Original Article
Epstein–Barr virus serology and PCR: conflicting results in an immunocompetent host. A case report and review of literature L. Cattoir1, V. Van Hende2, P. De Paepe3, E. Padalko1,4 1
Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium, Department of Hematology, Ghent University Hospital, Ghent, Belgium, 3Department of Pathology, AZ Sint-Jan Bruges-Ostend AV, Bruges, Belgium, 4School of Life Sciences, Hasselt University, Diepenbeek, Belgium
2
We present the case of a 27-year-old immunocompetent man who progressively developed a generalized lymphadenopathy and B symptoms. Results of Epstein–Barr virus (EBV) serology were suggestive for a past infection, but the EBV viral load in whole blood was high. Also, core needle biopsy of the largest lymph node showed an image which could fit an EBV-driven reactive lymphoproliferation. Despite the absence of an immune disorder, all medical evidence points to an EBV-driven lymphoproliferative proces. In immunocompetent patients, it seems extremely uncommon to detect a high EBV viral load in the absence of serological evidence of an acute EBV infection or reactivation. We reviewed literature on this topic and on the selection of the appropriate sample type for EBV PCR, as this is known to be a critical point. Serological testing for the diagnosis of EBV infection is the gold standard in immunocompetent patients. Measuring EBV viral load is only recommended when dealing with immunocompromised patients. Although extremely rare, this case report shows that there is a place for EBV PCR in certain situations in immunocompetent patients. Besides, there is still no consensus regarding the specimen of choice for the determination of the EBV viral load. The preferred specimen type seems to depend on the patient’s underlying condition. Keywords: Epstein–Barr virus, Serology, PCR, Immunocompetent, Specimen type
Introduction Epstein–Barr virus (EBV) is a double stranded DNA virus of the herpesvirus family. EBV is widely present, more than 90% of the adults are EBVseropositive. In children, primary EBV infection is often asymptomatic, but adolescents and adults frequently present with the classical symptoms of acute infectious mononucleosis. Primary infection is usually self-limited, but the virus remains in a latent stage in a small fraction of the memory B lymphocytes throughout life, making viral reactivation possible. EBV is also associated with several malignant disorders, such as Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin, and non-Hodgkin lymphomas. Both reactivation and EBV-associated lymphoproliferative disorders (for example, posttransplant lymphoproliferative disorder, PTLD) most commonly occur in immunocompromised patients. In these patients, EBV infection can cause serious disease and is associated with high rates of morbidity and mortality. Correspondence to: E. Padalko, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185 (2P8), B-9000 Ghent, Belgium. E-mail: [email protected]
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ß Acta Clinica Belgica 2014 DOI 10.1179/2295333714Y.0000000037
We report a rare case of a young, immunocompetent man who was diagnosed with an EBV-lymproproliferative disorder, despite non-suggestive EBV serology. EBV viral load measured in whole blood using a quantitative real-time PCR was high, and lymph node biopsy fitted an EBV-driven reactive lymphoproliferation. This article focuses on the diagnosis of EBV-related disorders in immunocompetent patients and on the preferred specimen type for EBV viral load measurement.
