Antifungal drug resistance pattern of Candida. spp isolated from vaginitis in Ilam-Iran during 2013-2014

Vaginal Candidiasis is the most common and important opportunistic fungal infection in women. By increasing use of antifungal drugs in recent years, it has caused drug resistance. This study aims to evaluate antifungal drugs susceptibility of Candida. spp isolated of women with vaginitis from Ilam-Iran during 2013-2014. samples were collected and cultured from 385 women with vaginitis, then Candida.spp was diagnosed by standard method. Antifungal drug susceptibility test for nystatin 100 unit/disk, fluconazole 10µg/disk, itraconazole 10µg/disk, ketoconazole 10µg/disk, amphotericinB 20µg/disk, clotrimazole 10µg/disk, posaconazole 5µg/disk, and voriconazole 1µg/disk were carried out by M44-A method(CLSI). From all culture positive samples, 150 isolates were Candida albicans and 89 isolates were non-albicans. The resistance to fluconazole, itraconazole, ketoconazole, clotrimazole, voriconazole, posaconazole, nystatin and amphotericin B was 76%, 62%, 72%, 55%, 6%, 7%, 1% and 0%. The highest resistance was seen for fluconazole , itraconazole, and the highest susceptible was seen for nystatin and amphotericin B. These results indicate nystatin and amphotericin B can be used as the first line for empirical therapy of vaginal candidiasis in the district.     

Expression of the complement-binding protein (MP60) of Candida albicans in experimental vaginitis

The expression of the Candida albicans complement-binding C3d protein (MP60) was investigated both in vitro and in vivo by immunogold labelling and electron microscopy. In vivo expression was determined in a rat vaginitis model. Reactivity of in vitro-grown cells to an anti-MP60 rabbit serum was associated with both cytoplasmic and cell wall sites. Immunostaining in the cell wall of both yeast and hyphae was most concentrated in the inner, electron-lucid layer. Immunogold stained preparations of C. albicans from vaginal smears of infected animals also showed intense localization of the MP60 in the inner cell wall, plasma membrane. However, immunogold label was also intense at the cell surface in these samples, mostly in the area of close adherence with the keratinocytes of the vaginal epithelia. These observations indicate that MP60 is expressed both in vitro and in vivo, but to a different degree in the different cell wall layers. This revised version was published online in August 2006 with corrections to the Cover Date.     

乳杆菌代谢物对白色念珠菌影响的体外试验研究

目的从阴道微生态平衡角度出发,观察乳杆菌(DM9811)代谢物对白色念珠菌的抑杀效果,并行组份分析,探索CV(candidal vaginitis,CV)的生态疗法.方法取CV患者阴道分泌物30例,分离培养,鉴定,保留白色念珠菌菌株.1.以提取液(以下简称提取液)Ⅰ和Ⅱ分别代替蒸馏水配制培养基SⅠ和SⅡ,将保留的白色念珠菌株传代5次后,配制菌液,接种于S、SⅠ和SⅡ中,培养后活菌计数.2.对提取液Ⅰ和Ⅱ倍比稀释,并代替蒸馏水配制多个培养基SⅠ和SⅡ,测定SⅠ和SⅡ中没有菌落生长的最低有效浓度(MIC).3.气相色谱测定提取液中挥发酸类成分并定量.4.选择提取液最低浓度加1的方法配制培养基,比较提取液和乙酸水溶液对白色念珠菌的抑制效果.结果1.白色念珠菌生长24 h后计活菌数,将SⅠ与S,SⅡ与S中活菌数相比较,非参数配对T检验P＜0.01.2.提取液Ⅰ最低有效浓度为1:4,提取液Ⅱ最低有效浓度为1:2.3.气相色谱测定提取液Ⅱ中挥发酸类主要成分为乙酸,浓度为0.235 9 S/100ml.4.提取液和乙酸对白色念珠菌的生长都有抑制作用,但提取液的抑制作用明显强于乙酸.结论乳酸杆菌DM9811代谢物对白色念珠菌有明显的抑菌和杀菌作用,可作为一种新型的生态制剂进行开发,用于治疗念珠菌阴道炎.     

