UP-PCR结合RFLP快速检测体液标本中病原菌的初步研究和应用

目的 建立通用引物聚合酶链反应(UP-PCR)结合限制性酶切片段长度多态性(RFLP)酶切图谱的方法,快速检测临床体液标本中常见病原菌。方法 根据体液标本中常见病原菌16S rRNA基因保守区设计的通用引物进行UP-PCR扩增,确定标本是否有细菌感染,然后对阳性标本的扩增产物进行RFLP分析,进一步鉴定细菌。利用该方法对204例体液标本进行了检测,并与常规细菌培养鉴定法进行比较。结果UP-PCR所有细菌均产生1032bp,扩增产物经RFLP法可将细菌进行鉴别。204份体液标本的检测中,与培养法相比,其灵敏度、特异性、阳性预测值、阴性预测值分别为95.7％、88.6％、88.0％和99.4％。结论 该方法具有敏感、特异的特点,可广泛应用于临床体液标本中病原菌的快速检测。     

阴道念珠菌的检验方法比较分析

目的：评价几种阴道念珠菌检验方法的临床价值。方法：同时用革兰染色法、培养法、盐水法和快速凝集法对临床拟诊的168例阴道分泌物进行检测,比较每种检测方法的检测结果。结果：快速凝集法的检出率最高,革兰染色法次之,盐水法的检出率最低,以培养法为标准,革兰染色法和培养法比较,P〉0.05,检测结果差异无统计学意义;盐水法和培养法比较,P〈0.01,快速凝集法和培养法比较,P〈0.05,快速凝集法和盐水法比较,P〈0.01,检测结果差异有统计学意义。结论：快速凝集法检测阴道念珠菌具有简便、检出率高等特点,培养法检出率也很高,并可将念珠菌鉴定到菌种,但用时较长,建议两法结合使用,可互补。     

念珠菌鉴定方法的研究进展

机会性真菌感染对于免疫缺陷患者有着较大的威胁,其中念珠菌属在真菌感染中占较大比例,而不同念珠菌对抗真菌药物的敏感性亦不尽相同,因此,如何快速、准确地检测和鉴定念珠菌便成为临床微生物分析鉴定工作者所面对的巨大挑战。本文就念珠菌鉴定工作中所涉及的酶学法、经过常规改进的鉴定方法以及分子生物学法进行扼要综述。     

临床血液检验中两种不同病菌鉴定法的应用分析

目的探讨血液检验中两种不同病菌鉴定方法的临床应用效果,为提高血液检验病菌鉴定准确性提供可靠依据。方法对100例各类感染患者均进行血液抽样,并分别实施药敏试验以及常规检测,观察并记录两种检测方法所得结果 ,进行鉴定符合率判断,给予统计学分析,得出结论。结果 直接药敏试验与常规检验法进行病菌鉴定符合率较高,为84.00%,且P>0.05,两种方法病菌检出结果 无显著差异。结论直接药敏试验法鉴定血液中病原菌即可节约医疗成本,又可快速提供结果 ,使患者及时进行临床治疗,保障其疗效及预后。     

不同方法对酵母样病菌分离培养的结果比较

目的 :调查近年来我院住院病人呼吸道酵母样病菌感染的情况 ,防止医院感染。方法 :应用CHROMager念珠菌显色培养法与沙保罗氏培养法进行对比鉴定 ,并对其病原体菌种进行分析。结果 :CHROMager念珠菌显色培养法阳性率34 2 % (421/1232) ,沙保罗氏鉴定法阳性率35 8 % (441/1232) ,x2=1.46 ,P>0 05 ;CHROMager念珠菌显色培养基上呈现的不同颜色的菌落判断的菌种同酵母样真菌同化试验鉴定板鉴定的菌种 ,两者的符合率为95 5 % (421/441)以上。结论 :CHROMager念珠菌显色培养基在快速鉴定常见念珠菌感染中非常有用 ,可对临床常见的念珠菌做出初步鉴定 ,缩短检测时间。     

科玛嘉显色培养基检测深部念珠菌感染538例分析

目的 对科玛嘉念珠菌显色培养基在深部念珠菌标本检测中应用的评价。方法 对直接镜检阳性的538例患者的便、痰、尿标本，采用科玛嘉显色培养25℃ 48—72h，根据培养基表面菌落不同的颜色，鉴定念珠菌的种类。结果 538例深部标本，白念珠菌266株，热带念珠菌105株，光滑念珠菌77株，克柔氏念珠菌59株。结论 科玛嘉念珠菌显色培养基优于常规沙氏生化法，且快速、简便、准确地分离鉴定各种念珠菌，可做为检测深部念珠菌的首选方法。     

