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Talaromyces marneffei is the only dimorphic species in its genus and causes a fatal systemic mycosis named talaromycosis. Our previous study indicated that knockdown of AcuD gene (encodes isocitrate lyase of glyoxylate bypass) of T. marneffei by RNA interference approach attenuated the virulence of T. marneffei, while the virulence of the AcuD knockout strains was not studied. In this study, T. marneffei-zebrafish infection model was successfully established through hindbrain microinjection with different amounts of T. marneffei yeast cells. After co-incubated at 28°C, the increasing T. marneffei inoculum doses result in greater larval mortality; and hyphae generation might be one virulence factor involved in T. marneffei-zebrafish infection. Moreover, the results demonstrated that the virulence of the ΔAcuD was significantly attenuated in this Zebrafish infection model.
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Gene Knockout Techniques , Hyphae , Isocitrate Lyase , Microinjections , Mortality , Rhombencephalon , RNA Interference , Talaromyces , Virulence , Yeasts , ZebrafishABSTRACT
Objective To investigate the effect of fungus Aspergillus fumigatus and Candida albicans on the expression of endocellular interferon-γ (IFN-γ) and interleukin-4 (IL-4) in cytokineinduced natural killer (NK) cells.Methods NK cells were cultured with Aspergillusfumigatus or Candida albicans by non-contact or direct-contact methods with a ratio of NK cells to fungus of 10 ∶ 1.The expressions of IFN-γ and IL-4 in NK cells were evaluated by flow cytometry after co-cultured for 6 h.Analysis of variance or SNK-q test was used to compare the expressions of IFN-γ and IL-4 among different groups.Results The IFN-γexpression rates in NK cells with direct contacting to Aspergillus fumigatus hyphae,or to different morphotypes of Candida albicans were (20.12 ± 0.53) ％,(20.69 ± 0.34) ％ and (20.8 ±0.37)％ respectively,while IFN-γexpression in NK cells with indirect contacting to fumigatus hyphae,or to different morphotypes of Candida albicans were (21.40 ± 0.53) ％,(20.57 ± 1.09) ％ and (20.20 ±0.51) ％ respectively,and all were significantly higher than that in the blank group [(15.11 ± 2.60) ％,all P ＞ 0.05].The IFN-γ expression rates in the Aspergillus fumigatus spores direct and indirect contacting groups were (14.33 ± 0.98) ％ and (14.97 ± 1.53) ％,which were not of significant difference compared with the blank group (P ＞ 0.05).The IL-4 expression rates in NK cells with direct contacting to different morphotypes of Aspergillus fumigatus and Candida albicans were (1.25 ± 0.06) ％,(1.21 ± 0.03) ％,(1.22 ± 0.46) ％ and (1.26 ± 0.11) ％,while those in indirect contacting groups were (1.21 ± 0.06) ％,(1.25 ±0.04)％,(1.27 ±0.03)％ and (1.26 ±0.1)％,which were not of significant difference compared with the blank group [(1.23 ± 0.05) ％,all P ＞ 0.05].Conclusion Fungus stimuli can reduce the secretion of IFN-γ in NK cells,but have not significant influence on the secretion of IL-4.
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Objective To evaluate the role of complement receptor 3 (CR3) on murine macrophages in the recognition of Penicillium marneffei.Methods RAW264.7 murine macrophage cells were cultured in vitro,and divided into four groups to be cocultured with inactivated and live Penicillium mameffei yeast cells as well as inactivated and live Penicillium marneffei conidia respectively at 37 ℃ in 5％ CO2 for one hour.The RAW264.7 cells incubated with phosphate-buffered saline (PBS) served as the blank control group.Then,reverse transcription-PCR was conducted to detect CR3 mRNA expression,Western blot to measure CR3 protein expression,flow cytometry to determine phagocytosis rate,enzyme-linked immunosorbent assay (ELISA) to quantify cytokine levels in culture supernatant.Some RAW264.7 macrophages were transfected with a specific siRNA targeting CR3 gene and cocultured with inactivated Penicillium marneffei conidia,subsequently,phagocytosis rate and supematant cytokine levels were determined.Data were processed by the SPSS 16.0 software,and one-way analysis of variance (ANOVA) was conducted for inter-group comparisons of these parameters.Results No significant differences were observed in the mRNA or protein expressions of CR3 among the four groups of RAW264.7 cells cocuhured with different forms of Penicillium marneffei (both P ＞ 0.05).The phagocytosis rate was 95.14％,89.56％,91.03％ and 90.78％ in RAW264.7 cells cocultured with inactivated conidia and yeast cells,as well as live conidia and yeast cells of Penicillium marneffei,respectively (P ＞ 0.05).The levels of interleukin (IL)-2,interferon (IFN)-γ,IL-4 and IL-10 in culture supernatant were increased at different degrees after one-hour coculture in the four coculture groups compared with the blank control group,but no statistical difference was noted among the four coculture groups in the supernatant levels of these cytokines (all P ＞ 0.05).After coculture with inactivated Penicillium marneffei conidia,the siRNA-transfected RAW264.7 cells showed a statistical decrease in phagocytosis rate (10.89％ vs.92.78％,P ＜ 0.05) and supernatant levels of IL-2,IFN-γ IL-4 and IL-10 compared with untransfected RAW264.7 cells.Conclusions In early stage of innate immunity,CR3 on macrophages may be one of the pattern recognition receptors participating in the recognition and mediation of phagocytosis of Penicillium marneffei.It's possible that both Thl-and Th2-type cytokines,such as IL-2,IFN-γ,IL-4 and IL-10,are involved in the immune response of macrophages against Penicillium marneffei.
