External Quality Assessment (EQA) for Molecular Diagnostics of Zika Virus: Experiences from an International EQA Programme, 2016⁻2018.

Quality Control for Molecular Diagnostics (QCMD), an international provider for External Quality Assessment (EQA) programmes, has introduced a programme for molecular diagnostics of Zika virus (ZIKV) in 2016, which has been continuously offered to interested laboratories since that time. The EQA schemes provided from 2016 to 2018 revealed that 86.7% (92/106), 82.4% (89/108), and 88.2% (90/102) of the participating laboratories reported correct results for all samples, respectively in 2016, 2017, and 2018. The review of results indicated a need for improvement concerning analytical sensitivity and specificity of the test methods. Comparison with the outcomes of other EQA initiatives briefly summarized here show that continuous quality assurance is important to improve laboratory performance and to increase preparedness with reliable diagnostic assays for effective patient management, infection and outbreak control.

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.

External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014 - 2016 Ebola outbreak in West Africa.

During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied.

Assessment of Recombination in the S-segment Genome of Crimean-Congo Hemorrhagic Fever Virus in Iran.

BACKGROUND: Crimean-Congo Hemorrhagic Fever Virus (CCHFV) belongs to genus Nairovirus and family Bunyaviridae. The main aim of this study was to investigate the extent of recombination in S-segment genome of CCHFV in Iran. METHODS: Samples were isolated from Iranian patients and those available in GenBank, and analyzed by phylogenetic and bootscan methods. RESULTS: Through comparison of the phylogenetic trees based on full length sequences and partial fragments in the S-segment genome of CCHFV, genetic switch was evident, due to recombination event. Moreover, evidence of multiple recombination events was detected in query isolates when bootscan analysis was used by SimPlot software. CONCLUSION: Switch of different genomic regions between different strains by recombination could contribute to CCHFV diversification and evolution. The occurrence of recombination in CCHFV has a critical impact on epidemiological investigations and vaccine design.

External quality assessment studies for laboratory performance of molecular and serological diagnosis of Chikungunya virus infection.

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.

Clinical implications of and lessons learnt from external assessment of Mers-CoV diagnostics.

With the emergence of new viral infections, it is necessary to set up new target-specific assays, based on existing molecular techniques such as real-time PCR, as quickly as possible. Without these diagnostic tools, the geographical spread of new infections, follow-up of the disease outbreak and analysis of the pathogenesis of the disease are not possible. Therefore, the genomic information of the emerging pathogen, diagnostic protocols and standards allowing quality control need to be available in a few days. This can only be implemented with good quality experienced laboratories having suitable infrastructure to establish in-house assays. Even though these molecular tools are available quickly, challenges still remain with what sample types to select for a proper diagnostic value.

First international external quality assessment of molecular diagnostics for Mers-CoV.

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.

International external quality assessment study for molecular detection of Lassa virus.

Lassa virus (LASV) is a causative agent of hemorrhagic fever in West Africa. In recent years, it has been imported several times to Europe and North America. The method of choice for early detection of LASV in blood is RT-PCR. Therefore, the European Network for Diagnostics of 'Imported' Viral Diseases (ENIVD) performed an external quality assessment (EQA) study for molecular detection of LASV. A proficiency panel of 13 samples containing various concentrations of inactivated LASV strains Josiah, Lib-1580/121, CSF, or AV was prepared. Samples containing the LASV-related lymphocytic choriomeningitis virus (LCMV) and negative sera were included as specificity controls. Twenty-four laboratories from 17 countries (13 European, one African, one Asian, two American countries) participated in the study. Thirteen laboratories (54%) reported correct results, 4 (17%) laboratories reported 1 to 2 false-negative results, and 7 (29%) laboratories reported 3 to 5 false-negative results. This EQA study indicates that most participating laboratories have a good or acceptable performance in molecular detection of LASV. However, several laboratories need to review and improve their diagnostic procedures.

