Detection and quantification of specific IgE antibodies against eight Malassezia species in sera of patients with atopic dermatitis by using an enzyme-linked immunosorbent assay

The lipophilic yeast Malassezia, a member of the cutaneous microflora, is an exacerbating factor in atopic dermatitis (AD). Of the 11 currently recognized species, M. globosa and M. restricta are found to frequently colonize the skin of AD patients. In this study, we attempted to quantify specific IgE antibodies against eight Malassezia species, namely, M. dermatitis, M. furfur, M. globosa, M. obtusa, M. pachydermatis, M. slooffiae, M. sympodialis, and M. restricta, in sera from AD patients by using an enzyme-linked immunosorbent assay (ELISA). The specific IgE value against M. restricta was greater than those against other Malassezia species. Competitive ELISA inhibition tests revealed that M. restricta contained species specific as well as shared antigens. Therefore, M. restricta could be considered as a candidate diagnostic antigen for detecting anti-Malassezia IgE in sera from AD patients.     

Isolation and characterization of a major antigenic component of Malassezia globosa to IgE antibodies in sera of patients with atopic dermatitis

Three major components of Malassezia globosa were isolated from 2-ME extracts of this fungus by ion-exchange column chromatography and are referred to as Malg46a, Malg46b and Malg67, respectively. IgE antibodies to these components in the sera of patients with AD were detected by immunoblots. In Western blot, IgE antibodies to Malg46b were most frequently detected in the sera of AD patients. Dot blot with the Malg46b-containing fraction immunologically reacted with 69% of the sera of the patients, and with 83% of the sera of the patients who were positive for IgE antibodies to the 2-ME extract of M. globosa in the Western blot. The intensities generated for each dot correlated well with the total intensities generated for the 2-ME extract of M. globosa in the Western blot (r=0.763). In the lectin blot, Con A reacted with both Malg46a and Malg46b but not with Malg67. The polyclonal antibody to Malg46b reacted strongly only with the 2-ME extract of M. globosa and reacted slightly with M. restricta. In conclusion, a glycoprotein, Malg46b of M. globosa, is dominantly expressed in this fungus and is a possible major antigen for IgE antibodies in patients with AD.     

Genotype analysis of Malassezia restricta as the major cutaneous flora in patients with atopic dermatitis and healthy subjects

Lipophilic yeasts of the genus Malassezia colonize the skin surface of humans and are an exacerbating factor in atopic dermatitis (AD). Two species, M. restricta and M. globosa are major cutaneous microflora in both AD patients and healthy subjects. We compared the DNA sequences of the intergenic spacer (IGS) region, located between the 26S and 5S rRNA genes of M. restricta colonizing the skin surfaces of 13 AD patients and 12 healthy subjects, and of three CBS stock strains as references. The IGS 1 sequences were divided into two major groups, corresponding to AD patients and healthy subjects. These findings suggest that a specific genotype of M. restricta plays a significant role in AD, although M. restricta commonly colonizes both AD patients and healthy subjects.     

Recent developments in the immunology and biology of Malassezia species

Malassezia spp. are members of the normal cutaneous flora, but are also associated with several cutaneous diseases. Recent studies of the interaction of Malassezia spp. with melanocytes, fibroblasts, keratinocytes and dendritic cells have highlighted their potential to modulate the immune response directed against them. In normal skin they may downregulate the inflammatory response, allowing them to live as commensals. In contrast, in atopic/eczema dermatitis syndrome and psoriasis, they may elicit an inflammatory response that contributes to the maintenance of lesions. Future research may define ways to influence this inflammatory cycle and hence to control or prevent exacerbations of these diseases.     

Chitin synthase 2 gene sequence of Malassezia species.

Nucleotide sequences of the chitin synthase 2 (CHS2) gene of seven species, Malassezia furfur, M. globosa, M. obtusa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS2 gene were amplified from these Malassezia species by polymerase chain reaction (PCR) and sequenced. The CHS2 nucleotide sequences of these Malassezia species showed more than 95% similarity between the species. A phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of seven Malassezia species revealed that the species were genetically distinct from each other.     

Quantitative analysis of cutaneous malassezia in atopic dermatitis patients using real-time PCR

We quantified the cutaneous Malassezia in patients with atopic dermatitis using a real-time PCR assay. Seven to 12 times more Malassezia colonized the head and neck compared to the trunk or limbs, and the species M. globosa and M. restricta accounted for approximately 80% of all Malassezia colonization at any body site.     

