Haugum Kjersti, Ravi Anuradha, Afset Jan Egil, Ås Christina Gabrielsen
Department of Medical Microbiology, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway.
Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, NTNU, Trondheim, Norway.
PLoS One. 2025 Sep 2;20(9):e0331288. doi: 10.1371/journal.pone.0331288. eCollection 2025.
Infectious gastroenteritis is a significant health issue globally. Identifying the causative pathogen is crucial for treatment, infection control and epidemiological surveillance. While PCR-based analyses are fast and sensitive, they only detect known pathogens. Clinical metagenomics can potentially identify novel or unexpected pathogens. This study aimed to evaluate shotgun metagenomics for detecting diarrhoeal pathogens in faecal samples from patients with infectious gastroenteritis and spiked samples from healthy donors, compared to PCR. DNA from clinical faecal samples (n = 12), spiked samples (n = 36), and control samples (n = 7) were analysed by PCR and shotgun metagenomics sequencing. Reads were taxonomically assigned, assembled, and binned into MAGs. MAGs were taxonomically assigned, and virulence genes were detected in bacterial assemblies and MAGs. Pathogens detected by PCR were also identified by taxonomic assignment of reads, though with lower sensitivity. Taxonomic assignment of MAGs identified 50% of bacterial pathogens and HAdV-F. Additional potential pathogens were observed in most samples. More bacterial virulence genes were detected in assemblies than in MAGs. In spiked samples, C. jejuni and HAdV-F were detected by both PCR and metagenomics, with significant correlation between Cq values and reads. Parasites were detected by few reads. Metagenomics has lower sensitivity compared to PCR but can provide supplementary information relevant for treatment. Challenges include additional potential pathogens, background microbiome, and introduced kitome, necessitating optimized extraction methods and strict quality controls.
感染性肠胃炎是全球一个重要的健康问题。确定致病病原体对于治疗、感染控制和流行病学监测至关重要。虽然基于聚合酶链反应(PCR)的分析快速且灵敏,但它们只能检测已知病原体。临床宏基因组学有可能识别新的或意外的病原体。本研究旨在评估鸟枪法宏基因组学在检测感染性肠胃炎患者粪便样本以及健康供体加标样本中腹泻病原体方面的效果,并与PCR进行比较。对临床粪便样本(n = 12)、加标样本(n = 36)和对照样本(n = 7)的DNA进行PCR和鸟枪法宏基因组学测序分析。读取的序列进行分类学注释、组装,并归类到宏基因组组装基因组(MAGs)中。对MAGs进行分类学注释,并在细菌组装体和MAGs中检测毒力基因。通过PCR检测到的病原体也通过读取序列的分类学注释得以识别,不过灵敏度较低。MAGs的分类学注释识别出了50%的细菌病原体和人腺病毒F型(HAdV-F)。在大多数样本中还观察到了其他潜在病原体。在组装体中检测到的细菌毒力基因比在MAGs中更多。在加标样本中,空肠弯曲菌和HAdV-F通过PCR和宏基因组学均被检测到,Cq值与读取序列之间存在显著相关性。通过很少的读取序列检测到了寄生虫。与PCR相比,宏基因组学的灵敏度较低,但可以提供与治疗相关的补充信息。挑战包括其他潜在病原体、背景微生物群和引入的试剂盒组,这需要优化提取方法和严格的质量控制。
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