International external quality control assessment for the serological diagnosis of dengue infections.

BACKGROUND: Dengue is endemic to the tropics and subtropics, and the most frequent of arthropod-borne viral diseases. Reliable diagnosis of dengue infection is important not only in clinical care but also in disease surveillance, the control of outbreaks, and the development of new vaccines. The diagnosis of dengue infection is usually established by a variety of commercial or in-house serological protocols. The European Network for the Diagnostics of Imported Viral Diseases (ENIVD) recognized the need to survey the accuracy of dengue serological diagnostics in current use, and organized an external quality assurance (EQA) study of dengue serological practice in diagnostic laboratories. METHODS: A 15-sample panel, consisting of sera reactive against dengue plus specificity and negative controls, was sent to 48 laboratories for serological testing. The results returned by the participating laboratories were anonymized, scored, and subjected to comparison and statistical analysis. RESULTS: Ten laboratories rated all samples correctly with regard to IgM, and only three achieved the full score for IgG detection. The main handicaps in assay performance were suboptimal sensitivity of in-house IgM detection protocols by comparison with better-performing commercial ELISA tests, and the presence of IgG cross-reactivity with heterologous flaviviruses. Differences of detail in the methodology of dengue IgG antibody detection appear to underlie the disparities in accuracy observed between laboratories. CONCLUSION: This EQA study demonstrates that there is room for many laboratories to improve sensitivity in the detection of anti-dengue virus IgM antibodies, against the benchmark set by commercial antibody capture ELISA tests. The EQA shows also that cross-reactivity is a continuing issue, and IgG detection protocols must be optimized to increase their specificity.

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).

International external quality assessment of molecular detection of Rift Valley fever virus.

Rift Valley fever (RVF) is a viral zoonosis that primarily affects animals resulting in considerable economic losses due to death and abortions among infected livestock. RVF also affects humans with clinical symptoms ranging from an influenza-like illness to a hemorrhagic fever. Over the past years, RVF virus (RVFV) has caused severe outbreaks in livestock and humans throughout Africa and regions of the world previously regarded as free of the virus. This situation prompts the need to evaluate the diagnostic capacity and performance of laboratories worldwide. Diagnostic methods for RVFV detection include virus isolation, antigen and antibody detection methods, and nucleic acid amplification techniques. Molecular methods such as reverse-transcriptase polymerase chain reaction and other newly developed techniques allow for a rapid and accurate detection of RVFV. This study aims to assess the efficiency and accurateness of RVFV molecular diagnostic methods used by expert laboratories worldwide. Thirty expert laboratories from 16 countries received a panel of 14 samples which included RVFV preparations representing several genetic lineages, a specificity control and negative controls. In this study we present the results of the first international external quality assessment (EQA) for the molecular diagnosis of RVF. Optimal results were reported by 64% of the analyses, 21% of the analyses achieved acceptable results and 15% of the results revealed that there is need for improvement. Evenly good performances were achieved by specific protocols which can therefore be recommended as an accurate molecular protocol for the diagnosis of RVF. Other protocols showed uneven performances revealing the need for improved optimization and standardization of these protocols.

