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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 × 104 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 × 104 IU/mL; after: 6.5 × 103 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 × 1011 IU/mL; after: 8.0 × 1011 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. ispartof: ESC HEART FAILURE vol:8 issue:4 pages:2723-2730 ispartof: location:England status: published

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 (Rt), 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 Rt of COVID-19. Next, we studied whether adding mobility trends to an environmental model improved thepredictive 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 Rt 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 Rt (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.

IntroductionImmunocompromised patients are prone to reactivations of multiple latent DNA viruses. Viral load monitoring by single-target quantitative PCRs (qPCR) is the current cornerstone for virus quantification. In this study, a metagenomic next-generation sequencing (mNGS) approach was used for identification and load monitoring of transplantation-related DNA viruses.MethodsLongitudinal plasma samples from six patients that were qPCR-positive for cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyoma virus (BKV, adenovirus (ADV), parvovirus B19 (B19V), and torque teno-virus (TTV) were sequenced using the quantitative metagenomic Galileo Viral Panel Solution (Arc Bio, LLC) reagents and bioinformatics pipeline combination. Qualitative and quantitative performance was analysed with focus on viral load ranges relevant for clinical decision-making.ResultsAll pathogens identified by qPCR were also identified by mNGS. In addition, BKV, CMV, and HHV6B were detected by mNGS which were not ordered initially but could be confirmed by qPCR. Viral loads determined by mNGS correlated with the qPCR results, with inter-method differences in viral load per virus ranging from 0.19 log10 IU/ml for EBV to 0.90 log10 copies/ml for ADV. TTV, analysed by mNGS in a semi-quantitative way, showed a mean difference of 3.0 log10 copies/ml. Trends over time in viral load determined by mNGS and qPCR were comparable, and clinical thresholds for initiation of treatment were equally indicated by mNGS.ConclusionThe Galileo Viral Panel for quantitative mNGS performed comparable to qPCR with regard to detection and viral load determination, within clinically relevant ranges of patient management algorithms.

BackgroundDiagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious aetiologies potentially involved. Viral metagenomic next-generation sequencing (mNGS) has the potential to detect any virus present in a patient sample and is increasingly being used for difficult to diagnose cases. The aim of this study was to analyse the performance of mNGS for viral pathogen detection in the clinical setting of international travellers returning with febrile illness.MethodsThirty-eight serum samples from international travellers returning with febrile illness and presenting at the outpatient clinic of the Leiden University Medical Center in the Netherlands in the time period 2015-2016 were selected retrospectively. Samples were processed for viral metagenomic sequencing using a probe panel capturing all known vertebrate viruses. Bioinformatic analysis was performed using Genome Detective software for metagenomic virus detection. Metagenomic virus findings were compared with viral pathogen detection using conventional methods.ResultsIn 8 out of the 38 patients (21%), a pathogenic virus was detected by mNGS. All viral pathogens detected by conventional assays were also detected by mNGS: dengue virus (n=4 patients), Epstein-Barr virus (n=2), hepatitis B virus (n=1). In addition, mNGS resulted in additional pathogenic findings in 2 patients (5%): dengue virus (n=1), and hepatitis C virus (n=1). Non-pathogenic viruses detected were: GB virus C (n=1) and torque teno viruses (n=3). High genome coverage and depth using capture probes enabled typing of the dengue viruses detected.ConclusionsViral metagenomics has the potential to assist the detection of viral pathogens and co-infections in one step in international travellers with a febrile syndrome. Furthermore, viral enrichment by probes resulted in high genome coverage and depth which enabled dengue virus typing.

BackgroundWe 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 (latitude: 52°N). 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.MethodsWe used meteorological, pollen/hay fever and mobility data from the Netherlands with its 17.4 million inhabitants. For the reproduction number of COVID-19 (Rt), we used data from the Dutch State Institute for Public Health. This Rt metric is a daily estimate that is based on positive COVID-19 tests in the Netherlands in hospitals and municipalities. For all datasets we selected the overlapping period of COVID-19 and the first allergy season: from February 17, 2020 till September 21, 2020 (total number of measurements: n = 218), the end of pollen season. Backward stepwise multiple linear regression was used to develop an environmental prediction model of the Rt of COVID-19. Next, we studied whether adding mobility trends to an environmental model improved the predictive power.ResultsBy means of stepwise backward multiple linear regression four highly significant (p value < 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 Rt of COVID-19 and has a good and highly significant fit: F(4, 213) = 374.2, p-value < 0.00001. The combined model had a better overall predictive performance compared to a solely environmental model, which still explains 77.3% of the variance of Rt, and a good and highly significant fit: F(4, 213) = 181.3, p < 0.00001.ConclusionsWe 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 mobility to indoor recreation locations with increased COVID-19 spread.HighlightsThe seasonality of COVID-19 can be well-explained by environmental factors and mobility.A combined model explains 87.5% of the variance of the reproduction number of COVID-19Inhibitors of the reproduction number of COVID-19 are higher solar radiation, and seasonal allergens/allergies.Mobility, especially to indoor recreation locations, increases the reproduction number of COVID-19.Temperature has no direct effect on the reproduction number of COVID-19, but affects mobility and seasonal allergens.Adding mobility trends to an environmental model improves the predictive value regarding the reproduction number of COVID-19.

Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories.MethodsMetagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analysed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analysed.ResultsOverall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection.ConclusionA wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

Objectives To determine whether children with asymptomatic carriage of rhinovirus in the nasopharynx before elective cardiac surgery have an increased risk of prolonged PICU length of stay. Study design Prospective, single-center, blinded observational cohort study. Setting PICU in a tertiary hospital in The Netherlands. Patients Children under 12 years old undergoing elective cardiac surgery were enrolled in the study after informed consent of the parents/guardians. Interventions The parents/guardians filled out a questionnaire regarding respiratory symptoms. On the day of the operation, a nasopharyngeal swab was obtained. Clinical data were collected during PICU admission, and PICU/hospital length of stay were reported. If a patient was still intubated 3 days after operation, an additional nasopharyngeal swab was collected. Nasopharyngeal swabs were tested for rhinovirus and other respiratory viruses with polymerase chain reaction. Measurements and main results Of the 163 included children, 74 (45%) tested rhinovirus positive. Rhinovirus-positive patients did not have a prolonged PICU length of stay (median 2 d each; p = 0.257). Rhinovirus-positive patients had a significantly shorter median hospital length of stay compared with rhinovirus-negative patients (8 vs 9 d, respectively; p = 0.006). Overall, 97 of the patients (60%) tested positive for one or more respiratory virus. Virus-positive patients had significantly shorter PICU and hospital length of stay, ventilatory support, and nonmechanical ventilation. Virus-negative patients had respiratory symptoms suspected for a respiratory infection more often. In 31% of the children, the parents reported mild upper respiratory complaints a day prior to the cardiac surgery, this was associated with postextubation stridor, but no other clinical outcome measures. Conclusions Preoperative rhinovirus polymerase chain reaction positivity is not associated with prolonged PICU length of stay. Our findings do not support the use of routine polymerase chain reaction testing for respiratory viruses in asymptomatic children admitted for elective cardiac surgery.