小鼠烟曲霉角膜炎的实验研究

目的 比较伊曲康唑和氟康唑对烟曲霉的体外抗菌活性,观察伊曲康唑对小鼠烟曲霉角膜炎的治疗作用.方法 通过角膜基质注射法建立烟曲霉角膜炎小鼠模型.造模后观察角膜病变,取角膜病变处分泌物做真菌镜检、真菌培养以证实造模成功.用药基法检测伊曲康唑和氟康唑对烟曲霉的最低抑菌浓度( MIC)和最低杀菌浓度(MFC).对烟曲霉角膜炎小鼠给予伊曲康唑治疗,治疗结束行临床评分、炎性评分、菌落形成单位测定以评价疗效.结果 伊曲康唑对烟曲霉的MIC和MFC分别为6.25 μg/mL、12.5 μg/mL;氟康唑对烟曲霉的MIC和MFC分别为500 μg/mL、1 000 μg/mL.伊曲康唑治疗组临床评分、炎性评分和测定的菌落数较对照组均明显减少(P＜0.05).结论 伊曲康唑对烟曲霉的体外抗菌活性优于氟康唑,并且对烟曲霉性角膜炎有明显疗效.     

Occurrence of Malassezia species in healthy and dermatologically diseased dogs

The presence of Malassezia spp. yeasts was investigated in dermatological specimens of 224 dogs, 164 dermatologically diseased and 60 normal dogs. Subjects included in the study were of different breed, age, sex and habitat. Malassezia spp. positive cultures were obtained in 142 (63.4%) specimens: 67.6% from dermatologically diseased subjects and 51.6% from healthy dogs. Malassezia pachydermatis, either as a pure culture or in association with lipid-dependent species, was identified in 138 (97%) specimens. Malassezia furfur was identified in 69 (48.6%) specimens and was associated with other Malassezia species in 68 dogs, as a pure culture in one subject: at the best of our knowledge, this species was identified before as the sole species from canine dermatitis. Malassezia sympodialis was identified in 11 (7.7%) specimens, always in association with other species: it was never isolated from kennel dogs. Statistical analysis of data showed a very significant difference (P < 0.01) in the prevalence of isolation of Malassezia spp. between animals with and without dermatological signs, and in the distribution of cultural burden between diseased and healthy dogs. A statistically significant difference (P<0.05) was also detected in the group of animals between 1- and 5-years of age. No significant difference was found between male and female dogs.     

A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects

Over the last few years, new Malassezia species have been found regularly in Japanese subjects. We isolated another new Malassezia species from a Japanese patient with seborrheic dermatitis (SD), and named it M. yamatoensis. In its physiological characteristics and the utilization of Tween by M. yamatoensis is similar to that of M. furfur and M. dermatis. It is distinguished by its growth temperature. To examine the distribution of the microorganism in the skin of patients with SD and atopic dermatitis (AD), and healthy subjects, we applied transparent dressings to the skin, and detected M. yamatoensis DNA using a non-culture-based method that consisted of nested PCR with specific primers. M. yamatoensis DNA was detected from 3 of 31 SD patients (9.7%), 5 of 36 AD patients (13.9%), and 1 of 22 healthy subjects (4.6%). Therefore, M. yamatoensis is a rare member of the cutaneous microflora.     

DNA base alignment and taxonomic study of genus Malassezia based upon partial sequences of mitochondrial large subunit ribosomal RNA gene

The sequences of the large subunit of mitochondrial ribosomal RNA (LsmtrRNA) gene of Malassezia species were analysed. The sequences of the seven species of Malassezia are well separated in each species. Therefore the LsmtrRNA gene is thought to be one of the gene targets for species identification in the genus Malassezia. The dendrogram obtained from this gene supports the previous study of Malassezia species based upon the chromosomal genes. This is the first report of taxonomic analysis of Malassezia species based upon the mitochondrial gene.     

