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

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

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 ( 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

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

Changes in enterovirus epidemiology after easing of lockdown measures

INTRODUCTION: Public health measures aimed at controlling transmission of SARS-CoV-2, otherwise known as "lockdown" measures, had profound effects on circulation of non-SARS viruses, many of which decreased to very low levels. The interrupted transmission of these viruses may have lasting effects. Some of the influenza clades seem to have disappeared during this period, a phenomenon which is described as a "funnel effect". It is currently unknown if the lockdown measures had any effect on the diversity of circulating viruses, other than influenza. Enteroviruses are especially interesting in this context, as the clinical presentation of an infection with a particular enterovirus-type may be clade-dependent.METHODS AND MATERIALS: Enteroviruses were detected in clinical materials using a 5'UTR-based detection PCR, and partial VP-1 sequences were obtained, using methods described before. All samples with EV detections from a large part of the Netherlands were included in the study. The samples originated from general practitioners, general hospitals, university hospitals and public health offices.RESULTS: Five EV-genotypes circulated in significant numbers before and after the lockdown, EV-D68, E-11, CV-A6, CV-B5 and CV-A2. All five genotypes showed decreased genetic diversity after the lockdown, and four indicate a significant number of sequences clustering together with a very high sequence homology. Moreover, children with E-11 and CV-B5 detections were significantly older after the lockdown than before.CONCLUSIONS: The reduced enterovirus transmission in the Netherlands during the pandemic, seems to have led to a decrease in genetic diversity in the five most commonly detected enterovirus serotypes.</p

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

Health Seeking Behaviour and Treatment Intentions of Dengue and Fever:A Household Survey of Children and Adults in Venezuela

BACKGROUND:Dengue in Venezuela is a major public health problem with an increasing incidence of severe cases. Early diagnosis and timely treatment influences the outcome of dengue illness, as delay in care-seeking is significantly associated with complications leading to severe dengue. We aimed to understand patterns of health seeking behaviour (HSB) in individuals exposed to high dengue incidence in order to improve early attendance to health centres. METHODS:Between September 2013 and February 2014 a cross-sectional household survey was performed in Maracay, Venezuela. Intended HSB of adults and children's parents/guardians was assessed with respect to fever or suspected dengue. Data was collected through structured questionnaires from 105 individuals. RESULTS:Most individuals felt at risk of dengue and believed it could be a deadly disease. In the case of suspected dengue, the majority (60%) would choose to first seek medical help versus first treating at home, in contrast to 11% in the case of fever. Amongst those who decided to visit a doctor, a suspected dengue infection would prompt them to search medical help earlier than if having only fever (p<0.001). Multivariate analysis modelling showed that the independent factors associated with the intention to firstly visit a doctor versus treating at home in the case of dengue were feeling at risk (OR = 3.29; p = 0.042) and being an adult (as opposed to caring for a child as a parent/guardian; OR = 3.33, p = 0.021), while having had a previous dengue infection (OR = 0.29; p = 0.031) and living in the neighbourhood Caña de Azúcar (OR = 0.28, p = 0.038) were negatively associated with seeking medical care as their first action. CONCLUSION:Knowledge of HSB related to dengue is scarce in the Americas, our study attempts to contribute to a better understanding of HSB in this region. Improving early dengue disease recognition and awareness may enhance prompt attendance to medical care in affected populations and thereby reduce mortality and severity of dengue. Especially for those with a previous dengue infection, efforts have to be made to promote prompt health centre attendance

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

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

Changes in enterovirus epidemiology after easing of lockdown measures

INTRODUCTION: Public health measures aimed at controlling transmission of SARS-CoV-2, otherwise known as "lockdown" measures, had profound effects on circulation of non-SARS viruses, many of which decreased to very low levels. The interrupted transmission of these viruses may have lasting effects. Some of the influenza clades seem to have disappeared during this period, a phenomenon which is described as a "funnel effect". It is currently unknown if the lockdown measures had any effect on the diversity of circulating viruses, other than influenza. Enteroviruses are especially interesting in this context, as the clinical presentation of an infection with a particular enterovirus-type may be clade-dependent.METHODS AND MATERIALS: Enteroviruses were detected in clinical materials using a 5'UTR-based detection PCR, and partial VP-1 sequences were obtained, using methods described before. All samples with EV detections from a large part of the Netherlands were included in the study. The samples originated from general practitioners, general hospitals, university hospitals and public health offices.RESULTS: Five EV-genotypes circulated in significant numbers before and after the lockdown, EV-D68, E-11, CV-A6, CV-B5 and CV-A2. All five genotypes showed decreased genetic diversity after the lockdown, and four indicate a significant number of sequences clustering together with a very high sequence homology. Moreover, children with E-11 and CV-B5 detections were significantly older after the lockdown than before.CONCLUSIONS: The reduced enterovirus transmission in the Netherlands during the pandemic, seems to have led to a decrease in genetic diversity in the five most commonly detected enterovirus serotypes.</p