直接药敏试验与常规药敏试验在临床血液细菌鉴定与检验中的应用比较

目的对比分析直接药敏试验与常规药敏试验在临床血液细菌鉴定与检验中的应用价值。方法 2017年4月至2017年10月选择390份血液标本作为实验对象,经血培养仪培养后发现157份阳性血液样本,均应用直接药敏试验法与常规药敏试验法进行细菌鉴定,比较两种方法的细菌鉴定结果以及药敏结果符合率。结果 (1)在细菌鉴定方面,直接法鉴定出革兰阳性(G~+)球菌28株、革兰阴性(G~-)杆菌106株,常规法鉴定出G~+球菌36株、G~-杆菌121株。两种方法细菌鉴定总符合率达到85.35%,其中G~+球菌鉴定符合率77.78%、G~-杆菌符合率87.60%。(2)两种方法对G~+球菌、G~-杆菌的敏感、中度敏感、耐药结果比较未见显著性差异(P>0.05)。(3)直接法检验结果报告所需时间为(9.97±2.69)h,明显短于常规法(71.52±9.01)h。结论直接药物试验与常规药敏试验在临床血液细菌鉴定与检验中的应用效果相似,但直接药敏试验能够缩短检测时间,快速明确病原菌与药敏结果,利于临床早期用药治疗,减少盲目治疗。     

临床血液检验中的两种不同细菌鉴定法应用分析

目的探究直接药敏试验与常规药敏试验在临床血液细菌鉴定与检验中的临床应用价值。方法对我院200例阳性血培养标本,分别给予直接法与常规法进行细菌鉴定和药敏试验对比。结果全部200份血液标本中阳性血液标本共计122份,22份G+球菌,100份G-杆菌,药敏试验结果显示,17份G+球菌,85份G-杆菌,一致率分别为77.27%和85%,两种方法总一致率为83.61%。两种检测方法药物敏感、中度敏感以及耐药差异不具有统计学意义(P>0.05)。结论阳性血培养标本进行直接药敏试验,其检验结果不仅操作简便、时间短,而且检测结果准确,可广泛应用于细菌感染的临床血液检验。     

科玛嘉念珠菌显色培养基的临床应用评价

目的：评价科玛嘉念珠菌显色培养基在临床念珠菌鉴定中的应用价值。方法：75株念珠菌分别用科玛嘉念珠菌显色培养基和API 20C AUX进行鉴定,并对两种方法鉴定结果做统计比较。结果：两种方法鉴定出的四种临床常见念珠菌——白色念珠菌、热带念珠菌、光滑念珠菌、克柔念珠菌差异无统计学意义,采用χ2检验,P〉0.05。其中科玛嘉念珠菌显色培养基有5株鉴定为其他念珠菌（培养基上菌落只显白色）,API 20C AUC有4株鉴定为其他念珠菌。科玛嘉念珠菌显色培养基与API 20C AUX鉴定符合率达98.5%。结论：科玛嘉念珠菌显色培养基能快速分离培养鉴定临床常见念珠菌,鉴定结果准确可靠。     

应用念珠菌显色培养基培养浅部真菌的研究

目的：探讨念珠菌显色培养基培养检测浅部真菌的应用价值。方法：分别使用直接镜检和念珠菌显色培养基培养检测法平行检测临床标本37例。结果：镜检和培养检测法结果差异无显著意义（P〉0.5）。如果以直接镜检作为确诊标准,则培养法的敏感度为77%,特异度为91%。在念珠菌显色培养基上念珠菌形成光滑的湿润菌落,其生长较快,一些念珠菌的颜色和形态有其特征性;而丝状真菌在念珠菌显色培养基上形成丝状菌落,生长较慢。结论：直接镜检结合念珠菌显色培养基培养有助于临床实验室鉴定和检测浅部真菌。     