INTRODUCTION: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. METHODS: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed. RESULTS: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. CONCLUSION: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases. ispartof: JOURNAL OF CLINICAL VIROLOGY vol:141 ispartof: location:Netherlands status: published

ObjectivesWe evaluated the effects of on-site rapid molecular testing at a drive-through sampling facility, deployment of mobile sampling teams and implementation of an online eHealth platform as supportive measures for general practitioners (GPs) during the COVID-19 pandemic.MethodsAn eHealth platform was developed that allowed GPs to either refer patients to a drive-through sampling facility or to request a home visit by a sampling team. Nasopharyngeal swab samples from patients marked as urgent (n=333) were tested immediately on-site using a GeneXpert System. Non-urgent samples (n=1,460) were sent once a day to a university hospital laboratory for routine testing. Time stamps starting from referral to the moment of test report sent were recorded to calculate the turnaround time.ResultsThe eHealth platform was rapidly adopted and used by a total of 517 GPs to test 1,793 patients in a period of 13 weeks. On-site rapid molecular testing reduced the median turnaround time to 03h:41m compared to 29h:15m for routine testing. Positive SARS-CoV-2 test results were identified amongst 84/1,477 (5.7%) and 33/316 (10.4%) patients sampled at the drive-through or at home, respectively. In the age category of >80 years, 80.4% of patients were tested by a mobile sampling team.ConclusionsThe combination of rapid molecular testing and eHealth reduced the time between referral and results sent back to the GP to less than four hours. In addition, mobile sampling teams helped in reaching non-mobile, elderly patient populations with a higher prevalence of COVID-19.

Introduction The SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness. Aim The aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness. Methods The performance of a viral metagenomic protocol in a clinical setting for the identification of novel coronaviruses was tested using clinical samples containing SARS-CoV-2, SARS-CoV, and MERS-CoV, in combination with databases generated to contain only viruses of before the discovery dates of these coronaviruses, to mimic virus discovery. Results Classification of NGS reads using Centrifuge and Genome Detective resulted in assignment of the reads to the closest relatives of the emerging coronaviruses. Low nucleotide and amino acid identity (81% and 84%, respectively, for SARS-CoV-2) in combination with up to 98% genome coverage were indicative for a related, novel coronavirus. Capture probes targeting vertebrate viruses, designed in 2015, enhanced both sequencing depth and coverage of the SARS-CoV-2 genome, the latter increasing from 71% to 98%. Conclusion The model used for simulation of virus discovery enabled validation of the metagenomic sequencing protocol. The metagenomic protocol with virus probes designed before the pandemic, can assist the detection and identification of novel coronaviruses directly in clinical samples.

Background: Diagnosis of congenital viral infection at birth is generally attempted by direct detection of the virus by PCR in various neonatal materials. How to reliably diagnose intrauterine infection with parvovirus B19 (B19 V) at birth is unknown.Objectives: To evaluate the performance of B19 V DNA detection in cord blood (CB) or neonatal dried blood spots (DBS) in diagnosing fetal infection. Study design: Two cohorts of children diagnosed prenatally with an intrauterine B19 V infection were included in this study. CB samples of intrauterine B19 V infections that were sent to a reference laboratory for congenital infections in Stuttgart, Germany in the period 1995-2014 were tested in triplicate for B19 V DNA by quantitative PCR. DBS from children with intrauterine B19 V infection that underwent IUT at the LUMC, Leiden, the Netherlands in the period 2009-2014 were tested for B19 V DNA by quantitative B19 V PCR in triplicate.Results: Fourteen of twenty (70 %) CB samples tested positive for B19 V DNA. The positivity rate was 40 % (4/10) in those with a prenatal diagnosis< 20 weeks gestation. When intrauterine B19 V infection was diagnosed thereafter, 100 % (10/10) samples were B19 V DNA positive. Of the thirteen available DBS, twelve (92 %) tested positive. Viral load in CB and DBS corresponded inversely with time from fetal diagnosis to birth.Conclusion: B19 V DNA can be detected in neonatal blood samples of children following intrauterine B19 V infection, although the possibility of false-negatives, even in severe infections, should be considered. B19 V viral load at birth correlates with timing of infection.

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.

Aim This study aimed to estimate long-term impairment attributable to congenital cytomegalovirus infection (cCMV). Method This nationwide cohort study retrospectively assessed cCMV in children born in 2008 in the Netherlands, testing 31 484 stored neonatal dried blood spots. Extensive medical data of cCMV-positive children (n=133) and matched cCMV-negative comparison children (n=274) up to 6 years of age were analysed. Results Moderate to severe long-term impairment was diagnosed in 24.8% (33 out of 133) of all cCMV-positive children (53.8% in symptomatic, 17.8% in asymptomatic), compared with 12.0% (33 out of 274) of cCMV-negative children. Sensorineural hearing loss was seen only in five cCMV-positive children (3.8%). Developmental delays were diagnosed more often in cCMV-positive children than cCMV-negative children: motor (12.0% vs 1.5%), cognitive (6.0% vs 1.1%), and speech–language (16.5% vs 7.3%). Long-term impairment in multiple domains was more frequent in symptomatic (19.2%) and asymptomatic (8.4%) cCMV-positive children than cCMV-negative children (1.8%). Interpretation Children with cCMV were twice as likely to have long-term impairment up to the age of 6 years, especially developmental delays and sensorineural hearing loss, than cCMV-negative comparison children, with a risk difference of 12.8%. These insights into the risk of cCMV-associated impairment can help optimize care and stimulate preventive measures. What this paper adds Congenital cytomegalovirus infection (cCMV) leads to impairment in 25% of cases. Fifty per cent of children with cCMV symptoms at birth have long-term impairment. The risk difference of moderate to severe long-term impairment between children with and without cCMV is 13%, attributable to cCMV. cCMV leads to motor, cognitive, and speech–language developmental delay in children.

