Genital ulcer disease (GUD) is clearly associated with increased risk of HIV acquisition and transmission. The genital ulcerations facilitate increased shedding and easier entry of HIV into the host [1] -[3] . Therefore, it is biologically plausible that presence of herpetic lesions would increase the risk of HIV acquisition. Herpes simplex virus type-2 (HSV-2) is one of the most prevalent sexually transmitted infections worldwide and the important leading cause of GUD ahead of syphilis and chancroid [4] -[6] . Epidemiological studies have established a strong association between prevalent HSV-2 and incident HIV infection [7] -[14] . Recent studies further suggest that incident HSV-2 infection increases the risk for new HIV infection [10] [12] [14] -[16] . In summary, underlying HSV-2 infection substantially elevates the risk of incident HIV infection. Therefore, prevalent and incident HSV-2 infection is indicated as a significant risk factor or co-factor for new HIV acquisition.

Fishing communities are composed of a sub-population of men and women with high-risk sexual behavior [17] biologically demonstrated by high HIV and/or HSV-2 prevalence [18] -[22] . Moreover, HIV incident rates are reportedly much higher among fishing communities than in the general population [23] . Although previous studies have evaluated risk factors for incident HSV-2 infection among individuals with high-risk sexual behavior [7] [14] [15] [24] , there is limited knowledge on HSV-2 incidence among fishing communities in Sub-Saharan Africa; the epicenter of the HIV epidemic. Knowledge of HSV-2 acquisition risk factors is needed in the design of targeted interventions to limit incident HSV-2 infection and potentially lowering rates of new HIV infection. In this study, we report the results of the first study to evaluate the incidence and risk factors for HSV-2 infection among fishermen on the shores of Lake Victoria, Kisumu, Kenya.

Of the 300 fishermen that were enrolled between September 2011 and December 2012, 254 (85%) returned for the scheduled annual exit visit. Enrolled men had median (IQR) age of 26.5 (21 - 32), median (IQR) 8 (5 - 15) number of sexual partners lifetime, and 2 (1 - 3) one year prior to study participation. The majority (91%) earned ≤ KShs 10,000 (110 USD)/month and only 24% were circumcised (Table 1). All men with baseline and study exit HSV-2 serology data were included in this study. At baseline, 169 men were detected with HSV-2. Of the 131 men who were negative for HSV-2 at baseline, 106 (81%) retuned for their exit visit and were significantly younger mean (SD) age 25.8 (5.84) years compared to men infected with HSV-2 at baseline (mean age 29.3, SD ± 6.37).

Baseline HSV-2 prevalence was 56.3% (95% CI: 50.7 - 62.0). Persons with prevalent HSV-2 infection were more likely to be older (>28 years old) (70% vs. 49%), married (62% vs. 34%), HIV-1 positive (76% vs. 50%), ever had an STI (71% vs. 51.1%) and co-infected with HPV (62% vs. 50%) as shown in Table 1. Bivariate analysis revealed that older age (OR, 2.41; 95% CI, 1.42 - 4.11), ever married (OR, 2.13; 95% CI, 1.02 - 4.93), HIV infection (OR, 3.06; 95% CI, 1.67 - 5.61), HPV infection (OR, 1.64; 95% CI, 1.03 - 2.60), history of STI (OR, 2.41; 95% CI, 1.38 - 4.22) and lack of condom use (OR, 5.31; 95% CI, 2.29 - 12.33) were associated with HSV-2 prevalence. Additionally, among married fishermen, most recent sexual act with partner other than the wife was associated with increased odds of HSV-2 infection; girlfriend (OR, 2.58; 95% CI, 1.17 - 5.71) or casual partner/sex worker (OR, 3.56; 95% CI, 1.49 - 8.62). On the multivariate model, lack of condom use (aOR, 6.34; 95% CI, 2.24 - 13.04), HIV-1 positive (aOR, 2.22; 95% CI, 1.17 - 4.22), and history of STI (aOR, 2.12; 95% CI, 1.19 - 3.91) were independently associated with HSV-2 prevalence (Table 2). Among men with baseline HSV-2 infection, 93 (55%) were positive for HPV DNA, of which 70 (75%) were at least infected with a HR HPV genotype. Similar HPV proportions were observed among HSV-2 negative men; 75% (42/56) of HPV positive men had at least one HR HPV genotype. Baseline HPV infection was not associated with HSV-2 prevalence (Table 2).

