Table_2_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
目前，用于检测肺炎链球菌(Streptococcus pneumoniae)定植的分子检测方法的特异性尚存争议。本研究提出一种用于定植监测与疫苗影响研究的实验流程，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
本研究采用培养法与实时荧光定量PCR(qPCR)，对来自荷兰（972份）、英格兰（577份）的1549份鼻咽拭子样本，以及仅取自319名荷兰成年人的配对口咽拭子样本进行肺炎链球菌及其血清型检测。上述鼻咽拭子样本采集自946名幼儿与603名成年人。对于初次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指导下的二次培养进行复检。以初次培养与qPCR指导培养分离得到的活肺炎链球菌作为参照，通过受试者工作特征(Receiver Operating Characteristic, ROC)曲线分析确定qPCR检测阳性的最优Cq阈值。
结果
对经培养（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具有近乎完美的一致性（科恩kappa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，且采用qPCR指导的培养法可显著提升可分离到活肺炎链球菌的鼻咽拭子与口咽拭子样本占比（p < 0.0001）。针对成年人的配对鼻咽拭子与口咽拭子样本，仅基于单一样本类型的检测方法，与初次培养联合qPCR指导培养的联合检测结果均未表现出良好一致性（科恩kappa系数：0.13~0.55）。然而，与单一鼻咽拭子或口咽拭子初次培养相比，采用培养富集的口咽拭子样本进行肺炎链球菌分子检测的灵敏度更高（p < 0.05）。
结论
结合qPCR与传统培养法（反之亦然），并通过传统培养与qPCR指导培养的结果解读qPCR数据，可大幅提升肺炎链球菌定植监测的准确性。基于受试者工作特征曲线分析的统计学流程，可增强分子检测活肺炎链球菌方法的特异性。本研究提出的实验流程可应用于未来的定植监测与疫苗影响研究，尤其可提升成年人肺炎链球菌定植的检测效能，并优化血清型定植检测的特异性。