Objective To evaluate the in vitro activity of seven imidazole antifungals against clinical isolates of common Candida species.Methods According to the Clinical and Laboratory Standards Institute (CLSI) microdilution method M27-A3,the in vitro activity of luliconazole,ketoconazole,miconazole,econazole,clotrimazole,sertaconazole and bifonazole was determined among 183 clinical isolates belonging to 5 species of Candida.Results The minimal inhibitory concentration range was 0.03-8 (geometric mean:0.067) mg/L for ketoconazole,0.03-16 (geometric mean:0.071 ) mg/L for miconazole,0.03-8 (geometric mean:0.207) mg/L for econazole,0.03-8 (geometric mean:0.061 ) mg/L for clotrimazole,0.03-16 (geometric mean:0.187) mg/L for sertaconazole and 0.03 -＞16 (geometric mean:1.050) mg/L for bifonazole. Luliconazole exhibited a superior activity against the 5 species of Candida in vitro,with the MIC range being 0.03-8 mg/L,geometric mean MIC 0.087 mg/L,MIC50 0.06 mg/L and MIC90 0.5 mg/L,respectively.However,some Candida isolates were identified to be relatively insensitive to these tested antifungals,including luliconazole.Conclusion All the tested imidazole antifungals,except for bifonazole,show an excellent activity against Candida species in vitro,but there exist a few Candida strains with relative insensitivity.

ObjectiveTo investigate the predisposing factors and pathogenic fungal species of Malassezia folliculitis in different geographical areas and body sites.MethodsTotally,241 patients diagnosed with Malassezia folliculitis were asked to complete a questionnaire.The content of hair follicles was obtained and subjected to fungal smear and culture examination.Fungal species were identified according to morphological,physiological and biochemical features.Results Of the 241 patients with Malassezia folliculitis,204 (84.65％) were positive for smear examination.A total of 259 specimens were collected from these patients,and fungal culture grew 213(82.24％) strains,of which,209 belonged to Malassezia species,4(1.54％) to Candida species.Among the 209 Malassezia strains,186 were activated and subjected to species identification which resulted in 6 species,including M.furfur (111 strains,59.68％ ),M.sloofiae (43 strains,23.12％ ),M.sympodialis (17 strains,9.14％),M.globosa (9 strains,4.84％),M.pachydermatis (4 strains,2.15％),and M.obtuse(2 strains,1.08％ ).Of the pathogenic fungi of Malassezia folliculitis,M.furfur predominated in the chest,back,abdomen,face and neck,M.sloofiae in the upper limbs,shoulders and vertex,M.globosa in the lower limbs.There were obvious differences in the distribution of pathogenic fungal species at different body sites in a same host,and M.furfur with M.sloofiae or M.sympodialis appeared to be the most common pathogens.ConclusionsIn this study,6 Malassezia species are identified in patients with Malassezia folliculitis in Nantong and Nanjing area,M.furfur and M.sloofiae appear to be the dominant pathogens.

ObjectiveTo dynamically observe the paradoxical effect (inhibitory at low concentratin but promotive at high concentration) of caspofungin and micafungin across Candida species in vitro.MethodsA broth microdilution testing was performed following the Clinical and Laboratory Standards Institute(CLSI) M27-A2 document to observe the paradoxical effect of caspofungin and micafungin across 85 Candida strains.The growth of Candida was observed on a daily basis for 7 days.ResultsAt 48 hours,the prevalence of paradoxical growth in C.albicans,C.glabrata,C.parapsilosis,C.tropicalis,C.dubliniensis and other species of Candida was 90％,20％,41.7％,37.5％,33.3％ and 28.6％ respectively after caspofungin treatment,and 5％,0,0,25％,33.3％and 0 respectively after micafungin treatment.The concentration range of caspofungin required for the paradoxical growth of C.albicans,C.glabrata,C.parapsilosis,C.tropicalis,C.dubliniensis and other species of Candida was 4-16,8-32,8-32,2-8,2-8,8-32 μg/ml respectively,and that of micafungin for the paradoxical growth of C.albicans,C.tropicalis and C.dubliniensis was 4-16,4-32 and 1-8 μg/ml,respectively.After 48 hours,the prevalence of paradoxical growth still increased in C.parapsilosis,C.glabrata,and other species of Candida following caspofungin treatment,and in C.albicans and C.glabrata following micafungin treatment.ConclusionsThe occurrence,and time of occurrence,of paradoxical effect of echinocandins is Candida speciesand drug-specific.The prevalence of paradoxical effect is higher for caspofungin than for micafungin,which seems unrelated to their MICs against Candida species.

