Ishihara Yuka, Okamoto Akira, Ohta Michio
Kansenshogaku Zasshi. 2015 May;89(3):375-81. doi: 10.11150/kansenshogakuzasshi.89.375.
Detection of Streptococcus pneumoniae colonized in the pharynx of healthy carriers currently relies on conventional culture methods of direct plating with pharyngeal swab specimens. The accurate measurement of the carriage of pneumococci, however, has not been necessarily achieved with these methods due to low density colonization and contamination of numerous oral streptococci that express α-hemolysis. A PCR-based detection method of pneumococci-specific for lytA as well as PCR serotyping of S. pneumoniae was recently developed and their effectiveness was confirmed. We modified the reaction conditions of these methods to improve the detection rate and applied them to the measurement of S. pneumoniae carried in healthy adults. Pharyngeal swab specimens obtained from 110 healthy volunteers over 40 and living in Nagoya were enriched for 5 hours with broth medium supplemented with rabbit serum and the template DNA for PCR was extracted from the mixed enriched culture. Of 110 specimens 36 (32.7%) were lytA-positive, the rate of which was much higher than the results of previous culture-based studies. The DNA template preparations were then used for PCR-based serotyping with primers specific for each of the types included in pneumococcal 23 valent vaccine (PPV23). We found that 28 out of 36 lytA-positive carriers were identified as being positive for the serotypes belonging to PPV23, although serotypes 6A and 6B were indistinguishable with the PCR method. The most frequent serotype was serotype 14, and serotypes 4, 18C, and 6A/B were also frequently identified. Five lytA-positive carriers were previously vaccinated with PPV23, and among them, 4 were positive for serotypes contained in PPV23. We recommend PCR-based identification and serotyping of S. pneumoniae in broth enrichment culture of pharyngeal swab specimens as a reliable method for the surveillance of healthy carriers with low density colonization.
目前，检测健康带菌者咽部定植的肺炎链球菌依赖于用咽拭子标本直接接种的传统培养方法。然而，由于肺炎链球菌定植密度低以及众多表达α溶血的口腔链球菌的污染，这些方法不一定能准确测量肺炎链球菌的携带情况。最近开发了一种基于PCR的肺炎链球菌特异性检测方法，该方法针对lytA基因，同时也开发了肺炎链球菌的PCR血清分型方法，并证实了其有效性。我们修改了这些方法的反应条件以提高检测率，并将其应用于测量健康成年人携带的肺炎链球菌。从110名居住在名古屋的40岁以上健康志愿者中采集咽拭子标本，用添加兔血清的肉汤培养基富集5小时，然后从混合富集培养物中提取用于PCR的模板DNA。在110份标本中，36份（32.7%）lytA呈阳性，该比率远高于以往基于培养研究的结果。然后将DNA模板制剂用于基于PCR的血清分型，使用针对肺炎球菌23价疫苗（PPV23）中包含的每种血清型的特异性引物。我们发现，36名lytA阳性携带者中有28名被鉴定为PPV23所属血清型呈阳性，尽管血清型6A和6B用PCR方法无法区分。最常见的血清型是血清型14，血清型4、18C和6A/B也经常被鉴定出来。5名lytA阳性携带者先前接种过PPV23，其中4名PPV23所含血清型呈阳性。我们建议在咽拭子标本的肉汤富集培养中对肺炎链球菌进行基于PCR的鉴定和血清分型，作为监测低密度定植健康携带者的可靠方法。
