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American Journal of Clinical Nutrition, Vol. 86, No. 4, 1152-1159, October 2007
© 2007 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Effect of high-dose vitamin A supplementation on the immune response to Bacille Calmette-Guérin vaccine1,2,3,4

Birgitte R Diness, Ane B Fisker, Adam Roth, Maria Yazdanbakhsh, Erliyani Sartono, Hilton Whittle, Jose E Nante, Ida M Lisse, Henrik Ravn, Amabelia Rodrigues, Peter Aaby and Christine S Benn

1 From the Bandim Health Project, INDEPTH Network, Bissau, Guinea-Bissau (BRD, ABF, JEN, A Rodrigues, and PA); the Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark (BRD, A Roth, HR, PA, and CSB); the Medical Research Council, Fajara, The Gambia (HW); the Department of Parasitology, Leiden University Medical Center, Leiden, the Netherlands (MY and ES); the Department of Pathology, Herlev University Hospital, Copenhagen, Denmark (IML); and the Medical Microbiology, Lund University, Malmö, Sweden (A Roth)

2 Bandim Health Project (BHP) is affiliated with Statens Serum Institut (SSI) which is a producer of PPD and BCG. However, SSI does not fund any of the studies performed by BHP. The sponsors of this study had no role in the study design, data collection, data analysis, interpretation, or the writing of the report.

3 Supported by the European Commission, International Cooperation (INCO) programme (contract no. ICA4-CT-2002-10053), The Danish Medical Research Council, the March of Dimes, the University of Copenhagen, and the Ville Heise Foundation. The Bandim Health Project received support from the Danish International Development Agency (DANIDA) and the Danish National Research Foundation. PA holds a research professorship grant from the Novo Nordisk Foundation.

4 Address reprint requests to BR Diness, Bandim Health Project, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark. E-mail: birgitte{at}diness.dk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Vitamin A supplementation (VAS) at birth has been associated with decreased mortality in Asia. Bacille Calmette-Guérin (BCG) vaccine is given at birth in tuberculosis-endemic countries. Previous studies suggest that VAS may influence the immune response to vaccines.

Objective: Our objective was to examine whether VAS influences the immune response to simultaneously administered BCG vaccine.

Design: Within a randomized trial of 50 000 IU vitamin A or placebo given with BCG vaccine at birth in Guinea-Bissau, 2710 infants were examined for BCG scar formation and delayed-type hypersensitivity (DTH) to purified protein derivative of Mycobacterium tuberculosis (PPD) at 2 and 6 mo of age. The ex vivo cytokine response to PPD was measured in 607 infants.

Results: At 2 mo of age, 39% (43% of the boys and 34% of the girls) responded to PPD. The prevalence ratio of a measurable PPD reaction for VAS compared with placebo recipients was 0.90 (95% CI: 0.80, 1.02) for all infants, 0.81 (95% CI: 0.69, 0.95) for boys, and 1.04 (95% CI: 0.86, 1.26) for girls. At 6 mo of age, 42% of the infants responded to PPD. No difference was observed between VAS and placebo recipients. The prevalence of BCG scar was not affected by VAS. The ex vivo interferon-{gamma} response to PPD was increased by VAS (means ratio: 1.40; 95% CI: 1.03, 1.91).

Conclusions: VAS with BCG vaccination does not appear to interfere with the long-term immune response to BCG. However, VAS temporarily altered the DTH reaction to PPD in boys at 2 mo of age, suggesting sex differences in the immunologic response to VAS given with BCG. This trial was registered at www.clinicaltrials.gov as #NCT00168597.

Key Words: Bacille Calmette-Guérin • BCG vaccination • vitamin A • PPD response • purified protein derivative of Mycobacterium tuberculosis response • randomized trial • sex-differential effects


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The World Health Organization recommends Bacille Calmette-Guérin (BCG) vaccination at birth in low-income countries. Two studies from Asia suggested that it might be beneficial to provide vitamin A supplementation (VAS) at birth (1, 2), whereas a recent study from Zimbabwe suggested no overall beneficial effect (3). The influence of simultaneous administration of VAS has been studied for most of the routine vaccines such as measles, oral polio vaccine, and tetanus. Although one study has raised concern about reduced seroconversion to measles vaccine after simultaneous administration of VAS (4), most studies found no effect or even a boosting effect on the immune response to vaccines (5). To our knowledge, the effect of VAS on the immune response to BCG vaccine has not been studied previously.

