In the recently reported proof-of-concept CAPRISA 004 trial, vaginal application of tenofovir 1% gel was demonstrated to confer protection from sexual acquisition of HIV infection among high-risk women in KwaZulu-Natal, South Africa, with an incidence rate ratio of 0.61 (P = 0.017) . An additional, surprising result from this groundbreaking microbicide trial was that among the 434 women documented to be seronegative for herpes simplex virus type 2 (HSV-2) at baseline, vaginal tenofovir was also associated with a decreased incidence of HSV-2 infection, with an incidence rate ratio of 0.49 (P = 0.003). Since HSV-2 is thought to be associated with increases in the acquisition, onward transmission, and clinical progression of HIV infection [2,3], such a reduction in HSV-2 incidence with tenofovir could have important benefits for both HIV-uninfected and HIV-infected individuals.
Oral administration of tenofovir disoproxil fumarate is routinely used in the treatment of HIV-infected individuals and is a first-line recommended treatment option in most current guidelines. It is unknown whether the oral use of tenofovir is similarly associated with decreases in HSV-2 activity. If so, then use of this antiretroviral drug could potentially offer secondary benefits to patients by reducing HSV-2 symptoms, asymptomatic shedding, and/or transmission. We report the impact of oral tenofovir use as part of combination antiretroviral therapy (ART) on asymptomatic HSV shedding in a cohort of HSV, HIV-1 co-infected adults in Toronto, Canada.
We enrolled HSV (type 1 and/or 2), HIV-1 co-infected individuals into an ongoing cohort study examining the impact of ART on HSV shedding at the Immunodeficiency Clinic of the Toronto General Hospital, Toronto, Canada. Patients were eligible for inclusion if they had documented HIV-1 and HSV (type 1 and/or 2) infection, were adults aged 18 years or older, had no history of either symptomatic herpes within the previous 4 months nor more than two episodes of herpes per year, had been using the same combination ART (cART) regimen for at least 90 days, had achieved plasma HIV RNA below 50 copies/ml, and were not using any medications with anti-HSV activity (including acyclovir, valacyclovir, famciclovir, ganciclovir, valganciclovir, cidofovir, foscarnet). Type-specific HSV serostatus was determined using the HerpeSelect gG-1 and gG-2 ELISA (Focus Technologies, Cypress, California, USA), a qualitative assay for detecting human IgG antibodies to glycoprotein G-1 (in HSV-1) or G-2 (in HSV-2), the major proteins which evoke type-specific responses. In cohorts of pregnant women and sexually active adults, the sensitivity is 91–96% and specificity 92–95% compared to Western blot [4–6].
Eligible participants were instructed on how to collect their own swab specimens from four anatomic sites (one oral, two sex-specific genital, one anal) daily for 28 consecutive days. Oral swabs were rubbed over the upper and lower gumlines and palate. Anal swabs were inserted 1 cm into the anus and rotated for three full rotations. Men rubbed one swab the entire surface of the shaft of the penis, and another swab over the urethral meatus. Women rubbed one swab over the surface of the labia majora and minora; another swab was inserted into the vagina, advanced until meeting resistance, and rotated for three full rotations. Each swab was inserted into a separate vial containing viral transport media and kept refrigerated; specimens were delivered in batches to the laboratory on a weekly basis. At each weekly visit, printed, written instructions on specimen collection and storage, including diagrams, were provided and instructions were reinforced verbally. Study participation was terminated early in the event of a clinically confirmed herpes outbreak. Plasma HIV RNA was repeated at the conclusion of the 28-day study period and if the participant had a detectable viral load at that time they were excluded from the analysis.
HSV-1 and HSV-2 shedding were detected qualitatively by polymerase chain reaction (PCR) using the LightCycler HSV 1/2 Detection Kit (Roche Diagnostics, Mannheim, Germany). The lower limit of detection of this assay is 1 copy/μl, and any signal was reported as positive. For cost reasons, genital and anal swabs were pooled prior to testing. Type-specific shedding rates were calculated as the proportion of days on which HSV PCR was positive among seropositive participants.
