Evaluation of oral HSV shedding among solid organ transplant recipients: a pilot study

Abstract

Background:

Herpes simplex viruses (HSV) frequently reactivate during immunosuppression and may be a risk factor for adverse outcomes after solid organ transplant. While suppressive antiviral therapy reduces the risk of symptomatic HSV reactivation, the kinetics of asymptomatic viral shedding with chronic immunosuppression after transplant are not well-understood. We report the characteristics of oral HSV shedding among fifteen HSV-1 seropositive solid organ transplant (SOT) recipients (n = 8 liver, n = 7 kidney, median age 58.5 years, median 20 months post-transplant) who were not taking daily antiviral suppressive therapy.

Methods:

Participants self-collected oral swabs 3 times daily for 6 weeks for HSV quantification and recorded the presence of oral symptoms or lesions in a diary.

Results:

Sample collection adherence was high (median 122 swabs/person, range: 85.7–101.6% of expected swabs). Most participants (n = 12, 80%) experienced at least 1 shedding episode, with a median shedding rate of 8.9% (range: 0–33.6%). There were 32 total shedding episodes, 24 (75%) of which occurred without symptoms or lesions. For episodes of known duration, median length was 21.8 hours (IQR: 10.8–46.1 hours).

Conclusion:

Most shedding episodes (78.1%) lasted >12 hours, suggesting that twice daily sampling may be sufficient to detect most episodes. These data show that self-collection of oral swabs is feasible for patients who have undergone solid organ transplant and can provide insight into the frequency of oral HSV reactivation, which can be used to design future studies in this population.

Graphical Abstract

Findings from a pilot study of oral swabs in SOT recipients found that subclinical HSV shedding is common. Shedding is frequent and can be long-lasting in immunocompromised hosts.

1. Introduction

Herpes simplex virus type 1 (HSV-1) causes lifelong infection, and most US adults are infected.¹ HSV-1 persists in the trigeminal ganglia and periodically reactivates from its latent state, leading to viral replication and detection of the virus on mucosal surfaces (“shedding”). While episodes of viral shedding may be associated with clinical symptoms or signs such as itching, tingling, and/or oral lesions, they are typically subclinical and occur without lesions. Prior research indicates that subclinical oral HSV-1 shedding occurs at least once per month in immunocompetent hosts.^2–4 Prior to the widespread use of antiviral prophylaxis, clinically-apparent HSV-1 disease was common in the first few weeks following transplantation among solid organ transplant (SOT) recipients, primarily due to transplant-related immunosuppression.⁵ While antiviral use reduces the incidence of clinical HSV-1 disease following transplantation,^6,7 the longer term effects of immunosuppression on subclinical viral shedding are less clear. In this pilot study, we characterized HSV replication kinetics among a cohort of solid organ transplant (SOT) recipients who were not taking daily antiviral suppressive therapy to understand whether we can conduct larger studies on the effect of HSV subclinical viral shedding in post-transplant populations in the future.

2. Materials and Methods

2.1. Participants and Setting

Individuals who had received an SOT at least three months prior to enrollment were recruited from the University of Washington (UW) in Seattle, Washington. Individuals were eligible for inclusion if they were age ≥ 18, planned to stay in the Seattle area throughout the study period, and tested positive for HSV-1 antibodies by UW HSV Western blot.⁸ This study was approved by the UW Institutional Review Board (IRB no. 41066 and IRB no. STUDY00001279, and IRB STUDY00020038), and all participants provided informed consent for study participation. One participant received acyclovir for an HSV lesion during the study; participants were not otherwise taking daily antiviral suppressive therapy.

2.2. Sample Collection

At enrollment, a physical exam was performed and each participant’s medical history and current medications, including antivirals, were reviewed. Participants were trained to self-collect oral swabs at home three times per day for a total of six weeks (126 swabs per person). Participants were supplied with Dacron swabs, storage vials with PCR buffer, and labels to record the date and time of swab collection. Oral samples were collected by rubbing a swab across the buccal mucosa and tongue, as previously described.² In addition, participants were provided with a diary card to log swab collection and record the presence or absence of oral symptoms consistent with HSV recurrence, such as itching or tingling and/or any visible lesions. At weekly follow-up visits, participants returned the swabs they had collected during the previous week, and were provided with a new collection kit. Study staff reviewed the diary entries from the prior week, discussed any changes in medications or health status with the participant, and provided additional reminders and training regarding oral swab collection methods.

