Infection with human herpesvirus-8 (HHV-8) is common among men who have sex with men (MSM). 1–8 Although no specific behavior has consistently been associated with acquisition of HHV-8 infection, HHV-8 may be transmitted through saliva. 9 HHV-8 DNA quantity is highest in saliva; oral epithelial cells are competent to act as a source of replication for the virus; and deep kissing has been associated with HHV-8 infection. 9 In a cohort of prospectively followed MSM, a dichotomous pattern of HHV-8 shedding was noted, with some men having HHV-8 detectable in the saliva on a majority of days and others failing to demonstrate detectable virus during the 15- to 75-day collection period. If oral contact is an important mode of transmission for HHV-8, understanding the determinants of oropharyngeal viral shedding may have important ramifications for prevention of disease. Therefore, we conducted a study to evaluate the predictors of HHV-8 shedding among HIV-seropositive MSM.
MATERIALS AND METHODS
Between July 1996 and December 1998, 275 HIV-infected MSM were enrolled into a study of HIV-1 and the development of anal dysplasia as previously described. 10 Men in Seattle, WA, were recruited from outpatient clinics and advertisements in the community. Written informed consent was obtained in accordance with a protocol approved by the University of Washington Human Subjects Division.
Participants were seen from 1–6 visits over a period ranging from 6 months–3 years. At each study visit, a self-administered questionnaire was used to obtain information regarding demographic characteristics, sexual behavior, and medical histories (including any medications taken during the week prior to the study visit). Specific questions were asked regarding the duration of HIV infection, number of sexual partners, and the history of sexually transmitted infections (STIs). General physical examinations were also performed.
Specimen Collection and Laboratory Testing
Serum and blood anticoagulated with ethylenediamine tetra-acetic acid (EDTA) were collected at each study visit. Oral secretions were collected at each visit with 10 mL of sterile saline and frozen for DNA extraction as previously described. 11
Serologic assays for HHV-8 were conducted using an immunofluorescence assay (IFA) with HHV-8-positive BCBL-1 lymphoma cells. 12 Seropositivity was defined as the presence of lytic antibodies, with or without latency-associated nuclear antigen at a serum dilution of at least 1:40. All were reacted against a BJAB cell line to detect nonspecific reactivity. The first and last serum samples from each participant were tested for antibodies to HHV-8, along with all available interval specimens from men who seroconverted.
HHV-8 DNA was measured quantitatively using a fluorescent probe-based polymerase chain reaction (PCR) assay (TaqMan assay, Applied Biosystems, Foster City, CA) as described previously. 9,13–15 Oral specimens were subjected to DNA extraction, purification, and amplification with primers to the orf73 gene (ORF73-F: 5´-CCA GGA AGT CCC ACA GTG TTC-3´, ORF73-R: 5´-GCC ACC GGT AAA GTA GGA CTA GAC-3´). All samples were run with positive and negative controls. Six specimens were selected at random to assess the uniqueness of each HHV-8 DNA amplimer using heteroduplex mobility assay (HMA) to a 672-basepair section of the K1 hypervariable region. 16 Two additional specimens, representing HHV-8 DNA amplified from the same participant at 2 visits separated by 3 months in time, was also subjected to HMA to assess the intraperson variability of detected strains. Primer set K1-A,B (TGC CCT GGA GTG ATT TCA AC and AGA TGC CAA ACG GTA ACA TTA TTT C) and K1-C,D (ATA ATG TTA CCG TTT GGC ATC TAC C and TGG CAC TGT TTT GTT TGA GTC A) amplified 305- and 416-basepair fragments, respectively, as previously described. 14 PCR cycle conditions were modified to an initial cycle of 96°C for 3 minutes followed by 50 cycles of 96°C (30 seconds), 54°C (30 seconds), and 72°C (30 seconds) and ending with a single cycle of 72°C for 5 minutes. PCR products K1-A,B and K1-C,D were analyzed by heteroduplex formation as described previously. 17 To generate probe, 20 ng QIAEX II–purified BCBL-1 PCR products were end-labeled with P32 by T4 polynucleotide kinase in a final volume of 20 μL. Four to 6 μL of each nonpurified PCR reaction was hybridized to 0.5 μL of probe. Each hybridization reaction consisted of 0.5 μL of probe, 4–6 μL of PCR reaction, 200 mM NaCl, 10 mM tromethamine hydrochloride (pH 8.0), and 2 mM EDTA. After denaturation at 95°C for 5 minutes and annealing at 55°C for 2 hours, the entire hybridization reaction was loaded on 1-mm-thick mutation detection enhancement (MDE) gel (FMC BioProducts, Rockland, ME) and electrophoresed for 20 hours at 500 V. The gel was dried and exposed to x-ray film. The hybridization reactions of BCBL-1 PCR reactions to the probes were used as the marker of homoduplex formation.
