Enterovirus D68 - The New Polio? Cassidy, Hayley; Poelman, Randy; Knoester, Marjolein ...
Frontiers in microbiology,
11/2018, Letnik:
9
Journal Article
Recenzirano
Odprti dostop
Enterovirus D68 (EV-D68) has emerged over the recent years, with large outbreaks worldwide. Increased occurrence has coincided with improved clinical awareness and surveillance of non-polio ...enteroviruses. Studies showing its neurotropic nature and the change in pathogenicity have established EV-D68 as a probable cause of Acute Flaccid Myelitis (AFM). The EV-D68 storyline shows many similarities with poliovirus a century ago, stimulating discussion whether EV-D68 could be ascertaining itself as the "new polio." Increasing awareness amongst clinicians, incorporating proper diagnostics and integrating EV-D68 into accessible surveillance systems in a way that promotes data sharing, will be essential to reveal the burden of disease. This will be a necessary step in preventing EV-D68 from becoming a threat to public health.
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.
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
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.
Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a ...challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.
Enterovirus infections are known to cause a diverse range of illnesses, even in healthy individuals. However, information detailing enterovirus infections and their severity in immunocompromised ...patients, such as transplant recipients, is limited. We compared enterovirus infections in terms of genotypes, clinical presentation, and severity between transplant and nontransplant patients. A total of 264 patients (38 transplant recipients) with 283 enterovirus infection episodes were identified in our hospital between 2014 and 2018. We explored the following factors associated with enterovirus infections: clinical presentation and diagnosis on discharge, length of hospital stay, symptom persistence, and infection episodes in both children and adults. We observed some differences in genotypes between patients, with enterovirus group C occurring mainly in transplant recipients (
< 0.05). EV-associated gastrointestinal infections were more common in patients with a transplant (children 71% and adults 46%), compared to nontransplant patients (
< 0.05). Additionally, nontransplant patients had a higher number of hospital stays (
< 0.05), potentially reflecting more severe disease. However, transplant patients were more likely to have symptom persistence after discharge (
< 0.05). Finally, children and adults with a transplant were more likely to have additional enterovirus infection episodes (
< 0.05). In our cohort, enterovirus infections did not seem to be more severe after transplantation; however, patients tended to present with different clinical symptoms and had genotypes rarely found in nontransplant recipients.
Despite the high prevalence of enteroviruses in the community and the increasing demand for transplants from an aging population, knowledge on enteroviruses in solid organ transplant recipients is currently limited. Transplant recipients represent a significant patient population and require additional considerations in patient management, particularly as they have an increased risk of disease severity. Enteroviruses are known to cause significant morbidity, with a diverse range of clinical presentation from over 100 different genotypes. In this study, we aimed to provide a more comprehensive overview of enteroviral infections in transplant recipients, compared to nontransplant patients, and to bridge some gaps in our current knowledge. Identifying potential clinical manifestation patterns can help improve patient management following enterovirus infections.
•PCR can produce non-specific traces if left at room temperature for over 30min.•This can lead to negative clinical samples being reported as weak false positives.•No effect on control Ct value ...however significant reduction of control fluorescence.•We suggest monitoring control fluorescence as well as Ct to detect any issues.
Non-specific amplification can arise in real-time PCR when temperatures are above 4°C during PCR set up. Pressure of high throughput tests, particularly in a clinical setting, can lead to short cuts being taken during PCR set up.
This study set out to evaluate the outcome of exposing a real-time PCR assay to increasing durations of room temperature prior to PCR amplification.
A real-time PCR assay was exposed to increasing durations of room temperature prior to PCR amplification.
We found that reactions left at room temperature for 30min or more produced non-specific traces in the negative controls which could be mistaken for weak positive traces. In addition we found that the fluorescence of positive control traces was significantly reduced indicating reduced reaction efficiency, however the Ct valves were comparable between all reactions highlighting that control Ct monitoring alone would not have detected this issue.
This study acts as a reminder for PCR users to set up reactions on ice/chill blocks prior to PCR amplification.
•European Non-polio Enterovirus Network established.•Collect respiratory, stool and CSF samples for EV testing from patient with neurological infection.•Sensitive PCR method should be used to ...diagnose EV infection.•Sequencing of VP1 capsid protein gene is recommended for EV typing.•Standardased laboratory diagnostics and characterisation key for effective surveillancce.
Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5′noncoding regions (5′NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5′NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden.
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.
BACKGROUNDEnteroviruses 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. OBJECTIVEWe aimed to provide genomic and relative frequency data on CVA22 from a regional hospital perspective between 2013-2020. STUDY DESIGNSanger 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. RESULTSA 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. CONCLUSIONSWe 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.
Rapid and sensitive diagnostic strategies are necessary for patient care and public health. Most of the current conventional microbiological assays detect only a restricted panel of pathogens at a ...time or require a microbe to be successfully cultured from a sample. Clinical metagenomics next-generation sequencing (mNGS) has the potential to unbiasedly detect all pathogens in a sample, increasing the sensitivity for detection and enabling the discovery of unknown infectious agents. High expectations have been built around mNGS; however, this technique is far from widely available. This review highlights the advances and currently available options in terms of costs, turnaround time, sensitivity, specificity, validation, and reproducibility of mNGS as a diagnostic tool in clinical microbiology laboratories. The need for a novel diagnostic tool to increase the sensitivity of microbial diagnostics is clear. mNGS has the potential to revolutionize clinical microbiology. However, its role as a diagnostic tool has yet to be widely established, which is crucial for successfully implementing the technique. A clear definition of diagnostic algorithms that include mNGS is vital to show clinical utility. Similarly to real-time PCR, mNGS will one day become a vital tool in any testing algorithm.
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