2.3. Laboratory Methods

Oral swab samples were tested for the presence and quantity of HSV DNA via PCR at the Molecular Diagnostic Laboratory at the University of Washington, as previously described.^9,10

2.4. Data Analysis

2.4.1. Number of Episodes and Shedding Rate

The overall number of HSV reactivations (episodes), per-person total oral HSV shedding rate, and subclinical shedding rate were calculated. Swabs with ≥150 copies/mL of HSV DNA detected were considered positive. Consistent with prior literature, the shedding rate was calculated by dividing the number of positive swabs by the total number of swabs collected throughout the study.^2,11 The subclinical shedding rate was calculated by dividing the number of positive swabs collected where no lesions were reported by the total number of swabs collected where no lesions were reported. Definitions are also provided in Supplemental Table 1.

2.4.2. Shedding Episodes

The total number of HSV episodes included those of known, estimated, and unknown duration, were quantified. As previously described, a shedding episode of known duration was defined as one or more sequential positive swabs that were both preceded and followed by two negative swabs.² Episodes of known duration could include single negative or missing swabs amidst a series of positive swabs. For these episodes, we estimated start times as the midpoint between the first positive swab and the prior negative swab, and end times as the midpoint between the final positive swab and first negative swab, rounded to the quarter hour. Shedding episodes of estimated duration were defined as those with a missing swab before or after the two negative swabs required to initiate or terminate a shedding episode. Because we anticipated that samples were collected approximately every eight hours, the durations of these episodes were estimated by taking the average of the minimum (assuming the swab was negative) and maximum (assuming the swab was positive) number of timepoints for the episode, multiplied by eight. Episodes of unknown duration were defined as those for which start or end times could not be determined due to multiple consecutive missing swabs, or when the episode may have extended beyond the observation window (i.e., if the initial or final swabs collected were positive). The minimum duration of these episodes was estimated by multiplying the number of timepoints for the episode by eight hours. Analyses were conducted in RStudio version 2022.12.0+353.

3. Results

3.1. Study Population

Fifteen participants were enrolled (8 liver transplant recipients and 7 kidney transplant recipients) (Table 1). All participants were HSV-1 seropositive, 4 (26.7%) were also HSV-2 seropositive, 7 (46.7%) were CMV seropositive, and 10 (66.7%) participants self-reported a prior history of oral HSV. The median age of participants was 58.5 years (range: 44–69 years), 9 (60%) were women, and 10 (66.7%) participants self-identified as white. The median time since transplant was 20 months (range: 10–32 months). The most common induction immunosuppression used among study participants was a combination of antithymocyte globulin (ATG) and methylprednisone (Table 2). All study participants were taking at least one immunosuppressive medication, and the most common regimens were tacrolimus/prednisone/mycophenolate (46.7%) and tacrolimus only (26.7%). Maintenance immunosuppression regimens did not change throughout the study. None of the participants experienced an episode of graft rejection during sample collection or in the six months prior to study entry.

Table 1:

Participant Demographics

	Overall (N=15)
Age (Years)
Mean (SD)	57.4 (6.51)
Median [Min, Max]	58.5 [44.5, 69.7]
Sex
Men	6 (40.0%)
Women	9 (60.0%)
Transplant Type (Organ)
Kidney Transplant	7 (46.7%)
Liver Transplant	8 (53.3%)
Months Since Transplant
Mean (SD)	20.1 (6.39)
Median [Min, Max]	20.0 [10.0, 32.0]
Self-Reported History of Oral Herpes
Yes	10 (66.7%)
No	5 (33.3%)
HSV-1 and HSV-2 Serostatus
HSV-1	11 (73.3%)
HSV-1 and HSV-2	4 (26.7%)
CMV Serostatus
Seronegative	8 (53.3%)
Seropositive	7 (46.7%)

Table 2:

