Patterns of human herpesvirus-8 oral shedding among diverse cohorts of human herpesvirus-8 seropositive persons

Between 1993 and 2011, 248 subjects met inclusion criteria and contributed 298 sessions (observation periods) to the primary analysis, with median duration of 30 days (range 14–136) per session [Fig. 1]. These cohorts included persons at high risk for HHV-8 infection, including heterosexuals from KS-endemic regions, men who have sex with men, and female sex workers. Overall, 152 (61 %) participants were men, and the median age was 38 (interquartile range, IQR: 31–45). One hundred forty-three participants (58 %) were HIV-infected, 35 (14 %) had KS, and 1 (0.4 %) had MCD. Among 143 persons with HIV, CD4 count was <200 in 9 (6 %), 200–499 in 31 (22 %), ≥500 in 17 (12 %) and not available in 86 (60 %). HIV RNA was <10⁴ copies/mL in 23 (16 %), and unmeasured in 70 (49 %). Sixteen (44 %) had endemic KS, and 20 (56 %) had HIV-associated KS. All participants from Peru and Cameroon were HIV-infected, while other sites enrolled both HIV-infected and uninfected persons [Table 1 and Fig. 2].

Overall, 10,557 swabs were analyzed for HHV-8 DNA, and HHV-8 was detected in 3,123 (29.5 %). Within the entire cohort, 137 persons (55 %) had HHV-8 DNA detected at least once. The median (IQR) shedding rate per session was 7 % (0–73 %) in the US, 0 % (0–33 %) in Peru, 18 % (0–56 %) in Cameroon, 0 % (0–34 %) in Uganda, 7 % (0–43 %) in Kenya, and 4 % (0–54 %) overall [Table 1]. Among US participants, 33 % of swabs had detectable HHV-8 DNA, 21 % in Peru, 31 % in Cameroon, 21 % in Uganda and 24 % in Kenya [Table 1]. In order to validate the utilization of samples from persons with and without HIV and KS, we compared shedding rate within the factorial design of the Ugandan cohort as previously described [4]. In 34 Ugandan persons with KS, the shedding rate was 25 % in HIV-associated cases and 40 % in endemic KS cases; Ugandans without KS had a shedding rate of 27 %. The Ugandan cohort was the only African cohort to include both men and women; HHV-8 was detected in 30 % of samples from men, compared with 6 % from women. Amongst all cohorts, HHV-8 was detected in 18 % of swabs from HIV-infected individuals and 18 % of swabs from HIV-uninfected individuals.

The median quantity of HHV-8 DNA in samples with HHV-8 detected was 4.7 (IQR: 3.7–5.6) log₁₀ copies/mL [Fig. 3]. Median HHV-8 quantity during the session correlated strongly with shedding rate (ρ = 0.72, p < 0.0001) [Fig. 4]. Maximum quantity of HHV-8 detected during a session also correlated highly with the shedding rate (ρ = 0.82, p < 0.0001) (data not shown).

We defined a shedding episode as a period of detectable HHV-8 of at least one day bounded by at least two preceding and subsequent negative swabs, and could include singular missed or negative swabs [15]. When initiation or termination of the shedding episode was not observed, interval-censoring survival was used estimate the episode duration. We dichotomized episodes by median quantity above or below the median quantity of all detectable samples and tested the association between quantity and duration for episodes with log rank tests. We evaluated up to 3 shedding episodes per session, for a total of 229 episodes in 116 persons. Duration was uncertain in 106 (46 %) episodes because of censoring at either limit of session observation. For 123 episodes of certain duration, the median duration was 1 day (IQR: 1–4 days). Using interval censoring survival analysis to include both episodes of certain and uncertain duration, 30 % of episodes were predicted to last less than 1 day, 50 % to last less than 9 days, and 60 % to last less than 20 days. One hundred-nineteen episodes (52 %) with a high median quantity (>4.5 log₁₀ copies/mL) had a predicted median duration of 42 days; for episodes with lower median quantity (≤4.5 log₁₀ copies/mL), the median duration was 3 days (p < 0.0001). We investigated the duration of shedding episodes and intervals without HHV-8 shedding between episodes. “Short” episodes were defined as ≤ 3 days (median duration of all episodes) and “long” episodes as > 3 days. Short episodes were preceded by a median 13-day interval without shedding whereas long episodes were preceded by a median 7-day interval (p = 0.035). A sensitivity analysis reclassifying episodes censored prior to 3 days (26 episodes) as “short” instead of “long” provided similar estimates.