The immune response to BCG vaccination is difficult to quantify because the induced immunity is mainly cellular. Instead, scar development and response to an immunogenic component of BCG, purified protein derivative of Mycobacterium tuberculosis (PPD), are used as measures of vaccine response. The response to PPD can be determined in vivo in skin tests [as delayed-type hypersensitivity (DTH)] (6) and ex vivo in cell cultures, and these tests were used in epidemiologic studies (7). Both scar development and in vivo PPD response were shown to correlate well with a history of BCG vaccination (8, 9). The correlation to vaccine efficacy is more uncertain (10, 11). For instance, boys develop scars and PPD responses more frequently than do girls (12), but there is no evidence that boys are better protected against tuberculosis after BCG vaccination. Ex vivo cytokine response after stimulation with PPD or other tuberculosis-associated antigens, particularly interferon-{gamma} (IFN-{gamma}), was suggested as a better marker of vaccine-induced tuberculosis immunity (13-15).

We aimed to investigate whether high-dose VAS given with BCG at birth influenced the response to BCG vaccination evaluated as scar formation and as response to PPD in vivo and ex vivo. This was done within a large randomized, placebo-controlled, double-blinded trial of the effect on mortality of vitamin A compared with placebo given at birth with BCG vaccine. Because previous studies have suggested sex-differential effects of vitamin A (1, 16, 17) and on response to vaccines (18), we conducted the analyses separately for each sex.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Setting
The Bandim Health Project runs a demographic surveillance system in 6 districts of Bissau, Guinea-Bissau. All houses are visited monthly to identify new pregnancies, births, and deaths. Once a newborn is identified, the infant is followed with a home visit every third month. The incidence of tuberculosis was estimated to be 470 per 100 000 person-years in the area (19). Cases of tuberculosis in the study area are registered and offered free treatment. In the present study, infants living in the same multifamily house as a patient with tuberculosis were considered to be exposed if living with the patient within the 6 mo preceding initiation of treatment. The prevalence of HIV-1 among pregnant women is <5.0% (Z de Silva, sentinel data, 2007), and a program to prevent vertical transmission conducted by a nongovernmental organization was ongoing in the area while the present study was conducted. The program included free HIV screening at first antenatal consultation, provision of antiretroviral medication to be taken intrapartum, and a recommendation to abstain from breastfeeding. The Ministry of Health in Guinea-Bissau and the Danish Central Ethical Committee approved the protocol for this study (624-02-0010).

Enrollment
Enrollment into the randomized trial of the effect of VAS at birth on overall rate of mortality and morbidity took place from 2002 to 2004. In brief, infants born at the maternity wards or reporting for their first vaccination at 2 of 3 health centers in the study area were enrolled if consent was obtained, if they weighed ≥ 2500 g, had no overt disease, and lived in the study area. For logistic reasons enrollment was not undertaken in the third health center. A total of 1452 (54%) of the infants participating in the present study were enrolled at birth or 1 d after birth, and most were enrolled within the first 3 wk of life. The infants were vaccinated intradermally in the upper left deltoid region with 0.05 mL BCG vaccine (Statens Serum Institut, Copenhagen, Denmark). After providing consent, the mother drew a lot from an envelope. The envelope was prepared by the study supervisor. It contained 100 lots, 50 marked "1," and 50 marked "2," indicating from which of 2 numbered bottles, "1" or "2," the infant should receive his or her supplement. A new envelope was not used before the previous envelope had been completely emptied. The randomization procedure was performed by the same, carefully trained assistant every day except during short vacations. The bottles were prepared at Skanderborg Pharmacy, Skanderborg, Denmark. They were made of dark glass and contained 10 mL vegetable oil, corresponding to 20 doses of 0.5 mL. Half of the bottles contained 50 000 IU vitamin A as retinyl palmitate and 20 IU vitamin E/0.5 mL oil, the other half contained only 20 IU vitamin E/0.5 mL oil. The code was kept at the pharmacy until 12 mo after the last infant was included.

