Introduction 1 2 3 4 5 6 1 7 8 9 10 11 12 13 5 We here studied the trend in HSV-1 and HSV-2 prevalence among homosexual men over a 20-year time period (1984–2003) and whether risk factors for infection changed during this period. Methods Study population In 1984 an open and prospective cohort study on HIV seroconversion and AIDS among sexually active HIV-negative and positive homosexual men was started. The Amsterdam Cohort Study (ACS) is still ongoing, although entry criteria with respect to HIV status and age have changed over time. From 1984 until May 1985, both HIV-positive and HIV-negative men were included. From May 1985 until February 1988, only HIV-negative men were allowed in the study. From February 1988 through 1994, HIV-positive and HIV-negative men could enter the study, but since 1995, they must be ≤30 year of age. At an ACS visit, a standardised questionnaire is administered regarding demographics, sexual behaviour, and medical history for sexually transmitted infections (STI). Blood samples are collected for immunologic and virologic testing and for storage. For this study, stored sera (collected at the first cohort visit) taken from ACS participants with at least two cohort visits (1847/2100 (88%)) were tested for HSV-1 and HSV-2. Laboratory methods 14 14 1 Fig. 1 Comparison of HSV-2 serology by ELISA and Western blot. 100 samples were tested with HSV-2 ELISA and re-tested with the Western blot to identify discrepancies between the two assays  Blood samples were tested for HSV-1 and HSV-2 at the Public Health Laboratory of the Health Service of Amsterdam. The Western blot was conducted at the Institute for Pathology and Medical Research (ICPMR) in Sydney, Australia. Blood samples are also tested for HIV antibodies by enzyme linked immunosorbent assay (ELISA) (Abbot Laboratories, North Chicago, Illinois, USA; Vironostika, Organon, Teknika, Boxtel, the Netherlands), and when positive, are confirmed by Western blot. Variables and statistical analyses The statistical analyses were based on the data collected at entry of the cohort. Variables used in this study were calendar year of ACS entry, HIV-status, age, nationality, education, age of first homosexual contact, lifetime sexual partners, and self-reported history of syphilis and gonorrhoea in the past 5 years. Variables concerning sexual practices included orogenital, anogenital and oroanal contact in the prior 6 months. Some changes in the questions were made in the questionnaires between 1984 and 2003 regarding the sexual practices. Oroanal contact was not asked for from 1889 to 1994, resulting in approximately 25% missing values on this variable. From 1995 onward, the most important difference was that orogenital contact with ejaculation was asked, while in the previous years orogenital contact in general was asked. The percentage of participants not having orogenital contact was somewhat higher for the years 1995 and 1996. 15 We tested whether risk factors changed over time by testing for interaction between variables under investigation and calendar time in the multivariate model. Calendar time therefore was categorised as 1984–1986, 1987–1991, 1992–1996, 1997–2003. P P For 77 MSM the HSV-1 index value was missing and 91 MSM had a missing HSV-2 index value. Participants with a missing index value for both HSV-infections were not included in the analyses. MSM with a missing index value for one HSV-infection, but with a known HSV status of the other HSV-infection were included in the analyses for the known HSV serostatus. Finally, sensitivity analyses for