Lasioglossins LLIII affect the morphogenesis of Candida albicans and reduces the duration of experimental vaginal candidiasis in mice

Lasioglossins are a group of peptides with identified antimicrobial activity. The inhibitory effects of two synthetic lasioglossin derivatives, LLIII and D‐isomeric variant LLIII‐D, on morphological changes in Candida albicans in vitro and the effect of local administration of LLIII during experimental murine candidiasis were investigated. C. albicans blastoconidia were grown in the presence of lasioglossin LLIII or LLIII‐D at concentrations of 11.5 μM and 21 μM, respectively, for 1, 2 and 3 days and their viability determined by flow cytometry using eosin Y staining. Morphological changes were examined by light and fluorescent microscopy. The Candida‐inhibitory effect of daily intravaginal administration of 0.7 or 1.4 μg of LLIII was assessed in mice with experimentally‐induced vaginal candidiasis. LLIII and LLIII‐D lasioglossins exhibited candidacidal activity in vitro (>76% after 24 hr and >84% after 48 hr of incubation). After 72 hr incubation of Candida with low concentration of lasioglossins, an increase in viability was detected, probably due to a Candida antimicrobial peptides evasion strategy. Furthermore, lasioglossins inhibited temperature‐induced morphotype changes toward hyphae and pseudohyphae with sporadic occurrence of atypical cells with two or enlarged nuclei, suggesting interference with mitosis or cytokinesis. Local application of LLIII reduced the duration of experimental candidiasis with no evidence of adverse effects. Lasioglossin LLIII is a promising candidate for development as an antimicrobial drug for treating the vaginal candidiasis.

     

Ultrastructural localization of the secretory aspartyl proteinase in Candida albicans cell wall in vitro and in experimentally infected rat vagina

Detection and ultrastructural localization of aspartyl proteinase (Sap) in Candida albicans experimentally infecting rat vagina were studied. Two Sap-positive (Sap+) and one Sap-negative (Sap-) strains of the fungus, endowed with high and low experimental vaginopathic potential, respectively, were used. Both Sap+ strains produced consistent Sap levels in the rat vagina, while the Sap- strain did not produce any measurable Sap. Electron microscopy of thin sections of chemically-fixed vaginal scrapings showed clear evidence of hyphae of proteolitic strains of C. albicans invading the keratinized epithelial cell layer of the vagina. The fungal cells exhibited a pronounced fibrillar layer on the cell wall with a marked intermixing of fungal and vaginal materials especially pronunced at the hyphal tip. Post-embedding immunogold techniques with the use of anti-Sap polyclonal and the specifically generated monoclonal antibody GF1 showed that Sap was essentially localized in the cell wall of C. albicans early during infection, in a cytological pattern mirroring Sap localization in C. albicans cells grown in Sap-inductive media in vitro. In summary, the data offer a new biochemical and ultrastructural evidence that Sap is actively secreted during experimental rat vaginitis by C. albicans. Cell wall localization of Sap is probably inherent to this active secretion process. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterisation of Candida albicans infections of haematogenous and mucosal origin in mice lacking the interferon gamma receptor protein

Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.     

Performing Vaginal Lavage,Crystal Violet Staining,and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.     

白念珠菌ALS3、SSA1基因表达在念珠菌性阴道炎免疫机制中的作用

目的探讨白念珠菌ALS3、SSA1基因缺失对阴道上皮细胞激发免疫反应的作用。方法培养白念珠菌野生株及ALS3、SSA1基因敲除株(SC5314、Δals3、Δssa1),对其进行形态测定。按不同MOI感染人阴道上皮细胞系VK2/E6E7细胞,通过台盼蓝染色观察和乳酸脱氢酶(LDH)活性检测,评价不同MOI白念珠菌对上皮细胞的损伤作用;使用酶联免疫吸附试验(ELISA)评估感染过程中炎性细胞因子及趋化因子在共培养上清中的差异。结果 ALS3基因的缺失对白念珠菌芽管长度影响差异无统计学意义,而SSA1基因的缺失与其他两个菌株相比芽管长度减少约30%～40%(P<0.001)。台盼蓝染色观察及LDH测定发现,3株菌在感染上皮细胞时,其细胞损伤能力均与菌载量成正比;与野生型相比,Δssa1突变体在相同比率感染上皮细胞时,细胞损伤能力明显降低,且差异有统计学意义(P<0.05),Δals3突变株影响较小,甚至略微升高。检测炎性细胞因子及趋化因子发现,突变株在诱导上皮细胞产生促炎因子及趋化因子(GM-CSF、G-CSF、IL-1α、IL-8)的能力上明显减弱,差异均有统计学意义(P<0.05)。结论 ALS3和SSA1基因表达在阴道上皮细胞抗白念珠菌感染的局部免疫应答过程中可能起到重要作用,且SSA1基因表达意义更大。     

