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Virus-specific cellular and humoral responses are major determinants for protection from critical illness after SARS-CoV-2 infection. However, the magnitude of the contribution of each of the components to viral clearance remains unclear. Here, we studied the timing of viral clearance in relation to 122 immune parameters in 102 hospitalised patients with moderate and severe COVID-19 in a longitudinal design. Delayed viral clearance was associated with more severe disease and was associated with higher levels of SARS-CoV-2-specific (neutralising) antibodies over time, increased numbers of neutrophils, monocytes, basophils, and a range of pro-inflammatory cyto-/chemokines illustrating ongoing, partially Th2 dominating, immune activation. In contrast, early viral clearance and less critical illness correlated with the peak of neutralising antibodies, higher levels of CD4 T cells, and in particular naïve CD4+ T cells, suggesting their role in early control of SARS-CoV-2 possibly by proving appropriate B cell help. Higher counts of naïve CD4+ T cells also correlated with lower levels of MIF, IL-9, and TNF-beta, suggesting an indirect role in averting prolonged virus-induced tissue damage. Collectively, our data show that naïve CD4+ T cell play a critical role in rapid viral T cell control, obviating aberrant antibody and cytokine profiles and disease deterioration. These data may help in guiding risk stratification for severe COVID-19. [ABSTRACT FROM AUTHOR]

Viral metagenomics is increasingly applied in clinical diagnostic settings for detection of pathogenic viruses. While several benchmarking studies have been published on the use of metagenomic classifiers for abundance and diversity profiling of bacterial populations, studies on the comparative performance of the classifiers for virus pathogen detection are scarce. In this study, metagenomic data sets (n = 88) from a clinical cohort of patients with respiratory complaints were used for comparison of the performance of five taxonomic classifiers: Centrifuge, Clark, Kaiju, Kraken2, and Genome Detective. A total of 1144 positive and negative PCR results for a total of 13 respiratory viruses were used as gold standard. Sensitivity and specificity of these classifiers ranged from 83 to 100% and 90 to 99%, respectively, and was dependent on the classification level and data pre-processing. Exclusion of human reads generally resulted in increased specificity. Normalization of read counts for genome length resulted in a minor effect on overall performance, however it negatively affected the detection of targets with read counts around detection level. Correlation of sequence read counts with PCR Ct-values varied per classifier, data pre-processing (R2 range 15.1–63.4%), and per virus, with outliers up to 3 log10 reads magnitude beyond the predicted read count for viruses with high sequence diversity. In this benchmarking study, sensitivity and specificity were within the ranges of use for diagnostic practice when the cut-off for defining a positive result was considered per classifier. [ABSTRACT FROM AUTHOR]

Background The relative incidence and clinical impact of individual respiratory viruses remains unclear among children presenting to the hospital emergency department with acute respiratory tract infection (ARTI). Methods During two winter periods, respiratory virus real-time multiplex PCR results were evaluated from children (< 18 years) presenting to the emergency department of a tertiary referral hospital with ARTI that had been sampled within 48 hours of hospital presentation. In an attempt to identify virus-specific distinguishing clinical features, single virus infections were correlated with presenting signs and symptoms, clinical findings and outcomes using multivariate logistic regression. Results In total, 274 children with ARTI were evaluated and most were aged < 3 years (236/274, 86%). PCR detected respiratory viruses in 224/274 (81.8%) children and included 162 (59%) single and 62 (23%) mixed virus infections. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) single virus infections were common among children aged < 3 years, but proportional differences compared to older children were only significant for RSV (95% CI 1.3-15). Clinical differentiation between viral ARTIs was not possible due to common shared presenting signs and symptoms and the high frequency of mixed viral infections. We observed virus-associated outcome differences among children aged < 3 years. Oxygen treatment was associated with RSV (OR 3.6) and inversely correlated with FLU (OR 0.05). Treatment with steroids (OR 3.4) or bronchodilators (OR 3.4) was associated with HRV. Severe respiratory complications were associated with HRV (OR 3.5) and inversely correlated with RSV (OR 0.24). Conclusions Respiratory viruses are frequently detected in young children presenting to the hospital emergency department with ARTI and require PCR diagnosis since presenting signs and symptoms are not discriminant for a type of virus. RSV and HRV bear a high burden of morbidity in the pediatric clinical setting. [ABSTRACT FROM AUTHOR]

