Blinded multiplex PCR analyses of middle ear and nasopharyngeal fluids from chinchilla models of single- and mixed-pathogen-induced otitis media
Blinded multiplex PCR analyses of middle ear and nasopharyngeal fluids from chinchilla models of single- and mixed-pathogen-induced otitis media
复制标题
DOI：
10.1128/cdli.5.2.219-224.1998
复制
发表时间：
1998-03-01
期刊：
影响因子：
--
通讯作者：
Ehrlich, GD
中科院分区：
文献类型：
--
作者：
Bakaletz, LO;White, GJ;Ehrlich, GD
细菌和病毒病原体的多重PCR分析进行了盲法的方式对33个档案标本，已知的文化状态，采购从龙猫模型的单一和混合病原体诱导的中耳炎和儿科患者。这些标本在-70摄氏度下保存了长达6年。评价的实验标本包括中耳积液、鼻咽灌洗液和中耳灌洗液，这些标本来自免疫学未处理、假免疫或用不可分型流感嗜血杆菌抗原主动免疫的动物。所用采样时间范围为细菌或病毒攻毒当天至攻毒后42天。对33例标本的初始PCR分析与24例（73%）的传统培养数据相匹配，正确鉴定了不可分型的H。流感、卡他莫拉菌、肺炎链球菌或腺病毒作为病原体。在4份培养阴性的动物模型标本（12%）中也获得了接种微生物的PCR阳性信号。两个培养阴性的人渗出液之一也是PCR阳性。因此，总体而言，通过盲法PCR获得的结果与传统培养方法的一致性为85%，或正确地指示了在4份无菌标本中引入的特定病原体。在任何情况下，都没有得到一个假阳性信号的任何五个病原体正在评估。我们的结论是，多重PCR分析是快速和准确的方法，当它们被用来回顾性地评估不同的档案标本的有限体积的中耳炎的实验模型。
Multiplex PCR analyses for both bacterial and viral pathogens were conducted in a blinded manner on 33 archival specimens, of known culture status, procured from chinchilla models of both single- and mixed-pathogen-induced otitis media and from a pediatric patient. These specimens had been maintained at -70 degrees C for up to 6 years. Experimental specimens evaluated included middle-ear effusions, nasopharyngeal lavage fluids and middle-ear lavage fluids from animals which were immunologically naive, sham-immunized or actively immunized with nontypeable Haemophilus influenzae antigens. Sampling times used ranged from the day of bacterial or viral challenge to 42 days after challenge. Initial PCR analyses of the 33 specimens matched the traditional culture data in 24 instances (73%), correctly identifying nontypeable H. influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, or adenovirus as the causative agent. A PCR-positive signal for the microbe(s) inoculated was also obtained in four animal model specimens (12%) which were culture negative. One of two culture-negative human effusions was also PCR positive. Thus, overall, results obtained by blinded PCR were 85% concordant with traditional culture methods or correctly indicated the specific pathogen introduced in four specimens that were sterile. In no instance was a false-positive signal obtained for any of the five etiologic agents being evaluated. We conclude that the multiplex PCR analyses are rapid and accurate methodologies when they are used to retrospectively evaluate diverse archival specimens of limited volume from experimental models of otitis media.