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Norovirus causes an estimated 18% of all cases of acute gastroenteritis worldwide and is found to be associated with mortality. To create a first overview of severe complications and chronic sequelae of norovirus infections, a systematic review of literature was performed. Of 3928 individual hits, 176 publications remained for data extraction. Study periods varied between 1974 and 2017, though strongly skewed towards the last decade (n = 122, 70%). Countries of studies were worldwide, though Africa, and Carribean, Central and South America were underrepresented. Strong evidence was found for chronic diarrhea in immunocompromised patients, affecting 9%-100% of investigated cohorts. The duration of chronic diarrhea varied from four weeks up to nine years, leading to either wasting, weight loss or failure to thrive in a third of the reported cases (224). Other complications with significant evidence were necrotizing enterocolitis (NEC) in preterm infants associated with norovirus infection (8 papers), and benign infantile convulsions with gastroenteritis (BICG; 19 papers). Studies on norovirus infection and inflammatory bowel disease (IBD) mostly concluded against this association (5 of 7). The remaining papers mentioned a large variety of possible sequelae or complications. Based on the available literature, chronic norovirus diarrhea is the major sequela of norovirus infection in primary immune deficient, oncologic and transplant patients. Norovirus infection - like other gastrointestinal pathogens - can cause a range of sequelae and complications, and should be considered in the differential diagnosis of these manifestations.

Highlights • Commercial SARS-CoV-2 RT-PCR analytical performance comparison. • Analytical sensitivity of 13 SARS-CoV-2 RT-PCRs varied between 3.3–330 RNA copies. • All but 1 assay had sensitivity within one order of magnitude of the reference assay. • Only 1 assay cross reacted with another coronavirus (MERS)., The emergence of a new coronavirus in Wuhan China has triggered a global need for accurate diagnostic assays. Initially, mostly laboratory developed molecular tests were available but shortly thereafter different commercial assays started to appear and are still increasing in number. Although independent performance evaluations are ongoing, available data is still scarce. Here we provide a direct comparison of key performance characteristics of 13 commercial RT-PCR assays. Thirteen RT-PCR assays were selected based on the criteria that they can be used following generic RNA extraction protocols, on common PCR platforms and availability. Using a 10-fold and 2-fold dilution series of a quantified SARS-CoV-2 cell-cultured virus stock, performance was assessed compared to our in house validated assay. Specificity was tested by using RNA extracted from cultured common human coronaviruses. All RT-PCR kits included in this study exhibited PCR efficiencies > 90%, except for the Sentinel Diagnostics B E-gene RUO assay (80%). Analytical sensitivity varied between 3.3 RNA copies to 330 RNA copies. Only one assay cross reacted with another human coronavirus (MERS). This study provides a technical baseline of 13 different commercial PCR assays for SARS-CoV-2 detection that can be used by laboratories interested in purchasing any of these for further full clinical validation.

Background Diagnostic requests for both Zika virus (ZIKV) and dengue virus (DENV) infections in returning travelers have significantly increased during the recent ZIKV outbreak in the Americas. These flaviviruses have overlapping clinical syndromes and geographical distribution, but diagnostic differentiation is important because of different clinical consequences. As flaviviruses are known to have a short viremic period, diagnostics often rely on serological methods, which are challenging due to extensive cross-reactive antibodies. Objective To re-evaluate the performance of DENV serological assays in laboratory confirmed ZIKV-infected travelers. Study design The extent of cross-reactivity of the DENV NS1 antigen, IgM and IgG ELISA was analyzed in 152 clinical blood samples collected from 69 qRT-PCR and 24 virus neutralization titer (VNT) confirmed ZIKV-infected travelers. Results The majority of travelers in the presented cohort returned to the Netherlands from Suriname and presented with symptoms of fever and rash. Twenty-three percent of the female travelers were pregnant. None of the 39 ZIKV RNA positive blood samples were cross-reactive in the DENV NS1 antigen ELISA. The rates of cross-reactivity of the DENV IgM and IgG ELISAs were 31% and 54%, respectively, after excluding travelers with (potential) previous DENV exposure. Conclusions Although the DENV NS1 antigen assay was highly specific in this cohort of laboratory confirmed ZIKV-infected travelers, we demonstrate high percentages of cross-reactivity of DENV IgM and IgG ELISAs of which diagnostic laboratories should be aware. In addition, the high rate of DENV IgG background of >25% complicates a proper serological diagnosis in this group.

