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Patterns of human herpesvirus-8 oral shedding among diverse cohorts of human herpesvirus-8 seropositive… Bender Ignacio, Rachel A; Goldman, Jason D; Magaret, Amalia S; Selke, Stacy; Huang, Meei-Li; Gantt, Soren; Johnston, Christine; Phipps, Warren T; Schiffer, Joshua T; Zuckerman, Richard A; McClelland, R. S; Celum, Connie; Corey, Larry; Wald, Anna; Casper, Corey Feb 10, 2016

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RESEARCH ARTICLE Open AccessPatterns of human herpesvirus-8 oralshedding among diverse cohorts ofhuman herpesvirus-8 seropositive personsRachel A. Bender Ignacio1,2,3*†, Jason D. Goldman1,2,3†, Amalia S. Magaret1,4,5, Stacy Selke5, Meei-Li Huang1,5,Soren Gantt6, Christine Johnston1,2, Warren T. Phipps1,2, Joshua T. Schiffer1,2, Richard A. Zuckerman7,R. Scott McClelland2,3,8, Connie Celum2,8, Larry Corey1,2,5, Anna Wald1,2,3,5 and Corey Casper1,2,3,8AbstractBackground: Human herpesvirus-8 (HHV-8), the etiologic agent of Kaposi sarcoma (KS), establishes lifelong latentinfection with periodic lytic replication (“shedding”) at mucosal sites, especially the oropharynx. Patterns of HHV-8shedding are not well understood, and require elucidation to better predict risk of HHV-8 related malignancies inthose infected. We sought to characterize patterns of HHV-8 oropharyngeal shedding among diverse cohorts thatenrolled HHV-8 seropositive persons.Methods: We quantified HHV-8 oral shedding using PCR among HHV-8 seropositive persons who collected at least14 days of oral swabs in 22 studies on 3 continents. We excluded persons taking antivirals during sampling or anyprior use of antiretrovirals in those who were HIV-infected.Results: 248 participants were enrolled from the US, Peru, Cameroon, Uganda, and Kenya; 61 % were men, 58 %were HIV seropositive, and 16 % had KS. Overall, 3,123 of 10,557 samples (29.6 %) had HHV-8 detected. Quantity ofvirus shed was highly correlated with shedding rate, (ρ = 0.72, p < 0.0001). HHV-8 was detected in ≥1 sample in 55 %of participants with a median of 7 % of days in the US and Kenya, 0 % in Uganda and Peru, and 18 % in Cameroon.Median episode duration was three days, and episodes with high median quantity lasted longer (42 vs 3 days,p < 0.0001). In persons with multiple observations over time, 66 % of shedding rate variance was attributable todifferences between individuals.Conclusions: In HHV-8 infected individuals from diverse settings, oral mucosal shedding rate, quantity, andduration were correlated; individual shedding was highly variable. Studies are needed to determine factorsaccounting for between-person variation and the relationship of HHV-8 shedding to development of associateddiseases.Keywords: Herpesviridae infections, Herpesvirus 8, Human, Kaposi sarcoma-associated herpesvirus (KSHV),Sarcoma, Kaposi, HIV infections, Virus replication* Correspondence: rbi13@uw.eduDr. Goldman and Dr. Bender Ignacio are first co-author.†Equal contributors1Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer ResearchCenter, Seattle, WA, USA2Division of Allergy and Infectious Diseases, Department of Medicine,University of Washington, Seattle, WA, USAFull list of author information is available at the end of the article© 2016 Bender Ignacio et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Bender Ignacio et al. Infectious Agents and Cancer  (2016) 11:7 DOI 10.1186/s13027-016-0052-2BackgroundHuman herpesvirus-8 (HHV-8), also known as Kaposisarcoma-associated herpesvirus (KSHV), establishes life-long latent infection, punctuated with periods of lyticreplication at mucosal sites (“shedding”) [1]. Shedding ismost commonly detected at the oropharynx, which isthought to be the site of transmission, primary HHV-8latency and reactivation [2–4]. Prevalence of HHV-8 in-fection is geographically variable, in >60 % of adults inUganda and other sub-Saharan African (SSA) countries[5, 6] and as low as 3.3 % in healthy Americans [7, 8].While in most people HHV-8 is asymptomatic, HHV-8replication