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Circulating Epstein–Barr virus (EBV) in HIV-infected patients and its relation with primary brain lymphoma
International Journal of Infectious Diseases (2007) 11, 172—178
http://intl.elsevierhealth.com/journals/ijid
Circulating Epstein—Barr virus (EBV) in HIV-infected patients and its relation with primary brain lymphoma Marı´a Dolores Fellner a,*, Karina Durand a, Rita Mariel Correa a, Liliana Redini b, Claudio Yampolsky c,d, Antonio Colobraro e,f, ´lica R. Teyssie ´ a, Jorge Benetucci b, Gustavo Sevlever e,f, Ange Marı´a Alejandra Picconi a a
´nicos, Instituto Nacional de Enfermedades Infecciosas (INEI)- ANLIS ‘‘Dr. Carlos G. Malbra ´n’’, Servicio Virus Oncoge ´rsfield 563, C 1282AFF Buenos Aires, Argentina Av. Velez Sa b Laboratorio de Retrovirus y Virus asociados, FUNDAI, Buenos Aires, Argentina c Servicio de Neurocirugı´a, Sanatorio Guemes, Buenos Aires, Argentina d ˜iz’’, Buenos Aires, Argentina Hospital de Enfermedades Infecciosas ‘‘Dr. F. Mun e Servicio de Patologı´a, Instituto FLENI, Buenos Aires, Argentina f Servicio de Patologı´a, Clı´nica Bazterrica, Buenos Aires, Argentina Received 24 November 2005; received in revised form 4 April 2006; accepted 6 April 2006 Corresponding Editor: Jane Zuckerman, London, UK
KEYWORDS EBV load; Blood compartments; HIV-infected population; Brain lymphoma
Summary Objective: To analyze Epstein—Barr virus (EBV) load at different HIV infection stages and its relation with brain lymphoma. Design: A cross-sectional study was conducted on 172 HIV-infected individuals: 62 asymptomatic HIV carriers (group A), 30 HIV progressors (group B), 73 AIDS patients (group C), seven AIDS patients with brain lymphoma (group C-BL); and 26 blood donors (group BD) as healthy carriers. EBV load was measured in peripheral blood mononuclear cells (PBMC) and plasma samples using a semi-quantitative PCR method. Results: PBMC-EBV levels in HIV-infected patients were higher than in the blood donors ( p < 0.05). No differences in PBMC-EBV loads were found in groups A, B, or C ( p > 0.05), while the C-BL group had significantly lower levels ( p < 0.05). Similar PBMC-EBV loads were seen in HIVinfected patients with CD4+ T cell counts higher than 50/mm3 ( p > 0.05), while significantly lower levels were found in cases with less than 50 cells/mm3 ( p < 0.05). In all HIV-infected patients, plasma-EBV load was lower than, or similar to, PBMC-EBV load, unlike 2/7 HIV-positive brain lymphoma patients. Conclusions: During HIV infection PBMC-EBV load rises in comparison to healthy carriers, but decreases when immunosuppression progresses and CD4+ T cell count becomes <50/mm3.
* Corresponding author. Tel.: +55 11 4301 7428/4302 5064; fax: +55 11 4302 5064/4303 2210. E-mail address: [email protected] (M.D. Fellner). 1201-9712/$32.00 # 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2006.04.001
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EBV in HIV-infected patients and primary brain lymphoma
Circulating EBV is mainly cell-associated in the HIV-infected population. Neither PBMC-EBV nor plasma-EBV loads would be useful to diagnose brain lymphoma in AIDS patients. # 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Introduction Epstein—Barr virus (EBV) is a widely spread human gammaherpesvirus, whose prevalence exceeds 95% of the adult human population.1 After the primary infection, as EBV establishes persistence in B lymphocytes, it can be detected both in lymphoid tissue and peripheral blood.2,3 In immunosuppressed patients, the virus—host imbalance favors EBV reactivation which in turn induces lymphoproliferation.4 Therefore, it has been proposed that these benign proliferations may become malignant lymphomas if they are not well controlled by the host immune system, although this is a rare event.5 EBV has been associated with a variety of malignancies due to its detection in malignant cells.6—9 The EBV-associated tumors described in the HIV-infected population include non-Hodgkin lymphomas, Hodgkin lymphomas, Burkitt lymphomas, and smooth muscle malignancies. The strength of association differs, being about 100% in cases of primary brain lymphomas, which appear when patients are profoundly immunosuppressed; in this sense, they are essentially analogous to post-transplant lymphoproliferative disorder (PTLD) lesions of the transplant group.4,10 In the transplant population a relationship was found between circulating EBV load and PTLD.11,12 Moreover, higher circulating EBV DNA levels precede PTLD diagnosis, which allows for early therapeutic intervention in patients with rising levels of EBV, thus preventing the establishment of this life-threatening entity; also other reports noted decreased EBV load with successful therapy.13—15 The EBV load has been much less studied in the HIVinfected group than in transplant individuals. As in transplant patients, frequent reactivation of EBV infection has been observed in HIV-infected individuals, reflected by increased EBV-specific antibody titers,16—18 and more recently, high numbers of circulating copies of EBV.19,20 However, controversy persists regarding the usefulness of measuring EBV levels in AIDS patients in relation to lymphoma risk, diagnosis, and management.21—25 The semi-quantitative PCR (SQ-PCR) method used to measure EBV load was developed in our laboratory to monitor transplant patients and in fact, its usefulness was demonstrated for the detection of differences in circulating levels of EBV DNA. Although different strategies such as competitive methods or real time PCR have proven to yield the best results, the SQ-PCR method is, for us, more affordable.26 The aim of our study was to analyze the EBV load in different HIV infection stages and its relation with primary brain lymphoma.
Materials and methods Study population One hundred and seventy-two HIV-infected patients from the ‘‘J.F. Mun ˜iz’’ Infectious Diseases Hospital (Buenos Aires,
Argentina) were included in this study. All of them were receiving antiretroviral therapy, with the exception of asymptomatic HIV-infected patients with CD4+ T cells counts above 350/mm3. Mean patient age was 26 years (range 16—61 years), male/female ratio was 2:3, and they were grouped according to the 1993 CDC classification,27 as follows: 62 asymptomatic HIV-infected patients (group A), 30 HIV progressors (group B), 73 AIDS patients (group C), and seven AIDS patients with a diagnosis of primary central nervous system lymphoma (PCNSL) (group C-BL). They were all typed as nonHodgkin lymphoma according to the WHO classification. Twenty-six blood donors (group BD), who were healthy EBV carriers from the same hospital, were also included. They were all negative for the usual blood bank tested infections; mean age of this group was 34 years (range 19—50 years). All the study population showed previous evidence of EBV infection (presence of IgG antibodies against viral capsid antigen (VCA) detected by indirect immune fluorescence assay). All participants gave their informed consent for inclusion in this study. A local ethics committee approved all procedures.
Samples EDTA anti-coagulated blood samples were collected and peripheral blood mononuclear cells (PBMC) separated from plasma after a Ficoll-Hypaque gradient centrifugation (Lymphoprep, GIBCO). Fresh brain biopsy tissue (BB) and in some cases cerebrospinal fluid (CSF) were obtained from C-BL patients. All samples were stored at 20 8C until used. Paraffin embedded lymphoma tissue was examined with in situ hybridization (ISH).
