Objective To determine whether Trichophyton rubrum isolated from different lesions in the same patient is of different strains. Methods DNA was extracted from the isolates, then subjected to a polymerase chain reaction-based typing which analyzed the number of repetitive elements in the non-transcribed spacer region of the ribosomal DNA gene repeats. Results Thirty-six strains of T. rubrum were isolated from 17 patients with fungal infection on multiple sites. All strains could be classified into 10 genotypes. The genotype distribution was unrelated to sites of infection. It happened to 8 of the 17 patients that multiple genotypes were involved in T. rubrum infection on different sites in the same body. Conclusion The study shows that multiple genotypes are involved in T. rubrum infection on different sites in the same patient, suggesting external sources of infection rather than infection from a different site in the same individual.

Objective To develop a murine model for infection by Cladosporium carrioni. Methods A total of 72 ICR mice were equally divided into 4 groups, group A (healthy mice inoculated by C. Carrioni suspension of 1 × 108 cfu conidia mL-1, group B (immune-suppressed mice inoculated by C. Carrioni sus pension of 1 × 108 cfu conidia mL-1), group C (immune-suppressed mice inoculated by C. Carrioni suspen-sion of 1 × 106 cfu conidia mL-1), group D (healthy mice inoculated by sodium chloride solution). C. Car-rioni suspension or sodium chloride solution was subcutaneously inoculated into foot pads of mice. On day 7, 30 and 60 after inoculation, 6 mice were killed in each group followed by the measurement of thickness of foot pads, pathology and mycology of skin samples taken from foot pads. Results In group A, B and C, there were swelling, blackening, ulceration and crusts at the inoculation site of all mice, with a morbidity of 100%. The thickness of foot pads in group A on day 30 was significantly higher than that on day 7 (2.40 ± 0.45 mm vs 2.85 ± 0.47 mm, P < 0.05), but lower than that on day 60 (1.64 ± 0.13 ram, P < 0.05). In group B, increased thickness of foot pads was observed on day 30 compared with that on day 7 and day 60 (2.19 ± 0.27 mm vs 1.80 ± 0.21 mm and 1.86 + 0.22 mm, respectively, both P < 0.05), which was the case with group C (1.98 ± 0.06 nun vs 1.51 ± 0.11 mm and 1.82 ± 0.09 mm, respectively, both P < 0.05). No significant changes occurred to the thickness of foot pads in group D from day 7 to day 60 (P > 0.05). Pathological changes in group A, B and C included necrosis, abscess and chronic granuloma formation; dark brown sclerotic bodies were observed on HE and PAS staining as well as on direct microscopy; cultures of tissue samples grew Cladosporium carrionii. The mice in group D remained uninfected. Conclusion Mouse model for chromoblastomycosis may be established by subcutaneous inoculation of Cladosporium carrionii suspension into foot pads of healthy or immuno-suppressed mice.

Objective To investigate the characteristic,diagnosis and treatment of the nipple discharge.Methods The clinical data of 174 cases who were diagnosed as nipple dlscharge from January 2001 to December2006 were collected and analyzed retrospectively.Results Among those 174 cases,136 cases were confirmed histopathologieally to be intraduetal papilloma.The confirmed diagnosis rate of intraduetal papilloma by galaetograghy was 85.00%.Carcinoma-charge rate of the intraduetal papilloma was 5.17%.Conclusion Nipple discharge was the most common symptom in the intraductal papillomatesis.The galaetography was a valuable method in the diagnosis of the intraduetal papilloma.There was carcinoma-charge possibility in the intraduetal papilloma.

Objectives To analyse the antifungal susceptibility of Candida spp. isolated from the patients with recurrent vulvovaginal candidiasis (RVVC), vulvovaginal candidiasis (VVC) and asymptomatic carriers and to study the correlation between different Candida strains and antifungal susceptibility. Method According to the NCCLS-M27-A scheme, the antifungal susceptibility of Candida spp. isolated from the above different groups was measured. Results Almost all the MICs of C. glabrata and C. krusei to 8 antifungal agents were higher than those of C. albicans. The average MIC of C. albicans isolated from RVVC patients was higher than that from asymptomatic carriers. The resistant strains were mainly isolated from the RVVC group. No resistant strains against itraconazole, fluconazole, ketoconazole, econazole and nystatin was found in asymptomatic carriers. Conclusions These results indicate that more attention has to be paid to the low susceptibility of non-Candida albicans in the treatment of vulvovaginal candidiasis, and the resistant strains may result from long-term or irregular antifungal treatment.

