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Factors contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis.

ABSTRACT: While viral load testing has gained widespread acceptance, a primary limitation remains the variability of results, particularly between different laboratories. While some work has demonstrated the importance of standardized quantitative control material in reducing this variability, little has been done to explore other important factors in the molecular amplification process. Results of 185 laboratories enrolled in the College of American Pathologists (CAP) 2009 viral load proficiency testing (PT) survey (VLS) were examined. This included 165 labs (89.2%) testing for cytomegalovirus (CMV), 99 (53.5%) for Epstein-Barr virus (EBV), and 64 (34.6%) for BK virus (BKV). At the time of PT, laboratories were asked a series of questions to characterize their testing methods. The responses to these questions were correlated to mean viral load (MVL) and result variability (RV). Contribution of individual factors to RV was estimated through analysis of variance (ANOVA) modeling and the use of backward selection of factors to fit those models. Selection of the quantitative calibrator, commercially prepared primers and probes, and amplification target gene were found most prominently associated with changes in MVL or RV for one or more of the viruses studied. Commercially prepared primers and probes and amplification target gene made the largest contribution to overall variability. Factors contributing to MVL and RV differed among viruses, as did relative contribution of each factor to overall variability. The marked variability seen in clinical quantitative viral load results is associated with multiple aspects of molecular testing design and performance. The reduction of such variability will require a multifaceted approach to improve the accuracy, reliability, and clinical utility of these important tests.

SUBMITTER: Hayden RT

PROVIDER: S-EPMC3264161 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Factors contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis.

Hayden R T RT Yan X X Wick M T MT Rodriguez A B AB Xiong X X Ginocchio C C CC Mitchell M J MJ Caliendo A M AM

Journal of clinical microbiology 20111123 2

While viral load testing has gained widespread acceptance, a primary limitation remains the variability of results, particularly between different laboratories. While some work has demonstrated the importance of standardized quantitative control material in reducing this variability, little has been done to explore other important factors in the molecular amplification process. Results of 185 laboratories enrolled in the College of American Pathologists (CAP) 2009 viral load proficiency testing ...[more]

PMID: 22116152

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Project description:The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to unprecedented social and economic disruption. Many nucleic acid testing (NAT) laboratories in China have been established to control the epidemic better. This proficiency testing (PT) aims to evaluate the participants' performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities. Two different concentrations of RNA samples (A, B) were used for quantitative PT. Pseudovirus samples D, E (different concentrations) and negative sample (F) were used for qualitative PT. 50 data sets were reported for qualitative PT, of which 74.00% were entirely correct for all samples. Forty-two laboratories participated in the quantitative PT. 37 submitted all gene results, of which only 56.76% were satisfactory. For qualitative detection, it is suggested that laboratories should strengthen personnel training, select qualified detection kits, and reduce cross-contamination to improve detection accuracy. For quantitative detection, the results of the reverse transcription digital PCR (RT-dPCR) method were more comparable and reliable than those of reverse transcription quantitative PCR (RT-qPCR). The copy number concentration of ORF1ab and N in samples A and B scattered in 85, 223, 50, and 106 folds, respectively. The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills. Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing, 95.65% of the laboratories with satisfactory quantitative results also judged the qualitative results correctly, while 85.71% of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments. Therefore, the quantitative ability is the basis of qualitative judgment. Overall, participants from hospitals reported more satisfactory results than those from enterprises and universities. Therefore, surveillance, daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.

Dataset Information

Factors contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis.

Publications

Factors contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis.

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