KILMARX, PETER H. MD*†; BLANCHARD, KELLY MSc‡; CHAIKUMMAO, SUPAPORN BHP†; FRIEDLAND, BARBARA A. BA‡; SRIVIROJANA, NUCHAREE MSc§; CONNOLLY, CATHY MPH∥; WITWATWONGWANA, PAISIT MD¶; SUPAWITKUL, SOMSAK MD**; MOCK, PHILIP A. MAppStats†; CHAOWANACHAN, THANYANAN MSc†; TAPPERO, JORDAN MD*†
DOZENS OF POTENTIAL MICROBICIDE products, topical preparations that could be used vaginally or rectally before sexual intercourse to prevent transmission of HIV and other sexually transmitted infections (STIs), are in various stages of development. Several products are currently in or will soon begin large-scale effectiveness testing.1,2
Carraguard (formerly PC-515) is the Population Council's lead noncontraceptive microbicide candidate. Carraguard is a gel formulation of a mixture of λ- and κ-carrageenan derived from red seaweed. Carrageenans have been used extensively in the food, pharmaceutical, and cosmetics industries as stabilizers and emulsifiers and have a long history of human use.3,4 Carrageenans have been shown in in vitro tests to prevent HIV-1 infection at concentrations significantly lower than those associated with cellular toxicity, presumably by nonspecific binding to pathogens or host cells to inhibit fusion or entry.5 In addition, in vivo studies in a herpes simplex virus type-2 (HSV-2) mouse model have shown that carrageenans are highly effective at blocking infection with HSV-2, an enveloped virus like HIV-1.6–9
Carraguard and closely related carrageenan formulations have been shown to be safe for vaginal use in women for short durations (7 days)10,11 and up to 12 months.12,13 In Phase I and II studies, women reported that using the gel was acceptable. Although women in these studies reported what their male partners said about the gel, men were not directly questioned about use of the product.
We conducted this study of the safety of Carraguard use among Thai couples primarily to establish the safety of exposure during intercourse for male partners and to evaluate acceptability among men. Male safety data are critical for microbicide development, both to assure researchers and policy makers that continued evaluation of a potential product is warranted and to provide information to potential consumers about product safety.
Men in diverse settings have reported that they will want specific information on the safety of microbicide exposure during intercourse.14,15 However, previous evaluations of male safety of microbicide products have generally required sexually abstinent men to apply the product directly to their penis.16–20 In contrast, we evaluated male safety after exposure during vaginal sex in actual use conditions to provide more accurate data on safety and acceptability of potential microbicides. To our knowledge, this study was the first to use this design to evaluate male safety and acceptability of a candidate microbicide.
We conducted a randomized, placebo-controlled, triple-blind trial to evaluate the safety and acceptability of Carraguard vaginal gel among low-risk heterosexual couples in Chiang Rai, Thailand. The risk of HIV transmission among married couples in Chiang Rai was relatively high in the early 1990s; HIV-1 seroprevalence peaked at 17.3% in male army conscripts and at 10.8% in young primigravid women. Infection rates had declined by the time of this clinical trial.21 Couples were recruited from family planning clinics and during community talks organized by health workers in and around Chiang Rai. In most cases, the female partner was the point of contact; each woman was asked to come to the clinic with her partner to be screened for the study if they were interested.
Consenting men and women were screened for eligibility separately, but were asked to come for screening and all subsequent study visits on the same day whenever possible. They were interviewed and examined separately at each visit and their data were not shared with their partner. To be eligible, both the male and female partner had to be at least 18 years old, in good general health, resident in the area for at least 1 year and planning to stay in the area for at least 6 months, willing to comply with the protocol, and able to achieve a score of 80% or better on true-false comprehension test of key study concepts. Women had to have a normal (Class I) Pap smear. In addition, to qualify as “low risk” for HIV infection both partners had to report no other sexual partners in the previous year and that they were not planning on new partners for the duration of the study, and needed to be free of any STI at screening. Women who were diagnosed with symptomatic bacterial vaginosis (BV) or yeast infection at screening could be enrolled after treatment. Women with asymptomatic BV or yeast infection were eligible for enrollment. Participants needed to be free of genital lesions with epithelial disruption at enrollment. To be eligible, couples could not be regular condom users (reporting condom use ≥25% of the time) and had to report having vaginal sexual intercourse at least once per week, on average.
