McClelland, R. Scott MD, MPH*†; Baeten, Jared M. MD, PhD†; Overbaugh, Julie PhD¶**; Richardson, Barbra A. PhD‡**; Mandaliya, Kishorchandra MBChB††; Emery, Sandra BS¶; Lavreys, Ludo MD, MSc†; Ndinya-Achola, Jeckoniah O. MBChB, MSc‡‡; Bankson, Daniel D. PhD,§; Bwayo, Job J. MBChB, PhD‡‡; Kreiss, Joan K. MD, MSPH*†
Nutritional deficiency is an important problem among people infected with HIV-1.1 Wasting syndrome and micro-nutrient deficiencies are common among vulnerable populations including injection drug users and people from less developed countries.1-3 Multiple factors including decreased nutrient intake, malabsorption, diarrhea, and altered metabolism may contribute to HIV-1-related malnutrition.4
Observational studies have demonstrated dramatic associations between micronutrient deficiencies, disease progression, and mortality.5-8 Higher micronutrient intake has been associated with delayed progression to AIDS and improved survival.9-12 There has also been interest in the relationships between nutritional status, genital HIV-1 shedding, and infectivity. Deficiencies of vitamin A and selenium have been associated with female genital HIV-1 shedding.13,14 Vitamin A deficiency has also been associated with mother-to-child transmission.15,16 However, a causal role cannot be inferred solely from observational studies.
Randomized trials have begun to address the role of micronutrient supplementation as an intervention to modify HIV-1 infectivity. A trial of vitamin A supplementation in Kenyan women showed that daily oral vitamin A had no effect on genital HIV-1 shedding, plasma viral load, or CD4 count.17 Another study evaluated micronutrient supplementation as a means of decreasing mother-to-child HIV-1 transmission in Tanzania.18-20 In this trial, women receiving a multivitamin including vitamins B, C, and E had higher postpartum CD4 and CD8 lymphocyte counts than those who received placebo. Infants of women receiving supplements had fewer adverse birth outcomes and improved survival at 24 months. In contrast, vitamin A supplementation had no effect on early mother-to-child transmission, adverse perinatal outcomes, or maternal health. Surprisingly, vitamin A increased breastfeeding transmission of HIV-1 compared with placebo. These unexpected results are an indication of how much remains to be learned about the effects of nutritional supplementation in HIV-1 infection.
To better understand the effects of micronutrient supplementation on HIV-1 infectivity, this randomized, double-blind, placebo-controlled trial was undertaken with the primary objective of determining the effects of micronutrient supplementation on cervical and vaginal shedding of HIV-1-infected cells and RNA. Secondary outcomes included plasma viral load and T-lymphocyte counts.
PATIENTS AND METHODS
Between September 1998 and June 2000, women attending outpatient clinics at Coast Provincial General Hospital in Mombasa, Kenya were offered HIV-1 counseling and testing. After written informed consent, women were tested for antibodies to HIV-1 and asked to return in 1 week for results. At the results visit, seropositive women 18-45 years old were offered participation in the study after written informed consent. Women were excluded if they had used vitamin supplements or oral contraceptives or had been pregnant during the previous 3 months. Those who had signs or symptoms of a sexually transmitted disease (STD) including abnormal vaginal discharge, purulent cervical discharge, pelvic pain, or cervical or vaginal ulcer were treated and excluded from further participation until 1 week after completion of effective treatment. All women were antiretroviral naive. The protocol was approved by the institutional review boards of the University of Nairobi and the University of Washington.
Participants were interviewed regarding demographic, sexual, obstetric, and medical history. Ethylenediamine tetraacetic acid-anticoagulated blood and serum were collected for lymphocyte subset analysis, quantitation of plasma HIV-1 RNA, and assays for selenium and vitamin E concentrations. A physical and pelvic examination were performed, including collection of vaginal and cervical specimens for HIV-1 detection. A dry swab and a swab in freezing medium (70% RPMI [Roswell Park Memorial Institute], 20% fetal calf serum, 10% dimethyl sulfoxide with added penicillin, streptomycin, and amphotericin B) were collected from each site. Vaginal secretions were sampled by rotating a polyethylene terephthalate swab 3 full turns against the lateral vaginal wall. Cervical secretions were sampled by inserting a polyethylene terephthalate swab 1 cm into the cervical os and rotating it 2 full turns. This sample collection technique is sensitive and reproducible for genital HIV-1-shedding studies in women.21
Women were randomly assigned to receive the micronutrient supplement (20 mg B1, 20 mg B2, 25 mg B6, 100 mg niacin, 50 μg B12, 500 mg vitamin C, 30 mg vitamin E, 0.8 mg folic acid, and 200 μg selenium) or placebo using a computer-generated block randomization scheme created by the study statistician (B.A.R.) in Seattle. Treatment assignments remained concealed to investigators in Mombasa until after completion of the study. Active micronutrient supplement (multivitamin with selenium) and micronutrient placebo were dispensed as hard gel capsules (Tishcon, Westbury, NY).
