Toscana, West Nile, Usutu and tick-borne encephalitis viruses: external quality assessment for molecular detection of emerging neurotropic viruses in Europe, 2017.

BackgroundNeurotropic arboviruses are increasingly recognised as causative agents of neurological disease in Europe but underdiagnosis is still suspected. Capability for accurate diagnosis is a prerequisite for adequate clinical and public health response.AimTo improve diagnostic capability in EVD-LabNet laboratories, we organised an external quality assessment (EQA) focusing on molecular detection of Toscana (TOSV), Usutu (USUV), West Nile (WNV) and tick-borne encephalitis viruses (TBEV).MethodsSixty-nine laboratories were invited. The EQA panel included two WNV RNA-positive samples (lineages 1 and 2), two TOSV RNA-positive samples (lineages A and B), one TBEV RNA-positive sample (Western subtype), one USUV RNA-positive sample and four negative samples. The EQA focused on overall capability rather than sensitivity of the used techniques. Only detection of one, clinically relevant, concentration per virus species and lineage was assessed.ResultsThe final EQA analysis included 51 laboratories from 35 countries; 44 of these laboratories were from 28 of 31 countries in the European Union/European Economic Area (EU/EEA). USUV diagnostic capability was lowest (28 laboratories in 18 countries), WNV detection capacity was highest (48 laboratories in 32 countries). Twenty-five laboratories were able to test the whole EQA panel, of which only 11 provided completely correct results. The highest scores were observed for WNV and TOSV (92%), followed by TBEV (86%) and USUV (75%).ConclusionWe observed wide variety in extraction methods and RT-PCR tests, showing a profound absence of standardisation across European laboratories. Overall, the results were not satisfactory; capacity and capability need to be improved in 40 laboratories.

Need for additional capacity and improved capability for molecular detection of yellow fever virus in European Expert Laboratories: External Quality Assessment, March 2018.

An external quality assessment of yellow fever virus (YFV) molecular detection in European laboratories was organised in rapid response to an increase in human cases in Brazil in 2018 with risk of import to Europe. Detection of YFV was assessed among 32 laboratories in 23/31 European Union (EU) and European Economic Area (EEA) countries and two laboratories in one non-EU/EEA country. Adequate capabilities were lacking in 10/23 countries; five did not participate as they lacked implemented assays.

External Quality Assessment for Zika Virus Molecular Diagnostic Testing, Brazil.

We conducted an external quality assessment of Zika virus molecular diagnostic tests in Brazil using a new Zika virus standard. Of 15 laboratories, 73% showed limited sensitivity and specificity. Viral load estimates varied significantly. Continuous quality assurance is needed to adequately estimate risk for Zika virus-associated disease and determine patient care.

Variable Sensitivity in Molecular Detection of Zika Virus in European Expert Laboratories: External Quality Assessment, November 2016.

Zika virus (ZIKV) infections are a significant public health concern. A strong capability for ZIKV detection is an absolute requirement for adequate preparedness and response strategies and individual patient care. The objective of this study was to assess and improve the capability of European expert laboratories for molecular testing for ZIKV through an external quality assessment (EQA) scheme. Laboratories were provided a panel of 12 samples which included negative samples, samples containing African- or Asian-lineage ZIKV at various concentrations (103 to 109 copies/ml), and samples containing dengue virus, yellow fever virus, or chikungunya virus. The results were analyzed on the basis of the outcomes of testing for the samples and the extraction and detection method used. Samples with a ZIKV RNA status scored correctly by >50% of the laboratories were designated the core sample. A total of 85 panel outcomes were submitted by 50 laboratories in 31 countries. The results designated all samples as core samples. Thirty-three percent (28/85) of the panel outcomes identified all samples. Analysis at the laboratory level showed that only 40% of the laboratories (20/50), representing 45% of the countries, scored sufficiently; i.e., they had at least one test operational that scored all core samples correctly. There is a need for improvement of the molecular detection of ZIKV in 60% of the participating laboratories. While the specificity of the tests was more robust, the results of the EQA showed large variation in test sensitivity. Improvements should focus on both nucleic acid extraction and ZIKV detection methods.

Diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies.

West Nile virus, genus Flavivirus, is transmitted between birds and occasionally other animals by ornithophilic mosquitoes. This virus also infects humans causing asymptomatic infections in about 85% of cases and <1% of clinical cases progress to severe neuroinvasive disease. The virus also presents a threat since most infections remain unapparent. However, the virus contained in blood and organs from asymptomatically infected donors can be transmitted to recipients of these infectious tissues. This paper reviews the presently available methods to achieve the laboratory diagnosis of West Nile virus infections in humans, discussing the most prominent advantages and disadvantages of each in light of the results obtained during four different External Quality Assessment studies carried out by the European Network for 'Imported' Viral Diseases (ENIVD).

A simple method for molecular detection of Swine-origin and human-origin influenza a virus.

Here we present a real-time one-step reverse transcriptase-polymerase chain reaction SYBR Green assay derived from the method reported by van Elden and collaborators ( 2001 ) that ensures the rapid, sensitive, and cost-effective detection of both seasonal influenza A virus and emerging (H1N1) swine-origin influenza A virus (S-OIV). In addition to this screening test, which successfully detected both seasonal influenza A virus and S-OIV in human clinical samples, we showed that the probe initially designed by van Elden and collaborators could detect seasonal influenza A virus, but not S-OIV; a new probe was designed and tested that specifically detects S-OIV, but not seasonal influenza A. Both probe-based assays were validated by testing human clinical samples and specifically detected either seasonal influenza virus or S-OIV. Finally, in silico analysis of databases predicted that minor modifications of the van Elden primers would facilitate the use of this assay for the broad spectrum detection of all currently characterized variants of influenza A virus, including avian strains.