8种抗真菌药物对浅表真菌病致病菌株体外敏感性检测

目的确定8种抗真菌药物对100株浅表致病真菌的体外药物敏感性。方法参考美国临床和实验室标准化研究所(Clinical and Laboratory Standards Institute,CLSI)微量液基稀释法M27-A3和M38-A2方案,采用8种抗真菌药物对100株常见浅表致病真菌进行体外药物敏感性测定。结果体外药敏实验结果显示,8种药物对皮肤癣菌表现出良好的体外药物敏感性,其中特比萘芬(GM MIC,0.011μg/mL,MIC范围,0.004~0.06μg/mL)具有最强的抗真菌活性。咪康唑(GM MIC,0.147μg/mL,MIC范围0.015~1μg/mL)具有最强的抗念珠菌活性。酮康唑(GM MIC,0.097μg/mL,MIC范围0.03~1μg/mL)则对马拉色菌具有最佳体外抗真菌效果。结论体外药敏实验结果表明常见浅表致病真菌对8种常用抗真菌药物表现出不同的敏感性。酮康唑具有广谱高效的特点,对临床常见皮肤癣菌、念珠菌、马拉色菌均具有良好的体外抗真菌活性。特比萘芬和萘替芬对皮肤癣菌具有最佳的抗真菌活性。     

10种中药单体对球形孢子丝菌的体外抗真菌作用研究

目的观察10种中药单体对球形孢子丝菌体外抑菌效果。方法参照CLSI的制M38-A2(3rd),采用微量液基稀释法,检测10种中药单体对20株球形孢子丝菌的作用。结果 10种中药单体最小抑菌浓度(MIC)的几何均数范围为53.82μg/mL～1024μg/mL,大蒜素、小檗碱、苦参碱、蛇床子素和丹皮酚的抑菌活性最佳,几何均数分别依次为53.82μg/mL、84.45μg/mL、103.97μg/mL、137.19μg/mL和284.05μg/mL,大蒜素的抗菌作用最强,MIC的范围为16～128μg/mL。最小杀菌浓度(MFC)的范围为64μg/mL～1024μg/mL。大蒜素的MFC值最低,几何均数为415.87μg/mL。结论大蒜素、小檗碱、苦参碱、蛇床子素和丹皮酚对球形孢子丝菌具有一定的抑菌活性,不同中药单体抑菌活性存在差异。     

4种中草药及其复方抗马拉色菌体的外药敏实验

目的观察苦参、当归、侧柏叶和白鲜皮及观察苦参、当归、侧柏叶和白鲜皮、混合药液与主要有效成分对4株CBS马拉色菌标准株的体外抑菌效果。方法通过液基稀释法,用含橄榄油的液体培养基测定各药液对马拉色菌的抑制作用。结果最小抑菌浓度（MIC）由小到大排列分别为：白鲜碱、苦参碱、苦参、侧柏叶、氧化苦参碱、侧柏叶＋当归（二仙丸）、混合药液（侧柏叶＋当归＋苦参＋白鲜皮）、白鲜皮、二仙丸＋苦参、苦参＋白鲜皮、二仙丸＋白鲜皮、槲皮素、当归、阿魏酸。结论4种中草药及其混合药液、主要有效成分均有抑制马拉色菌的作用。     

东南亚青年人群中马拉色菌菌种构成分析

目的：研究部分东南亚国家青年人群中马拉色菌菌种构成。方法采集285名青年人（分别来源于中国、尼泊尔、印度、巴基斯坦）面部正常皮肤及皮损区标本,接种于 Leeming ＆amp; Notman 培养基后进行培养分离,生化及形态学方法鉴定到种。结果共分离出8个菌种,共计501株马拉色菌,以球形马拉色菌为主,占40.5℅（203/501）,其次为合轴马拉色菌19.0℅（95/501）、糠秕马拉色菌16.0℅（80/501）、限制马拉色菌11.0℅（55/501）、斯洛菲马拉色菌6.2℅（31/501）等。各国家间未见明显的地理学差异。结论东南亚地区青年正常人群及马拉色菌相关疾病患者中的主要菌种为球形马拉色菌。     