This trial was conducted partially in parallel with a trial of vitamin A supplementation.17 The control groups of the 2 trials overlapped during the period when both studies were enrolling (Fig. 1). After 100 women had enrolled in the vitamin A trial (ratio of vitamin A to placebo = 1:1), the micronutrient intervention was initiated. An additional 450 patients were enrolled while both studies were ongoing (ratio of vitamin A to micronutrient to placebo = 1:1:1). After completion of the vitamin A trial, 100 women were enrolled to complete the micronutrient trial (ratio of micronutrient to placebo = 1:1). Thus, the micronutrient trial included 400 women. None of the participants received both vitamin A and the micronutrient supplement.
Blinding was maintained by using numbered medication bottles and capsules that were identical in appearance for the active and placebo arms of the study. Enrollees were instructed to take 1 capsule each day and to bring any remaining capsules to the follow-up visit. During the period of overlap between the 2 trials, women in the micronutrient study also received a vitamin A study placebo (soft gel capsule) while women in the vitamin A trial received a micronutrient study placebo (hard gel capsule). Thus, women in both studies took 2 capsules each day (one hard gel and one soft gel) during the period of parallel enrollment, so all of the regimens were identical in appearance. Study medication was dispensed with an electronic alarm vial (RemindRx, IBV Technologies, Seattle, WA), which has been shown to improve adherence in this population.22
At the 6-week follow-up, a brief history, physical examination, and collection of blood and genital tract specimens were repeated. The procedures were identical to the enrollment visit. The number of capsules remaining was recorded.
Serologic testing for HIV-1 was performed using an enzyme-linked immunosorbent assay (ELISA; Detect HIV 1/2, BioChem Immunosystems, Montreal, Canada) and confirmed with a second ELISA (Recombigen, Cambridge Biotech, Worcester, MA). Sera were tested for vitamin E by high-pressure liquid chromatography.23 Serum selenium was determined by graphite furnace atomic absorption spectrophotometry.24,25 Samples were tested in duplicate using an autosampler. The average of the 2 values was reported. If selenium concentrations for duplicates did not agree within 10%, the assay was repeated. Lymphocyte subsets were counted using a semi-automated system (Zymmune CD4/CD8 Cell Monitoring Kit, Bartels, Inc., Issaquah, WA). The lower limit of quantitation was 25 cells/μL.
The presence of Trichomonas vaginalis and vaginal yeast were determined using microscopy of a saline wet preparation and bacterial vaginosis was diagnosed using Gram stain criteria.26 Culture for Neisseria gonorrhoeae was performed on modified Thayer-Martin media. Testing for Chlamydia trachomatis was not performed because we have previously found a very low prevalence among the women at this hospital.27
The quantity of HIV-1 RNA was determined using the Gen-Probe quantitative HIV-1 assay (Gen-Probe, Inc., San Diego, CA). The lower limit of quantitation is 3 copies/reaction (12 copies/mL for plasma and 15 copies/swab for genital samples).28 HIV-1 proviral DNA was detected using nested polymerase chain reaction amplification of the gag gene.29 The assay is able to detect a single copy of HIV-1 DNA.
The sample size of 400 was calculated to give 80% power to detect a 3-fold difference in vaginal HIV-1 DNA using a 2-sided test with a type 1 error rate of 0.05, 15% loss to follow-up, and 14% baseline detection of HIV-1 DNA.13 This sample size allowed even greater power to distinguish differences between the micronutrient and placebo groups for the other primary outcomes.
