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Epstein-Barr virus (EBV) establishes lifelong latent infection. Almost every adult (about 90%) acquires EBV and will be seropositive to this virus.1,2 The majority of patients with primary infections are usually asymptomatic except for acute infectious mononucleosis seen in adolescents and young adults. In some patients the host-virus balance is disturbed, resulting in chronic active Epstein-Barr virus infection (CAEBV). In general, CAEBV is characterized by chronic or recurrent infectious mononucleosis-like symptoms, an abnormal pattern of anti-EBV antibodies with high titers to EBV-capsid antigen (VCA), early antigen (EA), and low titers to EBV nuclear antigen (EBNA).3,4 Although CAEBV has been previously reported to be associated with an aggressive clinical course,5,6 the pathogenesis and pathophysiology of CAEBV are not well characterized. It remains unclear which factors on the virus side and which factors on the host side predispose the development of this potentially fatal infection. Abnormal proportions of lymphocyte subsets have been detected in most of the patients with CAEBV. Such immunological abnormalities have also been detected in some of their parents, suggesting that CAEBV might be associated with certain genetic background related immunological disorders.7 Meanwhile, severe EBV disease has been observed with higher incidences in individuals with congenital immunodeficiency syndromes.8 These findings indicate that impaired function of immune cells may eliminate EBV-infected cells or disturbe immunoregulatory functions and the presence of certain genetic factors may play some role in development of CAEBV.
Infectious mononucleosis is common in western countries. In Asia, the majority of clinical cases with fulminant EBV infection were reported in Japan. It has been suggested that CAEBV in western countries is usually milder than in Asian countries.9 Thus, it is likely that the immunological function which controls EBV infection may differ from one ethnic population to another.
METHODS
Patients
The study group comprised 53 pediatric patients who were admitted to Beijing Children′s Hospital from 2003 to 2007. All the patients satisfied the following diagnostic criteria. (1) The duration of illness must be more than 3 months. EBV-related illness or symptoms including fever, persistent hepatitis, extensive lymphadenopathy, hepato- splenomegaly, pancytopenia, interstitial pneumonia, and hydroa vaccinifrome. (2) Increases in quantities of EBV were found in either affected tissues or peripheral blood. The amount of EBV was defined as increased when one or more of the following criteria were met: (a) EBV DNA was detected in tissues or peripheral blood by Southern blot hybridization; (b) EBV-encoded small RNA (EBER) positive cells were detected in tissues; (c) the illness was associated with high titers of IgG antibody to EBV-VCA and with little or no antibody to EBNA. (3) No evidence of any prior immunologic abnormalities or any other recent infection that might explain the condition.
Procedures
For each patient, family medical records, the patient′s medical history, age of onset, forewarning signs, symptoms, complications, laboratory data at diagnosis, EBV-specific antibodies, virus loads, and outcomes were collected and analyzed. All the patients tested negative for human immunodeficiency virus antibodies in serum. In this study, quantitative real-time polymerase chain reaction (PCR) was used to detect EBV DNA in serum. We used the EBV PCR Fluorescence Diagnosis Kit (Daan, Guangzhou, China), which has a EBV DNA detection range of 5.0×102 to 5.0×108 copies/ml. PCR products were detected by an ABI Prism 7300 Sequence Detection System (PE Biosystems, USA).
Statistical analysis
The differences among the mean values of white blood cell counts, haemoglobin, platelets, aminotransferase and albumin between living and deceased patients were analyzed using an independent-sample t test (SPSS Inc, Chicago, IL, USA). The results are presented as mean ± standard deviation (SD). P <0.05 was considered statistically significant.
