Objective To observe the ultrastructure of dimorphic Penicillium marneffeiisolates from wild bamboo rats (Rhizomys pruinosus) in Guangxi region as well as from a patient with penieilliosis marneffei,and to compare their biological characteristics and anti-oxidative mechanisms.Methods Two Penicillium marneffei strains,including one isolated from wild bamboo rats (Rhizomys pruinosus) in Guangxi region and one from a patient with penicilliosis marneffei,were cultured with or without the presence of 2.0 mmol/L hydrogen peroxide in potato dextrose agar (PDA) at 25 ℃ and in brain-heart infusion (BHI) broth at 37℃ for seven days.The shape of colony and growth of both strains were observed.Light microscopy was carried out to study the morphology,and transmission electron microscopy to observe the ultrastructure,of both isolates.Results Ater incubation with hydrogen peroxide,there was a slowdown in the growth of both Penicillium marneffei isolates at both mycelial phase and yeast phase,with an increase in the production of pigment at mycelial phase at 25℃.No obvious changes were observed at 37 ℃ in the morphology of either the clinical isolate or the bamboo rat isolate when cultured with hydrogen peroxide compared with those cultured without hydrogen peroxide.Light microscopy showed attenuated spore formation by the clinical isolate when cultured at 25 ℃ with hydrogen peroxide,crenation of both isolates when cultured at 37 ℃ with hydrogen peroxide.Under a transmission electron microscope,the mycelial cells of both isolates exhibited smooth cell walls,intact cell membranes,with nuclei,mitochondria,endoplasmic reticulum,lipid body,vacuoles of various sizes in the cytoplasm at 25 ℃,and even microbodies at 37 ℃,when cultured without the presence of hydrogen peroxide.After incubation with hydrogen peroxide,the cell wall of both isolates became incomplete with defects in some areas and uneven thickness,the cell membrane discontinuous with shrinkages and projections,and the cytoplasm was inhomogeneous with obvious phagocytosis and numerous phagocytic vacuoles.Conclusions The clinical and bamboo rat isolates of Penicillium marneffei experience different biological and morphological changes under oxidative stress,hinting differences in antioxidative mechanism between them.

Objective To observe ultrastructural changes in a clinical isolate of Penicillium marneffei(PM) before and after treatment with amphotericin B or voriconazole by using scanning electron microscopy (SEM)and transmission electron microscopy (TEM). Methods A microdilution method was performed to determine the minimum inhibitory concentration (MIC)of amphotericin B and voriconazole against a clinical isolate of PM. Then, the PM isolate was treated with amphotericin B or voriconazole at their MICs and 10-fold MICs for 24, 48 and 72 hours. The ultrastructural changes in this isolate before and after the treatment were observed by using SEM and TEM. Results After the treatment with amphotericin B, SEM showed that the conidia or yeast cells of the PM isolate were gradually damaged, and their outer layers experienced detachment, shrinkage, breakage and adhesion with the increase in treatment duration and concentrations of amphotericin B; TEM also showed degenerated mitochondria, broken nuclei and cell walls, and shrunken cytoplasmic membrane with disappearance of cytoplasmic organelles. Similarly, the damage, shrinkage, shriveling and collapse of PM cells were seen by using SEM, and TEM showed many high-density electron-dense granules in cytoplasm, degeneration of mitochondria, roughening of cell wall surface, damage and shrinkage of cytoplasmic membrane, and disappearance of cytoplasmic organelles after voriconazole treatment. Conclusions Amphotericin B and voriconazole both had a strong antifungal effect on PM, and could induce evident ultrastructural changes, which were positively associated with treatment duration and concentrations. Moreover, amphotericin B caused more severe damage to PM compared with voriconazole.

ObjectiveTo investigate the relationship between HLA-DRB1 alleles and chronic urticaria with positive autologous serum skin test (ASST) in Guangxi Zhuang Autonomous Region.MethodsASST was conducted in 144 patients with chronic urticaria,who were subsequently divided into two groups according to the test result:positive group (n =62) and negative group (n =82).PCR amplification with sequence-specific primers was used to determine the genotypes of HLA-DRB1 alleles in the patients and 199 normal human controls.Chi-square test was performed to analyse the difference in the frequency of HLA-DRB1 alleles between the 3 groups by using the SPSS 13.0 statistical software package.ResultsThere were significant differences in the frequency of HLA-DRB1*01,*1401 and *16 alleles among the patients with positive and negative ASST and the controls (x2 =10.92,Pc =0.032;x2 =35.34,Pc ＜ 0.01 ;x2 =12.69,Pc =0.032).Paired comparison revealed significant differences in the frequency of HLA-DRB1*1401 allele between the patients with positive ASST and controls(RR =17.09,Pc ＜ 0.01 ) as well as between the patients with positive and negative ASST (RR =7.20,Pc ＜ 0.01).ConclusionHLA-DRB1*1401 allele may be,or be linked to,the predisposing gene of chronic urticaria with positive ASST in Guangxi Zhuang Autonomous Region.