Data were analyzed using SPSS 11.0 (SPSS, Inc., Chicago, IL). Comparisons were performed using χ2 tests for binary data and independent-samples t tests for continuous data. Logistic and linear regression were used for multivariate analyses, which were prespecified for this study. Each outcome was adjusted for its own baseline value. In addition, variables in Table 1 and genital tract infections were individually evaluated as potential confounders and retained if they produced a ≥10% change in the regression coefficient for the micronutrient effect. Viral loads were log10-transformed to better satisfy the regression modeling assumptions. All analyses were performed on the basis of intention to treat.
It was hypothesized prior to study initiation that the micronutrient effect might be greater in patients with more advanced immunosuppression or baseline nutritional deficiency. Multivariate models were used to test for effect modification by CD4 lymphocyte depletion (<200 cells/μL), low serum selenium (<85 μg/L), vitamin E deficiency (<5 mg/L), and low body mass index (BMI <20).
Screening for HIV-1 was performed in 2021 women, and 1026 (50.8%) were seropositive. Of the 857 seropositive women who returned for results, 207 were excluded from further participation; 131 declined participation and the remainder were excluded because of age >45 years (n = 12), pregnancy (n = 14), oral contraceptive use (n = 10), serious illness (n = 19), and other causes (n = 21).
Of the 650 women who were randomized, 250 were included in a study of vitamin A supplementation (Fig. 1).17 The remaining 400 women were randomly assigned to receive micronutrients vs. placebo (n = 200 in each group). Overall, 179 women (89.5%) in the micronutrient group and 178 (89.0%) in the placebo group returned for follow-up.
Baseline characteristics of the micronutrient and placebo groups are shown in Table 1. In general, women in this trial were poor (<10% had running water at home) and had at least 1 child. They had symptomatic HIV-1 disease with moderate to severe immunosuppression. The 2 groups were similar in their demographic, obstetric, gynecologic, and nutritional characteristics. The CD4 and CD8 counts were slightly higher in the micronutrient group than in the placebo group. Vaginal shedding of HIV-1-infected cells was somewhat higher, while vaginal HIV-1 RNA was slightly lower in the micronutrient vs. placebo group.
Three hundred and fifty-seven women returned for follow-up after a mean of 45 (SD ± 10) days. The 43 women lost to follow-up were not significantly different from those who returned with respect to demographic, obstetric, gynecologic, nutritional, baseline plasma viral load, or baseline genital HIV-1 shedding characteristics presented in Table 1. However, the group lost to follow-up had lower CD4 counts (mean 221 vs. 285 cells/μL, P = 0.05) compared with women who returned. Five women were pregnant at follow-up (4 micronu-trient and 1 placebo) and were included in the analysis. Exclusion of these women did not significantly alter the results.
There was good adherence to the study regimen. A total of 306 (85.7%) of the 357 women returned their medication bottles, and 288 of 306 (94.1%) had [H11349]2 capsules (<5% of doses) remaining. Of the 51 women who did not return the bottles, 44 (86.3%) reported [H11349]2 capsules remaining. Overall, 168 of 179 (93.7%) micronutrient women and 164 of 178 (92.1%) placebo women took [H11350]95% of scheduled doses (P = 0.5).
Effect of Multivitamin Plus Selenium Supplementation on Genital Shedding of HIV-1
After 6 weeks, vaginal HIV-1 shedding was significantly higher among women who received micronutrients than among those who received placebo (Table 2). HIV-1-infected cells were detected in the vaginal secretions of 56 micronutrient (31.3%) vs. 30 placebo women (16.9%) (odds ratio 2.2, P = 0.002). The quantity of HIV-1 RNA in vaginal secretions was also higher among women who received the micronutrient supplement than among placebo recipients (3.1 vs. 2.9 log10 copies/swab, P = 0.1). There was little difference in cervicalHIV-1 shedding between the 2 groups.
Multivariate analyses were performed to adjust for baseline differences between the micronutrient and placebo groups (Table 2). After adjustment, micronutrients resulted in a 2.5-fold greater likelihood of detection of HIV-1-infected cells (P = 0.001) and a 0.37 log10 copies/swab higher quantity of HIV-1 RNA (P = 0.004) in vaginal secretions compared with placebo. Cervical HIV-1-infected cells and RNA were also higher among multivitamin vs. placebo recipients, though these findings were not statistically significant.