RESULTS
Clinical manifestations and outcomes
The study group consisted of 36 boys and 17 girls with ages ranging from 6 months to 15 years (mean (6.3±3.8) years) and a male:female ratio of 2.12:1.00, as shown in Figure 1, the age at onset of CAEBV infection was 2 months to 14.6 years (mean (5.3±3.3) years). At the time of onset, 23 out of 53 patients (43.4%) had an infectious mononucleosis-like illness. Three patients (5.7%) had a past history of hypersensitivity to mosquito bites (HMB). None of the patients had a background of hereditary disease. Most of the pediatric patients came from Northern China, and 39 patients (73.6%) came from rural areas. No seasonal or yearly variation in the frequency of CAEBV was detected. Figure 2 shows major clinical symptoms and incidence of forewarning signs of this disease as a percentage. Most of the patients with CAEBV presented with high fever (92.5%), hepatomegaly (81.1%) and splenomegaly (77.4%). More than two-third (69.9%) of the patients had lymphadenectasis and about half of the patients (47.2%) had anemia. Life-threatening complications including hemophagocytic syndrome (24.5%), interstitial pneumonia (24.5%), hepatic failure (15.1%), malignant lymphoma (11.3%), cardiovascular lesions (9.4%) in which coronary artery aneurysms and valvular disease were observed in one case of each, central nervous system involvement (9.4%), and disseminated intravascular coagulopathy (3.8%) were observed. Digestive tract ulcers and acute leukemia were each seen in one patient. Only 5 patients experienced remission of 1 to 3 years after the onset in the 42 patients who were followed. Note that no follow-up data were available for 11 patients. A majority of the patients with CAEBV generally had continuous symptoms during the observational periods, although the intensity of the symptoms tended to fluctuate among the patients. During the observational period, 11 of 42 patients (26.2%) died 7 months to 3 years after the disease onset. Virus-associated hemophagocytic syndrome, hematological malignancies, hepatic failure or/and cardiac failure were the main causes of death.
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Figure 1. Age distribution of 53 children at the onset of CAEBV. |
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Figure 2. Clinical presentations in pediatric patients with CAEBV at the time of diagnosis. |
Hematological and imaging findings at diagnosis
The laboratory findings at the time of diagnosis are shown in Table 1. White blood cell count in 53 patients ranged from 0.8×109/L to 32.2×109/L. Serum biochemical examination showed an elevation of aminotransferase levels. A reduction in serum albumin was observed in most of the patients. Some patients showed high IgG and IgE concentration while the total numbers of T cells and NK cell decreased. The differences in some laboratory findings at the diagnosis of CAEBV infection between living and deceased patients are shown in Table 2.
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Table 1. Laboratory findings at the time of diagnosis |
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Table 2. Differences of laboratory data at diagnosis of CAEBV between living and deceased patients |
EBV-related antibody titers at the time of diagnosis
There were 6 different types of antibody response against EBV in CAEBV patients (Table 3). Serum titers of antibodies to VCA-IgG ranged widely from 80 to 20 480; 20 out of 49 patients (40.8%) presented high titer levels (≥5120), while 41 out of 49 patients (83.7%) had titers levels ≥2560 and 6 out of 49 patients (12.2%) had titers lower than 640. Forty-seven out of 51 patients (92.2%) were anti-EA-IgG positive, and 6 out of 51 patients (11.8%) were anti-VCA-IgM positive. Only 18 out of 51 patients (35.3%) were anti-EBNA-IgG negative.
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Table 3. Types of antibody response against EBV in patients |
Histopathological findings at the time of diagnosis
Seven patients had taken liver biopsy and eighteen patients had taken lymph node biopsy. In situ hybridization with the EBER probe showed infiltration of small lymphocytes positive for EBER in the portal area in all the 7 patients who underwent liver biopsy. Sixteen out of 18 patients who underwent lymph node biopsies showed similar histological findings with a large number of EBER positive cells.
Bone marrow aspirates were analyzed at least once for each patient. Some patients showed normocellular marrow without any abnormality; others showed hypocellular marrow with mild or moderate hemophagocytosis. Nevertheless, in all cases, results from bone marrow analysis showed an absence of hematologic malignant disorders at the diagnosis of CAEBV.
Virus load detection at the time of diagnosis
Virus load was measured in 23 patients using quantitative PCR. The serum EBV DNA level in those patients with CAEBV ranged from 5.05×102 to 4.60×106 copies/ml, except for 4 patients who showed negative values. The average copy number of EBV-DNA in serum was 103.7 copies/ml.
DISCUSSION
CAEBV presented in all ages from infants to adolescents in our 53 Chinese pediatric patients. The majority of the patients (64.7%) were positive for anti-EBNA antibodies. It suggests that most patients with CAEBV have already suffered from primary EBV infection before the onset of CAEBV, although the precise timing of EBV infection is unclear.
The pathogenesis of CAEBV remains unknown10 although EBV-infected T cells and NK cells have been found to play a central role.11-13 In this study, nineteen patients (39.6%) and thirteen patients (27.1%) had elevated levels of serum IgG and IgE. The ratio of CD4+/CD8+ T cells was inverted in 15 patients (32.6%) and there were 16 patients (34.8%) who had low NK cell counts. These results suggest that both cellular and immunological abnormalities may be associated with patients who develop CAEBV. Thus, the exact causes of CAEBV are likely to be heterogeneous, and may involve subtle host immune dysregulation, viral factors, or both. A possible pathophysiology of this disease could be that the balance between immunological function and viral pathogenesis is disrupted.
