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).
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ABSTRACT |
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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 -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-8seropositive 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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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-8seropositive 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-8associated diseases in HHV-8seropositive individuals, other than being of the male sex, ethnic origin, and being immunocompromised, are still elusive.
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Manuscript received November 23, 2004; revised May 4, 2005; accepted May 19, 2005.
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