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Human Herpesvirus 8 DNA in Serum During Seroconversion in Allogeneic Bone Marrow Transplant Recipients

Giuseppe Gentile, Angela Capobianchi, Antonio Volpi, Giorgio Palù, Francesca Pica, Arianna Calistri, Maria Angela Biasolo, Pietro Martino

Affiliations of authors: Department of Cellular Biotechnology and Hematology, Univ. "La Sapienza," Rome, Italy (GG, AC, PM); Department of Public Health, Univ. "Tor Vergata," Rome, Italy (AV, FP); Department of Histology, Microbiology, and Medical Biotechnology, Univ. Padova, Padova, Italy (GP, AC, MAB)

Correspondence to: Giuseppe Gentile, MD, Department of Cellular Biotechnology and Hematology, University "La Sapienza," Via Benevento 6, 00161 Rome, Italy (e-mail: gentile{at}bce.uniroma1.it).


    ABSTRACT
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To determine the prevalence of human herpesvirus 8 (HHV-8) infection, the rate of HHV-8 seroconversion, and the presence of serum HHV-8 DNA after bone marrow transplantation (BMT), we evaluated sera from 187 Italian BMT donor–recipient pairs. Antibodies to lytic and latent HHV-8 antigens were detected by immunofluorescence. Sera of donor–recipient pairs who seroconverted were examined by real-time polymerase chain reaction (RT-PCR). Before BMT, 24 (13%) of 187 donors and 20 (11%) of 187 recipients were seropositive; after BMT, 28 (15%) of 187 recipients were seropositive. Seroconversion occurred in 19 (11%) of 167 recipients seronegative at baseline: 14 (9%) from 149 seronegative donors and five (28%) from 18 seropositive donors (relative risk of seroconversion with BMT from a seropositive donor = 2.96, 95% confidence interval = 1.21 to 7.25; P = .02, two-sided Fisher's exact test). One donor and two recipients who seroconverted after BMT were positive for HHV-8 by RT-PCR. No HHV-8–related complications were observed after a median follow-up of 6 years. BMT-associated HHV-8 seroconversion is relatively common in seronegative recipients from seropositive donor, but factors other than BMT may also contribute to seroconversion.


Human herpesvirus 8 (HHV-8, also known as Kaposi sarcoma–associated herpesvirus) has been associated with Kaposi sarcoma (classic, endemic, AIDS-associated Kaposi sarcoma, and iatrogenic Kaposi sarcoma in immunosuppressed patients), body cavity lymphomas, and Castleman disease (1). Kaposi sarcoma occurs in 0.2%–5% of patients after solid organ transplantation (2) but is rare in allogeneic bone marrow transplantation (BMT) recipients. However, nonneoplastic complications (cytopenias, splenomegaly, and marrow failure) associated with HHV-8 infection after BMT have been described previously (3,4). HHV-8 reactivation, after immunosuppressive treatments, and primary HHV-8 infection in patients who have undergone solid organ transplantation (8) have been reported. HHV-8 may be transmitted through saliva (5) or sexual contact (6,7), but it is uncertain whether bloodborne transmission occurs (911), even if HHV-8 DNA can be detected in blood mononuclear cells (CD8+ T cells, B cells, and monocytes) [for review, see (12)]. Tests for antibodies to lytic and latent HHV-8 antigens are useful to monitor HHV-8 infection in transplant patients (13), but their meaning is still poorly elucidated (14). Detection of either HHV-8 DNA or mRNA in serum or plasma provides a better marker for replicating virus (15,16).

The current study was designed to investigate seroprevalence of HHV-8 infection in BMT recipients and donors at our institution. After BMT, we determined the rate of HHV-8 seroconversion and the presence of HHV-8 DNA in serum of recipients seroconverting and in the serum of their respective donors.

This retrospective study included 187 pairs of BMT donors and their recipients who lived in Central and South Italy and who underwent allogeneic BMT between January 1, 1991, and January 31, 2000, in the Department of Cellular Biotechnology and Hematology, University "La Sapienza," in Rome. This study was approved by the institutional review board of the University "La Sapienza," Rome. Serum samples taken from an archival collection were anonymized.

