Shigella sp., Salmonella sp., Campylobacter sp., Giardia lamblia, Entamoeba histolytica, and hepatitis A virus (4, 27, an urban STD clinic.

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1991, p /91/ $2./ Copyright C) 1991, American Society for Microbiology Vol. 29, No. 1 Characterization of Risk Factors for Helicobacter pylori Infection among Men Attending a Sexually Transmitted Disease Clinic: Lack of Evidence for Sexual Transmission LOUIS B. POLISH,"2t JOHN M. DOUGLAS, JR.,12,3* ARTHUR J. DAVIDSON,2'3 GUILLERMO I. PEREZ-PEREZ,4 AND MARTIN J. BLASER4 Division of Infectious Diseases, Department of Medicine,' and Department of Preventive Medicine,3 University of Colorado School of Medicine, Denver, Colorado 8262; Denver Disease Control Service, Denver Department of Health and Hospitals, Denver, Colorado ; and Division of Infectious Diseases, Department of Medicine, and Division of Infectious Diseases, Vanderbilt University School of Medicine, and Veterans Affairs Medical Center, Nashville, Tennessee Received 4 January 1991/Accepted 27 June 1991 The mechanism of transmission of Helicobacter pylori is unknown. To investigate the role of sexual behavior and demographic factors in the acquisition of H. pyloni infection, we evaluated the seroprevalence of antibody to H. pylori in 37 men attending an urban sexually transmitted diseases clinic. Sera from the following three groups were analyzed by enzyme-linked immunosorbent assay for H. pyloni-specific immunoglobulin G: 78 human immunodeficiency virus (HIV)-seropositive homosexual men, 12 HIV-seronegative homosexual men, and 19 HIV-seronegative heterosexual men. Overall, the seroprevalence of H. pyloni was 1 of 37 men (27%), with rates of 18% in HIV-seropositive homosexual men and 2% in HIV-seronegative homosexual men versus 35% in heterosexual men (P <.5, x2 test). By ethnic group, 21 (12%) of 181 Caucasian men, 4 (41%) of 97 black men, and 37 (43%) of 87 Hispanic men were seropositive (P <.1, X2 test). Multivariate analysis revealed that race was associated with H. pyloni seropositivity independent of HIV status, sexual preference, or age. There was no relationship between H. pylori seropositivity and the number of lifetime sexual partners or previous sexually transmitted diseases. Three HIV-seropositive men with H. pylori immunoglobulin G had essentially identical antibody titers over 8 to 16 months of follow-up. In conclusion, black and Hispanic men have significantly higher H. pylori seroprevalence rates than do Caucasian men, but neither sexual behavior nor HIV infection influences the presence or persistence of H. pylori antibody. Further evaluation of the factors associated with these ethnic differences may lead to a better understanding of H. pyloni acquisition and transmission. Epidemiologic investigation of Helicobacter pylori (formerly Campylobacter pylori) (12) infection has largely been based on detection of the bacteria from specimens obtained at the time of endoscopy. Infection is chronic, lasting for years or decades and possibly for life (22). Infected individuals mount an antibody response to H. pylori (17, 29), and recent refinements permit sufficiently high specificity and sensitivity of immunoglobulin G (IgG) and IgA assays for use as diagnostic tests (7, 8, 25). Given this degree of accuracy, serologic diagnosis has become an important tool to further define the epidemiology of H. pylori infection (24). The mechanism of H. pylori transmission remains unknown. Marshall and colleagues (17) found that H. pylori antibody in women attending a sexually transmitted disease (STD) clinic is more common in those women with reactive serologic tests for syphilis than in those without reactive serologic tests for syphilis. In addition, Aceti et al. (1) found that antibody levels were higher in homosexual men than in age-matched heterosexual men. Both studies suggest that sexual behavior could be important in the transmission of this organism. These observations are consistent with the known potential for oral-anal sexual transmission in homosexual men of a variety of enteric pathogens including * Corresponding author. t Present address: Hepatitis Branch, Division of Viral Diseases, Centers for Disease Control, Atlanta, GA Shigella sp., Salmonella sp., Campylobacter sp., Giardia lamblia, Entamoeba histolytica, and hepatitis A virus (4, 27, 28). To investigate the role of sexual behavior and demographic characteristics in the acquisition of H. pylori, we evaluated the seroprevalence of infection in 37 men attending an urban STD clinic. MATERIALS AND METHODS Population. Sera from 2,168 men collected from May 1987 through July 1988 in the course of studies of human immunodeficiency virus (HIV) seroprevalence in the Denver Metro Health Clinic for STD were available for evaluation. Of these, a