Table_7_It Takes Two to Tango: Combining Conventional Culture With Molecular Diagnostics Enhances Accuracy of Streptococcus pneumoniae Detection and Pneumococcal Serogroup/Serotype Determination in Carriage.docx

BackgroundThe specificity of molecular methods for the detection of Streptococcus pneumoniae carriage is under debate. We propose a procedure for carriage surveillance and vaccine impact studies that increases the accuracy of molecular detection of live pneumococci in polymicrobial respiratory samples.
MethodsCulture and qPCR methods were applied to detect pneumococcus and pneumococcal serotypes in 1,549 nasopharyngeal samples collected in the Netherlands (n = 972) and England (n = 577) from 946 toddlers and 603 adults, and in paired oropharyngeal samples collected exclusively from 319 Dutch adults. Samples with no live pneumococci isolated at primary diagnostic culture yet generating signal specific for pneumococcus in qPCRs were re-examined with a second, qPCR-guided culture. Optimal Cq cut-offs for positivity in qPCRs were determined via receiver operating characteristic (ROC) curve analysis using isolation of live pneumococci from the primary and qPCR-guided cultures as reference.
ResultsDetection of pneumococcus and pneumococcal serotypes with qPCRs in cultured (culture-enriched) nasopharyngeal samples exhibited near-perfect agreement with conventional culture (Cohen’s kappa: 0.95). Molecular methods displayed increased sensitivity of detection for multiple serotype carriage, and implementation of qPCR-guided culturing significantly increased the proportion of nasopharyngeal and oropharyngeal samples from which live pneumococcus was recovered (p < 0.0001). For paired nasopharyngeal and oropharyngeal samples from adults none of the methods applied to a single sample type exhibited good agreement with results for primary and qPCR-guided nasopharyngeal and oropharyngeal cultures combined (Cohens kappa; 0.13–0.55). However, molecular detection of pneumococcus displayed increased sensitivity with culture-enriched oropharyngeal samples when compared with either nasopharyngeal or oropharyngeal primary cultures (p < 0.05).
ConclusionThe accuracy of pneumococcal carriage surveillance can be greatly improved by complementing conventional culture with qPCR and vice versa, by using results of conventional and qPCR-guided cultures to interpret qPCR data. The specificity of molecular methods for the detection of live pneumococci can be enhanced by incorporating statistical procedures based on ROC curve analysis. The procedure we propose for future carriage surveillance and vaccine impact studies improves detection of pneumococcal carriage in adults in particular and enhances the specificity of serotype carriage detection.

背景
肺炎链球菌（Streptococcus pneumoniae）定植检测的分子方法特异性尚存争议。本研究提出一套用于定植监测与疫苗影响研究的流程，可提升多微生物呼吸道样本中活肺炎链球菌分子检测的准确性。

方法
本研究对荷兰（n=972）与英格兰（n=577）采集的1549份鼻咽拭子样本（来自946名幼儿与603名成人），以及仅从319名荷兰成人采集的配对口咽拭子样本，采用培养法与定量聚合酶链反应（qPCR）检测肺炎链球菌及其血清型。对于原代诊断培养未分离到活肺炎链球菌，但qPCR检测呈现肺炎链球菌特异性信号的样本，采用qPCR引导的二次培养进行复检。以原代培养及qPCR引导培养分离得到的活肺炎链球菌为参照，通过受试者工作特征（Receiver Operating Characteristic, ROC）曲线分析确定qPCR检测阳性的最佳Cq临界值。

结果
对经培养富集的鼻咽拭子样本采用qPCR检测肺炎链球菌及其血清型，与传统培养法的检测结果几乎完全一致（科恩κ系数：0.95）。分子检测方法对多重血清型定植的检测灵敏度更高；实施qPCR引导培养后，可分离到活肺炎链球菌的鼻咽拭子与口咽拭子样本占比显著提升（p < 0.0001）。针对成人的配对鼻咽与口咽拭子样本，仅采用单一样本类型的检测方法，与原代培养及qPCR引导的鼻咽、口咽联合培养结果均未达到良好一致性（科恩κ系数：0.13~0.55）。然而，相较于单一鼻咽或口咽原代培养，经培养富集的口咽拭子样本的肺炎链球菌分子检测灵敏度显著提升（p < 0.05）。

结论
将qPCR与传统培养法互为补充，或通过结合传统培养与qPCR引导培养的结果解读qPCR数据，可大幅提升肺炎链球菌定植监测的准确性。基于ROC曲线分析的统计流程可增强分子检测活肺炎链球菌方法的特异性。本研究提出的这套用于未来定植监测与疫苗影响研究的流程，可显著改善成人肺炎链球菌定植的检测效果，并提升血清型定植检测的特异性。