菌率达11.3%,口腔念珠菌病患者为20.8%。Li等认为白色念珠菌和光滑念珠菌共同导致的感染比单独由白色念珠菌引起的感染更不容易治愈。同样Redding等也发现HIV感染者和癌症患者的白色念珠菌混合光滑念珠菌感染的相关口咽念珠菌病比白色念珠菌单独导致的感染更严重和更难治疗。
HIV感染者易感染口腔念珠菌病的机制较复杂,除宿主免疫因素外,念珠菌本身的毒力变化也可能参与致病过程。与念珠菌相关的毒力因素主要有粘附性、侵袭性的酶、溶血性、形态发生和表型转换等。念珠菌通过粘附来识别定植；通过其利于侵入的侵袭性酶而破坏黏膜完整性,增强毒力；通过溶血性获得营养；通过形态发生及酵母相和菌丝相之间的转换而使其利于侵入宿主,并对该微生物的适应性有利。
HIV感染者口腔白色念珠菌和光滑念珠菌混合感染导致的口咽念珠菌病更难治是否与念珠菌的毒力有关值得关注。此外,从细菌在宿主定植的机制上看,宿主的免疫状况和细菌的毒力亦密切相关。而光滑念珠菌的检出率逐渐增加是否与它的毒力增强相关,它是否具有比白色念珠菌更强的致病力,这也是本实验关注的问题。因此本文从是否混合感染和CD4细胞计数这两个因素出发,选取HIV感染者口腔白色念珠菌和光滑念珠菌各40株,研究并比较其对颊黏膜上皮细胞(Human buccal epithelial cells,BECs)的粘附性、体外磷脂酶活性和溶血性,来探讨其毒力的变化规律。
复苏冻存的HIV感染者口腔白色念珠菌和光滑念珠菌临床分离株,从感染类型和CD4细胞计数两因素研究并比较其对BECs的粘附性、体外磷脂酶活性和溶血性,探讨不同菌种、机体免疫力、感染类型与念珠菌毒力之间的关系。
从冻存的菌株中挑选白色念珠菌40株。单一白色念珠菌感染的20株为单一感染组,其中CD4细胞计数>200个/mm3的菌株10株,CD4细胞计数<200个/mm3的菌株10株；与光滑念珠菌混合感染的20株为混合感染组,其中CD4细胞计数>200个/mm3的菌株10株,CD4细胞计数<200个/mm3的菌株10株。从冻存的菌株中挑选光滑念珠菌40株。单一光滑念珠菌感染的20株为单一感染组,其中CD4细胞计数>200个/mm3的菌株10株,CD4细胞计数<200个/mm3的菌株10株；与白色念珠菌混合感染的20株为混合感染组,其中CD4细胞计数>200个/mm3的菌株10株,CD4细胞计数<200个/mm3的菌株10株。
将-70℃保存的HIV感染者口腔白色念珠菌和光滑念珠菌临床分离株复苏,并接种到沙保琼脂培养基上,37℃培养18-24 h。
0.5 ml菌悬液和0.5 ml BECs液混匀,37℃水浴振荡1 h,12μm的微孔滤膜过滤,PBS冲洗,置于载玻片上晾干,进行革兰氏染色。计算每100个BECs上粘附的念珠菌个数,用H值表示。
将10μl菌悬液滴于蛋黄培养基表面,37℃培养48 h后观察结果。磷脂酶阳性的菌株在蛋黄培养基上的菌落周围会形成一个与菌落同圆心的沉淀圈(环),测定菌落直径和总直径(菌落+沉淀圈)。用Pz值表示磷脂酶的活力,Pz值=菌落直径／总直径。
将10μl菌悬液滴于羊血培养基上,37℃、5% CO2培养48 h。溶血性阳性的菌株,在透射光下观察菌落周围,可观察到半透明的与菌落同圆心的溶血环,测定菌落直径和总直径(菌落+溶血环)并计算溶血指数：溶血指数=菌落直径/总直径。
白色念珠菌粘附性结果：经析因方差分析结果显示是否混合感染和CD4细胞计数两因素间无交互作用(F=1.990,P=0.167),只需分析主效应。单一感染组和混合感染组对BECs的粘附性差异有统计学意义,白色念珠菌混合感染组比单一感染组粘附性强(F=14.939,P=0.000)；不同CD4水平间差异无统计学意义(F=0.336,P=0.566)。
光滑念珠菌粘附性结果：经析因方差分析结果显示是否混合感染和CD4细胞计数两因素间无交互作用(F=0.758,P=0.390),只需分析主效应。单一感染组和混合感染组对BECs的粘附性差异有统计学意义,光滑念珠菌混合感染组比单一感染组粘附性强(F=10.112,P=0.003)；不同CD4水平间差异有统计学意义,CD4细胞计数低组比CD4细胞计数高组粘附性强(F=5.031,P=0.031)。
白色念珠菌和光滑念珠菌对BECs的粘附性比较结果：当感染类型为单一感染时,存在交互效应(F=7.092,P=0.012),需分析单独效应。CD4细胞计数不同水平上,光滑念珠菌的粘附性均高于白色念珠菌(P<0.05)；当感染类型为混合感染时,两因素间无交互效应(F--0.042,P=0.840),光滑念珠菌的粘附性高于白色念株菌(F=5.661,P=0.023)；当CD4细胞计数>200个/mm3时,两因素间无交互效应(F=1.390,P=0.246),光滑念珠菌的粘附性高于白色念株菌(F=7.614,P=0.009)。当CD4细胞计数<200个/mm3时,两因素间无交互效应(F=1.286,P=0.264),光滑念珠菌粘附性高于白色念株菌(F=11.167,P=0.002)。总之无论是单一感染还是混合感染,无论是CD4细胞计数>200个/mm3还是CD4细胞计数<200个/mm3,光滑念珠菌对BECs的粘附性均高于白色念珠菌。
