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.     

Interlaboratory comparison of ability to detect nucleic acid of Mycoplasma gallisepticum and Mycoplasma synoviae by polymerase chain reaction.

In recent years polymerase chain reaction (PCR) assays have become widely used as methods to confirm the presence of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry flocks, but there has been limited standardization of the protocols used. Thirteen laboratories from five different countries participated in an interlaboratory comparison of detection of M. gallisepticum and M. synoviae DNA by PCR in samples that contained 10-fold dilutions of these bacteria. The concentration of bacteria ranged from 10(5) to 10(2) genome copies/100 microl sample, as quantified by real-time PCR, and the samples were supplied on dry cotton swabs. Each laboratory was asked to use its standard method for PCR testing of these pathogens. A questionnaire was supplied with the samples to obtain details of the methods that were used in testing. One-half of the laboratories used a commercially available test kit, while the others used an in-house protocol. The protocols used for DNA extraction varied greatly, even among those using commercially available test kits. Two laboratories had developed the primers for nucleic acid amplification themselves, and one of these used real-time PCR for amplification. While the majority of the laboratories detected M. synoviae down to the 100 copy limit of the comparison, the detection limit for M. gallisepticum was somewhat higher. Furthermore, different results were obtained from laboratories that used the same commercial test kit. To the best of our knowledge this is the first investigation of its kind in the field of avian diseases.     

The first external quality assessment of isolation and identification of influenza viruses in cell culture in the Asia Pacific region, 2016

BackgroundThe isolation and propagation of influenza viruses from clinical specimens are essential tools for comprehensive virologic surveillance. Influenza viruses must be amplified in cell culture for detailed antigenic analysis and for phenotypic assays assessing susceptibility to antiviral drugs or for other assays.ObjectivesTo conduct an external quality assessment (EQA) of proficiency for isolation and identification of influenza viruses using cell culture techniques among National Influenza Centres (NICs) in the World Health Organisation (WHO) South East Asia and Western Pacific Regions.Study designTwenty-one NICs performed routine influenza virus isolation and identification techniques on a proficiency testing panel comprising 16 samples, containing influenza A or B viruses and negative control samples. One sample was used exclusively to determine their capacity to measure hemagglutination titer and the other 15 samples were used for virus isolation and identification.ResultsAll NICs performed influenza virus isolation using Madin Darby canine kidney (MDCK) or MDCK-SIAT-1 cells. If virus growth was detected, the type, subtype and/or lineage of virus present in isolates was determined using immunofluorescence, RT-PCR and/or hemagglutination inhibition (HI) assays. Most participating laboratories could detect influenza virus growth and could identify virus amplified from EQA samples. However, some laboratories failed to isolate and identify viruses from EQA samples that contained lower titres of virus, highlighting issues regarding the sensitivity of influenza virus isolation methods between laboratories.ConclusionThis first round of EQA was successfully conducted by NICs in the Asia Pacific Region, revealing good proficiency in influenza virus isolation and identification.     

GEMHEP multicenter quality control study of PCR detection of GB virus C/hepatitis G virus RNA in serum.

PCR is, to date, the only available tool for the detection of GB virus C (GBV-C) and hepatitis G virus (HGV) RNAs. Twenty-two French laboratories participated in a quality control study to assess the sensitivity and specificity of their procedures. The panel included 13 positive controls and 7 negative controls. The laboratories used either in-house PCR techniques adapted from the literature or partly standardized commercial tests. Three laboratories performed faultlessly with the entire panel. Most laboratories had excellent specificity (100% in 20 of 22 laboratories). Sensitivity was acceptable (85 to 100%) in 15 centers and insufficient (38 to 77%) in 7. As with nonstandardized in-house PCR, the commercial assays gave discrepant performances in different laboratories. These results suggest that laboratories willing to use PCR for detection of GBV-C/HGV RNA for research or diagnostic purposes should participate in multicenter quality control trials.     

External quality assurance studies for the serological and PCR diagnostics of tick-borne encephalitis virus infections

Improved diagnostics are critical for optimally detecting and managing tick-borne encephalitis (TBE) infections, and therefore quality control measures are essential for both serological and molecular diagnostics. Two external quality assurance (EQA) studies were performed to assess the quality of serological and molecular diagnostics of TBE infections. For the EQA of serological diagnostics, each participating laboratory received a proficiency panel of freeze-dried human sera containing 7 TBE-positive samples, 3 negative samples, and 2 samples positive for heterologous flaviviruses. For the EQA of molecular diagnostics, panels of prepared human plasma samples comprising 8 samples spiked with RNA from different TBE virus strains of European, Siberian, and Far Eastern subtypes, 2 specificity controls with heterologous flaviviruses, and two negative control samples were distributed. A total of 46 invited expert laboratories from 28 European and non-European countries participated at least in one of these studies. Applying proficiency criteria, the number of participating laboratories who passed the minimum requirements for successful participation was 60% for the EQA of serological (both IgM and IgG detection) and 48% for the EQA of PCR diagnostics. The EQA studies provide information on the diagnostic quality of the participating laboratories and indicate that most of them need to improve their assays.     