Among the 131 men negative for HSV-2 at baseline, 106 had exit data and 25 (23.6%) incident cases were identified during the one year follow-up period. This resulted in an incidence rate of 23.6 cases/100 pyr. Incidence rates were higher among men with a history of STI (25.0 vs 23.3/100 pyr), positive for HIV (28.6vs 22.8/100 pyr), HPV infection (31.9 vs 16.9/100 pyr), those whose most recent sexual act was with a casual partner/sex worker (44.8 vs 15.6/100 pyr)and those with multiple sexual partners in the last 6 months (31.3 vs. 17.2/100 pyr), 12 months, (33.3 vs. 9.3/100 pyr), and >5 sexual partners lifetime (31.3 vs. 10.3/100 pyr). The incidence rate was much lower among men not engaging in transactional sex (3.8 vs 30.0/100 pyr) and those who

reported condom use on the last sexual act (7.1 vs 33.3/100 pyr) or with new sexual partner (14.5 vs 36.4/100 pyr) as shown in Table 3. Bivariate analysis revealed that the number of sexual partners in the last 12 months (IRR, 3.58; 95% CI, 1.32 - 9.71), number of lifetime sexual partners (IRR, 3.06; 95% CI, 1.13 - 8.26) and most recent sexual act with casual partner/sex worker (IRR, 4.40; 95% CI, 1.53 - 12.82) were associated with HSV-2 incidence. Condom use with new sexual partner (IRR, 0.30; 95% CI, 0.10 - 0.83) and lack of transactional sex (IRR, 0.09; 95% CI, 0.01 - 0.71) were protective against incident HSV-2 infection. In multivariate analysis, lack of condom use with new sexual partner (aIRR, 2.53; 95% CI, 1.12 - 7.38), no transactional sex (aIRR, 0.15; 95% CI, 0.08 - 0.95), multiple sexual partners in the last 12 months (aIRR, 4.77; 95% CI, 1.12 - 11.38) and most recent sexual act with casual partner/sex worker (aIRR, 3.03; 95% CI, 1.17 - 8.58) remained significantly associated with incident HSV-2 infection (Table 3).

Of the 25 cases with incident HSV-2 infection, 15 (60.0%) were infected with persistent HR HPV genotypes compared to 29.6% (24/81) among men who remained HSV-2 negative at study exit. Therefore men with persistent HR HPV infection were 3.6 times more likely to acquire new HSV-2 infection compared to those without (aIRR, 3.35; 95% CI, 1.21 - 11.37). However, baseline HIV infection and exclusive infection with persistent LR HPV genotypes were not associated with HSV-2 incidence (Table 3). Among 92 men eligible for incidence evaluation for both HIV and HSV-2, 3 of 5 men with new HIV infection had incident HSV-2 infection compared to 18 of 87 men without new HIV infection. Therefore, men with new HSV-2 infection were 5 times more likely to acquire new HIV infection (IRR, 5.07; 95% CI, 0.91 - 28.37) compared to those without. However, the association between incident HSV-2 infection and HIV acquisition was on the borderline (P = 0.076).

Fishermen in this study had a high burden of HSV-2 infection. The estimated baseline HSV-2 prevalence of 56% is over two times higher than the estimated HSV-2 prevalence of 26.3% for men drawn from the general national adult population in Kenya [25] [26] , 26.5% among young men in Kisumu Kenya [16] , 22% among high risk men for HIV in Mombasa, Kenya [15] and 20.3% among MSM in the US [13] . Similar high prevalence (43% - 80%) was previously observed among fishermen along Lake Victoria in Kenya [23] , among patients attending STD clinic in India [10] , among women at high risk for HIV infection in Mozambique [27] and among female sex workers in Mombasa, Kenya [15] [24] . Risk factors for prevalent HSV-2 infection (Table 2) found in this study (older age, marital status, inconsistent condom use, history of STI, and HIV infection) were consistent with results from other studies [10] [15] [23] [24] .

The HSV-2 incidence rate of 23.6/100 pyr observed in this study, to our knowledge is the highest for HSV-2 among men observed anywhere in the world. Similar HSV-2 incident rates have only been documented among women with high risk sexual behavior. These studies found HSV-2 incidence rate of 23/100 pyr among female sex workers in Mombasa, Kenya [24] , 21.9/100 pyr among female sex workers in China [28] 22.1/100pyr among women at high risk for HIV infection in Mombasa, Kenya [15] and 20.5/100 pyr among women with high risk sexual behavior in Beira, Mozambique [27] . HSV-2 incidence of 11.4/100 pyr was shown among patients attending STD clinic in India [10] and 9.0/100 pyr found among men at high risk for HIV infection in Mombasa, Kenya [15] .