白色念珠菌体外磷脂酶活性：40株白色念珠菌均为磷脂酶阳性。经析因方差分析,结果显示是否混合感染和CD4细胞计数两因素之间无交互作用(F=0.823,P=0.370),只需分析主效应。白色念珠菌单一感染组和混合感染组磷脂酶活性差异有统计学意义(F=6.319,P=0.017),白色念珠菌混合感染组比单一感染组磷脂酶活性高；不同CD4水平间差异无统计学意义(F=0.440,P=0.512)。
光滑念珠菌体外磷脂酶活性：40株光滑念珠菌均为磷脂酶阳性。是否混合感染和CD4细胞计数这两因素之间存在交互效应(F=38.841,P=0.000),需进行单独效应分析。当感染类型为单一感染时,显示CD4不同水平间无差异(t=1.292,P=0.213),感染类型为混合时,CD4细胞计数低组磷脂酶活性显著高于CD4细胞计数高组(t=8.715,P=0.000)；当CD4细胞计数高时,不同感染类型间无差异(t=0.526,P=0.605),当CD4细胞计数低时,混合感染组磷脂酶活性显著高于单纯感染组(t=11.379,P=0.000)。
白色念珠菌与光滑念珠菌磷脂酶活性比较结果：当感染类型为单一感染时,菌种和CD4细胞计数水平之间不存在交互效应(F=1.251,P=0.271),光滑念珠菌的磷脂酶活性高于白色念珠菌(F=6.268,P=0.017)；当为混合感染时,存在交互效应(F--13.708,P=0.001),需分析单独效应,不同CD4细胞计数水平上,高时,白色念珠菌和光滑念珠菌体外磷脂酶活性无差异(t=-1.182,P=0.261)低时,光滑念珠菌的磷脂酶活性高于白色念珠菌(t=4.308,P=0.001)；当CD4细胞计数>200个/mm3时,存在交互效应(F=4.759,P=0.036),需分析单独效应。不同感染类型,白色念珠菌和光滑念珠菌的磷脂酶活性均无差异(P>0.05)；CD4细胞计数<200个/mm3时,存在交互效应(F=10.703,P=0.002),需分析单独效应。不同感染类型,单一感染时,光滑念珠菌和白色念珠菌的磷脂酶活性无统计学意义(t=1.372,P=0.187),混合感染时,光滑念珠菌磷脂酶活性高于白色念珠菌(t=4.308,P=0.001)。
白色念珠菌溶血性结果：40株白色念珠菌均为溶血性阳性,且均有alpha溶血和beta溶血。经析因方差分析,结果显示是否混合感染和CD4细胞计数这两因素之间无交互效应(F=2.435,P=0.127),只需分析主效应。不同感染类型间白色念珠菌的溶血活性无统计学意义(F=0.021,P=0.886)；不同CD4细胞计数水平间无统计学意义(F=0.408,P=0.527)。
光滑念珠菌溶血性结果：40株光滑念珠菌均为溶血性阳性,且均有alpha溶血和beta溶血。经析因方差分析,结果显示是否混合感染和CD4细胞计数这两因素之间无交互效应(F=0.124,P=0.727),只需分析主效应。不同感染类型间光滑念珠菌的溶血活性无统计学意义(F=0.007,P=0.934)；不同CD4水平间无统计学意义(F=0.194,P=0.662)。
白色念珠菌和光滑念珠菌的溶血性比较结果：当单一感染时,无交互效应(F--0.095,P=0.759),光滑念珠菌的溶血性高于白色念珠菌(F=l 1.921,P=0.001)；当混合感染时,无交互效应(F=1.816,P=0.186),光滑念珠菌的溶血性高于白色念珠菌(F=11.994,P=0.001)；CD4细胞计数>200个/mm3时,无交互效应(F=0.532,P=0.471),光滑念珠菌的溶血性高于白色念珠菌(F=8.520,P=0.006)；CD4细胞计数<200个/mm3时,无交互效应(F=0.920,P=0.344),光滑念珠菌的溶血性高于白色念珠菌(F=15.925,P=0.000)。总之,无论是单一感染还是混合感染,无论是CD4细胞计数>200个/mm3还是CD4细胞计数<200个/mm3,光滑念珠菌的溶血性均高于白色念珠菌。
1.在HIV感染者口腔白色念珠菌和光滑念珠菌混合感染时,菌株的磷脂酶和粘附性比在单一感染时要高,说明混合感染时的菌株毒力比单一感染时高,这可能是混合感染时念珠菌病更难治疗的原因之一。
2.与白色念珠菌相比,HIV感染者口腔光滑念珠菌分离株的粘附性、磷脂酶活性和溶血性均较高,说明光滑念珠菌的毒力比白色念珠菌高,这可能也是白色念珠菌与光滑念珠菌混合感染所致的口咽念珠菌病比单一白色念珠菌感染更难治疗的另一原因。
3.白色念珠菌和光滑念珠菌三种毒力因素的结果分别不同,两菌种毒力比较的结果也有所不同,这说明粘附性、磷脂酶活性和溶血性是相互独立的。且从结果提示,在念珠菌粘附定植、侵入并进入组织深部过程中,菌株内部和菌种之间的竞争可能逐渐趋于缓和。