Feasibility of Histological Scoring and Colony Count for Evaluating Infective Severity in Mouse Vaginal Candidiasis

Qualitative measurement of the infective level is relatively difficult in experimental vaginal candidiasis. Female BALB/c mice aged 8 to 10 weeks were randomly divided into E1, E2 and E0 groups, which received subcutaneous injection of 0.05 mg, 0.1 mg of estradiol benzoate or 0.1 ml soybean oil 3 days before vaginal inoculation, respectively, and hormone treatment continued every other day thereafter. Each group was further divided into infected and noninfected subgroups. The infected mice were inoculated intravaginally with 10 µl (5 × 10⁴ conidia) of Candida albicans suspension, while the noninfected mice were inoculated with 10 µl phosphate-buffered saline. Direct microscopic examination, colony count and vaginal histopathology including infection degree and inflammation extent were performed at 3, 7 and 14 days post inoculation. Estrogen treatment increased the vaginal fungal burden and extent of infection and inflammation compared with the control group, and 0.3 mg/week estrogen generally induced more severe infection and inflammation than 0.15 mg/week estrogen did. Colony count peaked on day 3 and decreased remarkably after 7 days. Infection score increased gradually during the first 7 days and decreased on day 14, while inflammation extent exacerbated progressively over the course of 14 days. This study demonstrates that the modified histological scoring system might be more feasible than colony count for evaluation of infectivity and dynamic change in experimental vaginal candidiasis.     

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the ‘‘stemness’’ of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.     

Antifungal evaluation of traditional herbal monomers and their potential for inducing cell wall remodeling in Candida albicans and Candida auris

Abstract

Traditional herbal monomers (THMs) are widely distributed in many traditional Chinese formulas (TCFs) and decoctions (TCDs) and are frequently used for the prevention and treatment of fungal infections. The antifungal activities of five common THMs, including sodium houttuyfonate (SH), berberine (BER), palmatine (PAL), jatrorrhizine (JAT) and cinnamaldehyde (CIN), and their potential for inducing cell wall remodeling (CWR), were evaluated against Candida albicans SC5314 and Candida auris 12372. SH/CIN plus BER/PAL/JAT showed synergistic antifungal activity against both Candida isolates. Furthermore, SH-associated combinations (SH plus BER/PAL/JAT) induced stronger exposure of β-glucan and chitin than their counterparts, while CIN triggered more marked exposure compared with CIN-associated combinations (CIN plus BER/PAL/JAT). Collectively, this study demonstrated the anti-Candida effect and the CWR induction potential of the five THMs and their associated combinations, providing a possibility of their in vivo application against fungal-associated infections.     

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. ¹H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.     

Expression of the fibrinogen binding mannoprotein and the laminin receptor of Candida albicans in vitro and in infected tissues

Abstract We have previously reported a 37 kDa laminin-binding protein (p37) and a 58 kDa fibrinogen-binding mannoprotein (mp58) on the surface of Candida albicans . A few yeast cells expressed both functional receptors at the surface while germ tubes expressed a functional mp58 fibrinogen but not a functional p37 laminin receptor. These receptors were heterogeneously dispersed at the surface as shown by binding of rabbit antiserum to mp58 (PAb anti-mp58) and antiserum to the human high affinity laminin receptor. In this report we have used a dual fluorescence technique to determine if the two receptors colocalize, perhaps as part of a receptor complex. Fibrinogen was used as a probe for mp58 and polyclonal antiserum generated to the p37 (PAb anti-p37) was used as a probe for the 37 kDa laminin-binding protein. Both receptors were heterogeneously distributed, but the receptors were not colocalized as the areas of concentration of each receptor were different. Immunohistochemical analysis of tissue sections from patients with disseminated and superficial candidiasis with PAb anti-p37 and PAb anti-mp58 revealed that both receptors were also expressed in infected tissues. The patterns of morphological expression were similar to the in vitro patterns detected by immunofluorescence.     