Objective To investigate the effects of Candida albicans on the expression of tumor necrosis factor-α(TNF-α)and activation of the intracellular signaling molecule p38 mitogen-activated protein kinase(p38MAPK)in a human acute monocytic leukemia cell line THP-1. Methods Some THP-1 cells were divided into several groups in vitro: two C. albicans groups treated with 105 CFU/ml and 106 CFU/ml heat-killed C. albicans respectively, a lipopolysaccharide (LPS)group treated with 100 μg/L LPS, a blank control group treated with RPMI 1640 medium, two dexamethasone-inhibited groups pretreated with 40 μg/L dexamethasone for 30 minutes followed by treatment with 106 CFU/ml heat-killed C. albicans and LPS respectively. After treatment for 1, 3 and 6 hours, real-time fluorescence-based quantitative PCR was performed to measure TNF-α mRNA expression in THP-1 cells in the above groups. Enzyme-linked immunosorbent assay(ELISA)was conducted to determine the level of TNF-α protein in the supernatant of THP-1 cells treated with 106 CFU/ml heat-killed C. albicans, 100 μg/L LPS or RPMI 1640 medium(blank control group)for 24 hours. Western blot was performed to measure the protein expression of p38MAPK and phosphorylated p38MAPK in THP-1 cells after treatment with 106 CFU/ml heat-killed C. albicans or RPMI 1640 medium (blank control group)for 30 and 60 minutes. Statistical analysis was carried out by using two-way analysis of variance, one-way analysis of variance and the least significant difference(LSD)-t test. Results Significant differences were observed in the mRNA expression level of TNF-α among the C. albicans groups, LPS group and blank control group (F = 110.98, P < 0.001). The mRNA expression level of TNF-α in THP-1 cells increased over time in a time-dependent manner after C. albicans treatment, with significant differences among different time points (F = 701.680, P < 0.001). Compared with the blank control group, both 106-CFU/ml C. albicans group and LPS group showed a significant increase in TNF-α protein expression (6385.70 ± 533.99 ng/L and 3212.06 ± 353.00 ng/L vs. 147.10 ± 0.53 ng/L, P < 0.001 and 0.005, respectively). An obvious increase was observed in the expression level of phosphorylated p38MAPK protein, but no significant changes were noted in that of p38MAPK protein, in THP-1 cells treated with 106 CFU/ml C. albicans for 30 and 60 minutes compared with the blank control group. The mRNA expression level of TNF-α significantly decreased in dexamethasone-pretreated 106-CFU/ml C. albicans group and LPS group compared with those without dexamethasone pretreatment(3.77 ± 0.62 vs. 208.50 ± 10.50, 6.20 ± 1.93 vs. 161.35 ± 1.65, both P < 0.001). Conclusions Heat-killed C. albicans can induce the activation of p38MAPK in and secretion of TNF-α by human THP-1 cells, which then participate in the innate immune response against C. albicans.

Objective To investigate whether human neutrophils kill Candida albicans through recognition of insoluble β-glucan in cell walls of C.albicans (CalG) by dectin-1,a C-type lectin receptor.Methods Neutrophils were obtained from peripheral blood of healthy human subjects and cultured in vitro.Real-time PCR was carried out to quantify the mRNA expressions of dectin-1 and Toll-like receptor 2 (TLR2) in neutrophils challenged with CaIG of 100 mg/L for 1,6,and 24 hours.A Fluoro hydrogen peroxide (H2O2) detection kit was used to determine H2O2 levels in some neutrophils exposed to CaIG (100 mg/L) for 15 minutes,2 hours,6 hours,as well as in some neutrophils preincubated with laminarin (a dectin-1 inhibitor) of 100 mg/L and 50 mg/L for 30 minutes followed by challenge with CaIG of 100 mg/L for 2 hours.Colony forming units (CFUs) were counted after the incubation of C.albicans with neutrophils pretreated with laminarin of 100 mg/L and 50 mg/L for 30 minutes.Results The relative mRNA expression level of dectin-1 was 2.8195 + 0.1669,5.4859 + 0.7244 and 3.6041 + 0.5372 in neutrophils challenged with CaIG for 1,6 and 24 hours,respectively,significantly higher than that in unchallenged neutrophils at these corresponding time points (all P ＜ 0.01).The level of H2O2 was (64.55 + 15.67),(290.34 + 30.56),and (208.54 ± 26.88) μ mol/L respectively in neutrophils treated with CaIG for 15 minutes,2 hours,and 6 hours respectively,compared to (22.05 ± 3.99) μmol/L in untreated neutrophils (all P ＜ 0.01).The pretreatment with laminarin of 100 and 50 mg/L attenuated the release of H2O2 in CaIG-treated neutrophils by 73％ ((80.45 + 22.41) μ mol/L,P＜ 0.01) and 45％ ((130.42 + 44.55) μmol/L,P＜ 0.01),respectively,compared with neutrophils treated with CaIG only.The fungicidal activity of neutrophils against C.albicans was also significantly inhibited by pretreatment with laminarin of 50 and 100 mg/L (both P＜ 0.01).Conclusions Dectin-1 may be involved in the secretion of H2O2 as well as killing of C.albicans by human neutrophils,which may provide a preliminary evidence for adoptive transfer of neutrophils as an approach to the treatment of systemic C.albicans infection.

