Chronic Active Epstein-Barr Virus (CAEBV) infection diagnosis relies on a combination of clinical evaluations and detailed virological assessments. Initially defined by diagnostic criteria proposed in 1988 and 1991 for “severe, chronic EBV infection” and “severe chronic active EBV infection syndrome”, these early frameworks highlighted persistent infectious mononucleosis-like symptoms, hematological abnormalities, significant organ involvement, and irregular EBV antibody levels [23, 24]. Further refinement came in 2005 with the Japanese Association for Research on EBV and Related Diseases suggesting quantitative EBV polymerase chain reaction (PCR) testing as a crucial laboratory tool for Caebv Diagnosis [7].
The diagnostic landscape evolved again in 2022 when the Ministry of Health, Labour and Welfare in Japan updated the CAEBV diagnostic criteria (Table 1). These revisions emphasize the necessity of confirming both elevated EBV genome counts and the presence of EBV-infected T/NK cells for a definitive CAEBV diagnosis. This shift reflects growing evidence linking CAEBV to the proliferation of EBV-infected T and Natural Killer (NK) cells. Let’s delve into the specifics of these laboratory tests crucial for accurate CAEBV diagnosis.
Virological Studies for CAEBV Diagnosis
Virological studies play a pivotal role in the diagnosis of CAEBV, encompassing several key areas: EBV-related antibody profiling, EBV DNA quantification, and identification of EBV-infected cell types. Each of these provides valuable insights into the presence and nature of CAEBV infection.
EBV-Related Antibodies in CAEBV Diagnosis
Historically, unusual EBV antibody patterns, specifically high titers of anti-viral capsid antigen (VCA)-IgG (≥ 640) and anti-diffuse and restricted early antigen (EA-DR)-IgG (≥ 160), measured using fluorescence antibody tests, were considered significant indicators of CAEBV. These findings were incorporated into earlier diagnostic criteria [23,24,25]. However, it’s now recognized that elevated VCA-IgG levels can be present in healthy individuals who are EBV-seropositive, independent of CAEBV. Furthermore, not all CAEBV patients exhibit these elevated antibody titers.
Data reveals that the geometric mean titer for VCA-IgG is significantly higher in T-cell CAEBV (2010) compared to NK-cell CAEBV (310). Similarly, anti-EA-DR-IgG titers show a stark contrast, with geometric means of 610 for T-cell CAEBV and only 70 for NK-cell CAEBV [26]. While unusual antibody patterns are more frequently seen in T-cell CAEBV, it’s crucial to understand that a subset of CAEBV patients, particularly those with NK-cell CAEBV, may not present with high EBV-related antibody titers. Therefore, current diagnostic protocols consider elevated antibody titers supportive but not mandatory for CAEBV diagnosis.
EBV DNA Quantification for CAEBV Diagnosis
Real-time PCR for EBV DNA load measurement has become a standard procedure in diagnosing and monitoring EBV-related diseases [27]. CAEBV patients often exhibit exceptionally high EBV DNA loads in their peripheral blood [28, 29]. This PCR analysis can be performed on whole blood or specific blood components like peripheral blood mononuclear cells (PBMCs) and plasma. PBMCs contain cell-associated EBV DNA, while plasma carries cell-free EBV DNA. The choice of blood component for testing depends on the specific EBV-associated disease and its activity phase.
During active CAEBV phases, EBV DNA load increases across all blood components. Conversely, plasma/serum EBV DNA might become undetectable during inactive phases [22, 28]. For CAEBV diagnosis, testing EBV DNA load in whole blood or PBMCs is generally preferred. However, plasma EBV DNA levels more accurately reflect disease activity in CAEBV, showing higher loads in active phases compared to inactive ones [29]. A recent study highlighted plasma EBV DNA’s superior accuracy over PBMCs in distinguishing disease activity, particularly in NK-cell-type CAEBV [30].
Although definitive threshold values for EBV DNA load in CAEBV diagnosis are still under investigation, the 2016 WHO International Standard for EBV DNA has standardized quantitative PCR, using international units (IU) for EBV DNA load reporting [31]. This standardization enhances comparability across different testing facilities. One study indicated that whole-blood EBV DNA loads exceeded 10,000 IU/mL in 94% of CAEBV patients, showing a strong correlation with PBMC EBV DNA loads [29]. Based on current data and ease of sample preparation, an EBV DNA load ≥ 10,000 IU/mL in whole blood is proposed as a diagnostic cutoff for CAEBV in updated guidelines. However, it’s essential to remember that elevated EBV DNA loads are not exclusive to CAEBV and can occur in other EBV-associated conditions and even in asymptomatic individuals. Therefore, EBV DNA load alone is insufficient for differentiating CAEBV from other conditions.