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Objective To evaluate the efficacy and safety of Qingpeng ointment in the treatment of eczema.Methods A multi-center,randomized,double-blind and placebo-controlled clinical trial was conducted.A total of 246 patients with eczema were randomly assigned with a ratio of 2∶1 to the treatment group and control group to topically apply Qingpeng ointment and placebo respectively twice daily for 3 weeks.Total symptom scores were calculated for the patients at the baseline,on week 1,2 and 3 during the treatment according to the individual scores for pruritus,lesions including erythema,papules,papulovesicles or vesicles,desquamation,crusting,infiltration and lichenification.The occurrence of adverse events was recorded.Results Totally,228 patients completed the trial,including 154 patients in the treatment group and 74 patients in the control group.After 3 weeks of treatment,a statistical difference was observed in the response rate (85.71％ vs.41.89％,Z=47.16,P＜ 0.01) and cure rate (31.82％ vs.12.16％,Z=12.30,P＜ 0.01) between the treatment and control group.There was no significant difference in the incidence of adverse events between the two groups (2.48％ vs.2.56％,x2 =0,P ＞ 0.05).Conclusion Qingpeng ointment displays a promising efficacy for the treatment of mild to moderate eczema with a rapid onset and high safety.
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Objective To make a clinical and mycological analysis of tinea capitis in Guangzhou region. Methods A retrospective analysis was performed on 241 cases of tinea capitis collected from Feb, 1997 to Aug, 2010 in the Department of Dermatology, Sun Yet-sen Memorial Hospital. Results Among the 241 cases, 179 (74.27%) were tinea alba, 34 (14.11%) tinea kerion, 28 (11.62%) black dot ringworm, and no favus was observed. The dominant pathogenic fungi in decreasing order were Microsporum canis (182,80.89%), Trichophyton violaceum (25, 11.11%), Trichophyton mentagrophytes (10, 4.44%), Trichophyton tonsurans (3, 1.33%), Trichophyton rubrum (2, 0.89%), Microsporum gypseum (2, 0.89%) and Trichophyton verrucosum (1, 0.44%). Children were the main population (39.00%) suffering from tinea capitis. Conclusions In Guangzhou region, tinea alba is the most common type of tinea capitis, Microsporum canis is the main causative pathogen, and children are the predominate population affected by tinea capitis.
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Objective To evaluate the performance of nested PCR in the detection of different fungi in paraffin wax embedded tissues. Methods Forty-four tissue samples were resected from rats infected with Fonsecaea monophora, patients with chromoblastomycosis, sporotrichosis or penicilliposis marneffei followed by preparation of paraffin wax embedded tissue sections for pathological examination and DNA extraction. Nested PCR was performed by using specific primers targeting the ribosomal DNA of Fonsecaea, Sporothrix and Penicillium marneffei, respectively. The sensitivity and specificity of nested PCR were analyzed and compared with those of pathological examination. Results The nested PCR showed positive results in 8 of 20 samples from rats with chromoblastomycosis, 7 of 10 samples from patients with sporotrichosis and all of the 10 samples from patients with penicilliposis marneffei, but not in the control samples. In the detection of Fonsecaea,Sporothrix schenki and Penicillium marneffei, the sensitivity was 40% ,70% and 100%, respectively, and the specificity was consistently 100%, for the nested PCR. Pathological examination revealed fungal elements in 95%, 70% and 80% of the corresponding samples, respectively. Conclusion Detection of fungal DNA in paraffin wax embedded tissue by nested PCR can be applied to the diagnosis of deep mycosis, especially to the diagnosis of penicilliposis marneffei.