Results Clinical and laboratory characteristics A 27-year-old man with a blank medical record consulted his general practitioner due to a swelling in his right axilla. Aside from this axillary lymph node, the patient also complained of malaise, fatigue, fever, and night sweats. Lymph node fine needle aspiration cytology showed a reactive presentation. The patient was treated with antibiotics for 5 days, however, without improvement of his complaints. During the next 2 months, the symptoms worsened and the patient developed significant weight loss and generalized lymphadenopathy. At that time, a haematologist was consulted. Laboratory analysis of peripheral
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blood showed a high erythrocyte sedimentation rate (.120 mm, normal 1–15 mm) and C-reactive protein (126.5 mg/dl, normal ,5 mg/dl). The red blood cell count was at low normal range 4.16106/mm3 (normal 4.1–5.76106/mm3) with a decreased haemoglobin level of 10.3 g/dl (normal 13.7–17.5 g/dl) and mean corpuscular volume of 78 fl (normal 80–100 fl). The white blood cell count (5.56103/mm3, normal 4.2–9.16103/mm3, with a normal differentiation) and platelet count (3176103/mm3, 140–4406103/mm3) were both within the normal range. Serum creatinine concentration, urea, uric acid, electrolytes, glucose, total protein, lactate dehydrogenase, and transaminases were within reference limits. The patient had increased blood levels of alkaline phosphatase (203 U/l, normal 40–130 U/l) and gamma-GT (106 U/l, normal ,60 U/l). Assessment of the iron status showed a decreased serum iron (26 mg/dl, normal 33–193 mg/dl) and transferrin (146 mg/dl, normal 200–360 mg/dl), and a highly increased ferritin concentration (2148 mg/l, normal 30–400 mg/l). Coagulation tests were clearly disturbed. Prothrombin time was decreased significantly to 44% (normal 70–100%), but with a normal activated partial thromboplastin time. Both fibrinogen and Ddimer were elevated to 759 mg/dl (normal 180– 400 mg/dl) and 1888 ng/ml (normal ,500 ng/ml), respectively. Results of cytomegalovirus and EBV serology were suggestive for a past infection. EBV serology was determined using EnzygnostH anti-EBV/ IgMII and EnzygnostH anti-EBV/IgG (Siemens Healthcare Diagnostics Products, Marburg, Germany) with negative IgM (0.26 ratio, positive if ratio .1)) and positive IgG (236 U/ml, positive if .30 U/ml) values. EnzygnostH anti-EBV/IgMII and EnzygnostH anti-EBV/IgG are ELISAs where a microtitration plate is coated with deactivated EBVantigens derived from EBV-infected lymphoblastoid cells containing viral capsid antigens (VCA), EBV
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nuclear antigens (EBNAs), and early antigens (EAs). Serological testing for the presence of antibodies to Bartonella henselae and mycobacterial culture was negative. Based on clinical presentation and laboratory results, the presence of a lymphoma were suspected.
Cytological characteristics A core needle biopsy of the largest axillary lymph node was performed. The biopsy showed extensive geographical necrosis surrounded by a blastic B-cell population (PAX5z/CD30z/partially CD15z). As the image resembled an EBV-driven lymphoproliferative disorder, an EBV staining was performed which confirmed the presence of a blastic EBVpositive B-cell proliferation (Fig. 1). The pathologist concluded that the image could fit either an EBVdriven lymphoproliferation or a classical Hodgkin’s lymphoma. Despite contradictory serology, when dealing with an immunocompromised patient, an EBV-mediated lymphoproliferative disorder would be preferred. A Hodgkin lymphoma also remains a possibility, but the extensive geographical necrosis and pronounced clinical signs present in this patient were not characteristic for this medical condition.
Additional serological and molecular diagnostics and specimen type Based on the pathology report, the haematologist requested more profound serological investigation and an EBV PCR. In order to determine specific antibody responses to individual EBV antigens, an immunoblotting test (recomLine EBV IgG; Mikrogen, Neuried, Germany) was performed. Nevertheless immunoblotting test is a qualitative method, the anti-EBV-VCA IgG antibody signal was strongly positive as demonstrated by the high intensity of specific antigen bands, whereas signals for anti-EBV-EA IgG and anti-EBV-EBNA IgG antibodies were weakly positive.
Figure 1 Immunohistochemistry of the lymph node. (A) CD30 staining (640). The large blasts are CD30-positive. (B) EBER/ EBV ISH staining (640). The large atypical cells contain EBV DNA.
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The EBV viral load in whole blood was high, 4.36104 copies/mg DNA, as measured using an inhouse quantitative real-time EBV PCR.1 Human immunodeficiency virus infection, common variable immunodeficiency, and hypogammaglobulinemia were excluded. B and T cellular maturation and natural killer cell levels were normal. Altogether, despite the absence of an immune deficiency, all objective medical evidence points to an EBV-driven lymphoproliferative disorder. In our hospital, EDTA whole blood is used as standard specimen for EBV PCR as EBV viral load is mainly employed to monitor transplant recipients at high risk of PTLD or under therapy for PTLD. Moreover, whole blood seems appropriate as it contains all types of EBV DNA (cell-free and cellassociated). However, periodical review of literature emerges in order to optimize current working practices based on latest research results and new developments.