There was a higher prevalence of vaginal HIV-1-infected cells in the micronutrient group than in the placebo group at baseline. Thus, it was somewhat surprising that the multivitamin produced an even greater effect on vaginal HIV-1-infected cells in the adjusted analyses. To evaluate the reason for this, we performed an analysis restricted to women who were not shedding vaginal infected cells at baseline. After supplementation, incident shedding was detected in 32 of 137 (23.4%) micronutrient vs. 13 of 144 (9.0%) placebo patients (odds ratio = 3.1, P = 0.001). Thus, supplementation significantly increased the incidence of vaginal shedding among women who were not shedding at baseline.
It was hypothesized a priori that the effect of the supplement might vary among women with baseline nutritional and immunologic differences. There was a significant interaction between baseline serum selenium and supplementation with respect to the vaginal HIV-1 RNA outcome (P for interaction = 0.007), and trends were observed for cervical HIV-1-infected cells (P for interaction = 0.09) and RNA (P for interaction = 0.1). Analyses are presented with stratification for baseline selenium status (Table 3). Among non-selenium-deficient women, micronutrient supplementation produced significantly higher levels of vaginal HIV-1 shedding compared with placebo, and there were trends for increased cervical shedding in the micronutrient group. In contrast, among selenium-deficient women, there were no significant differences in genital shedding between women who received micronutrients vs. placebo. The effect of the supplement on genital HIV-1 shedding did not differ significantly in relation to vitamin E deficiency, low BMI, or CD4 count <200 cells/μL (P for interaction all >0.2).
Effect of Multivitamin Plus Selenium Supplementation on T-Lymphocyte Subsets and Plasma HIV-1
Micronutrient supplementation resulted in higher CD4 and CD8 lymphocyte counts (Table 4). In multivariate analyses, micronutrient-supplemented patients had higher CD4 (+23 cells/μL) and CD8 (+74 cells/μL) counts than placebo patients after adjustment for baseline CD4 and CD8 count, respectively. There was no difference in plasma viral load between the groups (Table 4). The effect of the micronutrient supplement on T-lymphocyte counts and plasma viral load did not differ significantly with respect to selenium deficiency, vitamin E deficiency, low BMI, or CD4 cell count <200 cells/μL.
In this randomized trial, micronutrient supplementation unexpectedly increased genital HIV-1 shedding. Women receiving micronutrients had nearly 3-fold higher likelihood of detection of HIV-1-infected cells and a significantly higher quantity of HIV-1 RNA in vaginal secretions compared with placebo recipients. The increase in genital HIV-1 shedding was even more pronounced in the subgroup of women with normal baseline serum selenium. The presence of HIV-1 in genital secretions has been associated with mother-to-child HIV-1 transmission,30,31 and a similar relationship is likely for sexual transmission.32 Thus, these findings suggest that multivitamin plus selenium supplementation may increase HIV-1 infectivity in women.
Vaginal shedding of HIV-1-infected cells was somewhat higher in the micronutrient group at baseline, so it was important to demonstrate that the observed effect was not simply related to this difference. Results were similar in multivariate analyses after adjusting for baseline differences, supporting the conclusion that the supplement caused the higher rate of shedding compared with placebo. Furthermore, micronutrient supplementation significantly increased detection of vaginal HIV-1-infected cells in women who were not shedding at baseline, providing an explanation for the increased effect observed in the adjusted analyses.
Micronutrient supplementation led to significantly higher CD4 and CD8 counts compared with placebo. These results are consistent with the findings of a randomized trial of vitamin supplementation in pregnant women18 and with observational research that has shown higher CD4 counts associated with higher intake of micronutrients including vitamins B1, B2, and E.9 Small studies have also demonstrated increased CD4 counts in patients receiving selenium or vitamin C plus N-acetylcysteine (which enhances antioxidant effects by replenishing glutathione).33,34
It is difficult to explain the observed effect of micronutrient supplementation on genital HIV-1 shedding. In general, differences in genital HIV-1 shedding may be attributable to systemic factors or to local alterations in the genital compartment.35 In this study, micronutrients increased CD4 counts and did not affect the plasma viral load. Higher CD4 counts have been associated with less genital HIV-1 shedding.13,36 Thus, these results do not suggest that systemic effects on viral replication led to the increase in HIV-1 shedding. A local effect mediated through changes in the number, activity, or adherence of HIV-1-infected cells in the genital tract could explain these findings.