DNA extraction DNA from PBMC, plasma, and BB was extracted with proteinase K lysis buffer and purified by a phenol—chloroform procedure.28 DNA from CSF was extracted as previously described.29
EBV quantification Viral load was determined applying a semi-quantitative method as we have previously described.26 This method was developed to detect high EBV loads, i.e., levels above those found in healthy carriers (latent state). Briefly, a nested PCR was performed, which amplifies a fragment of 122 bp from the BWRF1 gene. For PBMC, DNA equivalent to 105 cells (366 ng) and its serial four-fold dilutions were amplified until limiting dilution was reached. The amplified products were run in a 2% agarose gel stained with ethidium bromide and the end point was converted into viral copies/105 PBMC by multiplying the sensitivity of the assay (1—16 copies) by the reciprocal of the limiting dilution (RLD). We subsequently applied
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Figure 1 Detection of PCR inhibitors. No inhibition was detected in DNA extracted from PBMC samples, while two DNA samples extracted from plasma exhibited inhibitors. Both samples were re-extracted, and when re-tested one showed no inhibitors so it was quantified, while the other was discarded since inhibitors could not be eliminated. (A) Detection of PCR inhibitors in DNA extracted from PBMC samples. Lanes 1—5: DNAPBMC from HIV positive patients. Lane 6: negative control. Lane 7: positive control. Lane 8: 100 bp ladder. (B) Detection of PCR inhibitors in DNA extracted from plasma samples. Lane 1: 100 bp ladder. Lanes 2—6: DNAplasma from HIV positive patients. Notice the presence of inhibitors in the plasma specimen run in lane 2. Lane 7: negative control. Lane 8: positive control.
the method to plasma samples; the intra- and inter-assay variation result was the same as for PBMC specimens, i.e., one four-fold dilution (data not shown). Thus, we considered the same sensitivity and reproducibility as was described for PBMC samples. For plasma specimens, DNA obtained from 30 mL (similar to the volume associated with 105 PBMC)14 and its serial four-fold dilutions were amplified and quantified as mentioned. The presence of PCR inhibitors was determined when EBV load was <1 copy/105 PBMC or 30 mL of plasma as follows: (A) Inhibitors in DNA extracted from PBMC samples were determined by amplifying the human b-globin gene using primer set PCO4 and GH20 to amplify a 268 bp DNA fragment as described by Bauer et al.30 (B) Inhibitors in DNA extracted from plasma samples were tested by spiking around 125 ng of DNA obtained from human fibroblasts onto DNA extracted from 30 mL of plasma, and then the human b-globin gene was amplified as described above (Figure 1).
EBV qualitative detection For EBV DNA qualitative determination, DNA extracted from 50 ul of CSF or about 300 ng of DNABB were amplified by the same nested PCR applied for quantification, using different cycling conditions in the first round: 3 min at 94 8C, and forty 1-min cycles at 94 8C, 30 s at 58 8C, 1 min at 72 8C; and 10 min at 72 8C.
EBERs detection To determine EBV association with PCNSL, a commercial in situ hybridization (ISH) method that detects EBV-encoded RNA (EBERs) transcripts was applied on paraffin-embedded lymphoma tissue sections mounted onto silane-coated glass slides. Peptide nucleic acid (PNA) fluorescein isothiocyanate (FITC)-labeled probes: EBER-specific, positive and negative
Figure 2 EBV detection and PBMC viral load in the study population. The limiting dilution value obtained for each patient was converted into viral copies/105 PBMC by multiplying the sensitivity of the assay (1—16 copies) by the reciprocal of the limiting dilution (RLD); e.g. if RLD = 4, after multiplying by 1 and by 16, the corresponding viral load ranges between 4 and 64. ND, not detected; dotted line represents the detection level with the SQ-PCR. Bars show the median RLD value with the confidence interval range (%) for each group.
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EBV in HIV-infected patients and primary brain lymphoma
Figure 3 EBV detection and load levels in HIV infected patients according to CD4+ T cell counts. The total number of patients included in this analysis is lower than that described in Figure 2 since CD4+ T cell counts were not available in 10 patients. ND, not detected; dotted line represents the detection level with the SQ-PCR. Bars show the median RLD value with the confidence interval range (%) for each group.
RNA controls (Dakocytomation, Glostrup, Denmark), followed by its detection with a PNA ISH detection kit (Dakocytomation, Glostrup, Denmark), were used according to manufacturers’ instructions.
Number of CD4+ and CD8+ lymphocytes These were determined by standard flow cytometry techniques.
EBV load and CD4+ T cells EBV detection was similar in all groups with CD4+ Tcell counts higher than 50/mm3 ( p > 0.05), and significantly higher than in patients with less than 50 CD4+ T cells/mm3 ( p < 0.05). When analyzing EBV levels, we found no statistically significant differences between groups with more than 50 CD4+ T cells/mm3. On the other hand, circulating EBV levels in patients with less than 50 CD4+ T cells/mm3 were significantly ( p < 0.05) lower than in those with CD4+ Tcells counts
Statistical analyses Categorical data were compared using Fisher’s exact test or the Chi-square test, while the non-parametric Mann—Whitney U test for unpaired data was used to analyze ordinal values. For the purpose of the analysis, not-detected (ND) RLD values were considered 0. The PEPI Program Finder version 4.0 was used for the analyses. Linear regression analysis was performed after RLD was converted into a natural logarithm (Ln) with the Analyse-it software for Excel 1997—2000.
Results EBV load and clinical HIV state HIV-infected individuals were found to exhibit a significantly higher detection rate and increased levels of circulating EBV DNA compared to blood donors ( p < 0.05). Similar results were obtained when comparing each HIV clinical set without lymphoma to the BD group (Figure 2). Also, no statistically significant differences ( p > 0.05) in EBV detection and EBV load were found between groups A, B, and C. The C-BL group was found to have a significantly ( p < 0.05) lower circulating EBV rate and levels compared to groups A and B.
Figure 4 Correlation between plasma-EBV and PBMC-EBV loads. A linear regression analysis of the natural logarithm of the reciprocal of the limiting dilution (RLD) values obtained from paired PBMC and plasma samples of 62 patients is represented (r = 0.173; p = 0.219).
176 Table 1
M.D. Fellner et al. Description of HIV-infected patients with primary central nervous system lymphoma
Patient
Histopathologic diagnosis
PCR
EBER ISH
PBMC-EBV load (copies/105 PBMC)
Plasma EBV load (copies/30 mL)
1 2 3 4 5 6 7
DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL
+ + + + + + + +
+ + + + + + NS
<1 <1 <1 1—16 <1 <1 <1
64—1024 <1 16—256 <1 <1 <1 <1
(BB) (BB) (BB) (BB) (BB) (BB) (BB) (CSF)
DLBCL, diffuse large B cell lymphoma; BB, brain biopsy; NS, no sample available.
between 50 and 500/mm3. Figure 3 illustrates the results of EBV quantification as regards the number of CD4+ T lymphocytes.
EBV load and CD8+ T cells No correlation was found between EBV circulating levels and the number of CD8+ T cells in the HIV-infected population studied (data not shown).
Relation between PBMC- and plasma-EBV load Plasma-EBV load was measured in about 20 patients of each HIV clinical set: A, B, and C (n = 55), and in all seven C-BL cases. The criterion for plasma selection was that all PBMCEBV load ranks were represented. Therefore, when analyzing EBV DNA in different peripheral blood compartments of all HIV groups, we observed no correlation between PBMC and plasma levels (Figure 4). Also, EBV load was lower in plasma samples than in PBMC for most HIV positive cases without lymphoma; only two patients (from groups A and C) had similar circulating EBV DNA copies in both samples. Two of the seven HIV-infected patients with PCNSL had higher EBV levels in plasma than in PBMC (patients 1 and 3, Table 1).
