Genomic characterization of coxsackievirus A22 from a regional university hospital in the Netherlands.

BACKGROUND: Enteroviruses are highly diverse with a wide spectrum of genotypes and clinical manifestations. Coxsackievirus A22 (CVA22) has been detected globally from sewage surveillance; however, currently there is limited information on its prevalence in patients, as well as available genomic data. OBJECTIVE: We aimed to provide genomic and relative frequency data on CVA22 from a regional hospital perspective between 2013-2020. STUDY DESIGN: Sanger sequencing was performed on all samples with a positive enterovirus RT-qPCR result (≤Ct 32). Viral targeted sequence capture (ViroCap) and next-generation sequencing (NGS) (Illumina NextSeq 500) was used to characterize and generate near-complete CVA22 genomes for enteroviruses without genotyping results from Sanger sequencing. Epidemiological and phylogenetic analysis was performed using maximum likelihood trees on MEGA-11. RESULTS: A total of twenty detections derived from fecal material from sixteen patients were observed between 2013- 2020. One transplant recipient had five different CVA22 infection episodes over five years, with phylogenetic analysis indicating possible reinfection with an additional prolonged infection (>3 weeks). Furthermore, we report the first two near-complete CVA22 sequences from Europe, which grouped with a strain previously isolated from Bangladesh in 1999. CONCLUSIONS: We show a highly diverse enterovirus genotype which causes infections annually, typically in autumn and winter, and is capable of recurrent infection in an immunocompromised patient. Furthermore, we highlight the use of NGS to complement conventional targeted Sanger sequencing.

Evaluation of the QIAstat-Dx RP2.0 and the BioFire FilmArray RP2.1 for the Rapid Detection of Respiratory Pathogens Including SARS-CoV-2.

Point-of-care syndromic panels allow for simultaneous and rapid detection of respiratory pathogens from nasopharyngeal swabs. The clinical performance of the QIAstat-Dx Respiratory SARS-CoV-2 panel RP2.0 (QIAstat-Dx RP2.0) and the BioFire FilmArray Respiratory panel RP2.1 (BioFire RP2.1) was evaluated for the detection of SARS-CoV-2 and other common respiratory pathogens. A total of 137 patient samples were retrospectively selected based on emergency department admission, along with 33 SARS-CoV-2 positive samples tested using a WHO laboratory developed test. The limit of detection for SARS-CoV-2 was initially evaluated for both platforms. The QIAstat-Dx RP2.0 detected SARS-CoV-2 at 500 copies/mL and had a positive percent agreement (PPA) of 85%. The BioFire RP2.1 detected SARS-CoV-2 at 50 copies/mL and had a PPA of 97%. Both platforms showed a negative percent agreement of 100% for SARS-CoV-2. Evaluation of analytical specificity from a range of common respiratory targets showed a similar performance between each platform. The QIAstat-Dx RP2.0 had an overall PPA of 82% (67-100%) in clinical samples, with differences in sensitivity depending on the respiratory target. Both platforms can be used to detect acute cases of SARS-CoV-2. While the QIAstat-Dx RP2.0 is suitable for detecting respiratory viruses within a clinical range, it has less analytical and clinical sensitivity for SARS-CoV-2 compared to the BioFire RP2.1.

Exploring a prolonged enterovirus C104 infection in a severely ill patient using nanopore sequencing.

Chronic enterovirus infections can cause significant morbidity, particularly in immunocompromised patients. This study describes a fatal case associated with a chronic untypeable enterovirus infection in an immunocompromised patient admitted to a Dutch university hospital over nine months. We aimed to identify the enterovirus genotype responsible for the infection and to determine potential evolutionary changes. Long-read sequencing was performed using viral targeted sequence capture on four respiratory and one faecal sample. Phylogenetic analysis was performed using a maximum likelihood method, along with a root-to-tip regression and time-scaled phylogenetic analysis to estimate evolutionary changes between sample dates. Intra-host variant detection, using a Fixed Ploidy algorithm, and selection pressure, using a Fixed Effect Likelihood and a Mixed Effects Model of Evolution, were also used to explore the patient samples. Near-complete genomes of enterovirus C104 (EV-C104) were recovered in all respiratory samples but not in the faecal sample. The recovered genomes clustered with a recently reported EV-C104 from Belgium in August 2018. Phylodynamic analysis including ten available EV-C104 genomes, along with the patient sequences, estimated the most recent common ancestor to occur in the middle of 2005 with an overall estimated evolution rate of 2.97 × 10-3 substitutions per year. Although positive selection pressure was identified in the EV-C104 reference sequences, the genomes recovered from the patient samples alone showed an overall negative selection pressure in multiple codon sites along the genome. A chronic infection resulting in respiratory failure from a relatively rare enterovirus was observed in a transplant recipient. We observed an increase in single-nucleotide variations between sample dates from a rapidly declining patient, suggesting mutations are weakly deleterious and have not been purged during selection. This is further supported by the persistence of EV-C104 in the patient, despite the clearance of other viral infections. Next-generation sequencing with viral enrichment could be used to detect and characterise challenging samples when conventional workflows are insufficient.

