JAIDS Journal of Acquired Immune Deficiency Syndromes 26:360364 2001 Lippincott Williams & Wilkins, Inc.

, Philadelphia

Selenium Deficiency Is Associated With Shedding of HIV-1Infected Cells in the Female Genital Tract
*Jared M. Baeten, *Sara B. Mostad, *Martin P. Hughes, Julie Overbaugh, *Daniel D. Bankson, Kishorchandra Mandaliya, Jeckoniah O. Ndinya-Achola, Job J. Bwayo, and *Joan K. Kreiss
*Departments of Epidemiology, Laboratory Medicine, and Medicine, University of Washington, Seattle, U.S.A.; Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, U.S.A.; Coast Provincial General Hospital, Mombasa, Kenya; and the Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya

Objective: To assess the relation between selenium deficiency and vaginal or cervical shedding of HIV-1infected cells. Design: Cross-sectional study of 318 HIV-1 seropositive women in Mombasa, Kenya. Methods: Vaginal and cervical swab specimens were tested for the presence of HIV-1 DNA by polymerase chain reaction. Multivariate logistic regression models, adjusting for CD4 count and vitamin A deficiency, were used. Results: Selenium deficiency (defined as levels <85 g/L) was observed in 11% of the study population. In unstratified multivariate analyses, there was no significant association between selenium deficiency and vaginal or cervical shedding. In stratified analyses, however, significant associations became apparent after excluding women with predictors of shedding with strong local effects on the genital tract mucosa. Among women who did not use oral contraceptives and who did not have vaginal candidiasis, selenium deficiency was significantly associated with vaginal shedding (adjusted odds ratio [AOR] 2.9, 95% confidence interval [CI] 1.08.8, p .05). Effect modification was also observed in the relation between selenium deficiency and cervical shedding, with a significant association seen among those women who were not using oral contraceptive pills or depot medroxyprogesterone acetate and who did not have Neisseria gonorrhoeae infection (AOR 2.8, 95% CI 1.17.0, p .02). Conclusions: We found selenium deficiency to be associated with a nearly threefold higher likelihood of genital mucosal shedding of HIV-1infected cells, suggesting that deficiency may increase the infectiousness of women with HIV-1. Nutritional interventions to prevent HIV-1 transmission warrant investigation. Keywords: HIV-1TransmissionSheddingSeleniumNutrition Deficiency.

Nutritional deficiencies are common in HIV-1 infected individuals (1,2). Antioxidant micronutrient deficiencies may influence HIV-1 pathogenesis, as sugAddress correspondence and reprint requests to Jared M. Baeten, University of Washington, 325 Ninth Avenue, Box 359909, Seattle, WA 98104-2499 U.S.A.; e-mail: jbaeten@u.washington.edu This study was supported in part by the U.S. National Institutes of Health through grant AI39996 and the Clinical Nutrition Research Unit (DK35816). J.M. Baeten and S.B. Mostad were scholars in the International AIDS Research and Training Program supported by the Fogarty International Center, National Institutes of Health (D43TW00007).

gested by evidence that oxidative stress enhances HIV-1 replication in vitro and is more common in infected individuals than in uninfected control subjects (3,4). Deficiencies in specific antioxidants (e.g., selenium and vitamin E) have been associated with faster progression to AIDS (5) and increased HIV-1 related mortality (6,7). The relation between antioxidant status and HIV-1 transmission has not been investigated, though there is some evidence that nutritional deficiency may play a role in transmission from mother to child (810). HIV-1infected women may shed the virus in vaginal 360

