兰州市城乡居民口腔假丝酵母菌分布与耐药性及口腔白假丝酵母菌实时荧光定量检测
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摘要
目的：
通过对兰州市城乡居民口腔假丝酵母菌的分离、鉴定及其药物敏感性检测,调查兰州市口腔假丝酵母菌种群分布及其对常用四种抗真菌药物的敏感性,为临床口腔假丝酵母菌病的预防及早期诊断和治疗提供参考。建立一种快速、灵敏、特异的鉴定口腔白假丝酵母菌实时荧光定量PCR方法。
方法：
对兰州市城乡居民采用含漱法取唾液样本,利用培养法和科玛嘉显色法分离、鉴定口腔假丝酵母菌。按不同年龄、性别、口腔卫生状况等分组,分析各组口腔假丝酵母菌的检出及分布情况,并采用药敏纸片法进行氟康唑、两性霉素B、制霉菌素、5-氟胞嘧啶药物敏感性测定。利用实时荧光定量PCR反应体系对口腔白假丝酵母菌进行检测。鉴定结果与临床常规鉴定方法对照,评价其敏感度、特异度及重复性。
结果：
口腔假丝酵母菌的检出率为36.31%(342/942),白假丝酵母菌为15.92%(150/942),光滑假丝酵母菌9.67%(91／942)、热带假丝酵母菌9.13%(86/942)、克柔假丝酵母菌3.40%(32/942)。不同分组中,21-60岁组、有牙结石组、男性组、有义齿组、城镇组口腔假丝酵母菌的检出率均分别高于其他组,有统计学意义(P<0.05)。
342株假丝酵母菌对氟康唑、两性霉素B、制霉菌素、5-氟胞嘧啶的总敏感率分别为61.2%、94.3%、89.6%、58.7%。口腔白假丝酵母菌对氟康唑、两性霉素B、制霉菌素、5-氟胞嘧啶的耐药率分别为35.7%、2.4%、4.8%、28.6%。
通过对40例样品的检测,结果显示实时荧光定量PCR法检测标本的鉴定结果与常规鉴定方法的结果对照,特异度与敏感度均为100%；最小能检测到10个拷贝数的重组质粒；批内重复实验和批间重复实验结果均与培养法结果相符。
结论：
兰州市城乡居民口腔假丝酵母菌的检出以白假丝酵母菌为主,对四种常见抗真菌药物均有不同程度的耐药性,两性霉素B、制霉菌素对常见口腔假丝酵母菌的敏感性较高。临床在治疗口腔假丝酵母菌菌感染时应结合菌种鉴定及药物敏感性实验结果合理选择抗假丝酵母菌药物。
实时荧光定量PCR法鉴定口腔白假丝酵母菌,特异度和敏感度高,重复性好,且快速、简便,该方法将有助于口腔白假丝酵母菌病的早期诊断和针对性治疗。
Objective:The purpose was to investigate and analyze the distribution of oral Saccharomyces spp. and the sensitivity to four antifungal agents among urban and rural residents in Lanzhou; So as to be helpful for the prevention, earlier diagnosis and treatment of oral candidosis. And to set up a Real-time Quantitative PCR assay with high sensitivity, specificity and rapidity to detect oral Saccharomyces albicans
Methods:Concentrated saliva samples from Lanzhou resident volunteers were used for Saccharomyces spp. to be isolated and identified by CHROMagar Saccharomyces culture medium. Its susceptibility to fluconazole, amphoterincin B, nystafungin and5-flurocytosine was detected by the method of fungal susceptibility paper. Volunteers were grouped according to age, gender, living location (urban and rural) and so on.And we used Real-time Quantitative PCR assay to detect oral Saccharomyces albicans and evaluated its sensitivity, specificity and repeatability。
Results:The whole detection rate of oral Saccharomyces spp. is36.31%(342/942), of which S. albicans is15.92%(150/942),S. glabrata is9.67%(91/942), S.tropicalis is9.13%(86/942),and S.krusei is3.40%(32/942) respectively. In age21-60group, calculus dentalis group, male group, denture group and urban group, the Saccharomyces spp detection rate however is higher than the controled groups in various groups (P<0.05). The sensitive of oral Saccharomyces spp. to fluconazole, amphoterincin B, nystafungin, and5-flurocytosine was61.2%,94.3%,89.6%and58.7%respectively. The resistance of oral S. albicans to fluconazole, amphoterincin B, nystafungin, and5-flurocytosine is35.7%,2.4%,4.8%and28.6%respectively. And the results obtained from the Real-time Quantitative PCR assay and the general culture were the same by detecting the samples of40cases. Its specificity was100%, and its sensitivity was100%. The lower limit of detection of this Real-time Quantitative PCR assay was10copies of recombinant plasmid DNA. The result within group and between groups were the same as the general culture.