Background: In patients with lower respiratory tract infection (LRTI), changes on chest radiography are rare but poorly characterised, especially in general practice.Aim: To describe the range of findings on chest radiographs and the associations between these findings and the aetiology of LRTI.Design Of Study: A prospective observational study.Setting: General practices in the Leiden region, The Netherlands.Method: Adult patients with a defined LRTI were included. Standard medical history and physical examination were performed. Sputum, blood, and throat swabs were collected for diagnostic tests. Chest X-ray findings were assessed in relation to the aetiology.Results: An abnormality on the chest X-ray was observed in 72 (55%) patients. Forty-five patients (35%) had changes due to infection, and 26 (20%) due to pneumonia. Pathogens were detected in 84 patients (33 single bacterial, 43 single viral, and 8 dual). Twelve (29%) patients with a bacterial infection (including dual infections) compared to four (9%) patients with viral infection had pneumonia on the chest X-ray (odds ratio [OR] = 4.0; 95% confidence interval [CI] = 1.2 to 13.8). Using the presence of pneumonia on chest X-ray as a test to predict a bacterial infection, the positive predictive value and the negative predictive value were 75% (CI = 48 to 93%) and 57% (CI = 45 to 69%), respectively.Conclusion: Pneumonia on the chest X-ray was found more frequently in patients with a bacterial infection than in patients with a viral infection. However, the sensitivity and the specificity are such that pneumonia on the chest X-ray is not a reliable test to discriminate between bacterial and non-bacterial LRTI in the general practice setting. [ABSTRACT FROM AUTHOR]

Background. Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation and may progress to life-threatening lymphoproliferative disease (EBV-LPD) in the absence of adequate EBV-specific T cell immunity. Quantification of EBV DNA load in asymptomatic individuals who are at risk is a useful (although not entirely predictive) indicator of progression to EBV-LPD and guide for preemptive treatment with CD20 antibodies. Methods. With the aim of improving the identification of patients at risk, we retrospectively analyzed, within a cohort of 25 consecutive allogeneic stem cell transplant recipients at risk for EBV-LPD, the pattern of T cell reconstitution during EBV reactivation in all preemptively treated patients (8 patients). Results. In 6 of 8 cases, a significant T cell reconstitution (i.e., a CD3+ T cell count of >300 cells/μL) was documented during EBV reactivation, which included an expansion of EBV-specific memory T cells, as shown by human leukocyte antigen class I tetramer analysis. Additional evidence for the antiviral potential of this T cell reconstitution was obtained prospectively from a cohort of 14 consecutive allogeneic stem cell transplant recipients at risk for EBV-LPD. EBV reactivation occurred in 3 patients. Preemptive treatment was successfully withheld for 2 of these patients in light of concurrent (EBV-specific) T cell recovery. Conclusion. We conclude that analysis of the level of (EBV-specific) T cell reconstitution during EBV reactivation is an important second parameter, in addition to quantification of EBV DNA load, that will be instrumental in a more accurate definition of patients at risk for EBV-LPD who, given their immunoincompetence, will be most certainly dependent on preemptive interventions. [ABSTRACT FROM AUTHOR]

OBJECTIVE -- Simultaneous pancreas-kidney (SPK) transplantation in type 1 diabetic patients requires immunotherapy against allo- and autoreactive T-cells. Cytomegalovirus (CMV) infection is a major cause for morbidity after transplantation and is possibly related to recurrent autoimmunity. In this study, we assessed the pattern of CMV viremia in SPK transplant recipients receiving either antithymocyte globulin (ATG) or anti-CD25 (daclizumab) immunosuppressive induction therapy. RESEARCH DESIGN AND METHODS -- We evaluated 36 SPK transplant recipients from a randomized cohort that received either ATG or daclizumab as induction therapy. Patients at risk for CMV infection received oral prophylactic ganciclovir therapy. The CMV DNA level in plasma was measured for at least 180 days using a quantitative real-time PCR. Recipient peripheral blood mononuclear cells were cross-sectionally HLA tetramer-stained for CMV-specific CD8+ T-cells. RESULTS -- Positive CMV serostatus in donors was correlated with a higher incidence of CMV viremia than negative serostatus. In patients at risk, daclizumab induction therapy significantly prolonged CMV-free survival. CMV viremia occurred earlier and was more severe in patients with rejection episodes than in patients without rejection episodes. CMV-specific CD8+ T-cell counts were significantly lower in patients developing CMV viremia than in those who did not. CONCLUSIONS -- Despite their comparable immunosuppressive potential, daclizumab is safer than ATG regarding CMV infection risk in SPK transplantation. ATG-treated rejection episodes are associated with earlier and more severe infection. Furthermore, high CMV-specific tetramer counts reflect antiviral immunity rather than concurrent viremia because they imply low viremic activity. These findings may prove valuable in the discussion on both safety of induction therapy and recurrent autoimmunity in SPK and islet transplantation. [ABSTRACT FROM AUTHOR]

This article describes the age distribution and clinical characteristics of 124 patients with serological evidence of recent cytomegalovirus (CMV) infection. As indicated by a high level of anti-CMV IgM and the presence of low-avidity anti-CMV IgG, in the absence of serological evidence of recent infection with Epstein-Barr virus, hepatitis A or B virus, or Toxoplasma gondii. The clinical characteristics of these 124 patients were evaluated retrospectively, yielding a wide range of symptoms, signs, and laboratory findings.