SELENIUM DEFICIENCY AND HIV SHEDDING and cervical secretions, and there is strong agreement between epidemiologic correlates of HIV-1 transmission and correlates of genital shedding of HIV-1 and HIV-1 infected cells (11,12). Epidemiologic studies have demonstrated that genital tract shedding is a risk factor for mother-to-child transmission of HIV-1 (13,14), and it has been postulated that women who shed HIV-1 in the genital tract are likely to be more infectious to sexual partners as well (11,12,15). Identification of correlates of shedding of HIV-1infected and HIV-1infected cells in genital tract secretions may suggest intervention strategies to prevent heterosexual and perinatal transmission. We previously reported the results of a large crosssectional study examining the correlates of vaginal and cervical shedding of HIV-1infected cells among HIV1infected women in Mombasa, Kenya (16). Because of increasing evidence suggesting the importance of antioxidant nutrients in HIV-1 disease, we decided to further investigate the association with selenium and vitamin E status in this cohort. METHODS
Between December 1994 and April 1996, 318 HIV-1 seropositive women attending a municipal sexually transmitted disease clinic in Mombasa, Kenya were enrolled, as previously described (16). The study protocol was approved by the institutional review boards of the University of Washington, Seattle, WA, U.S.A. and the University of Nairobi, Nairobi, Kenya. After providing informed consent, each participant was interviewed, using a standardized questionnaire. Pelvic examinations were performed by a single investigator (S.B.M.), and included screening for sexually transmitted diseases and collection of genital tract samples. Venipuncture was performed to obtain serum and EDTA-anticoagulated blood. Vaginal secretions for detection of HIV-1 DNA were collected by rolling a Dacron swab (Hardwood Manufacturing, Guilford, ME, U.S.A.) three turns on the lateral vaginal wall, avoiding pooled secretions. Endocervical samples were collected by inserting a Dacron swab into the cervical os and rotating two turns. Swabs were placed in dry cryovials on ice and transferred within 4 hours to a -70C freezer. Vaginal secretions were examined for candidiasis by wet mount and cervical secretions were cultured for Neisseria gonorrhoeae. Sera were tested for antibodies to HIV-1 by two enzyme immunoassays (DetectHIV, BioChem Immunosystems, Montreal, Canada and Recombigen, Cambridge Biotech, Galway, Ireland). CD4+ T-lymphocytes were quantified in EDTA-anticoagulated blood using a manual method (Cytosphere, Coulter, Hialeah, FL, U.S.A.). Light-protected sera were tested for vitamins A and E by high-pressure liquid chromatography and for selenium by graphite furnace atomic absorption spectrophotometry (17,18). Deficiency was defined as serum concentrations below 30 g/dl for vitamin A and 5 mg/L for vitamin E, according to standard definitions (19). Because the definition of selenium deficiency is not well standardized (20), we chose to define levels <85 g/L as deficient, given that serum concentrations less than this have been shown to be strongly predictive of HIV-1 related mortality (6,7). Cryovials containing swabs were shipped on dry ice to the University of Washington and stored at -70C. Samples were tested for HIV-1

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DNA by nested PCR amplification of the gag gene, as described previously (21). Statistical analysis was done with SPSS version 8.0 (SPSS, Chicago, IL, U.S.A.). Fisher exact test, 2 test, Student t-test, and Spearman correlation coefficient were used for univariate comparisons and logistic regression for multivariate analyses. In the previous report from this cohort (16), CD4 lymphocyte depletion, infection with N. gonorrhoeae, oral contraceptive pill use, and depot medroxyprogesterone acetate (DMPA) use were significantly associated with detection of HIV-1 DNA in cervical swabs, and CD4 lymphocyte depletion, vaginal candidiasis, oral contraceptive use, and vitamin A deficiency were significantly associated with detection in vaginal swabs. For the present investigation, multivariate models were adjusted for CD4 depletion and vitamin A deficiency. In addition, we performed stratified analyses which excluded those women with previously identified correlates of viral detection that were likely to work at a local level (namely, oral contraceptive use, DMPA use, N. gonorrhoeae infection, or vaginal candidiasis). These strong locally-acting correlates may increase the likelihood of genital mucosal shedding to such a degree that elevated risk estimates associated with antioxidant deficiencies might only be apparent among those women unexposed to these predictors.