Discussion In this project, we noted that in HIV-infected patients EBV in peripheral blood is higher than in healthy individuals. Thus, higher EBV DNA detection rates were found in PBMC of HIVinfected patients compared to healthy individuals, in agreement with the report by Ling et al.20 Contrary to this finding, Dehee et al. described a similar EBV positivity rate in HIVinfected patients and asymptomatic HIV negative volunteers.19 In the latter case, the percentage of EBV DNA detection in HIV negative individuals was strikingly high compared to other reports.20,21,29,31 These differences could be due to the control groups, the methodological strategies, or the sensitivity of the techniques. Also, increased PBMCEBV load was found in all the clinical HIV infection categories (groups A, B, and C), indicating that the imbalance between EBV infection and the host immunologic control starts early in the course of HIV clinical infection, and continues as the infection progresses. In a prospective study, Piriou et al.
proved that PBMC-EBV load increases rapidly following HIV seroconversion, and remains high even five years later, both in asymptomatic carriers and AIDS progressors.24 Since CD4+ T cell depletion is the immunologic hallmark of HIV-related disease progress,32,33 we also analyzed the relation of circulating EBV levels and the progress of immunological deterioration, according to CD4+ T cell counts by the CDC classification. In agreement with reports by Dehee et al.,19 and Ling et al.,20 when the number of CD4+ T cells is greater than 50/mm3, higher levels of circulating PBMC-EBV are seen in HIV-infected patients, compared to healthy carriers. This situation may reflect the loss of EBV infection control due to progressive immunosuppression, similar to the situation widely described for transplant patients subjected to severe immunosuppression.14,34—36 However, in HIV-infected patients with CD4+ T cell counts under 50/mm3 we observed lower levels of circulating PBMC-EBV, which counters the findings of Dehee et al. who found increased PBMC-EBV DNA levels even in this group (<50 LT CD4+/mm3).19 Our observations could be explained if we consider that copies of peripheral EBV have been detected only in memory B lymphocytes,37,38 cells generated after a germinal center reaction.39,40 Thus, the decline in the PBMC-EBV load could be due to the decrease in circulating memory B lymphocytes observed during HIV infection.41,42 This reduction may be due to hyperactivation-induced memory B cell exhaustion, increased apoptosis, on top of an abnormal naı¨ve B cell activation/ differentiation process.43 Therefore, we conclude that it would be interesting to analyze EBV level variation relative to the quantity of B lymphocytes at different stages of HIV infection. Like Dehee et al.,19 we found no correlation between total CD8+ Tcell count and PBMC-EBV load. It should be pointed out that van Baarle et al., studying HIV-infected patients, analyzed the EBV-specific CD8+ T cell population (which represents a small proportion of total CD8+ T lymphocytes), and observed a normal number, although with decreased functional capacity, compared to HIV negative individuals.44 Also, our study shows that in HIV-infected patients, the EBV found in peripheral blood is carried mainly in PBMC (cellassociated virus), in agreement with Stevens et al.,23 although our study analyzed a substantially larger number of patients. Moreover, we observed no correlation between PBMC-EBV and plasma-EBV loads, as reported by van Baarle et al.22 and Righetti et al.45 Further, the plasma-EBV levels of
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EBV in HIV-infected patients and primary brain lymphoma HIV-infected patients without lymphoma were lower or similar to those found in PBMC which could indicate that the EBVplasma load results from the lysis of latently-infected circulating B cells (necrosis or apoptosis), or that peripheral lytic infection (either the presence of circulating EBV-virions and/ or lysis of circulating EBV-activated B cells) is not the main cause of increased circulating EBV, or both. It would be interesting for future studies to establish whether plasmaEBV load corresponds to encapsulated virions or nude viral DNA. Different authors have described variable circulating EBV DNA levels when lymphoma is diagnosed in HIV-infected patients.21,31 Also, prospective studies by van Baarle et al. have shown that HIV-infected patients with non-Hodgkin lymphomas have EBV levels that fluctuate over time, being at the time of lymphoma diagnosis lower, similar to, or higher than those previously detected.22,44 Recently, Fan et al. described significantly higher plasma-EBV levels in HIVinfected patients with EBV-associated lymphomas compared to HIV-infected patients with non-EBV-associated lymphomas, or without lymphomas. However, plasma-EBV load varied in the samples collected before, during, and after lymphoma diagnosis, although EBV DNA was detectable at least in one sample. It was noted that in primary brain lymphoma cases, the EBV load was mainly not detectable at the time of diagnosis.25 In this study, the PBMC-EBV load in AIDS patients with PCNSL (group C-BL) was lower than that found in the initial stages of HIV infection (groups A and B), albeit similar to the levels found in AIDS patients without lymphoma (group C). Further, PBMC-EBV levels were undetectable or low in all patients with PCNSL diagnosis, seeming to indicate that EBV load determination would not be a useful indicator of lymphoma, even though only few cases were analyzed. Since PCNSL is a pathology found in HIV-infected patients with advanced immunological deterioration, our findings could be accounted for, as previously mentioned, by the decrease in memory B lymphocytes, described in the course of HIV infection. Moreover, although most of the lymphoma patients showed undetectable plasma-EBV load, it was higher, even more than PBMC-EBV levels, in two cases, suggesting increased lysis of latently-infected circulating B cells, and/or peripheral lytic infection, at least in some of these patients. In this sense, it would also be interesting to determine whether plasma-EBV load in HIV-infected patients with lymphoma corresponds to virions or to nude DNA. In conclusion, EBV found in peripheral blood of HIVinfected patients is mainly cell-associated. Also, during the course of HIV infection, when the CD4+ T cell count is above 50/mm3, the PBMC-EBV levels are increased, which would reflect the loss of EBV infection control due to progressive immunosuppression. When immunological deterioration progresses and the CD4+ T cell count is below 50/ mm3, the PBMC-EBV load decreases; perhaps because of altered B lymphocytes (memory and naı¨ve) distribution, and inappropriate activation/differentiation that have been described in the course of HIV infection. Also, the determination of the PBMC-EBV load in AIDS patients with PCNSL would not be a useful indicator of lymphoma, while the meaning of EBV DNA in these patients’ plasma remains to be clarified.
Acknowledgments The authors are grateful to Drs L. De Benedetti, M. Corti, C. Trione, C. Bonifacio, N. Zala, and S. Camino (Hospital Mun ˜iz) for their generous help during sampling, and to Drs J. Go ´mez and Sara Vladimirsky (Instituto Malbra ´n) for their epidemiological counseling. This project was partially supported by grants from the Mosoteguy and Bunge & Born Foundation (Buenos Aires, Argentina). Conflict of interest: No conflict of interest to declare.