Women and men were evaluated for eligibility at up to 3 screening visits. Couples who consented to enrollment were randomized to use either Carraguard gel (a gel formulation of mostly λ carrageenan mixed with a small amount of κ carrageenan) or a placebo gel (2.5% methyl cellulose gel). The placebo looks and feels the same as Carraguard; both are odorless, tasteless, clear gels, and have a viscosity between 30,000 and 34,000 centipoise. The methyl cellulose placebo was chosen based on its lack of microbicidal activity in in vitro and in vivo tests.6,22 Both gels were packaged in opaque Microlax-type single-use applicators (Norden Pac International AB, Kalmar, Sweden). Each applicator contained 7 mL of gel and was designed to dispense 4 mL of gel at each application.23 To preserve the study blinding, couples were randomized to receive boxes labeled with 1 of 4 shapes: square, circle, triangle, or pentagon. Two shapes were assigned to Carraguard and 2 to placebo gel. Neither the women nor the study staff knew which shape contained which study gel. The randomization scheme was generated in advance using the pseudorandom number generator in SPSS (Version 7.0, Chicago, IL) at the Population Council headquarters in New York. Couples were provided with boxes of 12 applicators of gel and were provided with and given instruction in using condoms if they desired.
Couples were asked to return to the clinic 14 days after their enrollment visit, 1 month after enrollment, and monthly thereafter for a total of 6 months. At every study visit, couples were interviewed by the study staff, had a complete naked-eye genital examination (including a pelvic exam for women), received safer sex counseling, and were given study product counseling and supplies (after enrollment). Clinicians used colposcopy for women and a magnifying lens for men to further evaluate findings visible to the naked eye. Photographs were taken of genital findings considered abnormal by a trained study staff member using a digital camera with ring flash.24
We collected endocervical and vaginal swabs (women) and urine specimens (men and women) to test for reproductive tract infections and pregnancy. At screening and each monthly visit women were tested for vaginal candidiasis and BV (Gram stain microscopy) and pregnancy (urine). At screening and months 3 and 6, we tested for chlamydial infection and gonorrhea (COBAS Amplicor PCR, Amplicor, Branchburg, NJ), and trichomoniasis (InPouch culture test, Biomed Diagnostics, San Jose, CA). At screening and month 6, we collected venous blood samples and tested for HIV (Genetic Systems HIV 1/2 Enzyme Immunoassay, Redmond, WA, with confirmation by NovaPath HIV-1 Immunoblot, BioRad Diagnostics Group, Hercules, CA) and syphilis (Macro-Vue rapid plasma regain card test, Becton-Dickinson, Franklin Lakes, NJ with confirmation by T. pallidum passive particle agglutination test, Fujirebio Diagnostics, Tokyo, Japan). Each woman had a Pap smear at screening and at month 6. For women who had symptoms of a reproductive tract infection at any visit, a vaginal swab was collected and immediately evaluated for presence of yeast, BV, or trichomoniasis and appropriate treatment was provided. A specimen was collected from the base of genital ulcers, when present, for multiplex PCR testing at CDC Atlanta for the presence of HSV-1, HSV-2, H. ducreyi, or T. pallidum.
We planned to collect approximately 300 couple-months of data: 150 couple-months of exposure to Carraguard gel and 150 couple-months of exposure to the placebo gel. We planned to enroll approximately 55 couples for 6 months to allow for an expected 10% loss-to-follow-up.
Our main safety outcome measure was occurrence of genital findings with epithelial disruption (abrasions, ulcers, and fissures). Study clinicians noted any genital findings and collected information on the location, color, and size of each finding and whether the epithelium was disrupted. We compared the proportions of participants in each study arm with genital findings (with and without epithelial disruption) separately for men and women. In all descriptive prevalence analyses, any finding present in more than 1 location was assigned to a single location using a hierarchical algorithm: for women, cervix (if present); vagina (if not present on the cervix); or external genitalia (if present on neither the cervix nor the vagina); and for men: penis shaft; penis head or foreskin; or scrotum, pubic area, perineal area, or other site. Compliance with gel and condom use instructions was ascertained by asking detailed questions about sexual activity and gel and condom use in face-to-face interviews. Women were encouraged to use coital diaries to document sex acts, gel, and condom use. The information recorded on the coital diary was used to complete the study forms as accurately as possible during the face-to-face interviews. Compliance with gel use was also measured by counting used and unused applicators at each study visit.
We calculated means for continuous variables and percentages for categorical variables. We compared the Carraguard and placebo groups using t-tests or Chi-square tests as appropriate. For nonparametric comparisons, we used the Wilcoxon rank sum test.
The Population Council Institutional Review Board, the US CDC Institutional Review Board, and the Thailand Ministry of Public Health Ethical Review Committee reviewed and approved the study protocol.