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 tested
Adherence 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.
引文
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Lachke1 mM CuSO4
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[6]Nikawa H, Egusa H, Makihira S, et al. A novel technique to evaluate the adhesion of Candida species to gingival epithelial cells. Mycoses,2003,46(9-10):384-389。
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[8]Samaranayake YH, Dassanayake RS, Jayatilake JA, et al. Phospholipase B enzyme expression is not associated with other virulence attributes in Candida albicans isolates from patients with human immunodeficiency virus infection. J Med Microbiol,2005,54(6): 583-593.
[9]Ribeiro MA, Miranda AE, Gambale W, et al. Prevalence and exoenzyme secretion by Candida albicans isolates from oral and vaginal mucosas of HIV-infected women. Mycopathologia,2004,157(3):255-261.
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[13]温旺荣,王德春,陈红等.白念珠菌的毒力研究——蛋白酶活力的测定.中国人兽共患病杂志,1999,15(6)：24-27.
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[52]Samaranayake YH, MacFarlane TW, Samaranayake LP, et al. The in vitro proteolytic and saccharolytic activity of Candida species cultured in human saliva. Oral Microbiol Immunol,1994,9(4):229-235.
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[55]Shuster A, Osherov N, Rosenberg M. Alcohol-mediated haemolysis in yeast. Yeast, 2004,21(16):1335-1342.
[56]Luo G, Samaranayake LP, Joyce Y.Y.Yau. Candida Species Exhibit Differential In Vitro Hemolytic Actibities.J Microbiol,2001,39(8):2971-2974.
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[58]Barnard JP, Stinson MW. The alpha-hemolysin of Streptococcus gordonii is hydrogen peroxide. Infect Immun,1996,64(9):3853-3857.
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Lachke1 mM CuSO4
[1]Anonymous.1993 revised classification system for HIV infection and expanded surveillance case difinition for AIDS among adolescents and adults. MMWR Recomm Rep, 1992,41(17):1-19.
[2]陈冰,廉翠红,刘维达。白色念珠菌的毒力因子研究新进展。国际皮肤性病学杂志,2006,32(2)：102-104.
