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Detection of Epstein-Barr Virus, but not Human Herpesvirus 8, DNA in Cervical Secretions From Swedish Women by Real-Time Polymerase Chain Reaction

ENBOM, MALIN MD, PhD*; STRAND, ANDERS MD; FALK, KERSTIN I. PhD* AND; LINDE, ANNIKA MD, PhD*

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Background Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8) are two related herpesviruses that may be sexually transmitted.

Goal To examine the presence of HHV-8 and EBV DNA in the female genital tract.

Study Design  Real-time polymerase chain reaction systems for quantification of DNA from HHV-8, EBV, and herpes simplex virus type 2 were developed and used for examination of cervical secretions from 112 Swedish women. HHV-8, EBV, and herpes simplex virus type 2 serology was also performed on samples from all subjects.

Results EBV DNA was found in 10 cervical secretion samples, sometimes in high amounts. No cervical secretion or leukocyte sample contained detectable HHV-8 DNA. Antibodies to HHV-8-latent and -lytic antigens were found in 2.7% and 24% of serum samples, respectively.

Conclusion This study supports a possible sexual route of transmission for EBV but not for HHV-8. The new real-time polymerase chain reaction systems could be valuable in future studies of relations between virus load and disease.

From the *Department of Virology, Swedish Institute for Infectious Disease Control and Microbiology and Tumor Biology Center, Karolinska Institute, Solna, Sweden; and the †Department of Medical Sciences, Dermatology and Venereology, Uppsala University Hospital, Uppsala, Sweden

Reprint requests: Malin Enbom, MD, PhD, Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden. E-mail: malin_enbom@hotmail.com

Received for publication July 11, 2000, revised September 6, 2000, and accepted September 13, 2000.

EPSTEIN-BARR VIRUS (EBV) and human herpesvirus 8 (HHV-8) are two related viruses classified as γ-herpesviruses. Human herpesvirus 8 was discovered in 1994 1 and is believed to be the causative agent of Kaposi sarcoma (KS). The virus is found in all forms of KS 2–5 and is associated with body cavity-based lymphoma 6 and multicentric Castelman disease. 7 Both EBV and HHV-8 are B-lymphotropic viruses, and as with all herpesviruses, they establish latency in their host after primary infection. The seroprevalence of HHV-8 is uncertain because different serologic assays give varying antibody frequencies and are shown to correlate poorly with when analyzing the same samples. 8,9 For northern Europe and the United States, different studies report antibody frequencies between 0.5% and 25%, 8,10–12 whereas the HHV-8 seroprevalence in Africa is probably higher (i.e., 30–60%). 13 Several studies strongly suggest that HHV-8 transmission among homosexual men occurs during sexual contact. 14–17 There are also indications of sexual transmission between heterosexuals, but not all studies confirm this finding. 18–21 However, the high seroprevalence among children in Africa suggests other routes of transmission, such as vertical, blood borne, or social.

EBV is a ubiquitous virus with a seroprevalence of more than 95% in the adult population. 22 It is known to spread through saliva, but a possible sexual transmission was suggested when EBV DNA sequences were detected in the uterine cervix of the female genital tract. 23,24 Primary infection is most commonly asymptomatic, but may cause infectious mononucleosis and reactivation in the immunocompromised host, and may be associated with lymphoproliferative disease. 22 The virus is known to spread through saliva, but a possible sexual transmission has also been suggested because viral DNA sequences have been detected in the male and female genital tract. 24,25 Sequence analysis has defined two strains of EBV named 1 and 2 (alternatively, type A and B) that differ mainly at the domains that code for EBV-latent proteins. Previous studies have reported variable frequencies of these EBV subtypes in blood or throat washings, but type 1 generally dominates in healthy individuals in Europe and the United States, whereas HIV-infected and African patients have a higher frequency of EBV-2. 26 Subtype 2 is suggested to be sexually transmitted, whereas subtype 1 is spread by saliva 27; however, the distribution of EBV subtypes in the genital tract has not been investigated.

The aim of this study was to investigate the presence of HHV-8 and EBV in the genital tract of Swedish women to further evaluate a sexual mode of transmission for these viruses. This investigation was done using real-time polymerase chain reaction (PCR) analysis of cervical secretions from 112 women to detect and quantify viral DNA. To further ascertain that the quality of the samples and DNA extraction was sufficient, we also performed real-time PCR for herpes simplex virus type 2 (HSV-2), which is known to be shed intermittently from the genital tract of infected women. 28 We also performed serologic assays to detect antibodies to HHV-8, EBV, and HSV-2 in all serum samples.