Scar and in vivo PPD response
In 4 of 6 study districts, infants were examined for BCG scar and in vivo PPD response when they reached 2 and 6 mo of age (scar and PPD visits). These 4 districts were served by the 2 health centers enrolling participants in the trial. At the visits a trained nurse documented health status, health care contacts, household characteristics, and vaccination status. She measured the size of the scar after BCG vaccination. Subsequently, 0.1 mL Tuberculin PPD RT23 (Statens Serum Institut, Copenhagen, Denmark) was injected intradermally on the ventral side of the forearm. Between 2 and 3 d later, the infant was visited again, and the induration was measured by a trained fieldworker using the track-ball technique (20). Size of scar and induration was defined as the average of the height and the width measured to the nearest millimeter with a transparent ruler. Infants with a measurable scar or PPD reaction (>1 x 1 mm) were categorized as "scar-reactors" or "PPD-responders," respectively. Infants who had taken chloroquine within 7 d before the visit were excluded from the PPD analysis, because chloroquine treatment is known to suppress PPD reaction (21). Those infants were retained in the analysis of scar reaction because we considered it unlikely that current chloroquine treatment would have affected the scar formation. We did not collect information on chloroquine treatment at the time of enrollment, but it is unlikely that more than a few of these young infants received chloroquine. Infants who had received BCG vaccination <30 d before the visit or with known exposure to tuberculosis in the household within the previous 6 mo were excluded. Infants with PPD reactions > 10 mm were referred to a medical doctor. The mothers of the infants were also tested with intradermal PPD, and mothers with large responses were similarly referred.

Ex vivo PPD response
During the trial, a subgroup of infants was selected for measurements of ex vivo PPD response. For logistic reasons, the subgroup consisted of infants enrolled in the randomized study between 13 April 2004 and 19 May 2004 and from 1 August 2004 until the end of enrollment on 28 November 2004. We attempted to visit the infants at 6 wk of age. A blood sample was collected by finger prick and analyzed for ex vivo cytokine responses.

For cytokine responses, heparinized whole blood was diluted 1:10 with RPMI-1640 (Invitrogen, Groningen, Netherlands) supplemented with 2 mmol glutamate/L, 1 mmol pyruvate//L, 100 IU penicillin, and 100 µg streptomycin/mL. Cultures were established in a total volume of 200 µL in 96-well U plate (Nunc, Roskilde, Denmark) with the presence of PPD (10 µg/mL; Statens Serum Institut) or medium only (control). Supernatant fluids were collected on day 3 and stored at –80 °C until and during transport to Leiden University Medical Center where the measurements were performed. Concentrations of interleukin-5 (IL-5), interleukin-10 (IL-10), interleukin-13 (IL-13), IFN-{gamma}, and tumor necrosis factor-{alpha} were measured simultaneously with the use of the commercial Luminex cytokine kit (Luminex Corporation, Austin, TX) and buffer reagent kit (BioSource, Camarillo, CA) and run on a Luminex-100 cytometer (Luminex Corporation), equipped with StarStation software (Applied Cytometry Systems, Dinnington, United Kingdom). The assay was performed with slight modification from the manufacturer's recommendation. Briefly, assays were done in a 96-well U plate at room temperature. Mixes of beads were incubated for 2 h with a standard, samples, or blank in a final volume of 50 µL for 2 h under continuous shaking. Beads were washed twice and incubated with a cocktail of biotinylated antibodies (25 µL/well) for 1 h. After removal of excess biotinylated antibodies, streptavidin-R-phycoerythin was added for 30 min. The plate was then washed and analyzed. The lower detection limit of the assays was 3 pg/mL for IL-5, 5 pg/mL for IL-10, 10 pg/mL for IL-13, 5 pg/mL for IFN-{gamma}, and 10 pg/mL for tumor necrosis factor-{alpha}. Samples with concentrations below the detection limit were given the value of this threshold. Infants were classified as "responders" if the cytokine concentration in the PPD-stimulated well exceeded the concentration in the control well. Only samples from infants vaccinated >30 d before sampling were considered in the analysis.

Statistical analysis
PPD response (yes or no), scar response (yes or no), and cytokine response (responder or nonresponder) were analyzed as prevalence data with the use of Poisson regression with robust variance estimates (22). Thus, the relative measure is a prevalence ratio (PR). Size of PPD reaction and scar was analyzed using t tests, and adjustments and interaction analysis of these outcomes were performed as a linear regression analysis after checking that the assumptions of normal distribution and equal variance were met.

