Comparison of a glucose consumption based method with the CLSI M38-A method for testing antifungal susceptibility of Trichophyton rubrum and Trichophyton mentagrophytes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The prevalence of dermatophytoses and the development of new antifungal agents has focused interest on susceptibility tests of dermatophytes. The method used universally for susceptibility tests of dermatophytes was published as document (M38-A) in 2002 by the Clinical and Laboratory Standards Institute (CLSI), dealing with the standardization of susceptibility tests in filamentous fungi, though not including dermatophytes especially. However, it is not a very practical method for the clinical laboratory in routine susceptibility testing. In this test, we developed a novel rapid susceptibility assay-glucose consumption method (GCM) for dermatophytes.</p><p><b>METHODS</b>In this study, we investigated the antifungal susceptibilities of dermatophytes to itraconazole (ITC), voriconazole (VOC), econazole nitrate (ECN) and terbinafine (TBF) by glucose consumption method (GCM), in comparison to the Clinical and Laboratory Standards Institute (CLSI) M38-A method. Twenty-eight dermatophyte isolates, including Trichophyton rubrum (T. rubrum) (n = 14) and Trichophyton mentagrophytes (T. mentagrophytes) (n = 14), were tested. In the GCM, the minimum inhibitory concentrations (MICs) were determined spectrophotometrically at 490 nm after addition of enzyme substrate color mix. For the CLSI method, the MICs were determined visually.</p><p><b>RESULTS</b>Comparison revealed best agreement for TBF against T. mentagrophytes and T. rubrum, since MIC range, MIC50, and MIC90 were identical from two methods. However, for ITC and VOC, GCM showed wider MIC ranges and higher MICs than CLSI methods in most isolates. For ECN against T. rubrum, high MICs were tested by GCM (0.125-16 microg/ml) but not M38-A method (0.5-1 microg/ml). The overall agreements for all isolates between the two methods within one dilution and two dilutions for ITC, VOC, ECN and TBF was 53.6% and 75.0%, 57.1% and 75.0%, 82.1% and 89.3%, and 85.7 and 85.7%, respectively.</p><p><b>CONCLUSION</b>Measurement of glucose uptake can predict the susceptibility of T. rubrum and T. mentagrophytes to ECN and TBF.</p>

In vitro susceptibility testing of Aspergillus spp. against voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>During recent years, the incidence of serious infections caused by opportunistic fungi has increased dramatically due to alterations of the immune status of patients with hematological diseases, malignant tumors, transplantations and so forth. Unfortunately, the wide use of triazole antifungal agents to treat these infections has lead to the emergence of Aspergillus spp. resistant to triazoles. The present study was to assess the in vitro activities of five antifungal agents (voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin) against different kinds of Aspergillus spp. that are commonly encountered in the clinical setting.</p><p><b>METHODS</b>The agar-based Etest MIC method was employed. One hundred and seven strains of Aspergillus spp. (5 species) were collected and prepared according to Etest Technique Manuel. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 hours at 35°C. MIC(50), MIC(90) and MIC range were acquired by Whonet 5.4 software.</p><p><b>RESULTS</b>The MIC(90) of caspofungin against A. fumigatus, A. flavus and A. nidulans was 0.094 µg/ml whereas the MIC(90) against A. niger was 0.19 µg/ml. For these four species, the MIC(90) of caspofungin was the lowest among the five antifungal agents. For A. terrus, the MIC(90) of posaconazole was the lowest. For A. fumigatus and A. flavus, the MIC(90) in order of increasing was caspofungin, posaconazole, voriconazole, itraconazole, and amphotericin B. The MIC of amphotericin B against A. terrus was higher than 32 µg/ml in all 7 strains tested.</p><p><b>CONCLUSIONS</b>The in vitro antifungal susceptibility test shows the new drug caspofungin, which is a kind of echinocandins, has good activity against the five species of Aspergillus spp. and all the triazoles tested have better in vitro activity than traditional amphotericin B.</p>

Inhibitory effect of dexamethasone on myeloid differentiation factor 88 and tumor necrosis factor-alpha expressions in mouse peritoneal macrophages / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the inhibitory effect of dexamethasone (DEX) on myeloid differentiation factor 88 (MyD88) and tumor necrosis factor-alpha (TNF-alpha) expression in mouse peritoneal macrophages in innate immune response to Penicillium marneffei (PM).</p><p><b>METHODS</b>Mouse peritoneal macrophages were cultured in the presence of heat-inactivated yeast-phase PM with or without DEX, and the protein and mRNA expressions of MyD88 in the macrophages were detected using Western blotting and real-time PCR, respectively. TNF-alpha in the cell culture supernatant was measured with enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>DEX suppressed TNF-alpha production by the macrophages co-cultured with PM. The expressions of MyD88 were up-regulated by PM stimulation, whose effect was inhibited by the application of DEX.</p><p><b>CONCLUSION</b>The inhibitory effect of DEX on PM-induced proinflammatory responses of the macrophage is directly associated with the inhibition of MyD88 expression.</p>

