Veldhuijzen, Nienke J. MD, PhD*; Dhont, Nathalie MD, PhD†,‡; Vyankandondera, Joseph MD‡,§; Gasarabwe, Ammiel MSc‡; Busasa, Rosetta BSc‡; Crucitti, Tania PhD¶; van de Wijgert, Janneke H. H. M. PhD*,‡
Transmission of sexually transmitted infections (STIs) depends on exposure level (sexual risk behavior) and the transmission potential per-coital-act. Transmission potential has been estimated at 0.04% per-coital-act for female-to-male HIV transmission, at 0.08% for male-to-female HIV transmission, and at 1% for herpes simplex virus type 2 (HSV-2) transmission. 1,2 The transmission potential of human papillomavirus (HPV) is less well defined. 3 A mathematical simulation model estimated a per-coital-act transmission probability of any HPV of 5% to 100% (median 40%). 4
In the absence of prospectively collected data, the transmission potential of an STI can be estimated by using its proxy of concordance in sexual partners. Concordance rates among steady couples reflect both the prevalence, the duration of infections, the duration of relationships, and the frequency of sexual intercourse.
HIV and HSV-2 are chronic infections and once a couple is concordant positive on serology it will remain so. On the contrary, the majority of HPV infections are self-limiting and couples may move between concordant positive, discordant, and concordant negative for HPV DNA over time. HIV concordance rates among cohabiting couples in Eastern Africa range from 2% to 20% with underlying population prevalence rates ranging from 3% to 24%. 5–7 HSV-2 transmission rates are higher than HIV transmission rates but few studies have investigated concordance in couples. Reported concordance rates range from 11% to 34%. 8,9 HPV concordance rates vary widely and also depend on sampling technique (especially in men), HPV assays used and definition of concordance (pooled-probe or type-specific). A recent meta-analysis estimated that overall 26% (95% confidence interval [CI]: 17%–36%) of couples were infected with one or more of the same HPV types. 10
HIV is known to increase incidence and persistence of HPV infection. 11 HSV-2 is associated with a 2- to 5-fold increased risk of HIV acquisition and is the most important risk factor for HIV transmission within couples. 8,12,13 Among HPV-infected women, HSV-2 has been implicated as a cofactor in cervical carcinogenesis. 14
Fewer data exist on STI concordance from East Africa, where HPV, HIV, and HSV-2 are highly prevalent. Data concerning HPV concordance rates in particular are lacking. Knowledge about concordance rates will improve our understanding of the burden of infections among partners and may influence public health policies. This paper describes the (type-specific) HPV, HIV, and HSV-2 prevalence and concordance among community-sampled heterosexual couples in Kigali, Rwanda.
Fertile couples participating in a case–control study assessing risk factors for infertility in Kigali, Rwanda (2007–2009) were included in these analyses. 15 The fertile controls were defined as nonpregnant women who delivered a baby in the previous 6 to 18 months, were between 21 and 45 years of age, had sex at least once in the 2 weeks before enrollment, and lived in 1 of 14 randomly selected neighborhoods of Kigali. Community mobilizers visited all families in those neighborhoods to identify potentially eligible candidates. They explained the study procedures and offered free cervical cancer screening and treatment (if needed). All eligible women were invited to enroll into the study. Male partners of the enrolled participants were also asked to participate, using a written invitation given to the woman at the end of her study visit. The male partners were seen on a separate occasion. The study was approved by the National Ethics Committee of Rwanda, the Ethics Committee of the University Hospital Ghent, Belgium and the Centre National de Lutte contre le Sida in Rwanda. Written informed consent was obtained from all study participants before study procedures.