HSV-2 were conducted by using the cut-off value of 1.1, as recommended by the manufacturer and by using the cut-off value of 3.5, excluding those with an index value in the grey area (between 0.9–1.1 and 0.9–3.5). However, time trends in prevalence and risk factors found were comparable to when 2.1 was the cut-off value (data not shown). Results General characteristics 1 Table 1 a Characteristics Total HSV-1-infection PRR (95% CI) P HSV-2-infection PRR (95% CI) P Total 1847 1207 (65)   759 (41)   Year of study entry    <0.0001   <0.0001 1984–1986 943 675 (72) 1  461 (49) 1  1987–1991 165 113 (68) 0.87 (0.78–0.97)  92 (56) 0.84 (0.72–0.98)  1992–1996 222 138 (43) 0.88 (0.79–0.96)  80 (36) 0.66 (0.56–0.77)  >1997 517 281 (54) 0.76 (0.70–0.84)  126 (24) 0.47 (0.40–0.55)  Index value <0.9 – 535   853   0.9–1.1 – 29   32   1.1–2.1 – 100   112   ≥2.1 – 1107   759   Missing  77   91   Age    <0.0001   <0.0001 <30 years 1002 570 1  252 1  ≥30 years 845 637 1.20 (1.13–1.29)  507 1.98 (1.75–2.23)  Nationality:    0.006   0.62 Dutch 1586  947 (60) 1  615 (39) 1  Northern/central Europe 116  72 (62) 1.04 (0.92–1.18)  54 (47) 1.08 (0.90–1.38)  Non-European 145  117 (81) 1.17(1.07–1.28)  75 (52) 1.04 (0.91–1.23)  Education    0.005   0.007 Low 96 77 (80) 1  57 (59) 1  Middle 669 408 (61) 0.85 (0.76–0.95)  239 (36) 0.72 (0.59–0.87)  High 971 574 (59) 0.81 (0.73–0.81)  387 (40) 0.79 (0.66–0.94)  Missing 115 77 (67)   54 (47)   Sexual partners in lifetime 1–20 860 497 (58) 1 <0.0001 277 (32) 1 0.51 21–200 531 360 (68) 1.12 (1.04–1.22)  212 (40) 2.30 (0.93–1.21)  >200 443 339 (77) 1.24 (1.15–1.34)  262 (59) 1.07 (0.95–1.20)  Age of first homosexual contact (median, IQR) 18 (15–20) 17 (15–20) 1.01(1.01–1.02)^ <0.0001 17 (15–20) c 0.32 HSV co-infection 568 568 1.29 (1.14–1.47) <0.0001 568 1.167 (1.08–1.24) <0.0001 HIV infection (%) 513  367 (72) 1.11 (1.03–1.18) 0.007 312 (61) 1.12 (1.00–1.24) 0.05 History of gonorrhoea in the past 5 years 1053  666 (63) 0.88 (0.83–0.93) 0.0005 424 (40) 0.88 (0.79–0.97) 0.02 History of syphilis in the past 5 years 278  219 (79) 1.15 (1.07–1.24) 0.001 197 (71) 1.51 (1.36–1.69) <0.0001 b 1363 892 (65) 0.99 (0.82–1.22) 0.46 565 (41) 0.69 (0.55–0.87) 0.01 b 1222 819 (67) 1.11 (1.14–1.60) 0.002 551 (45) 1.36 (1.16–1.60) 0.0002 b 1081 700 (65) 1.00 (0.91–1.09) 0.99 438 (41) 0.98 (0.85–1.12) 0.80 a b c Prevalence of HSV-1 and HSV-2 over time 1 2 2 P 2 P 2 Table 2 Multivariate model of risk factors associated with HSV-1 infection  HSV-1 P (a) HSV-1 infection Year of study entry  <0.0001 1984–1986 1  1987–1991 0.91 (0.70–1.18)  1992–1996 0.83 (0.66–1.02)  >1997 0.75 (0.63–0.90)  Age 1.13(1.07–1.18) <0.0001 HIV serostatus Negative 1 0.01 Positive 1.10 (1.02–1.18)   Nationality Dutch 1 0.0006 Northern or Central European 1.05 (0.92–1.20)  Non European 1.62 (1.12–1.36)   Education   0.25 Low 1  Middle  0.90 (0.79–1.00)  High 0.84 (0.76–1.06)  Sexual partners in lifetime 1–20 1 0.003 21–200 1.13 (1.05–1.25)  >200 1.13 (1.04–1.23)  History of Gonorrhoea in the past 5 years 0.97 (0.90–1.03)  0.11 History of Syphilis in the past 5 years   Orogenital contact in the past 6 months 1.12 (0.88–1.43) 0.42 Anogenital contact in the past 6 months 1.08 (0.97–1.20)  0.20 Oroanal contact in the past 6 months 1.02 (0.70–1.13) 0.81  HSV-2 P (b) HSV-2 infection Year of study entry  <0.0001 1984–1986 1  1987–1991 0.86 (0.70–1.06)  1992–1996 0.58 (0.48–0.71)  >1997 0.47 (0.39–0.56)  HIV serostatus Negative 1 <0.0001 Positive 1.50 (1.37–1.68)  HSV coinfection 1.15 (1.02–1.30) 0.02 History of Syphilis in the past 5 years 1.21 (1.08–1.36) 0.001 Orogenital contact in the past 6 months 0.69 (0.56–0.84) <0.0001 Anogenital contact in the past 6 months 1.20(1.08–1.42) 0.02 Oroanal contact in the past 6 months 1.00(0.87–1.15) 0.72 Fig. 2 a b  P 2 P P Risk factors 1 2 2 Changing risk factors over time Different interaction terms were included in the model. It appeared that the effect of calendar year differed between HIV-infected and HIV-uninfected MSM for both HSV-1 and HSV-2. 