A 5-year-old Mongolian girl from Inner Mongolia presented with a painless,round skin lesion on the left cheek for 10 months.Several weeks prior to the development of lesions,her left cheek was seratched by a dog.Subsequently small asymptomatic erythematous papules developed at the scratched site and gradually enlarged.Direct microscopy of scales from the lesions revealed septate,branching hyphae and fungal culture grew Eurotiom amstelodami(perfect stage of aspergillus).She was initially diagnosed as cutaneous aspergillosis and treated with itraconazole 200 mg per day for 2 months,but limited improvement was achieved.Histopathological examination with Wright-Giemsa staining of skin biopsies revealed abundant leishman Donovani hodies intracellulatry and extracellularly in the dermis and subcutaneous tissue; PAS staining of tissue specimens showed no fungal element and fungal culture was negative.She was finally diagnosed with cutaneous leishmaniasis.After treatment with terbinafine 125 mg per day for 2 months,the lesion subsided.

Objective To evaluate in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against Exophiala dermatitidis (E.dermatitidis).Methods The minimum inhibitory concentrations (MICs) of itraconazole and terbinafine against 12 strains of E.dermatitidis were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M38-A2 Document).A broth microdilution checkerboard method was used to evaluate the in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against E.dermatitidis.Results The MIC ranges of terbinafine and itraconazole against E.dermatitidis were 0.060-0.125 mg/L and 0.5-1 mg/L,respectively.The combination of tacrolimus with terbinafine showed synergistic inhibitory effects against 5 strains of E.dermatitidis,while the combination of tacrolimus with itraconazole revealed synergistic effects against 10 strains of E.dermatitidis.No antagonism was observed in either of the two combinations.Conclusion In vitro combination of tacrolimus with itraconazole or terbinafine can enhance the antifungal activity of itraconazole or terbinafine against E.dermatitidis.

Objective To investigate the action mechanism of glutamate-mediated signaling pathway in malignant melanoma. Methods WM451LU melanoma cells in log phase were classified into 6 groups, negative control group treated with PBS (100 μl), MK801 group treated with the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (100 μmol/L), CPCCOEt group treated with non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonist CPCCOEt, MAP2 group transfected with adenovirus vector containing microtubule associated protein 2a (Ad-MAP2a), MK801 + MAP2 group treated with MK801 of 100 μmol/L and transfected with Ad-MAP2a, CPCCOEt + MAP2 group treated with CPCCOEt of 10 μmol/L and transfected with Ad-MAP2a. Western blot was performed to detect the expression of an ionotropic glutamate receptor, i.e., N-methyl-D-aspartate receptor type 2A (NMDAR2A) in WM451LU cells transfected with Ad-MAP2a. Scratch motility assay and cell invasion assay were conducted in vitro to detect the changes in migration and invasion ability of WM451LU cells after treated with Ad-MAP2a, MK-801, CPCCOEt alone or in combination. In vivo study was carried out to compare the inhibitory effect of the above treatments on melanoma. Results Western blot revealed a decrease in the expression of NMDAR2A in WM451LU cells after transfected with Ad-MAP2a. The scratch motility assay showed that the number of migrating cells per high power field was 117.04 ± 2.76 in MAP2 group,107.64 ± 6.50 in MK801 group,97.36 ± 4.79 in CPCCOEt group, 43.28 ± 3.02 in MK801 + MAP2 group,30.76 ± 3.97 in CPCCOEt + MAP2 group,significantly different from that in the negative control group (152.3 ± 5.75,all P ＜ 0.01 ). Cell invasion assay demonstrated that the average number of invading cells per high power field in the negative control was significantly higher than that in MAP2 group, MK801 group, CPCCOEt group, MK801+MAP2 group and CPCCOEt + MAP2 group (170.43 ±8.72 vs. 98.26 ± 3.84, 97.22 ± 5.54, 112.23 ± 7.21, 42.89 ± 5.06, 58.25 ± 6.68, P ＜ 0.05, 0.05, 0.05, 0.01and 0.01, respectively).A significant decrease was observed in the average volume of experimental melanoma in mice of MAP2 group, MK801 group, MK801 + MAP2 group, CPCCOEt group and CPCCOEt + MAP2 group compared with the negative control group (224.02 ± 46.19 mm3, 160.33 ± 33.91 mm3, 91.49 ± 21.48 mm3,202.30 ± 52.37 mm3, 111.13 ± 69.81 mm3 vs. 342.70 ± 60.92 mm3, all P ＜ 0.01 ). Conclusions To block the glutamate signaling pathway in vitro can inhibit the invasion and migration of melanoma cells, and to block the pathway in vivo can inhibit the growth of malignant melanoma and alter the morphology of melanoma cells.