HIV-1 RNA was measured using the AMPLICOR Monitor HIV-1 Test (Roche, Alameda, CA). Oral inflammation was assessed semiquantitatively with the use of leukocyte esterase chemical indicator strips (Bayer, Elkhart, IN).
Infection with HHV-8 was defined as either a positive serologic assay or the presence of HHV-8 DNA detected from saliva by PCR. Highly active antiretroviral therapy (HAART) was defined as the use of ≥3 antiretroviral agents with at least 1 protease inhibitor or nonnucleoside reverse transcriptase inhibitor at the time of the study visit. Differences between HHV-8–seropositive and seronegative MSM were assessed using χ2 tests (dichotomous variables), Mantel-Haenszel test for trend (ordered categorical variables), or t tests (continuous variables).
The analysis of HHV-8 shedding over time included all samples from seropositive subjects. Because HAART use is indicated by factors such as low CD4 count or high plasma HIV-1 RNA level, the observed association between HAART and HHV-8 shedding may be confounded by the indication for HAART use. To address this, we used inverse probability of treatment-weighted (IPTW) estimation and generalized estimating equations (PROC GENMOD, SAS Institute, Cary, NC) with a logit link to obtain parameter estimates and robust estimates of the variance. 18,19 IPTW uses the predicted probability of actual HAART received (or lack of), conditional on the potential confounders of CD4 count and plasma HIV-1 RNA level to increase or decrease the weight given to visits that might be under- (i.e., HAART use in the setting of high CD4 counts) or overrepresented (i.e., HAART use among men with low CD4 counts or high plasma HIV-1 RNA level) due to confounding. A 2-sided 0.05 level test determined statistical significance for all analyses.
Baseline Study Characteristics
A total of 196 (71%) of 275 participants had specimens suitable for this analysis. At enrollment, 134 (68%) of 196 participants were HHV-8 seropositive (Table 1). HHV-8–seropositive persons were more likely to have used antiviral medications in the past (P = 0.003), were HIV-infected for a greater number of years (mean 9.8 vs. 8.4 years, P = 0.05), and had more advanced HIV disease with lower CD4 counts (mean 386 vs. 483 cells/mm3, P = 0.02). No significant differences were found in the number of sexual partners (P = 0.83) or history of STI (P = 0.20) between HHV-8–seronegative and –seropositive persons. An additional 9 (15%) of 62 participants seroconverted to HHV-8 during the course of follow-up.
Frequency of HHV-8 Shedding
HHV-8 DNA was detected in oral specimens from 43 (22%) of 196 participants, including 39 (27%) of 134 HHV-8– seropositive, 4 (8%) of 53 persistently seronegative subjects, and 5 (56%) of 9 men who seroconverted to HHV-8 (P = 0.06 for seropositive vs. seronegative, P = 0.003 for seroconverters vs. seronegative, P = 0.002 for seroconverters vs. seropositive). Of men with detectable HHV-8 DNA, 10 (23%) had HHV-8 detected at 1 visit, 18 (42%) at 2 visits, 8 (19%) at 3 visits, 5 (12%) at 4 visits, 1 (2%) at 5 visits, and 1 (2%) at 6 visits. Overall, HHV-8 was detected in 101 (15%) of 696 oral specimens including 84 (17%) of 481 samples from seropositive men, 11 (31%) of 35 specimens from men who seroconverted, and 6 (3%) of 180 specimens from seronegative men.
Correlates of HHV-8 Shedding
HHV-8 DNA was recovered more frequently from the oropharynx at visits when an individual was not receiving HAART (25 vs. 16%, odds ratio [OR] 2.4, 95% CI 1.0–6.0, P = 0.06), had a CD4 count of >200 cells/mm3 (20 vs. 5%, OR 4.8, 95% CI 1.0–22.8, P = 0.05), or had detectable leukocyte esterase in the oropharynx (21 vs. 7%, OR 5.0, 95% CI 2.0–12.5, P < 0.01) (Table 2). No significant association was found between HHV-8 shedding and duration of HIV infection (P = 0.39), age (P = 0.41), number of sexual partners in the past year (P = 0.07), or HIV-1 plasma RNA level (P = 0.63).