Background Clinical signs and symptoms of different airway pathogens are generally indistinguishable, making laboratory tests essential for clinical decisions regarding isolation and antiviral therapy. Immunochromatographic tests (ICT) and direct immunofluorescence assays (DFA) have lower sensitivities and specificities than molecular assays, but have the advantage of quick turnaround times and ease-of-use. Objective To evaluate the performance of a rapid molecular assay, ARIES FluA/B & RSV, using laboratory developed RT-PCR assays (LDA), ICT (BinaxNOW) and DFA. Methods Analytical and clinical performance were evaluated in a retrospective study arm (stored respiratory samples obtained between 2006–2015) and a prospective study arm (unselected fresh clinical samples obtained between December 2015 and March 2016 tested in parallel with LDAs). Results Genotype inclusivity and analytical specificity was 100%. However, ARIES was 0.5 log, 1–2logs and 2.5logs less sensitive for fluA, RSV and fluB respectively, compared to LDA. In total, 447 clinical samples were included, of which 15.4% tested positive for fluA, 9.2% for fluB and 26.0% for RSV, in both LDA and ARIES. ARIES clinical sensitivity compared to LDA was 98.6% (fluA), 93.3% (fluB) and 95.1% (RSV). Clinical specificity was 100% for all targets. ARIES detected 10.6% (4 fluA, 8 fluB, 11 RSV) and 26.9% (7 fluA, 3 fluB, 22 RSV) more samples compared to DFA and ICT, all confirmed by LDA. Conclusion Although analytically ARIES is less sensitive than LDA, the clinical performance of the assay in our tertiary care setting was comparable, and significantly better than that of the established rapid assays.

Highlights • Clinical specificity of Panther Fusion® is between 96 %–100 %, compared to LDT. • Clinical sensitivity Panther Fusion® is between 71.9 %–100 %, compared to LDT. • Overall linear regression showed good correlations between LDT and Panther Fusion® for all viruses, except RV and PIV-4. • The Panther Fusion® provides a random-access system with continuous loading and shorter sample-to-answer times compared to LDT., Background Respiratory tract infections are among the most common infections during winter season. Rapid diagnostics is required for clinical decision making regarding isolation of patients and appropriate therapy. Objectives The aim of this study was to evaluate the analytical and clinical performance characteristics of the Panther Fusion® respiratory panel using published laboratory-developed real-time PCR assays (LDT). Study design Analytical sensitivity of Panther Fusion® Flu A/B/RSV was assessed by testing dilutions of cell culture isolates. Clinical performance assessment included the complete Panther Fusion® respiratory panel (Flu-A/B/RSV, PIV 1-4 and AdV/hMPV/RV) and consisted of a retrospective and a prospective study-arm. The retrospective evaluation included 201, stored (−80 °C) samples collected between February 2006 and January 2017. Prospective evaluation was performed on 1045 unselected pretreated respiratory tract samples from patients presented to our hospital between November 2017 and May 2018. Results Analytical sensitivity was generally slightly lower for the Panther Fusion® assays. Clinical specificity and sensitivity was between 96 %–100 % and 71.9 %–100 %, respectively. Discrepant results were found in 146 samples of which 88 samples tested LDT positive / Panther Fusion® negative and 58 samples were LDT negative / Panther Fusion® positive. A total of ten discrepant samples with Ct-values

Highlights • The here studied rapid antigen detection test (RADT) BinaxNOW RSV has a high sensitivity and positive predictive value. • RADT BinaxNOW Influenza A&B has a relatively low sensitivity and positive predictive value. • We advise a restricted use of RADT BinaxNOW Influenza A&B in a tertiary paediatric care setting., Background Rapid antigen detection tests (RADTs) are increasingly used to detect influenza viruses and respiratory syncytial virus (RSV). However, their sensitivity and specificity are a matter of debate, challenging their clinical usefulness. Objectives Comparing diagnostic performances of BinaxNow Influenza AB® (BNI) and BinaxNow RSV® (BNR), to those of real-time reverse transcriptase PCR (RT-PCR), virus isolation and direct immunofluorescence (D-IF) in paediatric patients. Study design Between November 2005 and September 2013, 521 nasal washings from symptomatic children (age

Highlights • A simple typing of MERS-CoV variants is crucial in monitoring the MERS-CoV outbreak. • The developed MERS-CoV typing assay provides an indication of the MERS-CoV variant. • It allows more informative initial screening in labs with basic sequencing capacity. • The data aid in informing effective international preparedness and response., Background Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes lower respiratory tract infection in humans. Camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. Human-to-human transmission occurs sporadically. The wide geographic distribution of MERS-CoV among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a MERS-CoV variant with efficient human-to-human transmission capabilities. Phylogenetic analysis of MERS-CoV has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples. Objective To develop a simple, reliable MERS-CoV variant typing assay to facilitate monitoring of MERS-CoV diversity in animals and humans. Study Design Phylogenetic analysis of presently known full-length MERS-CoV genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable MERS-CoV variant typing. RT-PCR assays targeting these regions were designed and optimized. Results A reverse-transcription PCR assay for MERS-CoV targeting a 615 bp spike fragment provides a phylogenetic clustering of MERS-CoV variants comparable to that of full-length genomes. The detection limit corresponds to a cycle treshold value of ∼35 with standard upE real time PCR assays on RNA isolated from MERS-CoV EMC. Nasal swabs from RT-PCR positive camels (Ct values 12.9–32.2) yielded reliable sequence information in 14 samples. Conclusions We developed a simple, reliable MERS-CoV variant typing assay which is crucial in monitoring MERS-CoV circulation in real time with relatively little investment on location.

