Correspondence

Absence of HHV-8 in Prostate and Semen

N Engl J Med 1996; 335:1237-1239October 17, 1996

Article

To the Editor:

Monini et al. (May 2 issue)1 reported very high rates of human herpesvirus 8 (HHV-8) detection by nested polymerase chain reaction (PCR) in semen from patients undergoing surgery for varicocele who were negative for the human immunodeficiency virus (HIV). The viral sequences were detected mainly in the nonspermatic fraction of semen and in specimens from prostate-gland biopsies, suggesting that the latter may be a site of latent or persistent virus in the infected host and that the sexual route is potentially implicated in the transmission of HHV-8. Monini et al. conclude that HHV-8 infection is ubiquitous, since it is present so often in semen from healthy Italian men.

We too were unable to detect HHV-8 by nested PCR in testicular samples obtained at autopsy from patients with AIDS-associated Kaposi's sarcoma, whereas the rate of viral detection in their prostate glands was 100 percent.2 However, our data do not show a general diffusion of HHV-8 in the male population. In fact, in 20 drug abusers with AIDS and in 8 HIV-negative subjects without Kaposi's sarcoma, HHV-8 was not detectable by PCR in either the prostate gland or other sites of latent or persistent virus in infected hosts (i.e., secondary lymphoid organs, bone marrow, normal skin, or paravertebral sensory ganglia).2-4

In addition, we screened for HHV-8 DNA sequences in sperm samples from 20 healthy HIV-negative donors from Milan (kindly provided by Dr. Augusto Semprini, San Paolo Hospital, Milan, Italy). Semen was studied in toto and after Percoll-gradient separation into three fractions (seminal plasma, motile spermatozoa, and round cells). The round cells, together with immature spermatozoa, contain mononuclear cells of the genital epithelium. Specimens were extracted by standard phenol–chloroform methods, and 1 μg of DNA was loaded in each PCR assay. The integrity of the DNA was assessed by human β-globin PCR amplification. To reduce the likelihood of false negative results (i.e., PCR “dropouts,” which may occur when amplifying target molecules present at the start in extremely low numbers of copies per test volume), each sample was tested at least in quintuplicate individual PCR runs or, if the amount of extracted DNA was not enough, until the sample had been exhausted. Under these conditions, the probability of a false negative PCR result, according to the Poisson distribution, is 0.67 percent.5 Nested, hot-start PCR was allowed to proceed for 40 plus 40 amplification cycles, as described elsewhere.2

None of the semen samples were positive for HHV-8. Thus, our findings do not support the conclusions drawn by Monini et al. about the ubiquitousness of HHV-8 infection.

It is well known that Mediterranean Kaposi's sarcoma is more frequently encountered in certain areas of Italy, such as Ferrara, than in others, such as Milan. This may explain the discrepancy between our results and those reported by Monini et al., although the large differences in the detection rate (0 percent in our study vs. 91 percent in their study) is difficult to reconcile. Assays based on PCR can have poor accuracy and performance, even in the diagnosis of HIV infection, for which PCR has been used extensively for nearly a decade.6

Large-scale serologic screening is therefore necessary to provide a conclusive answer about the true prevalence of HHV-8 infection in the general population, especially in countries such as Italy, where one finds both the Mediterranean and AIDS-associated forms of Kaposi's sarcoma.

Mario Corbellino, M.D.
Giovanna Bestetti, M.D.
Massimo Galli, M.D.
University of Milan

Carlo Parravicini, M.D.
Luigi Sacco Hospital, 20157 Milan, Italy

6 References

1. 1

   Monini P, de Lellis L, Fabris M, Rigolin F, Cassai E. Kaposi's sarcoma-associated herpesvirus DNA sequences in prostate tissue and human semen. N Engl J Med 1996;334:1168-1172
   Full Text | Web of Science | Medline

2. 2

   Corbellino M, Poirel L, Bestetti G, et al. Restricted tissue distribution of extralesional Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS patients with Kaposi's sarcoma. AIDS Res Hum Retroviruses 1996;12:651-657
   CrossRef | Web of Science | Medline

3. 3

   Corbellino M, Parravicini C, Aubin JT, Berti E. Kaposi's sarcoma and herpesvirus-like DNA sequences in sensory ganglia. N Engl J Med 1996;334:1341-1342
   Full Text | Web of Science | Medline

4. 4

   Corbellino M, Poirel L, Aubin JT, et al. Human herpesvirus-8 DNA sequences and Epstein-Barr virus in the pathogenesis of giant lymph-node hyperplasia (Castleman's disease). Clin Infect Dis (in press).
   