The cross-sectional study was conducted at the women's health clinic at the Kigali University Teaching Hospital. Standardized questionnaires were administered by a female nurse interviewer to female participants and by a male nurse interviewer to male participants. Blood samples were taken for HIV and HSV-2 serology. Vaginal specimens were collected for the detection of Trichomonas vaginalis, bacterial vaginosis, Neisseria gonorrhoeae, and Chlamydia trachomatis. Endocervical specimens using a Cervex Brush (Rovers Medical Devices, the Netherlands) were collected for HPV genotyping. Cervical smears for conventional cervical cytology were obtained using an Ayre spatula. Men underwent a genital examination with the collection of specimens from the penile shaft, scrotum, glans/sulcus corona, and foreskin (in uncircumcised men) using saline-wetted swabs. The penile/scrotal area was lightly brushed with an emery paper prior the specimen collection. 16 The endocervical brush and male genital swabs were agitated in PreservCyt medium (Cytyc Corp, Londonderry, USA).
Women were initially screened for cervical abnormalities using visual inspection with acetic acid and were offered appropriate treatment if applicable. Women with abnormal cytology, who had not been treated based on the results of visual inspection with acetic acid, were recalled for colposcopy of the cervix and vagina by a gynecologist and were offered appropriate treatment if applicable. Participants with curable STIs received treatment and were offered partner notification. Participants who tested HIV-positive were referred to HIV care.
HPV genotyping was performed at the Institute of Tropical Medicine (Antwerp, Belgium) using the Linear Array (LA) HPV Genotyping Test (Roche Molecular Systems, USA) according to manufacturer's instructions. In brief, PreservCyt samples (Cytic Corp, Londonderry, USA) were stored at −80°C until testing. An aliquot of 250 μL was used for DNA extraction by AmpliLute Liquid Media Extraction Kit (Roche Molecular Systems, USA). HPV genotype targets were amplified using standard polymerase chain reaction (PCR). LA applies PGMY09/11 L1 as consensus primer system and coamplifies human β-globulin. LA identified 37 high- (HR) and low-risk (LR) HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66–73, 81, 82 subtype IS39, 82, 83, 84, CP108 and a mixed probe for types 33/35/52/58). An in-house real-time PCR (RT-PCR) was established at the Institute of Tropical Medicine to identify DNA of HPV genotype 52 in the mixed probe positive samples. Briefly, DNA was prepared using the NucliSens miniMAG (BioMérieux, Boxtel, The Netherlands). DNA templates were amplified in a qualitative RT-PCR assay. HPV52-F/R and hydrolysis probe HPV52 labeled at the 5′ with fluorescein amidite and at the 3′ with black hole quencher-1 (Eurogentec, Belgium) were used for amplification and detection. 17 Amplification was performed using the Absolute Blue QPCR mix kit (Thermo Scientific, United Kingdom), 500 nm of each primer and probe, and 10 μL of DNA template, in a final reaction volume of 30 μL. The RT-PCR was performed on the Rotor-Gene 6000 platform (Corbett Life Science, USA) using the following thermal cycling conditions: initial enzyme activation at 95°C for 15 minutes; followed by 60 cycles of 95°C for 15 seconds, 58°C for 45 seconds, and 65°C for 45 seconds. Detection of the fluorescence signal was recorded using the setting for hydrolysis probes (470 nm, 510 nm) following the extension phase cycle by cycle.
Other Laboratory Testing
Laboratory testing was conducted in Rwanda unless noted otherwise. HIV was diagnosed by First Response (Premier Medical Corporation, Daman, India) and Uni-Gold HIV1/2 (Trinity Biotech Plc, Ireland) rapid tests, and Capillus HIV1/2 (Trinidity Biotech Plc, Ireland) as tiebreaker if needed. HerpeSelect-2 enzyme-linked immuno sorbent assay (Focus Technologies, USA) was used for HSV-2 antibody testing. An index value of ≥3.5 was considered positive, an index value ≤1.1 negative and values in between equivocal. Equivocal samples were retested and reported as equivocal if both results were equivocal. These were excluded from the analysis (n = 2). A vaginal swab was placed in 3 mL universal transport medium (Copan Diagnostics, California) and stored at −80°C. Multiplex PCR for C. trachomatis and N. gonorrhoea (Abbott Laboratories, Illinois) was performed by Ghent University Hospital, Belgium. Conventional cervical cytology was also performed at Ghent University Hospital, using the Bethesda system for classification. 18 Trichomonas vaginalis was diagnosed using wet mount microscopy and bacterial vaginosis by Gram stain Nugent scoring (with quality control by Ghent University Hospital).
