Vitamin D as Supplementary Treatment for Tuberculosis

A Double-blind, Randomized, Placebo-controlled Trial

Christian Wejse1,2, Victor F. Gomes1, Paulo Rabna1, Per Gustafson1,3, Peter Aaby1, Ida M. Lisse4,
Paul L. Andersen2, Henning Glerup5, and Morten Sodemann1,6
1
Bandim Health Project, INDEPTH Network, Statens Serum Institute, Bissau, Guinea-Bissau; 2Infectious Disease Research Unit, Aarhus University
Hospital, Skejby, Denmark; 3Infectious Diseases Research Group, Department of Clinical Sciences, Lund University, Malmo
, Sweden; 4Department of
5
Pathology, Herlev University Hospital, Copenhagen, Denmark; Department of Internal Medicine, Aarhus University Hospital, Silkeborg, Denmark;
and 6Department of Infectious Diseases, Odense University Hospital, Odense, Denmark

Rationale: Vitamin D has been shown to be involved in the host

immune response toward Mycobacterium tuberculosis.
Objectives: To test whether vitamin D supplementation of patients
with tuberculosis (TB) improved clinical outcome and reduced
mortality.
Methods: We conducted a randomized, double-blind, placebocontrolled trial in TB clinics at a demographic surveillance site in
Guinea-Bissau. We included 365 adult patients with TB starting
antituberculosis treatment; 281 completed the 12-month followup. The intervention was 100,000 IU of cholecalciferol or placebo at
inclusion and again 5 and 8 months after the start of treatment.
Measurements and Main Results: The primary outcome was reduction
in a clinical severity score (TBscore) for all patients with pulmonary
TB. The secondary outcome was 12-month mortality. No serious
adverse effects were reported; mild hypercalcemia was rare and
present in both arms. Reduction in TBscore and sputum smear
conversion rates did not differ among patients treated with vitamin
D or placebo. Overall mortality was 15% (54 of 365) at 1 year of
follow-up and similar in both arms (30 of 187 for vitamin D treated
and 24 of 178 for placebo; relative risk, 1.19 [0.581.95]). HIV
infection was seen in 36% (131 of 359): 21% (76 of 359) HIV-1,
10% (36 of 359) HIV-2, and 5% (19 of 357) HIV-112.
Conclusions: Vitamin D does not improve clinical outcome among
patients with TB and the trial showed no overall effect on mortality in
patients with TB; it is possible that the dose used was insufficient.
Clinical trial registered with www.controlled-trials.com/isrctn
(ISRCTN35212132).
Keywords: vitamin D; tuberculosis; randomized clinical trial; clinical
score; HIV

Vitamin D was used for treatment of tuberculosis (TB) in the

preantibiotic era (13) and before then cod liver oil, rich in
vitamin D, was used as well as sun exposure (4). Vitamin D has
been attributed an important role in host immune defense against
Mycobacterium tuberculosis (5, 6) and observational studies have
found evidence of an association with vitamin D deficiency
(VDD) and active tuberculosis (710). Vitamin D is a low-cost

(Received in original form April 16, 2008; accepted in final form January 23, 2009)
Supported by Aarhus University Hospital (Ph.D. scholarship); the Danish Research
Council for Developmental Research; the SSAC; and the Skejby Hospital, Segel,
and Beckett Foundations. The funding sources and the provider of cholecalciferol
had no role in study design, data collection, data analysis, data interpretation, or
writing of the report. The principal investigator (corresponding author) had full
access to all the data and had final responsibility for the decision to submit for
publication.
Correspondence and requests for reprints should be addressed to Christian
Wejse, M.D., Ph.D., Infectious Disease Research Unit, Aarhus University Hospital,
Skejby, Brendstrupgaardsvej, 8200 Aarhus N, Denmark. E-mail: wejse@
dadlnet.dk
Am J Respir Crit Care Med Vol 179. pp 843850, 2009
Originally Published in Press as DOI: 10.1164/rccm.200804-567OC on January 29, 2009
Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject

Vitamin D insufficiency is associated with impaired immune

function and increased risk of active tuberculosis (TB).
Vitamin D has been used in the preantibiotic era in the
treatment of TB. It has also been suggested as a supplementary/prophylactic treatment for TB, but only two small trials
have assessed this.
What This Study Adds to the Field

Vitamin D can be given safely to patients with TB. Our

study suggests no overall effect on clinical outcome or
mortality with the doses used. The study raises the question
of whether vitamin D has a differential effect depending on
immune status.