5. 5

   Greenfield L, White TJ. Sample preparation methods. In: Persing DH, Smith TF, Tenover FC, White TJ, eds. Diagnostic molecular microbiology: principles and applications. Washington, D.C.: American Society for Microbiology, 1993:122-37.
   

6. 6

   Owens DK, Holodniy M, Garber AM, et al. Polymerase chain reaction for the diagnosis of HIV infection in adults: a meta-analysis with recommendations for clinical practice and study design. Ann Intern Med 1996;124:803-815
   Web of Science | Medline

To the Editor:

Monini and colleagues report that with the use of nested PCR, Kaposi's sarcoma–associated herpesvirus (KSHV) was found to be ubiquitous in urogenital and prostate tissues and sperm from healthy Italian adults. We performed a study to detect KSHV in normal prostatic specimens from 10 Italian men and 10 men from the United States, as well as in 32 prostatic-, 30 vulvar-, and 20 cervical-cancer specimens from patients in the United States. None of the subjects had a history of HIV infection. The quality of DNA extracted from paraffin-embedded tissue was tested by amplifying for an endogenous gene (glyceraldehyde-3-phosphate dehydrogenase), which is present at two copies per diploid genome.

The samples were tested by nested PCR (35 plus 35 cycles). We used the same nested primers used by Monini et al., resulting in a 160-bp band. The sensitivity of this assay was determined by a dilution study, which was performed by diluting KSHV DNA from the KS1 cell (a primary-effusion-lymphoma cell line estimated to have 16 copies of KSHV per cell)1 in DNA from a K562 myeloblastic cell line. The nested PCR could detect about 2.4 copies of KSHV sequences on a background of 1 μg of DNA from the K562 cell line, which means that our assay can detect 1 copy of KSHV in 62,500 KSHV-negative cells.

All samples studied were negative for KSHV sequences (Figure 1Figure 1Nested-PCR Analysis of KSHV Sequences in DNA from Urogenital Specimens Obtained from Italian and American Subjects.). Southern blot analysis of all nested-PCR samples, with the use of an internal ³²P-labeled probe, was also negative (data not shown). Therefore, we cannot confirm the finding reported by Monini and colleagues that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between our results and those of Monini et al. is unlikely to be explained by either ethnic or environmental factors. False positive results can easily occur with nested-primer PCR because of contamination. Our results argue against the suggestion that KSHV is a widespread virus in urogenital and prostate tissues in healthy persons.

Taizo Tasaka, M.D.
Jonathan W. Said, M.D.
H. Phillip Koeffler, M.D.
Cedars–Sinai Research Institute, Los Angeles, CA 90048

1 References

1. 1

   Said W, Chien K, Takeuchi S, et al. Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) in primary effusion lymphoma: ultrastructural demonstration of herpesvirus in lymphoma cells. Blood 1996;87:4937-4943
   Web of Science | Medline

Author/Editor Response

The authors reply:

To the Editor: Tasaka et al. rule out ethnic or environmental factors in the epidemiology of HHV-8 infection. On the other hand, Corbellino et al. speculate that the high prevalence of HHV-8 DNA in sperm from donors in Ferrara, Italy, may be related to a high risk of Mediterranean, or classic, Kaposi's sarcoma. We have now analyzed new sperm samples from a total of 15 persons with AIDS or AIDS-associated Kaposi's sarcoma and normal donors from Rome, where classic Kaposi's sarcoma is infrequent. We detected viral DNA in semen from only one of five patients with Kaposi's sarcoma. Subsequently, we received sperm samples from 13 consecutive male donors in Ferrara who had clinical conditions such as varicocele or infertility; those with a known history of HIV infection were not included.

Each of two operators processed a set of eight sperm samples in such a way that three specimens were independently processed twice. An aliquot of DNA from salmon sperm was extracted for every two human sperm samples. When the DNA was diluted for PCR, one negative-control sample was included for every three DNA samples. When PCR was performed, one negative-control assay was done for every three DNA assays.