Conclusion:S. albicans is a dominant species in the oral microorganism detection among urban and rural residents in Lanzhou. Oral Saccharomyces spp. displays different resistance to four antifungal agents. The sensitive of oral Saccharomyces spp. to amphoterincin B and nystafungin is higher than others. Clinical oral candidosis therapy should be conducted along with the results of pathogen identification and antifungal susceptibility testing. The Real-time Quantitative PCR assay can detect oral Saccharomyces albicans, sensitively, specifically, rapidly, simply and stably, and will be useful to early diagnosis and target treatment of the diseases caused by Saccharomyces albicans.
通过对兰州市城乡居民口腔假丝酵母菌的分离、鉴定及其药物敏感性检测,调查兰州市口腔假丝酵母菌种群分布及其对常用四种抗真菌药物的敏感性,为临床口腔假丝酵母菌病的预防及早期诊断和治疗提供参考。建立一种快速、灵敏、特异的鉴定口腔白假丝酵母菌实时荧光定量PCR方法。
方法：
对兰州市城乡居民采用含漱法取唾液样本,利用培养法和科玛嘉显色法分离、鉴定口腔假丝酵母菌。按不同年龄、性别、口腔卫生状况等分组,分析各组口腔假丝酵母菌的检出及分布情况,并采用药敏纸片法进行氟康唑、两性霉素B、制霉菌素、5-氟胞嘧啶药物敏感性测定。利用实时荧光定量PCR反应体系对口腔白假丝酵母菌进行检测。鉴定结果与临床常规鉴定方法对照,评价其敏感度、特异度及重复性。
结果：
口腔假丝酵母菌的检出率为36.31%(342/942),白假丝酵母菌为15.92%(150/942),光滑假丝酵母菌9.67%(91／942)、热带假丝酵母菌9.13%(86/942)、克柔假丝酵母菌3.40%(32/942)。不同分组中,21-60岁组、有牙结石组、男性组、有义齿组、城镇组口腔假丝酵母菌的检出率均分别高于其他组,有统计学意义(P<0.05)。
342株假丝酵母菌对氟康唑、两性霉素B、制霉菌素、5-氟胞嘧啶的总敏感率分别为61.2%、94.3%、89.6%、58.7%。口腔白假丝酵母菌对氟康唑、两性霉素B、制霉菌素、5-氟胞嘧啶的耐药率分别为35.7%、2.4%、4.8%、28.6%。
通过对40例样品的检测,结果显示实时荧光定量PCR法检测标本的鉴定结果与常规鉴定方法的结果对照,特异度与敏感度均为100%；最小能检测到10个拷贝数的重组质粒；批内重复实验和批间重复实验结果均与培养法结果相符。
结论：
兰州市城乡居民口腔假丝酵母菌的检出以白假丝酵母菌为主,对四种常见抗真菌药物均有不同程度的耐药性,两性霉素B、制霉菌素对常见口腔假丝酵母菌的敏感性较高。临床在治疗口腔假丝酵母菌菌感染时应结合菌种鉴定及药物敏感性实验结果合理选择抗假丝酵母菌药物。
实时荧光定量PCR法鉴定口腔白假丝酵母菌,特异度和敏感度高,重复性好,且快速、简便,该方法将有助于口腔白假丝酵母菌病的早期诊断和针对性治疗。
Objective:The purpose was to investigate and analyze the distribution of oral Saccharomyces spp. and the sensitivity to four antifungal agents among urban and rural residents in Lanzhou; So as to be helpful for the prevention, earlier diagnosis and treatment of oral candidosis. And to set up a Real-time Quantitative PCR assay with high sensitivity, specificity and rapidity to detect oral Saccharomyces albicans
Methods:Concentrated saliva samples from Lanzhou resident volunteers were used for Saccharomyces spp. to be isolated and identified by CHROMagar Saccharomyces culture medium. Its susceptibility to fluconazole, amphoterincin B, nystafungin and5-flurocytosine was detected by the method of fungal susceptibility paper. Volunteers were grouped according to age, gender, living location (urban and rural) and so on.And we used Real-time Quantitative PCR assay to detect oral Saccharomyces albicans and evaluated its sensitivity, specificity and repeatability。