In this sub-analysis, HSV type-specific shedding rates were compared between tenofovir users and nonusers using the Wilcoxon Rank Sum test. Participants were also classified according to the presence or absence of any type-specific HSV shedding during the study period, with results compared between groups using the chi-square test. All tests were two-sided, and a threshold of alpha = 0.05 was used to define statistical significance. Since the findings from the CAPRISA 004 trial relate to the impact of tenofovir gel on HSV-2 acquisition, we were primarily interested primarily in the impact of oral tenofovir use on HSV-2 shedding among HSV-2 seropositive individuals. However, because HSV-1 and HSV-2 share important virologic, pathophysiologic and clinical similarities , we conducted exploratory analyses regarding the impact of oral tenofovir on HSV-1 shedding and on shedding of either HSV type.
Ethical approval was obtained prior to study initiation from the University Health Network Research Ethics Board.
Forty individuals were enrolled, of which 30 were HSV-2 seropositive and included in the primary analysis. There were 32 participants who were HSV-1 seropositive, and 22 were both HSV-1 and HSV-2 seropositive. Swabs were obtained on all 28 study days by 38 participants; one participant inadvertently forgot to collect specimens on day 9, and another participant aborted specimen collection after 14 days due to an outbreak of herpes on the buttocks for which laboratory confirmation was not obtained. All participants maintained plasma HIV RNA below 50 copies/ml at post-28-day follow-up.
The baseline characteristics of the entire cohort are shown in Table 1. Most participants were men, and tenofovir was part of the cART regimen in 22 of 40 (55%) participants overall and 17 of 30 (57%) of HSV-2-infected participants. Tenofovir users had been using their current cART regimen for less time than nontenofovir users but there were no other significant differences between groups.
The HSV-2 shedding rate among HSV-2 seropositive participants was low, with shedding detected on a median [interquartile range (IQR)] of 7.1 (0, 14.3) percentage of specimen collection days, and did not differ between tenofovir users and nonusers (P = 0.36, Table 2). The maximum shedding rate was 46.4% of collection days, observed in a female tenofovir-using participant. There was no difference in the number of HSV-2 shedders in the tenofovir and nontenofovir using groups over the 28-day observation period (59 vs. 46%; P = 0.49).
In univariate analyses, no statistically significant differences were seen in HSV-2 shedding rates according to sex or history of symptomatic herpes. No correlation was observed between shedding rates of HSV-1, HSV-2 or both, with baseline CD4 cell count, years since HIV diagnosis, years on cART, or years on the current cART regimen.
Rates of shedding for HSV-1 alone, and for either HSV-1 or HSV-2, were also similar between tenofovir users and nonusers (Table 2). Similarly, the number of HSV-1 shedders and of HSV shedders overall did not differ between groups (P = 0.65 and P = 0.82, respectively). No statistically significant differences were seen in these shedding rates according to baseline clinical characteristics.
Tenofovir was hypothesized to have an anti-HSV-2 effect in the CAPRISA 004 trial because of its shared molecular chemistry with the anticytomegalovirus agent cidofovir . Both compounds, along with adefovir, are classified as acyclic nucleoside phosphonates (ANPs), and are derivatives of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), itself originally developed as a broad-spectrum anti-DNA virus agent . Although topical tenofovir gel was associated with a significant decrease in HSV-2 acquisition among high-risk women in the recent CAPRISA 004 trial, we did not observe an impact of tenofovir on HSV-2 or HSV-1 shedding rates in this cohort of 40 HIV, HSV co-infected asymptomatic adults.
The most likely explanation for these discrepant results is that in contrast to topical application, oral tenofovir achieves inadequate genital tract concentrations to block HSV-2 acquisition or replication. Taken as part of a suppressive HAART regimen, tenofovir achieves excellent penetration of both the female and male genital tract. In a sample of 16 HIV-infected women, for instance, the median [95% confidence interval (CI)] cervicovaginal fluid (CVF) tenofovir concentration at the end of the dosing interval was 84.03 (24.47–288.56) ng/ml, giving a CVF-to-plasma ratio of 5.15 (1.18–22.6) . Similarly, in a study of 15 HIV-infected men, the median seminal fluid tenofovir concentration at the midpoint between drug intake and specimen collection was 250 ng/ml, giving a semen to plasma ratio of 3.3 (range 0.9–49.2) . This pharmacologic feature is being exploited in ongoing clinical trials of oral tenofovir as pre-exposure prophylaxis for HIV, but such levels are low in comparison with tenofovir's in-vitro EC50 for HSV-2 of 10000 ng/ml . In comparison, CVF tenofovir concentrations achieved among tenofovir gel users who remained HIV negative throughout the CAPRISA trial was 520 (range 0–1.3 million) ng/ml . A related possibility is that different concentrations of tenofovir may be required to inhibit HSV replication and acquisition. Neither this study nor the CAPRISA trial directly assessed this question.