RESULTS Characteristics of the Study Population The median age of the 318 study participants was 28 years (range 1746). Thirty-six women (11%) were using oral contraceptive pills and 55 (17%) were using DMPA. Twenty-four women (8%) had gonorrhea and 61 (19%) had vaginal candidiasis. The mean CD4+ Tlymphocyte count was 451 (standard deviation [SD] 263) cells/l. Of the participants, 37% had counts >500 cells/ l, 46% had counts between 200 and 499 cells/l, and 17% had counts <200 cells/l. The mean serum concentration of vitamin A was 37 (SD 15) g/dl and 34% (108/318) were deficient (<30 g/dl). The mean serum selenium concentration was 110 (SD 23) g/L and 11% (35/314) of the women were deficient (<85 g/L). The mean CD4 count was not significantly different among women with selenium deficiency compared with those who were not deficient (454 vs. 427, p .6), and there was no significant correlation between CD4 count and selenium concentrations (r 0.03, p .5). Women with selenium deficiency were significantly more likely to have vitamin A deficiency compared with women who were not deficient in selenium (54% vs. 32%, p .007). The mean serum concentration of vitamin E was 8 (SD 3) mg/L and 4% (12/318) were deficient (<5 mg/L). There were too few cases of vitamin E deficiency to evaluate its relation with CD4 count or vitamin A or selenium deficiencies.

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362 Vaginal and Cervical Shedding of HIV-1Infected Cells

J. M. BAETEN ET AL. CD4 count and vitamin A deficiency (AOR 2.8, 95% CI 1.1-7.0, p 0.02). Because of the low prevalence of vitamin E deficiency, there was insufficient power to assess its relation with genital tract shedding. DISCUSSION In this large cross-sectional study of 318 women in Mombasa, we found that selenium deficiency was associated with a significantly increased prevalence of genital mucosal shedding of HIV-1infected cells. In the subgroup of women who did not have a strong, locallyacting predictor of shedding, selenium deficiency was associated with an threefold increase in HIV-1 DNA detection in both vaginal and cervical secretions. That the effects of selenium deficiency were not apparent in the subgroup with strong, locally-acting predictors of genital tract shedding may be a result of the higher prevalence of shedding among these women, which could overwhelm demonstration of elevated risk estimates. Molecular studies suggest there is biologic plausibility for our findings. Oxidative stress stimulates HIV-1 replication in vitro (22) and addition of antioxidant micronutrients (23), including selenium (24), inhibits this activation. Evidence that HIV-1 encodes viral selenoproteins has led to speculation that HIV-1 regulates its own replication based on selenium levels, with greater replication occurring when selenium concentrations are low

HIV-1 DNA was detected in 44 (14%) of 318 vaginal swabs. In univariate analysis, vaginal shedding was significantly more common in women who were deficient in selenium than in those who were not deficient (26% vs. 12%, p .04) (Table 1). In multivariate analysis, there remained a trend for increased shedding after adjustment for CD4 count and vitamin A deficiency (p .09). Using stratified analysis, we found that oral contraceptive use and vaginal candidiasis (correlates from our previously published analysis that exert an effect at the local level) modified the effect of selenium deficiency on vaginal shedding of HIV-1infected cells. In analysis excluding women with either of these two factors, selenium deficiency was significantly associated with shedding, independent of vitamin A deficiency and CD4 count (AOR 2.9, 95% CI 1.08.8, p .05). HIV-1 DNA was detected in 161 (51%) of 315 endocervical swabs. In univariate and multivariate analysis of the entire study population, selenium deficiency was not significantly associated with cervical shedding of HIV1infected cells (Table 1). However, in a stratified analysis that excluded women with a strong locally-acting predictor of cervical HIV-1 DNA (i.e., oral contraceptive use, DMPA use, or N. gonorrhoeae infection), selenium deficiency was associated with shedding (63% vs. 39%, p .02), and this relation remained after adjustment for