is considered to be integral to the develop-ment and maintenance of Kaposi sarcoma (KS). Othersequelae of the infection include Multicentric CastlemanDisease (MCD), Primary Effusion Lymphoma as well asthe recently described KHSV Inflammatory CytokineSyndrome [9–12].Despite high seroprevalence of HHV-8 in many regions,HHV-8 associated diseases are relatively uncommon, ex-cept in the endemic regions of SSA, or in persons with ad-vanced immunosuppression. KS remains the second mostprevalent cancer in men and the fifth most prevalent can-cer in women in the WHO Africa region [13]. Previouswork by our group demonstrated that HHV-8 mucosalshedding is associated with presence of KS and male sex,but not with HIV infection [4]. Prior studies assessing oralHHV-8 shedding evaluated small numbers of participantsand were limited in geography [14]. The patterns of HHV-8 shedding, such as duration of shedding episodes, inter-val between episodes, and variability between persons havenot yet been characterized. Due to the heterogeneityof cohort composition, we describe shedding charac-teristics and patterns across all persons, avoiding pre-diction of shedding by geographic, demographic orclinical covariates. We assembled diverse cohorts ofHHV-8 infected individuals to describe patterns ofHHV-8 oral mucosal shedding including rate, quan-tity, duration, and within- versus between-person vari-ability over time.ResultsStudy participantsBetween 1993 and 2011, 248 subjects met inclusion cri-teria 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 in-cluded persons at high risk for HHV-8 infection, includ-ing heterosexuals from KS-endemic regions, men whohave sex with men, and female sex workers. Overall, 152(61 %) participants were men, and the median age was38 (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 personswith HIV, CD4 count was <200 in 9 (6 %), 200–499 in31 (22 %), ≥500 in 17 (12 %) and not available in 86(60 %). HIV RNA was <104 copies/mL in 23 (16 %), andunmeasured in 70 (49 %). Sixteen (44 %) had endemicKS, and 20 (56 %) had HIV-associated KS. All partici-pants from Peru and Cameroon were HIV-infected,while other sites enrolled both HIV-infected and unin-fected persons [Table 1 and Fig. 2].Fig. 1 Flow diagram of study participant inclusion. Numbers listed are n = number of participants (“sessions” = number of observation periodscontributed). Abbreviations: ART, antiretroviral therapy; US: United StatesBender Ignacio et al. Infectious Agents and Cancer  (2016) 11:7 Page 2 of 9Table 1 Cohort, participant, and shedding characteristicsParticipant Location, N (%)a USA Peru Cameroon Uganda Kenya Total102 (41 %) 7 (3 %) 32 (13 %) 68 (27 %) 39 (16 %) 248 (100 %)Participant Demographic and Clinical Data:Male, n (%) 101 (99 %) 7 (100 %) 0 (0 %) 44 (65 %) 0 (0 %) 152 (61 %)HIV-infected, n (%) 46 (46 %) 7 (100 %) 32 (100 %) 37 (54 %) 21 (54 %) 143 (58 %)CD4 countbCD4 < 200 9 (20 %) 0 (0 %) 0 (0 %) – – 9 (6 %)CD4 200–499 17 (37 %) 1 (14 %) 13 (41 %) – – 31 (22 %)CD4 500+ 12 (26 %) 0 (0 %) 5 (16 %) – – 17 (12 %)Unknown 8 (17 %) 6 (86 %) 14 (44 %) 37 (100 %) 21 (100 %) 86 (60 %)HIV-1 RNAb<104 12 (26 %) 4 (57 %) – 7 (18 %) – 23 (16 %)≥104 22 (48 %) 3 (43 %) – 25 (68 %) – 50 (35 %)Unknown 12 (26 %) 0 (0 %) 32 (100 %) 5 (14 %) 21 (100 %) 70 (49 %)Kaposi sarcoma: 2 (2 %) 0 (0 %) 0 (0 %) 34 (50 %) 0 (0 %) 36 (15 %)HIV-associated: 1 (1 %) – – 19 (28 %) – 20 (8 %)HSV-2 seropositive, n/tested (%) 62/97 (64 %) 7 (100 %) 31 (97 %) ND ND 100/135 (74 %)Cohort Shedding Characteristics:Sessions,Number: 152 7 32 68 39 298Observations per session:Median # of days (range) 33 (15–136) 56 (27–57) 19 (14–23) 29 (14–32) 29 (26–29) 30 (14–136)Overall shedding:Positive swabs: 2,213 75 183 389 263 3,123Total swabs: 6,660 359 590 1834 1,114 10,557Percent positive: (33 %) (21 %) (31 %) (21 %) (24 %) (30 %)Participant shedding rate per session,Median % (IQR) 7 (0, 73) 0 (0, 33) 18 (0, 56) 0 (0, 34) 7 (0, 43) 4 (0, 54)Persons with any shedding 60 (59 %) 3 (43 %) 21 (66 %) 26 (38 %) 27 (69 %) 137 (55 %)Quantity, median (IQR) of positive swabs, log10 copies/mL 4.8 (3.9–5.6) 7.4 (5.0–7.8) 3.2 (2.7–3.8) 5.2 (3.9–6.0) 4.4 (3.5–5.5) 4.7 (3.7–5.6)Abbreviations. US United States, ND Not determined, IQR Interquartile Range, HSV Herpes simplex virusaPercentages are out of those within the given countrybPercentages for CD4 count and HIV RNA are of HIV-infected participantsFig. 