We tested serum samples stored at –80°C, one from each donor and three from each recipient, collected before BMT and 30 and 180 days after BMT, or earlier in case of death. Serologic testing was carried out in blinded fashion, as previously described (17). Antibodies to lytic and latent antigens of HHV-8 were detected by using one immunofluorescence assay for each antigen; these assays were based on body cavity B-cell lymphoma lines and on BCP-1 cell lines, respectively. Samples reactive at 1 : 40 dilution in the antilytic test and at 1 : 100 dilution in the antilatent test were considered positive. Positive control serum and negative control serum were included in each experiment. Maximum HHV-8 antibody dilutions were determined by an end-point immunofluorescence.

Persons with a positive result for at least one antigen (lytic or latent) were considered to be HHV-8 seropositive, because the combination of the two immunofluorescence assays showed high sensitivity and specificity (89.1% and 94.9%, respectively) in a multicenter study (18). Those recipients whose sample before BMT was negative and whose samples after BMT were positive were classified as seroconverters. Seroreversion was defined when a negative result followed a positive result in an earlier sample.

The amount of HHV-8 DNA present in 300 µL of serum was determined by use of real-time polymerase chain reaction (RT-PCR; with TaqMan probe, Applied Biosystems, Foster City, CA) of open reading frame 26 (ORF26), as previously described (19). The nucleotide sequence targeted by the primers and probes is highly conserved among the three major subgroups of HHV-8 (20). To standardize the assay, we used a plasmid containing part of ORF26. Serial dilutions of this plasmid ranging from 5 to 5 000 000 copies were used to characterize the linearity, precision, specificity, and sensitivity of the RT-PCR. The detection threshold was 40 genomic copies per 1 mL of serum. Amplification of human genomic {beta}-globin DNA was also used to assess the absence of PCR-inhibitory substances. All P values and confidence intervals (CIs) were from two-sided statistical tests.

Demographic characteristics and HHV-8 serostatus of donors and recipients before BMT are reported in Table 1. HHV-8 seropositivity before BMT was similar between donors (24 [13%] of 187 donors) and recipients (20 [11%] of 187 recipients) (P = .6, two-sided Fisher's exact test), but after BMT the percentage of HHV-8 seropositive recipients increased (28 [15%] of 187 recipients) (Table 1). HHV-8 seroprevalence among recipients and donors did not differ statistically significantly with respect to age and sex and was comparable to that found in the general population in the same geographic areas of Central and South Italy (Table 1). An HHV-8–seropositive recipient was more likely to receive a BMT from a seropositive donor than from a seronegative donor (relative risk [RR] of HHV-8 seropositive recipients to receive the graft from seropositive donors = 2.9, 95% CI = 1.24 to 6.84; P = .027, two-sided Fisher's exact test).


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Table 1.  Characteristics of bone marrow transplantation (BMT) recipients and their respective donors*

 
Among the 167 recipients seronegative before BMT, seroconversion occurred in 19 recipients (11%); 14 (9%) of these 19 recipients received BMT from one of the 149 HHV-8–seronegative donors and five (28%) received BMT from one of the 18 seropositive donors (Table 2). Ten recipients seroconverted within 30 days after BMT, and the remaining nine seroconverted within 180 days (Table 3). None of the seroconverters received intravenous gamma globulins. Among seroconverters, 28% of recipients from seropositive donor seroconverted compared with 9% from seronegative donor (relative risk of seroconversion for recipients from seropositive donor = 2.96, 95% CI = 1.21 to 7.25; P = .02, two-sided Fisher's exact test). Among those who converted, seroconversion was detected at 30 days in five of the five recipients from seropositive donor but in only five of 14 recipients from a seronegative donor (P = .032, two-sided Fisher's exact test). One donor and two recipients who seroconverted after BMT were positive by RT-PCR (range = 840–8625 copies per milliliter) (Table 3). We determined whether serum HHV-8 DNA was present in donors at the time of donation and in recipients at seroconversion. HHV-8 DNAemia did not persist, and we detected HHV-8 DNAemia in any BMT recipient only one time. Seroconversion and presence of HHV-8 DNA were not associated with manifestations of HHV-8 infection in our recipients during a median follow-up of 6 years (range = 1 month–12 years). In transplantation patients, molecular studies have shown that a renal graft can be the source of HHV-8 infection (21). A limitation of our study was that molecular studies in our population, however, were hampered by the lack of HHV-8 DNA–positive donor–recipient pairs.


