Limited Correlation of Shotgun Metagenomics Following Host Depletion and Routine Diagnostics for Viruses and Bacteria in Low Concentrated Surrogate and Clinical Samples

ORIGINAL RESEARCH published: 23 October 2018 doi: 10.3389/fcimb.2018.00375 Limited Correlation of Shotgun Metagenomics Following Host Depletion and Routine Diagnostics for Viruses and Bacteria in Low Concentrated Surrogate and Clinical Samples Corinne P. Oechslin 1,2,3 , Nicole Lenz 1,2 , Nicole Liechti 1,3,4 , Sarah Ryter 1 , Philipp Agyeman 2,5 , Rémy Bruggmann 4 , Stephen L. Leib 2 and Christian M. Beuret 1* 1 Biology Division, Spiez Laboratory, Swiss Federal Office for Civil Protection, Spiez, Switzerland, 2 Institute for Infectious Diseases, University of Bern, Bern, Switzerland, 3 Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland, 4 Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of Bern, Bern, Switzerland, 5 Infectious Diseases Division, Department of Paediatrics, University Hospital Bern, Bern, Switzerland Edited by: Ran Nir-Paz, Hebrew University of Jerusalem, Israel Reviewed by: Sharon Amit, Hadassah Medical Center, Israel David William Waite, University of Auckland, New Zealand *Correspondence: Christian M. Beuret Received: 08 June 2018 Accepted: 05 October 2018 Published: 23 October 2018 Citation: Oechslin CP, Lenz N, Liechti N, Ryter S, Agyeman P, Bruggmann R, Leib SL and Beuret CM (2018) Limited Correlation of Shotgun Metagenomics Following Host Depletion and Routine Diagnostics for Viruses and Bacteria in Low Concentrated Surrogate and Clinical Samples. Front. Cell. Infect. Microbiol. 8:375. doi: 10.3389/fcimb.2018.00375 The etiologic cause of encephalitis, meningitis or meningo-encephalitis is unknown in up to 70% of cases. Clinical shotgun metagenomics combined with host depletion is a promising technique to identify infectious etiologies of central nervous system (CNS) infections. We developed a straightforward eukaryotic host nucleic acid depletion method that preserves intact viruses and bacteria for subsequent shotgun metagenomics screening of clinical samples, focusing on cerebrospinal fluid (CSF). A surrogate CSF sample for a CNS infection paradigm was used to evaluate the proposed depletion method consisting of selective host cell lysis, followed by enzymatic degradation of the liberated genomic DNA for final depletion with paramagnetic beads. Extractives were subjected to reverse transcription, followed by whole genome amplification and next generation sequencing. The effectiveness of the host depletion method was demonstrated in surrogate CSF samples spiked with three 1:100 dilutions of Influenza A H3N2 virus (qPCR Ct-values 20.7, 28.8, >42/negative). Compared to the native samples, host depletion increased the amount of the virus subtype reads by factor 7127 and 132, respectively, while in the qPCR negative sample zero vs. 31 (1.4E-4 %) virus subtype reads were detected (native vs. depleted). The workflow was applied to thirteen CSF samples of patients with meningo-/encephalitis (two bacterial, eleven viral etiologies), a serum of an Andes virus infection and a nose swab of a common cold patient. Unlike surrogate samples, host depletion of the thirteen human CSF samples and the nose swab did not result in more reads indicating presence of damaged pathogens due to, e.g., host immune response. Nevertheless, previously diagnosed pathogens in the human CSF samples (six viruses, two bacteria), the serum, and the nose swab (Human rhinovirus A31) were detected in the depleted and/or the native samples. Unbiased evaluation of the taxonomic profiles supported the diagnosed pathogen in two native CSF samples and the native and depleted serum and nose swab, while detecting Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 October 2018 | Volume 8 | Article 375 Oechslin et al. Viral and Bacterial Metagenomic Diagnosis various contaminations that interfered with pathogen identification at low concentration levels. In summary, damaged pathogens and contaminations complicated analysis and interpretation of clinical shotgun metagenomics data. Still, proper consideration of these issues may enable future application of metagenomics for clinical diagnostics. Keywords: central nervous system infection, CSF, diagnostics, viruses, bacteria, NGS, shotgun metagenomics, host depletion INTRODUCTION bacterial or viral DNA, or are only based on RNA sequencing targeting bacterial 16S or viral RNA (Allander et al., 2001; Hall et al., 2014; Jensen et al., 2015; Kohl et al., 2015; Lewandowska et al., 2015; Ruppé et al., 2017; Sabat et al., 2017). This study aimed to establish a straightforward shotgun metagenomics workflow to simultaneously screen for both, bacteria and viruses in liquid clinical samples, focusing on human CSF. A simple host nucleic acid depletion method was developed to minimize the overwhelming host NA proportion, thus enriching presumed viral and bacterial NA within a patient’s sample. Surrogate CSF samples were developed to model inflammatory CSF of patients with meningo-/encephalitis to validate the shotgun metagenomics workflow comparing depleted to native aliquots. The novel approach was applied to clinical CSF samples, a human nose swab and a serum sample. The proposed host NA depletion method successfully increased viral and bacterial reads in surrogate CSF samples, which was not reproducible with clinical samples. The previously diagnosed pathogens were detected by shotgun metagenomics in the majority of samples but evident etiology was affected by low concentrations of pathogens, numerous contaminations and curation and extent of reference databases for bioinformatics analysis. Nonetheless, our shotgun metagenomics workflow is a novel approach to simultaneously detect RNA and DNA viruses as well as bacteria applying a total host nucleic acid depletion method. Various known pathogens, including viruses, bacteria, fungi and parasites, cause severe infections of the central nervous system (CNS) accounting for 30–50% of cases of meningitis, encephalitis, and meningo-encephalitis (Glaser et al., 2006; Mailles and Stahl, 2009; Granerod et al., 2010). Mortality can be high as observed with the DNA virus Herpes simplex (treated 25%, untreated 70%), which causes the most lethal viral CNS infections endemic in the USA (George et al., 2014; Whitley, 2015). The etiology of meningo-/encephalitis cannot be identified in up to 70% of the total cases (Cizman and Jazbec, 1993; Sivertsen and Christensen, 1996; Khetsuriani et al., 2002; Glaser et al., 2006). Diagnosis of CNS infections, which is routinely performed in cerebrospinal fluid (CSF), remains a great challenge. Patients are usually in a severe state of health, thus broad-spectrum antibiotics and antivirals are frequently administered prior to CSF sampling affecting the diagnostic outcome. Moreover, long lasting routine liquor cultivation remains difficult as the majority of species are non-cultivable. Finally, fast routine molecular diagnostics by polymerase chain reaction (PCR) is restricte (...truncated)