结核病临床分离株及痰标本中embB基因型的快速测定

目的建立快速测定结核病耐乙胺丁醇(EMB)分离株和痰标本中结核分枝杆菌EMB耐药基因型的方法，以期为患者提供及时、有效地化验结果。方法应用16S rDNA聚合酶链反应(PCR)-单链构象多态性(SSCP)和PCR-限制性片段长度多态性(RFLP)对11株耐EMB分离株和46例结核病痰标本进行分子菌种鉴定和embB基因突变的部位与性质分析。结果以结核分枝杆菌H37Rv标准株为对照，11株耐EMB分离株和46例临床痰标本经16S rDNA PCR-SSCP分析电泳图谱均与结核分枝杆菌标准株相同；限制性内切酶NlaⅢ消化embB基因扩增产物显示，11株耐EMB分离株中4株(36．4％)不被NlaⅡ消化；46例结核病痰标本中10例(21．7％)不被NlaⅡ消化。结论部分结核分枝杆菌耐EMB是由于embB基因突变所致。采用PCR-SSCP和PCR-RFLP方法可直接快速测定结核病耐EMB分离株和痰标本中结核分枝杆菌耐EMB基因型。     

137例阴道真菌感染及耐药性分析

目的了解女性阴道中真菌感染及耐药情况。方法将白带常规中涂片镜检真菌阳性患者的阴道分泌物的标本进行培养、鉴定和药敏试验。结果 137份送检标本中,白色假丝酵母菌(84株,61.3%)检出率最高,其次是光滑球拟酵母菌(19株,13.9%),热带假丝酵母菌(15株,10.9%),克柔假丝酵母菌(7株,5.1%),其他真菌(12株,8.8%)。分离菌株对氟胞嘧啶,两性霉素B和伏立康唑的敏感率均>90%,伊曲康唑敏感率为84.7%,氟康唑敏感率为70.8%。结论女性阴道真菌感染的白色假丝酵母菌为主。氟胞嘧啶,两性霉素B和伏立康唑敏感率较高,氟康唑和伊曲康唑敏感率较低,但仍然有效。     

Identification of a novel antifungal nonapeptide generated by combinatorial approach

It is becoming clear that antimicrobial peptides are important components of the innate defences of all species of life. They kill very rapidly, do not easily select resistant mutants and are synergistic with potentially toxic conventional therapeutic agents against microbes. This paper describes an attempt to expand a lead hexapeptide motif synthesized through combinatorial approach. A cationic peptide H-Arg-Trp-Trp-Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH2 was found to be active with a therapeutic index of >17. I was proposed that the combination of peptide with known antifungal agents may identify synergistic combinations that would ideally reduce the dosage of conventional antifungals as well as their associated toxicity. Nine different pathogenic strains and species of Candida and two of Cryptococcus neoformans were employed in chequerboard method and in time kill assays to evaluate the synergistic effect of the lead peptide in combination with amphotericin B, 5-flucytosine, ketoconazole and fluconazole. We found synergistic interaction between the peptide and all four drugs against Cryptococcus isolates whilst both synergistic and additive combinations occurred when Candida isolates were used.     

In vitro interactions of anidulafungin with azole antifungals, amphotericin B and 5-fluorocytosine against Candida species

Anidulafungin, an echinocandin, is in late stage development for the treatment of fungal infections. We investigated the activity of anidulafungin in combination with other antifungal agents (fluconazole, itraconazole, ketoconazole, amphotericin B and 5-fluorocytosine) against four isolates each of Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis, and two isolates of Candida krusei using a macrobroth chequerboard method with interactions evaluated by fractional inhibitory concentration indices (FICIs). Additive activity (FICI > 0.5 to 1) or indifference (FICI > 1 to < 4) was observed in 85 of 90 interactions of anidulafungin with another antifungal agent. Synergy with itraconazole (FICIor=4), a drug rarely used systemically, was noted for four strains of C. tropicalis. The combination of anidulafungin and amphotericin B demonstrated additive activity for each of the 18 isolates of Candida tested. These results suggest additional studies are warranted, for example in animal models, to evaluate further the potential of combination antifungal therapy with anidulafungin for Candida infections.     

血液真菌感染的菌群分布及耐药性分析

目的分析本院近3年来血液真菌的分离率、菌种分布和耐药特性。方法收集自2004年8月～2007年7月3年间住院患者的血液标本,经BacT／ALERT3D血培养仪培养,沙保罗培养基分离培养真菌,柯玛嘉显色培养基和VITEK-60YBC卡进行菌种鉴定,Rosco纸片扩散法测定药敏。结果8707份血液标本中共分离出93株真菌,分离率为1．07％,其中白念珠菌41株,占44．09％,热带念珠菌16株,占17．20％,光滑念珠菌13株,占13．98％,对氟康唑的耐药率分别为：2．4％、6．3％和23．1％,对两性霉素B和制霉菌素均敏感。结论我院血液真菌感染的病原菌以白念珠菌为主,热带念珠菌和光滑念珠菌次之,白念珠菌和热带念珠菌对唑类药物敏感性较高,光滑念珠菌对唑类药物的耐药率较高,两性霉素B和制霉菌素的抗菌活性最强。     