Review of Literature and Discussion Serological testing for the diagnosis of EBV infection is the gold standard in immunocompetent patients.2 Measuring EBV viral load is only recommended when dealing with immunocompromised patients as these patients may have defective antibody production or receive therapeutic immunoglobulin preparations, which both could affect serological test results.2–6 EBV viral load is a laboratory marker mainly used in transplant recipients to predict, diagnose and follow-up treatment of EBV-driven PTLD.2,6–28 Monitoring EBV viral load has led to a decrease in the incidence of PTLD.7 This case report shows that the above described approach is questionable in certain clinical situations. There are at least a number of patients who are, or appear to be, immunocompetent where EBV PCR
may have an additional diagnostic value. Complete exclusion of the involvement of an EBV infection solely based on serological results should be discouraged. For each patient individually, the physician should take into account all available information and decide whether additional laboratory investigation, including an EBV PCR, may be needed. In determining the EBV viral load in a specific patient, selecting the appropriate sample type is critical. Despite a lack of consensus regarding this issue, the preferred specimen type seems to depend on the patient’s underlying condition.6–19 In healthy carriers, EBV DNA is located in the intracellular compartment of the blood as EBV persists in memory B cells after primary infection. Therefore, to detect EBV DNA by PCR, one should use whole blood samples.6,8–12 In contrast, there are certain medical conditions (Hodgkin lymphoma, EBV-associated haemophagocytic syndrome, chronic active EBV infection, nasopharyngeal carcinoma) in which EBV viral load in serum/plasma is preferred over whole blood. For example, in Hodgkin lymphoma, only serum/plasma viral load is associated with diagnosis and response to therapy.6,8,9,13 In this setting, EBV viral load in serum/plasma is not routinely measured, but according to literature, it can serve as a surrogate marker for disease activity, therapeutic response, and prognosis.6,8,9,13 In patients with EBV-driven PTLD, both intracellular and cell-free EBV DNAs are increased.6–28 Authors remain to disagree on the optimal specimen type. Table 1 gives a summary of the studies which compared different specimen types in the context of PTLD. Initially, everyone used peripheral blood mononuclear cells (PBMCs) as most EBV is cellassociated and EBV DNA is detected at high copy numbers in PBMCs.8 However, compared to PBMCs, the extraction of EBV DNA from whole
Table 1 Summary of literature concerning the specimen of choice for EBV PCR in the context of post-transplant lymphoproliferative disorder Author
Year Patient population
Sample type
Ruf and Wagner9 Gequelin et al.13 Shihara et al.22 Tsai et al.20 Kullberg-Lindh et al.28 Kimura et al.8 Bakker et al.26 Kinch et al.21 Wada K et al.19 Hakim H et al.27 Fafi-Kremer et al.17 Wadowsky et al.16 Wagner et al.23 Wagner et al.24 Stevens et al.25 Row et al.29
2013 2011 2011 2008 2008 2008 2008 2007 2007 2007 2004 2003 2002 2001 2001 2000
No consensus No consensus PBMC, P P WB, P P WB, S No consensus WB WB, P WB WB, P P WB, P WB PBMC, WB, P WB PBMC, WB, P WB and P are equivalent WB, P WB PBMC, P P PBMC, P P WB, S WB WB
Review article Review article 17 kidney Tx; children 35 SOT or SCT and 31 controls; adults 222 SOT and SCT; children and adults Review article 25 heart–lung Tx; children and adults 39 SCT 27 SCT and 19 liver Tx; children and adults 122 SCT; children 100 patients, including 8 Tx patients 44 SOT; children 46 heart Tx and 21 controls; adolescents and adults 96 kidney Tx and 20 controls; children and adolescents 46 patients, including 4 lung Tx Review article
Note: WB: whole blood; S: serum, P: plasma; SOT: solid organ transplant; SCT: stem cell transplant.
