Streptococcus Laboratory, Respiratory Disease Branch, Division of Bacterial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd., NE, Mailstop C02, Atlanta, GA 30329, USA.
J Clin Microbiol. 2010 May;48(5):1611-8. doi: 10.1128/JCM.02243-09. Epub 2010 Mar 10.
The measurement of pneumococcal carriage in the nasopharyngeal reservoir is subject to potential confounders that include low-density and multiple-strain colonization. To compare different methodologies, we picked a random sampling of 100 nasopharyngeal specimens recovered from infants less than 2 years of age who were previously assessed for pneumococcal carriage and serotypes by a conventional method that used direct plating from the transport/storage medium (50 specimens were culture negative and 50 specimens were culture positive for pneumococci). We used a broth enrichment approach and a conventional PCR approach (with and without broth enrichment) to determine pneumococcal carriage and serotypes, and the results were compared to the initial conventional culture-based results. Additionally, we used a lytA-targeted real-time PCR for pneumococcal detection. Broth enrichment for both the culture-based and the PCR-based methods enhanced the isolation of pneumococci and detection of serotype diversity, with the most effective serotype deduction method being one that used broth enrichment prior to sequential multiplex PCR. Similarly, we also found that broth enrichment followed by the lytA-specific real-time PCR was the most sensitive for the detection of apparent pneumococcal carriage. The broth enrichment, conventional multiplex PCR, and real-time PCR approaches used in this study were effective in detecting pneumococcal carriage in the 50 specimens that were negative by conventional direct plating from transport medium (range of numbers of positive specimens, 8/50 to 22/50 [16 to 44%]), and the three different serotyping approaches that used broth enrichment increased the number of serotype identifications from the 100 specimens (12 to 29 additional serotype identifications to be positive). A PCR-based approach that employed a broth enrichment step appeared to best enhance the detection of mixed serotypes and low-density pneumococcal carriage.
鼻咽部携带状态的肺炎球菌检测可能受到包括低密度和多菌株定植等潜在混杂因素的影响。为了比较不同的方法，我们随机选择了 100 个鼻咽标本，这些标本均来自年龄小于 2 岁的婴幼儿，之前使用直接从运输/储存介质中接种平板的常规方法评估了肺炎球菌携带状态和血清型（50 个标本培养阴性，50 个标本培养阳性）。我们使用肉汤富集方法和常规 PCR 方法（包括和不包括肉汤富集）来确定肺炎球菌携带状态和血清型，并将结果与初始的基于培养的常规结果进行比较。此外，我们还使用 lytA 靶向实时 PCR 检测肺炎球菌。基于培养和基于 PCR 的方法的肉汤富集均增强了肺炎球菌的分离和血清型多样性的检测，最有效的血清型推导方法是在顺序多重 PCR 之前使用肉汤富集。同样，我们还发现肉汤富集后进行 lytA 特异性实时 PCR 是检测明显肺炎球菌携带最敏感的方法。本研究中使用的肉汤富集、常规多重 PCR 和实时 PCR 方法在 50 个通过常规直接从运输培养基中接种平板而呈阴性的标本中均有效（阳性标本数量范围为 8/50 至 22/50[16%至 44%]），并且使用肉汤富集的三种不同血清型鉴定方法增加了 100 个标本的血清型鉴定数量（12 至 29 个额外的阳性血清型鉴定）。使用肉汤富集步骤的基于 PCR 的方法似乎最能增强对混合血清型和低密度肺炎球菌携带的检测。