Three specimens were found to be positive with the pair of nested-PCR primers used in our previous study.1 One of the positive samples belonged to the three specimens that had been processed twice and tested positive with both operators. A second set of primers, amplifying a DNA region far away on the viral genome, was used in a double-blind analysis. The same three samples were positive. None of the other specimens or control samples were positive. In another analysis, two of seven hyperplastic-prostate specimens (29 percent) from patients without Kaposi's sarcoma were positive with two different sets of primers.

Besides contamination, a discrepancy in the results of different studies can be due to differences in enrollment criteria, sample processing, PCR performance, or other factors. For example, we analyzed relatively large urogenital-tissue fragments, whereas Tasaka et al. processed micrometric sections from paraffin-embedded tissues. This is an important difference, because persistent viral infection can be restricted to small cell foci. In addition, PCR has poor results with fixed tissues. When archival specimens are analyzed, the sensitivity of PCR should be determined with positive-control samples fixed and embedded accordingly.

A recent report describes the presence of HHV-8 DNA in peripheral-blood mononuclear cells (PBMCs) from normal Italian donors.2 Indeed, the prevalence of HHV-8 DNA in PBMCs from normal donors may differ between the United States and Italy (13 percent in the United States [Ensoli B: personal communication] and 6 percent in Italy2). In contrast to the findings of Corbellino et al., HHV-8 was also detected in two of nine bone marrow samples from normal U.S. donors, as well as in PBMCs from patients with AIDS but without Kaposi's sarcoma3,4 (and Colombini S, et al.: personal communication). The presence of HHV-8 DNA in semen from patients who have AIDS with or without Kaposi's sarcoma and from normal donors was first reported in Atlanta by Lin et al.5 These data suggest that, at least in some geographic areas, HHV-8 is not restricted to patients with clinical conditions associated with viral infection — an observation that has been made with other human herpesviruses.

Paolo Monini, Ph.D.
Laura de Lellis, Ph.D.
Enzo Cassai, Ph.D.
University of Ferrara, 44100 Ferrara, Italy

5 References

1. 1

   Monini P, de Lellis L, Fabris M, Rigolin F, Cassai E. Kaposi's sarcoma-associated herpesvirus DNA sequences in prostate tissue and human semen. N Engl J Med 1996;334:1168-1172
   Full Text | Web of Science | Medline

2. 2

   Bigoni B, Dolcetti R, de Lellis L, et al. Human herpesvirus 8 is present in the lymphoid system of healthy persons and can reactivate in the course of AIDS. J Infect Dis 1996;173:542-549
   CrossRef | Web of Science | Medline

3. 3

   Humphrey RW, O'Brien TR, Newcomb FM, et al. Kaposi's sarcoma (KS)-associated herpesvirus-like DNA sequences in peripheral blood mononuclear cells: association with KS and persistence in patients receiving anti-herpesvirus drugs. Blood 1996;88:297-301
   Web of Science | Medline

4. 4

   Decker LL, Shankar P, Khan G, et al. The Kaposi sarcoma-associated herpesvirus (KSHV) is present as an intact latent genome in KS tissue but replicates in the peripheral blood mononuclear cells of KS patients. J Exp Med 1996;184:283-288
   CrossRef | Web of Science | Medline

5. 5

   Lin J-C, Lin S-C, Mar E-C, et al. Is Kaposi's-sarcoma-associated herpesvirus detectable in semen of HIV-infected homosexual men? Lancet 1995;346:1601-1602
   CrossRef | Web of Science | Medline

Citing Articles (18)

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   Elisa Martró, Anna Esteve, Thomas F. Schulz, Julie Sheldon, Gemma Gambús, Rafael Muñoz, Denise Whitby, Jordi Casabona. (2007) Risk factors for human Herpesvirus 8 infection and AIDS-associated Kaposi's sarcoma among men who have sex with men in a European multicentre study. International Journal of Cancer 120:5, 1129-1135
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   J.D. Montgomery, L.P. Jacobson, R. Dhir, F.J. Jenkins. (2006) Detection of humanherpesvirus 8 (HHV-8) in normal prostates. The Prostate 66:12, 1302-1310
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   Slvia de Sanjos, Vickie Marshall, Judit Sol, Virgilio Palacio, Rosa Almirall, James J. Goedert, F. Xavier Bosch, Denise Whitby. (2002) Prevalence of Kaposi's sarcoma-associated herpesvirus infection in sex workers and women from the general population in Spain. International Journal of Cancer 98:1, 155-158
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   Lawrence Corey, Scott Brodie, Meei-Li Huang, David M. Koelle, Anna Wald. (2002) HHV-8 infection: a model for reactivation and transmission. Reviews in Medical Virology 12:1, 47-63
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6. 6