intervention that is easy to administer in resource-poor settings,

and has been suggested as prophylaxis in TB household contacts
(11) as well as in treatment of bacterial infections (12). A possible
mechanism of vitamin Dmediated effect on TB treatment
efficacy is that 1,25-dihydroxyvitamin D has been shown to induce
antimycobacterial activity in macrophages in vitro, to upregulate
protective innate host responses, and to trigger antimicrobial
peptides such as cathelicidin (5). No data are available on dosedependent effects of vitamin D on mycobacterial activity, but it
has been speculated that pharmacologic doses of vitamin D may
elevate serum 25-hydroxyvitamin D [25(OH)D] concentrations
to levels saturating the ability of vitamin Dbinding protein to
bind vitamin D metabolites, leading to an increase in biological
availability of 1,25-dihydroxyvitamin D in infected tissues (13).
Two small randomized studies (14, 15) have suggested beneficial
effects on weight gain and time to sputum conversion without an
initial screening for VDD. One review concluded there is
evidence that vitamin D modulates antimycobacterial immunity
and called for double-blind, randomized, placebo-controlled
trials in patients with active TB (13).
We aimed to test whether vitamin D supplementation could
improve clinical response to treatment or reduce mortality among
patients with TB. Vitamin A is known to be a beneficial intervention to children irrespective of vitamin A status (16), and
therefore we hypothesized that vitamin D, regardless of initial
vitamin D status, would improve immunologic capacities against
M. tuberculosis. We report the findings from a community-based
trial (the VDTB Trial) testing vitamin D supplementation for
patients with TB in Guinea-Bissau. We first reported these results
as a poster at the Royal Society of Hygiene and Tropical Medicine
Centennial Conference in London, September 2007 (17).

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METHODS
Study Site
The Bandim Health Project, a disease surveillance site located in
Guinea-Bissau, West Africa. A poor, urban population of 92,000 is
under continued epidemiologic surveillance, and we have previously
reported a high TB incidence of 470 per 100,000 in this area (18).

Study Design
We conducted a randomized, double-blind, placebo-controlled trial.
Trial inclusion criteria included either a diagnosis of TB by sputum
examination (smear microscopy; no culture was available) or by World
Health Organization (WHO, Geneva, Switzerland) clinical criteria (19),
age 15 years or more, and residence in the study area. There were no
exclusion criteria.

Patients
Field assistants daily identified new patients with TB initiating tuberculosis chemotherapy at the three health centers and at the national TB
hospital situated in the study area, inviting patients to be included in the
trial the next day. All received antituberculosis treatment consisting of
2 months of daily observed treatment with ethambutol (E), isoniazid (H),
rifampicin (R) and pyrazinamide (Z) followed by 6 months of H1E
collected by the patient twice per month. Adherence to daily observed
treatment was noted daily by the nurse at the health center; patients were
referred to sputum examinations after 2, 4, and 6 weeks of treatment in
addition to the regular sputum examinations provided by the national
tuberculosis program at 2, 5, and 8 months of treatment.

Enrollment Procedures
Patients were randomized to either 100,000 IU of cholecalciferol or
identical placebo ampoules at inclusion. Cholecalciferol and placebo
(vegetable oil without cholecalciferol) were given in ampoules with
drinkable content. At inclusion patients received 100,000 IU of cholecalciferol or placebo, and this was repeated 5 and 8 months after inclusion.
Hence patients completing treatment received in total 300,000 IU of
cholecalciferol or three placebo doses. The rationale behind the choice of
dosage and time points was a concern that vitamin D might induce hypercalcemia, and therefore no intervention was given when patients were
seen at 2 months; instead, samples were collected for calcium measurement and hypercalcemia was assessed at the first interim analysis. We
chose administration of a few large doses so as not to further increase the
substantial pill burden on the patients and because it was a simple
intervention applicable to low-resource settings. A single large dose has
been shown as effective as daily administration, and the half-life is
2 months (20). The dosage was known to be efficient for treatment of
vitamin D deficiency and also safe to give with no deficiency present and
even in pregnancy (21).

Follow-up
Patients were invited to clinical examinations after 2, 5, and 8 months
of treatment, followed by a household visit 12 months after initiation of
treatment, or until death or moving out of the study area.

Randomization Procedures
The random allocation sequence was computer generated; a list of
continuous study numbers was generated with a random allocation to
treatment 1 or 2. Study numbers were consecutive and given to patients
by the field assistant at inclusion, and patients were recorded in a book
with prewritten study numbers and allocation sequence numbers 1 or 2.
Study medicine was provided in identical containers labeled lot 204
(allocation sequence number 1) or lot 205 (allocation sequence number 2).
A physician gave the trial information, obtained patients consent, and
conducted the clinical examination; a trial nurse administered study
medicine according to sequence number.