Selected HHV-8 Strain Typing
All participants selected for HMA analysis had unique HHV-8 strains amplified from their oropharynx (Fig. 1). Neither of the 2 randomly selected PCR-negative samples had detectable HMA bands, and the 2 samples obtained from the same patient over a 3-month period had identical HMA banding patterns.
HHV-8 seroprevalence was high among this cohort of HIV-positive MSM, a group that has the highest prevalence of HHV-8 infection in North America and Europe. Approximately 50% of HIV-infected MSM without Kaposi sarcoma (KS) were determined to have serum antibodies to HHV-8 by IFA in 2 previous studies. 20,21 In our study, 68% of participants were HHV-8 seropositive upon enrollment, a fact that may be accounted for by the high proportion of men reporting multiple sexual partners or a history of STIs. The proportion of HHV-8–seropositive MSM with HHV-8 detectable in the oropharynx was similar to that seen in a previous study, 9 but our study is the first to note the nearly twice as high a rate of oropharyngeal shedding among HHV-8 seroconverters as compared with persons chronically infected with HHV-8. Detection of HHV-8 DNA in the oropharynx in most men with serologic evidence of recent HHV-8 infection supports the early colonization of this site in the course of HHV-8 infection. The observation of 4 persistently seronegative participants with HHV-8 DNA detected in the oropharynx suggests that development or detection of antibody may not be universal in infected persons, or that seroconversion may lag by many months after acquisition. 22 The use of stringent PCR methods, the failure to detect HHV-8 DNA in any negative controls, and the HMA results argue against false-positive PCR reactions. Two other studies have documented negative HHV-8 serologic tests in the setting of detectable HHV-8 DNA in the blood 23 and saliva. 24
We found that lack of HAART use and higher CD4 counts are correlated independently with increased frequency of HHV-8 shedding, but found no association with HIV-1 plasma viral load or other antiviral medication use. Since the introduction of HAART, a dramatic decline in the number of cases of KS has been observed. 25 The lower incidence rates of KS in the United States since 1996 do not seem to be explained by a drop in HHV-8 seroprevalence, 26 and the immune reconstitution afforded by potent antiretroviral therapy has been offered as one explanation for the decline in KS. A recent report found that HHV-8 viremia was significantly lower after the initiation of HAART among MSM with KS. 27 HAART may influence HHV-8 infection among HIV-positive persons in a number of ways: the action of Tat or Vpr, 28,29 changes in the cytokine milieu, 30,31 or the exertion of a direct antiviral effect on HHV-8 by ≥1 HAART medicines. 32–34
HHV-8 shedding was more frequent among men with higher CD4 counts, which is similar to what has been described among a cohort of HIV-positive women, 35 and is consistent with studies of the other human gamma-herpesvirus, Epstein-Barr virus, 36,37 and the beta-herpesvirus, human herpesvirus-6. 38 It is possible that persons with increased CD4 counts may have increased amounts of inflammatory cytokines, which are permissive for HHV-8 replication. 30
We also found an association between inflammation in the oral cavity and HHV-8 shedding, a relationship that has not been previously described. No relationship between HHV-8 vaginal or cervical shedding and clinical evidence of genital inflammation or ulceration was found in a small number of Zimbabwean women with KS. 11 However, HIV-1 is shed with higher frequency from urethral secretions in persons with urethritis, 39 and HIV-1 shedding in cervical secretions has been strongly correlated with tumor necrosis factor-α and interleukin-6 concentrations. 40 It is unclear from the current study whether inflammation is a consequence of HHV-8 infection or if HHV-8 shedding is more common from inflamed oral mucosa; future research should be aimed at determining the temporal relationship between these 2 factors.
This project was not initially designed to study HHV-8 infection and is limited by the infrequent sampling of oropharyngeal secretions. We have demonstrated in studies of other herpesvirus infections that daily sampling at mucosal sites is required to provide an accurate picture of the burden of viral replication in MSM. 41,42 Future studies with daily sampling will be important to more completely characterize the relationship between HIV-1 and HHV-8 oropharyngeal shedding.
If mucosal shedding frequency or quantity is important in determining the risk of acquiring HHV-8 from an infected sexual partner, then our data imply that the use of HAART therapy could decrease HHV-8 transmission. Recent HIV treatment guidelines suggest deferring HAART therapy until a substantial decrease in CD4 count. 43 Additional studies are needed to determine the effect of these recommendations on HHV-8 incidence rates in the future.
We are indebted to Stacy Selke for her careful review of laboratory data and analysis files. Support: NIH Grants 5R01CA055488-09, P01 AI30731 and U19 AI31448 and the Joel Meyer Infectious Disease Scholarship Grant.
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