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O.6.1 Human Herpesvirus type 6 external quality assessment evaluation among 51 laboratories in Europe
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Oral presentations, ESCV 2009, Amsterdam / Journal of Clinical Virology 44 Suppl. 1 (2008) S5–S16 O.6.3 Is it the cause? Robert Koch & viruses in the 21st century
Session 6. Quality related issues O.6.1 Human Herpesvirus type 6 external quality assessment evaluation among 51 laboratories in Europe Pagter1 ,
Loon1 ,
Vos1 ,
MacKay2 ,
Pandit2 ,
P.J. de A.M. van N.M. de W.G. A. R. Schuurman1 . 1 UMC Utrecht, Utrecht, Netherlands, 2 Quality Control for Molecular Diagnostics, Glasgow, United Kingdom
Purpose: The detection and quantification of Human Herpesvirus type 6 (HHV-6) DNA lacks standardization. We evaluated the performance of laboratories by external quality assessment (EQA). Methods: The HHV-6 EQA panel consisted of eight samples containing various concentrations of HHV-6 type A (strain GS) or type B (strain Z-29), two samples containing other herpesviruses (i.e. CMV and EBV) and two HHV-6 negative samples. Panel samples were aliquoted in normal human plasma, heat-inactivated, and lyophilised. Panel distribution, data management and analysis were coordinated by QCMD. Results: Fifty-one laboratories from 20 countries participated and submitted 57 datasets. Eleven (19.3%) datasets were generated using conventional inhouse assays, 11 (19.3%) datasets using commercial real-time PCR assays and 35 (61.4%) datasets using in-house real-time PCR assays. All datasets recorded correct qualitative results for the samples with viral loads exceeding 3.8log10 cp/mL and >80% of datasets recorded correct qualitative results for the sample of 2.3 log10 cp/mL. The false-positivity rate was 0.8% for both the negative samples and the samples containing CMV or EBV. The majority (23 of 33; 69.7%) of quantitative datasets were generated using in-house real-time PCR assays. The standard deviations for each of the samples, based on the geometric means calculated with all datasets, ranged between 0.5 and 0.7 log10. Conclusion: Results of this first EQA distribution for HHV-6 are encouraging. Currently used assays for HHV-6 detection are sensitive and specific. Further standardization of HHV6 quantification is needed to allow elucidating the clinical role of HHV-6 viral load. O.6.2 Evaluation of seven DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infection J.J.C. de Vries, E.C.J. Claas, A.C.M. Kroes, A.C.T.M. Vossen. Leiden University Medical Center, Leiden, Netherlands Purpose: To optimize DNA extraction for dried blood spots (DBS) followed by CMV PCR for diagnosing congenital CMV infection. Sensitivity, specificity, and suitability of the methods for high-throughput usage were assessed for seven extraction methods. Methods: Guthrie cards were spotted with CMV-positive whole blood with a broad range of CMV plasma loads (n=15). DNA was extracted from DBS using the following extraction methods: Dynabeads Silane (Invitrogen), QIAamp DNA modified Investigator Kit (Qiagen), NucliSens easyMAG (bioMeri´eux), MagNA Pure LC (Roche Diagnostics), Barbi et al. (Journal of Clinical Virology 17:2000;159–165), QIAsymphony (Qiagen), BioRobot Universal System (Qiagen). DNA extraction was followed by CMV amplification by means of an internally controlled real-time PCR. Results: DBS with high CMV DNA loads (whole blood loads over 10,000 c/ml, n = 6) were positive in six out of seven extraction methods tested. However, using DBS with lower CMV loads (whole blood loads 1000−10 000 c/ml, n = 7), sensitivity ranged from 30 to 100% depending on the method used. DBS with low CMV DNA loads (whole blood loads less than 1000 c/ml, n = 2) were negative in almost all extraction methods tested. Specificity of the seven extraction methods tested was 100%. The methods Barbi et al., QIAsymphony, and BioRobot Universal System were suitable for high-throughput applications using a 96-wells format. Conclusions: The most sensitive high-throughput DNA extraction methods for DBS tested were Barbi et al. and BioRobot Universal System. Both highly sensitive and high-throughput methods are required when considering nationwide newborn screening for congenital CMV infection.
C.R. Madeley. Stocksfield, United Kingdom At the end of the 19th century, Robert Koch published his Postulates to define criteria to confirm that a micro-organism was the cause of an illness. With the discovery of viruses, especially HIV, it has become increasingly difficult and even impossible to fulfil the Postulates in virus diseases. Moreover, highly sensitive diagnostic tests using amplification of viral nucleic acids raise new problems. How can the most sensitive test be validated (both in specificity and in interpretation), and does every positive result confirm currently significant activity? This paper will present the dilemma posed by finding small copy numbers of viruses both common and rare, comparing new diagnostic methods with older ones and suggesting an approach to defining what is significant by using longitudinal studies to find the appropriate cut-off level.
