Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Comparison of the performance of two targeted metagenomic virus capture probe-based methods using reference control materials and clinical samples.

Viral enrichment by probe hybridization has been reported to significantly increase the sensitivity of viral metagenomics. This study compares the analytical performance of two targeted metagenomic virus capture probe-based methods: (i) SeqCap EZ HyperCap by Roche (ViroCap) and (ii) Twist Comprehensive Viral Research Panel workflow, for diagnostic use. Sensitivity, specificity, and limit of detection were analyzed using 25 synthetic viral sequences spiked in increasing proportions of human background DNA, eight clinical samples, and American Type Culture Collection (ATCC) Virome Virus Mix. Sensitivity and specificity were 95% and higher for both methods using the synthetic and reference controls as gold standard. Combining thresholds for viral sequence read counts and genome coverage [respectively 500 reads per million (RPM) and 10% coverage] resulted in optimal prediction of true positive results. Limits of detection were approximately 50-500 copies/mL for both methods as determined by ddPCR. Increasing proportions of spike-in cell-free human background sequences up to 99.999% (50 ng/mL) did not negatively affect viral detection, suggesting effective capture of viral sequences. These data show analytical performances in ranges applicable to clinical samples, for both probe hybridization metagenomic approaches. This study supports further steps toward more widespread use of viral metagenomics for pathogen detection, in clinical and surveillance settings using low biomass samples. IMPORTANCE: Viral metagenomics has been gradually applied for broad-spectrum pathogen detection of infectious diseases, surveillance of emerging diseases, and pathogen discovery. Viral enrichment by probe hybridization methods has been reported to significantly increase the sensitivity of viral metagenomics. During the past years, a specific hybridization panel distributed by Roche has been adopted in a broad range of different clinical and zoonotic settings. Recently, Twist Bioscience has released a new hybridization panel targeting human and animal viruses. This is the first report comparing the performance of viral metagenomic hybridization panels.

A comparison of five Illumina, Ion Torrent, and nanopore sequencing technology-based approaches for whole genome sequencing of SARS-CoV-2.

Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies. A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Correlation of coverage with PCR Ct values varied per protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 log10 at disbalanced positions in samples with high viral loads (Ct values ≤ 23). Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. In conclusion, the studied protocols differed on a variety of the studied metrics. This study provides data that assist laboratories when selecting protocols for their specific setting.

Pre-COVID-19 international travel and admission to hospital when back home: travel behavior, carriage of highly resistant microorganisms, and risk perception of patients admitted to a large tertiary care hospital.

BACKGROUND: When people who recently travelled abroad are admitted to a hospital back home, there is a risk of introducing highly resistant microorganisms (HRMO) into the hospital. To minimize this risk, a feasible infection prevention strategy should be developed. In this study, we investigated patients' travel history and behavior during travel and analyzed whether this was correlated to HRMO carriage at admission. METHODS: From May 2018 until August 2019, adult patients admitted to a large tertiary care center in the Netherlands were asked upon hospital admission to participate in the study. Included patients received a questionnaire about risk perception, travel history in the last year, and behavior during travel, and were screened for HRMO carriage at admission using a perianal swab. RESULTS: Six hundred and eight questionnaires were handed out, of which 247 were returned (40.6%). One hundred and thirty (52.6%) patients did not travel abroad in the last year, of whom eight (6.2%) were HRMO carrier at admission. One hundred seventeen (47.4%) patients travelled in the preceding year, of whom seven patients (6.0%) were HRMO carrier at admission. Thirty patients (12%) travelled outside of Europe; in this group HRMO prevalence was 13.3% (4 out of 30). The majority of patients (71.3%) were aware that international travel could lead to carriage of HRMO, and an even larger majority (89.5%) would support a screening strategy upon hospital admission in case of a travel history, to minimize the risk of introducing HRMO. CONCLUSIONS: We identified that half of admitted patients to a large tertiary care hospital travelled abroad in the last year, with only a small percentage outside Europe. We discuss several screening strategies and propose a strategy of screening and preemptive isolation of patients who travelled to Asia or Africa in the 2 months before their hospital admission; a strategy that patients would support.

Acquisition of multidrug-resistant Enterobacterales during international travel: a systematic review of clinical and microbiological characteristics and meta-analyses of risk factors.

BACKGROUND: International tourism increased from 25 million tourist arrivals in 1950 to over 1.3 billion in 2017. These travelers can be exposed to (multi) resistant microorganisms, may become colonized, and bring them back home. This systematic review aims to identify the carriage rates of multidrug-resistant Enterobacterales (MDR-E) among returning travelers, to identify microbiological methods used, and to identify the leading risk factors for acquiring MDR-E during international travel. METHODS: Articles related to our research question were identified through a literature search in multiple databases (until June 18, 2019) - Embase, Medline Ovid, Cochrane, Scopus, Cinahl, Web of Science, and Google Scholar. RESULTS: Out of 3211 potentially relevant articles, we included 22 studies in the systematic review, and 12 studies in 7 random-effects meta-analyses. Highest carriage rates of MDR-E were observed after travel to Southern Asia (median 71%), followed by travel to Northern Africa (median 42%). Carbapenemase-producing Enterobacterales (CPE) were identified in 5 out of 22 studies, from a few patients. However, in only eight out of 22 studies (36.4%) the initial laboratory method targeted detection of the presence of CPE in the original samples. The risk factor with the highest pooled odds ratio (OR) for MDR-E was travel to Southern Asia (pooled OR = 14.16, 95% confidence interval [CI] = 5.50 to 36.45), followed by antibiotic use during travel (pooled OR = 2.78, 95% CI = 1.76 to 4.39). CONCLUSIONS: Risk of acquiring MDR-E while travelling increases depending on travel destination and if antibiotics are used during travel. This information is useful for the development of guidelines for healthcare facilities with low MDR-E prevalence rates to prevent admission of carriers without appropriate measures. The impact of such guidelines should be assessed.

Various neurocognitive deficits and conversion risk in individuals at clinical high risk for psychosis.

AIM: Individuals at clinical high risk for psychosis (CHR) exhibit neurocognitive deficits in multiple domains. The aim of this study is to investigate whether several components of neurocognition are predictive of conversion to psychosis. METHODS: Fifty-two CHR individuals were assessed with the Structured Interview for Psychosis Risk Syndromes and completed a battery of neurocognitive tests at baseline including measures of executive functioning, attention, working memory, processing speed and reaction time. Neurocognitive functioning at baseline was scored based on an external normative control group. Most subjects were followed for 2.5 years to determine conversion status. RESULTS: Significant differences in neurocognitive functioning between CHR individuals and the control group were present in all domains. Twenty-six per cent of the participants converted to psychosis within 9.8 (standard deviation = 8.0) months on average (median 9 months), but there were no significant differences in neurocognition converters and non-converters. CONCLUSIONS: Individuals at CHR have deficits in neurocognitive functioning, but such deficits do not appear to be related to conversion risk.