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blood or serum/plasma is easier to perform, less timeconsuming, and less expensive.9,17,32,35 Therefore, researchers started to focus on other sample types. Quickly, whole blood was accepted as an alternative for PBMCs as several studies demonstrated a good correlation between EBV DNA levels in both sample types.16,17,27,30 Later on, other sample types (serum and plasma) were also included in the studies. We found four review articles on this subject. Rowe et al. and Kimura et al., the two earliest review articles, recommend whole blood as the specimen type of choice.8,29 This opinion is supported by several individual articles which compared the use of whole blood with serum/plasma. Stevens et al., Wadowsky et al., Hakim et al., Wada et al., and Bakker et al., all conclude that the use of serum/ plasma results in a loss of sensitivity when compared to whole blood.16,19,25–27,35 Stevens et al. even failed to detect significantly elevated EBV DNA loads in serum of transplant recipients, despite highly elevated EBV DNA loads in simultaneously obtained whole blood specimens.25 The peripheral blood cell-associated viral load in patients with PTLD is 10- to 1000fold greater than the cell-free viral load. As a result, EBV DNA becomes detectable in whole blood earlier than in plasma. It also remains detectable for longer periods after initiation of antiviral therapies.29 Moreover, whole blood also best reflects the absolute EBV burden in the circulation as it contains all compartments that may harbor EBV.16,25 These considerations make whole blood more suitable for the monitoring of EBV infection in the immunocompromised host. The use of serum/plasma can lead to an underestimation of the total EBV viral load as the degree of lysis may vary depending on the EBVspecific cytotoxic T-cell response, the action of antiCD20 monoclonal antibodies administered for treatment of PTLD, and the manipulation and storage of the sample.16 In contrast, Gequelin et al. and Ruf et al., the two most recent review articles, and Fafi-Kremer et al. and Kullberg-Lindh et al., two individual articles, conclude that there is still no consensus on the optimal specimen type to monitor EBV DNA in transplant patients.9,13,17,28 Both whole blood and plasma can be valuable, but neither is optimal. As mentioned earlier, whole blood contains higher levels of EBV DNA. This entails several advantages: a higher sensitivity leading to a more accurate exclusion of PTLD, a better reproducibility of the results, and a facilitation of the monitoring of therapy and preventive measures. Serum has the advantage of being less sensitive to changes in lymphocyte count, avoiding the need for normalization to a human gene.28,34 Moreover, the absence of EBV DNA in serum/plasma in normal circumstances makes it more
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specific in comparison with whole blood. For this reason, the quantification of EBV viral load in serum/ plasma may be better to differentiate between patients with an asymptomatic chronic high viral load and patients at risk for PTLD, and to identify evolutions of EBV viral load in high load carriers.9,28 Finally, there are five individual articles which recommend the use of serum/plasma.20–24 Three of these studies compared plasma tot PBMCs, two of them compared plasma to whole blood. The most important reason to choose serum/plasma over PBMCs or whole blood is its superior specificity (while maintaining sensitivity).15,18,20,23,31,32 During treatment and remission of PTLD, EBV DNA in PBMCs or whole blood clears less effectively compared with plasma. EBV DNA levels often remain positive during treatment, regardless of the clinical response, making it an inferior approach for follow-up of patients with PTLD.20,24 Moreover, whole blood cannot always discriminate between patients with and without symptoms.21 In contrast, according to Ishihara et al. and Wagner et al., the detection of cell-free EBV DNA in plasma is highly specific (100%) and sensitive (100%) for diagnosis of EBV-positive PTLD using an assay-specific cutoff.22,24 An additional advantage of serum/plasma is that testing can be carried out on stored samples and is probably more easily standardized which facilitates comparison between different laboratories.15 Preiksaitis et al. demonstrated significant interlaboratory variability in the reported EBV DNA viral load on even identical specimens. They hypothesize that this variability could impact the quality of programs associated with PTLD prevention, diagnosis and monitoring.33 These contradictory findings may partially be explained by differences in sensitivity between EBV PCR techniques used in the various studies.17 As whole blood contains all types of EBV DNA, EBV DNA is present at high copy numbers and is therefore detected with both the sensitive and less sensitive PCR techniques. That can be reflected in the fact that earliest studies on this matter, containing probably less sophisticated molecular techniques, put forward whole blood as a preferential specimen type. In contrast, only the most sensitive PCR techniques detect EBV DNA in plasma as this sample type only contains cell-free EBV DNA. In conclusion, today the sensitivity of the EBV viral load measurement in an individual laboratory seems to be contributing to the choice of the specimen type in the context of PTLD. Ideally, one should take into account the clinical information (e.g. underlying condition, differential diagnosis) when selecting the specimen type for EBV viral load measurement. In order to select the optimal
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specimen for certain pathological conditions, ongoing interdisciplinary dialogue between clinician, pathologist, and laboratory specialist is essential for the optimal care of the patient. In addition, laboratories should regularly review their guidelines and if necessary adapt them (after careful validation) to current best practice according to their specific patients’ population.
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Disclaimer Statements Contributors None. Funding None. Conflicts of interest None declared. Ethics approval Not required.