伊曲康唑治疗复发性外阴阴道念珠菌病38例临床观察

目的了解国产伊曲康唑（商品名“美扶”）治疗复发性外阴阴道念珠菌病的疗效。方法收集38例复发性外阴阴道念珠菌病为治疗组,口服伊曲康唑200mg,1次／d,连续7d,以后每次月经第1天口服伊曲康唑200mg,1次／d,连续6个月经周期停药,而对照组20例则单子硝酸咪康唑栓200mg阴道外用,方法同前。两组完成冲击治疗后1周、3个月、6个月评价疗效。结果1周后治疗组总有效率为92．1％,对照组为90％,两组相比无统计学差异。3个月、6个月后治疗组的复发率分别为3．2％、6．7％,对照组为28．6％、38．5％,两组相比有统计学意义。结论伊曲康唑短程冲击治疗加长期间断给药对复发性外阴阴道念珠菌病的治疗和预防复发效果满意。     

伊曲康唑联合全厚皮片移植术治愈着色芽生菌病1例

患者男,47岁.颈部皮损5a,皮损直接镜检可见多个棕色硬壳小体,皮损组织真菌培养为卡氏枝孢瓶霉,皮损组织病理PAS染色可见棕色厚壁孢子.诊断为着色芽生菌病,给予患者口服伊曲康唑胶囊治疗8个月,皮损形成瘢痕,之后采取全厚皮片移植术切除瘢痕,术后继续口服伊曲康唑治疗2个月,皮损痊愈.     

甲真菌病患者生活质量及口服伊曲康唑间歇冲击疗法对其的影响

目的研究伊曲康唑治疗对甲真菌病患者生活质量的影响。方法221例患者应用伊曲康唑冲击治疗前后填写调查表,其中43例患者完成伊曲康唑3个疗程随访。结果全甲营养不良型甲真菌病患者生活质量下降较为明显的指标为精神、心理状态4．79±2．81,日常生活、工作能力3．33±2．11（P〈0．05）。伊曲康唑治疗后甲真菌病患者生活质量分值随疗程延长逐渐降低。结论甲真菌病患者生活质量受到影响,伊曲康唑冲击治疗能改善患者生活质量。     

Evaluation of the levels of specific IgE against Cryptococcus diffluens and Cryptococcus liquefaciens in patients with atopic dermatitis

Cryptococcus diffluens and Cryptococcus liquefaciens, 2 basidiomycetous yeasts, frequently colonize the skin of patients with atopic dermatitis (AD). In this study, we investigated the presence of specific IgE antibodies against C. diffluens and C. liquefaciens in the sera of AD patients by using an enzyme immunoassay . Of the 122 AD serum samples tested, 43 (35.2%) and 50 (41.0%) were positive for specific IgE antibodies against C. diffluens and C. liquefaciens, respectively. The levels of specific IgE against the C. diffluens antigen and that against the C. liquefaciens antigen were strongly correlated (r=0.96). In contrast, no remarkable correlation was observed between the levels of specific IgE against the 2 Cryptococcus species and that of specific IgE against Malassezia restricta. Competitive enzyme-linked immunosorbent assay (ELISA) inhibition tests revealed that C. diffluens and C. liquefaciens shared common antigens. This finding was consistent with the IgE immunoblotting data which demonstrated that several IgE-binding proteins with molecular masses of 77, 54, and 30 kDa were recognized in both C. diffluens and C. liquefaciens antigens . These results suggest that fungal components from C. diffluens and C. liquefaciens may act as allergens and play a role in the pathogenesis of AD.     

Report from the in vitro micronucleus assay working group

At the Washington “2nd International Workshop on Genotoxicity Testing” (25–26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth “3rd International Workshop on Genotoxicity Testing” (28–29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern:

1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test.

2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information.

3. Treatment schedules for cell lines and lymphocytes.

4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly).

5. Duplicate cultures and number of cells to be scored.

6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative.

7. Statistics: statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.

     

伊曲康唑对皮肤癣菌形态学影响研究

目的观察皮肤癣菌在伊曲康唑作用下的形态学变化。方法应用美国CLSI制订的标准M38-A方案进行伊曲康唑对皮肤癣菌的体外药敏试验,测定最小抑菌浓度（MIC）,将伊曲康唑作用前后的皮肤癣菌分别制成标本,在光学显微镜、扫描电子显微镜和透射电子显微镜下观察形态学变化。结果伊曲康唑作用于皮肤癣菌后,在光学显微镜下菌丝变得弯曲、短粗,顶端和局部出现膨大;扫描电子显微镜下菌丝变得弯曲、短粗、干瘪,顶端和局部出现膨大,有不规则分支,表面粗糙,有大小不等的凹陷;透射电子显微镜下菌丝变得皱缩,有凹陷,双层细胞壁结构消失或不完整,细胞膜不连续,皱缩细胞膜和细胞壁之间及胞浆内出现许多小的高电子密度颗粒,细胞器也变得不清晰。结论伊曲康唑使皮肤癣菌的形态发生明显变化。     