Neuraminidase-inhibitor resistance testing for pandemic influenza A (H1N1) 2009 in Ontario,Canada

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 × 10⁴ copies/ml but the detection rate was decreased to 68% for 1.2 × 10³ 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.     

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 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.     

Against the proposition: for the diagnosis of viral infections, commercial assays provide more reliable results than do in-house assays

There are no differences inherent in the design of commercial or in-house assays and their early development is similar. The same principles apply and it is on the same criteria of accuracy, reproducibility and clinical relevance of results that all assays are judged. However, if there is sufficient uptake of a commercial assay, its strengths and any flaws soon become apparent and it will only be the best commercial assays that remain in the market. For the in-house assays it is through comparability studies and external quality assessment (EQA) schemes that the best can be demonstrated, albeit this information is only accessible initially to the EQA provider and the laboratories using the assays. The EQA results described here support my supposition that, for the diagnosis of viral infections, commercial assays do not provide more reliable results than do in-house assays.     

Evaluation of the efficiency of external assessment of the quality of determining antibodies to hepatitis C virus in the network of screening laboratories

A regional external quality assessment (EQA) system for determining anti-hepatitis C virus (HCV) was developed and introduced. For this, a control panel comprising 14 samples with anti-HCV and 6 samples without anti-HCV was tested in the reference laboratory of Moscow Infectious Hospital One. A total of 6 sessions were conducted with the participation of 8 laboratories. The regional EQA system covering a limited number of laboratories was shown to significantly improve the quality of detection of hepatitis C virus antibodies. The optimal multiplicity of sessions for the developed EQA system was ascertained to be once fortnight. It is possible to define the current rating of screening laboratories in the sensitivity criterion and to elaborate address organizational-and-methodic measures according to the results of control tests.     

Multicenter External Quality Assessment of Molecular Methods for Detection of Human Herpesvirus 6

The purpose of this study was to evaluate the performance of laboratories for the detection and quantification of human herpesvirus 6 (HHV-6) by an external quality assessment (EQA) evaluation. The HHV-6 EQA panel consisted of eight samples containing various concentrations of HHV-6 type A (strain GS) or type B (strain Z29), two samples containing other herpesviruses (i.e., human cytomegalovirus [HCMV] and Epstein-Barr virus [EBV]), and two HHV-6-negative samples. Panel samples were prepared in human plasma, heat inactivated, and lyophilized. Panel distribution, data management, and analysis were coordinated by Quality Control for Molecular Diagnostics (QCMD), Glasgow, United Kingdom. Fifty-one laboratories participated and submitted 57 data sets. Eleven (19.3%) data sets were generated using conventional in-house assays, 11 (19.3%) data sets using commercial real-time PCR assays, and 35 (61.4%) data sets using in-house real-time PCR assays. The presence of HHV-6 DNA at viral loads exceeding 6,000 copies/ml was detected by all participants, and over 80% of the participants still reported correct qualitative results for the sample containing just over 200 copies/ml. The false-positivity rate was 1.8% for both the negative samples and the samples containing HCMV or EBV DNA. The majority (23/33; 69.7%) of quantitative data sets were generated using in-house real-time PCR assays. The standard deviations of the geometric means of the samples ranged from 0.5 to 0.7 log₁₀. The results of this first international EQA demonstrate encouraging analytical sensitivity for the detection of HHV-6-DNA in human plasma, although we observed extensive interlaboratory variation of quantitative HHV-6 DNA results. Standardization needs to be improved to allow further elucidation of the clinical significance of HHV-6 loads.Human herpes virus type 6 (HHV-6) is a member of the Betaherpesvirus subfamily (genus Roseolovirus). Two distinct variants have been described: HHV-6 type A (HHV-6A) and HHV-6B. HHV-6 type B infection is recognized as the cause of a febrile disease and exanthem subitum in early childhood. Over 90% of the population worldwide is infected within the first 18 months of life (5, 12). After primary infection, HHV-6 establishes life-long persistence in the host and is detectable in multiple tissues, similar to other herpesviruses, such as human cytomegalovirus (HCMV) (6). HHV-6 infection rarely causes severe disease in healthy children, but viral reactivation in immunocompromised patients is associated with severe morbidity (encephalitis and acute graft-versus-host disease) and increased mortality (11). The majority of HHV-6 infections are caused by HHV-6 type B (2, 9). Although HHV-6 type A is rarely detected, this variant is suggested to act as a more neurotropic pathogen in the context of severe central nervous system infections (4).Because of their speed, sensitivity, and specificity, molecular diagnostic assays are increasingly used for the detection of HHV-6 DNA to diagnose viral disease. Various commercial and in-house assays are currently available. Due to the lack of any available well-characterized reference reagents, most of these assays lack standardization, both in performance as well as in viral load calculation.This lack of standardization causes difficulties in comparing results between laboratories and, as a consequence, complicates the clinical interpretation of laboratory results for HHV-6. In an attempt to compare the results for HHV-6 DNA detection and quantitation between laboratories, a first international external quality assessment (EQA) study was organized.     