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Materials and Methods

Patient Samples

One hundred and twelve women attending a venereologic clinic at Uppsala University Hospital, Uppsala, Sweden participated in the study. Data regarding demographics and sexual contacts were collected through a questionnaire. The median age of participants was 24 years (range, 18–49 years). All but two participants of African and the Middle Eastern descent were European, and none was known to be HIV positive. The median number of lifetime sexual partners was seven (range, 1–25, data available from 81 subjects) and the median number of sexual partners the past 12 months was two (range, 0–10, data available from 106 subjects). After obtaining informed consent, serum, whole-blood, and cervical secretions were collected from each subject.

The whole-blood sample was separated into one peripheral mononuclear cell (PBMC) fraction and one granulocyte fraction using Ficoll-Hypaque (Pharmacia Biotech AB, Uppsala, Sweden). Erythrocyte cells were lysed with a lysis buffer containing 0.15 mol/l NH4Cl, 10 mmol/l KHCO3, and 10 mmol/l EDTA. The cervical secretions were collected with a Cytobrush and suspended in 1 to 2 ml saline. The suspension was centrifuged at 1500 rpm for 5 minutes and pellet and supernatant were stored separately. All samples were processed on the collection day and stored at −20 °C until analyzed.

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HHV-8 Serology

The HHV-8 serology was performed with immunofluorescence assays for antibodies to latent and lytic HHV-8 antigens, as described previously. 29 Two B-cell lines (BCP-1 and BCBL-1) latently infected with HHV-8 were used, and expression of lytic antigens was induced by adding phorbol 12-tetradecanoate 13-acetate (TPA) at 20 ng/ml for 48 hours. The cells were spotted on masked slides, air dried, and fixed in water-free acetone for 5 minutes. The slides were incubated with serum dilutions (1:20–1:1280) for 1 hour at 37 °C, washed, and incubated with a FITC-labeled conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at 37 °C before they were mounted and read in a fluorescence microscope.

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HSV-2 Serology

The HSV-2-specific seroreactivity was detected using an enzyme-linked immunosorbent assay with synthetic peptides from HSV-2 glycoprotein G-2 and a small amount native glycoprotein G-2 as antigen, as described previously. 30,31

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EBV Serology

Antibodies to EBV viral capsid antigen (VCA) and early antigens (EA) were detected by immunofluorescence, as described previously. 32 Immunoglobulin G reactivity to Epstein-Barr nuclear protein-1 was determined using an enzyme-linked immunosorbent assay with a synthetic peptide of the protein (p107) as antigen using a previously published method. 33

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Pretreatment of Samples Before PCR

The DNA was extracted from the cervical secretion pellet using the QiAmp Blood Kit (Qiagen Gmbh, Hilden, Germany) following the manufacturer’s instructions. A negative control was included each time DNA extraction was performed to control for possible contamination.

The PBMCs were diluted to 20,000 cells/μl in a lysis buffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA, 0.5% Nonidet P 40, 0.5% Tween 20, 300 μg/ml proteinase K) and incubated at 37 °C overnight. Before PCR analysis, the lysate was heated to 95 °C for 10 minutes to inactivate proteinase K.

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HHV-8–Nested PCR

A nested PCR amplifying a fragment of the HHV-8 ORF26 gene was performed on PBMC and cervical secretions from all patients. The outer set of primers were 5′-AGCCGAAAGGATTCCACCA-3′ and 5′-TCCGTGTTGTCTACGTCCAG-3′, and the inner set of primers were 5′-ACGGATTTGACCCCGTGTTC-3′ and 5′-AATGACACATTGGTGGTATA-3′, amplifying a final product of 160 bp. In each tube, 10-μl samples were used, giving a total volume of 50 μl and reaction concentrations of 10 mmol/l Tris-HCl, 50 mmol/l NaCl, 1.5 mmol/l MgCl, 0.2 mmol/l dNTP, 0.2 μmol/l, and 50 nm outer primers/200 nm inner primers. Thermal cycle conditions were 94 °C for 4 minutes, 35 cycles of 94 °C for 45 seconds, 55 °C for 1 minute 15 seconds, and 72 °C for 1 minute 15 seconds, and 72 °C for 2 minutes for step one. For step two, the conditions were 94 °C for 4 minutes, 25 cycles of 94 °C for 45 seconds, 60 °C for 1 minute 15 seconds, and 72 °C for 1 minute 15 seconds, and 72 °C for 2 minutes. All samples were tested in duplicates. For each sample, a third tube was prepared in which 1 μl positive control was added to the sample to be analyzed to control for possible inhibitory substances in the sample giving a false-negative result. If a sample result was positive in one of the duplicates, it was retested; only repeatedly positive samples were considered true-positive results.

