Identification of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> in Paraffin-Embedded Tissue Using Nested PCR

Penicillium marneffei is one of the unique thermally dimorphic fungi in Penicillium species that causes a disseminated, progressive and life threatening infection in immunocompromised patients. The diagnosis of Penicilliosis marneffei depends on culture that may delay the treatment due to the time-consuming process. In the present study, we evaluated the specificity and sensitivity of nested PCR to identify Penicillium marneffei from paraffin-embedded tissue. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA of Penicillium marneffei. The outer primers (RRF1 and RRH1) were specific to fungi. The inner primers (Pm1 and Pm2) were specific to Penicillium marneffei. The specific fragment of approximately 400 bp was amplified from all paraffin-embedded tissues from 14 patients with Penicilliosis marneffei and 10 bamboo rats. The detectable DNA concentration of single PCR and nested PCR were 14 pg/μl and 14 fg/μl, respectively. Further studies are required in order to use nested PCR for early diagnosis of the disease.     

Random amplified polymorphic DNA (RAPD) analysis ofPenicillium marneffei strains isolated from AIDS patients in Thailand

Results of random amplified polymorphic DNA (RAPD) analysis using three different primers showed that 16 strains ofPenicillium marneffei isolated from AIDS patients in Thailand belonged to a genetically homogenous group, but different slightly from an isolate from bamboo rat in China. Six PCR fragments (from about, 200 to 600 bp) that were commonly observed in the RAPD fingerprint of all strains were extracted and sequented. Usefulness of this sequence information for identification ofP. marneffei is discussed.     

The mitochondrial genome of the thermal dimorphic fungus Penicillium marneffei is more closely related to those of molds than yeasts

We report the complete sequence of the mitochondrial genome of Penicillium marneffei, the first complete mitochondrial DNA sequence of a thermal dimorphic fungus. This 35 kb mitochondrial genome contains the genes encoding ATP synthase subunits 6, 8, and 9 (atp6, atp8, and atp9), cytochrome oxidase subunits I, II, and III (cox1, cox2, and cox3), apocytochrome b (cob), reduced nicotinamide adenine dinucleotide ubiquinone oxireductase subunits (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), ribosomal protein of the small ribosomal subunit (rps), 28 tRNAs, and small and large ribosomal RNAs. Analysis of gene contents, gene orders, and gene sequences revealed that the mitochondrial genome of P. marneffei is more closely related to those of molds than yeasts.     

Genomic and experimental evidence for a potential sexual cycle in the pathogenic thermal dimorphic fungus Penicillium marneffei

All meiotic genes (except HOP1) and genes encoding putative pheromone processing enzymes, pheromone receptors and pheromone response pathways proteins in Aspergillus fumigatus and Aspergillus nidulans and a putative MAT-1 alpha box mating-type gene were present in the Penicillium marneffei genome. A putative MAT-2 high-mobility group mating-type gene was amplified from a MAT-1 alpha box mating-type gene-negative P. marneffei strain. Among 37 P. marneffei patient strains, MAT-1 alpha box and MAT-2 high-mobility group mating-type genes were present in 23 and 14 isolates, respectively. We speculate that P. marneffei can potentially be a heterothallic fungus that does not switch mating type.     

Rapid identification and characterization of Penicillium marneffei using multiplex ligation-dependent probe amplification (MLPA) in paraffin-embedded tissue samples

Penicillium marneffei infection is a deadly disease and early diagnosis leads to prompt and appropriate antifungal therapy. To develop a sensitive method to diagnose P. marneffei infection, a multiplex ligation-dependent probe amplification (MLPA) assay was adapted. This method can rapidly and specifically detect P. marneffei DNA in cultured cells and paraffin-embedded tissue samples. Three pairs of probes were designed for amplifying the internally (intergenic) transcribed spacer (ITS) region of P. marneffei rRNA using a systematic phylogenetic analysis. These three probe sets produced three amplicons of 198, 166, and 152 bp, respectively, specific for P. marneffei. In contrast, there was only one 198 bp amplicon produced for Talaromyces stipitatus, and one 152 bp amplicon for P. funiculosum, T. intermedius and T. derxii. The probes did not amplify any other reference strains. An array of 40 P. marneffei strains isolated from human patients, bamboo rat, and the local environment was tested by using MLPA, and all were positively identified. Most importantly, P. marneffei in paraffin-embedded tissue specimens from infected human patients was positively amplified by MLPA. The sensitivity and specificity of the MLPA assay could be a useful tool for prompt diagnosis, pathogen characterization, and epidemiological studies of fungal infections.     

The Penicillium commune Thom and Penicillium clavigerum Demelius Fungi Producing Fumigaclavines A and B

The type strains Penicillium clavigerum VKM F-447 and P. commune VKM F-3233 are found to produce fumigaclavines A and B. Of the seven other strains of these species, only two strains, P. commune VKM F-3088 and F-3491, possess the ability to synthesize these alkaloids. It is suggested that the five other strains under study either lost such an ability or require very specific conditions for the synthesis of these alkaloids.     