total of 37 serum samples were selected from among the following three clinical groups: HIV-seropositive homosexual men (n = 78), HIV-seronegative homosexual men (n = 12), and HIV-seronegative heterosexual men (n = 19). In order to achieve a broad demographic distribution within each group, specimens were selected chronologically from available samples on the basis of age group (<19, 2 to 29, 3 to 39, or.4 years) and ethnicity (Caucasian, black, Hispanic, or other). Information regarding age, sexual preference, and HIV antibody status was available for all 37 men, and information regarding ethnicity was available for 368 men. For 86 subjects whose sera were evaluated for HIV antibody in anonymous seroprevalence studies, identifying information was destroyed and further clinical or demographic data other than age, ethnicity, and sexual preference Downloaded from on July 13, 218 by guest

2 214 POLISH ET AL. were not available. The remaining 284 patients had participated in voluntary HIV seroprevalence studies, and the available clinic records were reviewed to determine the number of lifetime sexual partners, as well as a history of various STDs, e.g., gonorrhea, nongonococcal urethritis, syphilis, hepatitis, genital herpes, and genital warts. Serologic methods. (i) ELISA for H. pylori. Sera were frozen at -2 C and were examined for H. pylori-specific IgG antibodies by an enzyme-linked immunosorbent assay (ELISA) as described previously (7, 8, 23, 25). The screening serum dilution was 1:8. A peroxidase conjugate of goat anti-human IgG (TAGO Inc., Burlingame, Calif.) was diluted 1:5,. All assays were done in duplicate on at least 2 separate days. The intra- and interassay variations were less than 5%, as estimated with positive and negative control sera. To establish a threshold for positivity in the IgG ELISA, we defined a positive result as a serum sample in which the optical density (OD) value was greater than the mean plus three intervals of standard deviation for the results obtained when 35 healthy children under 1 years old were tested. We constructed a standard curve using the calculated thresholds obtained for the 35 children on 1 different occasions and the mean OD of four positive control serum samples run each time under the same conditions. Using the regression line, we calculated the threshold for each day's run on the basis of the mean OD value of the four control serum samples. The results for each serum sample are expressed as the ratio of the OD value of the serum sample to the calculated threshold for that day's run. Therefore, each sample with a ratio higher than 1. was considered positive. We used a positive IgG assay as evidence for H. pylori infection, as has been validated in previous studies (7, 8, 24, 25). A single serum specimen from each of 367 men was evaluated. For three HIV-seropositive homosexual men whose initial serum specimen showed H. pylori-specific IgG, subsequent serum specimens were obtained; these specimens were serially diluted, and an exact antibody titer was determined for IgA as well as IgG by using previously described methods (7, 25). (ii) HIV antibody. Sera were screened for HIV antibody by ELISA (Organon Teknika, Durham, N.C.), and repeatedly reactive specimens were confirmed as positive by Western blot (Immunodot; Dupont, Wilmington, Del.). Statistical methods. Analysis of data was performed by using Prodas Statistical Software (Conceptual Software, Inc., Houston, Tex.). Categorical variables were compared by a chi-square test with the Yates correction or a two-tailed Fisher exact test, as appropriate. Data collection for the number of total lifetime sexual partners was limited to two digits (if there were.98 partners, the total was recorded as 98); thus, data are presented as median values and comparisons were made by the Wilcoxon rank sum test (3). For logistic regression analysis, the number of lifetime partners was dichotomized to greater or less than the median value for the particular group. Multiple logistic regression analysis was performed to identify factors related to seropositivity for H. pylori; all factors, including age, race, sexual preference, HIV serostatus, previous STD, and number of lifetime sexual partners (dichotomized), were entered into the model. A stepwise forward procedure with maximum logarithmic-likelihood estimates and likelihood ratio tests identified the best model. Interaction terms were evaluated for factors noted to be significant in earlier models. All beta coefficients were converted to exponential values to approximate an adjusted odds ratio by controlling for all other a) VA HIV- nomosexual >4 HIV- Heterosexual N=38 CI 3 2 C' < 2D 1 < > 4 Age in Years FIG. 1. Seroprevalence of H. pylori infection by age, sexual preference, and HIV status among 37 men attending the