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Objective To compare the diagnostic value of fluorescent staining versus KOH wet-mount microscopy in detecting superficial fungal infection.Methods Totally,600 specimens from cases of clinically diagnosed superficial fungal infections and 102 from cases of clinically diagnosed Malassezia infection (including 54 cases of pityriasis versicolor and 48 cases of Malassezia folliculitis) were collected from the dermatology clinic of Tenth People's Hospital of Tongji University between July 2017 and February 2018.These specimens were subjected to fluorescent staining and KOH wet mount separately followed by direct microscopy,and the positive rate and average time for slide reading were compared between the two methods.Culture served as the gold standard method,and the missed diagnosis rate was compared between the two methods.Statistical analysis was carried out using chi-square test or Fisher's exact test for comparing enumeration data,and paired t test for comparing emeasurement data.Results Of the 600 specimens from clinically diagnosed superficial fungal infection cases,fungi were detected in 546 (91.00％) and 489 (81.50％) by fluorescent staining and KOH wet-mount microscopy respectively (x2 =22.83,P ＜ 0.05).Fluorescent staining showed significantly shorter average reading time (73.67 ± 13.56 s)compared with KOH wet-mount microscopy (87.12 ± 15.83 s,t =14.60,P ＜ 0.05).Among the 54 specimens from pityriasis versicolor cases,fluorescent staining and KOH wet-mount microscopy positive results in 51 (94.44％) and 50 (92.59％) specimens respectively (adjusted x2 =0,P ＞ 0.05),with the average reading time being 38.36 ± 8.79 s and 41.25 ± 15.67 s respectively (t =1.14,P ＞ 0.05).Of the 48 specimens from Malassezia infection cases,43 (89.58％) and 11 (22.92％) specimens were detected to be positive for fungi by fluorescent staining and KOH wet-mount microscopy respectively (x2 =43.34,P ＜ 0.05),and fluorescent staining showed shorter average reading time (42.14 ± 12.61 s) compared with KOH wet-mount microscopy (103.56 ± 9.48 s,t =17.83,P ＜ 0.05).Among the 600 specimens from superficial fungal infection cases,culture yielded fungi in 479.Moreover,476 specimens were found positive by fluorescent staining,and 3 were found negative (0.63％),while KOH wet-mount microscopy showed 465 positive results and 14 negative results (2.92％).There was a significant difference in the missed diagnosis rate between the two methods (x2 =7.25,P ＜ 0.05).Conclusion Compared with KOH wet-mount microscopy,fluorescent staining can increase the detection rate,reduce missed diagnosis rate and shorten reading time.
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<p><b>OBJECTIVE</b>To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.</p><p><b>METHODS</b>The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration. Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry. CD4(+)T cells and CD4(+)CD25(+)CD127low Treg cells were purified from peripheral blood mononuclear cells (PBMCs) by Magnetic cell sorting. After exosomes-like vesicles cultured with CD4(+)T cells or CD4(+)CD25(+)CD127low Treg cells, cell proliferation and apoptosis were assayed. Phosphorylated β-catenin level in Wnt signaling by phosflow.</p><p><b>RESULTS</b>Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-II molecule, costimulatory molecules CD86 etc. After co-cultured with CD4(+) T cells, exosomes-like vesicles inhibited the proliferation of the T cells in a dose-dependent manner. After Treg cells cultured with exosomes-like vesicles for 14 days, the survival rate of the Treg cells was 57.07%, while that of the control Treg was 30.91%. Frizzled receptors 2, 3, 4and LRP6 gene mRNA expressed (the relative gray value was 48.50, 34.84, 23.85, 49.73) in the Treg cells by RT-PCR, and Wnt molecular expressed in exosomes-like vesicles. After Treg cells co-cultured with exosomes-like vesicles, the MFI of phosphorylated β-catenin decreased (from 20.06 ± 2.99 to 12.41 ± 2.08), and the expression of Bcl-2 mRNA was upregulated significantly (the relative gray value from 0.45 to 84.97).</p><p><b>CONCLUSIONS</b>Exosomes-like vesicles existed in human plasma and express immune regulatory molecules. They can suppress the proliferation of activated CD4(+) T cells induce their apoptosis and pro-long the survival of natural Treg cells via Wnt signaling pathway.</p>