The natural history of HSV-2 infection and the temporal relationship between HSV-2 and HIV shedding in a population of high-risk women, Tanzania Final report June 2009 Dr Deborah Watson-Jones Current address: Department of Infectious & Tropical Diseases London School of Hygiene & Tropical Medicine Keppel Street, London Email: Deborah.watson-jones@lshtm.ac.uk Previous address: AMREF, P.O. Box 1482 Mwanza, Tanzania. Dr Clare Tanton Current address: Centre for Sexual Health & HIV Research University College London Mortimer Market Centre Off Capper Street, London Email: ctanton@gum.ucl.ac.uk Previous address: National Institute for Medical Research, Mwanza, Tanzania / London School of Hygiene & Tropical Medicine, Keppel Street, London. - 1 -
Index 1. Summary... 3 1.1. Aims... 3 2. Collaborators... 3 2.1. Collaborating organisations... 3 2.2. Principal Investigator & Co-investigators... 3 2.3. Funding... 4 3. Overview of the study... 4 4. Results... 4 4.1. Recruitment & follow-up... 4 4.2. Study population... 5 4.3. Aim: to describe the natural history of HSV-2 infection in HIV seronegative and HIV seropositive women... 6 4.4. Aim: to examine the variability in HSV-2 and HIV shedding and any modifying effect of HSV suppressive therapy... 8 4.5. Aim: to examine the temporal relationship between HSV-2 and HIV genital shedding... 12 5. Conclusions... 13 6. References... 13-2 -
1. Summary This report summarises the work carried out on a small sub-study (NIMR/HQ/R.8a/Vol. IX/367) nested within a randomised controlled trial conducted in north-west Tanzania. This is the final report of this study, the fieldwork for which was completed in October 2005. 1.1. Aims The aims of this sub-study were to: describe the natural history of HSV-2 infection in HIV seronegative and HIV seropositive high-risk women in Tanzania examine the variability in HSV-2 and HIV shedding in high-risk women in Tanzania and any modifying effect of HSV suppressive therapy (with aciclovir) examine the temporal relationship between HSV-2 and HIV genital shedding by comparing HIV shedding during and immediately following a period of HSV-2 shedding and between periods of HSV shedding 2. Collaborators 2.1. Collaborating organisations The following organisations are all collaborators for this study: African Medical & Research Foundation (AMREF), Tanzania National Institute for Medical Research (NIMR), Tanzania London School of Hygiene and Tropical Medicine (LSHTM), UK Université Pierre & Marie Curie (UPMC), France 2.2. Principal Investigator & Co-investigators The principal investigator for this study is: Dr Deborah Watson-Jones, AMREF, Mwanza. The Co-investigators / collaborators are: Dr Clare Tanton, LSHTM, UK & NIMR, Tanzania Mr John Changalucha, NIMR, Tanzania Dr Helen Weiss, LSHTM, UK Professor Richard Hayes, LSHTM, UK Professor David Ross, LSHTM, UK Professor Laurent Belec, Inserm U743, France Dr Jerome Legoff, Inserm U743, France - 3 -
2.3. Funding This study was funded by the Medical Research Council (MRC), UK. 3. Overview of the study This sub-study was nested within a double-blind placebo-controlled trial of HSV suppressive therapy with aciclovir (400mg b.i.d) which was carried out in the Mwanza, Shinyanga and Mara Regions in northwest Tanzania. The trial s aims were to determine the effect of aciclovir 400mg b.i.d on both HIV acquisition, among women who were initially HSV-2 seropositive but HIV-1 seronegative, and genital shedding of HIV, among women who were initially seropositive for both HSV-2 and HIV. This trial found no effect of aciclovir on either HIV acquisition [1] or genital HIV shedding (Tanton et al, submitted). For this sub-study, a sample of women already enrolled in this RCT and living at two of the sites (Geita Town and Kakola) were seen three times per week for four weeks to collect cervical, vaginal and external skin swabs. Data were collected between August and October 2005. Swabs were tested using PCR for quantity of HIV-1 RNA (cervical and vaginal swabs) and HSV DNA (cervical, vaginal and vulval/perineal/perianal). 4. Results 4.1. Recruitment & follow-up Of the 124 women given information on the sub-study at their 18 month appointment, 78 were enrolled at the later enrolment appointment. Of the 77 women who attended at least 2 visits and therefore whose data is used in this analysis, 50 were HIV negative (26 on placebo and 24 on aciclovir) and 27 were HIV positive (13 on placebo and 14 on aciclovir). Follow-up by treatment arm and HIV status is shown in Table 1. Follow-up was 95% or over in each group. Table 1 Follow-up of the study population by treatment allocation Total no. of visits attended Proportion of expected visits attended No. / % HIV negative Aciclovir No. / % No. / % HIV positive Aciclovir No. / % 309 275 155 159 99% 95% 99% 95% - 4 -