马尔尼菲青霉的分类学研讨

马尔尼菲青霉能引起人和鼠类的马尔尼菲青霉病,1956年由Capponi等分离自越南的竹鼠,后由Segretain (1959)正式发表为新种.DiSalvo等(1973)首次报道此菌对人的自然感染,也分离了菌种。Pitt (1980'1979')承认Segretain的菌株为马尔尼菲青霉,但把DiSalvo的菌株(ATCC 24100)由于具明显的黄色菌丝而与岐生青霉金黄色变种(ATCC 10438)合并,成立了樱草黄青霉新种。近年来一些中国的研究者对广西的竹鼠作了大量的调查,证明竹鼠普遍携带此菌,并分离了大量菌株。作者对来自广西的34株分离物(2株来自患者,32株来自银星竹鼠)根据形态和培养特征作了鉴定,并用IMI 68794(来自模式),ATCC 24100和ATCC 10438作了对比。全部34株分离物尽管在菌落外观有明显不同(许多菌株产生黄色具饰菌丝,影响菌落外观)但根据其帚状枝及瓶梗的特征,产生紫红色素以及在370C形成酵母状细胞诸特点,作者认为都是马尔尼菲青霉。ATCC 24100也同样具有马尔尼菲青霉的特征。而ATCC 10438虽然也产生明显的黄色菌丝,但其帚状枝和瓶梗与马尔尼菲青霉不同,不产生紫红色素,在370C不生长,因此不是同一个种。ATCC 24100应保持原来的种名马尔尼菲青霉。     

Exocellular hydrolases from Penicillium ulaiense

Penicillium ulaiense exhibited carboxymethylcellulase, pectinase, protease, amylase and phenolase activities, while no xylanase, cellulase, lipase or ligninase activities were found. Pectinolytic action was studied in liquid medium, showing low levels of pectinesterase and pectinase production. No mycotoxins were detected by thin-layer-chromatography.The authors are with INIQUI, Universidad Nacional de Salta, Buenos Aires 177, 4400 Salta, Argentina     

Production and partial purification of naringinase by Penicillium decumbens PTCC 5248

Penicillium decumbens PTCC 5248 produced naringinase when grown in a medium contained naringin as a source of carbon. Rhamnose also induced production of naringinase. Prunin disappeared as the time of enzymatic reaction increased. On fractionation with isopropanol 24-fold purification was achieved. Optimum pH and temperature for naringinase activity were determined to be 4.5 and 55 °C respectively. The K_m value of the enzyme with respect to naringin was found to be 1.7 mM. Citric acid, glucose, Ca²⁺, Mg²⁺, Zn²⁺ all inhibited naringinase activity.     

不同交配型马尔尼菲篮状菌的抗真菌药物敏感性

马尔尼菲篮状菌是马尔尼菲篮状菌病的致病菌,具有不同交配型,且交配型分布具有地域差异.交配型是影响某些真菌药物敏感性的因素之一,但是否影响马尔尼菲篮状菌的药物敏感性不详.为了解马尔尼菲篮状菌交配型和其药物敏感性的关系,本研究检测了不同交配型马尔尼菲篮状菌对7种抗真菌药物的敏感性.结果显示,与MAT-1型马尔尼菲篮状菌相比...     

Pectinase production by Penicillium frequentans

High activities of extracellular pectinase with viscosity-diminishing and reducing groups-releasing activities were produced by Penicillium frequentans after 48 h at 35°C, in agitated cultures supplemented with 0.5% citrus pectin and initial pH of 2.5. Under these conditions the fungus also produced high activity of pectinesterase. At an initial pH of 7.0 or 8.0, pectin lyase activity was also detected. Enzyme activity releasing reducing sugars was more stable at 50°C than viscosity-diminishing activity. Both activities were maximal at pH 2.5 to 5.2 and at 55°C.The authors are from the Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café, s/n^o, Bairro Monte Alegre, 14.049 Ribeirão Preto, S.P., Brazil.     

Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

树突状细胞与马尔尼菲青霉酵母体外相互作用的研究

本文探讨了树突状细胞(DCs)在抗马尔尼菲青霉感染免疫中的作用.用细胞因子rhGM-CSF和rhIL-4诱导人外周血单核细胞分化为树突状细胞,观察DCs的形态,并用流式细胞仪进行DCs的表型测定,ELISA方法检测培养上清液IL-12p70的浓度,混合淋巴细胞反应检测DCs刺激T淋巴细胞的增殖能力.实时荧光定量PCR检测趋化因子受体CCR7、CXCR4的mRNA 的表达.倒置显微镜下可见诱导获得的DCs细胞形态不规则,表面伸展出大量树突.与马尔尼菲青霉酵母共同培养24 h后DCs的胞内含有大量的酵母细胞;细胞表型CD86、CD83、HLA-DR和CD40的表达明显增高;刺激T淋巴细胞增殖的能力增强;趋化因子受体CCR7和CXCR4的mRNA表达量增加且能够产生IL-12p70但产生的量低于LPS刺激组.DCs能吞噬加热灭活的马尔尼菲青霉酵母,并趋于成熟,抗原呈递能力增加,但是产生IL-12p70的量较低,可能造成宿主抗马尔尼菲青霉酵母的细胞免疫功能的不足.     