References 1. Young LS, Rickinson AB. Epstein—Barr virus: 40 years on. Nat Rev Cancer 2004;4:757—68. 2. Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson DA. Identification of the site of Epstein—Barr virus persistence in vivo as resting B cell. J Virol 1997;71:4882—91. 3. Babcock GJ, Decker LL, Volk M, Thorley-Lawson DA. EBV persistence in memory B cells in vivo. Immunity 1998;9:395—404. 4. Rickinson AB, Kieff E. Epstein—Barr virus. In: Knipe DM, Howley PM, editors. Fields virology, 4th ed., Vol. 1. Philadelphia, USA: Lippincott Williams & Wilkins; 2001. p. 2575—627. 5. Thorley-Lawson DA, Gross A. Persistence of the Epstein—Barr virus and the origins of associated lymphomas. N Engl J Med 2004;350:1328—37. 6. Weiss LM, Movahed LA, Warnke RA, Sklar J. Detection of Epstein— Barr viral genomes in Reed—Sternberg cells of Hodgkin’s disease. N Engl J Med 1989;320:502—6. 7. Magrath I. The pathogenesis of Burkitt’s lymphoma. Adv Cancer Res 1990;55:133—270. 8. Pallesen G, Hamilton-Dutoit SJ, Zhou X. The association of Epstein—Barr virus (EBV) with T cell lymphoproliferations and Hodgkin’s disease: two new developments in the EBV field. Adv Cancer Res 1993;62:179—239. 9. Young LS, Murray PG. Epstein—Barr virus and oncogenesis: from latent genes to tumors. Oncogene 2003;22:5108—21. 10. Paya CV, Fung JJ, Nalesnik MA, Kieff E, Green M, Gores G, et al. Epstein—Barr virus-induced post-transplant lymphoproliferative disorders. Transplantation 1999;68:1517—25. 11. Riddler SA, Breinig MC, McKnight LC. Increased levels of circulating Epstein—Barr virus (EBV)-infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of post-transplant lymphoproliferative disease in solid-organ transplant recipients. Blood 1994;84:972—84. 12. Kenagy DN, Schlesinger Y, Weck K, Ritter JH, Gaudrealt-Keener MM, Storch GA. Epstein—Barr virus DNA in peripheral blood leukocytes of patients with post-transplant lymphoproliferative disease. Transplantation 1995;60:547—54. 13. Green M, Reyes J, Jabbour N, Yunis E, Putnam P, Todo S, Rowe D. Use of quantitative PCR to predict onset of Epstein—Barr viral infection and post-transplant lymphoproliferative disease after intestinal transplantation in children. Transplant Proc 1996; 28:2759—60. 14. Rowe DT, Webber S, Schauer EM, Reyes J, Green M. Epstein—Barr virus load monitoring: its role in the prevention and management of post-transplant lymphoproliferative disease. Transpl Infect Dis 2001;3:79—87. 15. Smets F, Sokal EM. Epstein—Barr virus related lymphoproliferation in children after liver transplant: role of immunity, diagnosis, and management. Pediatr Transplantation 2002;6: 280—7. 16. Rahman MA, Kingsley LA, Atchison RW, Belle S, Breinig MC, Ho M, et al. Reactivation of Epstein—Barr virus during early infection
178
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
M.D. Fellner et al. with human immunodeficiency virus. J Clin Microbiol 1991;29: 1215—20. Yao QY, Tierney RJ, Croom-Carter D, Dukers D, Cooper GM, Ellis CJ, et al. Frequency of multiple Epstein—Barr virus infections in Tcellimmunocompromised individuals. J Virol 1996;70:4884—94. O’Sullivan CE, Peng R, Cole KS, Montelaro RC, Sturgeon T, Jenson HB, et al. Epstein—Barr virus and human immunodeficiency virus serological responses and viral burdens in HIV-infected patients treated with HAART. J Med Virol 2002;67:320—6. Dehee A, Asselot C, Piolot T, Jacomet C, Rozenbaum W, Vidaud M, et al. Quantification of Epstein—Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR. J Med Virol 2001;65:543—52. Ling PD, Vilchez RA, Keitel WA, Poston DG, Peng RS, White ZS, et al. Epstein—Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy. Clin Infect Dis 2003;37:1244—9. Laroche C, Drouet EB, Brousset P, Pain C, Boibieux A, Biron F, et al. Measurement by the polymerase chain reaction of the Epstein—Barr virus load in infectious mononucleosis and AIDSrelated non-Hodgkin’s lymphomas. J Med Virol 1995;46:66—74. van Baarle D, Wolthers KC, Hovenkamp E, Niesters HG, Osterhaus AD, Miedema F, et al. Absolute level of Epstein—Barr virus DNA in human immunodeficiency virus type 1 infection is not predictive of AIDS-related non-Hodgkin lymphoma. J Infect Dis 2002; 186:405—9. Stevens SJ, Blank BS, Smits PH, Meenhorst PL, Middeldorp JM. High Epstein—Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy and quantitative EBV serology. AIDS 2002;16:993—1001. Piriou ER, van Dort K, Nanlohy NM, Miedema F, van Oers MH, van Baarle D. Altered EBV viral load setpoint after HIV seroconversion is in accordance with lack of predictive value of EBV load for the occurrence of AIDS-related non-Hodgkin lymphoma. J Immunol 2004;172:6931—7. Fan H, Kim SC, Chima CO, Israel BF, Lawless KM, Eagan PA, et al. Epstein—Barr viral load as a marker of lymphoma in AIDS patients. J Med Virol 2005;75:59—69. Fellner MD, Durand K, Correa M, Bes D, Alonio LV, Teyssie AR, et al. A semiquantitative PCR method (SQ-PCR) to measure Epstein—Barr virus (EBV) load: its application in transplant patients. J Clin Virol 2003;28:323—30. Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Centers for Disease Control and Prevention.1993. Morb Mortal Wkly Rep 1992;41:1—19. Harvard Medical School. Preparation and analysis of DNA. In: Ausubel FM, Brent R, Kingston R, Moore D, Seidman J, Smith J, et al. editors. Short protocols in molecular biology. 2nd ed. USA: Greene Publishing Associates and John Wiley & Sons; 1992. p. 4—5. Casas I, Powell L, Klapper PE, Cleator GM. New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay. J Virol Methods 1995;53:25—36.
30. Bauer H, Greer C, Manos M. Determination of genital human papillomavirus infection by consensus polymerase chain reaction amplification. In: Herrington C, McGree J, editors. Diagnostic molecular pathology: a practical approach. Oxford, UK: IRL Press; 1992. p. 131—52. 31. Stevens SJ, Vervoort MB, van den Brule AJ, Meenhorst PL, Meijer CJ, Middeldorp JM. Monitoring of Epstein—Barr virus DNA load in peripheral blood by quantitative competitive PCR. J Clin Microbiol 1999;37:2852—7. 32. Douek DC. Disrupting T-cell homeostasis: how HIV-1 infection causes disease. AIDS Rev 2003;5:172—7. 33. Alimonti JB, Ball TB, Fowke KR. Mechanisms of CD4+ T lymphocyte cell death in human immunodeficiency virus infection and AIDS. J Gen Virol 2003;84:1649—61. 34. Jabs WJ, Hennig H, Kittel M, Pethig K, Smets F, Bucsky P, et al. Normalized quantification by real time PCR of Epstein—Barr virus load in patients at risk for post-transplant lymphoproliferative disorders. J Clin Microbiol 2001;39:564—9. 35. Stevens SJ, Verschuuren EA, Verkuujlen SA, Van Den Brule AJ, Meijer CJ, Middeldorp JM. Role of Epstein—Barr virus DNA load monitoring in prevention and early detection of posttransplant lymphoproliferative disease. Leuk Lymphoma 2002; 43:831—40. 36. Gross TG, Loechelt BJ. Epstein—Barr virus associated disease following blood or marrow transplant. Pediatr Transplant 2003;7:44—50. 37. Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA. Epstein—Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients. J Exp Med 1999;190:567—76. 38. Rose C, Green M, Webber S, Ellis D, Reyes J, Rowe D. Pediatric solid-organ transplant recipients carry chronic loads of Epstein— Barr virus exclusively in the immunoglobulin D-negative B-cell compartment. J Clin Microbiol 2001;39:1407—15. 39. MacLennan IC. Germinal centers. Ann Rev Immunol 1994; 12:117—39. 40. Liu YJ, Arpin C. Germinal center development. Immunol Rev 1997;156:111—26. 41. De Milito A, Morch C, Sonnerborg A, Chiodi F. Loss of memory (CD27) B lymphocytes in HIV-1 infection. AIDS 2001;15:957—64. 42. Nagase H, Agematsu K, Kitano K, Takamoto M, Okubo Y, Komiyama A, et al. Mechanism of hypergammaglobulinemia by HIV infection: circulating memory B-cell reduction with plasmacytosis. Clin Immunol 2001;100:250—9. 43. De Milito A. B lymphocyte dysfunctions in HIV infection. Curr HIV Res 2004;2:11—21. 44. Van Baarle D, Hovenkamp E, Callan MF, Wolthers KC, Kostense S, Tan LC, et al. Dysfunctional Epstein—Barr virus (EBV)-specific CD8(+) T lymphocytes and increased EBV load in HIV-1 infected individuals progressing to AIDS-related non-Hodgkin lymphoma. Blood 2001;98:146—55. 45. Righetti E, Ballon G, Ometto L, Cattelan AM, Menin C, Zanchetta M, et al. Dynamics of Epstein—Barr virus in HIV-1-infected subjects on highly active antiretroviral therapy. AIDS 2002;16: 63—73.