4.2. Study population Characteristics of the population are shown in Table 2. The mean age was 29 years and approximately half of the women were divorced or separated. Median age at first sex was 16 years and median lifetime number of sexual partners was 5. A history of genital ulcers was reported by 30% of the women and 21% reported an episode of genital ulcer disease in the last year. HIV negative and HIV positive women were similar for most characteristics. Table 2 Key characteristics of the women enrolled in the sub-study, by HIV-1 status 1 Characteristic HIV seronegative (N=50) HIV seropositive (N=27) All participants (N=77) Socio-demographic characteristics Median age (IQR), yrs 29 (26-32) 30 (25-34) 29 (26-33) Marital status Single 6 (12.0) 4 (14.8) 10 (13.0) Married / living as married 16 (32.0) 4 (14.8) 20 (26.0) Divorced / separated 24 (48.0) 15 (55.6) 39 (50.7) Widowed 4 (8.0) 4 (14.8) 8 (10.4) Behavioural characteristics Median age at first sex (IQR), yrs 16 (15-18) 16 (15-17) 16 (15-18) Median no. lifetime partners 4 (3-5) 6 (4-20) 5 (3-7) (IQR), yrs No. of times cleanses vagina per day Doesn t cleanse 20 (40.0) 9 (33.3) 29 (37.7) 1-2 13 (26.0) 8 (29.6) 21 (27.2) 3 17 (34.0) 10 (37.0) 27 (35.1) Clinical characteristics History of genital ulcers / 17 (34.0) 6 (22.2) 23 (29.9) blisters Episode of GUD in the last yr 12 (24.0) 4 (15.4) 16 (21.1) Biological characteristics Mean plasma HIV RNA load 2 (SD), log 10 copies/ml - 4.62 (1.13) - Cervico-vaginal HIV-1 RNA 2 detected - 14 (58.3) - 1 Variables collected at enrolment to the main trial; 2 Among those HIV positive at enrolment to the main trial. Reported adherence during the sub-study was good with most women self-reporting taking the correct number of tablets at each visit: 84% (42/50) of HIV negative women and 85% (23/27) of HIV positive women. During the 3 month interval of the main trial within which this one month sub-study was nested, 52% of HIV negative women and 48% of HIV positive women had adherence of 90% or more. - 5 -
4.3. Aim: to describe the natural history of HSV-2 infection in HIV seronegative and HIV seropositive women The natural history of HSV-2 infection is described in the 26 HIV negative women in the placebo arm and the 13 HIV positive women in the placebo arm. 4.3.1. Frequency of HSV shedding In HIV negative women, HSV DNA was detected on 33 (11%) of days when samples were taken compared to 30 (19%) of days in the HIV positive women. Of the HIV negative women, 50% had HSV DNA detected from at least one swab during the follow-up period while 70% of HIV positive women had HSV DNA detected at least once (Figure 1). Figure 1 Graph to show proportion of visits women had HSV DNA detected by treatment allocation percent 0 20 40 60 80 HIV negative HIV positive None <25% 25-<50% 50-<75% There were a total of 19 episodes of HSV shedding (where HSV DNA was detected from at least one site at consecutive visits) in HIV negative women and 16 in HIV positive women. Most episodes lasted only one visit in both HIV negative and HIV positive women. Among those women with HSV DNA detectable during at least one visit, the total number of episodes of HSV DNA shedding ranged from 1-3 in both HIV negative and HIV positive women. - 6 -