Effect of Penicillium marneffei on TLR-2, TLR-4, and Dectin-1 expression and TNF-alpha production in macrophage / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of heat-killed Penicillium marneffei (PM) on the expressions of toll-like receptor-4 (TLR-4), toll-like receptor-2 (TLR-2) and dendritic cell associated C-type lectin-1 (Dectin-1)and the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). in mouse peritoneal macrophages.</p><p><b>METHODS</b>Mouse peritoneal macrophages were cultured in the presence of heat-killed yeast-phase PM for 24 h, and the average fluorescence intensity of TLR-2, TLR-4, and Dectin-1 in the macrophages was detected using flow cytometry. Fluorescent staining of the macrophages was performed to observe the fluorescence of TLR-2, TLR-4, and Dectin-1 with confocal microscopy. TNF-alpha mRNA in the cell culture supernatant was measured with real-time PCR, and TNF-alpha protein detected using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The average fluorescence intensity of TLR-2, TLR-4 and Dectin-1 in the macrophages was increased in response to a 24-h PM stimulation, and the stimulated macrophages produced large amounts of TNF-alpha.</p><p><b>CONCLUSION</b>PM up-regulates the expression of TLR-2, TLR-4 and Dectin-1 in mouse peritoneal macrophages, and their expressions are directly associated with macrophage activation.</p>

Differential expression of isocitrate lyase in P. marneffei phagocytized by nonstimulated and stimulated murine macrophages / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the differential expression of isocitrate lyase in Penicillium marneffei phagocytized by nonstimulated and stimulated murine macrophages, and explore the role of glyoxylate pathway in pathogenesis of Penicilliosis marneffei.</p><p><b>METHODS</b>Penicillium marneffei conidia and Raw264.7 cells were incubated in 16 cultures, which were divided to 4 groups for treatment with N-monomethyl-L-arginine (LNMMA, CI group), murine interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) (T group), IFN-gamma plus LPS and LNMMA (TI group), or the same volume of culture medium (C group). The transcriptional levels of isocitrate lyase were detected using real-time RT-PCR, and its expression levels detected biochemically.</p><p><b>RESULTS</b>The transcriptional levels of isocitrate lyase in C, CI, T, TI groups were 1.00, 1.42, 33.09, and 74.88 (P<0.05), while the expression levels were 0.06, 0.07, 0.18, and 0.93, respectively (P<0.05). The content of nitric oxide in T group was significantly higher than that in the other groups (P<0.01), but the CFU of T group was the lowest (P<0.01).</p><p><b>CONCLUSION</b>Reactive nitrogen intermediates induced by stimulated murine macrophages restrain the expression of isocitrate lyase of Penicillium marneffei and development of Penicillium marneffei, in which process the glyoxylate pathway may play an important role.</p>

Epidemiological study on pathogens of deep fungal infection / 中华检验医学杂志

Objective To investigate the dynamic distribution changes of fungi responsible for the deep infection and antifungal susceptibility to provide a basis for the empirical antifungal treatment.Methods Medical records were reviewed from cases suspectedof deep fungal infection at our hospital from January 1998 to December 2004.3122 isolates of 13 species were analyzed with SPSSll.0.Etest was used for the antifungal susceptibility test.Results Candida albicans was the most commonly isolated organism,while the prevalence of Candida albicans decreased(76.3% vs 66.8%,x~2=34.33,P

The Interaction Between Dendritic Cells and Penicillium marneffei Yeasts / 微生物学通报

To assay the influence of dendritic cells(DCs)on the function of anti-infective immunity to Penicillium marneffei.DCs were generated from peripheral blood mononuclear cells(PBMC)and pulsed with Penicillium marneffei yeasts.DCs morphology was observed by the inverted microscope and cell surface markers of DCs were analyzed by flow cytometry.The concentrations of IL-12p70 were detected by ELISA. Mixed lymphocyte reaction was performed to assay the proliferation of T cells.The mRNA of CCR7 and CXCR4 were detected by the Real-time PCR quantifications.The acquired DCs exhibited irregular appearance and numerous long dendrites under light microscope.DCs and Penicillium marneffei yeasts were co-cultured for 24 h,numerous yeasts were observed inside the cells;an enhanced expression of the cell sur-face markers CD86、CD83、HLA-DR and CD40 were demonstrated;the expression of CCR7 and CXCR4 mRNA were also increased;the improved proliferation of T cells were observed in the mixed lymphocyte reaction.Yeasts-pulsed DCs secreted more IL-12p70 than that of non-pulsed,but less than that of LPS-pulsed DCs.DCs can engulf the Penicillium marneffei yeasts.When pulsed with Penicillium marneffei yeasts,DCs improved their expression of the co-stimulatory molecules and chemokine receptor CCR7、CXCR4,enhanced their capacity to process antigen.DCs play an important role in host defense against Penicillium marneffei infection.But the low level of the IL-12p70 production may lead to deficiency in the cell-mediated immunity against Penicillium marneffei.