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Table 2.  HHV-8 serology of recipients and their donors before and after bone marrow transplantation (BMT)

 

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Table 3.  Characteristics of the 19 pairs of recipients and donors who seroconverted*

 
In our study population of 19 seroconverters, seroconversion was more frequent and nearer to receipt of the transplant among the five seronegative recipients from seropositive donors. Moreover, seroconversion in the presence of serum HHV-8 DNA was detected in one recipient 180 days after BMT, and 10 seroconversions were detected among seronegative recipients from seronegative donors. Thus, sources other than the graft, such as blood products (911) or saliva (22), may be involved in transmission of HHV-8 to BMT recipients and should evaluated further in future molecular studies.

The fact that 18 of 19 seroconverters were male deserves to be mentioned. Even if surprising, a similar trend was observed in a cohort of 287 renal transplant recipients (male/female ratio = 1.8) in which five of six HHV-8 seroconverters were male (23). It is of interest that none of the recipients in that cohort or in our population developed Kaposi sarcoma. Whether this observation is related to the receipt of an organ transplant or due to chance requires additional research.

Seroreversion after BMT occurred in 11 (55%) of 20 recipients who were HHV-8 seropositive before BMT (Table 2). Seroreversion was detected in eight recipients by the use of antibodies to lytic antigen, in two by the use of antibodies to latent antigen, and in one recipient by the use of antibodies to both antigens. Antibody to lytic antigens could be detected at a dilution of 1 : 40 (in nine recipients), and antibody to latent antigens could be detected at a dilution of 1 : 100 (in three recipients). The rate of seroreversion was similar to that already reported in another cohort of BMT recipients, by use of an immunofluorescence assay for antibodies to latent antigens (24).

In our study population, six recipients who became HHV-8 seropositive 30 days after BMT were seronegative at day 180 (Table 3). Seroreversion to HHV-8 antigens has been previously described (25) in renal transplant recipients, in allogeneic stem cell transplant recipients (13), and in HIV patients with or without Kaposi sarcoma (2629). Quinlivan et al. (27) reported that, in a longitudinal analysis of in an HIV cohort, seroreversion (81% using an immunofluorescence assay and 42% using an enzyme-linked immunosorbent assay) was detected 4.7 years before a high rate of Kaposi sarcoma was observed. Possible explanations for the frequency of this observation in our recipients include factors relating to the sensitivity of serologic tests, to serum storage, to potential differences in the immunologic function in our recipients, or to variability in immunoreactivity to HHV-8 antigens (28,29) in allogeneic BMT recipients. Longitudinal studies on HIV- and HHV-8–seropositive patients with and without Kaposi sarcoma reported changing profiles of antibody reactivity to HHV-8 over time, suggesting an incomplete recognition of latent or lytic epitopes after primary (28) and reactivated (29) infection. Furthermore, seroreversion was observed in HIV-positive patients with detectable HHV-8 DNA in oral fluid and/or in peripheral blood mononuclear cells (29). Unfortunately, the natural history of serologic reactivity to HHV-8 over time in asymptomatic healthy seropositive subjects is not clearly understood, because, to our knowledge, there have been no longitudinal studies on HHV-8 serology in populations that are not at risk of developing Kaposi sarcoma or not immunocompromised.

To our knowledge, this is the first description of seroconversion with detection of HHV-8 DNA in serum of allogeneic BMT recipients. This finding is not completely unexpected in immunocompromised patients because cell-free HHV-8 DNA in serum has been detected close to the time of seroconversion in 10% of the primary HHV-8 infections among homosexual HIV-infected and HIV-uninfected individuals (30). However, HHV-8 DNA has rarely been detected in plasma or serum from individuals without Kaposi sarcoma (31,32) or blood donors in whom the presence of serum HHV-8 DNA has been reported almost exclusively in African subjects (1433).

In conclusion, we provide evidence that HHV-8 seroconversion is relatively common among seronegative BMT recipients who received a BMT from a seropositive donor. However, there is also a possibility that factors other than receipt of a BMT may contribute to seroconversion in BMT recipients. Fortunately, seroconversion does not appear to be associated with overt clinical manifestations, at least in our series of recipients. Thus, cofactors that contribute to the development of Kaposi sarcoma and other HHV-8–associated diseases in HHV-8–seropositive individuals, other than being of the male sex, ethnic origin, and being immunocompromised, are still elusive.


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Preliminary data from this study were presented in part at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC; San Diego, CA), Washington, DC: American Society for Microbiology, 2002 (abstract V-930).


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Manuscript received November 23, 2004; revised May 4, 2005; accepted May 19, 2005.



             
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