The σC gene of ARV S1133 was designed to amplify by reverse transcription chain reaction(RTPCR).The σC gene was inserted into the vector pMD19-T,identified by PCR method and restriction enzyme,and sequenced.It showed that the insert cloned gene fragment was the σC gene of ARV.Then the gene was inserted intc the pET32a(+) and indicated that fusion expression vector pET32a-σC was constructed.The recombinant fusion protein was highly expressed in E.coli BL21 induced by 1.0 mmol/L IPTG for 5 hours in the form of inclusion bodies.The weight of recombinant fusion protein molecular is 54 000.Western-blot with ARV antibodies against the fusion protein showed the recombinant protein has a favourable reactivity.

ObjectiveTo test the susceptibility of Penicilliosis marneffei (PM) isolates from Guangxi bamboo rats and patients to voriconazole and several commonly used antifungal agents.MethodsAccording to the Clinical and Laboratory Standards Institute(CLSI) M27-A2 and M38-A document,a microdilution method was used to determine the minimum inhibitory concentration(MIC) of voriconazole,itraconazole,terbinafine,amphotericin B,and fluconazole against mycelial phase (25 ℃) and yeast phase (37 ℃) of 14 PM isolates from Guangxi Bamboo rats and 25 PM isolates from patients.The difference in MIC of the antifungals was assessed by two-sample t test between Bamboo rat PM isolates and clinical PM isolates,and by paired t test between the mycelial and yeast phase of PM isolates.Results The MIC ranges of voriconazole,itraconazole,terbinafine,amphoteriein B and fluconazole were 0.0313-0.1250,0.1250-1.0000,0.0313-0.5000,0.2500-4.0000,2.0000-8.0000 mg/L,respectively for mycelial phase of Bamboo rat PM isolates,0.0078-0.2500,0.0313-0.5000,0.0313-1.0000,0.2500-2.0000,1.0000-8.0000 mg/L,respectively for yeast phase of Bamboo rat PM isolates,0.0313-0.2500,0.0625-1.0000,0.0313-1.0000,0.2500-4.0000,2.0000-32.0000 mg/L,respectively for mycelial phase of clinical PM isolates,0.0039-0.2500,0.0313-0.5000,0.0313-2.0000,0.1250-2.0000,2.0000-16.0000 mg/L,respectively for yeast phase of clinical PM isolates.None of the PM isolates was resistant to any of the antifungals.The MIC of voriconazole was found to be the lowest for PM isolates from both Bamboo rats and patients at the same temperature (37 ℃ or 25 ℃),followed by itraconazole,terbinafine,amphotericin B and fluconazole.Statistical difference was found in the MIC values of itraconazole,terbinafine,amphotericin B between the yeast and mycelial phase of the same PM isolate,but not found in antifungal MIC values between Bamboo rat isolates and clinical isolates at the same phase.ConclusionsOf the tested drugs,voriconazole shows the strongest antifungal potency. The PM isolates from Guangxi Bamboo rats are similar to clinical PM isolates in the sensitivity to voriconazole,itraconazole,terbinafine,amphotericin B and fluconazole.The phase of PM isolates may affect their susceptibility to itraconazole,amphotericin B and terbinafine.