The scar and PPD analyses were done both with and without adjustment. In the adjusted analysis, linear regression was used for continuous variables and Poisson regressions for binary variables. The following potential confounders were adjusted for: assistant conducting the scar and PPD reading, season of PPD application, age in days at BCG vaccination, and time since BCG vaccination at visit. In addition, we decided to adjust for prior diphtheria, tetanus toxoid, and acellular pertussis (DTP) vaccination at the 2-mo visit and prior measles vaccine at the 6-mo visit because some infants had been enrolled in a trial of early measles vaccination at 4.5 mo of age (23). We also analyzed the prevalence of PPD reaction with the method of generalized estimating equation, taking into account the repeated measurements. The results were virtually identical and did not warrant separate presentation.

To evaluate the results of the cytokine study quantitatively, we calculated the ex vivo PPD-induced response by subtracting the cytokine concentrations in the control wells from the concentrations in the PPD-stimulated wells. Furthermore, we calculated a stimulation index as the ratio between the concentrations in the stimulated and the control wells. Unadjusted linear regression was performed on ln-transformed stimulation indexes; thus, the resulting back-transformed exponential function estimates represent ratios of means. These ratios express the percentage of difference associated with receiving vitamin A; eg, a means ratio of 1.40 for IFN-{gamma} indicates that infants in the vitamin A group have 40% higher stimulation indexes than do infants in the placebo group.

All analyses were conducted separately for each sex. Interactions between treatment groups and sex were investigated in a model that included both variables and tested in the relevant regression model. Analyses were made with STATA 9.2 (Stata Corporation, College Station, TX).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 2710 infants participated in the scar and in vivo PPD study, 1313 from the vitamin A group, 1397 from the placebo group. Background characteristics and age at visits were comparable between the 2 treatment groups (Table 1Go).


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  		TABLE 1. Background factors according to treatment group in Guinea-Bissau1

 
The participant flow is summarized in Figure 1Go. At 723 (14.2%) visits the infant was not met, mostly because of traveling. At 8 (0.2%) visits the parent did not want the infant to participate. Of 4355 successful interviews, 382 were excluded from the scar analysis [185 (8.8%) of 2113 in the vitamin A group and 197 (8.8%) of 2242 in the placebo group] because of 1) tuberculosis exposure within the past 6 mo (168 visits); 2) <30 d passed since BCG vaccination (141 visits); and 3) the fieldworker had failed to register the presence of scar, its dimensions, or both (73 visits). At 1241 visits a PPD reading was not obtained or was excluded (606 in the vitamin A group, 635 in the placebo group) because of chloroquine consumption (598 visits), tuberculosis exposure (168 visits), <30 d since vaccine (141 visits), and a valid reading was not obtained because the infant was not met in time for the reading of the reaction (334 visits). The reasons for nonparticipation were equally distributed in the 2 treatment groups.


Figure 1
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  		FIGURE 1.. Flow chart of infants included in the study of response to Bacille Calmette-Guérin (BCG) vaccination when administered with vitamin A or placebo. At late entry, 67 infants in the vitamin A group and 68 infants in the placebo group were not visited at 2 mo of age, because they were not registered as living in the areas where the purified protein derivative of Mycobacterium tuberculosis (PPD) and scar trial was conducted at the time the visits were planned. Infants met but not included in the scar analysis are those vaccinated <30 d before the visit, exposed to tuberculosis, or with missing information. Infants included in the scar analysis but not in the PPD analysis are infants not seen for a valid PPD reading and infants who consumed chloroquine during the 7 d before the reading.

 
The mean age (±SD) of the infants at visits did not differ between the groups (2-mo visit: 64 ± 11 d in the vitamin A group and 65 ± 11 d in the placebo group; 6-mo visit: 188 ± 13 d for both groups).

