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Background: Since the re-emergence of Chikungunya virus (CHIKV) in Reunion in 2005 and the recent outbreak in the Caribbean islands with an expansion to the Americas the CHIK diagnostic became very important., Objectives: We evaluate the performance of laboratories regarding molecular and serological diagnostic of CHIK worldwide., Study Design: A panel of 12 samples for molecular and 13 samples for serology were provided to 60 laboratories in 40 countries for evaluating the sensitivity and specificity of molecular and serology testing., Results: The panel for molecular diagnostic testing was analysed by 56 laboratories returning 60 data sets of results whereas the 56 and 60 data sets were returned for IgG and IgM diagnostic from the participating laboratories. Twenty-three from 60 data sets performed optimal, 7 acceptable and 30 sets of results require improvement. From 50 data sets only one laboratory shows an optimal performance for IgM detection, followed by 9 data sets with acceptable and the rest need for improvement. From 46 IgG serology data sets 20 provide an optimal, 2 an acceptable and 24 require improvement performance. The evaluation of some of the diagnostic performances allows linking the quality of results to the in-house methods or commercial assays used., Conclusion: The external quality assurance for CHIK diagnostics provides a good overview on the laboratory performance regarding sensitivity and specificity for the molecular and serology diagnostic required for the quick and reliable analysis of suspected CHIK patients. Nearly half of the laboratories have to improve their diagnostic profile to achieve a better performance., (Copyright © 2016 Z. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

On August 8 2014, the World Health Organization (WHO) declared the outbreak of Ebola Virus Disease (EVD) evolving in West Africa since December 2013, a Public Health Emergency of International Concern (PHEIC). It is expected that the outbreak of Ebolavirus Disease (EVD) in West Africa will lead to increased testing of individuals in Europe for EVD. The severity of the situation in West Africa warranted a critical appraisal of the laboratory preparedness and response for EVD, with a focus on information needs for laboratories involved in diagnostics of rare viral diseases associated with the European Network for the Diagnostics of "Imported" Viral Diseases", ENIVD. Essential knowledge and knowledge gaps for an adequate laboratory response focusing on virus properties, infection kinetics, tests specifics and field performances were identified. An inventory of the laboratory capacity for EVD diagnostics among ENIVD laboratories was made.

Yellow fever (YF) vaccine is considered safe but vaccine-associated complications have also been encountered. We report neurological symptoms after YF-vaccination in a previously healthy Finnish male. Other concomitant infections or causes for the symptoms could not be identified., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: WNV epidemics occur worldwide, new WNV isolates were isolated in southern-east Europe belonging to WNV lineage 2. A first international proficiency study on WNV indicted that some laboratories were not able to detect WNV lineage 2 virus genome by their PCR diagnostic assays. Therefore an actual External Quality Assessment with both virus lineages was performed to monitor the improvements in molecular diagnostics., Objectives: To asses the proficiency of laboratories to detect West Nile virus with molecular diagnostic tests., Study Design: A test panel of different WNV isolates and virus dilutions was given to 26 laboratories to test the samples with their routine diagnostic methods., Results: Twenty-one participating laboratories provided 28 data set results. WNV lineage 1 was detected with high overall efficiency of 92% (67.9-100%) but two different WNV lineage 2 strains were detected at lower rates (mean = 73%, 67.9-75%) by the different PCR assays. 93% of the laboratories were able to detect a WNV lineage 1 with a concentration of 1.2×10(4)copies/ml but the detection rate was decreased to 68% for 1.2×10(3)copies/ml. One laboratory generated false-positive result from the non-virus control samples and 29% of the datasets showed false-positive results for non-WNV flavivirus samples., Conclusions: The WNV EQA showed an improved proficiency of laboratories as compared to the first EQA. However, the data suggest that problems in the detection of both lineages were still present since the first proficiency test was performed in 2006. Further proceedings versus the detection of both lineages are needed particularly for in-house assays., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: We cannot predict when an influenza pandemic will occur or which variant of the virus will cause it. Little information is currently available on the ability of laboratories to detect and subtype influenza viruses including the avian influenza viruses., Objectives: To assess the ability of laboratories to detect and subtype influenza viruses., Study Design: In 2006 QCMD distributed an External Quality Assessment panel for the molecular detection and haemagglutinin subtyping of influenza viruses to 87 laboratories in 34 countries Worldwide, which were given 6 weeks to return results. These data were analysed to assess laboratory performance., Results: Influenza virus positive panel samples were correctly identified by 35-98% of laboratories. The correct haemagglutinin subtype was reported by 32-87% of laboratories that detected the virus: incorrect subtyping results included the reporting of avian influenza viruses as human strains and vice versa. Twelve laboratories reported false positives with some avian influenza viruses reported., Conclusions: These data suggest that improvements are needed in the molecular detection of influenza viruses and influenza virus A haemagglutinin subtyping. Only rapid and accurate identification of circulating pandemic influenza virus will ensure that the maximum time is available for intervention.