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Epstein–Barr virus serology and PCR: conflicting results in an immunocompetent host. A case report and review of literature L. Cattoir1, V. Van Hende2, P. De Paepe3, E. Padalko1,4 1
Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium, Department of Hematology, Ghent University Hospital, Ghent, Belgium, 3Department of Pathology, AZ Sint-Jan Bruges-Ostend AV, Bruges, Belgium, 4School of Life Sciences, Hasselt University, Diepenbeek, Belgium
2
We present the case of a 27-year-old immunocompetent man who progressively developed a generalized lymphadenopathy and B symptoms. Results of Epstein–Barr virus (EBV) serology were suggestive for a past infection, but the EBV viral load in whole blood was high. Also, core needle biopsy of the largest lymph node showed an image which could fit an EBV-driven reactive lymphoproliferation. Despite the absence of an immune disorder, all medical evidence points to an EBV-driven lymphoproliferative proces. In immunocompetent patients, it seems extremely uncommon to detect a high EBV viral load in the absence of serological evidence of an acute EBV infection or reactivation. We reviewed literature on this topic and on the selection of the appropriate sample type for EBV PCR, as this is known to be a critical point. Serological testing for the diagnosis of EBV infection is the gold standard in immunocompetent patients. Measuring EBV viral load is only recommended when dealing with immunocompromised patients. Although extremely rare, this case report shows that there is a place for EBV PCR in certain situations in immunocompetent patients. Besides, there is still no consensus regarding the specimen of choice for the determination of the EBV viral load. The preferred specimen type seems to depend on the patient’s underlying condition. Keywords: Epstein–Barr virus, Serology, PCR, Immunocompetent, Specimen type
Introduction Epstein–Barr virus (EBV) is a double stranded DNA virus of the herpesvirus family. EBV is widely present, more than 90% of the adults are EBVseropositive. In children, primary EBV infection is often asymptomatic, but adolescents and adults frequently present with the classical symptoms of acute infectious mononucleosis. Primary infection is usually self-limited, but the virus remains in a latent stage in a small fraction of the memory B lymphocytes throughout life, making viral reactivation possible. EBV is also associated with several malignant disorders, such as Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin, and non-Hodgkin lymphomas. Both reactivation and EBV-associated lymphoproliferative disorders (for example, posttransplant lymphoproliferative disorder, PTLD) most commonly occur in immunocompromised patients. In these patients, EBV infection can cause serious disease and is associated with high rates of morbidity and mortality. Correspondence to: E. Padalko, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185 (2P8), B-9000 Ghent, Belgium. E-mail: [email protected]
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ß Acta Clinica Belgica 2014 DOI 10.1179/2295333714Y.0000000037
We report a rare case of a young, immunocompetent man who was diagnosed with an EBV-lymproproliferative disorder, despite non-suggestive EBV serology. EBV viral load measured in whole blood using a quantitative real-time PCR was high, and lymph node biopsy fitted an EBV-driven reactive lymphoproliferation. This article focuses on the diagnosis of EBV-related disorders in immunocompetent patients and on the preferred specimen type for EBV viral load measurement.