苦豆子生物碱抗烟曲霉、须癣毛癣菌和新生隐球菌的作用研究

目的对比研究苦豆子总碱、槐果碱、苦参碱、槐定碱、氧化苦参碱体外抗烟曲霉、须癣毛癣菌和新生隐球菌的敏感性,为苦豆子进一步开发利用奠定基础。方法分别用管碟扩散法和微量液基稀释法,测定苦豆子总碱、槐果碱、苦参碱、槐定碱、氧化苦参碱对烟曲霉、须癣毛癣菌和新生隐球菌的抗菌活性。结果管碟法显示上述5种生物碱体外对烟曲霉、须癣毛癣菌和新生隐球菌均有不同程度的抗菌活性。前4种生物碱对烟曲霉的最低杀菌浓度(MFC)分别为25.0、4.25、5.0、3.125 mg·mL~(-1),氧化苦参碱对烟曲霉MFC500 mg·mL~(-1);对须癣毛癣菌的MFC分别为3.125、2.125、5.0、1.5625、125 mg·mL~(-1);对新生隐球菌的MFC分别为3.125、4.25、10、6.25、1000 mg·mL~(-1)。结论槐果碱、苦参碱及槐定碱其抗真菌活性与苦豆子总碱相当,氧化苦参碱的作用不显著。     

正常人耵聍中马拉色菌菌种构成研究

目的研究正常人耵聍中马拉色菌菌种构成及同一宿主耵聍中菌种是否一致。方法采集45名健康志愿者双侧耵聍,0.1%曲拉通X-100溶解稀释后接种于含菜籽油培养基,生化及形态学方法鉴定到种,同时提取菌种DNA,用真菌通用引物ITS1/ITS4做PCR扩增并测序鉴定。结果有44例(97.78%)双侧耵聍中均培养出马拉色菌(共分离出88株菌),菌种构成:糠粃马拉色菌29株(32.95%)、斯洛菲马拉色菌23株(26.14%)、合轴马拉色菌18株(20.45%)、球形马拉色菌11株(12.50%)、限制性马拉色菌7株(7.95%)。44例(88株菌)中双侧耵聍菌种相同者有38例(76株菌)(一致率86.36%)。结论正常人耵聍中马拉色菌菌种分布较广,主要菌种为糠秕马拉色菌。同一宿主双侧耵聍中马拉色菌菌种具有一定的一致性。     

2种中药单体对念珠菌的体外药敏研究

目的探讨人参茎叶皂甙、三七总皂甙、氟康唑、伊曲康唑体外抗念珠菌的敏感性。方法采用CLSI公布的M-27A方案微量液体稀释法分别测定氟康唑、伊曲康唑、人参茎叶皂甙、三七总皂甙对60株临床分离念珠菌的体外敏感性。结果氟康唑、伊曲康唑、人参茎叶皂甙、三七总皂甙对受试菌的MIC50分别为0.25、0.25、7.812和15.625 mg/L,MIC90分别为64、16、250和250 mg/L。60株念珠菌临床分离株中有10株对伊曲康唑、人参茎叶皂甙、三七总皂甙交叉耐药。结论人参茎叶皂甙、三七总皂甙在体外对念珠菌均有不同程度抑制作用,CLSI M27-A方案可用于对中药抗念珠菌活性的评价。     

头颈部特应性皮炎患者面部马拉色菌的菌种分布

目的探讨马拉色菌(Malassezia)在头颈部特应性皮炎(atopic dermatitis, AD)患者中面部菌种分布以及与严重程度之间的关系。方法 2020年9月—12月共招募45例AD患者和50名健康志愿者,无菌棉签对患者和志愿者面部皮损区和非皮损区分别进行皮肤鳞屑取样,用荧光实时定量PCR的方法检测两组人群的7种马拉色菌的拷贝数,进行菌种分布以及数量的横断面分析。结果头颈部AD患者面部皮损区限制马拉色菌和球形马拉色菌的拷贝数分别为健康志愿者的3.13倍(P0.05)和6.69倍(P0.05),两者皆与患者AD病情严重度相关(P0.05)。结论头颈部AD患者面部皮损区限制马拉色菌和球形马拉色菌含量均高于健康人,两者含量均与AD患者病情严重程度相关。     