Off-season circulation and characterization of enterovirus D68 with respiratory and neurological presentation using whole-genome sequencing.

To explore an off-season enterovirus D68 (EV-D68) upsurge in the winter season of 2019/2020, we adapted a whole-genome sequencing approach for Nanopore Sequencing for 20 hospitalized patients with accompanying respiratory or neurological presentation. Applying phylodynamic and evolutionary analysis on Nextstrain and Datamonkey respectively, we report a highly diverse virus with an evolutionary rate of 3.05 × 10-3 substitutions per year (entire EV-D68 genome) and a positive episodic/diversifying selection with persistent yet undetected circulation likely driving evolution. While the predominant B3 subclade was identified in 19 patients, one A2 subclade was identified in an infant presenting with meningitis. Exploring single nucleotide variations using CLC Genomics Server showed high levels of non-synonymous mutations, particularly in the surface proteins, possibly highlighting growing problems with routine Sanger sequencing for typing enteroviruses. Surveillance and molecular approaches to enhance current knowledge of infectious pathogens capable of pandemic potential are paramount to early warning in health care facilities.

Application of shotgun metagenomics sequencing and targeted sequence capture to detect circulating porcine viruses in the Dutch-German border region.

Porcine viruses have been emerging in recent decades, threatening animal and human health, as well as economic stability for pig farmers worldwide. Next-generation sequencing (NGS) can detect and characterize known and unknown viruses but has limited sensitivity when an unbiased approach, such as shotgun metagenomics sequencing, is used. To increase the sensitivity of NGS for the detection of viruses, we applied and evaluated a broad viral targeted sequence capture (TSC) panel and compared it to an unbiased shotgun metagenomic approach. A cohort of 36 pooled porcine nasal swab and blood serum samples collected from both sides of the Dutch-German border region were evaluated. Overall, we detected 46 different viral species using TSC, compared to 40 viral species with a shotgun metagenomics approach. Furthermore, we performed phylogenetic analysis on recovered influenza A virus (FLUAV) genomes from Germany and revealed a close similarity to a zoonotic influenza strain previously detected in the Netherlands. Although TSC introduced coverage bias within the detected viruses, it improved sensitivity, genome sequence depth and contig length. In-depth characterization of the swine virome, coupled with developing new enrichment techniques, can play a crucial role in the surveillance of circulating porcine viruses and emerging zoonotic pathogens.

A discussion of syndromic molecular testing for clinical care.

Current molecular detection methods for single or multiplex pathogens by real-time PCR generally offer great sensitivity and specificity. However, many infectious pathogens often result in very similar clinical presentations, complicating the test-order for physicians who have to narrow down the causative agent prior to in-house PCR testing. As a consequence, the intuitive response is to start empirical therapy to treat a broad spectrum of possible pathogens. Syndromic molecular testing has been increasingly integrated into routine clinical care, either to provide diagnostic, epidemiological or patient management information. These multiplex panels can be used to screen for predefined infectious disease pathogens simultaneously within a 1 h timeframe, creating opportunities for rapid diagnostics. Conversely, syndromic panels have their own challenges and must be adaptable to the evolving demands of the clinical setting. Firstly, questions have been raised regarding the clinical relevance of some of the targets included in the panels and secondly, there is the added expense of integration into the clinical laboratory. Here, we aim to discuss some of the factors that should be considered before performing syndromic testing rather than traditional low-plex in-house PCR.

First detection of porcine respirovirus 1 in Germany and the Netherlands.

Porcine respirovirus 1, also referred to as porcine parainfluenza virus 1 (PPIV-1), was first detected in deceased pigs from Hong Kong in 2013. It has since then been found in the USA, Chile and most recently in Hungary. Information on the pathogenicity and global spread is sparse. However, it has been speculated to play a role in the porcine respiratory disease complex. To investigate the porcine virome, we screened 53 pig samples from 26 farms within the Dutch-German border region using shotgun metagenomics sequencing (SMg). After detecting PPIV-1 in five farms through SMg, a real-time reverse transcriptase PCR (RT-qPCR) assay was designed, which not only confirmed the presence of the virus in 1 of the 5 farms but found an additional 6 positive farms. Phylogenetic analysis found the closest match to be the first detected PPIV-1 strain in Hong Kong. The Dutch-German region represents a significant area of pig farming within Europe and could provide important information on the characterization and circulation of porcine viruses, such as PPIV-1. With its recent detection in Hungary, these findings suggest widespread circulation of PPIV-1 in Central Europe, highlighting the need for further research on persistence, pathogenicity and transmission in Europe.