Data were double-entered, and analyzed using STATA 10.1 (StatCorp, Texas).
Definition of HPV Outcomes.
HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, and IS39 were considered HR-HPV types. 19–21 All other types were considered LR HPV. “Any HPV” included HR and/or LR-HPV. β-globulin negative samples were considered inadequate and excluded from analysis. The comparison group in all HR-HPV analyses included participants without HR-HPV, irrespective of LR-HPV detection. Grouped type-specific HPV concordance was defined per HPV risk group (HR or LR), as both partners sharing at least 1 HPV type(s) from that risk group, regardless of the presence or absence of other HPV type(s) from the same or the other risk group. In this paper, this is referred to as HR-HPV or LR-HPV concordance.
The McNemar χ2 statistic was used to compare type-specific HPV prevalence rates in male and female partners. Couple HPV, HIV, and HSV-2 status was categorized as concordant negative, concordant positive, or discordant. Couple (positive) concordance rates were evaluated among all couples and among exposed couples only (among whom at least 1 partner was infected). Expected HPV concordance, assuming independence of infection between partners, was calculated following the method described by Burchell et al. 22 Briefly, expected HPV type-specific concordance was calculated as the product of type-specific HPV prevalence in women (Pft) and men (Pmt). The probability that partners do not share type-specific infection is equal to 1 − (Pft × Pmt). The probability that partners do not share any HPV types is the product of the expected concordance for all the types included in a risk group: Πt1-tn(1 − (Pft × Pmt). The probability that couples are concordant for at least 1 HPV type is equal to 1 − (Πt1-tn (1 − (Pmt × Pft)). Concordance due to chance (expected concordance) was compared with observed concordance using a t test. The ratio of observed concordance over expected concordance gives a further indication of transmissibility, independent of the underlying prevalences. Similar methods were used to calculate expected concordance of HIV and HSV-2.
Four hundred seven potential female participants were identified in the community, 352 (86%) came to the clinic and 312 were confirmed eligible and enrolled. One hundred eighty-nine male partners (61%) agreed to participate. The median visit interval between women and men was 10 days (interquartile range: 5–36).
Women whose partner did not participate were significantly younger, more often not married (although the majority reported a steady partner), less likely to use hormonal contraceptives, and more likely to be infected with C. trachomatis, HR-HPV, and LR-HPV. Furthermore, they spent significantly fewer nights per week with their steady partner/husband (data not shown). None of the female samples, and 8 male samples (4.2%) were β-globulin negative and were excluded from these analysis. In addition, 15 men had missing HPV results. The final analytical sample therefore consisted of 166 couples for whom complete HPV, HIV and HSV-2 results were available. Demographic and (sexual) behavioral characteristics are described in Table 1.
HPV, HIV, and HSV-2 Prevalence
The prevalence of HR-HPV among women was 19.9% (CI: 13.7–26.0) compared with 26.5% (CI: 19.7–33.3) in their male partners (P = 0.078) (Fig. 1). The most prevalent HR types among women were HPV52 (6.0%; CI: 2.3–9.7), HPV16 and HPV68 (each 3.6%; CI: 0.7–6.5) and HPV33, HPV45, and HPV58 (each 3.0%; CI: 0.4–5.6). The most prevalent HR types among men were HPV18 and HPV51 (each 5.4%; CI: 1.9–8.9), HPV16 (4.2%; CI: 1.1–7.3) and HPV35 and HPV58 (each 3.6%; CI: 0.7–6.5). HPV 16 and/or HPV 18 were detected in 5.4% (CI: 1.9–8.9) of women and 8.4% (CI: 4.2–12.7) of men. Coinfection with LR-HPV was detected among 48.5% (16/33) of women with HR-HPV and among 56.8% (25/44) of men with HR-HPV.