Background: The diagnosis of tick borne encephalitis (TBE) is mainly based on the demonstration of specific antibodies in serum when neurological disease is manifested. Improving diagnostics is the most important step in detecting and dealing with these pathogens. Quality control measures are essential for TBE diagnosis., Objective: To assess an external quality assurance (EQA) program for the serologic diagnosis of TBE infections., Study Design: A panel of 12 serum samples was sent out to be tested for the presence of TBE virus-specific IgM and IgG. This panel contained seven TBE-positive samples for IgM and/or IgG; three negative samples; two samples positive either for West Nile virus (WNV) or Dengue virus (DENV)., Results: Fourty-two laboratories from 25 European and 2 non-European countries participated in this EQA. The correct answer by each laboratory for all samples ranked between 58 and 96% and sera with IgM antibody positive for TBE were correctly recognized by 46-88% of the laboratories. Sera with IgG antibody positive for TBE were correctly recognized by 83-95% of the laboratories. False TBE-positive results were obtained with DENV, WNV or negative sera only for IgG-based assays., Conclusion: Correct results for at least 90% of the samples were obtained by 33 of 40 participating laboratories for IgM and for 16 of 42 laboratories for IgG.

Background: Reverse transcriptase-polymerase chain reaction (RT-PCR) is an efficient method for the early detection of tick-borne encephalitis virus (TBEV) RNA in blood and serum samples taken prior to the appearance of antibodies. Improved diagnostics are critical for optimally detecting and managing TBE infections and quality control measures are therefore essential., Objective: To assess the diagnostic quality of laboratories by performing an external quality assurance (EQA) programme for the molecular detection of TBE infections., Study Design: A panel of 12 prepared human plasma samples were distributed and tested for the presence of TBEV-specific RNA. The panel comprised eight samples spiked with different TBEV strains of European, Siberian and Far Eastern subtypes, and included a 10-fold dilution series. Two specificity controls consisted of a sample with Louping ill virus (LIV) and a sample with a pool of four other flaviviruses, and two negative control samples were further included., Results: Twenty-three laboratories from 16 European and 2 non-European countries participated in this EQA programme. Only two participants correctly identified all samples. Nine laboratories correctly identified 75.0-91.7% of the samples; seven laboratories correctly identified 54.5-66.7% and five laboratories correctly identified < or =50%., Conclusions: The EQA programme provides information on the quality of the RT-PCR methods used by the participating laboratories and indicates that most of these need to improve sensitivity and specificity of their molecular assays for TBEV.

Yellow fever vaccine-associated viscerotropic disease (YEL-AVD) is a recently described severe adverse event after yellow fever vaccination, and some cases have been reported in different countries [Anonymous. Effects of yellow fever and vaccination. Lancet 2001;358(9296):1907-9]. Herein we describe a YEL-AVD case in a young woman, who died after vaccination with 17D-204 strain. Clinical, serological and immunochemical analysis as well as virus detection, quantification, sequence analysis and cytokine release, were performed. Further investigations on yellow fever vaccine adverse events, and carefully analysis of the immune response elicited are important tasks for the future.

To confirm an infection with the new coronavirus (SARS-CoV) causing the severe acute respiratory syndrome (SARS) diagnostic assays for detection of SARS-CoV specific antibody are necessary. To evaluate the diagnostic performance of laboratories an external quality assurance (EQA) study was performed in 2004. Participating laboratories (9/20) correctly detected anti-SARS antibodies in serum samples without false positive results in an immunofluorescence assay. In contrast, only 4/13 laboratories detected most of the anti-SARS antibody positive samples without false positive results using enzyme immunoassays (EIA) and/or immunoblot. The overall results clearly demonstrate that serological diagnosis of SARS-CoV remains at an early stage of development, with further technical improvements required, particularly with respect to the use of SARS specific EIAs.