Results Clinical and laboratory characteristics A 27-year-old man with a blank medical record consulted his general practitioner due to a swelling in his right axilla. Aside from this axillary lymph node, the patient also complained of malaise, fatigue, fever, and night sweats. Lymph node fine needle aspiration cytology showed a reactive presentation. The patient was treated with antibiotics for 5 days, however, without improvement of his complaints. During the next 2 months, the symptoms worsened and the patient developed significant weight loss and generalized lymphadenopathy. At that time, a haematologist was consulted. Laboratory analysis of peripheral
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blood showed a high erythrocyte sedimentation rate (.120 mm, normal 1–15 mm) and C-reactive protein (126.5 mg/dl, normal ,5 mg/dl). The red blood cell count was at low normal range 4.16106/mm3 (normal 4.1–5.76106/mm3) with a decreased haemoglobin level of 10.3 g/dl (normal 13.7–17.5 g/dl) and mean corpuscular volume of 78 fl (normal 80–100 fl). The white blood cell count (5.56103/mm3, normal 4.2–9.16103/mm3, with a normal differentiation) and platelet count (3176103/mm3, 140–4406103/mm3) were both within the normal range. Serum creatinine concentration, urea, uric acid, electrolytes, glucose, total protein, lactate dehydrogenase, and transaminases were within reference limits. The patient had increased blood levels of alkaline phosphatase (203 U/l, normal 40–130 U/l) and gamma-GT (106 U/l, normal ,60 U/l). Assessment of the iron status showed a decreased serum iron (26 mg/dl, normal 33–193 mg/dl) and transferrin (146 mg/dl, normal 200–360 mg/dl), and a highly increased ferritin concentration (2148 mg/l, normal 30–400 mg/l). Coagulation tests were clearly disturbed. Prothrombin time was decreased significantly to 44% (normal 70–100%), but with a normal activated partial thromboplastin time. Both fibrinogen and Ddimer were elevated to 759 mg/dl (normal 180– 400 mg/dl) and 1888 ng/ml (normal ,500 ng/ml), respectively. Results of cytomegalovirus and EBV serology were suggestive for a past infection. EBV serology was determined using EnzygnostH anti-EBV/ IgMII and EnzygnostH anti-EBV/IgG (Siemens Healthcare Diagnostics Products, Marburg, Germany) with negative IgM (0.26 ratio, positive if ratio .1)) and positive IgG (236 U/ml, positive if .30 U/ml) values. EnzygnostH anti-EBV/IgMII and EnzygnostH anti-EBV/IgG are ELISAs where a microtitration plate is coated with deactivated EBVantigens derived from EBV-infected lymphoblastoid cells containing viral capsid antigens (VCA), EBV
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nuclear antigens (EBNAs), and early antigens (EAs). Serological testing for the presence of antibodies to Bartonella henselae and mycobacterial culture was negative. Based on clinical presentation and laboratory results, the presence of a lymphoma were suspected.
Cytological characteristics A core needle biopsy of the largest axillary lymph node was performed. The biopsy showed extensive geographical necrosis surrounded by a blastic B-cell population (PAX5z/CD30z/partially CD15z). As the image resembled an EBV-driven lymphoproliferative disorder, an EBV staining was performed which confirmed the presence of a blastic EBVpositive B-cell proliferation (Fig. 1). The pathologist concluded that the image could fit either an EBVdriven lymphoproliferation or a classical Hodgkin’s lymphoma. Despite contradictory serology, when dealing with an immunocompromised patient, an EBV-mediated lymphoproliferative disorder would be preferred. A Hodgkin lymphoma also remains a possibility, but the extensive geographical necrosis and pronounced clinical signs present in this patient were not characteristic for this medical condition.
Additional serological and molecular diagnostics and specimen type Based on the pathology report, the haematologist requested more profound serological investigation and an EBV PCR. In order to determine specific antibody responses to individual EBV antigens, an immunoblotting test (recomLine EBV IgG; Mikrogen, Neuried, Germany) was performed. Nevertheless immunoblotting test is a qualitative method, the anti-EBV-VCA IgG antibody signal was strongly positive as demonstrated by the high intensity of specific antigen bands, whereas signals for anti-EBV-EA IgG and anti-EBV-EBNA IgG antibodies were weakly positive.
Figure 1 Immunohistochemistry of the lymph node. (A) CD30 staining (640). The large blasts are CD30-positive. (B) EBER/ EBV ISH staining (640). The large atypical cells contain EBV DNA.
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The EBV viral load in whole blood was high, 4.36104 copies/mg DNA, as measured using an inhouse quantitative real-time EBV PCR.1 Human immunodeficiency virus infection, common variable immunodeficiency, and hypogammaglobulinemia were excluded. B and T cellular maturation and natural killer cell levels were normal. Altogether, despite the absence of an immune deficiency, all objective medical evidence points to an EBV-driven lymphoproliferative disorder. In our hospital, EDTA whole blood is used as standard specimen for EBV PCR as EBV viral load is mainly employed to monitor transplant recipients at high risk of PTLD or under therapy for PTLD. Moreover, whole blood seems appropriate as it contains all types of EBV DNA (cell-free and cellassociated). However, periodical review of literature emerges in order to optimize current working practices based on latest research results and new developments.