Highlights • We describe results of the first HCoV-MERS external quality control panel. • 85% of the 189 returned results had a 100% score. • 8.1% of laboratories produced false positive results. • ORF1b target is not recommended for screenings PCR., Background Since the discovery of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, diagnostic protocols were quickly published and deployed globally. Objectives We set out to assess the quality of MERS-CoV molecular diagnostics worldwide. Study design Both sensitivity and specificity were assessed using 12 samples containing different viral loads of MERS-CoV or common coronaviruses (OC43, 229E, NL63, HKU1). Results The panel was sent to more than 106 participants, of which 99 laboratories from 6 continents returned 189 panel results.Scores ranged from 100% (84 laboratories) to 33% (1 laboratory). 15% of respondents reported quantitative results, 61% semi-quantitative (Ct-values or time to positivity) and 24% reported qualitative results. The major specific technique used was real-time RT-PCR using the WHO recommended targets upE, ORF1a and ORF1b. The evaluation confirmed that RT-PCRs targeting the ORF1b are less sensitive, and therefore not advised for primary diagnostics. Conclusions The first external quality assessment MERS-CoV panel gives a good insight in molecular diagnostic techniques and their performances for sensitive and specific detection of MERS-CoV RNA globally. Overall, all laboratories were capable of detecting MERS-CoV with some differences in sensitivity. The observation that 8% of laboratories reported false MERS-CoV positive single assay results shows room for improvement, and the importance of using confirmatory targets.

Background: Following the recognition of a measles case in a hospital in The Netherlands, health care workers (HCW) from the premises were screened by a commercial enzyme immunoassay (EIA) for measles IgG to identify persons at risk for measles. At least 10% of the HCW were tested measles IgG-negative. As this was considered an unusually high proportion, we hypothesized suboptimal sensitivity of EIAs, especially in medical personnel that had vaccine-induced immunity rather than antibodies resulting from natural infection. Objectives: To determine (vaccine-induced) measles immunity in HCW, using different EIAs compared to the plaque reduction neutralization (PRN) test, the best surrogate marker for vaccine efficacy and immune protection. Study design: Sera from HCW were tested for measles IgG antibodies in three commercial EIAs, in a bead-based multiplex immunoassay (MIA) and in the PRN test, and evaluated against age and vaccination history of the HCW. Results: Of the 154 HCW, born between 1960 and 1995, 153 (99.4%) had protective levels of measles antibodies (PRN > 120 ml U/ml). The three EIAs failed to detect any measles IgG antibodies in approximately 10% of the HCW, while this percentage was approximately 3% for the MIA. Negative IgG results rose to 19% for individuals born between 1975 and 1985, pointing to an age group largely representing vaccinated persons from the first measles vaccination period in The Netherlands. Conclusion: The results show limitations in the usefulness of current EIA assays for determining protective measles antibodies in persons with a vaccination history. (C) 2015 Elsevier B.V. All rights reserved.

Noroviruses are recognized as the most common cause of outbreaks of acute gastroenteritis. Yet, diagnostic testing for norovirus is based mostly on RNA detection by RT-PCR, which is not widely available. While antigen detection tests (ELISAs) are easier to perform, they are in general less sensitive.Our aim was to provide a scientific basis for declaring norovirus as the causative agent of an outbreak of acute gastroenteritis.Statistical analysis used binomial distribution to determine the minimal number of positive samples, and the probability of detecting the required number of positive samples, for different tests, required to assign norovirus as the causative agent of an outbreak of acute gastroenteritis.For either a standard RT-PCR or a commercially available ELISA, finding only 1 sample positive out of 2, 3 or 4 samples is sufficient to assign norovirus as the causative agent of an outbreak of acute gastroenteritis. However, when ELISA is used, the probability of detecting this required minimum number of positive samples is low when small numbers of samples are tested (57% when 2 samples are tested; 72% when 3 samples are tested). In order to reach a 90% probability of detecting a norovirus outbreak (false negativity at outbreak level10%), at least 3 samples should be tested using RT-PCR, and 6 samples when using an ELISA.The sensitivity for NoV outbreak diagnosis will increase from 57% to 92%, or from 84% to 96%, for ELISA or RT-PCR respectively, when sample size increases from 2 to 6. Thus, using ELISA instead of RT-PCR for the detection of norovirus in stool samples will result in considerable numbers of false negative outbreaks unless a minimum of 6 samples are tested per outbreak.