10种中药提取物抗念珠菌体外药敏实验

目的观察10种中药提取物对念珠菌体外抗菌效果。方法参照CLSI的M27-A3方案,采用微量液基稀释法,检测10种中药提取物抗念珠菌的作用。结果 10种中药提取物中白念珠菌对三七总皂甙、人参茎叶总皂甙较敏感,24h MIC均为62.5μg/mL,48h MIC均为125μg/mL;24h时克柔念珠菌对三七总皂甙和柠檬酸较敏感,MIC均为1.96μg/mL,48h时克柔念珠菌对三七总皂甙较敏感,MIC为31.25μg/mL;近平滑念珠菌对人参茎叶皂苷和麝香草酚较敏感,24h MIC均为3.91μg/mL,近平滑念珠菌在48h对人参茎叶皂甙较敏感,MIC为15.63μg/mL。结论三七总皂甙、人参茎叶皂甙、柠檬酸和麝香草酚对念珠菌具有一定的抗菌活性,不同中药提取物针对念珠菌不同菌种的抗菌作用存在一定差异。     

江西地区花斑癣患者马拉色菌菌种分析

目的了解江西地区花斑癣患者马拉色菌菌种分布情况。比较传统吐温试验和改良吐温试验。方法对141例临床典型、真菌镜检阳性的花斑癣患者,采用Leeming和Notman培养基培养皮屑。以标准菌株为对照,按形态学和生理生化特点进行分类,分析马拉色菌菌种构成情况。同时比较传统吐温试验和改良吐温试验优缺点。结果培养到95株马拉色菌,分离出5个菌种:合轴马拉色菌62株,糠秕马拉色菌17株,球形马拉色菌9株,钝形马拉色菌6株,限制马拉色菌1株。结论合轴马拉色菌占有明显优势(65.26%)。改良吐温试验易于操作、费时短,尤其适合多标本的流行病学调查研究,值得推广。     

球形孢子丝菌致婴儿固定型孢子丝菌病1例及相关实验研究

报道1例由球形孢子丝菌所致的婴儿固定型孢子丝菌病。患儿女,3个月,因左眼下内侧皮损2个月就诊,皮损脓液标本进行真菌培养,对培养获得菌株进行形态学、生理学和分子生物学鉴定,并进行药物敏感性检测。真菌培养阳性,镜下可见典型的套袖样菌丝。钙调蛋白基因序列分析鉴定为球形孢子丝菌。药敏试验显示特比萘芬和伊曲康唑对该菌株的菌丝相最低抑菌浓度（minimal inhibitorycon centration,MIC）分别为0．5μg／mL和0．5μg／mL;对该菌株的酵母相的MIC值分别为0．25μg／mL和0．5μg／mL。给予患者口服特比萘芬32．5mg／d治疗10周后皮损消退呈瘢痕化修复。依据临床及实验室检查确诊该病例为球形孢子丝菌所致固定型孢子丝菌病,特比萘芬治疗本病例显示较好疗效。     

红树植物木榄有机溶剂提取物对球形棕囊藻的抑制效应

基于化感作用原理,利用有机溶剂对红树植物木榄(Bruguiera gymnorrhiza)叶片中的活性物质进行连续提取分离,分别得到正己烷相、乙酸乙酯相、正丁醇相和水相提取物。利用这些提取物进行抑藻实验,通过测定球形棕囊藻(Phaeocystis globosa)的细胞密度发现:四种组分提取物对球形棕囊藻均具有显著的化感抑制作用;正己烷相和乙酸乙酯相提取物对球形棕囊藻的抑制效果明显优于正丁醇相和水相提取物的抑制效果。正己烷相提取物和乙酸乙酯相提取物对球形棕囊藻的48hEC₅₀分别为14.90mg/L和12.18mg/L。研究表明,藻细胞初始接种密度影响提取物的抑藻效应,低接种密度时抑制率极高,而随着接种密度的升高抑制率下降;接种密度极高时,提取物不但不会抑制甚至还会促进藻细胞的生长。     