汉防己甲素及其联合氟康唑对白念珠菌细胞周期影响的初步研究

目的 探讨汉防己甲素联合氟康唑对白念珠菌细胞周期的影响.方法 将白念珠菌CA-1菌悬液与汉防己甲素和（或）氟康唑共培养12h,应用流式细胞仪测定空白对照组、汉防己甲素组、氟康唑组及汉防己甲素联合氟康唑组DNA含量.比较细胞周期各期DNA含量变化并计算增殖抑制率PI％,分析汉防己甲素及其联合氟康唑对白念珠菌细胞周期的影响.结果 汉防己甲素、氟康唑组与对照组S期DNA含量相比,分别能增加17.25％ （P =0.018）与6.54％ （P ＞0.05）,其联合运用效果更明显,S期DNA含量可增加31.52％ （P =0.002）.这说明汉防己甲素能将白念珠菌细胞阻滞在S期.结论 汉防己甲素能抑制白念珠菌细胞DNA合成,阻断白念珠菌细胞周期进程,抑制细胞分裂,其与氟康唑联用时阻滞作用更显著.     

阴道乳杆菌胶囊对产后6周妇女特异性阴道炎防治作用的临床探讨

目的探讨微生物制剂（阴道乳杆菌胶囊）对产后6周妇女特异性阴道炎（细菌性阴道病、滴虫性阴道炎、外阴阴道念珠菌病）的防治作用。方法采用前瞻性研究方法。自2009年6月至2011年8月先后收集895例产后6周特异性阴道炎妇女,包括细菌性阴道病535例、外阴阴道念珠菌病339例和滴虫性阴道炎21例,随机分成三组,第一组（n=200）阴道局部用乳杆菌胶囊（定君生）250 mg/d,连用10 d;第二组（n=200）阴道局部用抗菌药（细菌性阴道病或滴虫性阴道炎：甲硝唑栓500 mg/d,外阴阴道念珠菌病：克霉唑栓150 mg/d,连用10 d）;第三组（n=200）阴道局部用抗菌药＋乳杆菌胶囊（细菌性阴道病或滴虫性阴道炎：甲硝唑栓500 mg/d＋定君生250 mg/d,外阴阴道念珠菌病：克霉唑栓150 mg/d＋定君生250mg/d,连用10 d）,三组均于停药1周后复查阴道分泌物以了解疗效,以及产后3、6、9、12个月分别复查阴道分泌物,以了解三组产后1年内阴道pH、乳杆菌数量变化情况及阴道炎复发率情况。结果 600例产后6周特异性阴道炎妇女中,单用定君生治疗组总有效率为76.5%;用甲硝唑栓或克霉唑栓治疗组总有效率为77.5%;两组总疗效比较差异无统计学意义（P〉0.05）。用抗菌药（甲硝唑栓或克霉唑栓）＋定君生治疗组总有效率为95.0%,与前两组比较差异有统计学意义（P〈0.05）。应用定君生治疗两组1年内阴道pH明显降低,阴道乳酸杆菌数量明显增多,以＋＋＋~＋＋＋＋比例为主,且追踪1年阴道炎的发生率均明显降低,与只用抗生素治疗组相比差异有统计学意义（P〈0.05）。结论阴道乳杆菌胶囊（定君生）是治疗特异性阴道炎的有效药物,与传统抗生素相比疗效差异无统计学意义,且定君生联合抗生素治疗特异性阴道炎,疗效更为显著,这不失为一个新的治疗途径。产后6周妇女特异性阴道炎选用微生态制剂（阴道乳?