Scar and in vivo PPD response
At 2 mo of age, 88% had a recognizable scar to BCG vaccination (89% of boys and 87% of girls). At 6 mo of age, the prevalence was 94% (95% of boys and 93% of girls). The percentage of infants with a measurable PPD response also increased: at the 2-mo visit 39% had a measurable PPD response (43% of boys and 34% of girls); at 6 mo of age the measurable PPD response was 42% in both boys and girls. At 2 mo the prevalence of a measurable PPD response was significantly higher in boys than in girls [prevalence ratio (PR): 1.25; 95% CI: 1.11, 1.42]. None of the other outcomes differed between the sexes. In infants not tested at 2 mo of age (not met or not enrolled at this time) who had a valid reading at 6 mo (n = 134), the prevalence of PPD response at 6 mo was 40%. Thus, the increased reactivity at 6 mo did not seem to be explained by boosting by prior PPD application.

At 2 mo of age, the proportion of PPD responders was lower among boys who received VAS than among boys who received placebo (PR: 0.81; 95% CI: 0.69, 0.95) (Table 2Go). The adjusted PR was 0.79 (95% CI: 0.67, 0.92) for boys and 1.02 (95% CI: 0.84, 1.24) for girls. The average size of reaction also tended to be reduced among VAS-supplemented boys at 2 mo of age. No differences were observed among girls, and no differences for scar development were observed in either sex. At 6 mo of age no differences in PPD response or scar among VAS and placebo recipients were observed overall or in either sex.


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  		TABLE 2. Effect of vitamin A supplementation given with Bacille Calmette-Guérin vaccine on scar and purified protein derivative of Mycobacterium tuberculosis (PPD) response at 2 and 6 mo of age (unadjusted) in Guinea-Bissau1

 
A measurable PPD response at 2 mo of age was more prevalent among infants of mothers who reacted to PPD (P = 0.01), but the effect of VAS on PPD response at 2 mo of age was similar after stratification by maternal PPD status (data not shown). None of the above results were changed by adjustment for potential confounders.

Ex vivo PPD responses
A total of 607 blood samples were available to measure ex vivo PPD response (292 in the vitamin A group, 315 in the placebo group). Of those samples, 571 were included in the analysis (Table 3Go). One sample was not included because the IFN-{gamma} value was an outlier; other exclusions were due to signs of contamination of the blood sample (n = 20) and <30 d since vaccination (n = 15). The ex vivo IFN-{gamma} response to PPD was significantly associated with the in vivo DTP response (data not shown).


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  		TABLE 3. Ex vivo cytokine response to purified protein derivative of Mycobacterium tuberculosis (PPD) of 6-wk-old infants in Guinea-Bissau1

 
The effect of VAS on the stimulation indexes for the tested cytokines is given in Table 3Go. VAS was associated with an IFN-{gamma} response to PPD. This was particularly apparent in boys (P = 0.04). The induced IFN-{gamma} production was 299 pg/mL among boys in the vitamin A group and 199 pg/mL among boys in the placebo group (Table 3Go). In girls the induced IFN-{gamma} production was 209 pg/mL in the vitamin group and 176 pg/mL in the placebo group. No differences were observed for other cytokines overall or in either sex. There was a tendency for interaction between VAS and sex for IL-5 response to PPD (P for interaction = 0.09); girls tended to have lower stimulation indexes after VAS, whereas the opposite was seen for boys. Otherwise, no indications of interactions were observed between treatment and sex for cytokine responses to PPD.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In line with previous studies in Bissau, by 6 mo of age a large proportion of infants had a scar (94%) and 42% reacted to PPD (8). Neonatal VAS was associated with increased IFN-{gamma} response to PPD at 6 wk of age. It was also associated with decreased in vivo PPD response at 2 mo of age in boys, whereas it was not associated with PPD responses at 6 mo of age in either sex. It did not affect the BCG scar reaction.

A limitation may be that few infants were vitamin A deficient in our study because of the enrollment criteria and the nutritional situation in the study area. We only included newborns with normal birth weight in our study, because BCG vaccine in Guinea-Bissau is postponed in newborns with low birth weight. Newborns with low birth weight may be more nutritionally compromised at birth. Furthermore, breast milk is the main source of vitamin A, and most infants in our study were breastfed. Hence, VAS could have effects on the response to BCG in vitamin A–deficient infants.

We did not expect the HIV prevalence in infants to be high because of the low prevalence among pregnant mothers and the ongoing vertical transmission program. HIV-positive women were recommended not to breastfeed, but only 15 (<1%) women in the present study stated that they would not breastfeed. Hence, the data do not allow any conclusions about a possible modifying effect of lack of breastfeeding, HIV infection, or both on the effect of VAS.