Objective To investigate the roles of glutamate signaling pathway in the activation of human peripheral blood lymphocytes(PBLs) and pathogenesis of vitiligo.Methods Peripheral blood lymphocytes (PBLs) isolated from 5 patients with generalized vitiligo and 5 healthy controls were cultured in vitro.Flow cytometry was performed to quantify the expression of CD25 and interferon-γ on PBLs derived from healthy controls and treated with MK801 (a non-competitive antagonist of N-methyl-D-aspartic acid receptor,NMDAR) at 100 μmol/L or phosphate buffered saline (PBS) for 48 hours,as well as the level of reactive oxygen species (ROS) in the controlderived PBLs treated with MK801 at 100 μmol/L,NMDA (an agonist of N-methyl-D-aspartic acid receptor) at 0.5 mmol/L or PBS for 48 hours.The protein and mRNA expressions of NMDAR1 and NMDAR2A were measured by flow cytometry and real-time PCR,respectively,in PBLs from the healthy controls and vitiligo patients.Immunohistochemistry was used to observe the expressions of NMDAR1 and NMDAR2A in tissue specimens from depigmented and postinflammatory hyperpigmentation lesions of the patients with vitiligo and from normal skin of the healthy controls.Results Compared with the PBS-treated PBLs from the healthy controls,the MK801-treated PBLs showed a downregulated expression of CD25 (7.28％ ± 0.18％ vs.16.02％ ± 0.42％,P ＜ 0.01),but an upregulated proportion of CD25+IFN-γ+ lymphocytes (1.79％ ± 0.09％ vs.0.78％ ± 0.06％,P ＜ 0.01),and the NMDA-treated PBLs displayed a higher ROS level (101.1 ± 3.50 vs.69.80 ± 2.08,P＜ 0.01 ).The protein expression of NMDAR1 in PBLs was significantly higher in vitiligo patients than in the healthy controls (3.85 ± 2.17 vs.0.97 ±0.55,P ＜ 0.05).Conclusion Glutamate signaling pathway may be involved in the immunopathogenesis of vitiligo via affecting the secretion of interferon-γ by,and ROS level in,activated lymphocytes.

Objective To investigate the value of Real-time PCR in the diagnosis of invasive aspergillosis(IA) and to compare it with galactomannan antigen assays.Methods A retrospective study was performed on 110 episodes of hospitalization of 88 patients who were at risk of invasive aspergillosis at Peking University First and Renmin Hospital from May 2008 to December 2010.23 cases with diagnosis and clinical diagnosis IA were classified as infection group and 87 cases with suspected diagnosis and non-IA were classified as non-infeciton group according to the international criterion.Real-time PCR and gaiactomannan antigen detections were performed on 257 serum samples.A receiver operating characteristic curve (ROC)was developed based on the quantitative cycle numbers and an optimal cut off value of quantification cycle (Cq) was determined.The sensitivity (Se),specificity (Sp),positive predictive value (PPV) and negative predictive value (NPV) were calculated under different considerations among which McNemar chi-square tests were used for statistical analyze.Results The area under ROC curve of Real-time PCR for the diagnosis of IA was 0.91 (95％ CI:0.825-0.995) and the optimal cut off value of Cq was 39.45.The Se and Sp of one positive PCR,two positive PCR,one positive GM and two positive GM were 87.0％,79.3％ ; 58.3％,97.8％ ; 78.3％,63.2％ ; and 58.3％,82.6％,respectively.When one positive PCR was considered as the diagnostic criterion of IA,Real-time PCR was able to diagnose 100％ and 84.2％ of proven and probable IA cases,respectively.The Sp of one/two positive PCR were statistically higher than one/two positive GM (P ＜ 0.05),respectively.The Sp of two positive PCR was statistically higher than one positive PCR (P ＜0.05).The Se and Sp were 65.2％,89.7％ and 100％,52.3％ for one positive PCR combined one positive GM and one PCR or GM positive,respectively.Conclusions Real-time PCR assays have better sensitivities and specificities than GM in the diagnosis of invasive aspergillosis.When two PCR positive were considered,better specificity and positive predictive value were achieved.

Objective To evaluate the in vitro antifungal activity of 4 antifungal agents alone or in combination against Exophiala dermatitidis (E.dermatitidis) biofilms.Methods E.dermatitidis biofilms were prepared by using a modified 96-well plate-based method.The in vitro antifungal activity of amphotericin B,voriconazole,itraconazole and caspofungin alone or in combination against E.dermatitidis biofilms were investigated via the broth microdilution checkerboard technique.Results The sessile minimum inhibitory concentration ranges resulting in 50％ (SMICS0) and 80％ inhibition (SMIC80) of E.dermatitidis biofilms were all ＞ 32 mg/L for itraconazole,voriconazole and caspofungin,and the SMIC50 and SMIC80 ranges of amphotericin B were 1-2 mg/L and 4-8 mg/L respectively.The combination of amphotericin B with voriconazole showed synergistic inhibitory effects against E.dermatitidis biofilms,while the combination of amphotericin B with itraconazole or caspofungin,as well as the combination of voriconazole with caspofungin,revealed no synergistic effects.No antagonistic effect was observed in any of the combinations.Conclusion Amphotericin B appears more active against E.dermatitidis biofilms,and the combination with voriconazole can enhance the anti-biofilm effects against E.dermatitidis biofilms.