Objective To compare broth microdilution and agar dilution methods for in vitro testing of activities of fluconazole,ketoconazole and itraconazole against clinical Malassezia isolates.Methods Broth microdilution and agar dilution methods were used to determine the minimal inhibitory concentration(MIC)of fluconazole,ketoconazole and itraconazole for 27 clinical strains(5 species)of Malassezia.Results The minimal inhibitory concentration(MIC)ranges of fluconazole,ketoconazole and itraconazole were 0.25-≥64 mg/L,≤0.03-0.5 mg/L and ≤0.03-0.125 mg/L respectively as shown by broth microdilution method,2-≥64 mg/L,≤0.03-0.5 mg/L and ≤0.03-0.25 mg/L respectively as revealed by agar dilution method.Both methods demonstrated that itraconazole possessed the strongest activity against Malassezia species,followed by ketoconazole and fluconazole.The agreement rate in MICs between the two methods was 78.8％,85.2％ and 88.9％,respectively for fluconazole,ketoconazole and itraconazole,with the intraclass correlation coefficients (ICCs)being 0.88,0.80 and 0.76 respectively.Conclusions Fluconazole,ketoconazole and itraconazole are highly active against Malassezia species in vitro,and itraconazole is the most active.Broth microdilution and agar dilution method coincide well in,and are applicable for,the antifungal susceptibility testing of Malassezia species in vitro.

Objective To investigate the effects of ischemic postconditioning on mitochondrial permeability transition and mitochondrial transmembrane potential(△Ψm)following hepatic ischemia-reperfusion(I/R)in rats.Methods Forty male SD rats weighing 220-260 g were randomly divided into 5 groups with 8 animals in each group:sham operation group(group S);atractyloside+sham operation group(group A+S);I/R group;ischemic postconditioning group(group IPO)and atractyloside+ischemic postconditioning group(group A+IPO).The animals were anesthetized with intramuscular injection of atropine 0.05 mg/kg.Hepatic I/R was produced by occlusion of hepatic blood flow for 60 min followed by 6 h reperfusion.In group A+S,atractyloside 5 mg/kg was injected intravenously before abdomen Was closed.In group IPO,the animals were subjected to 3 cycles of 1 min reperfusion interspersed with 1 min hepatic isehemia at the end of 60 min hepatic ischemia.In group A+IPO,atractyloside 5 mg/kg was injected intravenously before reperfusion. Venous blood samples were collected for determination of serum ALT and AST activities immediately before ischemia and at 6 h of reperfusion. The animals were then sacrificed.Their livers were removed for microscopic examination, detection of apoptosis and determination of cytochrome c (Cyt c) expression, △Ψm and mitochonerial permeability transition pore (MPTP)activity. Apoptosis index (AI) was calculated. Results There was no significant difference in serum ALT and AST activities, AI, Cyt c expression, △Ψm and MPTP activity between S and A + S groups (P＞0.05). Compared with group S, serum ALT and AST activities and AI were significantly increased, Cyt c expression was up-regulated, △Ψm was decreased and MPTP activity was increased in groups I/R, IPO and A+IPO(P＜0.05).Compared with group I/R, serum ALT and AST activities and AI were significantly decreased,Cyt c expression was down-regulated, △Ψm was increased and MPTP activity was decreased in group IPO(P＜0.05), while no significant change was found in group A+IPO(P＞0.05).Compared with group IPO,serum ALT and AST activities and AI were significantly increased, Cyt c expression was up-regulated, △Ψm was decreased and MPTP activity was increased in group A + IPO(P＜ 0.05).Microscopic examination showed that hepatic injury was reduced in group IPO compared with group I/R, while aggravated in group A+ IPO compared with group IPO. Conclusion Ischemic postconditioning can protect liver from I/R injury by attenuating the I/R-induced increase in MPTP opening and decrease in △Ψm in rats.