Background: Yellow fever remains one of the great burdens for public health in the endemic regions in Africa and South America. The under reporting of yellow fever cases in the respective regions and lack of international interest leads to an underestimation of the constant danger in these areas. Non-vaccinated travelers take a high risk without the effective protection of YFV 17D vaccination., Objectives: Two YF cases were imported to Europe in the last 4 years. We characterized two yellow fever virus (YFV) isolates from severely infected patients coming back from Africa, Ivory Coast and The Gambia, by genome sequencing and phylogenetic analysis., Study Design: The virus infections in different organs were analyzed with pathological, immunohistological, electronmicroscopical and quantitative real-time PCR methods., Results and Conclusion: High virus loads in spleen and liver (2.4 x 10 (6) to 3 x 10 (7)GE/mL) demonstrated by real time PCR show massive virus replication leading to extraordinary progression of the disease in these patients. Immunohistological and electronmicroscopical analysis confirms virus particles in liver tissue. In all other organs no virus could be detected. A fast, specific and sensitive virus PCR detection is recommended for diagnostic of acute infections. The further sequence alignments show that the new isolates belong to the type II West African strain with great homology to over 40-year old YF isolates from Senegal and Ghana. The divergence observed was on average 3.3%, ranging from 0.0% to 5.0% in the coding region of Gambia 2001 strain and 2.9 %, ranging from 0.0% to 4.3% in the coding region of the Ivory C 1999 strain. Most mutations (5.0%/4.3%, respectively) occurred in the envelope protein.

Background: Parvovirus B19 (PVB19) is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild to more severe outcomes, dependent on the haematological and/or immunological status of the host. Reports of PVB19 infection as a causative agent of paediatric or adult inflammatory cardiac diseases, or of cardiac transplant rejection are rare., Objectives: To identify PVB19 and other cardiotropic viruses in the myocardium of heart transplant (HTx) recipients and multi-organ donors (MOD). Furthermore, to assess the prevalence of cardiotropic viral infection in inflammatory heart disease., Study Design: Heart tissue samples from 110 explants were analysed for PVB19 using primers and a 5'-nuclease probe designed to amplify a 160-basepair PCR product from the VP1/NS1 gene region. Samples tested included those obtained from patients undergoing HTx or from MODs. The findings were correlated with clinical course, histologic analysis and serologic testing. Confirmation of the positive PCR-results was done by sequencing and in situ hybridisation., Results: The new assay described here allows precise quantitation of viral load over 7 orders of magnitude (10(6) to 10(0)IU/assay). Measurable amounts of parvoviral genomes were detected in 4/56 (7%) explanted HTx-hearts and in 5/54 (9%) explanted MOD-hearts., Conclusions: The newly developed real-time PCR is a rapid, sensitive and specific method to detect PVB19 infection in heart tissue. It will be a useful tool to address important questions regarding viral infections transmitted by transplantation, acute infections, relapses and complications involving late or chronic rejection.

Background: Increased travelling to countries endemic for dengue fever (DF) demands efficient laboratory diagnostics. Nucleic acid amplification techniques (NAT) are now frequently used for rapid diagnosis of imported viral diseases. Different PCR systems are available., Objectives: In order to assess the quality of molecular diagnostics of dengue virus infections, an external quality assurance (EQA) in PCR diagnostics was conducted., Study Design: A panel of 10 human plasma samples was prepared and spiked with dengue virus types DEN-1 to DEN-4. In addition, a 10-fold dilution series (1:10-1:10(4) ) of DEN-3 virus was included. The panel was pre-tested by nested RT-PCR, in-house real-time PCR, and a commercial real-time PCR kit. The samples were inactivated by gamma irradiation and shipped in freeze dried state. Thirteen laboratories, within the European network for the diagnostics of imported viral diseases (ENIVD) took part using either single-round, nested, or real-time RT-PCR methods. Two laboratories used two methods in parallel, summarising up to 15 comparable results., Results: 33-100% correct results were achieved. All laboratories detected DEN-2 correctly, followed by DEN-1 (14 positive results of 15), DEN-3 (12/15) and DEN-4 (11/15). Testing of the serial dilution revealed low sensitivity in many labs, with results ranging from 33 to 80% of correctly tested samples., Conclusion: The EQA gives a feedback of the quality of the RT-PCR system used by each respective laboratory. The different test systems and amplification conditions demonstrate the importance of external quality control measures.