2 Heterogeneity of participant characteristics within the cohorts included in this studyBender Ignacio et al. Infectious Agents and Cancer  (2016) 11:7 Page 3 of 9HHV-8 shedding rate and quantityOverall, 10,557 swabs were analyzed for HHV-8 DNA,and HHV-8 was detected in 3,123 (29.5 %). Within theentire cohort, 137 persons (55 %) had HHV-8 DNA de-tected at least once. The median (IQR) shedding rateper session was 7 % (0–73 %) in the US, 0 % (0–33 %)in Peru, 18 % (0–56 %) in Cameroon, 0 % (0–34 %) inUganda, 7 % (0–43 %) in Kenya, and 4 % (0–54 %)overall [Table 1]. Among US participants, 33 % ofswabs 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 samplesfrom persons with and without HIV and KS, we com-pared shedding rate within the factorial design of theUgandan cohort as previously described [4]. In 34Ugandan 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 %. TheUgandan cohort was the only African cohort to includeboth men and women; HHV-8 was detected in 30 % ofsamples from men, compared with 6 % from women.Amongst all cohorts, HHV-8 was detected in 18 % ofswabs from HIV-infected individuals and 18 % of swabsfrom HIV-uninfected individuals.The median quantity of HHV-8 DNA in samples withHHV-8 detected was 4.7 (IQR: 3.7–5.6) log10 copies/mL [Fig. 3]. Median HHV-8 quantity during the sessioncorrelated strongly with shedding rate (ρ = 0.72, p < 0.0001)[Fig. 4]. Maximum quantity of HHV-8 detected during asession also correlated highly with the shedding rate (ρ =0.82, p < 0.0001) (data not shown).Shedding episodesWe defined a shedding episode as a period of detect-able HHV-8 of at least one day bounded by at least twopreceding and subsequent negative swabs, and couldinclude singular missed or negative swabs [15]. Wheninitiation or termination of the shedding episode wasnot observed, interval-censoring survival was used esti-mate the episode duration. We dichotomized episodesby median quantity above or below the median quantityof all detectable samples and tested the association be-tween quantity and duration for episodes with log ranktests. We evaluated up to 3 shedding episodes persession, for a total of 229 episodes in 116 persons.Duration was uncertain in 106 (46 %) episodes becauseof censoring at either limit of session observation. For123 episodes of certain duration, the median durationwas 1 day (IQR: 1–4 days). Using interval censoringsurvival analysis to include both episodes of certain anduncertain duration, 30 % of episodes were predicted tolast 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 episodeswith lower median quantity (≤4.5 log10 copies/mL), themedian duration was 3 days (p < 0.0001). We investigatedthe duration of shedding episodes and intervals withoutHHV-8 shedding between episodes. “Short” episodes weredefined as ≤ 3 days (median duration of all episodes) and“long” episodes as > 3 days. Short episodes were precededby a median 13-day interval without shedding whereaslong episodes were preceded by a median 7-day interval(p = 0.035). A sensitivity analysis reclassifying episodescensored prior to 3 days (26 episodes) as “short” instead of“long” provided similar estimates.To illustrate patterns of HHV-8 shedding, data fromparticipants who collected swabs for 30–60 days and whoshed at least once were included in a heatmap [Fig. 5]. Wedid not observe a singular pattern of shedding, but ratherFig. 3 Quantity of HHV-8 DNA (Log10 copies/mL) within swabs positivefor HHV-8. 2.17 Log10 copies/mL represents the lower limit of accuratedetection (150 copies/mL). The label for each bin represents a rangeincluding the value listed and all values up to but not including thevalue listed in the next highest binFig. 