Our study has limitations that warrant consideration. First, the methods of specimen sampling may not have captured all anatomic reactivations of HSV infection, particularly given recent reports that many HSV reactivations last only hours , and errors or nonadherence with specimen self-collection by study participants may have reduced the diagnostic yield. However, self-collection was used in order to increase the acceptability of the study, and sampling instructions were regularly reinforced with participants using both verbal and written methods. In addition, since sampling methods were the same for all participants, any bias related to study methodology would be expected to be similar in those taking and those not taking tenofovir. Finally, because this study was primarily designed to study a different clinical question and is still enrolling patients, our sampling period of 28 days and sample size of 30 participants were relatively small. Therefore, although the difference in shedding we observed was in the opposite direction of that expected, with more HSV-2 shedding observed in tenofovir users than in nonusers, our sample size may have been inadequate to observe the hypothesized attenuating effect of tenofovir use on HSV-2 shedding.
We were unable to demonstrate an effect of oral tenofovir on shedding of either HSV-2 or HSV-1 in this cohort of Canadian co-infected adults on suppressive HAART. Future in-vivo work is needed to confirm these preliminary null findings. Given the negative synergy between HSV-2 and HIV, if orally administered tenofovir exhibited similar anti-HSV-2 effects, this could have additional public health benefits for decreasing the onward transmission of both viruses from HIV-infected persons.
All authors participated in the original design of the cohort study, read drafts of the manuscript and provided critical comments. D.T., R.K. and S.W. enrolled patients into the study. D.T. and J.R. performed sample size calculations and statistical analyses. D.T. wrote the original draft of the manuscript. S.W. oversaw the conduct of the study.
The work is supported by grants from the Ontario HIV Treatment Network and the Canadian Institutes of Health Research (#HET-85518). D.T. receives research fellowship support from the Canadian Institutes of Health Research. R.K. receives salary support from the Canadian Research Chair Programme. J.R. and S.W. receive salary support from the Ontario HIV Treatment Network.
1. Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, Mansoor LE, et al
. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 2010; 329:1168–1174.
2. Glynn JR, Biraro S, Weiss HA. Herpes simplex virus type 2: a key role in HIV incidence. AIDS 2009; 23:1595–1598.
3. Corey L, Wald A, Celum CL, Quinn TC. The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: a review of two overlapping epidemics. J Acquir Immune Defic Syndr 2004; 35:435–445.
4. Strick L, Wald A. Type-specific testing for herpes simplex virus. Exp Rev Molec Diagn 2004; 4:443–453.
5. Ashley RL. Sorting out the new HSV type specific antibody tests. Sex Transm Infect 2001; 77:232–237.
6. Wald A, Ashley-Morrow R. Serological testing for herpes simplex virus (HSV)-1 and HSV-2 infection. Clin Infect Dis 2002; 35:S173–182.
7. Tan DH, Kaul R, Walsmley S. Left out but not forgotten: should closer attention be paid to coinfection with herpes simplex virus type 1 and HIV? Can J Infect Dis Med Microbiol 2009; 20:e1–e7.
8. De Clercq E. The acyclic nucleoside phosphonates from inception to clinical use: historical perspective. Antiviral Res 2007; 75:1–13.
9. Kwara A, Delong A, Rezk N, Hogan J, Burtwell H, Chapman S, et al
. Antiretroviral drug concentrations and HIV RNA in the genital tract of HIV-infected women receiving long-term highly active antiretroviral therapy. Clin Infect Dis 2008; 46:719–725.
10. Lowe SH, van Leeuwen E, Droste JA, van der Veen F, Reiss P, Lange JM, et al
. Semen quality and drug concentrations in seminal plasma of patients using a didanosine or didanosine plus tenofovir containing antiretroviral regimen. Ther Drug Monit 2007; 29:566–570.
11. Kashuba AD, Abdool Karim SS, Kraft E, White N, Sibeko S, Werner L, et al.Do systemic and genital tract tenofovir concentrations predict HIV seroconversion in the CAPRISA 004 tenofovir gel trial?
Vienna, Austria. Abstract TUSS0503. 2010.
12. Mark KE, Wald A, Magaret AS, Selke S, Kuntz S, Huang ML, Corey L. Rapidly cleared episodes of oral and anogenital herpes simplex virus shedding in HIV-infected adults. J Acquir Immune Defic Syndr 2010; 54:482–488.