Participant Characteristics and Oral HSV Shedding Data

Participant Number	Agea	Sex	Tx Type	CMV Serostatus (Recipient/Donor)	Months Since Tx	Self-Reported History of Oral HSV	Oral HSV Episode in Past 12 Months	Total Swabs	Positive Swabs	OSRc	Subclinical OSRd	Shedding Episodes	Immunosuppressants
													Induction	Maintenance
1	50’s	M	L	− / +	17	No	No	126	0	0	0	0	ATG x 3, M	T
2	40’s	M	K*	− / +	19	Yes	No	124	5	4.0	4.0	1	ATG x 4	T, P, MMF
3	40’s	F	K	+ / −	25	Yes	No	128	6	4.7	4.7	1	ATG x 4, M	T, P, MMF
4	50’s	F	L	+ / ?	20	Yes	No	113	38	33.6	33.6	4	ATG x 3	T, P
5	60’s	M	L	+ / ?	14	No	No	118	0	0	0	0	ATG x 3, M	MMF
6	60’s	M	K*	+ / +	26	Yes	No	113	24	21.2	21.2	4	B x 2, M	T, P, MMF
7f	60’s	F	L	− / +	11	Yes	Yesh	108	17	15.7	16.0	4	ATG x 3, M	T, MMF
8	60’s	M	L	− / +	10	No	No	116	1	0.9	0.9	1	B	T
9	50’s	F	K	− / −	18	Yes	Yesg	127	13	10.2	10.2	5	B x 2, M	T, P, MMF
10	50’s	F	L	+ / ?	21	No	No	128	6	4.7	4.7	1	ATG x 3, M	T, P, MMF
11	50’s	M	K	− / −	25	Yes	No	114	12	10.5	10.5	2	ATG, M	T, P, MMF
12	60’s	F	L	− / +	25	Yes	Yesi	124	11	8.9	NA e	3	ATG x 3, M	T
13	60’s	F	K	− / +	32	Yes	Yesg	122	29	23.8	21.2	3	B x 2, M	T, P
14	50’s	F	L	+ / +	26	Yes	No	113	0	0	0	0	B x 2, M	T
15	40’s	F	K	+ / +	13	No	No	124	18	14.5	14.5	3	ATG x 3, M	T, P, MMF

^1.Maintenance immunosuppressants: T = tacrolimus, P = prednisone, MMF = mycophenolate mofetil

^2.Induction immunosuppressants: ATG = antithymocyte globulin (ATG dose = 100mg (ex: “ATG x 3” = 300mg ATG)), B = basiliximab, M = methylprednisone

^aAge presented in decades to protect identity

^bTx type = transplant type (L = liver, K = kidney). Asterisk (*) indicates participants also had prior SOT.

^cOSR = oral shedding rate

^dSubclinical OSR = subclinical oral shedding rate

^eSubclinical OSR could not be calculated for Participant 12 due to missing diary data for 3 episodes

^fParticipant was given acyclovir for lesion on day 20 of sampling

^gParticipants had one oral HSV recurrence in past year, none in past 3 months

^hParticipant had one oral HSV recurrence in past year that was within the past 3 months

ⁱParticipant had three oral HSV recurrences in past year, one in past 3 months

3.2. Adherence to Swabbing Protocol

A total of 1,798 swabs were collected by study participants. Participants collected a high percentage of the expected study swabs (median 96.8%, range: 85.7% - 101.6%), with a median of 122 swabs (range: 108–128) collected per person. Of the 13 participants who did not have 100% sample collection according to protocol, 6 (46.2%) continued to collect swabs beyond day 42. One participant did not attend the last follow-up visit but completed swab collection through day 42.

3.3. Number and Duration of Episodes, Oral HSV Shedding Rate

Twelve participants (80%) experienced at least one oral HSV shedding episode during the observation period, most of whom had a prior history of oral herpes (n = 9, 75%). Among the five participants with no prior history of oral HSV, three (60%) experienced at least one shedding episode. The median per-person oral shedding rate was 8.9% (range: 0–33.6%), and the median subclinical shedding rate was 7.5% (range: 0–33.6%). Shedding patterns for all participants are presented in Figure 1. Throughout the study, 32 HSV shedding episodes of any type or duration occurred, with a median of 2 episodes per person across all participants (range: 0–5 episodes). Among those who shed, the median number of episodes was 3, for an expected reactivation rate of 2.1 episodes per 30 days. Overall, 27 shedding episodes occurred without lesions (84.4%; 9.4% missing diary entries). Four (26.7%) participants reported symptoms at any time during the study period, and 2 (13.3%) experienced oral lesions. There were 18 episodes of known duration, with a median duration of 21.8 hours (IQR: 10.8–46.1 hours) and 6 episodes of estimated duration, with a median duration of 42 hours (IQR: 30–72 hours). There were also 8 episodes of unknown duration, for which only a minimum duration per episode (range: 24–216 hours) could be estimated.