Background: We recently showed that seasonal patterns of COVID-19 incidence and Influenza-Like Illnesses incidence are highly similar, in a country in the temperate climate zone, such as the Netherlands. We hypothesize that in The Netherlands the same environmental factors and mobility trends that are associated with the seasonality of flu-like illnesses are predictors of COVID-19 seasonality as well., Methods: We used meteorological, pollen/hay fever and mobility data from the Netherlands. For the reproduction number of COVID-19 (R t ), we used daily estimates from the Dutch State Institute for Public Health. For all datasets, we selected the overlapping period of COVID-19 and the first allergy season: from February 17, 2020 till September 21, 2020 (n = 218). Backward stepwise multiple linear regression was used to develop an environmental prediction model of the R t of COVID-19. Next, we studied whether adding mobility trends to an environmental model improved the predictive power., Results: Through stepwise backward multiple linear regression four highly significant (p < 0.01) predictive factors are selected in our combined model: temperature, solar radiation, hay fever incidence, and mobility to indoor recreation locations. Our combined model explains 87.5% of the variance of R t of COVID-19 and has a good and highly significant fit: F(4, 213) = 374.2, p < 0.00001. This model had a better overall predictive performance than a solely environmental model, which explains 77.3% of the variance of R t (F(4, 213) = 181.3, p < 0.00001)., Conclusions: We conclude that the combined mobility and environmental model can adequately predict the seasonality of COVID-19 in a country with a temperate climate like the Netherlands. In this model higher solar radiation, higher temperature and hay fever are related to lower COVID-19 reproduction, and higher mobility to indoor recreation locations is related to an increased COVID-19 spread., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Meningoencephalitis patients are often severely impaired and benefit from early etiological diagnosis, though many cases remain without identified cause. Metagenomics as pathogen agnostic approach can result in additional etiological findings; however, the exact diagnostic yield when used as a secondary test remains unknown., Areas Covered: This review aims to highlight recent advances with regard to wet and dry lab methodologies of metagenomic testing and technical milestones that have been achieved. A selection of procedures currently applied in accredited diagnostic laboratories is described in more detail to illustrate best practices. Furthermore, a meta-analysis was performed to assess the additional diagnostic yield utilizing metagenomic sequencing in meningoencephalitis patients. Finally, the remaining challenges for successful widespread implementation of metagenomic sequencing for the diagnosis of meningoencephalitis are addressed in a future perspective., Expert Opinion: The last decade has shown major advances in technical possibilities for using mNGS in diagnostic settings including cloud-based analysis. An additional advance may be the current established infrastructure of platforms for bioinformatic analysis of SARS-CoV-2, which may assist to pave the way for global use of clinical metagenomics.