We screened 84 couples to enroll 55 eligible couples in the study; 28 couples were randomized to the Carraguard group and 27 couples to the methyl-cellulose placebo gel group. The most common reason for ineligibility at first screening was an abnormal Pap smear (Class II or above; 18 women). Other reasons for ineligibility at first screening included: failure to return to study clinic (2 couples); presence of an STI or symptomatic vaginal infection (1 woman); cessation of enrollment because the enrollment target had been reached (2 couples), positive HIV test (1 couple and 1 man); genital abnormality (1 woman); health condition (1 man; 2 women). Study screening began in June 2001 and study follow-up was completed in June 2002. Fifty-two couples completed the full study; 1 couple (in the Carraguard group) withdrew early because they relocated to another province. One couple and 1 man in the Carraguard group were lost-to-follow-up. We collected a total of 296.4 couple-months of data (145.2 couple-months in the Carraguard group and 151.2 couple-months in the placebo group, P = 0.6).
Reported sociodemographic characteristics were similar in the Carraguard and placebo groups (Table 1), except that women reported less education in the Carraguard group compared with the placebo group. All couples reported that they were married. There were no significant differences in sexual behavior, family planning methods used, and vaginal product use by women between groups at screening (Table 2). Overall, men and women reported similar frequencies of sex and condom use at the baseline (data not shown). Genital findings with intact epithelium were relatively common. At first screening, 27.3% of women had a genital finding with intact epithelium, with no significant difference between the 2 groups. Among men, 34% had a genital finding with intact epithelium at first screening, significantly more in the Carraguard group (54%) than in the placebo group (15%, P = 0.004). Most of these findings with intact epithelium were on the scrotum, pubis, or perineum, not on the penis. All of the men were uncircumcised. As required by the study eligibility criteria, none of the participants had a genital lesion with disrupted epithelium at enrollment. There were no statistical differences in laboratory findings between the 2 groups at screening or enrollment—presence of yeast or evidence of BV with vaginal swab microscopy.
The median total number of penile-vaginal sex acts reported by the women during the study was 58 with a 25% to 75% interquartile range of 40 to 72 acts. The median number with gel use only was 53 (interquartile range, 38–69); with neither gel nor condoms, median—0, 90 percentile—4, maximum—59; with condoms only, 90 percentile—0, maximum 38; with gel plus condoms, 90 percentile—0, maximum 7. These reported gel and condom use practices were not significantly different between study arms overall or at any study visit.
During the 6-month study, a total of 7 women had a lesion with epithelial disruption at any time (Table 3). All 7 were in the placebo group (P = 0.004). In all 7 cases, the lesions were on the external genitalia, superficial, and sized less than or equal to 3 by 3 mm. Five of the 7 women had the finding at only 1 visit whereas 2 women had the finding at 2 visits (1 at consecutive visits month 4 and month 5; 1 at month 2 and month 6 only). In contrast, genital findings with intact epithelium were more common, but were nearly equally distributed between the 2 study groups. Most women, 19 (70%) in the placebo group and 19 (68%) in the Carraguard group had at least 1 such finding during follow-up, generally on the cervix, followed by external genitalia, and vaginal walls (all P = NS).
Ten men had a genital finding with epithelial disruption at some time during the 6-month study (Table 3). Six were in the Carraguard group and 4 were in the placebo group and (21% vs. 15%, P = 0.6). Six men in the Carraguard group had genital findings with intact epithelium during follow-up, compared with 3 men in the placebo group (21% vs. 11%, P = 0.5). Most findings in men were on the scrotum, pubis, or perineum, followed by the glans or foreskin, with relatively few on the penis shaft.
There were no significant differences between the 2 study groups in any other reported genital symptoms or clinical exam findings (Table 4). There was no pain on bimanual exam or adnexal mass at any study visit for the women. Study clinicians observed the presence of study gel during at least 1 follow-up visit in 3 women in each group. Colposcopy was done at least once during follow-up for 23 women in the placebo group and 22 women in the Carraguard group (85% vs. 79%, P = 0.7). During study follow up, none of the men reported urethral discharge, and the study clinicians did not observe any cases of balanitis, urethral discharge, or pain on physical exam of the men.
During the course of the study, there were no incident cases of chlamydial infection, gonorrhea, syphilis, HIV infection, or pregnancy. We also did not find any difference between the Carraguard and placebo groups in incidence or persistence of BV or candidiasis (data not shown). Seven women in the placebo group and 3 women in the Carraguard group had an incident case of BV during the study. Multiplex PCR results from 2 genital ulcers in women were negative and were positive for HSV from 3 of 6 ulcers in men. According to the eligibility criteria, all women had a Class I Pap smear at screening. Six women in the placebo group (22%) and 2 women in the Carraguard group (7.4%) had a Class II Pap smear at month 6 (P = 0.14).
