Exploring molecular diagnostic approaches in clinical virology:from point-of-care to next-generation sequencing

Following the SARS-CoV-2 pandemic and the re-emergence of monkeypox (mpox) virus, the need to monitor viral trends inside and outside the hospital has been made abundantly clear in recent years. Coincidentally, molecular virological methods have witnessed an unprecedented boost in this timeframe. While conventional Sanger sequencing provides a cheap and sturdy backbone, the emergence of point-of-care panels hints at a future in which dozens of viruses can be detected and quantified at the patient’s bedside. At the same time, next-generation sequencing approaches have the potential to sequence millions of nucleic acid fragments in parallel to screen all microbes and the host within an all-in-one assay. While this thesis focuses on the diagnostics of viral infections, with a special interest in enteroviruses, the molecular applications and recommendations presented here are highly applicable to emerging infectious diseases. Enterovirus infections are widespread within the community and responsible for thousands of hospital admissions every year, particularly in children. We investigated enterovirus D68 in particular due to several outbreaks reported worldwide, with accompanying severe respiratory and neurological presentation. In this thesis, we combined the available clinical and epidemiological data to provide several layers of information to help uncover the whole clinical picture. Our findings provide a groundwork for the early detection and higher resolution of viral infections which may prove crucial during outbreak scenarios

Enterovirus Infections in Solid Organ Transplant Recipients:a Clinical Comparison from a Regional University Hospital in the Netherlands

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 (P < 0.05). EV-associated gastrointestinal infections were more common in patients with a transplant (children [71%] and adults [46%]), compared to nontransplant patients (P < 0.05). Additionally, nontransplant patients had a higher number of hospital stays (P < 0.05), potentially reflecting more severe disease. However, transplant patients were more likely to have symptom persistence after discharge (P < 0.05). Finally, children and adults with a transplant were more likely to have additional enterovirus infection episodes (P < 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. IMPORTANCE 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

Future potential of metagenomics in clinical laboratories

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: The need for a novel diagnostic tool to increase the sensitivity of microbial diagnostics is clear. mNGS has the potential to revolutionise 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

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

Enterovirus D68-The New Polio?

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

A Measurement Invariance Analysis of the Interpersonal Needs Questionnaire and Acquired Capability for Suicide Scale in Autistic and Non-Autistic Adults.

Background: Autistic adults are more likely to engage in suicidal thoughts and behaviors, but there is little research to explore the underlying reasons. It is unclear whether self-report suicide scales that have been designed for non-autistic people accurately measure suicide risk constructs in autistic people. Therefore, this study explored, for the first time, whether the measurement properties of the self-report scales of the Interpersonal Theory of Suicide are equivalent in autistic and non-autistic adults. Methods: In this study, responses from 342 autistic and 353 non-autistic people on the Interpersonal Needs Questionnaire-10 (INQ-10) and Acquired Capability for Suicide Scale-Fearlessness about Death (ACSS-FAD) were compared by using measurement invariance analysis. Data were gathered through an online cross-sectional survey of the self-report measures. Results: Results suggest that measurement properties of the INQ-10 were different in autistic people. Autistic characteristics, such as different theory of mind and preference for concrete language, may have led the scale items to load differently on the factors in the autistic group than in the non-autistic group. The measurement properties of the ACSS-FAD were invariant between autistic and non-autistic people. Conclusions: Scores on the INQ-10 cannot be meaningfully compared between autistic and non-autistic people due to different measurement properties. Future research could explore how autistic people experience the concepts of burdensomeness and belonging, to consider how measures could accurately capture this. This would allow researchers to explore the role of these constructs in the development of suicidal thoughts and behaviors in autistic people. Clinicians should be aware that suicide risk factors may present differently in autistic people. Scores on the ACSS-FAD can be meaningfully compared, but the negatively worded scale items may pose similar response difficulties to autistic and non-autistic people

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

Understanding suicide risk in autistic adults : comparing the interpersonal theory of suicide in autistic and non-autistic samples

This study explored whether the Interpersonal Theory of suicide informs our understanding of high rates of suicidality in autistic adults. Autistic and non-autistic adults (n = 695, mean age 41.7 years, 58% female) completed an online survey of self-reported thwarted belonging, perceived burden, autistic traits, suicidal capability, trauma, and lifetime suicidality. Autistic people reported stronger feelings of perceived burden, thwarted belonging and more lifetime trauma than non-autistic people. The hypothesised interaction between burdensomeness and thwarted belonging were observed in the non-autistic group but not in the autistic group. In both groups autistic traits influenced suicidality through burdensomeness/thwarted belonging. Promoting self-worth and social inclusion are important for suicide prevention and future research should explore how these are experienced and expressed by autistic people