4 Correlation of HHV-8 DNA quantity and shedding rate: Scatterplotof HHV-8 quantity (median HHV-8 DNA copy number by qPCR duringshedding session) vs. HHV-8 shedding rate (number of days HHV-8 isdetectable at any level by qPCR divided by number of days of sheddingsession). Best-fit regression line is shown (ρ= 0.72, p< 0.0001)Bender Ignacio et al. Infectious Agents and Cancer  (2016) 11:7 Page 4 of 9a gradation was noted from infrequent and low copy num-ber shedding to very frequent and high copy numbershedding. For example, some participants shed virus insingle-day episodes, while others had intermittent epi-sodes of both high and low quantities, and a few partici-pants shed virus at high quantities throughout the session.Participants with KS and HIV were dispersed amongstparticipants when sorted by shedding rate [Fig. 5].Variation in shedding rate over timeStability of shedding rate over time was evaluated in par-ticipants contributing multiple sessions. Thirty-nine par-ticipants, all of whom were from the United States, had atleast one additional session within two years of the first:19 persons had 2, 9 persons had 3, 8 persons had 4, and 3persons had 5, totaling 112 sessions. The median sheddingrate of first sessions was 50 %. Over time, 4 persons(10.3 %) had shedding rate increase by at least 30 % on anabsolute scale, 7 persons (18.0 %) had shedding ratedecrease by the same amount, and 28 (71.8 %) hadno consistent pattern or a stable shedding rate [Fig. 6].Using variance components, we examined the variation inFig. 5 Patterns of HHV-8 Shedding. Included participants had HHV-8 detectable by PCR on at least one day during the first 60 days of observationand were observed for a minimum of 30 days. Each row represents one participant, ordered by shedding rate from lowest to highest over theshedding session. Sixty participants with at least 30 days of observation who never shed were omitted. Time is represented on the x-axis (in days)such that each box represents the HHV-8 PCR result from a single daily swab for an individual. DNA quantity is graded by color (in log10 copies/mL). Asterisks (*) denote participants with Kaposi Sarcoma and plus signs (+) denote participants with HIV. White indicates no swab collected;other colors as per legendFig. 6 Variation in oral shedding rate of HHV-8 by participant acrossmultiple shedding sessions. All cohort participants with 2–5 sessionswithin two years were included in the analysis of shedding variationover time, representing 39 participants with 112 total sessions. Fourpersons had shedding rates increase by at least 30 % on an absolutescale over time (red), 7 persons had rates decrease by the sameamount (blue), and 28 had no consistent pattern (black)Bender Ignacio et al. Infectious Agents and Cancer  (2016) 11:7 Page 5 of 9shedding rates over time within the population that wasdue to inter-person versus intra-person differences. Wefound that 66 % of the variance was due to inter-persondifferences and the remainder due to change in an individ-ual’s shedding rate over multiple sessions.DiscussionThis is the largest study of HHV-8 shedding publishedto date, evaluating more than 10,000 oral swab samplescollected from geographically and demographically di-verse HHV-8 seropositive persons not receiving anti-viral or antiretroviral therapy. HHV-8 shedding was acommon event, although nearly half of participantsnever shed while under observation. We found a strongcorrelation between shedding rate, quantity, and dur-ation of oropharyngeal shedding within an individual,but significant variability between individuals.Our findings are consistent with earlier studies ofHHV-8 oropharyngeal shedding that observed 15 %-32 % shedding rates [2, 16, 17]. In our large cohort, weobserved a gradation in shedding rate and quantityacross participants. In comparison to HSV-2, a herpes-virus with well-characterized shedding patterns, wefound several differences in mucosal reactivation pat-terns. HSV-2 reactivation is