Molecular Parameters for Precise Diagnosis of Asymptomatic Epstein-Barr Virus Reactivation in Healthy Carriers

Abstract

Asymptomatic Epstein-Barr virus (EBV) reactivations periodically occur in oral mucosa-associated lymphoid tissues. Until now, EBV reactivation has been diagnosed by serologic profiles that suggest virus replication. Serologic responses, however, are delayed and do not necessarily indicate ongoing replicative activity. The aim of the present study was to establish in healthy carriers parameters for a molecular diagnosis of reactivated EBV infection. Recent studies emphasized the association of an increase in peripheral-B-cell viral load with replicative activity at remote sites. Therefore, real-time PCR was used to quantitate EBV genomes in the peripheral blood mononuclear cells (PBMC) (viral load) and plasma samples (viremia) of 22 healthy EBV-seropositive blood donors over a period of 15 months. Furthermore, transcription of the immediate-early gene encoding BZLF1 was investigated in the PBMC of all volunteers. Serology suggested reactivation in nine donors, of whom all but one showed at least once a significant increase in viral load. Another five individuals also exhibited significant changes in viral load but no serologic response. Of the 13 volunteers with significant increases in viral load, 6 had a period of viremia accompanying the rise in viral load. A stable viral load without viremia and negative serology was seen in eight adults. BZLF1 mRNA was undetectable throughout. We conclude that for healthy subjects serology underestimates the frequency of asymptomatic EBV reactivations. Prospective examination of peripheral viral load and viremia is suitable for the exact diagnosis of EBV reactivation, which might be of advantage for immunocompromised patients in whom EBV reactivations are considerably harmful.

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

“…However, it must be kept in mind that there may be individual variations due to individual differences in kinetics, and viral load may increase after an initial decline, and in some cases, it may take as long as a year or more before it reaches stably low levels. Finally, even when this level is reached, the blood of a healthy carrier contains 1-50 copies of EBV DNA per million white blood cells, whereas EBV-DNA is almost always undetectable in plasma or serum [82,85,[93][94][95][96] . The presence of plasma/serum EBV-DNA is therefore considered a sign of primary infection [13] or reactivation, and the viral load correlates with disease severity [85,88,92] .…”

“…Patients with latent infection have an almost constant number of circulating infected B cells in peripheral blood and, in the case of reactivation, these differentiate into plasma cells, leading to the start of the replicative cycle and increased EBV DNA levels in PBMCs and serum/plasma. It has been reported that a search for EBV DNA in PBMCs and serum/plasma is important for an immediate diagnosis of reactivation but, although this is true in the patients who are followed up over time in order to detect any changes in viral load, the finding of EBV DNA in a single sample should not necessarily be seen as a sign of reactivation [95] . Consequently, as EBV DNA is present in cases of reactivation or primary infection, it is unlikely that testing one sample will be able to distinguish the two situations [75,95] .…”

“…It has been reported that a search for EBV DNA in PBMCs and serum/plasma is important for an immediate diagnosis of reactivation but, although this is true in the patients who are followed up over time in order to detect any changes in viral load, the finding of EBV DNA in a single sample should not necessarily be seen as a sign of reactivation [95] . Consequently, as EBV DNA is present in cases of reactivation or primary infection, it is unlikely that testing one sample will be able to distinguish the two situations [75,95] . Persistent or reactivated EBV infection is characterized by high titers of EA (D) IgG, especially in immunocompromised patients [26,29] .…”

“…Consequently, a search for VCA IgA is considered useful only in the diagnosis and management of patients with nasopharyngeal carcinoma [133][134][135][136] . EA (R) IgG, EA IgM, EA IgA, VCA IgG-3 and the EBNA-1/EBNA-2 ratio have also been used as markers of reactivation in some studies [18,95,114,[137][138][139] but, as many of these antibodies are also present in primary infections, and some even in past infections, the usefulness of a single sample in patients with the simultaneous presence of EBNA-1 IgG, VCA IgG and IgM has yet to be evaluated.…”

Ege Üniversitesi Hastanesi'ne başvuran hastalarda enzim işaretli floresan test ile elde edilen Epstein-Barr virüsü serolojik test sonuçlarının değerlendirilmesi

“…Sonuç olarak; EBV DNA hem re-aktivasyon hem primer enfeksiyonda kanda pozitif bulunmakla birlikte bu ikisinin ayrımı tek bir örnekten çalışılan test ile mümkün değildir (12). …”