Background: Hepatitis E virus (HEV) is the major etiologic agent of enterically transmitted viral hepatitis in much of the developing world. Evidence provided in recent years shows that HEV is also prevalent in very low numbers in non-endemic countries. Recently, a cluster of three patients with acute hepatitis E but no history of travel to endemic countries was discovered in the geographical area provided with service by the Public Health Laboratory Groningen and Drenthe, The Netherlands.Objective: This lead to the question whether hepatitis E is a cause of unexplained hepatitis in this district.Study design: The prevalence of anti-HEV IgG and IgM among 209 patients with clinical signs of hepatitis, negative test for hepatitis A-C, no history of foreign travel and no other cause of hepatocellular damage was compared with a matched control group of 209 individuals.Results: We found a significant difference in seroprevalence between the two groups for IgG anti-HEV as determined with the Abbot HEV ETA (6.2% versus 0.5%); however this difference could not be confirmed with the Genelabs Diagnostics HEV IgG ELISA (6.7% versus 3.8%). For confirmed cases of IgM anti-HEV we also detected a significant difference between the two groups (3.3% versus 0.5%). Remarkably, the combination of IgG and IgM anti-HEV was only found among hepatitis patients.Conclusion: This study provides evidence of locally acquired hepatitis E in The Netherlands. Therefore, in cases of unexplained acute hepatitis. the diagnosis of hepatitis E should be considered even in the absence of foreign travel. (C) 2004 Elsevier B.V. All rights reserved.

) Under the Section ‘6. Diagnosis of arbovirus infections’ of the above paper paragraph five should be as below: The detection of IgM antibodies in the CSF also implies recent infection. Interpretation of the results requires knowledge about the specific method used, and on patient background, for instance traveland vaccination history. IgM and IgG antibodies can cross-react within serogroups and cause false positive results when using ELISA or IFA for diagnosis. Typically, flavivirus IgG antibodies are also prone to cross-react with other serogroups within its genus. Infections or vaccinations may also trigger cross-reactive antibodies.112,113 VNT can be used if further confirmation of a specific viral infection is required. ) Table 2 should be

No: 1476 Presentation at ESCV 2015: Poster 1 Severity of mumps disease is related to viral shedding in urine S. Gouma1,∗, D. Gijselaar1, S. Hahne1, M. Koopmans2, R. van Binnendijk1 1 National Institute for Public Health and the Environment (RIVM), Netherlands 2 Erasmus MC, Netherlands Background: Mumps often starts with non-specific symptoms such as fever and is followed in the majority of cases by parotitis. Orchitis is the most common complication among males. Despite introduction of the measles, mumps, and rubella (MMR) vaccine in 1987, recently various mumps outbreaks occurred among vaccinatedpersons in several countries, including theNetherlands.Here, we studymumps virus shedding in urine in relation to clinical data, to investigateapossible correlationbetweensystemicmumpsvirus infection and severity of disease in both unvaccinated and twice MMR vaccinated mumps patients. Methods: We investigated shedding of mumps virus in urine from 379 laboratory confirmed caseswith 2MMR vaccinations and from 150 laboratory confirmed cases with no MMR vaccination. Data on parotitis side, orchitis and fever were available for a subset of these patients (n=359, n=265 and n=365, respectively). Data were compared using the chi-square test. Results: Mumps virus shedding in urine was detected in 64.7% of the unvaccinated patients and in 44.3% of the twice MMR vaccinatedpatients (p

Background: Norovirus P2 domain is commonly used to extrapolate transmission within an outbreak (OB) setting. The current definition is that transmission among cases is considered to be proven when no sequence variation is found. Objectives: Previous studies have shown a high mutation rate and errors during replication of the norovirus genome, therefore the validity of this criterion must be evaluated. Study design: Sequences of the P2 domain were obtained from patients and health care workers sampled during 4 prospectively GII.4 outbreaks. Fecal samples were tested by RT-PCR for presence of norovirus RNA against a standard control preparation to allow quantification. Estimated time of onset of shedding was derived from shedding kinetics modeled on data from sequential sampling. Thereby P2 sequence variation could be linked to estimated total virus excretion in individual subjects. Results: In all the outbreaks, P2 domain variation was found that resulted in unique codon changes in some patients. Mutations were found in 14% of initial samples and >50% of follow-up samples taken from patients involved in an outbreak. In three patients, aa mutations was observed in or near sites involved in host or antigen binding. Conclusions: We concluded that P2 domain variation increases with duration of virus shedding, but was unrelated to total amounts of virus shed. Therefore, we propose that cluster identification based on identical sequences should be relaxed to accommodate minor sequence variation. When using sequence data to support outbreak investigations, sequence diversity should be interpreted in relation to timing of sampling since onset of illness. (c) 2012 Elsevier B.V. All rights reserved.