Aims: Parvovirus B19 (B19V) is often assumed to be a cause of dilated cardiomyopathy (DCM), based on the quantification of B19V DNA in endomyocardial biopsies (EMB). Whether the presence of B19V DNA correlates with active infection is still debated. Application of the enzyme endonuclease to blood samples results in degradation of B19V DNA remnants but leaves viral particles intact, which enables differentiation between active and past infection. In this study, the susceptibility to degradation by endonuclease of B19V DNA in blood was compared between DCM patients and a control group of recent B19V infections., Methods and Results: Twenty blood samples from 20 adult patients with DCM, who previously tested positive for B19V DNA in EMB and/or blood, were tested with B19V PCR before and after application of endonuclease to the samples. Six blood samples tested positive for B19V DNA with a mean viral load of 2.3 × 10 4  IU/mL. In five samples, B19V DNA became undetectable after endonuclease (100% load reduction); in one sample DNA load showed a 23% log load reduction (viral load before endonuclease: 9.1 × 10 4  IU/mL; after: 6.5 × 10 3  IU/mL). Presence of cardiac inflammation did not differ between patients with B19V DNAemia (1/4) and patients without B19V DNAemia (6/14) (P value = 1.0). In all 18 control samples of proven recent B19V infections, DNA remained detectable after application of endonuclease, showing only a mean log load reduction of 2.3% (mean viral load before endonuclease: 8.1 × 10 11  IU/mL; after: 8.0 × 10 11  IU/mL). Load reduction differed significantly between the DCM group and the control group; indicating the presence of intact viral particles in the control group with proven active infection and the presence of DNA remnants in the DCM group (P value = 0.000)., Conclusion: During recent B19V infection, viral DNA levels in blood were unaffected by endonuclease. In contrast, B19V DNA in blood in patients with DCM became undetectable or strongly reduced after application of endonuclease. Circulating viral DNA in this subset of patients with presumed parvovirus-associated DCM does not consist of intact viral particles. Viral replicative activity cannot be assumed from demonstrating B19V DNA in cardiac tissue or in blood in DCM patients., (© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables unbiased detection of all potential pathogens. To apply mNGS in viral diagnostics, sensitive and simultaneous detection of RNA and DNA viruses is needed. Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. The sequencing protocol and bioinformatics analysis were designed and optimized, including exogenous internal controls. Subsequently, the protocol was retrospectively validated using 25 clinical respiratory samples. The developed protocol using Illumina NextSeq 500 sequencing showed high repeatability. Use of the National Center for Biotechnology Information's RefSeq database as opposed to the National Center for Biotechnology Information's nucleotide database led to enhanced specificity of classification of viral pathogens. A correlation was established between read counts and PCR cycle threshold value. Sensitivity of mNGS, compared with PCR, varied up to 83%, with specificity of 94%, dependent on the cutoff for defining positive mNGS results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow, were influenza C, KI polyomavirus, cytomegalovirus, and enterovirus. Sensitivity and analytical specificity of this mNGS protocol were comparable to PCR and higher when considering off-PCR target viral pathogens. One single test detected all potential viral pathogens and simultaneously obtained detailed information on detected viruses., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: There are few investigations into the aetiology of lower respiratory tract infections (LRTIs) in general practice., Aim: To describe the aetiology of LRTI among adult patients in general practice in The Netherlands., Design of Study: Prospective observational study., Setting: General practices in the Leiden region, The Netherlands., Method: Adult patients with a defined LRTI were included. Standard medical history and physical examination were performed. Sputum, blood and throat swabs were collected for diagnostic tests. Aetiological diagnosis, categorised as definite or possible, was based on the results of bacterial and viral cultures, serological techniques, and on polymerase chain reaction. Proportions of pathogens causing LRTI were assessed in relation to chest X-ray findings., Results: A bacterial cause was established in 43 (30%), and a viral cause in 57 (39%) of the 145 patients with a LRTI. Influenza virus A was the most frequently diagnosed microorganism, followed by Haemophilus influenzae, and Mycoplasma pneumoniae. Streptococcus pneumoniae was found in 6% of the patients., Conclusions: Pathogens were found in two-thirds of the patients. In half of these patients there was a viral cause. Influenza virus A was the most frequently found pathogen. The treatment with antibiotics of at least one-third of the patients with LRTI was superfluous. This observation should result in changes in the prescription of antibiotics in LRTI.

Background: The majority of patients with lower respiratory tract infections (LRTIs) are treated with antibiotics; some of them are unnecessary because of a viral cause. Information on prediction of the aetiology, especially in a general practice setting, is missing., Aim: To differentiate between viral and bacterial LRTI on simple clinical criteria, easily obtained at the bedside., Design of Study: Prospective observational study., Setting: General practices in the Leiden region of The Netherlands., Method: Adult patients with LRTI were included. Standard medical history and physical examination were performed. Sputum, blood and throat swabs were collected for diagnostic tests. According to microbiological findings, patients were classified as bacterial, viral, dual infection and unknown cause. In a logistic regression model independent predictors were determined. Scoring systems were developed. The accuracies of the diagnostic rules were tested by using receiver operating characteristic (ROC) curves., Results: One-hundred and forty-five patients were classified as having bacterial (n = 35), viral (n = 49), or dual infection (n = 8), or infection of unknown cause (n = 53), respectively. Independent predictors for bacterial infection were fever (odds ratio [OR] = 8.0; 95% confidence interval [CI] = 0.9 to 71.0), headache (OR = 4.3; 95% CI = 1.0 to 19.1) cervical painful lymph nodes (OR = 8.7; 95% CI = 1.1 to 68.0), diarrhoea (OR = 0.3; 95% CI = 0.1 to 1.0) and rhinitis (OR = 0.3; 95% CI = 0.1 to 0.9). As an additional independent predictor, an infiltrate on chest X-ray (OR = 5.0; 95% CI = 1.2 to 20.5) was found. The diagnostic rules developed from these variables classified the aetiology of LRTI with a ROC curve area of 0.79 (clinical score), 0.77 (simplified score) and 0.83 (extended score)., Conclusions: A diagnostic rule was developed, based on information that is easy to obtain at the bedside, to predict a bacterial infection. This diagnostic rule may be a tool for general practitioners in their management of patients with LRTI.