Objective To investigate the expression of TLR 4 and its downstream factor TNF-αin the patients with human immunodeficiency virus and Mycobacterium tuberculosis ( HIV/MTB) co-infection. Methods A total of 119 subjects including 32 patients with HIV infection (HIV group), 30 patients with HIV/MTB co-infection (HIV/MTB group), 28 patients with MTB infection (MTB group) and 29 healthy subjects ( control group ) were recruited continuously from the Fourth People′s Hospital of Nanning City , Guangxi.The expression of TLR4 in peripheral blood mononuclear cells (PBMCs) from the patients was de-termined by flow cytometry .ELISA was performed to detect TNF-αin plasma samples .The HIV-1 viral load was determined by standard method .Results The mean fluorescence intensity ( MFI) for TLR4 expression in PBMCs from HIV, HIV/MTB, MTB and control groups were 21.62±4.67, 18.29±3.87, 16.79±4.45, and 22.85±5.80, respectively, showing significant differences among four groups (F=8.105, P<0.01). The TLR4 levels in MTB and HIV/MTB groups were significantly lower than those in control group ( both P<0.01) and HIV group (P<0.01, P=0.014).The plasma concentrations of TNF-αin HIV, HIV/MTB, MTB and control groups were 15.892 (10.494-21.646) pg/ml, 13.142 (8.014-22.038) pg/ml, 16.284 (11.916-24.005) pg/ml, and 26.657 (16.321-34.541) pg/ml, respectively, that were significantly dif-ferent from each other (F=4.350, P=0.006).The levels of TNF-αin plasma from patients with HIV and HIV/MTB infection were significantly lower than those of healthy subjects (P=0.009 and P=0.001).The viral load in patients from HIV/MTB group (5.113 ±1.018 copies/ml) was significantly higher than that from HIV group (4.416±1.020 copies/ml) (t=3.449, P=0.001).Conclusion MTB infection might promote HIV replication by inhibiting the expression of TLR 4.HIV infection might increase host′s suscepti-bility to MTB infection by reducing the production of TNF-α.Suppressed expression of TLR and TNF-αpro-duction could contribute to the occurrence of HIV /MTB co-infection .

Objective To explore the expressions of Toll-like receptor 2 (TLR2 ) and the downstream proteins in patients with human immunodeficiency virus /Mycobacterium tuberculosis (HIV /M TB) co-infection .Methods A total of 119 subjects were randomly enrolled .The subjects were divided into four groups :HIV group (n = 32) ,HIV /M TB group (n = 30) ,M TB group (n = 28) and healthy control group (n= 29) .Peripheral venous blood was collected and the HIV-1 viral load was determined by standard method .The expression levels of TLR2 mRNA in peripheral blood mononuclear cells (PBMC) were determined by real-time quantitative PCR (qPCR) and mean fluorescent intensity (MFI) of TLR2 protein was detected by flow cytometry .The plasma interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels were measured with enzyme-linked immunosorbent assay kits .The data were statistically analyzed by chi-square test ,students t test ,analysis of variance and rank sum test when appropriate .Results The viral load in HIV /M TB group ([5 .113 ± 1 .018] lg copy/mL ) was significantly higher than that in HIV group ([4 .416 ± 1 .020] lg copy/mL ; t = 3 .449 , P< 0 .01) .The TLR2 mRNA expressions in PBMC
among HIV ,HIV/M TB ,M TB and healthy control groups were 1 .397 ± 0 .601 ,1 .463 ± 0 .702 ,1 .429 ± 0 .630 ,and 0 .970 ± 0 .488 ,respectively ,which was significantly different among the 4 groups (F =4 .197 , P= 0 .007) .The MFI of TLR2 protein expressions on PBMC among HIV ,HIV /M TB ,M TB and healthy control groups were 28 .12 ± 4 .55 ,38 .11 ± 11 .77 ,31 .13 ± 12 .10 and 23 .33 ± 5 .14 ,respectively . The TLR2 protein expression levels were significantly different among 4 groups (F= 13 .976 ,P< 0 .01) . The plasma IL-6 and TNF-α concentrations were significantly different among 4 groups (Z = 19 .088 , 15 .475 ,both P< 0 .01) .The IL-6 concentrations in three patient groups were higher than that in healthy control group ,but the TNF-α concentrations were lower than healthy control group .Conclusions The co-infection of HIV-1 and M TB may enhance the activation of TLR2 signaling pathway ,which leads to the increased expression of IL-6 .