Oral presentations, ESCV 2009, Amsterdam / Journal of Clinical Virology 44 Suppl. 1 (2008) S5–S16 O.6.3 Is it the cause? Robert Koch & viruses in the 21st century
Session 6. Quality related issues O.6.1 Human Herpesvirus type 6 external quality assessment evaluation among 51 laboratories in Europe Pagter1 ,
Loon1 ,
Vos1 ,
MacKay2 ,
Pandit2 ,
P.J. de A.M. van N.M. de W.G. A. R. Schuurman1 . 1 UMC Utrecht, Utrecht, Netherlands, 2 Quality Control for Molecular Diagnostics, Glasgow, United Kingdom
Purpose: The detection and quantification of Human Herpesvirus type 6 (HHV-6) DNA lacks standardization. We evaluated the performance of laboratories by external quality assessment (EQA). Methods: The HHV-6 EQA panel consisted of eight samples containing various concentrations of HHV-6 type A (strain GS) or type B (strain Z-29), two samples containing other herpesviruses (i.e. CMV and EBV) and two HHV-6 negative samples. Panel samples were aliquoted in normal human plasma, heat-inactivated, and lyophilised. Panel distribution, data management and analysis were coordinated by QCMD. Results: Fifty-one laboratories from 20 countries participated and submitted 57 datasets. Eleven (19.3%) datasets were generated using conventional inhouse assays, 11 (19.3%) datasets using commercial real-time PCR assays and 35 (61.4%) datasets using in-house real-time PCR assays. All datasets recorded correct qualitative results for the samples with viral loads exceeding 3.8log10 cp/mL and >80% of datasets recorded correct qualitative results for the sample of 2.3 log10 cp/mL. The false-positivity rate was 0.8% for both the negative samples and the samples containing CMV or EBV. The majority (23 of 33; 69.7%) of quantitative datasets were generated using in-house real-time PCR assays. The standard deviations for each of the samples, based on the geometric means calculated with all datasets, ranged between 0.5 and 0.7 log10. Conclusion: Results of this first EQA distribution for HHV-6 are encouraging. Currently used assays for HHV-6 detection are sensitive and specific. Further standardization of HHV6 quantification is needed to allow elucidating the clinical role of HHV-6 viral load. O.6.2 Evaluation of seven DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infection J.J.C. de Vries, E.C.J. Claas, A.C.M. Kroes, A.C.T.M. Vossen. Leiden University Medical Center, Leiden, Netherlands Purpose: To optimize DNA extraction for dried blood spots (DBS) followed by CMV PCR for diagnosing congenital CMV infection. Sensitivity, specificity, and suitability of the methods for high-throughput usage were assessed for seven extraction methods. Methods: Guthrie cards were spotted with CMV-positive whole blood with a broad range of CMV plasma loads (n=15). DNA was extracted from DBS using the following extraction methods: Dynabeads Silane (Invitrogen), QIAamp DNA modified Investigator Kit (Qiagen), NucliSens easyMAG (bioMeri´eux), MagNA Pure LC (Roche Diagnostics), Barbi et al. (Journal of Clinical Virology 17:2000;159–165), QIAsymphony (Qiagen), BioRobot Universal System (Qiagen). DNA extraction was followed by CMV amplification by means of an internally controlled real-time PCR. Results: DBS with high CMV DNA loads (whole blood loads over 10,000 c/ml, n = 6) were positive in six out of seven extraction methods tested. However, using DBS with lower CMV loads (whole blood loads 1000−10 000 c/ml, n = 7), sensitivity ranged from 30 to 100% depending on the method used. DBS with low CMV DNA loads (whole blood loads less than 1000 c/ml, n = 2) were negative in almost all extraction methods tested. Specificity of the seven extraction methods tested was 100%. The methods Barbi et al., QIAsymphony, and BioRobot Universal System were suitable for high-throughput applications using a 96-wells format. Conclusions: The most sensitive high-throughput DNA extraction methods for DBS tested were Barbi et al. and BioRobot Universal System. Both highly sensitive and high-throughput methods are required when considering nationwide newborn screening for congenital CMV infection.
C.R. Madeley. Stocksfield, United Kingdom At the end of the 19th century, Robert Koch published his Postulates to define criteria to confirm that a micro-organism was the cause of an illness. With the discovery of viruses, especially HIV, it has become increasingly difficult and even impossible to fulfil the Postulates in virus diseases. Moreover, highly sensitive diagnostic tests using amplification of viral nucleic acids raise new problems. How can the most sensitive test be validated (both in specificity and in interpretation), and does every positive result confirm currently significant activity? This paper will present the dilemma posed by finding small copy numbers of viruses both common and rare, comparing new diagnostic methods with older ones and suggesting an approach to defining what is significant by using longitudinal studies to find the appropriate cut-off level.