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay

The application of a modified colorimetric bioassay for the evaluation of the biological effects of mycotoxins is reported. Using three different monolayer cell lines (swine kidney, Madin Darby canine kidney, HeLa) the influence of nine different mycotoxins on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated. The yellow tetrazolium salt MTT is converted by mitochondrial dehydrogenases of metabolically active cells to an insoluble purple formazan product, which was then solubilized with dimethylsulfoxide. The optical density of this homogeneous solution was suitable for a precise spectrophotometric measurement by a plate reader at a wavelength of 510 nm. Nine mycotoxins were simultaneously tested in all three cell lines, from which the swine kidney cell line proved to be the most sensitive. The effects of additional 35 mycotoxins were therefore tested using swine kidney monolayers as target cells. A total of 28 toxins of the 44 mycotoxins tested proved to be cytotoxic in the MTT-bioassay. Most of them belong to the group of trichothecene mycotoxins. Concentrations ranged between 0.01 µg and 100 µg/ml of cell culture medium. The MTT cleavage assay was found to be a quick (24 hours) and easy to perform system for the evaluation of the biological activity of many different mycotoxins and may also provide a useful tool for the testing of a large variety of sample materials.     

PCR法评价伊曲康唑注射液治疗播散性白念珠菌病兔模型疗效

目的通过静脉内接种的方法,构建播散性白念珠菌感染的兔模型,并用PCR评价伊曲康唑注射液治疗播散性念珠菌病的疗效。方法在接种后24h,用伊曲康唑注射液5rag／kg对兔模型进行治疗,1次／d,共14d。在不同的时间段取兔模型的静脉血,进行血培养和真菌通用引物以及白念珠菌特异性引物的PCR检测,监测伊曲康唑注射液治疗播散性白念珠菌感染的疗效。结果在接种白念珠菌后1h、6h,外周血中用PCR方法就能检测到白念珠菌,且能持续到8—10d;实验兔外周血血培养1h后阳性,持续到18h。实验结束后解剖实验兔,治疗组较对照组内脏器官的组织培养阳性率及菌落数低。结论PCR是一种快速和敏感的检测播散性念珠菌病的方法,伊曲康唑注射液治疗播散性白念珠菌病有效,但是真菌的清除率特别是肾脏组织的真菌清除率并不理想,治疗结束7d后,组织匀浆真菌培养仍然阳性。     

伊曲康唑联合特比萘芬治疗孢子丝菌病疗效及其对病原真菌体外抗菌活性研究

目的 初步探讨伊曲康唑和特比萘芬联合治疗孢子丝菌病的疗效,评价两药体外联合对申克孢子丝菌菌丝相和酵母相的抗菌活性.方法 口服伊曲康唑200mg/d和特比萘芬250mg/d治疗孢子丝菌病;体外联合药敏试验采用棋盘微量稀释法,计算分数抑菌浓度(FIC)指数判定两药相互作用具有协同、拮抗或无关作用.结果 伊曲康唑和特比萘芬联...     

Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia

Vulvovaginal candidiasis is a common mucosal infection caused by opportunistic yeasts of the Candida genus. In this study, we isolated and identified the yeast species in the vagina of patients treated in the gynecology clinic and tested in vitro activities of fluconazole and itraconazole against 227 clinical yeast isolates by the NCCLS microdilution method. C. albicans (87.6%) was the most frequently identified species followed by C. glabrata (6.2%) and C. krusei (2.2%). Almost thirteen percent of yeast strains were resistant to fluconazole and 18.5% were resistant to itraconazole. Cross-resistance analyses of C. albicans isolates revealed that fluconazole resistance and itraconazole resistance were also associated with decreased susceptibilities to other azole derivatives mainly to ketoconazole and miconazole. At the same time no cross-resistance to polyene antibiotics amphotericin B and nystatin was observed. These results support the notion that antifungal agents used to treat vaginitis may be contributing to the drug resistance problem by promoting cross-resistance to a range of clinically used antifungals.