Unexpectedly, the increase in genital HIV-1 shedding with micronutrient supplementation was greatest among women with higher serum selenium levels. Statistical tests for interaction were significant for vaginal shedding of HIV-1 RNA and of borderline significance for cervical HIV-1 RNA and infected cells. In contrast, there was no interaction for vaginal HIV-1-infected cells. These findings suggest that the interaction between selenium deficiency and micronutrient supplementation varied by the site (cervical vs. vaginal) and shedding outcome measured (infected cells vs. RNA). This is consistent with studies that have shown distinct determinants of genital HIV-1 RNA and infected cells, as well as distinct co-factors for vaginal and cervical shedding.37,38 Because the supplement in this study included vitamins B, C, E, and selenium, it is impossible to determine the contributions of individual micronutrients to the observed effects. One explanation is that giving supplemental selenium to nondeficient patients might increase HIV-1 replication by providing a substrate for viral selenoproteins. 39 Notably, low serum selenium has previously been associated with higher levels of genital HIV-1 shedding.14 Further research could help to explain these paradoxical findings and provide insight about the role of selenium in HIV-1 transmission.
An important strength of this study was the randomized trial design, which offers protection from confounding due to factors that are related to both the exposure (micronutrients) and the outcome (genital HIV-1 shedding). While randomization does not guarantee baseline equivalence, there are standard analytic techniques to adjust for baseline differences that result in a true assessment of effect. This study has been carefully adjusted for baseline differences, and the effect of the supplement remains strong in the adjusted analyses. There was good adherence to the study regimen and the follow-up rate was nearly 90%. These factors support the validity of the findings.
This cohort was recruited from an ideal population in which to study micronutrient supplementation. Participants were HIV-1-seropositive women from a developing country. They had a high prevalence of symptomatic disease, borderline nutritional status (mean BMI = 22), moderate to severe immunosuppression, and no use of antiretrovirals. These women appeared to be a group with great potential to benefit from nutritional supplementation.
There are limitations to this study. First, supplementation was continued for only 6 weeks. Further changes might occur with prolonged supplementation. Second, these findings cannot be generalized to other doses or combinations of micronutrients. Third, the effect of micronutrient supplementation on HIV-1 infectivity could be different in other populations, for example, patients on antiretroviral therapy or pregnant women. Notably, supplementation of HIV-1-seropositive women in Tanzania with vitamins B, C, and E had no effect on mother-to-child transmission but reduced infant mortality, supporting the authors' recommendation to consider providing these supplements to pregnant and breast-feeding women.20
There remains an urgent need for simple, safe, and inexpensive means of decreasing infectivity, particularly in countries with limited resources. This study evaluated micronutrient supplementation as a possible means of decreasing genital HIV-1 shedding. The findings, surprisingly, demonstrate that micronutrients can increase genital shedding and therefore may actually increase infectivity. These results raise challenging questions about the relative risks vs. benefits of micronutrient supplementation in HIV-1-seropositive women. About half of the HIV-1-seropositive individuals in developed countries use micronutrient supplements.9,40 There is little information regarding the prevalence of nutritional supplementation among HIV-1-seropositive individuals from resource-limited countries, but it has been suggested that micronutrient supplements may play an important role in these settings.2,12 With widespread use of nutritional supplements, even a modest increase in infectivity could have important public health implications.
The authors wish to acknowledge the excellent work and valuable contributions made to this study by our clinic staff (Mary Wamugunda, Florence Murigi, and Virginia Njuki), laboratory staff (Bhavna Chohan, Khamis Mwinyikai, Amina Abdalla, Gladwell Maina, and Dana Panteleeff), and administrators (Jeanne Gow and Chris Kealy). Del Landicho provided assistance with nutritional testing performed at the University of Washington's Clinical Nutrition Research Unit. The authors thank Coast Provincial General Hospital for allowing us to use their clinical facilities. Finally, we would like to express our gratitude to the women who participated in this study, without whose time and effort this research would not have been possible.
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© 2004 Lippincott Williams & Wilkins, Inc.