HIV prevalence among women was 13.3% (CI: 8.0–18.5) and 7.8% (CI: 3.7–12.0) among their male partners. HSV-2 prevalence was 40.2% among women (CI: 32.7–47.8) and 36.7% among men (CI: 29.3–44.2).
HIV was associated with higher HR-HPV prevalence in both women and men. HSV-2 was associated with HR-HPV detection in men, but not in women (Table 2). Partner's HIV status was a predictor of male HR-HPV detection, but not of female HR-HPV detection. Controlling for own HIV did not change this gender-specific association between HR-HPV and partner's HIV status (Table 2).
HPV, HIV and, HSV-2 Concordance
In 14 couples, partners shared the same HR-HPV type(s). Ten couples shared one type, 1 couple 2 types, 2 couples 3 types and 1 couple 8 types. HR-HPV concordance was therefore 8.4% (CI: 4.2–12.7) (Table 3). Concordance was highest for HPV68 (3.0%; CI: 0.4–5.6). LR-HPV concordance was 11.5% (CI: 6.6–16.3). Observed concordance was significantly higher than concordance due to chance for both HR- and LR-HPV (Table 3). In 50% of HR-HPV concordant couples (7/14 couples), the partners also shared type-specific LR-HPV infection. Of the total of 44 HR-HPV discordant couples, men were the index case in 25 couples (64.1%), women in 14 couples (31.8%; P <0.001), and men and women were infected with all different HR-HPV types in 5 couples. HPV concordance among HIV concordant negative couples was 7.1% (CI: 2.8–11.4) for HR-HPV, 8.5% (CI: 3.8–13.2) for LR-HPV, and 12.8% (CI: 7.2–18.3) for any HPV. This was significantly higher than concordance rates due to chance (data not shown). Only 10 couples were HIV concordant positive and HPV concordance in these couples was not calculated. Among HR-HPV exposed couples (couples in whom at least 1 partner was HR-HPV infected) positive concordance was 24% (CI: 20.3–45.2).
In 10 couples, both partners were HIV infected—HIV-positive concordance was therefore 6.0% (CI: 2.4–9.7). This was significantly higher than concordance due to chance (P = 0.003). Among the 15 HIV discordant couples, women were more often the index case (12 women) than men (3 men). HIV concordance among HIV-exposed couples was 40%.
HSV-2 positive concordance among all couples was 25%, and this was significantly higher than concordance due to chance (P = 0.008). Among the HIV-negative concordant couples (n = 139), 22 couples (16%) had positive concordant HSV-2 serology results and 38 couples (27%) had discordant HSV-2 serology results (data not shown). Women were the index case in 20 HSV-2 discordant couples and men in 18 couples. Among HIV concordant negative couples, HR-HPV prevalence in men and women was not significantly influenced by HSV-2 couple status. The prevalence of HSV-2 concordance among HSV-2 exposed couples was 49% and HSV-2 couple status was significantly associated with HIV couple status (P < 0.001).
The ratio of observed concordance over expected concordance was highest for HR-HPV, followed by HIV and lowest for HSV-2 (Table 3).
This study is the first to describe type-specific prevalence and concordance of HPV, HIV, and HSV-2 among steady heterosexual couples in an East African country, where all three viral STIs all highly prevalent.
We found a high prevalence of all 3 viral infections and concordance rates in couples were all higher than expected by chance. The HIV prevalence was higher than compared to previously reported population-based prevalence rates among women and men in Kigali (8% and 5%, respectively), but agrees with reported HIV prevalence in antenatal clinics in Kigali (13.2%). 5,23
As expected for STIs, HPV, HIV, and HSV-2 concordance rates in heterosexual couples were higher than expected by chance alone. Of the 3 viral STIs, positive concordance was highest for HSV-2 (25%), followed by any HPV (15.7%), HR-HPV (8.4%), and HIV (6%). Concordance rates among steady couples are influenced by the underlying prevalence rates, transmission potential, the duration of infection, the duration of the relationship, and the frequency of sexual intercourse. Relationship duration is likely to influence the concordance rates for the chronic infections, HSV-2 and HIV, the most but, unfortunately, we did not collect this information. Only 3 men self-reported to have a casual partner, and it was therefore not possible to assess the influence of extramarital sexual partners on concordance rates.