The rate of new HSV-2 infection in this study was almost 3-5 times that observed (4.9 - 7.4 /100 pyr) in studies evaluating HSV-2 incidence among young men drawn from the general population (in Kenya and Uganda) [16] [29] , and adolescents in the US [30] . Additionally, this study found much higher HSV-2 incidence rate compared to 2.4/100 pyr (with HIV negative partner) - 5.0/100 pyr (with HIV positive partner) for all anal intercourse observed among MSM in the US [13] and 4.0/100 pyr among injection drug users (IDU) in Chiang Mai, Thailand [31] .

In this study, engagement in transactional sex had a borderline association (P = 0.047) with HSV-2 incidence, although the majority of fishermen reported recent transactional sexual activity. Transactional sex as a risk for HSV-2 acquisition was also observed in other studies [10] [25] . Moreover, recent sex with casual partners, multiple sexual partners and inconsistent condom use; found to be independently associated with HSV-2 incidence in this study, were also demonstrated elsewhere [10] [13] [27] [30] . A greater (≥3) number of sexual partners in the 12 months prior to study participation was a stronger risk factor for HSV-2 acquisition compared to higher (≥6) number of sexual partners lifetime (Table 3). Similarly, Brown et al. (2006) found an increased risk of HSV-2 acquisition strongly associated with a higher number of sexual partners (≥6) and multiple (≥5) unprotected sexual acts, six months prior to study participation [13] . This emphasized that it is not just a temporary sequence of events cumulated in a lifetime but rather the intensity of high-risk sexual events occurring in the period immediately preceding HSV-2 seroconversion.

To our knowledge, this is the first study to assess HPV infection as a risk factor for incident HSV-2 infection. Therefore, the observed association between persistent HR HPV and new HSV-2 infection warrant further studies and should be interpreted with caution. However, several studies have found that HPV infection increases the risk for HIV acquisition by 1.8 - 3.5 times [32] -[34] . Lissouba et al. (2013) in their meta-analysis review found that HR HPV infection was associated with almost a 2-fold increased risk for incident HIV infection, while LR HPV infection had only a borderline association [35] . Hypothetically, persistent HR HPV infection is biologically necessary for development of HPV disease that may be associated with breach of epithelial membranes and infiltration with pro-inflammatory cytokines and other immune cells. These potentially provide an enabling environment for HSV-2 or HIV entry and replication. Just like HSV-2, HPV is believed to be more infectious than HIV, with prevalence always exceeding that of HIV and the most infectious of the three viruses [36] . Therefore, if the interaction between HSV-2 and HPV found in this study is confirmed by other studies, HPV vaccination would be a recommended strategy for HSV-2 prevention in addition to safer sexual practices.

HSV-2 and HIV have a common route for transmission and therefore possibly share common factors onhigh-risk sexual behavior. Numerous prevalence studies have shown a strong association between the two sexually transmitted viral infections [37] . Several incidence studies have found prevalent (baseline) HSV-2 infection an important risk factor for new HIV acquisition, with the strongest association emerging between HIV incident and recent or incident HSV-2 infection [10] [12] [13] [15] [38] . In these studies, the majority of incident HSV-2 infection events occurred concurrently with or close to the HIV seroconversion events. Therefore, the high HSV-2 incidence rate observed in our current study is likely a strong biological indicator for a rapidly growing HIV epidemic in this population.

This study had some limitations. Evaluation of the independent role of incident HSV-2 infection on HIV acquisition was limited by the study power, resulting from the small numbers of HIV serocoversion events observed in this study. Additionally, the incidence of these two infections was determined at the end of the study and therefore we could not establish whether HSV-2 incident infection preceded HIV acquisition.

Fishermen are highly burdened and at increased risk for HSV-2 infection characterized by the highest incidence rate ever reported among men, but only comparable to rates documented among female sex workers. Persistent HR HPV infection was independently associated with increased risk for HSV-2 acquisition in addition to factors related to high risk sexual behavior. Without an efficacious HSV-2 vaccine in sight, well-tailored intervention programs promoting safer sex for prevention of HSV-2, HPV and subsequently HIV infections are urgently needed.

We gratefully acknowledge the study participants and the Beach Management Units on the study beaches for their commitment and cooperation during this study. We feel indebted to all the research assistants who worked tirelessly in the field successfully implementing this study. We sincerely appreciate the Director KEMRI and the Director Centre for Microbiology Research (CMR) KEMRI for their unwavering institutional support. This study was fully funded by the Consortium for National Health Research (CNHR) Kenya, Research Leadership Grant (RLG) 026/2009.

The authors declare that there is no conflict of interests regarding the publication of this paper.