PDF(Pubmed)
Abstract：
The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires\' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires\' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.
摘要：
全基因组测序（WGS）的前所未有的精度和分辨率可以为流行病学暴发跟踪提供明确的传染病原鉴定。WGS接近，然而,经常受到从可用的原始样本或不可培养的样本中回收低病原体DNA的阻碍。开发了一种经济有效的混合捕获测定法，用于直接在主要标本上进行嗜肺军团菌WGS分析。用这种方法富集了各种痰和尸检标本的DNA，该标本对嗜肺乳杆菌血清群1（LPSG1）呈PCR阳性，并进行了WGS。确定所有测试的标本都富含军团菌读数（高达209,000倍），当没有临床分离株可用时，显著提高了比较亲缘关系的辨别能力。我们发现，与匹配的培养分离株的WGS数据相比，来自某些富集标本的WGS数据差异不到五个单核苷酸多态性（SNP）。此测试和分析回顾性地提供了以前未经证实的与痰液和尸检肺组织的临床标本的环境来源的联系。后者提供了确定与纽约市南布朗克斯2015军团病（LD）调查相关的这些文化阴性病例的来源所需的其他信息。这种新方法为在暴发调查期间结合WGS和生物信息学分析的未来直接临床标本混合捕获富集提供了概念证明。IMPORTANCELgionnaires病（LD）是一种严重且可能致命的肺炎类型，主要由人造水或冷却系统吸入军团菌污染的气溶胶引起。由于它是一种罕见的肺炎形式，因此LD仍然诊断不足，并且依赖于临床医生将其包括在差异中并要求进行专门测试。此外,从LD病例中获取临床下呼吸道标本是具有挑战性的，如果有的话，文化需要专门的媒介和生长条件，并非所有微生物实验室都有。在目前的研究中,开发了一种通过RNA诱饵杂交捕获嗜肺军团菌的方法，这使我们能够从嗜肺乳杆菌血清群1PCR阳性临床标本中产生足够的基因组分辨率。这种新方法为监测未来LD爆发提供了额外的工具，在这些疾病中无法隔离军团菌，并且可能有助于解决过去LD调查中以前未解决的问题。