First international external quality assessment of molecular detection of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis caused by a Nairovirus of the family Bunyaviridae. Infection is transmitted to humans mostly by Hyalomma ticks and also by direct contact with the blood or tissues of infected humans or viremic livestock. Clinical features usually include a rapid progression characterized by hemorrhage, myalgia and fever, with a lethality rate up to 30%. CCHF is one of the most widely distributed viral hemorrhagic fevers and has been reported in Africa, the Middle East and Asia, as well as parts of Europe. There is no approved vaccine or specific treatment against CCHF virus (CCHFV) infections. In this context, an accurate diagnosis as well as a reliable surveillance of CCHFV infections is essential. Diagnostic techniques include virus culture, serology and molecular methods, which are now increasingly used. The European Network for the Diagnostics of "Imported" Viral Diseases organized the first international external quality assessment of CCHVF molecular diagnostics in 2011 to assess the efficiency and accurateness of CCHFV molecular methods applied by expert laboratories. A proficiency test panel of 15 samples was distributed to the participants including 10 different CCHFV preparations generated from infected cell cultures, a preparation of plasmid cloned with the nucleoprotein of CCHFV, two CCHFV RNA preparations and two negative controls. Forty-four laboratories worldwide participated in the EQA study and 53 data sets were received. Twenty data sets (38%) met all criteria with optimal performance, 10 (19%) with acceptable performance, while 23 (43%) reported results showing a need for improvement. Differences in performance depended on the method used, the type of strain tested, the concentration of the sample tested and the laboratory performing the test. These results indicate that there is still a need for improving testing conditions and standardizing protocols for the molecular detection of Crimean-Congo hemorrhagic fever virus.

First international external quality assessment study on molecular and serological methods for yellow fever diagnosis.

OBJECTIVE: We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis. STUDY DESIGN: For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included. RESULTS: Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols. CONCLUSION: This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles.

Second external quality assessment of the molecular diagnostic of West Nile virus: are there improvements towards the detection of WNV?

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.

rapidSTRIPE H1N1 test for detection of the pandemic swine origin influenza A (H1N1) virus.

The rapidSTRIPE H1N1 test, based on a nucleic acid lateral-flow assay, has been developed for diagnosis of a swine-origin influenza A (H1N1) virus. This test is simple and cost-effective and allows specific detection of the S-OIV A (H1N1) virus from swab sampling to final detection on a lateral-flow stripe within 2 to 3 h.

2nd International external quality control assessment for the molecular diagnosis of dengue infections.

BACKGROUND: Currently dengue viruses (DENV) pose an increasing threat to over 2.5 billion people in over 100 tropical and sub-tropical countries worldwide. International air travel is facilitating rapid global movement of DENV, increasing the risk of severe dengue epidemics by introducing different serotypes. Accurate diagnosis is critical for early initiation of preventive measures. Different reverse transcriptase PCR (RT-PCR) methods are available, which should be evaluated and standardized. Epidemiological and laboratory-based surveillance is required to monitor and guide dengue prevention and control programmes, i.e., by mosquito control or possible vaccination (as soon as an effective and safe vaccine becomes available). OBJECTIVE: The purpose of the external quality assurance (EQA) study described is to assess the efficiency and accuracy of dengue molecular diagnosis methods applied by expert laboratories. STUDY DESIGN: A panel of 12 human plasma samples was distributed and tested for DENV-specific RNA. The panel comprised 9 samples spiked with different DENV serotypes (DENV-1 to DENV-4), including 10-fold dilution series of DENV-1 and DENV-3. Two specificity controls consisted of a sample with a pool of 4 other flaviviruses and a sample with chikungunya virus. A negative control sample was also included. RESULTS: Thirty-seven laboratories (from Europe, Middle East Asia, Asia, the Americas/Caribbean, and Africa) participated in this EQA study, and reports including 46 sets of results were returned. Performance among laboratories varied according to methodologies used. Only 5 (10.9%) data sets met all criteria with optimal performance, and 4 (8.7%) with acceptable performance, while 37 (80.4%) reported results showed the need for improvement regarding accomplishment of dengue molecular diagnosis. Failures were mainly due to lack of sensitivity and the presence of false positives. CONCLUSIONS: The EQA provides information on each laboratory's efficacy of RT-PCR techniques for dengue diagnosis and indicates for most laboratories an urgent need to improve sensitivity and specificity.

Quality control assessment for the serological diagnosis of tick borne encephalitis virus infections.

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.

Quality control assessment for the PCR diagnosis of tick-borne encephalitis virus infections.

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.

External quality control assessment in PCR diagnostics of dengue virus infections.

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.

Quality control assessment for the serological diagnosis of dengue virus infections.

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.