characterized by shorterepisodes with 20 % lasting >9 days [18], whereas wefound longer episodes of HHV-8 shedding, with 40 %ongoing at 20 days. In contrast to HSV-2, which hasrapid expansion and decay, we observed variable pat-terns of HHV-8 shedding, with many episodes showinga long plateau without observed termination. Additionally,quantity of HHV-8 detected tended to be more homoge-neous compared to HSV-2, with the vast majority ofswabs clustered around 4.5 log copies/mL, whereas HSV-2 is frequently detected at both lower and much higherquantities [18, 19]. Our analysis found similar sheddingrates in HIV-infected and uninfected individuals, an intri-guing finding that is dissimilar to other herpesviruses,including HSV, CMV, and EBV [20–24].HHV-8 shedding in relationship to KS and HIV hasbeen studied and results are inconsistent. In Uganda, KSpatients were more likely to have HHV-8 detected thanthose with asymptomatic HHV-8 infection, while HIV ser-ostatus was not associated with detection of HHV-8 in theoral mucosa [4]. Another cross-sectional study found nodifference in detection of HHV-8 shedding in the oral mu-cosa of HIV-infected men with and without KS [25];persons with KS in our study were not observed prior toonset of disease. Our data do not address the risk ofasymptomatically-infected persons developing HHV-8-associated diseases, nor compare shedding patternsbetween persons with HHV-8 related diseases and asymp-tomatic infection; the understanding of viral sheddingpatterns gained from our work may be used to design lon-gitudinal studies to address questions of HHV-8 transmis-sion and disease association.Our study was limited by performance characteristics ofthe HHV-8 serologic assays used to include and excludeparticipants, which have sub-optimal sensitivity and speci-ficity [26, 27]. We may have excluded persons from ourcohort who were HHV-8 infected but seronegative, or in-cluded those with false positive serology. Using PCR toquantitate oral virus may not strictly detect lytic replicat-ing virus, as latent cell-associated virus may also bepresent in these samples. However, that the large majorityof samples were negative in HHV-8 seropositive personssuggests that latent virus in oral epithelial cells is generallynot detected by this collection method and PCR tech-nique. Additionally, we were limited in our ability tomodel demographic and clinical risk factors for HHV-8shedding due to both measured and unmeasured co-founders in these heterogeneous cohorts, especially withrespect to available HIV clinical data. Conversely, the di-versity of participants in this large cohort allowed us todescribe HHV-8 shedding patterns unrestricted to anynarrow population or geographic area, as has frequentlybeen done up to this point. We benefitted from long ses-sions and serial observations on many participants; priorstudies have not been able to evaluate viral replication pat-terns in individuals over time. Other strengths of thisstudy include robust methods of swab collection, labora-tory, and statistical analysis concordant with methodsused by our group in studies of herpesvirus shedding overthe last three decades. While different PCR methods wereused in our reference laboratory during the time periodover which our cohorts span, the standardization wasconsistent across all cohorts, was supervised by the samevirologist, and quality control showed high correlation be-tween assays.ConclusionsWe did not detect a consistent or singular pattern toHHV-8 shedding, and substantial variability was observedbetween persons. We demonstrated that rate and quantityof HHV-8 shedding are highly correlated and that personsexperiencing longer and higher quantity shedding episodescan be predicted to shed more frequently in the future.Patterns of HHV-8 shedding are distinctly different fromother well-characterized herpesvirus infections, such asHSV-2. This work provides a foundation to assess mucosalshedding of HHV-8 as a possible biomarker to assess riskof HHV-8 transmission or disease. Longitudinal