红树植物干粉和新鲜组织水提物对两种赤潮藻的化感抑制效应

通过测定藻细胞密度,研究了5种红树植物木榄、秋茄、海漆、海芒果、小花老鼠簕水提物对赤潮藻球形棕囊藻和赤潮异弯藻的化感抑制效应,探讨了高温处理对化感抑藻效应的影响.研究结果表明,红树植物干粉水提物和新鲜组织水提物对两种赤潮藻均显示出显著的化感抑制作用.第5 d,红树植物干粉水提物对2种赤潮藻的抑制效果依次为:海漆>秋茄>小花老鼠簕>木榄>海芒果.新鲜组织水提物抑藻效果最强的红树植物是木榄和秋茄.秋茄和海漆水提物中的抑藻化感物质对高温相对不稳定,木榄和小花老鼠簕水提物中的抑藻化感物质对高温相对比较稳定.     

Isolation and characterization of a major antigenic component of Malassezia globosa to IgE antibodies in sera of patients with atopic dermatitis

Three major components of Malassezia globosa were isolated from 2-ME extracts of this fungus by ion-exchange column chromatography and are referred to as Malg46a, Malg46b and Malg67, respectively. IgE antibodies to these components in the sera of patients with AD were detected by immunoblots. In Western blot, IgE antibodies to Malg46b were most frequently detected in the sera of AD patients. Dot blot with the Malg46b-containing fraction immunologically reacted with 69% of the sera of the patients, and with 83% of the sera of the patients who were positive for IgE antibodies to the 2-ME extract of M. globosa in the Western blot. The intensities generated for each dot correlated well with the total intensities generated for the 2-ME extract of M. globosa in the Western blot (r=0.763). In the lectin blot, Con A reacted with both Malg46a and Malg46b but not with Malg67. The polyclonal antibody to Malg46b reacted strongly only with the 2-ME extract of M. globosa and reacted slightly with M. restricta. In conclusion, a glycoprotein, Malg46b of M. globosa, is dominantly expressed in this fungus and is a possible major antigen for IgE antibodies in patients with AD.     

Genetic diversity of Ocimum gratissimum L. based on volatile oil constituents, flavonoids and RAPD markers

Morphological, chemical and genetic differences of 12 tree basil (Ocimum gratissimum L.) accessions were studied to determine whether volatile oils and flavonoids can be used as taxonomical markers and to examine the relationship between RAPDs to these chemical markers. Eugenol, thymol, and geraniol were the major volatile oil constituents found in Ocimum gratissimum. Xantomicrol and cirsimaritin were the major external flavones. The accessions morphologically described as O. gratissimum var. gratissimum contained eugenol as the major volatile oil constituent, and cirsimaritin as the major flavone. Ocimum gratissimum var. macrophyllum accessions contained thymol as the major volatile oil constituent, and xantomicrol as the major flavone. A distinct essential oil and flavone chemotype (producing geraniol and a mixture of the flavones cirsimaritin, isothymusin, xanthomicrol, and luteolin) was found in an accession genetically more distant from the other two groups when analyzed by molecular markers. The accessions could be divided based on volatile oil constituents into six groups: (1) thymol: alpha-copaene (ot24, ot25, ot26, and ot28); (2) eugenol:spathulenol (ot17, ot63, and ot52); (3) thymol:p-cymene (ot65); (4) eugenol:gamma-muurolene (ot27 and ot29); (5) eugenol:thymol: spathulenol (ot85); and (6) geraniol (ot84). Cluster analysis of RAPD markers showed that there are three groups that are distinct genetically and highly correlated (r=0.814) to volatile oil constituents.     