Results:The whole detection rate of oral Saccharomyces spp. is36.31%(342/942), of which S. albicans is15.92%(150/942),S. glabrata is9.67%(91/942), S.tropicalis is9.13%(86/942),and S.krusei is3.40%(32/942) respectively. In age21-60group, calculus dentalis group, male group, denture group and urban group, the Saccharomyces spp detection rate however is higher than the controled groups in various groups (P<0.05). The sensitive of oral Saccharomyces spp. to fluconazole, amphoterincin B, nystafungin, and5-flurocytosine was61.2%,94.3%,89.6%and58.7%respectively. The resistance of oral S. albicans to fluconazole, amphoterincin B, nystafungin, and5-flurocytosine is35.7%,2.4%,4.8%and28.6%respectively. And the results obtained from the Real-time Quantitative PCR assay and the general culture were the same by detecting the samples of40cases. Its specificity was100%, and its sensitivity was100%. The lower limit of detection of this Real-time Quantitative PCR assay was10copies of recombinant plasmid DNA. The result within group and between groups were the same as the general culture.
Conclusion:S. albicans is a dominant species in the oral microorganism detection among urban and rural residents in Lanzhou. Oral Saccharomyces spp. displays different resistance to four antifungal agents. The sensitive of oral Saccharomyces spp. to amphoterincin B and nystafungin is higher than others. Clinical oral candidosis therapy should be conducted along with the results of pathogen identification and antifungal susceptibility testing. The Real-time Quantitative PCR assay can detect oral Saccharomyces albicans, sensitively, specifically, rapidly, simply and stably, and will be useful to early diagnosis and target treatment of the diseases caused by Saccharomyces albicans.
引文
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[2]P. Lewis White,David W. Williams, Tomoari Kuriyama, Shamim A. Samad, Michael A. O. Lewis, and Rosemary A. Barnes Detection of Candida in Concentrated Oral Rinse Cultures by Real-Time PCR. [J].JOURNAL OF CLINICAL microbiology 2004:42:2101-2107
[3]Pfaller MA, Diekema DJ. Epidemiology of invasive candidiases:a persistent public health problem. Clin[J]. Microbiol Rev 2007:20:133-163
[4]廖万清,侵袭性真菌感染的实验室诊断,[J].检验医学2010 35(7)：503-505
[5]Baixench M T, Taillandier A, Paugam A. Clinical and experimental evaluation of a new chromogenic medium (OCCA, Oxoid) for direct identification of Candida albicans, C. tropicalis and C. krusei[J]. Mycoses,2006,49:311
[6]Cornelia Lass-Florl. The changing face of epidemiology of invasive fungal disease in Europe[J]. Mycoses,2009,52:197-205
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