In vitro antifungal susceptibilities of Candida species isolated from the blood

In vitro susceptibility of 158 strains of Candida species isolated from blood cultures to amphotericin B, flucytosine and fluconazole was evaluated. The broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS, M27-P) was adapted to the microdilution method. For 43 tests of standard strains, there were excellent agreement (within one doubling dilution) of minimum inhibitory concentrations (MICs) of amphotericin B and flucytosine with macrodilution reference. For fluconazole, 100% agreement was found using MICs scored 2 (prominent decrease in turbidity; MIC-2s) at 24 h reading, and much lower agreement using MICs scored 1 or at 48 h reading. In testing of the clinical isolates, amphotericin B and flucytosine demonstrated a very narrow range of MICs. On the other hand, fluconazole MICs showed a broad range and shifted substantially from higher level to lower level when MIC endpoints changed from the most strict MICs (complete absence of growth, MIC-0s) to a less stringent MIC-2s. However, MIC-2s distinguished C. glabrata as resistant compared with the other species. Also, MICs increased after prolonged incubation, particularly for fluconazole. Almost all Candida blood culture isolates tested were very susceptible to flucytosine. However, higher prevalence of fluconazole resistance was noted in our blood culture isolates than those in previous studies. The present study validated that broth microdilution method is an adequate tool for antifungal susceptibility testing following the recommendations provided by the NCCLS, and the MIC results might be determined as early as 24 h after incubation.     

In vitro antifungal activity of itraconazole, a new triazole antifungal agent, against clinical isolates from patients with systemic mycoses]

The in vitro antifungal activity of itraconazole (ITZ), a new oral triazole antifungal agent, against clinical isolates from patients with systemic mycoses were compared with those of existing systemic antifungals, viz. ketoconazole (KCZ), miconazole or amphotericin B. The studies were performed with 65 isolates of pathogenic yeasts and 13 isolates of Aspergillus spp. using the agar dilution method on casitone agar. ITZ showed the most potent antifungal activities against isolates of pathogenic yeasts including several Candida spp. (Candida parapsilosis, Candida krusei, Candida guilliermondii), Cryptococcus neoformans, Trichosporon cutaneum (MIC less than or equal to 0.08 micrograms/ml) and Aspergillus spp. including Aspergillus fumigatus (MIC less than or equal to 5 micrograms/ml). On the other hand, activities of ITZ against isolates of other Candida spp. such as Candida albicans and Candida glabrata were lower than those of KCZ and other reference drugs. Some isolates of C. albicans and C. tropicalis were not completely inhibited by ITZ even at concentrations above 10 micrograms/ml on casitone agar. However, in the micro-broth dilution method using synthetic amino acid medium, fungal as the test medium, ITZ completely inhibited the growth of all these isolates at drug concentrations of less than or equal to 0.20 micrograms/ml.     

Itraconazole: increased activity by chlorhexidine

Chlorhexidine increases the activity of itraconazole against Candida isolates; itraconazole-chlorhexidine combinations show synergistic activity in culture media. The activity of itraconazole is discussed.     

Voriconazole activity against clinical yeast isolates: a multicentre Italian study

The activity of voriconazole was tested in vitro against 1996 clinical yeast isolates collected in 20 Italian microbiology laboratories. Voriconazole susceptibility testing was carried out with the broth microdilution (NCCLS M27-A2), Etest and disk diffusion methods. The minimum inhibitory concentrations at which 90% of the isolates were inhibited (MIC90) obtained with the NCCLS method were 0.03 mg/L for Candida albicans, 0.5 mg/L for Candida non-albicans and 0.25 mg/L for other genera; those obtained with Etesting were, respectively, 0.032 mg/L, 0.125 mg/L and 0.125 mg/L. With the disk diffusion method, the majority of isolates (92.3%) showed inhibition zone diameters between 21 mm and 40 mm. Using a tentative MIC cut-off of 1mg/L as indicative of in vitro susceptibility, 98.1% of the isolates tested in our study would be classified as susceptible, and only 28 (1.4%) of the isolates, with MICs higher than 2mg/L, would be classified as resistant to the drug. Our findings confirm the broad-spectrum in vitro activity of voriconazole against yeasts, including Candida species that are generally less susceptible to other azoles.