BackgroundBK polyomavirus (BKPyV)–associated nephropathy (BKPyVAN) is a major threat for kidney transplant recipients (KTRs). The role of specific BKPyV genotypes/serotypes in development of BKPyVAN is poorly understood. Pretransplantation serotyping of kidney donors and recipients and posttransplantation genotyping of viremic recipients, could reveal the clinical relevance of specific BKPyV variants.MethodsA retrospective cohort of 386 living kidney donor-recipient pairs was serotyped before transplantation against BKPyV genotype I–IV viral capsid protein 1 antigen, using a novel BKPyV serotyping assay. Replicating BKPyV isolates in viremic KTRs after transplantation were genotyped using real-time polymerase chain reaction and confirmed by means of sequencing. BKPyV serotype and genotype data were used to determine the source of infection and analyze the risk of viremia and BKPyVAN.ResultsDonor and recipient BKPyV genotype and serotype distribution was dominated by genotype I (>80%), especially Ib, over II, III and IV. Donor serotype was significantly correlated with the replicating genotype in viremic KTRs (P < .001). Individual donor and recipient serotype, serotype (mis)matching and the recipient replicating BKPyV genotype were not associated with development of viremia or BKPyVAN after transplantation.ConclusionsBKPyV donor and recipient serotyping and genotyping indicates the donor origin of replicating BKPyV in viremic KTRs but provides no evidence for BKPyV genotype–specific virulence.

Background Parvovirus B19 (B19V) DNA can be detected in blood over a long period after acute infection. Several reports associate the presence of B19V DNA with disease, irrespective of timing of the initial B19V infection. Objectives This study aims to analyze the properties of B19V DNA in blood, differentiating between bare, non-infectious strands of DNA and B19V DNA in viable virions. Study design Ten blood donors with asymptomatic acute B19V infection were followed and sampled up to 22 months after infection. The samples were treated with and without an endonuclease and tested for B19V DNA, to distinguish between DNA in virions and naked DNA. Results In the acute phase of infection, high levels of B19V DNA were detected, concurrent with B19V IgM antibodies. B19V DNA apparently was encapsidated, as indicated by resistance to endonuclease degradation. Subsequently, B19V DNA remained detectable for more than one year in all donors at low levels ( 5 IU/mL). Approximately 150days after infection B19V DNA became degradable by an endonuclease, indicating that this concerned naked DNA. In some donors a second endonuclease-resistant peak occurred. Discussion Detection of B19V DNA in blood by PCR does not necessarily imply that B19V replication takes place and that infectious B19V virions are present. We propose that remnant B19V DNA strands can be released from tissues without active replication. This finding urges to reconsider an assumed role of B19V infection mainly based on B19V DNA detection in blood, a much debated subject in clinical syndromes such as myocarditis and arthritis.

Introduction: Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables the unbiased detection of all potential pathogens in a clinical sample, including variants and even unknown pathogens. To apply mNGS in viral diagnostics, there is a need for sensitive and simultaneous detection of RNA and DNA viruses. In this study, the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections, with single tube DNA and RNA sample-pre-treatment and potential for automated pan-pathogen detection was studied. Materials and Methods: The sequencing protocol and bioinformatics analysis was designed and optimized including the optimal concentration of the spike-in internal controls equine arteritis virus (EAV) and phocine-herpes virus-1 (PhHV-1).The whole genome of PhHV-1 was sequenced and added to the NCBI database. Subsequently, the protocol was retrospectively validated using a selection of 25 respiratory samples with in total 29 positive and 346 negative PCR results, previously sent to the lab for routine diagnostics. Results: The results demonstrated that our protocol using Illumina Nextseq 500 sequencing with 10 million reads showed high repeatability. The NCBI RefSeq database as opposed to the NCBI nucleotide database led to enhanced specificity of virus classification. A correlation was established between read counts and PCR cycle threshold value, demonstrating the semi-quantitative nature of viral detection by mNGS. The results as obtained by mNGS appeared condordant with PCR based diagnostics in 25 out of the 29 (86%) respiratory viruses positive by PCR and in 315 of 346 (91%) PCR-negative results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow were influenza C, KI polyomavirus, and cytomegalovirus. Conclusions: Sensitivity and analytical specificity of this mNGS protocol was comparable with PCR and higher when considering off-PCR target viral pathogens. All potential viral pathogens were detected in one single test, while it simultaneously obtained detailed information on detected viruses.

We report a second case of pigeon paramyxovirus type 1 infection, which has pigeons and doves as reservoir, causing fatal respiratory disease in a human patient under immunosuppressive therapy. We recommend testing for this virus in respiratory disease cases where common respiratory pathogens cannot be identified., The characteristics and risk factors of pigeon paramyxovirus type 1 (PPMV-1) infection in humans are poorly known. We performed virological, pathological, and epidemiological analyses of a Dutch case, and compared the results with those of a US case. Both infections occurred in transplant patients under immunosuppressive therapy and caused fatal respiratory failure. Both virus isolates clustered with PPMV-1, which has pigeons and doves as reservoir. Experimentally inoculated pigeons became infected and transmitted the virus to naive pigeons. Both patients were likely infected by contact with infected pigeons or doves. Given the large populations of feral pigeons with PPMV-1 infection in cities, increasing urbanization, and a higher proportion of immunocompromised individuals, the risk of severe human PPMV-1 infections may increase. We recommend testing for avian paramyxovirus type 1, including PPMV-1, in respiratory disease cases where common respiratory pathogens cannot be identified.