Denver Metro Health Clinic. factors for that exposure variable (31). Dummy variables were created to evaluate the various ethnic groups. RESULTS J. CLIN. MICROBIOL. The study population had a mean age of 31.2 years and included 181 (49%) Caucasians, 97 (26%) blacks, 87 (24%) Hispanics, 3 (1%) of other ethnic origin, and 2 (.5%) of unknown ethnicity. The mean age of the 78 HIV-seropositive homosexual men was 32 years (range, 19 to 52 years) and included 56 (72%) Caucasians, 6 (8%) blacks, 15 (19%) Hispanics, and 1 (1%) Asian. The mean age of the 12 HIV-seronegative homosexual men was 33 years (range, 16 to 7 years) and included 68 (67%) Caucasians, 12 (12%) blacks, 19 (19%) Hispanics, 1 (1%) Native American, and 2 (1%) of unknown ethnicity. The mean age of the heterosexual men was 3 years (range, 15 to 61 years) and included 57 (3%) Caucasians, 79 (41%) blacks, 53 (28%) Hispanics, and 1 (.6%) Asian. Of the 37 serum samples, 1 (27%) showed H. pylorispecific IgG. The results of serology according to sexual orientation and HIV status are shown in Fig. 1. Overall, the H. pylori infection rate among HIV-seropositive homosexual men (18%) was similar to that among HIV-seronegative homosexual men (2%), but both were significantly lower than the rate in heterosexual men (4%) (P <.5). For all age groups, seropositivity rates were higher in heterosexual men than they were in homosexual men. While heterosexual men older than 4 years had a higher rate of infection (39.5%) than did those younger than 4 years (33.6%), this difference was not statistically significant, and no other significant age-related differences were noted among any of the three groups. The association of H. pylori seropositivity and risk factors for STD are described in Table 1. There was no significant relationship between H. pylori infection and the median number of lifetime sexual partners for any of the three groups. In addition, for no group was there a significant association between H. pylori infection and a history of any previous STD or of gonorrhea, nongonococcal urethritis, syphilis, hepatitis, or genital herpes, specifically; a history of genital warts was inversely associated with H. pylori infection among heterosexual men, but not homosexual men. As expected, among the three groups, the median number of lifetime sexual partners and the rates of any past STD were Downloaded from on July 13, 218 by guest

3 VOL. 29, 1991 H. PYLORI INFECTION IN MEN ATTENDING AN STD CLINIC 2141 TABLE 1. Group and H. pylori Association of H. pylori seropositivity with number of lifetime sexual partners and history of previous STDs Median no. of % with history of the following STD: lifetime sexual antibody statusa partnersb Gonorrhea urethritis Syphilis Hepatitis Genital Genital Any urethritis ~~~~herpes warts HIV-seropositive homosexual men HP' (n = 7) 25 (3) HP- (n = 36) 6 (11) HIV-seronegative homosexual men HP' (n =11) 98 (3) HP- (n = 45) 25 (1) HIV-negative heterosexual men HP' (n = 66) 9 (23) C 5 HP- (n = 119) 15 (28) ' b HP', H. pylori antibody positive; HP-, H. pylori antibody negative. Numbers in parentheses are number of patients indicating that they did not know the number of lifetime sexual partners. c P <.5 for H. pylori seropositive versus H. pylori seronegative patients by Fisher's exact test. significantly lower for heterosexual men, the group with the highest rate of H. pylori seropositivity, than for homosexual men (P <.1 for both comparisons). In contrast to the lack of association with age or risk factors for STD, H. pylori seropositivity varied markedly by ethnicity, with significantly lower rates in Caucasians (12%) than in blacks (41%) or Hispanics (43%) (Fig. 2). The same trend was consistently noted for each age group that was evaluated. To determine the relative importance of sexual preference, ethnicity, and other factors in H. pylori seropositivity, a multivariate analysis was conducted (Table 2). Of all the characteristics evaluated, only ethnicity was independently associated with H. pylori seropositivity (odds ratio = 5.7, 95% confidence interval = 3.1 to 1.3); this factor alone resulted in the best model by using likelihood ratio tests. The higher seroprevalence noted in heterosexual men than that noted in homosexual men thus appears to be a result of the small proportion of homosexual men available for evaluation who were non-caucasian in comparison with the number of non-caucasian heterosexual men. Rates for non-caucasian men were similar among HIV-seropositive homosexual (27%), HIV-seronegative homosexual (44%), and heterosexual (44%) men and were higher than those for Caucasian men in each group (14, 9, and 12%, respectively). Sequential serum samples were evaluated for three of the > 6~._D oq 5Q. a) cn 4-._= a 3- c 2 La. 1 O N=17 O White 3 Black *} Hispanic N-S Age