TABLE 1. Selenium deficiency and shedding of HIV-1infected cells in vaginal and cervical secretions Univariate analysis Number/total (%) with HIV-1 DNA detected Vaginal shedding All women Selenium Not deficient Deficient Subgroup Selenium Not deficient Deficient Cervical shedding All women Selenium Not deficient Deficient Subgroup Selenium Not deficient Deficient OR (95% CI) p value Multivariate analysis* AOR (95% CI) p value

34/279 (12) 9/35 (26) 17/196 (9) 6/27 (22)

1.0 2.5 (1.06.2) 1.0 3.0 (0.99.1)

.04

1.0 2.1 (0.95.2) 1.0 2.9 (1.08.8)

.09

.04

.05

137/276 (50) 21/35 (60) 71/182 (39) 17/27 (63)

1.0 1.5 (0.73.3) 1.0 2.7 (1.16.7)

.2

1.0 1.6 (0.73.4) 1.0 2.8 (1.17.0)

.2

.02

.02

* Multivariate logistic regression model adjusting for CD4 count and vitamin A deficiency. Subgroup excluding women using oral contraceptive pills or with vaginal candidiasis. Subgroup excluding women using oral contraceptive pills or depot medroxyprogesterone acetate or with N. gonorrhoeae infection.

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SELENIUM DEFICIENCY AND HIV SHEDDING (25,26). Enhanced HIV-1 replication could lead to higher numbers of HIV-1infected cells in the genital tract, and we suggest that deficiency in selenium increases shedding through this mechanism. Clinical studies suggest that there may be a role for antioxidant supplementation in HIV-1 disease. One small trial found a significant increase in CD4/CD8 ratio after 12 weeks of selenium supplementation (27). Another showed a trend for a decrease in HIV-1 plasma viral load during 3 months of high-dose supplementation with vitamins E and C (28). Higher intake of some antioxidant micronutrients (vitamins E and C) has been associated with slower progression to AIDS in two observational studies (29,30). In general, however, the potential for supplementation with selenium or any other antioxidant to decrease HIV-1 replication requires further study. One limitation of our study is that its cross-sectional design cannot establish if selenium deficiency is truly responsible for increased genital shedding of HIV-1 infected cells. Other investigators have found lower selenium levels to be associated with more advanced HIV-1 disease stage (31). Though we found selenium deficiency to be associated with vaginal and cervical shedding in multivariate models controlling for CD4 count, it is possible that more active HIV-1 infection alone may be the cause of both lower selenium levels and increased genital shedding. However, in this population there was no significant correlation between selenium levels and CD4 counts, making this unlikely. Despite our large sample size, the relatively low prevalence of selenium deficiency limited study power. In addition, the very low prevalence of vitamin E deficiency prohibited analysis of the relation between deficiency of this antioxidant and genital tract shedding. Higher prevalences of vitamin E deficiency have been reported in other populations of HIV-1infected individuals (1,2), and further investigations may be worthwhile. These data, along with our earlier report of a strong relationship between vitamin A deficiency and vaginal shedding of HIV-1infected cells, suggest that micronutrient deficiencies may play a role in transmission of HIV-1. Micronutrient supplementation may have the greatest potential impact in the developing world, where wasting is a common manifestation of HIV-1 infection, the prevalence of micronutrient deficiency is high, and antiretroviral therapy is generally not available. Given that 95% of new HIV-1 infections occur in the developing world, it is critical to develop intervention strategies that are inexpensive and widely available (9,32). The results of our study suggest that selenium deficiency may be a determinant of infectivity in HIV-1 infected

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women. If other observational studies confirm our results, a randomized clinical trial of selenium supplementation would establish whether there is a causal relation between selenium deficiency and genital shedding of HIV-1.
Acknowledgments: We thank the women who participated in this study, the research clinic and laboratory staff, and the Municipality of Mombasa and the administration of Coast Provincial General Hospital for provision of clinical and laboratory space.

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