In Malawi, Floyd et al (24) found that each person may have a specific "propensity" to react with a DTH response to PPD. However, that individual preference had the least importance at the peak of reactivity 2–3 mo after vaccination. The PPD response at 2 mo of age in our study may have been measured before the peak of reactivity. Thus, it may depend more on the propensity to react in the population than on the immunogenicity of the vaccine. Earlier studies from Guinea-Bissau confirm that the reactivity to PPD increases between 2 and 6 mo after vaccination, suggesting that the peak reactivity period in this population is at 6 mo of age (25). The increase in reactivity was not confined to infants boosted by PPD application at 2 mo. Hence, the reduced PPD response in boys at 2 mo of age may be due to a sex-differential effect of VAS on the individual immunologic setting. If the immunogenicity of the vaccine is indeed most adequately evaluated at the peak of reactivity, it was unchanged in the present study.

In the present study IFN-{gamma} response was marginally increased by VAS in both sexes, although the in vivo PPD response was lower among boys who received VAS. This may seem contradictory, because IFN-{gamma} production is considered an important mediator of the DTH reaction (13), and IFN-{gamma} production was indeed associated with DTH reaction in the present study. These observations may suggest that VAS did not disturb the biologic association between IFN-{gamma} production and DTH but rather affected the 2 outcomes by separate mechanisms. Similarly, other researchers have reported that varying the conditions of the administration of BCG vaccination (eg, age at administration, route, or dose) affects the various measures of induced immunity in a differentiated manner (13-15). It was suggested that the different testing methods and assays measure unique aspects of tuberculosis immunity (26).

Infants of mothers displaying a measurable PPD response were more likely to respond to PPD themselves. This is likely to be explained by a combination of shared environment, shared genetic disposition to DTH reactivity, and prenatal transfer of passive immunity from mother to infant (27). Previously, it was suggested that VAS may interfere with measles vaccine responses in infants with maternal measles antibodies (4). To investigate whether the observed depressed DTH response to PPD at 2 mo of age could be driven by a similar interaction, we stratified by maternal PPD reactivity. However, the suppression by VAS of PPD response in boys was, if anything, slightly less pronounced in infants of reacting mothers, thus discarding the idea that passive immunity could explain the decreased PPD response in boys in the present study.

The unaffected scar reaction, the moderately increased IFN-{gamma} response to PPD ex vivo, and the unaltered in vivo PPD reaction at 6 mo of age support the interpretation that long-term tuberculosis immunity is unhampered by the transient immunologic changes induced by VAS at birth. In the present study VAS affected boys at 2 mo of age but not girls. A sex-differential response is in line with earlier published results of VAS at birth (1, 2, 28). Our group has previously reported increased antibody response in boys after VAS administered with measles vaccine at 9 mo of age (18). Others examined the in vitro proliferative responsiveness of T lymphocytes to tetanus toxin and PPD after VAS with a DTP booster in children aged 6–13 y previously vaccinated with BCG. Intriguingly, they found no change in the DTH reaction to tetanus toxoid (the simultaneously administered vaccine) but found an increased responsiveness to PPD in girls after VAS (29).

In conclusion, the results of the present study suggest that VAS does not influence the immunogenicity of BCG vaccination. If simultaneous VAS and BCG vaccination are undertaken in areas with a high prevalence of tuberculosis, further studies seem warranted of the efficacy of the vaccine in a longer perspective and with tuberculosis mortality and morbidity as outcomes. The analysis of such studies should be conducted separately for each sex.


   ACKNOWLEDGMENTS
 
The author's responsibilities were as follows—CSB, PA, MY, HW, A Roth, IML, and A Rodrigues: designed the study and obtained funding; CSB, JEN, and PA: initiated enrollment in the randomized trial; A Roth: initiated PPD and scar registration; IML, ABF, MY, and ES: collected and analyzed blood samples; BRD and ABF: supervised fieldwork and data entry; HR provided statistical support; BRD wrote the first draft of this paper. All authors contributed to the final version. None of the authors had a conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication February 26, 2007. Accepted for publication June 13, 2007.




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