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Objective To detect the mutations in connexin genes in a family with hidrotic ectodermal dysplasia(HED)complicated by pseudo-ainhum.Methods Peripheral blood samples were collected from a 20-year-old patient with HED complicated by pseudo-ainhum,and from his unaffected sister.Total DNA was extracted from these samples,and PCR was performed to amplify the partial coding region of GJB2,GJB5 and GJB6 genes.Subsequently.PCR products were bidirectionally sequenced in both subjects.Results No mutation was detected in GJB5 or GJB6 gene in either subjects.Two mutations (V27I and V37I)were detected in the GJB2 gene in the patient but not in his sister.Conclusion The mutation in the GJB6 gene may be absent in patients with HED;there might be other genes involved in the pathogenesis.
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Objectives To identify the route and time of transmission by Candida species from mothers' vagina to their neonates' mouth.Methods Specimens for fungal cultures were obtained from vaginal discharge of mothers just before delivery and also from the mouth of their offspring just after birth.Eleven mother-infant pairs were investigated.Candida species was identified based on morphology,biochemical analysis,and sequencing of D1/D2 domain of the large subunit of ribosomal DNA (LSUrDNA).Electrophoretic karyotyping (EK) and random amplified polymorphic DNA analysis (RAPD) were performed to search for DNA homology.Results Candida isolates (16 strains) from 8 mother-infant pairs were identified as Candida albicans by 100% homology of their D1/D2 sequences with reference strain C.albicans Y-12983 (GenBank access No.U45776).Similarly,4 strains from two mother-infant pairs and 2 isolates from the other pair were identified as Candida glabrata and Candida krusei,respectively,by 100% homology in sequences alignment of the domains with reference strains,C.glabrata Y-65(U44808) and C.krusei Y-5396 (U76347).The same EK profiles were found for each C.albicans or C.krusei strain pair from both mother and her neonate.Although different EK bands with various molecular size were generated for each C.glabrata isolate pair,they were still considered to be homologous based on the fact that main EK bands were identical.Each isolate pair from mother and her infant presented almost the same RAPD profile,except for one pair,isolates F7n and F7m,which showed minor diverse DNA bands.Conclusion Eleven Candida isolates from neonates have identical molecular characteristics with their mother's isolates.Vertical transmission may be the main pathway of Candida spp.from mothers to their neonates.
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Objective To analyze the incidence of the disease, clinical features, diagnostic criteria, therapy and prognosis of Penicilliosis marneffei found in Guangdong province. Methods To analyze patients data, clinical features, laboratory findings, response to therapy, and prognosis of 15 cases Penicilliosis marneffei found in Guangdong province of China. Results The male was predominant compared with the female (ratio 2 to 1) and without occupational preference, but the patients with AIDS as underlying disease were mostly drivers and the unemployed. Thirteen patients were immunocompromised such as AIDS, connective tissue disease, and kidney transplant. Clinical features showed different manifestations, such as high fever, loss of weight, skin lesion, and respiratory system symptoms. Biopsy of the skin lesion showed PAS stain positive yeast-like, or sausage-form spores. Four patients were localized infection of the skin, eleven patients were systemic infection. Nine patients died, five recovered, 1 patient refused to be treated. Fifteen isolates from different anatomic sites of the patients were identified to be Penicillium marneffei by morphology and dimorphism in the culture, and eleven isolates among these 15 isolates were also confirmed by DNA sequence analysis. Conclusion The incidence rate of Penicilliosis marneffei become higher in the recent years and many patients were accompanied with AIDS in Guangdong province. Attention should be paid to the disease.
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Objective To investigate the relationship between the concentration of plasma (1→3)-?-D-glucan (?-D-glucan) and deep fungal infection. Methods Thirteen patients were recruited in this study, who were suspected with deep fungal infection. G-test TE reagent for ?-D-glucan measurement was used to detect the plasma (1→3) ?-D-glucan in the patients by using UV-2450 spectrophotometer at 545 nm wavelength. The final concentrations were calculated according to concentration conversion formula. Results Nine of thirteen patients were confirmed as deep fungal infection by positive tissue culture, in whom high concentrations of ?-D-glucan were detected, the highest concentration was 352.94 pg/mL (mixed infection), with a mean value of 203.47 pg/mL. In the other four patients with negative culture, the ?-D-glucan concentration was over 54.40 pg/mL in three patients and 16.16 pg/mL in the another. Our results showed that the sensiti-vity, specificity, positive predictive value and negative predictive value of this test were 92.31%, 100%, 100% and 98.36%, respectively. Conclusion G-test TE method is a simple and rapid test and may be used for the diagnosis of patients with deep fungal infection.