To illustrate patterns of HHV-8 shedding, data from participants who collected swabs for 30–60 days and who shed at least once were included in a heatmap [Fig. 5]. We did not observe a singular pattern of shedding, but rather a gradation was noted from infrequent and low copy number shedding to very frequent and high copy number shedding. For example, some participants shed virus in single-day episodes, while others had intermittent episodes of both high and low quantities, and a few participants shed virus at high quantities throughout the session. Participants with KS and HIV were dispersed amongst participants when sorted by shedding rate [Fig. 5].

Stability of shedding rate over time was evaluated in participants contributing multiple sessions. Thirty-nine participants, all of whom were from the United States, had at least one additional session within two years of the first: 19 persons had 2, 9 persons had 3, 8 persons had 4, and 3 persons had 5, totaling 112 sessions. The median shedding rate of first sessions was 50 %. Over time, 4 persons (10.3 %) had shedding rate increase by at least 30 % on an absolute scale, 7 persons (18.0 %) had shedding rate decrease by the same amount, and 28 (71.8 %) had no consistent pattern or a stable shedding rate [Fig. 6]. Using variance components, we examined the variation in shedding rates over time within the population that was due to inter-person versus intra-person differences. We found that 66 % of the variance was due to inter-person differences and the remainder due to change in an individual’s shedding rate over multiple sessions.

HHV-8 seropositivity was determined by positive test result on either immunofluorescence assay (IFA) against latent or lytic antibodies [39] or whole-virus enzyme immunoassay (EIA) [27]. Swabs were collected into lysis buffer containing 100 mM KCl (or NaCl), 25 mM EDTA (pH 8.0), 10 mM Tris (pH 8.0), and 1 % Igepal CA-630 and stored at −20 °C until testing, a process validated to be stable for extended storage [40, 41]. Quantitative polymerase chain reaction (qPCR) was performed to quantitate HHV-8 DNA using previously described and validated methods [1, 16, 38, 42]. Three qPCR assays were used using the study period: the KS330Bam₂₃₃ region of orf26 (a viral capsid protein)¹, orf73 (latency-associated nuclear antigen-1)² or a multiplex assay using orf73 plus T07-K12 (kaposin)³. Briefly, early samples (orf26 assay) were extracted from swabs with traditional phenol-chloroform method and all other samples were extracted using Qiagen column (QIAmap DNA blood kit), then purified, and amplified with the given primers-probe sets on the Taqman qPCR platform (Applied Biosystems, Foster City, CA). Compared to qPCR with orf73 as the gold standard, orf26 had sensitivity 83.1 %, specificity 98.8 %. To minimize the variance in sensitivity at low end of detectable range, a cut-off of ≥150 copies/mL (≥3 copies/PCR reaction) were considered positive for HHV-8 detection [43]. Applying this cut-off, the correlation between orf26 and orf73 results was R² = 0.75. Compared to qPCR with orf73 and T07-K12 multiplex as the gold standard, orf73 had a sensitivity of 94.1 %, specificity of 97.5 %. Applying the minimum cut-off for positivity, the correlation between the latter two primer sets was R² = 0.96. Quantitation standard using the cloned PCR product of KS330Bam₂₃₃ primers was consistent throughout all cohorts. Quality control using standardization and positive and negative controls for each batch were supervised by M.L.H. in the same Seattle-based reference laboratory for all cohorts. Overall, 92 % of samples were run with the orf73 assay, 5 % with the orf26 assay and 3 % with the orf73 and T07.K12 multiplex assay.

Sessions are defined as discrete periods of contiguous swab collection ≥14 days and separated in time from other observation periods. For participants contributing multiple sessions, the first and second sessions were included in the primary analysis. For each session, shedding rate was defined as number of swabs with HHV-8 DNA detection, divided by total swabs. Quantity of HHV-8 was log₁₀ transformed for analysis and was assessed as maximum and median log₁₀ copies/mL. We used Pearson correlations to evaluate the association of shedding rate with maximum and median log₁₀ copy number within sessions.

To test the association of episode duration with time between episodes, we classified episodes as “short” or “long” by dichotomizing at the median length of all episodes and used log rank tests. First, we assumed episodes not observed to terminate were “long” and then then performed a sensitivity analysis reclassifying these episodes as “short”. We excluded Cameroonian samples from episode duration analysis since swabbing was not conducted daily.

Participants with two to five sessions were included in the longitudinal shedding analysis. The proportion of variability in shedding rate ascribed to the individual was evaluated with variance components.