http://intl.elsevierhealth.com/journals/ijid
Circulating Epstein—Barr virus (EBV) in HIV-infected patients and its relation with primary brain lymphoma Marı´a Dolores Fellner a,*, Karina Durand a, Rita Mariel Correa a, Liliana Redini b, Claudio Yampolsky c,d, Antonio Colobraro e,f, ´lica R. Teyssie ´ a, Jorge Benetucci b, Gustavo Sevlever e,f, Ange Marı´a Alejandra Picconi a a
´nicos, Instituto Nacional de Enfermedades Infecciosas (INEI)- ANLIS ‘‘Dr. Carlos G. Malbra ´n’’, Servicio Virus Oncoge ´rsfield 563, C 1282AFF Buenos Aires, Argentina Av. Velez Sa b Laboratorio de Retrovirus y Virus asociados, FUNDAI, Buenos Aires, Argentina c Servicio de Neurocirugı´a, Sanatorio Guemes, Buenos Aires, Argentina d ˜iz’’, Buenos Aires, Argentina Hospital de Enfermedades Infecciosas ‘‘Dr. F. Mun e Servicio de Patologı´a, Instituto FLENI, Buenos Aires, Argentina f Servicio de Patologı´a, Clı´nica Bazterrica, Buenos Aires, Argentina Received 24 November 2005; received in revised form 4 April 2006; accepted 6 April 2006 Corresponding Editor: Jane Zuckerman, London, UK
KEYWORDS EBV load; Blood compartments; HIV-infected population; Brain lymphoma
Summary Objective: To analyze Epstein—Barr virus (EBV) load at different HIV infection stages and its relation with brain lymphoma. Design: A cross-sectional study was conducted on 172 HIV-infected individuals: 62 asymptomatic HIV carriers (group A), 30 HIV progressors (group B), 73 AIDS patients (group C), seven AIDS patients with brain lymphoma (group C-BL); and 26 blood donors (group BD) as healthy carriers. EBV load was measured in peripheral blood mononuclear cells (PBMC) and plasma samples using a semi-quantitative PCR method. Results: PBMC-EBV levels in HIV-infected patients were higher than in the blood donors ( p < 0.05). No differences in PBMC-EBV loads were found in groups A, B, or C ( p > 0.05), while the C-BL group had significantly lower levels ( p < 0.05). Similar PBMC-EBV loads were seen in HIVinfected patients with CD4+ T cell counts higher than 50/mm3 ( p > 0.05), while significantly lower levels were found in cases with less than 50 cells/mm3 ( p < 0.05). In all HIV-infected patients, plasma-EBV load was lower than, or similar to, PBMC-EBV load, unlike 2/7 HIV-positive brain lymphoma patients. Conclusions: During HIV infection PBMC-EBV load rises in comparison to healthy carriers, but decreases when immunosuppression progresses and CD4+ T cell count becomes <50/mm3.
* Corresponding author. Tel.: +55 11 4301 7428/4302 5064; fax: +55 11 4302 5064/4303 2210. E-mail address: [email protected] (M.D. Fellner). 1201-9712/$32.00 # 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2006.04.001
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EBV in HIV-infected patients and primary brain lymphoma
Circulating EBV is mainly cell-associated in the HIV-infected population. Neither PBMC-EBV nor plasma-EBV loads would be useful to diagnose brain lymphoma in AIDS patients. # 2006 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Introduction Epstein—Barr virus (EBV) is a widely spread human gammaherpesvirus, whose prevalence exceeds 95% of the adult human population.1 After the primary infection, as EBV establishes persistence in B lymphocytes, it can be detected both in lymphoid tissue and peripheral blood.2,3 In immunosuppressed patients, the virus—host imbalance favors EBV reactivation which in turn induces lymphoproliferation.4 Therefore, it has been proposed that these benign proliferations may become malignant lymphomas if they are not well controlled by the host immune system, although this is a rare event.5 EBV has been associated with a variety of malignancies due to its detection in malignant cells.6—9 The EBV-associated tumors described in the HIV-infected population include non-Hodgkin lymphomas, Hodgkin lymphomas, Burkitt lymphomas, and smooth muscle malignancies. The strength of association differs, being about 100% in cases of primary brain lymphomas, which appear when patients are profoundly immunosuppressed; in this sense, they are essentially analogous to post-transplant lymphoproliferative disorder (PTLD) lesions of the transplant group.4,10 In the transplant population a relationship was found between circulating EBV load and PTLD.11,12 Moreover, higher circulating EBV DNA levels precede PTLD diagnosis, which allows for early therapeutic intervention in patients with rising levels of EBV, thus preventing the establishment of this life-threatening entity; also other reports noted decreased EBV load with successful therapy.13—15 The EBV load has been much less studied in the HIVinfected group than in transplant individuals. As in transplant patients, frequent reactivation of EBV infection has been observed in HIV-infected individuals, reflected by increased EBV-specific antibody titers,16—18 and more recently, high numbers of circulating copies of EBV.19,20 However, controversy persists regarding the usefulness of measuring EBV levels in AIDS patients in relation to lymphoma risk, diagnosis, and management.21—25 The semi-quantitative PCR (SQ-PCR) method used to measure EBV load was developed in our laboratory to monitor transplant patients and in fact, its usefulness was demonstrated for the detection of differences in circulating levels of EBV DNA. Although different strategies such as competitive methods or real time PCR have proven to yield the best results, the SQ-PCR method is, for us, more affordable.26 The aim of our study was to analyze the EBV load in different HIV infection stages and its relation with primary brain lymphoma.
Materials and methods Study population One hundred and seventy-two HIV-infected patients from the ‘‘J.F. Mun ˜iz’’ Infectious Diseases Hospital (Buenos Aires,
Argentina) were included in this study. All of them were receiving antiretroviral therapy, with the exception of asymptomatic HIV-infected patients with CD4+ T cells counts above 350/mm3. Mean patient age was 26 years (range 16—61 years), male/female ratio was 2:3, and they were grouped according to the 1993 CDC classification,27 as follows: 62 asymptomatic HIV-infected patients (group A), 30 HIV progressors (group B), 73 AIDS patients (group C), and seven AIDS patients with a diagnosis of primary central nervous system lymphoma (PCNSL) (group C-BL). They were all typed as nonHodgkin lymphoma according to the WHO classification. Twenty-six blood donors (group BD), who were healthy EBV carriers from the same hospital, were also included. They were all negative for the usual blood bank tested infections; mean age of this group was 34 years (range 19—50 years). All the study population showed previous evidence of EBV infection (presence of IgG antibodies against viral capsid antigen (VCA) detected by indirect immune fluorescence assay). All participants gave their informed consent for inclusion in this study. A local ethics committee approved all procedures.
Samples EDTA anti-coagulated blood samples were collected and peripheral blood mononuclear cells (PBMC) separated from plasma after a Ficoll-Hypaque gradient centrifugation (Lymphoprep, GIBCO). Fresh brain biopsy tissue (BB) and in some cases cerebrospinal fluid (CSF) were obtained from C-BL patients. All samples were stored at 20 8C until used. Paraffin embedded lymphoma tissue was examined with in situ hybridization (ISH).