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).

Objective To evaluate the cell-mediated immune tissue reactions of mice model of chromoblastomycosis. Methods First we developed a murine model of chromoblastomycosis subcutaneous infection with F. pedrosoi inoculated into the footpads using immunocompetent and immunocompromised mice immuned by CTX, and then by immunohistochemistry methods analyzed the expression of cytokines IL-4, IL-10, TNF-α and IFN-γ at the seventh and thirtieth day, the expression of these cytokines at the same time in the healthy mice footpads were used as control. Results In the immunocompetent mice, The expression of IL-4, IL-10 and TNF-α was significantly higher when compared with healthy control at the seventh day,showing an up-regulation pattern of Th2 and Th1 type cellular immune functions with a predominance of the Th2 response. At the 30th day, the expression of IL-10 was significantly lower when compared with the 7th day and no difference with healthy controls, while IFN-γand TNF-α were gradually increased, the T cellmediated immune drives to Th1 response from Th2. In the immunocompromised mice, the expression of IL4 and IL-10 were significantly higher, meanwhile IFN-γwas lower than those in immunocompetent or healty mice, the levels of TNF-α was not significantly different fiom healty control at the 7th day, it showed that Th2 response was more increased with the Th1 responses was significantly inhibited in the early immune reactions. At the 30th day, the Th1 type cytokines IFN-γ and TNF-α were highly significantly higher than early stage but still lower than immunocompetent levels, the level of Th2 type cytokine IL-10 rradually decreased although it was still above the other two rroups. Conclusion In different immune state, there was an immune defence translation from the predominance of Th2 type cellular immunity to Th1 type in the process of murine model of chromoblastomycosis. Thl type cytokines which favors resistance to fungal disease, played a major role at controlling the development of chromoblastomycosis.

Objective To investigate the immunization effect of influenza A/H1N1 vaccine in health care workers (HCW) in Inner Mongolia Greater Khingan Mountains area. Methods Five hundred and five HCW who received A/H1N1 influenza vaccination (immunized group) and 129 staffs who didn't receive the vaccination (unimmunized group) were randomly sampled for semiquantitative testing of serum H1N1 antibody (IgG) levels by enzyme-linked immunosorbent assay (ELISA).Results were analyzed and stratified by age, sex, occupation and the time interval between the time of vaccination and serum sample collection. The antibody positive rates of the two groups were compared by x2test. Results There were 401 (79. 4%) HCW whose H1N1 antibody were positive and 50 (9.9%) whose antibody were weak positive among 505 immunized HCW. While among 129 unimmunized HCW, there were 59 (45.7%) whose antibody were positive and 15 (11.6%) whose antibody were weak positive. The seroconversion rates of specific antibody were not significantly different among the different age groups after receiving A/H1N1 influenza vaccine (P＞ 0.05).However, there were statistical differences of the seroconversion rates among different sex groups (men 95.7% vs women 87.4% in immunized group, x2=6.40, P＜0.05; and men 73.3% vs women 52.5% in unimmunized group, x2 =4.07, P＜0.05) and different occupation groups (doctor 86.0% vs nurse 94.5% in immunized group, x2 = 9. 16, P＜0.01; and doctor 43. 8% vs nurse 75.0% in unimmunized group, x2=12.61, P＜0.01 ). The seroconversion rate was 81.5% after 80 to 89 days of vaccination, which was significantly lower than those after 30 to 39, 50 to 59 days and 60 to 69 days of vaccination, which was 100.0%, 94.7% and 93.6%, respectively (x2 =3.96, P ＜0.05; x2=7.15, P ＜0. 01; x2 = 9. 98, P＜0. 01). Conclusions A/H1N1 influenza vaccination can induce effective immune response in HCW in Greater Khingan Mountains area of Inner Mongolia. However,the level of specific antibody significantly reduces after 80 to 89 days of vaccination.