title: Surveying the virome of paediatric patients with sepsis using Next Generation Sequencing
Creators: name: Thrupp, Christopher G
Publisher: name: Griffith University
Year: 2021
Subjects: subject: next generation sequencing
subject: identify
subject: viral pathogens
subject: sepsis

description: Background: Modern day science has enabled the understanding and ability to treat many of society’s prevalent diseases. Sepsis, however, is not one of these diseases. Within the developed world 2.5 million people are admitted to hospital with sepsis each year, and approximately 650,000 people die from sepsis each year. Globally, sepsis results in approximately 19 million cases per year, and an estimated 5 million deaths [1]. Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection according to the 2016 sepsis 3 definition [2]. Current treatment for sepsis includes supportive therapies and antibiotic administration [3], however problems arise when the pathogenic agent is viral and not bacterial, as antibiotics are ineffective against viral infections. Consequently, novel therapies are required to treat viral sepsis. Next generation sequencing (NGS) offers researchers the ability to identify multiple organisms in a complex system without culturing or a priori knowledge of the microorganism’s nucleic acid sequences. This system provides a means of characterising the population of viruses that cause sepsis, thus paving the way for the development of rapid detection kits, as well enabling clinicians the confidence to distinguish viral from bacterial sepsis. Methodology: Whole blood and plasma samples were filtered to remove host/bacterial contaminates. To further purify nucleic acids for sequencing the samples were subjected to DNase enzyme treatment followed by phenol-chloroform extraction coupled to sodium acetate precipitation. Sample DNA content was then quantified using the high sensitivity Quant-iT assay (Invitrogen). Samples with insufficient content were not sequenced. Sample library preparation was carried out using the Nextera DNA Flex kit and the prepared sample library quality was assessed using the bioanalyzer 2100 high sensitivity DNA assay (Agilent). Samples were then sequenced via the Illumina Miseq platform using 5% phi x as an internal positive control. Results: After performing bioinformatic analysis on the generated reads, respiratory syncytial virus (RSV), adenovirus, anellovirus, chikungunya (CHIV), enterovirus, sindbis virus (SINV) and cytomegalovirus (CMV) were identified within the sequenced samples. Though not all identified viruses have been documented to cause sepsis, all have been documented to be associated with immune incompetence. It was also discovered that bacteriophages pertaining to sepsis causing bacteria were found within the samples (i.e. phage typing), thus indicating a potential future area of study. Conclusions: This study demonstrates how next generation sequencing can be utilised to identify viral pathogens that cause sepsis, in the absence of a prior knowledge of the pathogens. Further this study highlights the viruses that can be associated with sepsis in children. Phages were shown to describe bacteria responsible for sepsis and this method may pave the way for future studies. Therefore, future studies should aim to take the information pertaining to the sepsis causing pathogens shown here and develop methods for rapid identification.