Figure 1: Oral HSV Shedding Patterns Among Participants Who Shed.

Data from the participants (n = 12, 80%) with detectable shedding during the study are shown. Most episodes (n = 24, 75%) occurred without either symptoms or oral lesions. Timepoints where symptoms such as itching, burning, or tingling were reported are noted with a triangle, and timepoints without symptoms are shown with open circles. Any gaps in the plot indicate missing swabs. Participant 12 had missing symptom data for 3 episodes, which are shown with an X. Participants 7 and 13 experienced oral lesions, which are indicated with an asterisk.

3.4. HSV DNA Quantity

Across all positive swabs (n = 180, 10% of all swabs), the median HSV DNA quantity was 4.02 log₁₀ c/mL (IQR: 3.21–4.99). For episodes of known duration, the pooled median DNA level across all positive swabs was 3.79 log₁₀ c/mL (IQR: 2.95–4.70) and the median per-episode maximum was 4.05 log₁₀ c/mL (IQR: 2.93–5.67). Similarly, for episodes of estimated duration, the pooled median across all positive swabs was 3.72 log₁₀ c/mL (IQR: 3.15–4.65) and the median per-episode maximum was 5.76 log₁₀ c/mL (IQR: 4.35–6.35). For episodes of unknown duration, the pooled median across all positive swabs was 4.50 log₁₀ c/mL (IQR: 3.63–5.12) and the median per-episode maximum was 6.52 log₁₀ c/mL (IQR: 6.22–6.67). For episodes of duration ≤ 8 hours, the median maximum per-episode DNA level was 3.16 log₁₀ c/mL (IQR: 2.74–3.57); for episodes of duration > 8 hours and ≤ 16 hours, the median maximum per-episode DNA level was 2.80 log₁₀ c/mL (IQR: 2.48–2.96); for episodes of duration > 16 hours and ≤ 24 hours, the median maximum per-episode DNA level was 5.19 log₁₀ c/mL (IQR: 4.95–5.59); and for episodes longer than 24 hours (including the episodes of unknown duration with estimated duration >24 hours), the median maximum per-episode DNA level was 6.23 log₁₀ c/mL (IQR: 5.26–6.59) (Figure 2).

Figure 2: Distribution of Episode Duration and Corresponding Median of Maximum Viral DNA Levels.

Episodes of known, estimated, and unknown duration > 24 hours are shown (n = 31 episodes). One episode of unknown duration (estimated duration equals 24 hours) is not shown. Within each hourly duration category, the maximum DNA level for each episode was calculated, and the medians of those maximums are reported here.

3.5. Clinical Events

3.5.1. CMV or EBV viremia

Three participants (Participants 8, 13, and 15) experienced an episode of CMV reactivation within six months of study entry. Participant 8 had CMV viremia about four months prior to swab collection, with no gastrointestinal or pulmonary involvement. Participant 15 had two episodes of CMV viremia prior to study entry, the most recent of which occurred about five months prior to swab collection. None of these episodes involved gastrointestinal or pulmonary CMV. Participant 13 experienced CMV syndrome, including multiple episodes of CMV viremia prior to study entry and one episode during the study a few weeks into swab collection. This episode consisted of low-level viral reactivation and was not treated. In addition, one participant (Participant 4) was taking valganciclovir about four months prior to study entry, although no episodes of CMV viremia were recorded in the medical record. No episodes of EBV viremia occurred within six months of entry or during the study period.

3.5.2. Other events

Two participants (Participant 15 and Participant 6) were hospitalized during the study period with klebsiella urinary tract infection and community acquired pneumonia, respectively. Participant 13 also experienced recurrent urinary tract infections during the study period.