Objective To evaluate the in vitro antifungal activity of osthole against Talaromyces marneffei (TM) in yeast phase,in order to provide an experimental reference for the clinical treatment of TM infection with Chinese medicine.Methods There were 20 TM strains,including 1 standard strain,2 fluconazole-spontaneously resistant strains,11 clinical isolates,and 6 isolates from wild bamboo rats.A microdilution method was used to prepare 96-well antifungal sensitivity test plates containing osthole,fluconazole,amphotericin B,itraconazole and voriconazole at different concentrations,which were incubated with (1-5) × 103 CFU/ml of tested TM strain suspensions at 37 ℃ for 48 hours.Meanwhile,TM strains cultured in the media without antifungal drugs served as positive (growth) control group,and culture media served as negative group.The minimum inhibitory concentrations (MICs) of antifungal drugs against yeasts were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M27-A2 Document).Fluconazole MIC was defined as the lowest drug concentration that resulted in ≥ 80％ growth inhibition,and MICs of other antifungal drugs were the lowest drug concentrations that resulted in 100％ growth inhibition,compared with growth control wells.Results The MICs among the quality control strains were all within the reference range,and TM grew well in the positive control wells.The MIC ranges of fluconazole,amphotericin B,itraconazole and voriconazole against TM strains were 2.0-8.0 mg/L,1.0-4.0 mg/L,0.03-0.25 mg/L and 0.06-0.25 mg/L respectively,and the MIC of fluconazole against fluconazole-spontaneously resistant strains was 128 mg/L.The MICs of osthole against the TM standard strain (FRR2161),fluconazole-spontaneously resistant strains and 1 isolate from wild bamboo rats were 16,32 and 128 mg/L respectively,and the MIC range of osthole against other 16 TM strains was 16-64 mg/L.The MICs of osthole at which 90％ and 50％ of the TM strains were inhibited were 64 and 32 mg/L respectively.Conclusion Osthole exhibits the antifungal activity against the yeast form of TM.

Objective:To evaluate the in vitro antifungal activity of berberine against Talaromyces marneffei (TM) in yeast phase. Methods:There were 21 TM strains, including l standard strain (ATCC22019), 10 clinical isolates and 10 isolates from wild bamboo rats. TM strain suspensions at a concentration of (1 - 5) × 10 3 colony-forming units/ml were incubated in microdilution plates containing difierent concentrations of berberine, fluconazole, itraconazole, voriconazole, amphotericin B or caspofungin at 37 ℃ for 48 hours. Meanwhile, the wells containing only culture media and TM strains but without antifungal drugs served as the positive control group, and those containing only culture media served as the negative control group. The minimum inhibitory concentrations (MICs) of antifungal drugs against TM yeasts were determined according to the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M27-A3 document) . Results:The MICs of the above antifungal drugs were all within the reference ranges for the quality control strain (ATCC22019), and TM strains grew well in the positive control wells. The MIC ranges of berberine, itraconazole, voriconazole, amphotericin B and caspofungin against TM strains were 32 - 64 mg/L, 0.06 - 0.125 mg/L, 0.06 - 0.125 mg/L, 1 - 2 mg/L and 16 - 32 mg/L respectively; the MIC range of fluconazole was 2 - 4 mg/L for non-resistant strains, and 128 mg/L for fluconazole-resistant clinical strains.Conclusion:Berberine exhibits antifungal activity against TM in yeast phase.

Objective To report 9 HIV-negative patients with talaromycosis marueffei (TSM)complicated by Sweet syndrome,and to analyze the relationship of the anti-interferon-γ (anti-IFN-γ)autoantibody with TSM complicated by Sweet syndrome.Methods HIV-negative patients with TSM complicated by Sweet syndrome were collected from the First Affiliated Hospital of Guangxi Medical University between 2013 and 2018.Their clinical and laboratory data were analyzed retrospectively.Meanwhile,19 HIV-positive patients with TSM and 107 health checkup examinees served as controls.Anti-IFN-γ autoantibody was detected in peripheral blood samples of the patients and controls.Results A total of 9 HIV-negative patients with TSM (5 males and 4 females) were included in this study,and the age of onset ranged from 38 to 60 years.The 9 patients all presented with disseminated infections,manifesting as long-term irregular fever,multiple lymph node enlargement,cough,emaciation and anemia.All of the 9 patients met the diagnostic criteria for classical Sweet syndrome,and microbiological examination of Sweet syndrome lesions was negative.Besides Talaromyces marneffei,6 patients also were infected with nontuberculous mycobacteria,4 with varicella-zoster virus,and 2 with Salmonella.All the 9 HIV-negative patients with TSM were positive for anti-IFN-γ autoantibody,while the 107 healthy controls and 19 HIV-positive patients with TSM were negative for anti-IFN-γ autoantibody.Conclusion Anti-IFN-γ autoantibody may be associated with HIV-negative TSM complicated by Sweet syndrome.