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Quantitative PCR

Real-time (TaqMan) PCR was used for detection and quantification of EBV, HHV-8, and HSV-2 DNA. This method is based on a primer pair and an oligonucleotide probe with a reporter-dye 6-carboxyfluorescein (FAM) attached to the 5′ end and a tetramethyl rhodamine (TAMRA) quencher linked to the 3′ end. 34 The exonuclease activity of the DNA polymerase releases the reporter molecule as elongation of the DNA chain occurs. This activity is seen as an increase in reporter fluorescence, which can be detected by a luminescence spectrometer. A threshold cycle value (Ct) is calculated for each sample by determining the point at which the fluorescence exceeds the threshold limit chosen for the specific plate.

Primers and probes were designed with the Primer Express software (primers and probe for the HSV-2 system designed by Dr. A. Allard, Department of Virology, Umeå University, Umeå, Sweden) (Table 1). To each well of a 96-well plate, 5 μl sample and 20 μl PCR mixture consisting of 12.5 μl Universal PCR Mastermix (PE-Applied Biosystems, Cheshire, UK) and primers and probe of various concentrations were added. The Mastermix contained uracil-DNA glycosylase (UNG), which eliminates previously amplified PCR products, thereby protecting against carryover contamination. For the EBV system, magnesium chloride was added to the reaction mixture to a final concentration of 6.5 mmol/l. Cycling parameters were as follows: 50 °C for 2 minutes, 95 °C for 10 minutes, 50 cycles of 95 °C for 15 seconds, and 60 °C for 1 minute A negative and a positive control were included in triplicates on each plate. The samples were analyzed in triplicates, including one inhibition control to which 0.5 μl positive control was added to control for inhibitory substances in the samples giving a false-negative result.

Table 1

Table 1

The number of genomes in each sample was calculated from an EBV DNA standard. The DNA standard used was extracted DNA (QiAmp Blood kit, Qiagen Gmbh) from the EBV-infected Burkitt lymphoma cell line Namalwa, which carries two integrated EBV genomes per cell. 35 Each cell has a total DNA content of 6.7 pg, 36 and by spectrophotometric measuring of the DNA amount in the extracted material, the number of EBV genomes/μl could be calculated. Four 10-fold dilutions (1 × 103–1 × 106 genomes/ml) of this DNA standard together with EBV primers and probe were included on each plate, giving a standard curve from which the number of genomes in all samples could be calculated. The standard curve was created automatically by the ABI 7700 Sequence Detection System software by plotting the Ct values against each known concentration of the EBV standard.

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Subtyping of EBV

For cervical secretions containing detectable EBV DNA, EBV subtyping was performed by PCR amplification and sequencing of a fragment of the EBNA-6 gene, as described previously. 37,38 This part of the EBV genome contains a n × 39 base pair repeat in EBV-1 isolates but not in EBV-2 isolates, which makes it possible to distinguish the two subtypes by analyzing the DNA sequence. 37

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Results

HHV-8 Serology

Among the 112 women tested, antibodies to HHV-8 latent antigens were found in 3 (2.7%) and antilytic antibodies were found in 27 (24%). The three women with antilatent antibodies had antilytic antibodies. The median titers were 20 (range, 20–40) for antilatent antibodies and 80 (range, 20–1280) for antilytic antibodies. There was no correlation between the number of sexual partners (total number or number in past 12 months) and HHV-8 serostatus (P > 0.05, Mann-Whitney U test).