   C. Boshoff, Y. Chang. (2001) K APOSI ' S S ARCOMA –A SSOCIATED H ERPESVIRUS : A New DNA Tumor Virus. Annual Review of Medicine 52:1, 453-470
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7. 7

   Pauk, John, Huang, Meei-Li, Brodie, Scott J., Wald, Anna, Koelle, David M., Schacker, Timothy, Celum, Connie, Selke, Stacy, Corey, Lawrence, . (2000) Mucosal Shedding of Human Herpesvirus 8 in Men. New England Journal of Medicine 343:19, 1369-1377
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8. 8

   M. A. Rubin, J. P. Parry, B. Singh. (1998) RE. The Journal of Urology505-506
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9. 9

   Maria Capuano, Ilaria Lesnoni La Parola, Paola Cattani, Francesca Cerimele, Francesco Sasso, Cinzia Masini, Giovanni Fadda, Decio Cerimele. (1998) RE. The Journal of Urology505
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10. 10

    M.A. Rubin, J.P. Parry, B. Singh. (1998) RE: KAPOSI'S SARCOMA ASSOCIATED HERPESVIRUS DEOXYRIBONUCLEIC ACID SEQUENCES. The Journal of Urology 160:2, 505-506
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11. 11

    Maria Capuano, Ilaria Lesnoni La Parola, Paola Cattani, Francesca Cerimele, Francesco Sasso, Cinzia Masini, Giovanni Fadda, Decio Cerimele. (1998) RE: KAPOSI'S SARCOMA ASSOCIATED HERPESVIRUS DEOXYRIBONUCLEIC ACID SEQUENCES. The Journal of Urology 160:2, 505
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12. 12

    Ruth M. Greenblatt. (1998) KAPOSI'S SARCOMA AND HUMAN HERPESVIRUS-8. Infectious Disease Clinics of North America 12:1, 63-82
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13. 13

    M.A. Rubin, J.P. Parry, B. Singh. (1998) KAPOSI'S SARCOMA ASSOCIATED HERPESVIRUS DEOXYRIBONUCLEIC ACID SEQUENCES: LACK OF DETECTION IN PROSTATIC TISSUE OF HUMAN IMMUNODEFICIENCY VIRUS-NEGATIVE IMMUNOCOMPETENT ADULTS. The Journal of Urology 159:1, 146-148
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14. 14

    Reinhold Munker, Taizo Tasaka, Dorothy Park, Carl W. Miller, H. Phillip Koeffler. (1997) HHV-8 (KSHV) does not establish latency in prostate cancer cell lines. The Prostate 33:4, 286-288
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15. 15

    Shahul H. Ebrahim, Thomas A. Peterman, Akbar A. Zaidi, Françoise F. Hamers. (1997) Geography of AIDS-associated Kaposiʼs sarcoma in Europe. AIDS 11:14, 1739-1745
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16. 16

    Jonathan W. Said, Taizo Tasaka, Sven de Vos, H Phillip Koeffler. (1997) Kaposiʼs sarcoma-associated herpesvirus/human herpesvirus type 8 encephalitis in HIV-positive and -negative individuals. AIDS 11:9, 1119-1122
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17. 17

    Enza Viviano, Francesco Vitale, Francesca Ajello, Anna Maria Perna, Maria Rosaria Villafrate, Filippa Bonura, Mario Aricò, Giovanni Mazzola, Nino Romano. (1997) Human herpesvirus type 8 DNA sequences in biological samples of HIV-positive and negative individuals in Sicily. AIDS 11:5, 607-612
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18. 18

    David J. Blackbourn, Jay A. Levy. (1997) Human herpesvirus 8 in semen and prostate. AIDS 11:2, 249-250
    CrossRef