Congenital Cytomegalovirus infection (cCMV) is the leading infection in determining permanent long-term impairments (LTI), and its pathogenesis is largely unknown due to the complex interplay between viral, maternal, placental, and child factors. The cellular activity, considered to be the result of the response to exogenous and endogenous factors, is captured by the determination of gene expression profiles. In this study, we determined whole blood transcriptomes in relation to cCMV, CMV viral load and LTI development at 6 years of age by using RNA isolated from neonatal dried blood spots (DBS) stored at room temperature for 8 years. As DBS were assumed to mainly reflect the neonatal immune system, particular attention was given to the immune pathways using the global test. Additionally, differential expression of individual genes was performed using the voom/limma function packages. We demonstrated feasibility of RNA sequencing from archived neonatal DBS of children with cCMV, and non-infected controls, in relation to LTI and CMV viral load. Despite the lack of statistical power to detect individual genes differences, pathway analysis suggested the involvement of innate immune response with higher CMV viral loads, and of anti-inflammatory markers in infected children that did not develop LTI. Finally, the T cell exhaustion observed in infected neonates, in particular with higher viral load, did not correlate with LTI, therefore other mechanisms are likely to be involved in the long-term immune dysfunction. Despite these data demonstrate limitation in determining prognostic markers for LTI by means of transcriptome analysis, this exploratory study represents a first step in unraveling the pathogenesis of cCMV, and the aforementioned pathways certainly merit further evaluation.

Congenital Cytomegalovirus infection (cCMV) is the most common cause of congenital infections worldwide causing permanent long-term impairment (LTI). cCMV immunopathogenesis remains largely unknown due to the complex interplay between viral, maternal, placental and child factors. The aim of this study was to determine the possible role of particular HLA antigens, of the number of HLA mismatches (mm) and non-inherited maternal antigens (NIMAs) in a large retrospective nation-wide cohort of children with cCMV and their mothers. HLA Class I (HLA-A, HLA-B and HLA-C) and HLA Class II (HLA-DR and HLA-DQ) were assessed in 96 mother-child pairs in relation to a control group of 5604 Dutch blood donors, but no significant differences were observed. Next, although these HLA antigens could not be assessed in relation to symptoms at birth, nor to LTI, due to the low number of cases, they could be evaluated in relation to CMV viral load. HLA-DRB1*04, and potentially HLA-B*51, was shown to have a protective role in the children as its frequency was increased in the low viral load group compared to the high viral load group, and this remained significant after correction. The number of HLA mm and of NIMAs were not associated to symptoms at birth nor to LTI or viral load. In conclusion, although none of the HLA alleles could be put forward as prognostic marker for long-term outcome, our findings give useful insights into cCMV pathogenesis, and identify potential HLAs that correlate with a better viral control.

Congenital CMV infection (cCMV) is the most common congenital infection causing permanent long-term impairments (LTI). cCMV immunopathogenesis is largely unknown due to the complex interplay between viral, maternal, placental, and child factors. In this study, a large retrospective nationwide cohort of children with cCMV and their mothers was used. HLA-C, HLA-E, and HLA-G were assessed in 96 mother-child pairs in relation to symptoms at birth and LTI at 6 years of age. The mothers were additionally typed for killer cell immunoglobulin-like receptors. The maternal HLA-G 14 bp deletion/deletion polymorphism was associated with a worse outcome, as the immunomodulation effect of higher protein levels may induce less CMV control, with a direct impact on placenta and fetus. The absence of maternal HLA-C belonging to the C2 group was associated with symptoms at birth, as activating signals on decidual NK may override inhibitory signals, contributing to a placental pro-inflammatory environment. Here, the increased HLA-E*0101 and HLA-C mismatches, which were associated with symptoms at birth, may enhance maternal allo-reactivity to fetal Ags, and cause suboptimal viral clearance. Finally, HLA-C non-inherited maternal antigens (NIMAs) were associated with LTI. The tolerance induced in the fetus toward NIMAs may indirectly induce a suboptimal CMV antiviral response throughout childhood. In light of our findings, the potential role of maternal-child HLA in controlling CMV infection and cCMV-related disease, and the clinical value as predictor for long-term outcome certainly deserve further evaluation.

In children, rhinovirus viremia has been associated with higher nasopharyngeal loads and increase in severity of clinical signs and symptoms.This study aims to detect rhinovirus viremia in adult patients and to establish potential correlations with the clinical course.Adult patients with rhinovirus strongly positive bronchoalveolar lavages (BAL, quantitation cycle, Cq values25) detected between 2008 and 2014 were studied retrospectively. Blood sampled between two weeks before and two weeks after BAL sampling was tested for rhinovirus RNA. Underlying conditions, symptoms, radiography, microbiological data, and disease outcome were analysed.Twenty-seven of 43 patients with rhinovirus positive BAL at Cq values25 had blood samples available within the prespecified time-frame (mean blood 3-4 samples per patient). Four of these 27 patients (15%) tested rhinovirus RNA positive in their blood (of whom one patient twice). Genotyping demonstrated rhinovirus A01, A24, B52 and B92 in these four immunocompromised patients. Viremic patients were not significantly different with regard to underlying conditions, respiratory symptoms, radiological findings, co-pathogens nor the number of blood samples tested for RV. However, patients with rhinovirus viremia had significant higher mortality rates compared to patients without viremia, as all four died as a consequence of respiratory problems (100%) versus 22% (5/23), p=0.007 (Fisher's exact).Rhinovirus viremia can occur in adult patients with a high viral load in BAL fluid. Rhinovirus viremia may be considered a negative prognostic factor, although a causative role with regard to the adverse outcome has yet to be demonstrated.

Congenital Cytomegalovirus infection (cCMV) is the most common cause of congenital infections worldwide that can cause long-term impairment (LTI). The metabolic alterations due to cCMV are largely unknown. This study aims to assess the metabolites included in the neonatal screening in relation to cCMV and cCMV outcome, allowing the identification of prognostic markers for clinical outcome. Essential amino acids, hormones, carnitines and enzymes from Dried Blood Spots (DBS) were analyzed of 102 children with cCMV and 179 children without cCMV, and they were related to symptoms at birth and LTI at 6years of age. In this cohort, the neonatal screening parameters did not change in relation to cCMV, nor to symptoms at birth or LTI. However, metabolic changes were observed in children born preterm, with lower concentrations of essential amino acids in premature infants with cCMV compared to premature controls. Finally, a higher concentration of palmytoilcarnitine (C16) in the group with higher viral load was observed. Though these data demonstrate limitations in the use of neonatal screening data as predictors for long-term cCMV outcome, the metabolism of preterm neonates with cCMV merits further evaluation.