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HSV-2 Serology

A total of 13 (12%) of 112 subjects had immunoglobulin G to HSV-2. The HSV-2–seropositive group was significantly older than the HSV-2–seronegative group (P = 0.02, Mann-Whitney U test), and among 49 women older than 25 years, 10 (20%) had HSV-2 antibodies compared with 3 of 63 (4.8%) women younger than 25 years (P = 0.01, Fisher exact test). There was no correlation between the number of sexual partners and HSV-2 serostatus (P > 0.05, Mann-Whitney U test).

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EBV Serology

Of the 112 subjects, antibodies to EBV VCA were found in 110 (98%) and antibodies to the EBNA peptide p107 were found in 108 (96%). The median titer was 1,280 (range, 0–10,240) for antibodies to VCA; the mean OD value in the p107 enzyme-linked immunosorbent assay was 1.0 (range, 0.12–1.4). Eighteen of 112 (16%) women had antibodies to EBV EA, with a median titer of 80 (range, 20–640). Anti-EA antibodies were significantly more common in women with detectable EBV DNA in cervical secretions [5/10 (50%)] than in those without detectable EBV DNA [13/107 (12%)] (P < 0.01, Fisher exact test). Presence or titer levels of antibodies to EBV VCA or p107 did not correlate significantly with detectable EBV DNA in cervical secretions.

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HHV-8, EBV, and HSV-2 PCR

All PBMC and cervical secretions from all women were negative in the nested HHV-8 PCR. The cervical secretions from HHV-8 seropositive women were also analyzed with the quantitative HHV-8 PCR, and were negative in that assay.

A total of 10 (8.9%) of 112 cervical secretions contained detectable EBV DNA. The median number of genomes was 5,700 (range, 990–3,000,000) genomes/ml. The patients with detectable EBV DNA did not have any obvious common symptoms. Acetowhite lesions were not routinely looked for.

Cervical secretions from 2 of the 13 HSV-2–seropositive subjects contained detectable HSV-2 DNA with the concentrations 190 genomes/ml and 860 genomes/ml, respectively. These two patients had clinical symptoms indicative of HSV reactivation, such as itching, stinging, and ulcers.

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EBV Subtyping

Because of insufficient amounts of DNA, only 5 of the 10 cervical secretions containing detectable EBV DNA could be subtyped. Of those five, three were type 1 and two were type 2.

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Relations Between Different Analyses

There was no significant correlation between presence of antibodies to HSV-2 and antibodies to HHV-8 (P > 0.05, Fisher exact test).

Six (60%) of the ten subjects with EBV DNA in cervical secretions had detectable antibodies to HHV-8 in serum, compared with 21 of 102 (21%) women without EBV in the cervix (P = 0.01, Fisher exact test, Table 2). No patient had both detectable EBV and HSV-2 DNA in cervical secretions.

Table 2

Table 2

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Discussion

The subjects included in this study represent consecutive patients attending a venereologic clinic, and were not selected because of high-risk behavior for acquiring sexually transmitted diseases. However, it is likely that these women have been exposed to more risk factors than female blood donors. In a previous study of HHV-8 seroprevalence among Swedish blood donors using the same serologic methods, we found that none of the females had antibodies to latent antigen, and 9 of 58 (16%) subjects had antibodies to HHV-8 lytic antigen. 8 In the current study, Swedish women of similar age tended to have a higher HHV-8 seroprevalence (P = 0.07, Fisher exact test) with 3 of 112 (2.7%) subjects having antibodies to latent antigen, and 27 of 112 (24%) subjects having antibodies to lytic antigen. There was no correlation between the number of sexual partners and HHV-8 serostatus, in contrast to what has been reported previously. 18,21 However, that data regarding total number of sexual partners were available from only 72% of the women may have affected this calculation.

The HHV-8 DNA was not detected in any cervical secretion sample with nested PCR or real-time PCR. In two previous studies, HHV-8 DNA was detected only rarely in cervical secretions from HIV-infected and HIV-uninfected women, 39,40 but in one Chinese study, HHV-8 DNA was found in 9.2% of cervical cells with normal histology. 41 It is possible that HHV-8 is shed only intermittently in infected persons, as is known for HSV-2, and the frequency of detectable HHV-8 DNA in the genital tract might be underestimated unless a study of consecutive samples from the same subjects is performed. The frequency with which HHV-8 DNA can be detected in semen samples has been debated, but recent reports agree on a low frequency of HHV-8 DNA in semen, even in KS patients. 42

It is possible that a larger group of samples, or a selection of persons with high-risk sexual behaviors, would have increased the borderline significance to show a difference in HHV-8 serostatus between the present group of women and female blood donors, as we reported in a previous study where men and women were included. 8 However, the serologic results together with the PCR findings give no support to a frequent sexual transmission of HHV-8.