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Objective To investigate the effects of compound bifonazole solution for the treatment of superficial mycosis.Methods The study groups were treated with compound bifonazole solution and the control group with clotrimazole solution in a double-blind controlled clinical trial.The solutions were applied to skin lesions once a day.The course of treatment was two weeks for tinea corporis and tinea cruris and four weeks for tinea manus and tinea pedis.The patients were followed up weekly for two weeks after cessation of treatment and evaluated with regard to erythema,papule,blister,scale,keratinization and pruritus.Mycologic examinations were performed before,during and right after treatment and two weeks after treatment.Results A total of434patients participated into the study.The clinical cure rates of study group were82.25%in tinea corporis and tinea cruris,and68.75%tinea manus and tinea pedis,with a total response rates of95.85%and92.5%in tinea corporis and tinea cruris,and92.5%in tinea manus and tinea pedis,respectively.The clinical cure rates of control group were58.6%in tinea corporis and tinea cruris,and44.7%in tinea manus and tinea pedis,with a total response rates of83.0%and87.2%in tinea corporis and tinea cruris,and in tinea manus and tinea pedis,respectively.The MICs to350clinical isolates of pathogenic fungi were1.6~2.5mg/L for compound bifonazole solution,and3.125~25mg/L for clotrimazole solution.Conclusions Compound bifonazole solution is a high-effective,broad-spectrum anti-fungal agent.It is keratolytic,well permeable and safe for relatively long term application.
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The incidence of invasive fungal infections has increased dramatically in recent years.However,the traditional methods used in routine practice for the diagnosis of invasive fungal infections could not meet the requirements.The detection of specific fungal antigens by serological methods will have a widely applications in near future.Fungal DNA detecting by molecular biological technique is the most promising technique and it will make high speed selecting to clinic specimens possible.
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Objective To elucidate the clinicopathological characteristic, differential diagnosis, treatment and prognosis of systemic amyloidosis. Methods An inpatient diagnosed as systemic amyloidosis was analyzed for clinical and pathological features as well as laboratory findings. The related literature was reviewed. Results The patient was confirmed to have amyloidosis of the muscle. Muscle involvement was the most prominent and first manifestation, and the patient had widespread visceral involvements, which included cardiovascular system, kidney, respiratory as well as gastrointestinal tracts and tongue. The biopsy of the muscle, mucosa of stomach and intestine, and cutaneous tissue revealed amyloid material deposited in the skeletal and smooth muscle as well as vessel walls. Conclusion Amyloid myopathy is a rare manifestation in systemic amyloidosis. Skeletal muscle weakness and stiffening may be an important clue to the diagnosis of systemic amyloidosis.
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Objective To report a case of chromomycosis caused by Phialophora verrucosa and explore the laboratory features of the pathogen. Methods Skin lesion was examined by histopathology and fungus culture. The morphology of the isolate was observed by microscopy and scanning electron microscopy. The coenzyme Q system of this isolate was analyzed by HPLC assay. The DNA sequences of LSU rDNA D1/D2 region of this isolate and a standard fungus strain were compared. Results The initial lesion was an erythematous papule that subsequently developed into one or multiple coalescing warty papules or plaques slowly. The bronze-colored spores could were observed in the dermis or macrophages. The isolate grew very slowly, requiring 4 weeks of incubation. Microscopically, no characteristic structures were found on Sabourand′s dextrose agar, while there were vase-like structures, which were referred to as phialides on potato dextrose agar (PDA) and corn meal agar I (CMA-I). The phialides on PDA mostly grew at the top of hypha, but on CMA-I they mostly grew on the side of hypha. The isolate contained coenzyme Q-10, and its DNA sequence of LSU rDNA D1/D2 region completely consistent with those of the standard strain. Conclusion Chromomycosis caused by Phialophora verrucosa is rare in China. It can be diagnosed by fungus culture and histopathological examination. Coenzyme Q system analysis and DNA sequencing can exclude the interference from different phenotypes.