Objective To evaluate the in vitro effects of photodynamic therapy alone or in combination with antifungal agents on the apoptosis of planktonic and biofilm cells of Exophiala dermatitidis (E.dermatitidis).Methods The planktonic suspensions of E.dermatitidis were prepared,and the biofilms of E.dermatitidis were prepared via a modified 96-well plate-based methods.Planktonic and biofilm cells of E.dermatitidis were separately divided into several groups:antifungal agent groups treated with antifungal agents alone,photodynamic therapy group receiving photodynamic therapy alone,combination groups receiving photodynamic therapy followed by the treatment with antifungal agents,and blank control group receiving no treatment.These antifungal agents included amphotericin B,posaconazole,voriconazole and itraconazole.The concentrations of these antifungal agents were all 1 mg/L,and the treatment with antifungal agents lasted 2 hours.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect the apoptosis of planktonic and biofilm cells of E.dermatitidis in all the groups.Results The antifungal agents and photodynamic therapy both affected the apoptosis of planktonic (both P ＜ 0.001) and biofilm cells (beth P ＜ 0.05) of E.dermatitidis.The apoptosis rates of E.dermatitidis planktonic cells in the control group,amphotericin B group,posaconazole group,voriconazole group and itraconazole group were 11.67％ ± 0.21％,13.30％ ± 1.78％,14.30％ ± 3.61％,14.51％ ± 1.91％and 36.17％ ± 4.00％ respectively.The apoptosis rate of E.dermatitidis planktonic cells was significantly higher in the itraconazole group than in the control group (P ＜ 0.05),but no significant differences were observed between the other 3 antifungal agent groups and control group (all P ＞ 0.05).The photodynamic therapy group also showed a significantly higher apoptosis rate of E.dermatitidis planktonic cells (41.37％ ±7.80％) compared with the control group (P ＜ 0.05).After the treatment with photodynamic therapy combined with amphotericin B,posaconazole,voriconazole or itraconazole,the apoptosis rates of E.dermatitidis planktonic cells were 29.23％ ± 6.71％,37.23％ ± 10.86％,43.57％ ± 6.42％ and 69.87％ ± 3.53％ respectively.Moreover,the photodynamic therapy + voriconazole group and photodynamic therapy + itraconazole group both showed significantly higher apoptosis rates compared with the voriconazole group and itraconazole group respectively (both P ＜ 0.05).The apoptosis rate of E.dermatitidis biofilm cells was significantly higher in the photodynamic therapy group than in the control group (32.00％ ± 0.43％ vs.25.30％ ± 1.31％,P ＜ 0.05),as well as in the photodynamic therapy + amphotericin B than in the amphotericin B group (P ＜ 0.05).Conclusion Photodynamic therapy combined with antifungal agents can markedly promote the apoptosis of planktonic and biofilm cells of E.dermatitidis.

To analyze the clinical characteristics and drug resistance in patients with non-tuberculous mycobacteria (NTM) pulmonary disease in Changsha Central Hospital of Hunan Province in recent three years.
 Methods: The clinical data of 153 patients with NTM pulmonary disease, who were diagnosed in Changsha Central Hospital of Hunan Province from February 2014 to May 2017, were retrospectively analyzed. According to the concentration of drug sensitivity test, the patients were divided into a low concentration group and a high concentration group. The status of drug sensitivity and drug resistance were examined.
 Results: Among 153 patients, 79 patients (51.63%) were male, 74 patients (48.37%) were female. The mean ages were (60.27±19.46) years. The NTM pulmonary disease mainly occurred in the individuals with bronchiectasis, and the course of disease was long (mean 7.8 years). The clinical symptoms were not specific and mostly misdiagnosed as pulmonary tuberculosis (92.81%). Mycobacterium avium-intracellulare (56.21%) and mycobacterium chelonae-abscess (20.92%) were the majority. The drug-resistance rate of the first-line and second-line anti-tuberculosis drugs was high. The majority was resistant to more than eight drugs, 38.56% patients in the low concentration group were resistant to total drugs, and 25.49% patients in the high concentration group were resistant to total drugs.
 Conclusion: The NTM pulmonary disease is easily misdiagnosed, and the drug resistance rate is high. Identification of mycobacterium species and detection of drug sensitivity play an important role in clinical diagnosis and treatment.
Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium Infections, Nontuberculous ; Nontuberculous Mycobacteria ; Retrospective Studies