The HSV-2 seroprevalence of 12% was somewhat lower than what might be expected, because previous studies found approximately 30% of Swedish women to have antibodies to HSV-2. 43 This finding might be due to the relatively low median age (24 years) of this group; the HSV-2–seropositive group was significantly older than the HSV-2–seronegative group, and among persons older than 25 years, 10 of 49 (20%) had HSV-2 antibodies compared with 3 of 63 (4.8%) women younger than 25 years. A correlation between HSV-2 and HHV-8 seropositivity has been reported by others, 20 but this could not be confirmed in this study. Rather, there was a tendency toward a higher HHV-8 seroprevalence in the HSV-2–seronegative group, but this difference was not significant.

Two (15%) of the thirteen women with antibodies to HSV-2 had detectable HSV-2 DNA in cervical secretions. This finding is similar to what has been reported previously, 28,44,45 which suggests that our samples and DNA extraction method were of sufficient quality to detect virus shed from the uterine cervix.

The EBV DNA was detected in 8.9% of the cervical secretions, sometimes in surprisingly high amounts. Whether this finding represents EBV infection of the epithelial cells in the uterine cervix or infected leukocytes in the mucosa cannot be judged, but presence of a high EBV load in the genital tract suggests that the virus could be transmitted through sexual contact. The virus is associated with a number of human neoplasms, but it is not known if presence of EBV in the cervix is related to any disease. An association with cervical tumor pathogenesis and acetowhite lesions on the vulva has been proposed, but not all studies have been able to confirm this finding. 46–48 Future studies using new real-time PCR could reveal if there is an association not only to presence but also to quantity of EBV genomes and disease. Our results also show that both EBV-1 and EBV-2 can be detected in the uterine cervix, but the small material does not allow any firm conclusions on whether the prevalence of the two subtypes differs in this location compared with peripheral blood or oropharynx. It would be of interest to compare EBV subtypes derived from, for example, blood, saliva, and the genital tract of the same person.

The EBV serology showed that, with the exception of two women, all subjects had signs of previous EBV infection, which is normal in adults. Antibodies to EBV EA are indicative of EBV reactivation. Interestingly, antibodies to EA were significantly more common in those who also had detectable EBV DNA in cervical secretions. Although this sample was small, it might indicate that the reactivation of EBV leading to raised antibody titers to EA might occur not only in the oropharynx, as known previously, but also in other locations such as the uterine cervix.

Six (60%) of the ten subjects with EBV DNA in cervical secretions had detectable antibodies to HHV-8 in serum, compared with 21 of 102 (21%) for women without EBV in the cervix. Serologic cross-reactions between EBV and HHV-8 have been excluded in several studies, and are unlikely to explain this finding. 8,11,12 Rather, it might point at common risk factors for contracting HHV-8 and shedding EBV in the genital tract, but this sample is too small for any firm conclusions.

The TaqMan system is a highly sensitive and specific method for DNA quantification that allows a high number of samples and is less labor intensive and more informative than an ordinary nested PCR. The closed system and the use of dUTP in the reaction mix also make the assay less sensitive to contamination problems. Quantification of viral load has been shown to be of clinical importance for several agents, such as HIV, hepatitis C, and EBV. 49,50 In this study, we describe new real-time PCRs for HHV-8, EBV, and HSV-2 that we think can be valuable for diagnostic and research purposes. It has been shown that increased EBV load in plasma can predict lymphoproliferative disorders in transplant patients, 50 and would be of interest to investigate whether quantification of HHV-8 genomes in immunosuppressed persons correlates to development of KS or other possibly HHV-8–associated malignancies.

In conclusion, we found no evidence for a frequent sexual transmission of HHV-8 in women. However, EBV was present in high amounts in the genital tract, indicating that a sexual route of transmission and relation to genital disease is possible. Future studies using real-time PCR will be useful in determining the role of different herpesviruses in human disease.

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