Identification of EBV-Infected Cells in CAEBV Diagnosis
Identifying the lineage of EBV-infected cells is crucial for CAEBV diagnosis and differentiating it from other EBV-associated disorders. Since CAEBV is primarily linked to EBV-infected T/NK cell proliferation, pinpointing these cell types is vital. In situ hybridization (ISH) for EBV-encoded small RNA (EBER) in tissue samples is a common method for detecting EBV-infected cells [32]. However, tissue biopsies are not always clinically feasible.
Alternatively, peripheral blood samples can be used to identify EBV-infected cells. Real-time PCR on PBMCs, separated into B, T, and NK cell fractions using immuno-bead methods, allows for lineage determination. The principle is that EBV DNA load will be higher in cell fractions of the infected lineage compared to uninfected fractions or unfractionated PBMCs. Comparing EBV DNA loads across sorted cell fractions helps identify the infected cell lineage, a technique used in CAEBV and other EBV-related disorder diagnoses [26, 33]. However, potential false positives due to low purity of sorted cells or persistent cell-free EBV DNA in uninfected lineages should be considered.
Flow cytometry with fluorescence in situ hybridization (flow-FISH) provides another approach to evaluate EBV-infected cell lineages [34,35,36]. This technique combines antibody-based staining for surface markers with EBER detection via FISH within a flow cytometry framework, enabling identification of EBV-infected cell subsets. EBERs are highly expressed in all EBV-infected cells, making them reliable markers [37]. Studies using flow-FISH in EBV-associated T/NK lymphoproliferative diseases, including CAEBV, found that 0.15–67% of PBMCs were EBER-positive T/NK cells [38]. While flow-FISH offers detailed phenotypic characterization of EBV-infected cells, it is less sensitive than real-time PCR on fractionated PBMCs and is not widely available in diagnostic labs.
Pathological Findings in CAEBV Diagnosis
Pathological examinations are a critical component of CAEBV diagnosis, particularly when correlated with virological findings. While no definitive morphological hallmarks of CAEBV exist, EBER-ISH studies become essential, capable of detecting even sparse EBV-positive cells in limited tissue samples.
CAEBV is classified as a latency type II infection, characterized by the expression of specific viral antigens: Epstein-Barr virus nuclear antigen 1 (EBNA1) and latent membrane proteins 1 and 2 (LMP1, LMP2). However, immunohistochemical staining often fails to detect these antigens in CAEBV patients. EBV-positive cells commonly infiltrate organs like the liver, spleen, lymph nodes, and bone marrow, with less frequent involvement of the heart, gastrointestinal system, and muscles [7]. The infiltrating lymphocytes typically range from small to medium in size, lacking overt signs of malignancy. Lymph node pathology frequently reveals paracortical hyperplasia and polymorphic lymphocyte proliferation mixed with other inflammatory cells [39, 40]. Disease progression in some CAEBV cases can lead to lymphoma or leukemia. Consequently, histological examinations can present a spectrum of cytological findings, from reactive appearances to frank leukemia/lymphoma [41].
The lymphocyte lineage in CAEBV is more commonly T-cell than NK-cell, typically expressing CD3ɛ and cytotoxic markers like TIA1 and granzyme B [22, 42]. Within T-lineage cells, TCRαβ and CD4 positivity are frequent, while a smaller subset expresses CD8 and/or TCRγδ [26]. CD56 expression is particularly common in NK-lineage cells. Notably, some CAEBV patients exhibit EBV infection in both T and NK cells [14].
Clonality analysis of EBV-positive cells, often using Southern blot hybridization targeting EBV terminal repeats, reveals that CAEBV typically presents with monoclonality. However, oligoclonal or polyclonal EBV populations are also observed in some patients [41]. This raises questions about whether CAEBV originates as a monoclonal lymphoproliferative disorder from a single cell. Recent research suggests that driver mutations in CAEBV patients can be shared across various cell lineages [14]. This indicates a potential model where EBV infects a common lymphoid progenitor, leading to clonal evolution involving multiple cell lineages [18].