Many aspects of acceptability were measured throughout the study. Tables 5 and 6 show tabulations of these data after 6 months of gel use for women and men, respectively. There were no significant differences in responses to any of the acceptability questions between the 2 study arms. All women reported at each study visit that they tried to squeeze all the gel out of the applicator, except for 1 woman in each group at 1 visit reported that she didn't know.
In this 6-month randomized, placebo-controlled study of Carraguard use in 55 low-risk, heterosexual couples in northern Thailand, we found that genital findings with epithelial disruption were uncommon in both men and women and tended not to persist. There were more findings with epithelial disruption in women in the placebo group, but the lesions were in a minority of women, and were small and on the external genitals. Among men, there was no significant difference between groups in the number of men with genital findings with epithelial disruption. We therefore conclude that use of Carraguard was not associated with genital epithelial disruption, and, whereas use of the methyl cellulose placebo was associated with genital epithelial disruption in this study, a causal relationship is unlikely. In two phase II studies in Thailand and South Africa, we did not find significantly increased rates of epithelial disruption among the control group of women using the same placebo. The sample sizes in those trials were substantially larger (Thailand 165 women, South Africa 400 women), and the duration of gel use was longer (12 months). With the relatively small number of findings with epithelial disruption in this study, the observed association could be due to a chance. The findings could also be related to a difference in HSV-2 infection, but we did not test women for HSV-2 antibodies and so cannot determine whether there were differences between the 2 groups or whether the women with epithelial disruption had herpes. Carraguard does have anti-HSV-2 activity in a mouse model, but that was for prevention of primary infection, not to prevent reactivation, which the small lesions in the current study may have represented.6 HSV-2 serologic testing in future studies could help address this issue.
Most women in both groups had genital findings with intact epithelium, but there was no significant difference between the 2 groups. This seemingly high rate of findings can be expected with meticulous exams conducted every month for 6 months. Genital findings with intact epithelium were not common among men and there were no significant differences between the placebo group and the Carraguard group. We therefore conclude that Carraguard use was not associated with intact genital findings.
This study was designed to detect potential adverse effects of gel use that are relatively frequent and manifest within several months of beginning gel use, and was conducted to determine whether Carraguard and the placebo gel could advance to phase III testing. Safety evaluation in women continues in phase III testing and will take place in men and women in postmarketing surveillance if the product proves effective and is licensed and distributed. This study was conducted in low-risk couples who used the gel on average 2 to 3 times per week and did not determine safety of more frequent use, such as by women engaged in commercial sex.
Reported use of study gel without condoms was similarly high in both groups throughout the trial, more than 95% of vaginal sex acts at most visits. Although there may have been some social-desirability bias in the face-to-face interviews, men and women in both groups reported that gel use was acceptable. At the 6-month visit, most participants reported that they liked the gel, they found the applicator to be appealing, and that using the gel increased sexual pleasure. None of the participants disliked the gel or found the applicator to be unappealing. As has been reported, we concluded that use of a candidate microbicide gel like Carraguard was acceptable to low-risk, heterosexual northern Thai couples.25 Considerable effort was required to recruit these couples, and the participants reported being motivated primarily by altruism. Consistency and acceptability of microbicide use outside of a clinical trial setting may differ. In addition, care must be taken in generalizing results from this northern Thai population to other geographic areas.
This study was the first to our knowledge to assess male safety and acceptability of a candidate microbicide through direct exposure during sex instead of through manual application to the penis. We successfully recruited and retained a cohort of sexually active couples. Men had frequent exposure to the study gels, and despite low reported condom use, no incident infections with HIV or other sexually transmitted pathogens were detected in this carefully screened, low-risk group. We believe this study has demonstrated the safety and the value of this approach. The design allowed us to collect data on safety and acceptability under close to real-life use conditions. We would recommend this design as a stand-alone study for male safety and couples' acceptability evaluation of other new microbicide candidates. Such a design could be included as part of a run-in to a phase III efficacy trial.
In this randomized, placebo-controlled trial in 55 heterosexual couples in northern Thailand, we found that use of the candidate microbicide gel Carraguard during vaginal sex an average of 2 to 3 times a week for 6 months did not cause significant genital irritation to men or women. These low-risk couples also found gel use to be highly acceptable. These results, together with extensive additional evaluations, provided support for proceeding with the South African phase III trial of the effectiveness of Carraguard use as for prevention of HIV and other STIs.26
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