Congenital cytomegalovirus (cCMV) infection is the most common congenital infection worldwide and can lead to long-term impairments such as developmental delay. It is currently unknown how this affects the daily life of children and their parents. Children For this study, children with cCMV were identified by testing stored dried blood spots of 31,484 five-year-old children born in 2008 in the Netherlands. Parents of 133 children with cCMV and 274 children without cCMV participated and filled in questionnaires on the child's development, the child's and parents' quality of life, care provided for the children and consequences of cCMV on daily life. School performance reports at 6 years of age were also investigated. Children with cCMV had delays in general and expressive language development more often, and they attended physical therapists more frequently than children without cCMV. School performance of children with cCMV and symptoms at birth was poorer than that of cCMV-negative children with similar symptoms at birth. The quality of life of children with long-term impairment was lower in children with cCMV than those without cCMV. Parents of children with cCMV and long-term impairments reported more physical and concentration problems than parents of children without cCMV. These findings indicate that cCMV has a considerable impact not only on the child's development and school performance but also on the daily life of children and their parents. The care for children with cCMV should therefore include support for motor and speech-language development as well as family-centered care.

International audience; Kidney transplant donors are not currently implicated in predicting BK polyomavirus (BKPyV) infection in kidney transplant recipients. It has been postulated, however, that BKPyV infection originates from the kidney allograft. Because BKPyV seroreactivity correlates with BKPyV replication and thus might mirror the infectious load, we investigated whether BKPyV seroreactivity of the donor predicts viremia and BKPyV-associated nephropathy (BKPyVAN) in the recipient. In a retrospective cohort of 407 living kidney donor-recipient pairs, pretransplantation donor and recipient sera were tested for BKPyV IgG levels and correlated with the occurrence of recipient BKPyV viremia and BKPyVAN within 1 year after transplantation. Donor BKPyV IgG level was strongly associated with BKPyV viremia and BKPyVAN (p < 0.001), whereas recipient BKPyV seroreactivity showed a nonsignificant inverse trend. Pairing of high-BKPyV-seroreactive donors with low-seroreactive recipients resulted in a 10-fold increased risk of BKPyV viremia (hazard ratio 10.1, 95% CI 3.5-29.0, p < 0.001). In multivariate analysis, donor BKPyV seroreactivity was the strongest pretransplantation factor associated with viremia (p < 0.001) and BKPyVAN (p = 0.007). The proportional relationship between donor BKPyV seroreactivity and recipient infection suggests that donor BKPyV seroreactivity reflects the infectious load of the kidney allograft and calls for the use of pretransplantation BKPyV serological testing of (potential) donors and recipients.

Introduction: Exacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations. Objectives: To study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS. Study design: 88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006–2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an Illumina library preparation protocol with in-house adaptations. Results: By mNGS, 24/88 samples tested positive. Sensitivity and specificity, as compared with PCR, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). Additional viral pathogens detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral normalized species reads (log value) (p = 0.002). Patients with viral pathogens had lower percentages of bacteriophages (p

Because maternal seropositivity for CMV is associated with substantial protection against congenital CMV infection, prevention measures have focused mainly on seronegative pregnant women for decades. However, population-wide insight in the contribution of nonprimary infection (reactivation and/or re-infection with a different strain) on the most common sequela, hearing loss, is missing. A population-based prediction model was developed to estimate the proportion of congenital CMV-related hearing loss resulting from nonprimary maternal infection. Incorporated was a meta-analysis of the risk of hearing loss, calculating pooled proportions of children with hearing loss after nonprimary and primary infection. Subsequently, the model was applied for worldwide present population seroprevalences (range 30-95%). It was estimated that, for all population seroprevalences, nonprimary maternal infections are responsible for the majority of congenital CMV infections. This proportion increased with seroprevalence, ranging from 57% (95%CI 24-85%) to 96% (95% CI 88-99%) for seroprevalences of 30% to 95%. Our meta-analysis (six reports) showed that the risk of hearing loss after nonprimary infection was 11% (28/253 children, 95% CI 7-15%) versus 13% (50/385 children, 95% CI 10-16%) after primary infection. Incorporating this risk into our model, we estimated that nonprimary infections also accounted for the majority of CMV-related hearing loss. This proportion ranged from 53% (95% CI 13-86%) to 95% (95% CI 62-99%) for seroprevalences of 30% to 95%. Our data underline the worldwide contribution of nonprimary infections in causing CMV-related hearing loss. These results imply that prevention research such as vaccine and hygiene studies should not only be directed at seronegative but also seropositive pregnant women. © 2013 John Wiley & Sons, Ltd.

Background Recently we showed that the level of BK polyomavirus (BKPyV) IgG seroreactivity in kidney donors predicted viremia and BKPyV-associated nephropathy in kidney transplant recipients (KTRs). This observation could be explained by assuming a direct association between BKPyV seroreactivity and the amount of persistent infectious virus in the renal allograft. Objectives Since the renal BKPyV reservoir is probably sowed by viremia during primary BKPyV infection, we systematically analysed the dynamics of BKPyV IgG seroreactivity in relation to preceding BKPyV viremia in KTRs and healthy individuals. Study design A cohort of 85 KTRs consisting of BKPyV viremic and nonviremic subjects was analysed for BKPyV IgG seroreactivity at five fixed time points until one year after transplantation. A cohort of 87 healthy blood donors (HBDs) was used as controls. Results Baseline BKPyV seropositivity was high in both KTRs and HBDs, and the baseline mean BKPyV IgG level comparable. BKPyV IgG levels in nonviremic KTRs and HBDs remained stable during follow-up, while a considerable increase was observed in viremic KTRs (p = 0.015). The increase of BKPyV seroreactivity in viremic KTRs was associated with the duration and peak level of BKPyV viremia. Conclusions BKPyV IgG seroreactivity was stable over time in immunocompetent subjects, which enables the use of this potential pretransplantation biomarker in kidney donors. The observed dose-dependent relationship of BKPyV IgG seroreactivity with preceding BKPyV replication is in agreement with the assumption that BKPyV seroreactivity reflects past BKPyV activity and correlates with the amount of latent BKPyV residing within a kidney allograft.

