Objective To establish a diagnosis method for fungal infection using two-round PCR,and to evaluate its sensitivity in the detection of clinical specimens suspected to be infected with fungi.Methods A total of 29 specimens of clinical sputum and alveolar wash solution were collected from patients with suspicious fungal infection.All specimens uaderwent direct microscopy with 10%KOH,fungal culture,one-round PCR and two-round PCR.The fungal universal primer targeting ITS regions of rDNA was used in PCR.The detection rate for fungi was compared between these methods.Results The detection rate for fungi was 20.69%by direct microscopy,37.9%by fungal culture,17.2%by one-round PCR,and 48.3%by two-round PCR.More than one species of fungus were detected in 6.9%(2/29),3.4%(1/29)and 24.1%(7/29)of these specimens by fungal culture.one-round PCR and two-round PCR, respectively.There was a significant difference in the detection rate between two-and one-round PCR(x2=6.34,P＜0.05).With regard to the detection rate for more than one species of fungus,two-round PCR was significantly higher than one-round PCR and fungal culture(x2=4.09,6.30.bom P＜0.05).Conclusion Two-round PCR may help to improve the sensitivity of molecular diagnosis of fungus-infected specimens.

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.

Objective To investigate the effect of electron transfer system on the hyphal formation of Candida albicans. Methods Candida albicans was cultured in RPMI 1640 supplemented with 10% new-born calf serum in 5% CO2 at 37 ℃ with or without the presence of inhibitors or activators of electron transfer system. Growth curve, morphology and percent of filamentation were observed for Candida albicans. MTT assay was used to assess the viability of Candida albicans. Results The solvents (chloroform and dimethyl sulfoxide) had no significant effect on the growth of and filamentation in Candida albicans. After incubation with thenoyltrifluoroacetone (TTFA) or benzhydroxamic acid for 24 hours, yeast cells of Candida albicans predominated in the culture. The growth of Candida albicans was significantly inhibited in log phase by the incubation with classic respiratory chain inhibitors such as rotenone, antimycin A, oligomycin, sodium azide, TTFA and sodium malonate, compared with the controls (all P < 0.01). Benzhydroxamic acid, an inhibitor of alternative oxidative pathway, also significantly inhibited the growth of Candida albicans in log phase (t = 10.92, P < 0.01). After incubation with rotenone, antimycin A, oligomycin, sodium azide, TTFA, sodium malonate, benzhydroxamic acid and disodium gnanylate, the percentage of filamentation in Candida albicans at 12 hours was 87.49 ± 0.52, 48.75 ± 4.44, 50.33 ± 8.50, 99.00 ± 1.00, 1.60 ± 0.53, 94.01 ± 0.99, 0.00 ± 0.00 and 92.33 ± 2.08, respectively, and the growth of Candida albicans at 7 hours was inhibited by (1.34 ± 0.15)%, (70.61 ± 1.02)%, (50.63 ± 5.38)%, (17.80 ± 7.89)%, (45.17 ± 1.27)%, (10.75 ± 3.62)%, (72.46 ± 1.14)% and -(5.96 ± 4.07)%, respectively. Conclusions Hyphal formation of Candida albicans could be suppressed by inhibitors of classic respiratory chain or alternative oxidative pathway, and is mainly regulated by alternative oxidative pathway.

Objective To report a case of vaginal colonization due to Trichosporon inkin. Methods A 34-year-old female presented with increased vaginal discharge accompanied by abnormal odor for 2 months. Clinical laboratory examination was carried out. Cultures of vaginal discharge yielded yeast-like colony. Subsequently, the isolate underwent the following mycological examinations: purification, slide micro-culture, temperature test, urea enzyme test, biochemistry identification, antifungal susceptibility test, and gene sequencing. Results Gynecological examination revealed white homogeneous secretions attached to mucous membrane of the vagina. Nugent scores of vaginal discharge amounted to 5-6. Two rounds of culture of vaginal discharge resulted in stramineous, reductus and yeast-like colony. The isolate could grow in 42 ℃. Appressorium on the top of hypha and typical sarcinae formed in slide microculture of corn agar, and yeast malt agar was the optimal growth medium for it. Urea enzyme test was positive. API 20C AUX biochemical test and gene sequencing revealed that the isolate was consistent with Trichosporon inkin. The isolate was sensitive to amphotericin B and azoles such as clotrimazole and fluconazole, but resistant to flucytosine and caspofungin. Conclusions It is the first report of vaginal colonization due to T. Inkin in China. The accu-rate identification of T. Inkin relies on synthetic analysis of phenotype characteristics, biochemistry test and molecular sequencing.

Objective To develop a PCR-RFLP method to rapidly identify filamentous fungi causing deep infection. Methods Universal fungal primers were used to amplify the internal transcribed spacer (ITS) region of Aspergillus fumigatus, Aspergillus Bavus, Aspergillus terreus, Aspergillus niger, Aspergillus versicolor, Aspergillus nidulans, Scedosporium apiospermum and Fusarium moniliforme followed by restriction fragment length polymorphism (RFLP) analysis with restrictive endonucleases Hha I, Hae III, Hinf I, Taq I and Msp I. Then, 22 clinical and 2 environmental fungal isolates were identified with the developed PCR-RFLP method. Results The RFLP analysis of PCR products with restrictive endonucleases Hha I and Hinf I allowed discrimination of 8 filamentous fungi causing invasive infection, and it took only 1 day to carry out the whole procedure from DNA extraction to PCR and restriction digestion. The identification results of 22 clinical strains and 2 environmental isolates with this PCR-RFLP method were completely consistent with those with conventional morphological method. Conclusion PCR-RFLP analysis is an efficient method for rapid identification of cultured filamentous fungi.