studies inasymptomatically infected persons are needed to furtherunderstand clinical correlates to patterns of HHV-8 shed-ding, and if replication is associated with HHV-8 relateddiseases.Bender Ignacio et al. Infectious Agents and Cancer  (2016) 11:7 Page 6 of 9MethodsWe evaluated 22 prospective cohorts in which HHV-8DNA quantification had been performed on oropharyn-geal swabs, including 18 cohorts from the VirologyResearch Clinic, University of Washington [14, 28–32],and 4 studies by affiliated investigators in Lima, Peru[33]; Kampala, Uganda [4]; Mombasa, Kenya [34]; andYaounde, Cameroon. The University of Washington In-stitutional Review Board and appropriate partner insti-tutions for international sites approved each study; allparticipants provided written informed consent in ac-cordance with the Declaration of Helsinki. Participantscollected daily samples of the oropharyngeal mucosawith a Dacron swab (three times weekly in Cameroononly) with similar technique across all studies, for aminimum of 14 days [35]. Each such observation periodis termed a “session”. We included HHV-8 seropositiveadults (age ≥18) with and without HIV, and with andwithout HHV-8 associated diseases including MCD andKS. We included HIV-infected participants only if anti-retroviral naïve to avoid confounding from immunerestoration and potential direct and indirect effects ofART on HHV-8 replication [36]. We excluded partici-pants receiving antiviral therapies including valganci-clovir, acyclovir, valacyclovir or famciclovir [31], andparticipants with ≤14 samples per session, as prior ana-lyses performed by our group demonstrate substantialinstability in herpesvirus shedding estimates with fewersamples [37]. Data from 216 of 298 HHV-8 sheddingsessions (72 %) included in this analysis were previouslypublished [1, 4, 14, 16, 31, 34, 38]. We aggregated per-sons from these diverse populations to more robustlydescribe associations between shedding rate, quantity,duration, and variability of shedding episodes in HHV-8 infected persons.Laboratory methodsHHV-8 seropositivity was determined by positive testresult on either immunofluorescence assay (IFA)against latent or lytic antibodies [39] or whole-virus en-zyme immunoassay (EIA) [27]. Swabs were collectedinto 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 processvalidated to be stable for extended storage [40, 41]. Quan-titative polymerase chain reaction (qPCR) was performedto quantitate HHV-8 DNA using previously described andvalidated methods [1, 16, 38, 42]. Three qPCR assays wereused using the study period: the KS330Bam233 region oforf26 (a viral capsid protein)1, orf73 (latency-associatednuclear antigen-1)2 or a multiplex assay using orf73 plusT07-K12 (kaposin)3. Briefly, early samples (orf26 assay)were extracted from swabs with traditional phenol-chloroform method and all other samples were extractedusing Qiagen column (QIAmap DNA blood kit), thenpurified, and amplified with the given primers-probe setson the Taqman qPCR platform (Applied Biosystems, Fos-ter City, CA). Compared to qPCR with orf73 as the goldstandard, orf26 had sensitivity 83.1 %, specificity 98.8 %.To minimize the variance in sensitivity at low end of de-tectable range, a cut-off of ≥150 copies/mL (≥3 copies/PCR reaction) were considered positive for HHV-8 detec-tion [43]. Applying this cut-off, the correlation betweenorf26 and orf73 results was R2 = 0.75. Compared to qPCRwith 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 correl-ation between the latter two primer sets was R2 = 0.96.Quantitation standard using the cloned PCR product ofKS330Bam233 primers was consistent throughout all co-horts. Quality control using standardization and positiveand negative controls for each batch were supervised byM.L.H. in the same Seattle-based reference laboratory forall cohorts. Overall, 92 % of samples were run with theorf73 assay, 5 % with the orf26 assay and 3 % with theorf73 and T07.K12 multiplex assay.Statistical analysisSessions are defined as discrete periods