4.3.2. Sites of HSV reactivation The most common site of HSV reactivation was the vulva/perineal/perianal area in both HIV negative and HIV positive women (Figure 2) followed by the vagina and then the cervix. Figure 2 Percentage of days HSV DNA was detected from each site, by HIV status 18 16 Percentage days positive 14 12 10 8 6 4 Cervical Vaginal Vulval/perineal/perianal 2 0 HIV negative HIV positive Table 3 shows the rates of HSV DNA shedding by site and by whether HSV symptoms were also present. Most shedding was sub-clinical (ie there were no ulcers present when the HSV DNA was detected). Ulcers were present on only 3 days in HIV negative women and 4 days in HIV positive women. During the 7 visits when ulcers were present, there was only one visit when HSV DNA was not detected. Table 3 Rates of total, clinical and subclinical HSV DNA detection by site sampled No. positive / No. visits sampled (%) HIV negative HIV positive Any Clinical 2/3 (66.7) 4/4 (100) Sub-clinical 31/306 (10.1) 26/151 (17.2) Total 33/309 (10.7) 30/155 (19.4) Cervical Clinical 2/3 (67) 4/4 (100) Sub-clinical 14/306 (4.6) 11/151 (7.3) Total 16/309 (5.2) 15/155 (9.7) Vaginal Clinical 2/3 (67) 4/4 (100) Sub-clinical 19/306 (6.2) 14/151 (9.3) Total 21/309 (6.8) 18/155 (11.6) Vulval/perineal/perianal Clinical 2/3 (67) 4/4 (100) Sub-clinical 29/306 (9.5) 22/151 (14.6) Total 31/309 (10.0) 26/155 (16.8) In HIV negative women, of the 33 days when HSV DNA was detected, detection was from all sites at 42% of these days. Of the 30 visits when HSV DNA was detected in HIV positive women, it was detected at all sites at 43% of visits (Figure 3). Most episodes of shedding - 7 -
involved the vulval/perineal/perianal area. There were few visits where HSV DNA was detected from only the cervix or the vagina. Figure 3 Sites from which HSV DNA was detected at the visits where HSV DNA was detected from at least one site HIV positive HIV negative All sites Vulval/perineal/perianal & cervix Vulval/perineal/perianal & vagina Vulval/perineal/perianal only Cervix & vagina Cervix only Vagina only 0% 20% 40% 60% 80% 100% 4.3.3. Clinical and sub-clinical HSV DNA detection Few HSV shedding visits were associated with genital ulcers. In HIV negative women, 31 out of 33 days when HSV was detected were subclinical. In HIV positive women 26 out of 30 episodes were sub-clinical. The overall rate of subclinical shedding was 10.1% in HIV negative and 17.2% in HIV positive women (Table 3). 4.4. Aim: to examine the variability in HSV-2 and HIV shedding and any modifying effect of HSV suppressive therapy 4.4.1. Description of genital HSV DNA shedding As described in section 0, HSV shedding varied greatly by individual and also occurred at a higher rate in HIV positive women. Aciclovir is expected to reduce HSV shedding. The effect of aciclovir on overall and site specific shedding is shown in Table 4. For HIV negative women there is a significant decrease in HSV shedding overall and at each site. However, for HIV positive women HSV shedding rates are similar in those on placebo and aciclovir. - 8 -
Table 4 Effect of aciclovir on overall and site specific shedding by HIV status (N=309) HIV negative Aciclovir (N=275) visits HSV DNA detected Any swab 33 (11) 9 (3) OR 1 (95%CI) (N=155) HIV positive Aciclovir (N=160) 0.28 30 (19) 23 (14) (0.12, 0.66) P interaction (treatment & HIV status) = 0.17 Cervical swabs 0.20 16 (5) 3 (1) 15 (10) 15 (9) (0.05, 0.82) P interaction = 0.09 Vaginal swabs 0.30 21 (7) 6 (2) 18 (12) 16 (10) (0.10, 0.95) P interaction = 0.22 Vulval/perineal/ 0.10 31 (10) 3 (1) 26 (17) 17 (11) perianal swabs (0.03, 0.35) P interaction = 0.05 1 OR for the effect of aciclovir on HSV DNA detection compared to placebo. OR 1 (95%CI) 0.69 (0.26, 1.85) 0.97 (0.29, 3.23) 0.85 (0.25, 2.88) 0.59 (0.18, 1.92) Similarly, in HIV negative women, aciclovir reduced the HSV viral load in visits where HSV was detected, but there was no reduction for HIV positive women (Figure 4). Figure 4 Effect of aciclovir on frequency of HSV DNA detection by HIV status HIV negative HIV positive Percent 0 20 40 60 80 None <25% 25-<50% 50-<75% None <25% 25-<50% 50-<75% Aciclovir The variability in HSV DNA detection and viral load is shown for each HIV negative woman with detectable HSV DNA in Figure 5. In the placebo arm, episodes of HSV shedding often involved shedding from more than one site, high viral loads and persisted for more than one visit, while in the aciclovir arm episodes tended to involve only one site and be shorter. - 9 -