in Years FIG. 2. Seroprevalence of H. pylori infection by age and ethnicity among 368 men attending the Denver Metro Health Clinic. N=41 HIV-seropositive homosexual men found to have H. pylori antibody (Table 3). Antibody titers remained elevated and essentially constant over 8 to 16 months in all three men. Data regarding T4 cell count and stage of HIV infection were not available. DISCUSSION This study was based on the use of a serologic assay to detect serum IgG to H. pylori as a means of determining the prevalence of infection. The assay has been validated previously in comparison with examination of H. pylori in gastric biopsy specimens and has sensitivity and specificity each exceeding 9% (7, 8, 11, 23, 32, 33). Therefore, serology is a reliable tool for diagnosing H. pylori infections in seroepidemiologic studies (6, 13, 18, 19, 22, 24, 26). The source of H. pylori as well as the mechanism of its transmission to humans remain unknown. Since H. pylori is present in low concentrations in gastric juice and has not been isolated from saliva, feces, or urethral or vaginal swabs, plausible modes of person-to-person transmission have not been proposed (5). Attempts to better define mechanisms of transmission through serologic studies have yielded conflicting results. Mentally retarded institutionalized residents were found to have higher seropositivity rates TABLE 2. Multivariate analysis of factors associated with H. pylori seropositivity No. (%) H. pylori: Adjusted 95% Factor Seropositive Seronegative odds confidence (n = 1) (n = 27) ratio intervals Non-Caucasian 72 (72) 11 (41) ethnicity Homosexual 34 (34) 146 (54) preference Age (yr) <2 19 (19) 42 (16) (28) 92 (34) (32) 81 (3) (21) 55 (2) HIV seropositive 14 (14) 64 (24) Downloaded from on July 13, 218 by guest

4 2142 POLISH ET AL. Patient TABLE 3. Persistence of H. pylori antibody in three HIV-infected individuals' Date of serum Reciprocal titer" collection (mo-day-yr) IgG IgA A B ,6 2 C , ,2 1 aeach patient was HIV antibody positive at the time that the first serum specimen was obtained. b Titration was performed by evaluating twofold serial dilutions of serum. Titer was defined as the last dilution showing an OD value exceeding the threshold value, as indicated in the text. J. CLIN. MICROBIOL. for H. pylori than age-matched blood donors, suggesting person-to-person transmission (3). Similarly, children in a Bangkok orphanage, where enteric infection is hyperendemic, were usually seropositive by the age of 3 years, a significantly higher prevalence than that in rural Thai children of the same age (24). Furthermore, family contacts of four children with proven H. pylori infection had an increased prevalence of antibody compared with those in controls (19), and both siblings and mothers of children with proven H. pylori infection were significantly more likely to be infected with H. pylori than were matched controls, suggesting intrafamilial clustering (8). In contrast, however, Jones et al. (14) found an antibody prevalence of only 17% among the household contacts of 4 patients from whom H. pylori was cultured from a gastric biopsy specimen. Although several previous reports (1, 17) have suggested that sexual behavior may be important in the transmission of this organism, our data failed to show any association between H. pylori infection and key selected risk factors for sexually transmitted infection. Overall, rates were no higher in homosexual men than they were in heterosexual men, despite a higher number of lifetime sexual partners and a more frequent history of past STDs, including syphilis, which is the STD serologically associated with H. pylori seropositivity by Marshall and colleagues (17). Furthermore, among homosexual men, H. pylori seroprevalence rates were similar for HIV-seropositive and HIV-seronegative men, despite the more frequent history of STDs in the former, findings which are consistent with those recently reported by Aceti et al. (2) in a population of Italian men. Although two studies in HIV-seropositive patients with upper gastrointestinal symptoms referred for endoscopy have suggested that H. pylori may be found less commonly in HIV-seropositive patients than in age- and sex-matched HIV-seronegative controls, the ethnicity of control subjects was not specified, nor was the sensitivity of the serologic assays evaluated (1, 16). While there is evidence that HIV infection impairs humoral immunity (15), creating a potential for false-negative serologic tests, antibody responses to previously recognized antigens may be persistently elevated in HIV-seropositive persons, as is observed with such chronic viral infections as cytomegalovirus and Epstein-Barr virus. The persistence of H. pylori titers in our serially evaluated HIV-seropositive men suggests that antibody levels to