of contiguousswab collection ≥14 days and separated in time from otherobservation periods. For participants contributing mul-tiple sessions, the first and second sessions were includedin the primary analysis. For each session, shedding ratewas defined as number of swabs with HHV-8 DNA detec-tion, divided by total swabs. Quantity of HHV-8 was log10transformed for analysis and was assessed as maximumand median log10 copies/mL. We used Pearson correla-tions to evaluate the association of shedding rate withmaximum and median log10 copy number within sessions.To test the association of episode duration with timebetween episodes, we classified episodes as “short” or“long” by dichotomizing at the median length of all epi-sodes and used log rank tests. First, we assumed epi-sodes not observed to terminate were “long” and thenthen performed a sensitivity analysis reclassifying theseepisodes as “short”. We excluded Cameroonian samplesfrom episode duration analysis since swabbing was notconducted daily.Participants with two to five sessions were included inthe longitudinal shedding analysis. The proportion ofvariability in shedding rate ascribed to the individualwas evaluated with variance components.Endnotes1ORF26-F: 5’- AGC CGA AAG GAT TCC ACC AT-3’,ORF26-probe: ATG TGG TAC ACC AAC AGC TGCTGC, ORF26-R: 5’- TCC GTG TTG TCT ACG TCCAG-3’.Bender Ignacio et al. Infectious Agents and Cancer  (2016) 11:7 Page 7 of 92ORF73-F: 5'-CCA GGA AGT CCC ACA GTG TTC-3', ORF73-probe: FAM-CAT CCG GGC TGC CAGCAT TTG-TAMRA, ORF73-R: 5'-GCC ACC GGT AAAGTA GGA CTA GAC-3'.3T07.K12-F: 5'-TCC CCC ACC GAG TGC TT-3',T07.K12-probe: FAM-AAT GCG GAG AGG AAT G-MGB, T07.K12-R: 5'-GCA CGC GGT GTC AAC CA-3'.Competing interestAll authors have reported no conflict of interest.Authors’ contributionsDesigned the current study: RBI, JDG, SG, ASM, AW and CCasper. Designed andexecuted the parent cohorts: CJ, WP, RSM, CCelum, LC, RZ, CCasper, and AW.Performed laboratory testing: MLH, SS. Managed data and compiled cohorts: SS.Performed the statistical analyses: ASM. Interpreted results: RBI, JDG, ASM, JS,AW, CCasper. Drafting of the manuscript: RBI, JDG. Revision of the manuscriptfor important intellectual content and final approval of manuscript: all authors.AcknowledgementsWe thank the patients for participation and the dedicated study staff whofacilitated these studies.FundingNIH: T32-AI-007140, T32-CA-080416, K23-AI-054162, K23-CA-150931, P30-AI-027757,P30-CA-015704, P01-AI-030731, R01-CA-086795, K24-AI-071113, U01-AI-038858,R37-AI-042528; GlaxoSmithKline R103; FHCRC Early Detection Initiative.Author details1Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer ResearchCenter, Seattle, WA, USA. 2Division of Allergy and Infectious Diseases,Department of Medicine, University of Washington, Seattle, WA, USA.3Department of Epidemiology, School of Public Health, Seattle, WA, USA.4Department of Biostatistics, School of Public Health, Seattle, WA, USA.5Department of Laboratory Medicine, University of Washington, Seattle, WA,USA. 6Division of Infectious Diseases, Department of Pediatrics, University ofBritish Columbia, Vancouver, BC, Canada. 7Section of Infectious Disease andInternational Health, Department of Medicine, Geisel School of Medicine atDartmouth, Lebanon, NH, USA. 8Department of Global Health, University ofWashington, Seattle, WA, USA.Received: 8 October 2015 Accepted: 28 December 2015References1. 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Shebl FM, Emmanuel B, Bunts L, Biryahwaho B, Kiruthu C, Huang ML, et al.Population-based assessment of kaposi sarcoma-associated herpesvirusDNA in plasma among Ugandans. J Med Virol. 2013;85(9):1602–10.43. Magaret AS, Wald A, Huang ML, Selke S, Corey L. Optimizing PCR positivitycriterion for detection of herpes simplex virus DNA on skin and mucosa.J Clin Microbiol. 2007;45(5):1618–20.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Bender Ignacio et al. Infectious Agents and Cancer  (2016) 11:7 Page 9 of 9


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