耐氟康唑念珠菌和耐伊曲康唑烟曲霉对泊沙康唑的敏感性测定

目的：测定耐氟康唑念珠菌和耐伊曲康唑烟曲霉临床分离株对泊沙康唑的敏感性。方法参照美国临床实验室标准化研究所制定的 M27-A3和 M38-A2方案,测定从临床获得的11株耐氟康唑的念珠菌和3株耐伊曲康唑烟曲霉对泊沙康唑的 MIC 值。结果对于氟康唑耐药的念珠菌,泊沙康唑的 MIC 范围是0.125-1μg/ mL。对于伊曲康唑耐药烟曲霉,泊沙康唑的 MIC 范围是0.06-0.5μg/ mL。结论11株耐氟康唑的念珠菌和3株耐伊曲康唑烟曲霉均对泊沙康唑有效。     

In vitro susceptibility of isolates of Sporothrix schenckii to amphotericin B, itraconazole, and terbinafine: comparison of yeast and mycelial forms

Forty-three clinical isolates of Sporothrix schenckii derived from humans and animals were evaluated in vitro for their susceptibility to amphotericin B, itraconazole, and terbinafine. MICs were determined by the method of micro dilution in liquid media, using protocols M27-A2 for the yeast form and M38-A for the mycelial form, both standardized by the Clinical Laboratory Standards Institute. In general, higher MICs were found for the mycelial form (intervals of up to two dilutions). In the case of amphotericin B, a significant difference in activity was observed, with higher values (p<0.05) found for the mycelial form. MICs for itraconazole and terbinafine were similar for both yeast and mycelial forms but slightly higher for mycelia. Although data presented here indicate different levels of susceptibility when both growth forms were compared, indicating an intrinsic difference between them, it is still difficult to draw a consensus as to which form correlates better with clinical findings. More studies are necessary to determine the criteria for in vitro tests that will lead to efficient therapeutic choices.     

Fractionation of a herbal antidiarrheal medicine reveals eugenol as an inhibitor of Ca2+-Activated Cl- channel TMEM16A

The Ca(2+)-activated Cl(-) channel TMEM16A is involved in epithelial fluid secretion, smooth muscle contraction and neurosensory signaling. We identified a Thai herbal antidiarrheal formulation that inhibited TMEM16A Cl(-) conductance. C18-reversed-phase HPLC fractionation of the herbal formulation revealed >98% of TMEM16A inhibition activity in one out of approximately 20 distinct peaks. The purified, active compound was identified as eugenol (4-allyl-2-methoxyphenol), the major component of clove oil. Eugenol fully inhibited TMEM16A Cl(-) conductance with single-site IC(50)~150 μM. Eugenol inhibition of TMEM16A in interstitial cells of Cajal produced strong inhibition of intestinal contraction in mouse ileal segments. TMEM16A Cl(-) channel inhibition adds to the list of eugenol molecular targets and may account for some of its biological activities.     

Advanced fibre optical scanning in thin-layer chromatography for drug identification

A reliable and sensitive method was developed for determination of thymol in human plasma by automated headspace solid-phase microextraction (SPME). After enzymatic cleavage of thymol sulfate thymol was extracted by a 65 microm polydimethylsiloxane-divinylbenzene crimped fiber (Supelco) after addition of sodium chloride and phosphoric acid (85%). Desorption of the fiber was performed in the injection port of a gas chromatograph at 220 degrees C (HP 5890; 50 m x 0.2 mm I.D., 0.2 microm HP Innowax capillary column; flame ionization detection). Fibers were used repeatedly up to 40 analysis. The recovery was 5% after 35 min of extraction. The calibration curve was linear in the range of 8.1-203.5 ng ml(-1) with a limit of quantitation (LOQ) of 8.1 ng ml(-1). The within-day and between-day precision and accuracy were < or = 20% at the LOQ and <15% at higher concentrations according to international guidelines for validation of bioanalytical methods. After administration of a thymol-containing herbal extract only thymol sulfate, no free thymol, could be detected in human plasma, thus analysis of thymol was after enzymatic cleavage of thymol sulfate. It is concluded that the newly developed automated method can be used in clinical trials on bioavailability and pharmacokinetics of thymol-containing herbal medicinal products.