Background: A major drawback of modern society's rapidly increasing mobility is the ease with which dangerous infections can be imported into Europe. Often these infections are not diagnosed because physicians are not familiar with the symptoms and laboratory tests are not always available in local diagnostic centres. Improving diagnostics is the most important step in detecting and dealing with these pathogens and quality control measures are, therefore, essential tools., Objectives: To assess the diagnosis of imported dengue virus infections in Europe by (1) running a pre-evaluation panel (four serum samples, sent out in 1999) and optimising sample preparation and shipping procedures and (2) initiating an External Quality Assurance (EQA) program (20 serum samples, sent out in 2002)., Study Design: All serum samples sent out were to be tested for the presence of dengue virus-specific IgM and IgG. For the pre-evaluation panel, four samples were distributed (one sample IgM+/IgG+, one sample IgM-/IgG+, two samples IgM-/IgG-) and for the EQA 20 samples (12 samples IgM+/IgG+, five samples IgM-/lgG+, one sample lgM+/IgG- two samples IgM-/IgG-). 13 laboratories took part in the pre-evaluation panel and 18 laboratories participated in the first EQA run., Results: For the pre-evaluation panel, the participants reported concurrent and correct results for 88% of the IgG-positive samples and for 100% of the IgG-negative samples. The results for the IgM-positive sample were correct in 91% of the reported tests and in 97% of the IgM-negative samples. For the EQA, the participants reported concurrent and correct results for 71% of the IgG-positive samples and 89% of the IgG-negative samples. 58% concurrent and correct results were reported for the IgM-positive samples and 97% for the IgM-negative samples., Conclusions: The results presented here demonstrate the importance of quality measures for imported viral pathogens like dengue viruses and clearly indicate the need for improving the existing test systems.

Background: With society's rapidly increasing mobility, patients infected with severe viral infections can become seriously ill at any place in Europe and elsewhere. Improving the diagnostics of these infections is the most important step in detecting the pathogens and dealing with them, and for this purpose, quality control measures are essential tools., Objectives: To assess the diagnostic reality for rare hantavirus infections in Europe by (1) running a pre-evaluation panel (four samples, sent out in 1999) to optimise sample preparation and shipping procedure and afterwards (2) starting an External Quality Assurance (EQA) program (20 samples, sent out in 2001)., Study Design: All samples sent out had to be tested for the presence of specific IgG and IgM antibodies against hantavirus. For the pre-evaluation panel, four samples were distributed (two samples IgG+/IgM-, one sample IgG-borderline/IgM-, one sample IgG-/IgM-), for the EQA 20 samples (six samples IgG+/IgM+, eight samples IgG+/IgM-, one sample IgG-borderline/IgM-, five samples IgG-/IgM-). Thirteen laboratories took part in the pre-evaluation panel, 18 laboratories participated in the first EQA run., Results: For the pre-evaluation panel, the participants reported correct results for 64% of the IgG-positive samples (85% excluding borderline-positive sample), and 92% for the IgG-negative sample. IgM testing was correctly negative in all laboratories. For the EQA, the participants reported correct results for 76% of the IgG-positive samples, and 97% correct results for the IgG-negative samples. For the IgM-positive samples, 53% correct results were reported, and 98% correct results for the IgM-negative samples., Conclusions: The results presented here prove the importance of quality measures also for viruses only rarely suspected, like hantavirus, and they clearly demonstrate the need for improvement of the existing test systems.

Background: Tick-borne encephalitis virus (TBEV) is a pathogenic human flavivirus endemic in some parts of Europe and Asia. Commercial enzyme immunoassays (EIA) for the detection of IgG antibodies are often used in TBEV-seroprevalence studies, as well as for the confirmation of a successful vaccination against TBEV. However, the detection of TBEV-specific antibodies can be biased by the cross-reactivity between different flavivirus genera., Objectives: To compare different EIA test systems for the detection of TBEV-IgG antibodies., Study Design: Six commercial EIA kits for the detection of TBEV-specific antibodies are compared, using serum panels (n=139) of subjects with a documented clinical history (109 sera from TBEV infected patients, 30 sera from people vaccinated against TBEV). For the analysis of possible cross-reactivities, 24 sera from yellow fever vaccinated people and 13 sera positive for Dengue virus-specific antibodies were also included., Results: The sensitivity of the different TBEV test systems ranges from 73 to 99%. However, when testing the yellow fever and Dengue virus positive specimens, problems with the flavivirus cross- reactivity become obvious, resulting in specificities between 14 and 81%., Conclusions: This study shows the necessity of further improvement of the existing TBEV test systems regarding both sensitivity and specificity.