Merck & Co., Inc., West Point, PA, USA.
Merck & Co., Inc., West Point, PA, USA.
J Immunol Methods. 2023 Aug;519:113516. doi: 10.1016/j.jim.2023.113516. Epub 2023 Jun 20.
Streptococcus pneumoniae is one of the most common microorganisms causing acute otitis media (AOM) in children. While bacterial culture of middle ear fluid (MEF) is the gold standard to detect the etiological organisms, several host and pathogen factors impact the survival of the organisms resulting in false negatives. To overcome this limitation, we have developed and validated an innovative multiplex immuno-molecular assay to screen and detect the S. pneumoniae 15-valent pneumococcal conjugate vaccine (PCV15; STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) vaccine serotypes in MEF. This novel in vitro approach involves two-step testing. First, the MEF specimens were tested for highly conserved pneumococcal genes, autolysin, lytA, and pneumolysin, ply using direct PCR to identify pneumococcus positive specimens. The pneumococcus positive specimens were screened for the presence of vaccine serotype specific pneumococcal polysaccharides using a 15-plex Pneumococcal Antigen Detection (PAD) assay, with specific capture and detection monoclonal antibodies. Due to the lack of availability of MEF samples, cerebrospinal fluid (CSF) was used as the surrogate matrix for the development and validation of the PCR-PAD assays. The PCR and PAD assays were separately evaluated for sensitivity and specificity. Subsequently, the PCR-PAD assays were cross-validated with human MEF samples (n = 39) which were culture confirmed to contain relevant bacterial strains. The combined PCR-PAD assays demonstrated high rate of agreement 94.9% (95% CI; 82.7, 99.4%) with historical Quellung serotype data of these MEF samples. This novel PCR-PAD assay demonstrates the feasibility of combining molecular and immunological assays to screen and identify PCV15 pneumococcal vaccine serotypes in AOM clinical samples.
肺炎链球菌是导致儿童急性中耳炎（AOM）最常见的微生物之一。虽然中耳液（MEF）的细菌培养是检测病原体的金标准，但一些宿主和病原体因素会影响病原体的存活，导致假阴性。为了克服这一限制，我们开发并验证了一种创新的多重免疫分子检测方法，用于筛选和检测 MEF 中的 15 价肺炎球菌结合疫苗（PCV15；STs 1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、22F、23F 和 33F）疫苗血清型。这种新的体外方法涉及两步检测。首先，使用直接 PCR 测试 MEF 标本中高度保守的肺炎球菌基因、自溶素、lytA 和肺炎球菌溶血素 ply，以鉴定肺炎球菌阳性标本。然后，使用 15 重肺炎球菌抗原检测（PAD）检测试剂盒，用特异性捕获和检测单克隆抗体，对肺炎球菌阳性标本进行疫苗血清型特异性肺炎球菌多糖的筛查。由于缺乏 MEF 标本，因此使用脑脊液（CSF）作为替代基质，用于开发和验证 PCR-PAD 检测方法。单独评估了 PCR 和 PAD 检测方法的敏感性和特异性。随后，将 PCR-PAD 检测方法与人类 MEF 标本（n=39）进行交叉验证，这些 MEF 标本的培养结果证实含有相关细菌株。PCR-PAD 联合检测方法与这些 MEF 标本的历史 Quellung 血清型数据具有高度一致性，达到 94.9%（95%CI；82.7，99.4%）。这种新型的 PCR-PAD 检测方法证明了结合分子和免疫检测方法筛选和鉴定 AOM 临床样本中 PCV15 肺炎球菌疫苗血清型的可行性。