Figure 5 Quantity of cervical, vaginal and perineal/perianal HSV DNA detected at each visit for each HIV negative participant with at least one visit with detectable HSV DNA 1 5 6 8 HSV DNA (log10 copies/swab) 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 10 11 12 14 15 16 21 23 0 7 14 21 28 0 7 14 21 28 0 7 14 21 28 25 0 7 14 21 28 Day of follow-up Cervical Perineal/perianal Vaginal GUD Acyclovir 29 30 31 HSV DNA (log10 copies/swab) 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 33 37 44 46 50 0 7 14 21 28 0 7 14 21 28 0 7 14 21 28 Day of follow-up Cervical Perineal/perianal Vaginal GUD The variability in HSV shedding in HIV positive women is shown in Figure 6. Unlike for the HIV negative women, there is no apparent difference by treatment arm. In both arms some episodes lasted only one visit while for other women, shedding episodes were longer in duration. Viral loads in both arms were similar. - 10 -
Figure 6 Quantity of cervical, vaginal and perineal/perianal HSV DNA detected at each visit for each HIV positive participant with at least one visit with detectable HSV DNA 52 54 56 HSV DNA (log10 copies/swab) 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 57 58 59 60 62 63 0 7 14 21 28 0 7 14 21 28 0 7 14 21 28 Day of follow-up Cervical Perineal/perianal Vaginal GUD Acyclovir 64 65 67 HSV DNA (log10 copies/swab) 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 68 69 72 73 76 77 0 7 14 21 28 0 7 14 21 28 0 7 14 21 28 Day of follow-up Cervical Perineal/perianal Vaginal GUD 4.4.2. Description of genital HIV RNA shedding In the following section, we describe HIV shedding in the 27 HIV positive women (13 on placebo and 14 on acyclovir). HIV RNA was detected during at least one visit in 84.6% of women in the placebo and 92.9% of women in the acyclovir arm. HIV shedding was less variable than HSV shedding. Rates and quantities of HIV shedding were similar in the placebo and acyclovir arms (Table 5). - 11 -
Table 5 Summary of cervical & vaginal HIV-1 RNA detection and viral load overall and by treatment allocation / Mean (SD) Acyclovir / Mean (SD) Cervical swabs Per woman N=13 N=14 HIV-1 RNA detection No visits 2 (15) 1 (7) <50% visits 5 (38) 6 (43) 50-99% visits 3 (23) 4 (29) All visits 3 (28) 3 (21) Per visit N=155 N=160 Visits HIV-1 RNA detected 81 (52) 85 (53) Mean quantity (±SD) log 10 1 copies/ml 2.95 (0.59) 2.97 (0.54) Vaginal swabs Per woman N=13 N=14 HIV-1 RNA detection No visits 2 (15) 6 (43) <50% visits 8 (62) 6 (43) 50-99% visits 2 (15) 2 (14) All visits 1 (8) 0 Per visit analysis N=155 N=160 Visits HIV-1 RNA detected 44 (28) 31 (19) Mean quantity (±SD) log 10 1 copies/ml 3.06 (0.69) 2.71 (0.43) 1 Among those swabs with detectable HIV-1 RNA 4.5. Aim: to examine the temporal relationship between HSV-2 and HIV genital shedding Data from the placebo arm were visually examined to describe the longitudinal relationship between HSV and HIV shedding. This is shown in Figure 7. There was no pattern in HIV RNA quantity in relation to HSV shedding. Figure 7 Variation in cervical and vaginal HIV-1 RNA loads in women with HSV DNA detected at one or more visits. Visits where HSV DNA was detected are marked with an x. 53 55 57 HIV-1 RNA (log10 copies/swab) 0 2 4 6 0 2 4 6 0 2 4 6 58 59 60 61 63 0 7 14 21 28 0 7 14 21 28 0 7 14 21 28 Day of follow-up Cervical Vaginal GUD HSV detected Menstrual blood present - 12 -
5. Conclusions Follow-up in this small sub-study nested within a randomised controlled trial was good. HSV reactivation (shedding) was found to vary greatly between individuals. HIV positive women were more likely to shed HSV than HIV negative women. Shedding was common over the 12 visits, 50% of HIV negative women and 70% of HIV positive women had HSV DNA detected at least once. Rates of shedding were similar to those observed in other studies [2]. Most HSV shedding was observed to be sub-clinical, ie not associated with symptoms. The use of episodic therapy for HSV recurrences within this population would therefore only affect a small proportion of total HSV shedding. In this sub-study, while acyclovir reduced HSV shedding rates in HIV negative women, it did not appear to have an impact in HIV positive women. Although adherence rates were similar in HIV negative and HIV positive women, adherence may not have been high enough for adequate HSV suppression in the HIV positive women. 6. References 1. Watson-Jones D, Weiss HA, Rusizoka M, et al. Effect of herpes simplex suppression on incidence of HIV among women in Tanzania. N Engl J Med 2008;358:1560-71 2. Nagot N, Foulongne V, Becquart P, et al. Longitudinal assessment of HIV-1 and HSV-2 shedding in the genital tract of West African women. J Acquir Immune Defic Syndr 2005;39:632-4 - 13 -