4. Discussion

We enrolled a cohort of 15 SOT recipients to determine the feasibility of characterizing patterns of oral HSV shedding among this population. Participants were able to adhere to the three times daily swab collection and short episodes of HSV reactivation were captured. The median per-person shedding rate was 8.9% of swabs and there was a median of 2 shedding episodes over 30 days, with a median episode duration of 21.8 hours for those of known duration. Episode duration and HSV DNA levels varied within and between participants, with individual episodes that ranged in length from a single positive timepoint to those that lasted multiple days. Most shedding episodes occurred without signs or symptoms.

Self-collected swabs are a reliable method for HSV detection and have been used in prior studies^2,12,13, including two^2,12 conducted by Mark et al. that characterized HSV oral shedding patterns by sampling four times per day among immunocompetent persons and persons with HIV (PWH). In the first study, the median shedding episode duration among immunocompetent persons was 24 hours; 39% of episodes lasted ≤12 hours and 45.5% lasted >24 hours. In the study among PWH (median CD4 count of 426 cells/mm³), 59% of oral episodes of known duration lasted ≤12 hours and 24% lasted >24 hours. In the current study, we found that more than 60% of episodes among SOT recipients lasted >24 hours. While our results are not directly comparable due to different sampling frequencies, the duration estimates for these (>24 hours) are less likely to be impacted by the differences in sampling schedules, suggesting that longer-duration episodes may be more common among SOT recipients. The HSV reactivation rate among SOT patients who shed in this study (2.1 episodes per 30 days) was also slightly higher than that observed among immunocompetent persons and PWH (1.4 per 30 days for both), suggesting that reactivations may also be more frequent among SOT recipients. In this study and the prior studies, subclinical HSV reactivation was common.

Oral HSV shedding is a key measure of viral reactivation, and understanding viral kinetics may have important implications for outcomes in transplant settings. HSV shedding may be associated with immune activation, and we hypothesize that viral reactivation may be associated with persistent local or systemic inflammation.¹⁴ Future studies will be required to evaluate the potential clinical significance of these subclinical shedding episodes. Furthermore, while our study only evaluated the presence of HSV DNA, HSV-1 shedding in saliva concurrent with other herpesviruses has been observed among SOT¹⁵ and hematopoietic stem cell transplant recipients¹⁶ which may be important to evaluate in future work.

There are some limitations to this pilot study. First, our results are subject to selection bias, as participants were recruited from a setting that may not be representative of the larger population of HSV-1 seropositive SOT recipients. Second, participants collected swabs at various timepoints after their SOT, and this heterogeneity combined with the small sample size makes it challenging to determine risk factors for shedding in this population. In the future, studying participants at a uniform time post-transplant, potentially at the height of immunosuppression and during/after stopping antiviral prophylaxis, may provide further insights about HSV reactivation. Future studies will also be required to determine if oral HSV shedding is associated with adverse outcomes in this population. Third, the small sample size limited our ability to evaluate potential differences in shedding rates and number of episodes between subgroups, such as comparing liver to kidney transplant recipients. Fourth, because sampling only occurred three times daily, it is possible that episodes of very short duration (i.e. < 8 hours) were not captured.

The current study provides a detailed description of HSV reactivation frequencies among SOT recipients, and our findings suggest that collecting multiple daily oral swabs is feasible among SOT recipients, as participants in this study showed a high rate of adherence to the sample collection protocol. In addition, this study provides important pilot data that can be used for sample size and power calculations when designing future clinical trials. Based on these data, twice daily sampling (i.e. every 12 hours) would be expected to capture 78.1% of shedding episodes. While twice daily sampling will likely miss shorter episodes and reduce the precision of episode duration estimates, it may be sufficient to detect most episodes, and may be easier for participants. These findings provide important data and considerations for future studies of HSV oral shedding among immunosuppressed populations.

Abbreviations

HSV

herpes simplex virus

HSV-1/HSV-2

herpes simplex virus type 1/type 2

PCR

polymerase chain reaction

PWH

people with HIV

SOT

solid organ transplant

UW

University of Washington

Data Availability Statement

Data are available upon request from the authors.