树突状细胞与马尔尼菲青霉酵母体外相互作用的研究

本文探讨了树突状细胞(DCs)在抗马尔尼菲青霉感染免疫中的作用。用细胞因子 rhGM-CSF 和rhIL-4诱导人外周血单核细胞分化为树突状细胞, 观察DCs的形态, 并用流式细胞仪进行DCs的表型测定, ELISA方法检测培养上清液IL-12p70的浓度, 混合淋巴细胞反应检测DCs刺激T淋巴细胞的增殖能力, 实时荧光定量PCR检测趋化因子受体CCR7、CXCR4的mRNA的表达。倒置显微镜下可见诱导获得的DCs细胞形态不规则, 表面伸展出大量树突。与马尔尼菲青霉酵母共同培养24 h后DCs的胞内含有大量的酵母细胞; 细胞表型CD86、CD83、HLA-DR和CD40的表达明显增高; 刺激T淋巴细胞增殖的能力增强; 趋化因子受体CCR7和CXCR4的mRNA表达量增加且能够产生IL-12p70但产生的量低于LPS刺激组。DCs能吞噬加热灭活的马尔尼菲青霉酵母, 并趋于成熟, 抗原呈递能力增加, 但是产生IL-12p70的量较低, 可能造成宿主抗马尔尼菲青霉酵母的细胞免疫功能的不足。     

电穿孔介导马尔尼菲青霉转化体系的建立和优化

目的 建立和优化马尔尼菲青霉电穿孔转化体系,为其基因功能研究提供良好平台.方法 直接使用马尔尼菲青霉缺陷株SPM4（pyrG-,niaD-）萌发孢子进行电穿孔转化,并通过改变孢子龄、孢子萌发时间、质粒浓度、电场强度等影响因素对体系进行优化.结果 适合SPM4电穿孔转化条件为：孢子龄为6d,孢子萌发时间为4h,电场强度为5 kV/cm.上述条件下分别使用1 μg环状或线性化质粒DNA转化SPM4,平均可得到21个和13个转化子.结论 马尔尼菲青霉电穿孔转化效率高,重复性好,针对SPM4萌发孢子,环状质粒较线性化质粒电穿孔转化效率高.     

Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

Penitrem and Thomitrem Formation by Penicillium crustosum

The levels penitrem A, B, C, D, E, F, roquefortine C and thomitrem A and Erecovered from extracts of 36 Norwegian, 2 American and one each of Japanese,German, South African, Danish and Fijian isolates of Penicillum crustosumThom were quantitatively determined using high performance liquid chromatography-mass spectrometry (HPLC-MS). Forty-two of the 44 isolates of penitrem-producing isolates grown on rice, afforded levels of thomitrem A and E comparable to that of penitrem A. Thomitrems A and E were also found, but at lower levels, when cultures were grown onbarley. No thomitrems were found when the isolates were grown on liquid media. Theeffects of time and temperature on mycotoxin formation were studied on rice over a 4week period at 10, 15 and 25 °C, respectively. No mycotoxins could be detectedafter 1 week at 10 °C, but after 2 weeks at 10 °C levels were similar tothose produced at 15 and 25 °C. Higher levels of thomitrems A and E weredetected when media were maintained at lower pH. The possibility that thomitremsA and E might be derived by acid promoted conversion of penitrems A and E wasexplored in stability trials performed at pH 2, 3, 4, 5 and 7 in the presence and absenceof media. Thomitrems were formed at pH 2, 3 and 4 but not at pH 5 and 7.     

结核分枝杆菌吡嗪酰胺耐药性检测方法的研究进展

吡嗪酰胺(pyrazinamide,PZA)是重要的一线抗结核药物,与异烟肼、利福平和乙胺丁醇构成治疗方案的核心。因其疗效较好,被广泛应用于结核病的治疗过程。然而,近年来随着耐多药结核病的出现,PZA耐药导致部分患者治疗失败,因此常规开展PZA药物敏感性试验对于减少耐药性的发生显得极为重要。由于PZA在酸性条件下才能发挥作用,而结核分枝杆菌在酸性环境下生长不良,故PZA耐药性检测一直是临床中的难题。本文结合国内外最新研究进展,就结核分枝杆菌PZA耐药性检测方法的研究进行阐述,期望能为更有效地诊治结核病提供新思路。