Multicentre validation study of nucleic acids extraction from FFPE tissues

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp–250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.     

Standardized hepatitis C virus RNA panels for nucleic acid testing assays.

BACKGROUND: Methods for the quantification of hepatitis C virus (HCV) RNA are useful in the clinical management of infected patients. However, the introduction of assays based upon various nucleic acid testing (NAT) technologies, each utilizing a different set of standards, creates the potential for misinterpretation of patient results. OBJECTIVE: In order to address the need for worldwide standardization of these assays, a HCV RNA quantification panel (NAP HCV-RNA) calibrated against the World Health Organization (WHO) First International Standard for HCV RNA was prepared. The eight-member HCV RNA quantification panel was evaluated utilizing a variety of commercially available and in-house NAT technologies. STUDY DESIGN: NAP HCV-RNA panels were tested and analyzed using the methods and data reduction protocols specific to each NAT technology employed in this study (bDNA, TMA and two PCR methods). Proprietary units of measure from each assay were compared to WHO International Units (IU) for each panel member (0, 50, 500, 5000, 50000, 200000, 500000 and 2000000 IU/ml). RESULTS: Evaluation of the NAP HCV-RNA in a variety of NAT procedures demonstrated linearity across the range of target concentrations detected in each assay and R(2) values ranged from 0.9929 to 0.9995 across the four technologies. As expected, the correspondence of assay specific proprietary units to IU differed depending upon the technology utilized. CONCLUSIONS: NAP HCV-RNA provides a consistent, standardized method for comparing results across laboratories and technologies and is useful in ensuring the quality of NAT testing for HCV RNA, independent of the methodology used.     

Evaluation of a new NASBA assay for the qualitative detection of hepatitis C virus based on the NucliSens Basic Kit reagents.

BACKGROUND: Direct detection of HCV RNA by nucleic acid amplification methods is an essential tool in the diagnosis of HCV infections. In-house developed methods based on reverse transcribed polymerase chain reaction (RT-PCR) are widely used but they are laborious and usually lack the standardization required by clinical laboratories. OBJECTIVES: To evaluate the sensitivity and the clinical performance of an HCV specific nucleic acid sequence based amplification (NASBA) assay based on the commercially available, NucliSens Basic Kit (bioMérieux) reagents. STUDY DESIGN: The analytical sensitivity of the Basic Kit-based HCV assay (BK-HCV) was determined using dilutions of the First World Health Organization International Standard for HCV RNA. The performance of the BK-HCV was evaluated at two study sites in comparison with in-house RT-nested PCR (RT-nPCR) by testing a total of 77 plasma specimens. Additional HCV laboratory tests such as Amplicor HCV v2.0 (Roche Diagnostics) and genotype were also included in the comparative analysis. RESULTS: The sensitivity of the BK-HCV was 100-150 IU/ml HCV RNA (85-100% hit rate). When evaluating the clinical performance, we found 96-100% correlation between BK-HCV and RT-nPCR, and 85-91% correlation between BK-HCV and Amplicor. The level of efficiency of the BK-HCV for detecting prevalent HCV genotypes was equal to in house RT-nPCR and Amplicor. CONCLUSIONS: The BK-HCV offers adequate sensitivity for diagnostic purposes and equivalent clinical performance to in-house RT-nPCR assays. The BK-HCV could become a suitable alternative to the in-house amplification methods, providing standardized reagents and procedures, plus rapid results to clinical laboratories.