Luu Laurence Don Wai, Rafique Raisa, Payne Michael, Octavia Sophie, Robson Jennifer, Sintchenko Vitali, Lan Ruiting
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia.
School of Life Sciences, University of Technology Sydney, Sydney, New South Wales, Australia.
J Clin Microbiol. 2024 Dec 11;62(12):e0117824. doi: 10.1128/jcm.01178-24. Epub 2024 Nov 4.
Whooping cough (pertussis) has re-emerged despite high vaccine coverage in Australia and many other countries worldwide, partly attributable to genetic adaptation of the causative organism, to vaccines. Therefore, genomic surveillance has become essential to monitor circulating strains for these genetic changes. However, increasing uptake of PCR for the diagnosis of pertussis has affected the availability of cultured isolates for typing. In this study, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of directly from respiratory swabs. We developed a nine-target mPCR amplicon assay that could accurately type major lineages [non-, , non- and epidemic lineages (ELs) 1-5] circulating in Australia. Validation using DNA from isolates and 178 residual specimens collected in 2010-2012 ( = 87) and 2019 ( = 91) showed that mPCR amplicon sequencing was highly sensitive with a limit of detection of 4.6 copies [IS cycle threshold (Ct) 27.3]. Shotgun metagenomic sequencing was successful in genotyping in 84% of clinical specimens with PCR Ct < 24 and was concordant with mPCR typing results. The results revealed an expansion of EL4 strains from 2010 to 2012 to 2019 in Australia and identified unrecognized co-circulating cases of . This study provides valuable insight into the circulating lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low, and paves the way for the genomic surveillance of in the era of culture-independent PCR-based diagnosis.
In this paper, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of directly in respiratory swabs. We first developed a novel targeted mPCR amplicon sequencing assay that can type major circulating lineages and validated its accuracy and sensitivity on 178 DNA extracts from clinical swabs. We also demonstrate the feasibility of using deep metagenomic sequencing for determining strain lineage and markers of virulence, vaccine adaptation, macrolide resistance, and co-infections. Our culture-independent typing methods applied to clinical specimens revealed the expansion of a major global epidemic lineage in Australia (termed EL4) just prior to the COVID-19 pandemic. It also detected cases of previously hidden co-infections from another species called . These findings offer valuable insight into the circulating pertussis lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low. It also provides comparative data for future surveillance as pertussis resurgence after the COVID-19 pandemic has been reported this year in Australia and many other countries. Overall, our paper demonstrates the utility, sensitivity, and specificity of mPCR amplicon and metagenomic sequencing-based culture-independent typing of , which not only paves the way for culture-independent genomic surveillance of but also for other pathogens in the era of PCR-based diagnosis.
尽管澳大利亚以及全球许多其他国家的疫苗接种率很高，但百日咳仍再度出现，部分原因是致病生物体对疫苗产生了基因适应性变化。因此，基因组监测对于监测流行菌株的这些基因变化至关重要。然而，用于百日咳诊断的PCR检测方法使用的增多，影响了用于分型的培养分离株的可获得性。在本研究中，我们评估了靶向多重PCR（mPCR）扩增子测序和鸟枪法宏基因组测序用于直接从呼吸道拭子中进行非培养分型的情况。我们开发了一种九靶点mPCR扩增子检测方法，该方法能够准确地对在澳大利亚流行的主要谱系（非、、非和流行谱系（ELs）1 - 5）进行分型。使用2010 - 2012年（ = 87）和2019年（ = 91）收集的分离株DNA以及178份残留标本进行验证，结果表明mPCR扩增子测序高度灵敏，检测限为4.6拷贝[插入序列循环阈值（Ct）27.3]。鸟枪法宏基因组测序在84%的PCR Ct < 24的临床标本中成功进行了基因分型，并且与mPCR分型结果一致。结果显示，在澳大利亚，EL4菌株从2010年至2012年到2019年有所扩张，并发现了未被识别的共循环病例。本研究为在新冠疫情大流行之前澳大利亚的流行谱系提供了有价值的见解，在此期间边境关闭和其他干预措施使百日咳病例降至历史最低水平，并为基于非培养PCR诊断时代的百日咳基因组监测铺平了道路。
在本文中，我们评估了靶向多重PCR（mPCR）扩增子测序和鸟枪法宏基因组测序用于直接在呼吸道拭子中进行百日咳非培养分型的情况。我们首先开发了一种新型靶向mPCR扩增子测序检测方法，该方法能够对主要流行谱系进行分型，并在178份临床拭子DNA提取物上验证了其准确性和灵敏度。我们还证明了使用深度宏基因组测序来确定菌株谱系以及毒力、疫苗适应性、大环内酯抗性和共感染标志物的可行性。我们应用于临床标本的非培养分型方法显示，在新冠疫情大流行之前，澳大利亚一种主要的全球流行谱系（称为EL4）有所扩张。它还检测到了来自另一种名为的物种的先前隐藏的共感染病例。这些发现为新冠疫情大流行之前澳大利亚的百日咳流行谱系提供了有价值的见解，在此期间边境关闭和其他干预措施使百日咳病例降至历史最低水平。它还为今年澳大利亚和许多其他国家报告的新冠疫情大流行后百日咳复苏的未来监测提供了比较数据。总体而言，我们的论文证明了基于mPCR扩增子和宏基因组测序的百日咳非培养分型的实用性、灵敏度和特异性，这不仅为百日咳的非培养基因组监测铺平了道路，也为基于PCR诊断时代的其他病原体监测铺平了道路。