DNA extraction DNA from PBMC, plasma, and BB was extracted with proteinase K lysis buffer and purified by a phenol—chloroform procedure.28 DNA from CSF was extracted as previously described.29
EBV quantification Viral load was determined applying a semi-quantitative method as we have previously described.26 This method was developed to detect high EBV loads, i.e., levels above those found in healthy carriers (latent state). Briefly, a nested PCR was performed, which amplifies a fragment of 122 bp from the BWRF1 gene. For PBMC, DNA equivalent to 105 cells (366 ng) and its serial four-fold dilutions were amplified until limiting dilution was reached. The amplified products were run in a 2% agarose gel stained with ethidium bromide and the end point was converted into viral copies/105 PBMC by multiplying the sensitivity of the assay (1—16 copies) by the reciprocal of the limiting dilution (RLD). We subsequently applied
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Figure 1 Detection of PCR inhibitors. No inhibition was detected in DNA extracted from PBMC samples, while two DNA samples extracted from plasma exhibited inhibitors. Both samples were re-extracted, and when re-tested one showed no inhibitors so it was quantified, while the other was discarded since inhibitors could not be eliminated. (A) Detection of PCR inhibitors in DNA extracted from PBMC samples. Lanes 1—5: DNAPBMC from HIV positive patients. Lane 6: negative control. Lane 7: positive control. Lane 8: 100 bp ladder. (B) Detection of PCR inhibitors in DNA extracted from plasma samples. Lane 1: 100 bp ladder. Lanes 2—6: DNAplasma from HIV positive patients. Notice the presence of inhibitors in the plasma specimen run in lane 2. Lane 7: negative control. Lane 8: positive control.
the method to plasma samples; the intra- and inter-assay variation result was the same as for PBMC specimens, i.e., one four-fold dilution (data not shown). Thus, we considered the same sensitivity and reproducibility as was described for PBMC samples. For plasma specimens, DNA obtained from 30 mL (similar to the volume associated with 105 PBMC)14 and its serial four-fold dilutions were amplified and quantified as mentioned. The presence of PCR inhibitors was determined when EBV load was <1 copy/105 PBMC or 30 mL of plasma as follows: (A) Inhibitors in DNA extracted from PBMC samples were determined by amplifying the human b-globin gene using primer set PCO4 and GH20 to amplify a 268 bp DNA fragment as described by Bauer et al.30 (B) Inhibitors in DNA extracted from plasma samples were tested by spiking around 125 ng of DNA obtained from human fibroblasts onto DNA extracted from 30 mL of plasma, and then the human b-globin gene was amplified as described above (Figure 1).
EBV qualitative detection For EBV DNA qualitative determination, DNA extracted from 50 ul of CSF or about 300 ng of DNABB were amplified by the same nested PCR applied for quantification, using different cycling conditions in the first round: 3 min at 94 8C, and forty 1-min cycles at 94 8C, 30 s at 58 8C, 1 min at 72 8C; and 10 min at 72 8C.
EBERs detection To determine EBV association with PCNSL, a commercial in situ hybridization (ISH) method that detects EBV-encoded RNA (EBERs) transcripts was applied on paraffin-embedded lymphoma tissue sections mounted onto silane-coated glass slides. Peptide nucleic acid (PNA) fluorescein isothiocyanate (FITC)-labeled probes: EBER-specific, positive and negative
Figure 2 EBV detection and PBMC viral load in the study population. The limiting dilution value obtained for each patient was converted into viral copies/105 PBMC by multiplying the sensitivity of the assay (1—16 copies) by the reciprocal of the limiting dilution (RLD); e.g. if RLD = 4, after multiplying by 1 and by 16, the corresponding viral load ranges between 4 and 64. ND, not detected; dotted line represents the detection level with the SQ-PCR. Bars show the median RLD value with the confidence interval range (%) for each group.
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EBV in HIV-infected patients and primary brain lymphoma
Figure 3 EBV detection and load levels in HIV infected patients according to CD4+ T cell counts. The total number of patients included in this analysis is lower than that described in Figure 2 since CD4+ T cell counts were not available in 10 patients. ND, not detected; dotted line represents the detection level with the SQ-PCR. Bars show the median RLD value with the confidence interval range (%) for each group.
RNA controls (Dakocytomation, Glostrup, Denmark), followed by its detection with a PNA ISH detection kit (Dakocytomation, Glostrup, Denmark), were used according to manufacturers’ instructions.
Number of CD4+ and CD8+ lymphocytes These were determined by standard flow cytometry techniques.
EBV load and CD4+ T cells EBV detection was similar in all groups with CD4+ Tcell counts higher than 50/mm3 ( p > 0.05), and significantly higher than in patients with less than 50 CD4+ T cells/mm3 ( p < 0.05). When analyzing EBV levels, we found no statistically significant differences between groups with more than 50 CD4+ T cells/mm3. On the other hand, circulating EBV levels in patients with less than 50 CD4+ T cells/mm3 were significantly ( p < 0.05) lower than in those with CD4+ Tcells counts
Statistical analyses Categorical data were compared using Fisher’s exact test or the Chi-square test, while the non-parametric Mann—Whitney U test for unpaired data was used to analyze ordinal values. For the purpose of the analysis, not-detected (ND) RLD values were considered 0. The PEPI Program Finder version 4.0 was used for the analyses. Linear regression analysis was performed after RLD was converted into a natural logarithm (Ln) with the Analyse-it software for Excel 1997—2000.
Results EBV load and clinical HIV state HIV-infected individuals were found to exhibit a significantly higher detection rate and increased levels of circulating EBV DNA compared to blood donors ( p < 0.05). Similar results were obtained when comparing each HIV clinical set without lymphoma to the BD group (Figure 2). Also, no statistically significant differences ( p > 0.05) in EBV detection and EBV load were found between groups A, B, and C. The C-BL group was found to have a significantly ( p < 0.05) lower circulating EBV rate and levels compared to groups A and B.
Figure 4 Correlation between plasma-EBV and PBMC-EBV loads. A linear regression analysis of the natural logarithm of the reciprocal of the limiting dilution (RLD) values obtained from paired PBMC and plasma samples of 62 patients is represented (r = 0.173; p = 0.219).
176 Table 1
M.D. Fellner et al. Description of HIV-infected patients with primary central nervous system lymphoma
Patient
Histopathologic diagnosis
PCR
EBER ISH
PBMC-EBV load (copies/105 PBMC)
Plasma EBV load (copies/30 mL)
1 2 3 4 5 6 7
DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL
+ + + + + + + +
+ + + + + + NS
<1 <1 <1 1—16 <1 <1 <1
64—1024 <1 16—256 <1 <1 <1 <1
(BB) (BB) (BB) (BB) (BB) (BB) (BB) (CSF)
DLBCL, diffuse large B cell lymphoma; BB, brain biopsy; NS, no sample available.
between 50 and 500/mm3. Figure 3 illustrates the results of EBV quantification as regards the number of CD4+ T lymphocytes.
EBV load and CD8+ T cells No correlation was found between EBV circulating levels and the number of CD8+ T cells in the HIV-infected population studied (data not shown).
Relation between PBMC- and plasma-EBV load Plasma-EBV load was measured in about 20 patients of each HIV clinical set: A, B, and C (n = 55), and in all seven C-BL cases. The criterion for plasma selection was that all PBMCEBV load ranks were represented. Therefore, when analyzing EBV DNA in different peripheral blood compartments of all HIV groups, we observed no correlation between PBMC and plasma levels (Figure 4). Also, EBV load was lower in plasma samples than in PBMC for most HIV positive cases without lymphoma; only two patients (from groups A and C) had similar circulating EBV DNA copies in both samples. Two of the seven HIV-infected patients with PCNSL had higher EBV levels in plasma than in PBMC (patients 1 and 3, Table 1).