Congenital CMV infection (cCMV) is the most common congenital infection that can cause long-term impairment (LTI). The pathogenesis of LTI is not completely understood. Fetal immunity may play a role in controlling the infection and preventing LTI, although immune activation may also contribute to fetal immunopathology. In this study, we analyzed various molecular markers of T and B cell numbers in neonatal dried blood spots of 99 children with cCMV and 54 children without cCMV: δRec-ψJα signal joints on TCR excision circles, intron recombination signal sequence k-deleting element signal joints on Igκ-deleting recombination excision circles, genomic intron recombination signal sequence k-deleting element coding joint, genomic Vδ1-Jδ1, and Vδ2-Jδ1 rearrangements. Of this cohort, clinical symptoms at birth and LTI at 6 y of age were recorded. Neonates with cCMV had fewer TCR excision circles in their blood than non-infected controls. Furthermore, cCMV infection was associated with increased numbers of γδ T cells and B cells, and these numbers were positively correlated with CMV viral load in the dried blood spots. Infected children with a better long-term outcome had higher numbers of B cells at birth than those who developed LTI; no difference in B cell replication was observed. The potential protective role of B cells in controlling cCMV-related disease and the clinical value of this marker as a predictor of long-term outcome merit further evaluation.

SUMMARYCongenital cytomegalovirus infection (cCMV) may lead to symptoms at birth and long-term consequences. We present a nationwide, retrospective cohort study on the outcome of cCMV up to age 6 years. For this study we identified cCMV, using polymerase chain reaction, by analysing dried blood spots, which are taken shortly after birth for neonatal screening. The group of children with cCMV were compared to a group of children who were cCMV negative at birth. Data were collected about their health and development up to age 6 years. Parents of 73 693 children were invited to participate, and 32 486 (44·1%) gave informed consent for testing of their child's dried blood spot for CMV. Of the 31 484 dried blood spots tested, 156 (0·5%) were positive for cCMV. Of these, four (2·6%) children had been diagnosed with cCMV prior to this study. This unique retrospective nationwide study permits the estimation of long-term sequelae of cCMV up to the age of 6 years. The birth prevalence of cCMV in this study was 0·5%, which is in line with prior estimates. Most (97·4%) children with cCMV had not been diagnosed earlier, indicating under-diagnosis of cCMV.

Host immunity dictates influenza A(H1N1)pdm09 infection outcome in hematology–oncology patients

Genotyping of cytomegalovirus (CMV) is useful to examine potential differences in the pathogenicity of strains and to demonstrate coinfection with multiple strains involved in CMV disease in adults and congenitally infected newborns. Studies on genotyping of CMV in dried blood spots (DBS) are rare and have been hampered by the small amount of dried blood available. In this study, two multiplex real-time PCR assays for rapid gB and gH genotyping of CMV in DBS were developed. Validation of the assays with 39 CMV-positive plasma samples of transplant recipients and 21 urine specimens of congenitally infected newborns was successful in genotyping 100% of the samples, with gB1 and gB3 being the most prevalent genotypes. Multiple gB and gH genotypes were detected in 36% and 33% of the plasma samples, respectively. One urine sample from a newborn with symptomatic congenital CMV was positive for gB1 and gB2. DBS of congenitally infected newborns ( n = 41) were tested using 9 μl of dried blood, and genotypes were detected in 81% (gB) and 73% (gH) of the samples, with gB3 being the most prevalent genotype. No clear association of specific genotypes with clinical outcome was observed. In conclusion, the CMV gB and gH PCR assays were found to be rapid, sensitive for detecting mixed infections, and suitable for direct usage on DBS. These assays are efficient tools for genotyping of CMV in DBS of congenitally infected newborns.

BACKGROUND Persistent infections with herpesviruses such as human cytomegalovirus (HCMV) frequently occur after solid organ or stem cell transplantation, and are due to either failure of the host to immunologically control the virus or emerging resistance of the virus to the antiviral drug(s) used. Antiviral therapy can be guided by viral drug susceptibility testing based on screening for known resistance-inducing mutations in the viral genome. Mass spectrometry-based comparative sequence analysis (MSCSA) might be advantageous for this purpose because of its suitability for semi-automation. OBJECTIVES The applicability of MSCSA to detect sequence polymorphisms and drug resistance-inducing mutations in the HCMV genome was investigated. STUDY DESIGN We analyzed the 3' part of the HCMV UL97 gene, which encodes the kinase that is activated by the commonly used anti-HCMV drug ganciclovir. Sequences obtained by MSCSA of material from HCMV-infected patients (43 samples) and the HCMV type strain were compared to conventional cycle sequencing results. RESULTS In 94.1% of all samples the results obtained by MSCSA of the UL97 gene were identical to those from conventional cycle sequencing. The threshold to detect mutant sequences in a mixture with wild-type material was 20% using either technique. Furthermore, MSCSA was successfully applied to study the development of drug resistance in a patient who developed encephalitis due to ganciclovir-resistant HCMV. CONCLUSIONS MSCSA was found to be equally accurate compared to conventional cycle sequencing in the analysis of UL97 of HCMV.

In this article, we review the virology, pathology, epidemiology and clinical spectrum of intrauterine human parvovirus B19 (B19V) infection, including intrauterine fetal death, non-immune hydrops fetalis, thrombocytopenia and neurological manifestations such as pediatric stroke and perivascular calcifications. In addition, we discuss the new insights into the neurodevelopmental outcome of intrauterine B19V infection. Current diagnosis and management of B19V infection is summarized, including a diagnostic and follow-up flowchart for practical clinical use. Copyright © 2011 John Wiley & Sons, Ltd.

Congenital cytomegalovirus (CMV) infection is an important public health problem with approximately 7 in 1,000 newborns infected and consequently at risk for hearing impairment. Newborn hearing screening will fail to detect this hearing impairment in approximately half of the cases because late onset hearing loss is frequent. Hearing impairment has profound impact on cognitive and social development of children and their families, determining most of the disease burden of congenital CMV infection. The potential value of newborn screening for congenital CMV is increasingly discussed. To date, many experts acknowledge the benefit of antiviral treatment in the prevention of hearing deterioration in newborns with neurological symptoms, and the benefit of early identification of late-onset hearing impairment by means of extensive audiological follow up of infected infants. These opinions imply that the potential of newborn screening for CMV would lie in the identification of the large proportion of asymptomatic congenitally infected newborns at risk for developing late-onset hearing loss. Experience with postnatal antiviral treatment of symptomatic newborns is encouraging, but has not been studied in asymptomatic congenitally infected newborns. A large-scale study on the safety and effectiveness of combined screening and antiviral therapy for congenital CMV infection is the necessary next step to take and should not be delayed.