Review of Literature and Discussion Serological testing for the diagnosis of EBV infection is the gold standard in immunocompetent patients.2 Measuring EBV viral load is only recommended when dealing with immunocompromised patients as these patients may have defective antibody production or receive therapeutic immunoglobulin preparations, which both could affect serological test results.2–6 EBV viral load is a laboratory marker mainly used in transplant recipients to predict, diagnose and follow-up treatment of EBV-driven PTLD.2,6–28 Monitoring EBV viral load has led to a decrease in the incidence of PTLD.7 This case report shows that the above described approach is questionable in certain clinical situations. There are at least a number of patients who are, or appear to be, immunocompetent where EBV PCR
may have an additional diagnostic value. Complete exclusion of the involvement of an EBV infection solely based on serological results should be discouraged. For each patient individually, the physician should take into account all available information and decide whether additional laboratory investigation, including an EBV PCR, may be needed. In determining the EBV viral load in a specific patient, selecting the appropriate sample type is critical. Despite a lack of consensus regarding this issue, the preferred specimen type seems to depend on the patient’s underlying condition.6–19 In healthy carriers, EBV DNA is located in the intracellular compartment of the blood as EBV persists in memory B cells after primary infection. Therefore, to detect EBV DNA by PCR, one should use whole blood samples.6,8–12 In contrast, there are certain medical conditions (Hodgkin lymphoma, EBV-associated haemophagocytic syndrome, chronic active EBV infection, nasopharyngeal carcinoma) in which EBV viral load in serum/plasma is preferred over whole blood. For example, in Hodgkin lymphoma, only serum/plasma viral load is associated with diagnosis and response to therapy.6,8,9,13 In this setting, EBV viral load in serum/plasma is not routinely measured, but according to literature, it can serve as a surrogate marker for disease activity, therapeutic response, and prognosis.6,8,9,13 In patients with EBV-driven PTLD, both intracellular and cell-free EBV DNAs are increased.6–28 Authors remain to disagree on the optimal specimen type. Table 1 gives a summary of the studies which compared different specimen types in the context of PTLD. Initially, everyone used peripheral blood mononuclear cells (PBMCs) as most EBV is cellassociated and EBV DNA is detected at high copy numbers in PBMCs.8 However, compared to PBMCs, the extraction of EBV DNA from whole
Table 1 Summary of literature concerning the specimen of choice for EBV PCR in the context of post-transplant lymphoproliferative disorder Author
Year Patient population
Sample type
Ruf and Wagner9 Gequelin et al.13 Shihara et al.22 Tsai et al.20 Kullberg-Lindh et al.28 Kimura et al.8 Bakker et al.26 Kinch et al.21 Wada K et al.19 Hakim H et al.27 Fafi-Kremer et al.17 Wadowsky et al.16 Wagner et al.23 Wagner et al.24 Stevens et al.25 Row et al.29
2013 2011 2011 2008 2008 2008 2008 2007 2007 2007 2004 2003 2002 2001 2001 2000
No consensus No consensus PBMC, P P WB, P P WB, S No consensus WB WB, P WB WB, P P WB, P WB PBMC, WB, P WB PBMC, WB, P WB and P are equivalent WB, P WB PBMC, P P PBMC, P P WB, S WB WB
Review article Review article 17 kidney Tx; children 35 SOT or SCT and 31 controls; adults 222 SOT and SCT; children and adults Review article 25 heart–lung Tx; children and adults 39 SCT 27 SCT and 19 liver Tx; children and adults 122 SCT; children 100 patients, including 8 Tx patients 44 SOT; children 46 heart Tx and 21 controls; adolescents and adults 96 kidney Tx and 20 controls; children and adolescents 46 patients, including 4 lung Tx Review article
Note: WB: whole blood; S: serum, P: plasma; SOT: solid organ transplant; SCT: stem cell transplant.