Discussion In this project, we noted that in HIV-infected patients EBV in peripheral blood is higher than in healthy individuals. Thus, higher EBV DNA detection rates were found in PBMC of HIVinfected patients compared to healthy individuals, in agreement with the report by Ling et al.20 Contrary to this finding, Dehee et al. described a similar EBV positivity rate in HIVinfected patients and asymptomatic HIV negative volunteers.19 In the latter case, the percentage of EBV DNA detection in HIV negative individuals was strikingly high compared to other reports.20,21,29,31 These differences could be due to the control groups, the methodological strategies, or the sensitivity of the techniques. Also, increased PBMCEBV load was found in all the clinical HIV infection categories (groups A, B, and C), indicating that the imbalance between EBV infection and the host immunologic control starts early in the course of HIV clinical infection, and continues as the infection progresses. In a prospective study, Piriou et al.
proved that PBMC-EBV load increases rapidly following HIV seroconversion, and remains high even five years later, both in asymptomatic carriers and AIDS progressors.24 Since CD4+ T cell depletion is the immunologic hallmark of HIV-related disease progress,32,33 we also analyzed the relation of circulating EBV levels and the progress of immunological deterioration, according to CD4+ T cell counts by the CDC classification. In agreement with reports by Dehee et al.,19 and Ling et al.,20 when the number of CD4+ T cells is greater than 50/mm3, higher levels of circulating PBMC-EBV are seen in HIV-infected patients, compared to healthy carriers. This situation may reflect the loss of EBV infection control due to progressive immunosuppression, similar to the situation widely described for transplant patients subjected to severe immunosuppression.14,34—36 However, in HIV-infected patients with CD4+ T cell counts under 50/mm3 we observed lower levels of circulating PBMC-EBV, which counters the findings of Dehee et al. who found increased PBMC-EBV DNA levels even in this group (<50 LT CD4+/mm3).19 Our observations could be explained if we consider that copies of peripheral EBV have been detected only in memory B lymphocytes,37,38 cells generated after a germinal center reaction.39,40 Thus, the decline in the PBMC-EBV load could be due to the decrease in circulating memory B lymphocytes observed during HIV infection.41,42 This reduction may be due to hyperactivation-induced memory B cell exhaustion, increased apoptosis, on top of an abnormal naı¨ve B cell activation/ differentiation process.43 Therefore, we conclude that it would be interesting to analyze EBV level variation relative to the quantity of B lymphocytes at different stages of HIV infection. Like Dehee et al.,19 we found no correlation between total CD8+ Tcell count and PBMC-EBV load. It should be pointed out that van Baarle et al., studying HIV-infected patients, analyzed the EBV-specific CD8+ T cell population (which represents a small proportion of total CD8+ T lymphocytes), and observed a normal number, although with decreased functional capacity, compared to HIV negative individuals.44 Also, our study shows that in HIV-infected patients, the EBV found in peripheral blood is carried mainly in PBMC (cellassociated virus), in agreement with Stevens et al.,23 although our study analyzed a substantially larger number of patients. Moreover, we observed no correlation between PBMC-EBV and plasma-EBV loads, as reported by van Baarle et al.22 and Righetti et al.45 Further, the plasma-EBV levels of
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EBV in HIV-infected patients and primary brain lymphoma HIV-infected patients without lymphoma were lower or similar to those found in PBMC which could indicate that the EBVplasma load results from the lysis of latently-infected circulating B cells (necrosis or apoptosis), or that peripheral lytic infection (either the presence of circulating EBV-virions and/ or lysis of circulating EBV-activated B cells) is not the main cause of increased circulating EBV, or both. It would be interesting for future studies to establish whether plasmaEBV load corresponds to encapsulated virions or nude viral DNA. Different authors have described variable circulating EBV DNA levels when lymphoma is diagnosed in HIV-infected patients.21,31 Also, prospective studies by van Baarle et al. have shown that HIV-infected patients with non-Hodgkin lymphomas have EBV levels that fluctuate over time, being at the time of lymphoma diagnosis lower, similar to, or higher than those previously detected.22,44 Recently, Fan et al. described significantly higher plasma-EBV levels in HIVinfected patients with EBV-associated lymphomas compared to HIV-infected patients with non-EBV-associated lymphomas, or without lymphomas. However, plasma-EBV load varied in the samples collected before, during, and after lymphoma diagnosis, although EBV DNA was detectable at least in one sample. It was noted that in primary brain lymphoma cases, the EBV load was mainly not detectable at the time of diagnosis.25 In this study, the PBMC-EBV load in AIDS patients with PCNSL (group C-BL) was lower than that found in the initial stages of HIV infection (groups A and B), albeit similar to the levels found in AIDS patients without lymphoma (group C). Further, PBMC-EBV levels were undetectable or low in all patients with PCNSL diagnosis, seeming to indicate that EBV load determination would not be a useful indicator of lymphoma, even though only few cases were analyzed. Since PCNSL is a pathology found in HIV-infected patients with advanced immunological deterioration, our findings could be accounted for, as previously mentioned, by the decrease in memory B lymphocytes, described in the course of HIV infection. Moreover, although most of the lymphoma patients showed undetectable plasma-EBV load, it was higher, even more than PBMC-EBV levels, in two cases, suggesting increased lysis of latently-infected circulating B cells, and/or peripheral lytic infection, at least in some of these patients. In this sense, it would also be interesting to determine whether plasma-EBV load in HIV-infected patients with lymphoma corresponds to virions or to nude DNA. In conclusion, EBV found in peripheral blood of HIVinfected patients is mainly cell-associated. Also, during the course of HIV infection, when the CD4+ T cell count is above 50/mm3, the PBMC-EBV levels are increased, which would reflect the loss of EBV infection control due to progressive immunosuppression. When immunological deterioration progresses and the CD4+ T cell count is below 50/ mm3, the PBMC-EBV load decreases; perhaps because of altered B lymphocytes (memory and naı¨ve) distribution, and inappropriate activation/differentiation that have been described in the course of HIV infection. Also, the determination of the PBMC-EBV load in AIDS patients with PCNSL would not be a useful indicator of lymphoma, while the meaning of EBV DNA in these patients’ plasma remains to be clarified.
Acknowledgments The authors are grateful to Drs L. De Benedetti, M. Corti, C. Trione, C. Bonifacio, N. Zala, and S. Camino (Hospital Mun ˜iz) for their generous help during sampling, and to Drs J. Go ´mez and Sara Vladimirsky (Instituto Malbra ´n) for their epidemiological counseling. This project was partially supported by grants from the Mosoteguy and Bunge & Born Foundation (Buenos Aires, Argentina). Conflict of interest: No conflict of interest to declare.