If one assumes that concordance reflects transmission, concordance rates should reflect something about transmission potential. However, comparison of concordance rates of different STIs in one population is limited by the different testing protocols. HIV and HSV-2 antibodies are tested by serology and remain positive for life. The presence of HPV DNA is diagnosed using cervical samples and the majority of these infections are transient. Nevertheless, the ratio between observed and expected concordance was highest for HPV, which fits a high transmission potential. The ratio we found was higher than the ratio previously reported among university students in Canada (a ratio of 3.0 for HR-HPV and 4.1 for LR-HPV), but only newly formed couples (<6 months) were included in the Canadian study. 22 Unexpectedly, we found a lower ratio for HSV-2 compared with HIV.
The 3 viral STIs strongly interact with each other. This is best documented for HSV-2 and HIV. HSV-2 positive persons are at increased risk of HIV acquisition and, HIV/HSV-2 coinfected persons are more infectious for HIV compared with HIV-infected persons. 12,24 The association between HSV-2 and HPV has mainly been as a cofactor in HPV persistence and cervicocarcinogenesis although a few studies reported an association with HPV acquisition. 14,25,26 In our study, we found a positive association between HSV-2 and HR-HPV in men but not in women. We also found positive associations between HIV and HR-HPV in both men and women, as has been documented by others. 11 Only one other study found an association between HIV and HPV concordance in heterosexual couples in South Africa. 27 Furthermore, HPV in male partners was influenced by their own HIV-positive status and by that of their female partners. However, HPV in female partners was only determined by their own HIV-positive status and not by that of their male partners. Here we reported similar findings. We did not have sufficient statistical power to stratify the analysis for HR-HPV status of the partner (which is on the causal pathway). Apart from differences in exposure, higher infectiousness of HIV-HPV coinfected persons as well as increased susceptibility of HIV-infected individuals may explain these associations. Studies have shown that HPV viral load in HIV-infected women is higher compared with HIV-negative women which may lead to higher transmissibility. 28,29 Whether HPV viral load in HIV-coinfected heterosexual men is also increased remains to be established.
The generalizability of our results may be limited by selection bias. Women whose partner did not participate had a higher sexual risk behavior and HPV prevalence than women whose male partner did participate. HPV prevalence and concordance rates may therefore be underestimated. Sexual risk behavior, HIV and HSV-2 prevalence were higher in infertile couples. 15 Moreover reduction in sperm mobility, viability, and count have been associated with male HPV infection. 30,31 However, this will only have limited impact on the generalizability because the prevalence of infertility in the general population is relatively low. Another limitation is that HPV samples were not taken on the same day for both partners (median interval 10 days [interquartile range: 5–36]). Almost the entire study population consisted of monogamous couples (by self-report), and we did not find a statistical association between sampling interval and HR-HPV concordance. However, we cannot exclude that reactivation of latent infections or clearance of HPV infections in one of the partners during the sampling interval had an impact on our concordance results. Finally, even if we assume that concordance reflects transmission, a valid measure of transmission potential can only be assessed in longitudinal studies.
In conclusion, more than half of exposed community-sampled heterosexual couples in Kigali, Rwanda, were discordant for HIV, HR-HPV, and/or HSV-2. Partner referral and testing would offer opportunities to reduce transmission within couples. To minimize transmission, discordant couples should learn about their infection status and about ways to prevent transmission (including condom provision) and be offered diagnoses and testing for other genital infection that may facilitate transmission. HIV and STI prevention programs should therefore target discordant couples.
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