Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Demultiplexed raw sequencing data files (fastq.gz

Metagenomic sequencing complements routine diagnostics in identifying viral pathogens in lung transplant recipients with unknown etiology of respiratory infection

BACKGROUND: Lung transplant patients are a vulnerable group of immunosuppressed patients that are prone to frequent respiratory infections. We studied 60 episodes of respiratory symptoms in 71 lung transplant patients. Almost half of these episodes were of unknown infectious etiology despite extensive routine diagnostic testing. METHODS: We re-analyzed respiratory samples of all episodes with undetermined etiology in order to detect potential viral pathogens missed/not accounted for in routine diagnostics. Respiratory samples were enriched for viruses by filtration and nuclease digestion, whole nucleic acids extracted and randomly amplified before high throughput metagenomic virus sequencing. Viruses were identified by a bioinformatic pipeline and confirmed and quantified using specific real-time PCR. RESULTS: In completion of routine diagnostics, we identified and confirmed a viral etiology of infection by our metagenomic approach in four patients (three Rhinovirus A, one Rhinovirus B infection) despite initial negative results in specific multiplex PCR. Notably, the majority of samples were also positive for Torque teno virus (TTV) and Human Herpesvirus 7 (HHV-7). While TTV viral loads increased with immunosuppression in both throat swabs and blood samples, HHV-7 remained at low levels throughout the observation period and was restricted to the respiratory tract. CONCLUSION: This study highlights the potential of metagenomic sequencing for virus diagnostics in cases with previously unknown etiology of infection and in complex diagnostic situations such as in immunocompromised hosts

Correction to: Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Abstract Following publication of the original article [1], the authors were alerted by a colleague of a column duplication in Table 1. Since the summary row was correct, though, the interpretation and the conclusion of the article were not affected

Persistent mammalian orthoreovirus, coxsackievirus and adenovirus co-infection in a child with a primary immunodeficiency detected by metagenomic sequencing: a case report

Background: We report a rare case of Mammalian orthoreovirus (MRV) infection in a child with a primary immunodeficiency (PID). Infections with Mammalian orthoreovirus are very rare and probably of zoonotic origin. Only a few cases have been described so far, including one with similar pathogenesis as in our case. Case presentation: The patient, age 11, presented with flu-like symptoms and persistent severe diarrhea. Enterovirus has been detected over several months, however, exact typing of a positive cell culture remained inconclusive. Unbiased metagenomic sequencing then detected MRV in stool samples from several time points. The sequencing approach further revealed co-infection with a recombinant Coxsackievirus and Adenovirus. MRV-specific antibodies detected by immunofluorescence proved that the patient seroconverted. Conclusion: This case highlights the potential of unbiased metagenomic sequencing in supplementing routine diagnostic methods, especially in situations of chronic infection with multiple viruses as seen here in an immunocompromised host. The origin, transmission routes and implications of MRV infection in humans merit further investigation

TTV and HHV-7 viral loads did not correlate with symptoms.

<p>TTV virus loads in blood samples (A), TTV viral loads in throat swabs (B) and HHV-7 viral loads in throat swabs (C). HC: healthy controls, B: baseline (time of transplantation) of baseline enrollment patients, S: symptomatic visits of both enrollment scenarios, NS: non-symptomatic visits of both enrollment scenarios. Undetermined values were set to -2 log10 copies/μl for plotting and statistical analysis. Error bars show median and interquartile ranges, <i>p</i>-values are calculated with the Mann-Whitney U test.</p

Virus reads reported by the bioinformatic pipeline.

<p>Virus reads reported by the bioinformatic pipeline.</p