A retrospective nationwide study on the use of intravenous (IV) zanamivir in patients receiving intensive care who were pretreated with oseltamivir in the Netherlands was performed. In 6 of 13 patients with a sustained reduction of the viral load, the median time to start IV zanamivir was 9 days (range, 4-11 days) compared with 14 days (range, 6-21 days) in 7 patients without viral load reduction (P = .052). Viral load response did not influence mortality. We conclude that IV zanamivir as late add-on therapy has limited effectiveness. The effect of an immediate start with IV zanamivir monotherapy or in combination with other drugs need to be evaluated.

Background. Cytomegalovirus (CMV) infections after transplantation are commonly treated using a prophylactic or preemptive regimen with (val)ganciclovir. It remains unclear, which approach is most effective in preventing CMV disease in D+R— patients. The aim of this retrospective study was to compare the treatment response and antiviral resistance in CMV infections between two treatment regimens in D+R— renal transplant recipients. Methods. Before 2006, a preemptive treatment regimen with valganciclovir was applied (42 patients). From 2006 onwards, patients first received prophylaxis with valganciclovir for 90 days, followed by a preemptive regimen (29 patients). CMV infections were monitored by regular determination of the CMV DNA load in plasma. Patient charts were reviewed for antiviral treatment data, and resistance was analyzed by nucleotide sequence analysis of the UL97 and UL54 genes in CMV DNA-positive samples. Results. Treatment failure, defined as a CMV DNA load more than or equal to 1000 copies/mL after at least 2 weeks of treatment, occurred less frequently in the prophylaxis cohort than in the preemptive cohort (14% vs. 71 %, P

Background: Congenital infections are associated with a wide spectrum of clinical symptoms, including lenticulostriate vasculopathy (LSV). Objective: To determine the relationship between LSV and congenital infections, as diagnosed by TORCH serology and viral culture for cytomegalovirus (CMV). Methods: All neonates with LSV admitted to our neonatal intensive-care unit from 2004 to 2008 were included in the study. Results of maternal and neonatal TORCH testing were evaluated. Results: During the study period, cranial ultrasound scans were performed in 2,088 neonates. LSV was detected in 80 (4%) neonates. Maternal and/or neonatal serological TORCH tests were performed in 73% (58/80) of cases. None of the mothers or infants (0 of 58) had positive IgM titres for Toxoplasma, rubella, CMV or herpes simplex virus. Additional urine culture for CMV was performed in 38 neonates. None of the infants (0 of 38) had a positive CMV urine culture test. Conclusions: Routinely applied efforts to diagnose congenital infections in cases presenting with LSV have a poor yield. Routine TORCH screening in neonates with LSV cases should only be regarded as mandatory once well-designed studies demonstrate a clear diagnostic benefit.

The factors that cause prolonged human influenza virus respiratory tract infection and determine its clinical impact and the development of drug-resistant viruses are unclear. During a 3-year period, symptomatic influenza virus excretion for 2 weeks was observed among 8 immunocompromised patients and found to be associated with lymphocytopenia at onset (8 of 8 patients) more often than with granulocytopenia (2 of 8 patients) or monocytopenia (2 of 8 patients). Six (75%) of 8 patients developed influenza lower respiratory tract infection (10 episodes), and receipt of oseltamivir treatment was significantly associated with clinical improvement (8 of 8 episodes vs. 0 of 2 untreated episodes; P = .02). Complete viral clearance was strongly correlated with lymphocyte reconstitution (P = .04) but was never observed during the first 2 weeks after oseltamivir treatment. Neuraminidase inhibitor-resistant influenza viruses emerged in 2 (67%) of 3 patients eligible for resistance analysis. In conclusion, prolonged influenza virus infection was associated with lymphocytopenia, influenza lower respiratory tract infection, and frequent development of drug resistance during antiviral therapy. Clinical improvement in influenza lower respiratory tract infection is observed during oseltamivir treatment, but complete viral clearance is dependent on lymphocyte reconstitution, irrespective of receipt of antiviral medication.

Increased vaccine doses and mid-season boosting may increase the proportion of residents with protective immunity from influenza in long-term care facilities. In a multi-center study (1997-1998), 815 residents from 14 long-term care facilities were assigned at random to receive 15 or 30 microg of inactivated influenza vaccine, followed by a 15 microg booster vaccine or a placebo vaccine at Day 84. Seroresponses were re-analyzed by hemagglutination-inhibition (> or =4-fold titer increases, protective titer > or =40, geometric mean titers. Forty percent of the participants had pre-vaccination titers > or =40. At Day 25 after vaccination, this increased to 66.3% after a 15 microg dose versus 73.3% after a dose of 30 microg (P = 0.049). Participants receiving a 30 microg dose followed by a 15 microg booster showed more > or =4-fold titer increases at Day 109 (43.6% vs. 35.4%, P = 0.003) and protective titers > or =40 (74.2% vs. 64.6%, P = 0.041), compared to those receiving only a 15 microg dose. Differences were most apparent in participants with low pre-vaccination titers. Booster vaccination after an initial 15 microg dose of the vaccine did not increase the protective rate (61.9% vs. 63.9% after placebo). The number of participants needed to vaccinate to protect one additional resident by a dose of 15 microg was 4, by a dose of 30 microg 3, and 15 when using a 30 microg dose instead of 15 microg. Doubling the dose of influenza vaccine increased protection-related responses among residents of long-term care facilities, especially in those with low pre-vaccination titers.