2 2 . 2 P P 3 P P Fig. 3 b  3 Discussion In the present study, we demonstrated an overall decrease in HSV-1 and HSV-2 prevalence among HIV-negative MSM, but not among HIV-positive MSM. In the 1984–2003 period, the association between HSV-2 and HIV among MSM became stronger over time, and HSV-1 prevalence increased in highly sexually active HIV-negative MSM. To our knowledge, this is the first study based on almost 20 years of HSV-1 and HSV-2 prevalence data among MSM. 3 HIV infection in this respect may reflect an epidemiological marker for sexual risk behaviour for HSV-1 transmission. HSV-1 prevalence did not decrease in those infected with HIV, and we consider that genital HSV-1 infection has a growing role in the acquisition of HIV. Likewise, HSV-2 prevalence did not decline over time among those infected with HIV, whereas a decline was noted among HIV uninfected MSM. Although HSV-2 is sexually transmitted, we did not find an association between a higher number of lifetime partners and HSV-2 infection. HSV-2 was also highly prevalent among MSM with 1–20 lifetime partners. Mainly between 1984 and 1995, there were no major differences in the proportion of MSM infected with HSV-2. This suggests that HSV-2 is highly sexual transmissible and when having a low number of life time partner the risk of receiving a HSV-2 infection is still very high. The results of this study show a protective effect of orogenital contact for HSV-2 infection, which might be explained by the fact that anogenital contact is a stronger predictor for HSV-2 infection. All variables measuring sexual practices are included in the analyses at the same time. Since most MSM practised all the techniques during the same time period these practices could not be analysed as independent risk factors. The stronger effect of anogenital contact might have overruled the effect of orogenital contact, resulting in a protective effect of orogenital contact. 16 18 1 10 19 20 21 22 One limitation of our study is its cross-sectional design. As a consequence, we cannot reveal the relation between HIV and HSV infection, being unable to determine which occurred first. As both HIV and HSV are sexually transmitted diseases, their association may well reflect shared sexual behavioural practices leading to transmission as well as a biological relation. Longitudinal studies, in which incident HIV and HSV cases are captured are therefore needed to give more insight into the relationship between HIV and HSV as affected by changes in sexual risk behaviour. The results of this study have two implications for HIV and HSV research among highly sexually active MSM. First, it appears that HSV-2 and HIV are now more strongly related than in the early days of the HIV epidemic. As a vaccine against HSV-2 for MSM is not yet available, a determination of the extent to which the prevention of HSV-2, specially aimed for MSM at high risk for HIV, can contribute to controlling the HIV epidemic is needed. Second, since the extent of sexual transmission of HSV-1 is rising, we need to clarify its potential role as a risk factor for HIV acquisition in longitudinal studies.