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blood or serum/plasma is easier to perform, less timeconsuming, and less expensive.9,17,32,35 Therefore, researchers started to focus on other sample types. Quickly, whole blood was accepted as an alternative for PBMCs as several studies demonstrated a good correlation between EBV DNA levels in both sample types.16,17,27,30 Later on, other sample types (serum and plasma) were also included in the studies. We found four review articles on this subject. Rowe et al. and Kimura et al., the two earliest review articles, recommend whole blood as the specimen type of choice.8,29 This opinion is supported by several individual articles which compared the use of whole blood with serum/plasma. Stevens et al., Wadowsky et al., Hakim et al., Wada et al., and Bakker et al., all conclude that the use of serum/ plasma results in a loss of sensitivity when compared to whole blood.16,19,25–27,35 Stevens et al. even failed to detect significantly elevated EBV DNA loads in serum of transplant recipients, despite highly elevated EBV DNA loads in simultaneously obtained whole blood specimens.25 The peripheral blood cell-associated viral load in patients with PTLD is 10- to 1000fold greater than the cell-free viral load. As a result, EBV DNA becomes detectable in whole blood earlier than in plasma. It also remains detectable for longer periods after initiation of antiviral therapies.29 Moreover, whole blood also best reflects the absolute EBV burden in the circulation as it contains all compartments that may harbor EBV.16,25 These considerations make whole blood more suitable for the monitoring of EBV infection in the immunocompromised host. The use of serum/plasma can lead to an underestimation of the total EBV viral load as the degree of lysis may vary depending on the EBVspecific cytotoxic T-cell response, the action of antiCD20 monoclonal antibodies administered for treatment of PTLD, and the manipulation and storage of the sample.16 In contrast, Gequelin et al. and Ruf et al., the two most recent review articles, and Fafi-Kremer et al. and Kullberg-Lindh et al., two individual articles, conclude that there is still no consensus on the optimal specimen type to monitor EBV DNA in transplant patients.9,13,17,28 Both whole blood and plasma can be valuable, but neither is optimal. As mentioned earlier, whole blood contains higher levels of EBV DNA. This entails several advantages: a higher sensitivity leading to a more accurate exclusion of PTLD, a better reproducibility of the results, and a facilitation of the monitoring of therapy and preventive measures. Serum has the advantage of being less sensitive to changes in lymphocyte count, avoiding the need for normalization to a human gene.28,34 Moreover, the absence of EBV DNA in serum/plasma in normal circumstances makes it more
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specific in comparison with whole blood. For this reason, the quantification of EBV viral load in serum/ plasma may be better to differentiate between patients with an asymptomatic chronic high viral load and patients at risk for PTLD, and to identify evolutions of EBV viral load in high load carriers.9,28 Finally, there are five individual articles which recommend the use of serum/plasma.20–24 Three of these studies compared plasma tot PBMCs, two of them compared plasma to whole blood. The most important reason to choose serum/plasma over PBMCs or whole blood is its superior specificity (while maintaining sensitivity).15,18,20,23,31,32 During treatment and remission of PTLD, EBV DNA in PBMCs or whole blood clears less effectively compared with plasma. EBV DNA levels often remain positive during treatment, regardless of the clinical response, making it an inferior approach for follow-up of patients with PTLD.20,24 Moreover, whole blood cannot always discriminate between patients with and without symptoms.21 In contrast, according to Ishihara et al. and Wagner et al., the detection of cell-free EBV DNA in plasma is highly specific (100%) and sensitive (100%) for diagnosis of EBV-positive PTLD using an assay-specific cutoff.22,24 An additional advantage of serum/plasma is that testing can be carried out on stored samples and is probably more easily standardized which facilitates comparison between different laboratories.15 Preiksaitis et al. demonstrated significant interlaboratory variability in the reported EBV DNA viral load on even identical specimens. They hypothesize that this variability could impact the quality of programs associated with PTLD prevention, diagnosis and monitoring.33 These contradictory findings may partially be explained by differences in sensitivity between EBV PCR techniques used in the various studies.17 As whole blood contains all types of EBV DNA, EBV DNA is present at high copy numbers and is therefore detected with both the sensitive and less sensitive PCR techniques. That can be reflected in the fact that earliest studies on this matter, containing probably less sophisticated molecular techniques, put forward whole blood as a preferential specimen type. In contrast, only the most sensitive PCR techniques detect EBV DNA in plasma as this sample type only contains cell-free EBV DNA. In conclusion, today the sensitivity of the EBV viral load measurement in an individual laboratory seems to be contributing to the choice of the specimen type in the context of PTLD. Ideally, one should take into account the clinical information (e.g. underlying condition, differential diagnosis) when selecting the specimen type for EBV viral load measurement. In order to select the optimal
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specimen for certain pathological conditions, ongoing interdisciplinary dialogue between clinician, pathologist, and laboratory specialist is essential for the optimal care of the patient. In addition, laboratories should regularly review their guidelines and if necessary adapt them (after careful validation) to current best practice according to their specific patients’ population.
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Disclaimer Statements Contributors None. Funding None. Conflicts of interest None declared. Ethics approval Not required.
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