Influenza and respiratory syncytial Virus (RSV) infections are responsible for considerable morbidity, mortality and health-care resource use. For the Netherlands, we estimated age and risk-group Specific numbers of antibiotics, otologicals and cardiovascular prescriptions per 10,000 person-years during periods with elevated activity of influenza or RSV, and compared these with peri-season rates. Data were taken from the University of Groningen in-house prescription database (www.iadb.nl) and Virological Surveillance for the period 1998-2006. During influenza and RSV periods excess antibiotic prescriptions were estimated for all age groups. In the age groups 0-1 and 2-4 years. excess antibiotic prescriptions during periods with elevated RSV activity (65% and 59% of peri-seasonal rates) exceeded the surpluses estimated during the influenza-activity periods (24% and 34% of peri-seasonal rates) while for otologicals excess prescriptions were higher for influenza (22% and 27%) than for RSV (14% and 17%). Among persons of 50 years and older, notably those without medical high-risk conditions, excess prescriptions for cardiovascular medications were estimated during the influenza periods at approximately 10% (this was also already seen in persons aged 45-49). Our results may have implications for influenza vaccination policies. In particular, extension of influenza vaccination to groups of non-elderly adults and young children may lower excess prescriptions during these influenza periods for all three types of drug prescriptions investigated. (C) 2008 Elsevier Ltd. All rights reserved.

Reports on infectious complications following reduced intensity conditioning (RIC) before allogeneic stem cell transplantation (allo-SCT) are equivocal. This prospective follow-up study compared the impact of cytomegalovirus (CMV) infections following RIC with fludarabine, ATG and busulphan or conventional myeloablative conditioning (MAC). Forty-eight RIC and 59 MAC patients were enrolled. The occurrence and severity of CMV infections within 100 days following allo-SCT were assessed, using plasma CMV DNA load kinetics. CMV DNAemia was observed in 21 RIC (60%) and in 19 MAC (44%) patients at risk for CMV. The mean CMV DNAemia free survival time was comparable following RIC and MAC: 70 days (95% (confidence interval) CI: 59-80 days) and 77 days (95% CI: 68-86 days), respectively (P=0.24). Parameters indicative for the level of CMV reactivation, including the area under the curve of CMV DNA load over time as well as the onset, the peak values and duration of CMV infection episodes, the numbers and duration of CMV treatment episodes and recurrent infections, were not different in both groups. During follow-up, none of the patients developed CMV disease. RIC with fludarabine, ATG and busulphan demonstrated safety comparable to conventional MAC with regard to frequency and severity of CMV infections within 100 days following T cell-depleted allo-SCT.

Adenovirus infections after allogeneic stem cell transplantation (SCT), particularly in children, may be severe and protracted. Up to 51 different serotypes of adenovirus are presently recognized but serotyping is usually limited to initial viral isolates.A systematic and sustained analysis of adenovirus serotypes in a cohort of adenovirus-infected pediatric SCT recipients, correlated to transplant-associated variables.Eighty-three consecutive pediatric SCT recipients were studied by culture of feces and adenoviruses isolated were serotyped by neutralization. Upon persistent viral excretion, serotyping was repeated for at least two isolates of any infectious episode, including initial and final isolates, and patients with single and multiple serotypes were compared. In a subset of cases, serotyping of fecal isolates was compared to genotypic analysis.In 33 patients, adenovirus was isolated at least once after SCT. Serotyping uncovered 49 different adenoviruses, including three isolates without an assigned serotype. In 16 patients, a single serotype was present for a sustained period, whereas 12 patients (36%) showed multiple serotypes. Comparison of these groups demonstrated more frequent non-malignant primary disease with multiple infections (p0.01), but otherwise no significant differences were observed, although single serotype infections had a lower survival rate. Remarkably, serotype 31 appeared initially in 7 out of 12 patients with multiple infections. Genotyping by sequencing confirmed neutralization assays at least at the species level in 14 of 18 isolates.In 36% of adenovirus infections after SCT more than one serotype could be detected by sequential analysis. Multiple serotypes occurred more often with non-malignant disorders. Adenovirus serotype 31 was often included. This finding is relevant for diagnostic purposes and immunotherapeutic interventions and provides insight into the pathogenesis of this problem.

Objectives: Facilitate risk assessment of vital complications in fetuses of pregnancies affected by acute parvovirus B19 (B19V) infection. Design: Study of the natural course of maternal B19V infection in four cases, from early pregnancy on. Setting: University Medical Center in the Netherlands. Population: Pregnant mothers attending obstetric services. Methods: Serial measurements of the maternal and fetal or neonatal viral load and antibody responses. Main Outcome Measures: Maternal and fetal/neonatal serum B19V viral DNA load and specific IgM and IgG antibodies in maternal serum. Results: Peak viral load levels occurred within 1 week after maternal infection and peak IgM levels were observed 1 week after the peak viral load levels. Approximation of IgG and IgM ratios usually took place 4 weeks after infection. Vertical transmission occurred 1–3 weeks after maternal infection, suggesting that fetal infection occurs during the maternal peak viral load. Conclusions: Maternal B19V DNA load levels and IgM responses are useful to estimate the risk of parvovirus B19-associated fetal complications. The maternal peak viral load directly precedes the onset of fetal infection and may be used to indicate the stage of intrauterine B19V infection.

Background and methods To study clinical symptoms, timing and consequences of human herpesvirus-6 (HHV-6) reactivation after pediatric allogeneic stem cell transplantation (SCT), HHV-6 was investigated by plasma polymerase chain reaction in a cohort of 106 pediatric SCT recipients. Results HHV-6 viremia was detected post-SCT in 48% of the patients with a median time of onset at 20 days after SCT. In week 3 and 4 post-SCT, HHV-6 is the most common infectious agent detected. In up to 30% of the patients with fever of unknown origin, HHV-6 was the only detected infectious agent to explain fever. Patients transplanted with an unrelated donor or receiving serotherapy were at increased risk of HHV-6 reactivation. The onset of HHV-6 reactivation coincided with the appearance of lymphocytes and monocytes in peripheral blood. Treatment with alemtuzumab (MabCampath) delayed both lymphocyte and monocyte engraftment and, concomitantly, onset of HHV-6 reactivation was delayed in those cases. HHV-6 reactivation was not associated with an increased incidence of acute graft-versus-host disease (GvHD). However, progression to grade II-IV GvHD was in 9 of 10 patients associated with HHV-6 reactivation before GvHD (P = 0.006) and HHV-6 was the only infection with such an association. Conclusions HHV-6 frequently reactivates after pediatric SCT around the time of mononuclear cell engraftment and is associated with an increased severity of GvHD. HHV-6 may explain fever of unknown origin in 30% of the patients early after SCT. Assessment of HHV-6 reactivation in patients early after SCT can be instrumental for clinical decision making.