Multicenter Comparison of Different Real-Time PCR Assays for Quantitative Detection of Epstein-Barr Virus

Abstract

Quantification of Epstein-Barr virus (EBV) in peripheral blood is important for the diagnosis and management of serious EBV diseases, including posttransplant lymphoproliferative disorder. A variety of PCRbased methods are currently in use; however, there is little information on their comparability. This study assessed the relative performance of different quantitative assays. A multicenter comparative study was performed at eight sites using three panels consisting of serial dilutions of quantified EBV DNA and extracts from a total of 19 whole-blood specimens. Samples were distributed and tested blindly. Instrumentation, probe chemistries, amplification targets, and other test-related aspects varied considerably between laboratories. Each laboratory's calibration curve indicated strong evidence of a consistent log-linear relationship between viral load and cycle threshold, suggesting that intralaboratory tracking of a given patient would yield similar relative quantitative trends among the participating test sites. There was strong concordance among laboratories with respect to qualitative test results; however, marked quantitative discordance was seen. For most samples, the across-laboratory interquartile range of the reported viral load (in copies/l) was roughly 0.6 log-units, and for one sample the overall range was approximately 4.2 log-units. While intralaboratory tracking of patients may yield similar results, these data indicate a need for caution when attempting to compare clinical results obtained at different institutions and suggest the potential value to be gained by more standardized testing methodology.

Serological diagnosis of Epstein-Barr virus infection: Problems and solutions

“…More recent studies indicate that real-time PCR is particularly sensitive [28] , and very useful for defining infection status, especially in immunocompromised patients [45,75,76] and those at risk of developing EBVrelated disorders [45] . However, there is still no consensus concerning the best material to use, units of measurement, or the quantitative levels requiring intervention or predicting prognosis [16,74,[77][78][79] . This means that particular care is necessary when comparing the data of different studies [73] : for example, the units of measurement include copies per milliliter, copies per microgram of DNA, copies per 100 000 leukocytes, and copies per positive cell [77] .…”

“…However, there is still no consensus concerning the best material to use, units of measurement, or the quantitative levels requiring intervention or predicting prognosis [16,74,[77][78][79] . This means that particular care is necessary when comparing the data of different studies [73] : for example, the units of measurement include copies per milliliter, copies per microgram of DNA, copies per 100 000 leukocytes, and copies per positive cell [77] . The targets used may also vary from one method to another: LMP2, BKRF1 or BamHI-W (EBNA-1), BNRF1 (membrane protein), BXLF1 (thymidine kinase), BZLF1 (ZEBRA), BALF5 (viral DNA polymerase) or BHRF-1 (transmembrane protein).…”

Prognostic role of plasma Epstein-Barr virus DNA load for nasopharyngeal carcinoma: a meta-analysis

“…Use of real-time PCR to measure EBV DNA was first reported in 1999 [16,44], and since then, the availability and diversity of reagents and protocols have expanded considerably [45]. Not unexpectedly, several studies have documented substantial variability in quantitative assays [46][47][48]. The World Health Organization (WHO) introduced an EBV DNA standard in 2011 [49], providing an opportunity for institutions to reduce the inter-institutional variability of their data.…”

Posttransplant Lymphoproliferative Diseases During Childhood: Review

“…Zaman içinde aynı hastanın aynı laboratuvarda tekrarlı değerlerle izleminin yapılması önerilmektedir. 80,81 Erken PTLH ve semptomatik EBV enfeksiyonunda kantitatif EBV viral yük analizinin duyarlılık ve öz-güllüğüne dair yapılan çalışmalar sınırlıdır. Yüksek riskli hasta grubu EBV viral yük tayini ile izlenmektedir.…”