Objective To analyze the gene expression of pathogenic factors in vaginal secretions of patients with vulvovaginal candidiasis by using Oligo chips. Methods RNA was extracted from vaginal secretions of 10 patients with vulvovaginal candidiasis and 3 asymptomatic carriers, and hybridized with oligonuscreened followed by a bioinformatic analysis. Results Comparing with the asymptomatic carriers, the patients showed a higher expression of 44 genes and lower expression of 17 genes. Of these differentially expressed (TLR) 4, HWP1, SAP2, SAP5, LIP4, EFG1 and CPH1 were highly expressed in more than 80% of the secretion samples from patients with an average ratio of 4.013, while LIP6 and WH11 were lowly expressed in more IFN-γ and TLR4 were associated with native immunity, HWP1 associated with hyphal adhesion and formation, SAP2, SAP5, LIP4 and LIP6 associated with extracellular hydrolysis, and EFG1, CPH1 and WH11 associated with phenotypic switching. Conclusions Both the host adaptive immunity deficiency and increased virulence of Candida species are involved in the pathogenesis of vulvovaginal candidiasis, and TLR4 possibly plays a certain role in the local immunity of patients with this entity.

Objective To determine the extracellular enzymatic activity of common pathogens for onychomycosis, in the hope of finding virulence factors associated with the pathogenesis of onychomycosis. Methods Strains tested in this study included standard strains of common dermatophyte and non-dermatophyte fungi as well as clinical isolates of Trichophyton rubrum from patients with onychomycosis. All the tested strains were cultured in medium containing nail fragments at 25 ℃ for 10 to 21 days followed by the determination of the nail fragment-containing medium, a significant increase was observed in the activities of esterase, esterase lipase, N-acetyl-β-glucosaminidase and α-mannosidase in dermatophytes compared with non-dermatophytes (all P ＜ 0.05 ), as well as in the activity of N-acetyl-β-glucosaminidase in Trichophyton rubrum compared with the other tested species of fungi (all P ＜ 0.05). No significant difference was noted in the activity of extracellular enzymes, except for that of naphthol-AS-BI-phosphohydrolase, between the isolates of Trichophyton rubrum from patients with different ranges of scoring clinical index for onychomycosis (SCIO). Conclusions In specific conditions, the extracellular enzymatic activity of fungi isolated from patients with onychomycosis is associated with fungal species, and may have a certain influence on the manifestations of anychomycosis.

Objective To estimate the application value of colony PCR in the detection of pathogenic filamentous fungi. Methods Colony PCR was established and performed to amplify the internal transcribed spacer (ITS) region of 19 species (strains) of filamentous fungus followed by sequencing analysis. At the same time, DNA extracts from 8 of the 19 species of filamentous fungus were subjected to conventional PCR. Hha I and Hinf I endonucleases were used for restriction fragment length polymorphism (RFLP) analysis of the conventional and colony PCR products. Comparison analysis was carried out between the colony and conventional PCR. Results Of the 19 strains, 16(84.2%) yielded positive results by colony PCR; sequence analysis of the PCR products of ITS region revealed a 96% - 100% similarity with the reference sequence (NCBI database)of corresponding fungi. The amplification product length and RFLP profile of these products from the 8 species of filamentous fungus, except for those from Aspergillus nidulans, were consistent between the colony and conventional PCR. Conclusions Compared with conventional PCR, colony PCR-based detection of filamentous fungi is easy to operate, time and labor-saving, with high accuracy and reliability, and can be applied to the rapid identification of filamentous fungi.

A 19-year-old man was admitted to the hospital for erythema and nodules on the face and postauricular region for 6 years. Microscopic examination of lesion scrapings revealed brown septate hyphae. A restricted, velvety and black colony grew on Sabouraud's dextrose agar. Slide culture on potato dextrose agar plate showed flask-shaped phialides at the apex of or around the hyphae with clear collarettes and flaring apex,mucilage-encapsuled, round to oval, semi-endogenous phialosporae accumulating at the apex of the phialides,giving a flower-like appearance. Anti-fungal susceptibility test showed that the fungus was sensitive to itraconazole, terbinafine and amphotericin B, but resistant to fluconazole. Sequence analysis of the ITS1-ITS4 region revealed a 98% consistency with the reference sequence of ITS1-ITS4 of Phialophora verrucosa. On the basis of above findings, the patient was diagnosed with cutaneous phaeohyphomycosis. Clinical improvement was seen after treatment with oral itraconazole (400 mg/d).