[3]Tsang CS, Samaranayake LP. Factors affecting the adherence of Candida albicans to human buccal epithelial cells in human immunodeficiency virus infection. Br J Dermatol, 1999,141(5):852-858.
[4]Sweet SP, Cookson S, Challacombe SJ. Candida albicans isolates from HIV-infected and AIDS patients exhibit enhanced adherence to epithelial cells. J Med Microbiol,1995,43(6): 452-457.
[5]Pereiro M Jr, Losada A.Adherence of Candida albicans strains isolated from AIDS patients. Comparison with pathogenic yeasts isolated from patients without HIV infection. Br J Dermatol,1997,137(1):76-80.
[6]Nikawa H, Egusa H, Makihira S, et al. A novel technique to evaluate the adhesion of Candida species to gingival epithelial cells. Mycoses,2003,46(9-10):384-389。
[7]Lyman CA, Garrett KF, Peter J, et al. Increased adherence of fluconazole- resistant isolates of Candida species to explanted esophageal mucosa. Eur J Clin Microbiol Infect Dis, 1999,18(3):213-216.
[8]Samaranayake YH, Dassanayake RS, Jayatilake JA, et al. Phospholipase B enzyme expression is not associated with other virulence attributes in Candida albicans isolates from patients with human immunodeficiency virus infection. J Med Microbiol,2005,54(6): 583-593.
[9]Ribeiro MA, Miranda AE, Gambale W, et al. Prevalence and exoenzyme secretion by Candida albicans isolates from oral and vaginal mucosas of HIV-infected women. Mycopathologia,2004,157(3):255-261.
[10]Kumar CPG, Kumar SSJ, Menon T. Phospholipase and proteinase activities of clinical isolates of Candida from immunocompromised patients. Mycopathologia,2006, z161(4): 213-218.
[11]Clancy CJ, Nguyen MH. Do in vitro susceptibility data predict the microbiologic response to amphotericin B? Results of a prospective study of patients with Candida fungemia.J Infect Dis,1998,177(2):425-430.
[12]Anil S, Samaranayake LP. Brief exposure to antimycotics reduces the extracellular phospholipase activity of Candida albicans and Candida tropicalis. Chemotherapy, 2003,49(5):243-247.
[13]温旺荣,王德春,陈红等.白念珠菌的毒力研究——蛋白酶活力的测定.中国人兽共患病杂志,1999,15(6)：24-27.
[14]Ollert MW, Wende C, Increased Expression of Candida albicans Secretory Proteinase,a Putative Virulence Factor, in Isolates from Human Immunodeficiency Virus-Positive Patients. J Clin Microbiol,1995,33(10):2543-2549.
[15]Luo G, Lakshman P.Samaranayake, Joyce Y.Y.Yau. Candida Species Exhibit Differential In Vitro Hemolytic Actibities. J Microbiol,2001,39(8):2971-2974.
[16]Luo G, Samaranayake LP, Cheung BPK. Reverse transcriptase polymerase chain reaction(RT-PCR) detection of HLP gene expression in Candida glabrata and its possible role in vitro haemolysin production. APMIS,2004,112(4-5):283-290.
[17]曹岩,张巨,沙春茂等白色念珠菌分泌型酸性蛋白酶和细胞外磷脂酶活性与其毒力的关系吉林大学学报(医学版)2007,33(3)：483-487.
[18]Vargas K, Messer SA, Pfaller M, et al. Elevated phenotypic switching and drug resistance of Candida albicans from human immunodeficiency viruspositive individuals prior to first thrush episode. J Clin Microbiol,2000,38(10):3595-3607.
[19]Samaranayake YH, Samaranayake LP, Yau JY, et al. Phenotypic diversity of oral C. albicans isolated on single and sequential visits in an HIV-infected Chinese cohort APMIS, 2003,111(2):329-337.
[20]Antony G, Ssralaya V, et.al. Phenotypic switching and its influence on expression of virulence factors by Candida albicans causing candidiasis in human immunodeficiency virus-infected patients. Indian J Med Microbiol,2007,25(3):241-244.
[21]Lachke SA, Srikantha T, Tsai LK, et al. Phenotypic Switching in Candida glabrata Involves Phase-SpecificRegulation of the Metallothionein Gene MT-Ⅱ and the Newly Discovered Hemolysin Gene HLP. Infect Immune,2000,68(2):884-895.