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 analyze the clinical characteristics, mycology and therapeutics of 5 cases of cutaneous zygomycosis collected in recent 3 years. Methods A retrospective study was performed using clinical data on 5 cases of cutaneous zygomycosis collected in recent 3 years. Also, previous reports of this entity were reviewed. Results There were 1 male and 4 females among the 5 patients with cutaneous zygo-mycosis confirmed by mycology and/or pathology. The onset of age varied from 5 to 49 years, and course of disease from 7 months to 16 years. Of the 5 patients, 1 presented with superficial cutaneous zygomycosis, and the other 4 with gangrenous cutaneous zygomycosis; 3 had a history of trauma or surgery, 2 had no obvious inducements. Eruptions were located in the face of 2 patients and in the extremities of 3 patients. The isolate was identified as Rhizomucor variabilis in 3 cases, and species remained unclear in 2 cases. Four patients were treated by amphotericin B, and 1 by oral flueonazole as well as oral and injected itraconazole. Finally, 2 patients were healed, 1 was improved, 1 experienced no obvious improvement, and 1 died. Con-clusions Cutaneous zygomycosis is a rare severe devastating deep fungal infection. The first choice of drug is amphoteracin B for it. To improve the understanding of this disease may benefit the early diagnosis and therapy of it.

Objective To genotype Trichosporon spp. with rDNA-ITSAGSl-RFLP analysis followed by cluster analysis, and attempt to apply this method to rapid species identification of human pathogenic Trichosporon spp.. Methods Fourteen strains of Trichosporon, which belonged to 8 species, were collected. The rDNA-ITS/IGSl regions were amplified by PCR and sequenced. Simultaneously, the amplicons were digested separately with restriction enzymes, including Hae III, Hha I , Hae IH and Hha I , Hinf I , Msp I and Taq I . Results The 8 species of Trichosporon could be classified into 4 subgroups with rDNA-ITS-RFLP, while inter-species identification of all the 14 strains from 8 species of Trichosporon could be realized with rDNA-IGSl-RFLP. Also, those genotypes of T. asahii which had relative long phylogenic distance could even be discriminated with rDNA-IGSl-RFLP. Conclusion The rDNA-ITS/IGSl-RFLP analysis is expected to be used in rapid interspecific identification of genus Trichosporon.

Three cases of swimming pool granuloma are reported.Case 1:a 40-year-old female presented with a 2-month history of nodules and plaques on the right hand and forearm.She was a tropical fish salesperson but denied trauma history.Skin examination revealed multiple irregularly sized,dark-red nodules and plaques on the joints of right fingers,wrist,and elbow,as well as multiple subcutaneous nodules simulating strings of beads on the right upper limb.Case 2:a 48-year-old female presented with a 2-month history of nodules and plaques on the left hand and forearm.There was a history of trauma due to tropical fish tank and filter cleaning.Physical examination showed multiple deep purple plaques and painless subcutaneous nodules scattered on the left hand,wrist,and upper limb.Case 3:a 39-year-old male presented with a 3-month history of nodules on the fingers of both hands.There was no history of trauma,but he was a tropical aquarist.Skin examination revealed multiple soybean-sized dark-red nodules on the extensor aspect of interphalangeal joints of both hands.Fungal examinations yielded negative results in the 3 cases,while histopathology revealed infectious granuloma with a mixed inflammatory cell infiltrate.All of the cases showed positive results in purified protein derivative (PPD)skin test.Mycobacterium marinum was isolated from the lesional tissue of Case 1 and 2,but not from Case 3.All the patients were diagnosed with swimming pool granuloma,and given anti-atypical mycobacterial therapy including oral rifampin and clarithromycin.The lesions disappeared after 1 to 3 months of treatment,with the treatment course varying from 2 to 5 months.No recurrence was observed during a 3- to 12-month follow-up.

The study was designed to explore the drug-drug interactions mechanisms mediated by OATP1B1 between traditional Chinese medicine Danshensu and rosuvastatin. First, the changes of rosuvastatin pharmacokinetics were investigated in presence of Danshensu in rats. Then, the primary rat hepatocytes model was established to explore the effects of Danshensu on the uptake of rosuvastatin by hepatocytes. Finally, HEK293T cells with overexpression of OATP1B1*a and OATP1B1*5 were established using a lentiviral delivery system to explore the effects of Danshensu on the uptake of rosuvastatin. Rosuvastatin pharmacokinetic parameters of C(max0, AUCO(0-t), AUC(0-∞) were increased about 123%, 194% and 195%, by Danshensu in rats, while the CL z/F value was decreased by 60%. Uptake of rosuvastatin in the primary rat hepatocytes was decreased by 3.13%, 41.15% and 74.62%, respectively in the presence of 20, 40 and 80 μmol x L(-1) Danshensu. The IC50 parameters was (53.04 ± 2.43) μmol x L(-1). The inhibitory effect of Danshensu on OATP1B1 mediated transport of rosuvastatin was related to the OATP1B1 gene type. In OATP1B1*5-HEK293T mutant cells, transport of rosuvastatin were reduced by (39.11 ± 4.94)% and (63.61 ± 3.94)%, respectively, by Danshensu at 1 and 10 μmol x L(-1). While transport of rosuvastatin was reduced by (8.22 ± 2.40)% and (11.56 ± 3.04)% and in OATP1B1*1a cells, respectively. Danshensu significantly altered the pharmacokinetics of rosuvastatin in rats, which was related to competitive inhibition of transport by OATPJBI. Danshensu exhibited a significant activity in the inhibition of rosuvastatin transport by OATP1B1*5-HEK293T, but not by OATP1B1*1a, suggesting a dependence on OATP1B1 sequence.