With advanced molecular techniques, such as the PCR and in situ hybridization targeted EBER, it becomes possible to detect the EBV genome more accurately. By using those laboratory techniques, in situ hybridization revealed a large number of EBER positive cells in the biopsy specimen for the 7 patients who had liver biopsy and 18 patients who had a lymph node biopsy. Previous studies showed that patients with CAEBV could have a marked increase in EBV DNA level in the peripheral blood.14 In addition, viral loads of more than 102.5 copies/μg DNA have been demonstrated in the PBMCs of patients with CAEBV.12 As CAEBV has been thought to be linked to viral replication, quantitative analysis of genes which are responsible for the lytic part of the life cycle of the virus may be useful in studying this disease.15 For this reason, virus loads in serum were analyzed for 23 pediatric patients with CAEBV in this study. The results showed noticeable increases in viral loads except for 4 patients, suggesting that quantitative PCR analysis of serum and tissue staining for EBV are valuable diagnostic methods in CAEBV. Based on these findings, we recommend that if a patient presents with a series of unexplained symptoms which fit in the spectrum of symptoms of CAEBV, EBV loads should be measured and tissue should be stained using in situ hybridization for EBER.
In this study, CAEBV patients did not always satisfy the proposed criteria defined by Straus.3 Six patients had duration of illness of less than 6 months, and some patients had rather low antibody titers. Previous report has also revealed that few patients showing clinical and laboratory findings died at less than 2 months after onset.16 Similar to those previously reported cases,2,6,12 not all patients in this study had high VCA-IgG titers levels (≥5120). However, those patients had typical symptoms of CAEBV, high EBV loads in their peripheral blood, and/or EBV-encoded small RNA (EBER) in their tissues. Kimura et al12 considered that a high titer of EBV-related antibodies is not always a prerequisite for diagnosis of CAEBV. Therefore, in agreement with other authors,12,17 we suggest that the diagnosis of CAEBV should be based on the constellation of both clinical and laboratory findings rather than being limited by disease duration or by laboratory parameters. Although high titers of antibody to EBV antigens and duration of illness should be included in the diagnostic criteria, a more flexible approach to those criteria is necessary, particularly for those cases showing typical clinical and pathological findings.
In this study, majority of the patients with CAEBV showed symptoms and forewarning signs similar to those having been reported in the Japanese studies.12,18,19 It confirms that this disease is often associated with virus- related hemophagocytic lymphohistiocytosis, interstitial pneumonitis and hepatic failure at some point during the course of the disease, consistent with the previous Japanese reports. However, prevalence of some signs and symptoms in this study differs slightly from those reported in Japan. In this study, hypersensitivity to mosquito bites as past history were only found in 3 out of 53 patients (5.7%), while higher occurrences (31.3% to 43.0%) were reported in Japan.12,18 Cardiovascular lesions, such as pericarditis, myocarditis, and mitral valve regurgitation, were comparatively low in our study. Those dissimilarities may be attributed to the differences in diet, environment and hygienic conditions, or pathological factors other than EBV. In addition, the fatality ratio in this study, 11 out of 42 patients (26.2%) was lower than those reported in Japan (43.0% to 61.5%).18,19 The difference may be attributed to the observational time in this study, which was shorter than in the Japanese study, or to some seriously ill patients who lived in poorer, remote rural regions who could not be contacted after they discharge from the hospital. Unfortunately, a sound comparison cannot be made with those cases reported in western countries. It is because there are not enough reported cases in the literature to allow a meaningful statistical summary for individual western countries.
The laboratory data shown in Table 2 suggest that the deceased patients were more likely to have had lower platelet counts and albumin levels than the living patients (P <0.05 for all comparisons). These results imply that thrombocytopenia and decreases in albumen might potentially be the risk factors of a poor prognosis.
In conclusion, this study reveals that CAEBV in Chinese pediatric patients has a severe clinical course and prognosis remains poor. Thrombocytopenia and decrease of albumin may be the risk factors of poor prognosis. EBV loads should be measured and tissue should be stained for hybridization for EBER if a patient presents with a series of unexplained symptoms which fit within the spectrum of symptoms of CAEBV. Further, prospective, well designed investigations concerning immunological aspects and a therapeutic approach towards CAEBV are recommended for improvement of both prevention and treatment of CAEBV.
Acknowledgments: We are grateful to Prof. ZHAORI Ge-tu for a critical reading of the manuscript. We are also grateful to the anonymous peer-reviewers whose constructive comments have significantly improved this paper.
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