this chronic bacterial infection may also persist over time. Finally, within none of our three clinical groups did we find a significant association between H. pylori infection and the number of lifetime sexual partners or a previous history of STD, suggesting that sexual practices are not important in the transmission of H. pylori for either homosexual or heterosexual men. The reason for the discrepancy between our data and those of Marshall et al. (17) and Aceti et al. (1) is not clear; however, in their reports, characterization of the study populations was limited and did not include ethnicity. While it is possible that risk factors for the acquisition of H. pylori may vary between ethnic or cultural groups, we found no association between H. pylori seropositivity and sexual behavior within either Caucasian or non-caucasian groups. Our findings are supported by a recent study of persons attending an infertility clinic in New York which also failed to suggest sexual transmission (26). Although several serologic and endoscopic studies have documented an age-related increase in the prevalence of H. pylori among adults in developed countries in predominantly Caucasian populations (7, 2, 21), age-related changes are less striking in developing countries, where seroprevalence rates reach a high plateau in early adulthood (18, 24). Studies from the Ivory Coast and Algeria demonstrated that seroconversion occurs at a young age and that by adolescence 8% of persons have been infected (18). In addition, El Salvadorean, Ethiopian, and Vietnamese refugees in Australia did not show increases in seroprevalence with age (9). Preliminary studies suggest that age-related trends in infection in developed countries may be related to ethnicity as well. An asymptomatic adult Hispanic population from Los Angeles showed an overall H. pylori endoscopic prevalence rate of 79% which did not increase with age (6). Our inability to detect significant age-related changes in H. pylori seroprevalence among Caucasians is in contrast to the results described in previous reports, but it likely reflects the relatively narrow age range we evaluated (95% of patients were between ages 18 and 48 years). The most striking finding from our study was the significantly higher rate of H. pylori seropositivity among blacks and Hispanics compared with that among Caucasians. These data are consistent with a recent study from Maryland where seropositivity rates for blacks aged 2 to 39 years was markedly higher than seropositivity rates for similarly aged Caucasians (13). The explanation for these ethnic differences is not clear. It may be that ethnicity is simply a marker for socioeconomic differences, such as household crowding, which might enhance person-to-person transmission of an enteric infection. Differences in dietary or other behavioral patterns between ethnic groups might also contribute to differential rates of infection, although the study in Maryland found no difference in the rates between Seventh-Day Adventists (vegetarians) and control groups (13). Finally, our data suggest that the persistence of H. pylori antibody over time, which is well documented in immunocompetent individuals (22), occurs in HIV-infected persons as well. This preliminary finding is of interest and indicates that, as a chronic mucosal bacterial infection, H. pylori may offer a useful model for the evaluation of the persistence of both the IgG and IgA immune response in HIV-infected patients. More detailed investigation will be necessary to assess the persistence of H. pylori antibody in HIV-infected individuals at different stages and to determine whether the loss of such antibody is associated with clinically significant disease. Downloaded from on July 13, 218 by guest

5 VOL. 29, 1991 H. PYLORI INFECTION IN MEN ATTENDING AN STD CLINIC 2143 In summary, we found that among men attending an STD clinic, blacks and Hispanics have a significantly higher seroprevalence of H. pylori infection than do Caucasians, regardless of sexual preference, HIV status, past history of STD, or number of lifetime sexual partners. Our data on sexual activity do not support the hypothesis that H. pylori infection is sexually transmitted. Further studies to confirm these findings and to investigate the factors responsible for the ethnic distribution of H. pylori infection may offer important clues in the search to define the source and mechanism of transmission of this infection. ACKNOWLEDGMENTS We gratefully acknowledge Jeanette Maez for typing the manuscript and Kim C. Le for technical assistance. This study was supported in part by the Medical Research Service of the Department of Veterans Affairs and by the Procter and Gamble Co. REFERENCES 1. Aceti, A., R. Attanasio, A. 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