Public Health Laboratory (GGD) Amsterdam, Department of infectious diseases, Amsterdam, The Netherlands.
Medical Microbiology Laboratory, Maasstad ziekenhuis, Rotterdam, The Netherlands.
PLoS One. 2020 Jun 4;15(6):e0233990. doi: 10.1371/journal.pone.0233990. eCollection 2020.
Typing of Chlamydia trachomatis (CT) is traditionally performed by characterising the ompA gene, resulting in more than a dozen different genovars, A to L. Type L is associated with Lymphogranuloma venereum (LGV) and commonly screened for using PCR, targeting the chromosomal pmpH gene. We aimed to develop and validate a new CT/LGV plasmid-based typing assay targeting the pgp3 gene, to increase sensitivity and thus reduce the number of non-typeable results.
The new pgp3 PCR assay using LNA probes to detect point mutations was analytically and prospectively validated in a routine diagnostic laboratory setting. For the analytical tests, quantified nucleotide constructs (gBlocks) were used to perform limit of detection analyses. Quality control panel samples from 2018 and 2019 for CT were also tested. For the clinical study patient samples which were collected in two months in 2018 were tested simultaneously using the pmpH PCR and the pgp3 PCR.
Analytically, the assay proved to be 100% specific relative to the previously used LGV typing assay targeting the single copy pmpH gene but it was much more sensitive to detect non-LGV CT. In the quality control panel 2 nonLGV samples and 7 LGV samples were solely positive with the pgp3 PCR and not with the pmpH PCR. None of the samples from analytical specificity panels were positive, indicating 100% specificity. In a prospective panel of 152 clinical samples, 142 (93%) were successfully typed with the pgp3 PCR compared to 78% with the pmpH PCR. The pgp3 PCR was fully concordant with the pmpH PCR to identify all LGV subtypes and detected an increased number of clinical samples of non-LGV subtype.
We developed and validated a sensitive and specific plasmid-based typing assay to discriminate LGV from non-LGV CT subtypes. This is useful in a clinical setting to quickly determine the optimal treatment for Chlamydia trachomatis infections.
沙眼衣原体（CT）的分型传统上是通过特征分析ompA 基因来完成的，这导致了 16 种以上不同的血清型，从 A 到 L。血清型 L 与性病淋巴肉芽肿（LGV）有关，通常使用针对染色体 pmpH 基因的 PCR 进行筛查。我们旨在开发和验证一种新的基于 CT/LGV 质粒的 pgp3 基因检测方法，以提高灵敏度，从而减少无法分型的结果数量。
新的 pgp3 PCR 检测方法使用 LNA 探针检测点突变，在常规诊断实验室环境中进行了分析和前瞻性验证。对于分析测试，使用定量核苷酸构建体（gBlocks）进行检测极限分析。还测试了 2018 年和 2019 年 CT 的质量控制面板样本。对于临床研究，同时使用 pmpH PCR 和 pgp3 PCR 检测了 2018 年两个月收集的患者样本。
分析表明，该检测方法与之前针对单拷贝 pmpH 基因的 LGV 分型检测相比具有 100%的特异性，但对非 LGV CT 的检测更为敏感。在质量控制面板中，2 个非 LGV 样本和 7 个 LGV 样本仅用 pgp3 PCR 检测呈阳性，而用 pmpH PCR 检测呈阴性。分析特异性面板中的样本均未呈阳性，表明特异性为 100%。在 152 个临床样本的前瞻性样本中，142 个（93%）成功用 pgp3 PCR 进行了分型，而用 pmpH PCR 分型的比例为 78%。pgp3 PCR 与 pmpH PCR 完全一致，可识别所有 LGV 亚型，并检测到更多非 LGV 亚型的临床样本。
我们开发并验证了一种敏感且特异性的质粒分型检测方法，用于区分 LGV 和非 LGV CT 亚型。这在临床环境中有助于快速确定沙眼衣原体感染的最佳治疗方法。