References 1. Young LS, Rickinson AB. Epstein—Barr virus: 40 years on. Nat Rev Cancer 2004;4:757—68. 2. Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson DA. Identification of the site of Epstein—Barr virus persistence in vivo as resting B cell. J Virol 1997;71:4882—91. 3. Babcock GJ, Decker LL, Volk M, Thorley-Lawson DA. EBV persistence in memory B cells in vivo. Immunity 1998;9:395—404. 4. Rickinson AB, Kieff E. Epstein—Barr virus. In: Knipe DM, Howley PM, editors. Fields virology, 4th ed., Vol. 1. Philadelphia, USA: Lippincott Williams & Wilkins; 2001. p. 2575—627. 5. Thorley-Lawson DA, Gross A. Persistence of the Epstein—Barr virus and the origins of associated lymphomas. N Engl J Med 2004;350:1328—37. 6. Weiss LM, Movahed LA, Warnke RA, Sklar J. Detection of Epstein— Barr viral genomes in Reed—Sternberg cells of Hodgkin’s disease. N Engl J Med 1989;320:502—6. 7. Magrath I. The pathogenesis of Burkitt’s lymphoma. Adv Cancer Res 1990;55:133—270. 8. Pallesen G, Hamilton-Dutoit SJ, Zhou X. The association of Epstein—Barr virus (EBV) with T cell lymphoproliferations and Hodgkin’s disease: two new developments in the EBV field. Adv Cancer Res 1993;62:179—239. 9. Young LS, Murray PG. Epstein—Barr virus and oncogenesis: from latent genes to tumors. Oncogene 2003;22:5108—21. 10. Paya CV, Fung JJ, Nalesnik MA, Kieff E, Green M, Gores G, et al. Epstein—Barr virus-induced post-transplant lymphoproliferative disorders. Transplantation 1999;68:1517—25. 11. Riddler SA, Breinig MC, McKnight LC. Increased levels of circulating Epstein—Barr virus (EBV)-infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of post-transplant lymphoproliferative disease in solid-organ transplant recipients. Blood 1994;84:972—84. 12. Kenagy DN, Schlesinger Y, Weck K, Ritter JH, Gaudrealt-Keener MM, Storch GA. Epstein—Barr virus DNA in peripheral blood leukocytes of patients with post-transplant lymphoproliferative disease. Transplantation 1995;60:547—54. 13. Green M, Reyes J, Jabbour N, Yunis E, Putnam P, Todo S, Rowe D. Use of quantitative PCR to predict onset of Epstein—Barr viral infection and post-transplant lymphoproliferative disease after intestinal transplantation in children. Transplant Proc 1996; 28:2759—60. 14. Rowe DT, Webber S, Schauer EM, Reyes J, Green M. Epstein—Barr virus load monitoring: its role in the prevention and management of post-transplant lymphoproliferative disease. Transpl Infect Dis 2001;3:79—87. 15. Smets F, Sokal EM. Epstein—Barr virus related lymphoproliferation in children after liver transplant: role of immunity, diagnosis, and management. Pediatr Transplantation 2002;6: 280—7. 16. Rahman MA, Kingsley LA, Atchison RW, Belle S, Breinig MC, Ho M, et al. Reactivation of Epstein—Barr virus during early infection
178
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
M.D. Fellner et al. with human immunodeficiency virus. J Clin Microbiol 1991;29: 1215—20. Yao QY, Tierney RJ, Croom-Carter D, Dukers D, Cooper GM, Ellis CJ, et al. Frequency of multiple Epstein—Barr virus infections in Tcellimmunocompromised individuals. J Virol 1996;70:4884—94. O’Sullivan CE, Peng R, Cole KS, Montelaro RC, Sturgeon T, Jenson HB, et al. Epstein—Barr virus and human immunodeficiency virus serological responses and viral burdens in HIV-infected patients treated with HAART. J Med Virol 2002;67:320—6. Dehee A, Asselot C, Piolot T, Jacomet C, Rozenbaum W, Vidaud M, et al. Quantification of Epstein—Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR. J Med Virol 2001;65:543—52. Ling PD, Vilchez RA, Keitel WA, Poston DG, Peng RS, White ZS, et al. Epstein—Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy. Clin Infect Dis 2003;37:1244—9. Laroche C, Drouet EB, Brousset P, Pain C, Boibieux A, Biron F, et al. Measurement by the polymerase chain reaction of the Epstein—Barr virus load in infectious mononucleosis and AIDSrelated non-Hodgkin’s lymphomas. J Med Virol 1995;46:66—74. van Baarle D, Wolthers KC, Hovenkamp E, Niesters HG, Osterhaus AD, Miedema F, et al. Absolute level of Epstein—Barr virus DNA in human immunodeficiency virus type 1 infection is not predictive of AIDS-related non-Hodgkin lymphoma. J Infect Dis 2002; 186:405—9. Stevens SJ, Blank BS, Smits PH, Meenhorst PL, Middeldorp JM. High Epstein—Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy and quantitative EBV serology. AIDS 2002;16:993—1001. Piriou ER, van Dort K, Nanlohy NM, Miedema F, van Oers MH, van Baarle D. Altered EBV viral load setpoint after HIV seroconversion is in accordance with lack of predictive value of EBV load for the occurrence of AIDS-related non-Hodgkin lymphoma. J Immunol 2004;172:6931—7. Fan H, Kim SC, Chima CO, Israel BF, Lawless KM, Eagan PA, et al. Epstein—Barr viral load as a marker of lymphoma in AIDS patients. J Med Virol 2005;75:59—69. Fellner MD, Durand K, Correa M, Bes D, Alonio LV, Teyssie AR, et al. A semiquantitative PCR method (SQ-PCR) to measure Epstein—Barr virus (EBV) load: its application in transplant patients. J Clin Virol 2003;28:323—30. Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Centers for Disease Control and Prevention.1993. Morb Mortal Wkly Rep 1992;41:1—19. Harvard Medical School. Preparation and analysis of DNA. In: Ausubel FM, Brent R, Kingston R, Moore D, Seidman J, Smith J, et al. editors. Short protocols in molecular biology. 2nd ed. USA: Greene Publishing Associates and John Wiley & Sons; 1992. p. 4—5. Casas I, Powell L, Klapper PE, Cleator GM. New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay. J Virol Methods 1995;53:25—36.
30. Bauer H, Greer C, Manos M. Determination of genital human papillomavirus infection by consensus polymerase chain reaction amplification. In: Herrington C, McGree J, editors. Diagnostic molecular pathology: a practical approach. Oxford, UK: IRL Press; 1992. p. 131—52. 31. Stevens SJ, Vervoort MB, van den Brule AJ, Meenhorst PL, Meijer CJ, Middeldorp JM. Monitoring of Epstein—Barr virus DNA load in peripheral blood by quantitative competitive PCR. J Clin Microbiol 1999;37:2852—7. 32. Douek DC. Disrupting T-cell homeostasis: how HIV-1 infection causes disease. AIDS Rev 2003;5:172—7. 33. Alimonti JB, Ball TB, Fowke KR. Mechanisms of CD4+ T lymphocyte cell death in human immunodeficiency virus infection and AIDS. J Gen Virol 2003;84:1649—61. 34. Jabs WJ, Hennig H, Kittel M, Pethig K, Smets F, Bucsky P, et al. Normalized quantification by real time PCR of Epstein—Barr virus load in patients at risk for post-transplant lymphoproliferative disorders. J Clin Microbiol 2001;39:564—9. 35. Stevens SJ, Verschuuren EA, Verkuujlen SA, Van Den Brule AJ, Meijer CJ, Middeldorp JM. Role of Epstein—Barr virus DNA load monitoring in prevention and early detection of posttransplant lymphoproliferative disease. Leuk Lymphoma 2002; 43:831—40. 36. Gross TG, Loechelt BJ. Epstein—Barr virus associated disease following blood or marrow transplant. Pediatr Transplant 2003;7:44—50. 37. Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA. Epstein—Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients. J Exp Med 1999;190:567—76. 38. Rose C, Green M, Webber S, Ellis D, Reyes J, Rowe D. Pediatric solid-organ transplant recipients carry chronic loads of Epstein— Barr virus exclusively in the immunoglobulin D-negative B-cell compartment. J Clin Microbiol 2001;39:1407—15. 39. MacLennan IC. Germinal centers. Ann Rev Immunol 1994; 12:117—39. 40. Liu YJ, Arpin C. Germinal center development. Immunol Rev 1997;156:111—26. 41. De Milito A, Morch C, Sonnerborg A, Chiodi F. Loss of memory (CD27) B lymphocytes in HIV-1 infection. AIDS 2001;15:957—64. 42. Nagase H, Agematsu K, Kitano K, Takamoto M, Okubo Y, Komiyama A, et al. Mechanism of hypergammaglobulinemia by HIV infection: circulating memory B-cell reduction with plasmacytosis. Clin Immunol 2001;100:250—9. 43. De Milito A. B lymphocyte dysfunctions in HIV infection. Curr HIV Res 2004;2:11—21. 44. Van Baarle D, Hovenkamp E, Callan MF, Wolthers KC, Kostense S, Tan LC, et al. Dysfunctional Epstein—Barr virus (EBV)-specific CD8(+) T lymphocytes and increased EBV load in HIV-1 infected individuals progressing to AIDS-related non-Hodgkin lymphoma. Blood 2001;98:146—55. 45. Righetti E, Ballon G, Ometto L, Cattelan AM, Menin C, Zanchetta M, et al. Dynamics of Epstein—Barr virus in HIV-1-infected subjects on highly active antiretroviral therapy. AIDS 2002;16: 63—73.