Blinding
Patients, staff, and researchers assessing outcome were blinded. Trial
medicine was available in two lots and for logistical reasons it was not
concealed whether a patient was on lot 1 or lot 2 during the trial.

2009

Blinding was not assessed among patients, but we assessed blinding

among 10 field assistants and investigators through tasting both lots.
Five voted in favor of lot 204 being vitamin D, five voted in favor of lot
205. The randomization code was broken by the primary investigator in
December 2006 on completion of data analysis.

Adverse Effects
We questioned the patients for the following adverse effects related to
hypercalcemia: nausea, vomiting, excessive thirst, anorexia, symptoms of
kidney stones, and confusion. We measured calcium concentrations in
120 patients seen at 2 months, and in 120 patients completing 8 months of
treatment. After the first interim analysis of calcium concentrations in
control samples taken at 2 months, we proceeded to give vitamin D at 5
and 8 months.

Measurements
To determine the body mass index, we used the following formula: body
mass index 5 weight/(height)2. Height was measured with a meter scale;
weight was measured in kilograms, using the same weight scale at each
patient visit. Midupper arm circumference was measured at the midpoint between the acromion and olecranon over the biceps of the nondominant arm, using a nonstretchable measuring tape (TALC, Guilford,
UK) to the nearest 0.2 cm (22).
Severity of TB disease was assessed by the TBscore, which counts
signs, symptoms, and anthropometry as described in OUTCOME. The TBscore
has been validated in another cohort and has been grouped in severity
classes as follows: I (05 points), II (6 or 7 points), or III (8 points or more)
(23).
We allocated patients into HIV-infected and HIV-uninfected groups,
but there was a high frequency of HIV-2-infected patients as shown in
Table 1. Despite milder clinical progression and lower mortality in HIV2infected than HIV-1infected individuals (24, 25), HIV-2 patients
were more comparable to HIV-1 patients than to uninfected patients:
79% (22 of 28) of HIV-2 patients had a CD41 T-lymphocyte count less
than 500 cells/ml and 12-month mortality was 17% (6 of 36).
We measured serum 25-hydroxy metabolites of vitamins D2 and D3
[25(OH)D213] simultaneously by isotope-dilution liquid chromatography
tandem mass spectrometry on an API 3000 mass spectrometer (Applied Biosystems, Foster City, CA), according to a method adapted
from Maunsell and colleagues (26). The inter- and intraassay coefficients
of variation were 9.4 and 9.7%. We defined VDD as serum 25(OH)D3
not exceeding 50 nmol/L and vitamin D insufficiency (VDI) as 25(OH)D3
not exceeding 75 nmol/L, according to Vieth (27).
Serum calcium and albumin were measured by absorbance (COBAS
Integra; Roche Diagnostics, Mannheim, Germany). We corrected total
serum calcium for individual variations in albumin by the following
equation: adjusted serum calcium (mmol/L) 5 serum calcium total
(mmol/L) 3 0.00086 3 (650 2 serum albumin [mmol/L]).
T-lymphocyte subsets were determined at the National Public Health
Laboratory, Bissau, by flow cytometry (FACStrak; Becton Dickinson,
San Jose, CA) with the use of three two-color immunofluorescence
reagents, CD45/CD14, CD3/CD4, and CD3/CD8 (Simultest; Becton
Dickinson). Leukocyte and differential counts were performed manually.
Because of technical problems on several occasions CD41 T-lymphocyte
counts were available only from 276 patients at inclusion and from 187
patients at 8 months.

Sample Size
To show the safety of vitamin D supplementation in patients with TB, we
investigated whether vitamin D increased serum calcium concentrations
significantly. A significant change in serum calcium was defined as
albumin-corrected serum calcium increasing to a concentration greater
than 2.75 mmol/L in a patient with no hypercalcemia before vitamin D
supplementation. Estimating no hypercalcemia before vitamin D supplementation and hypercalcemia in 15% afterward, 60 patients in each
group would be needed to reject the null hypothesis with a power of 80%
and a 5% significance level.
For vitamin D supplementation we used the clinical end point of
weight increase and mortality for sample size assessment; the primary
end point TBscore was still under development at the time of sample size
calculation, but weight was known to be a major component. We
estimated that a weight increase of more than 10% would be seen for
70% of the patients, and with intervention 8085% (relative risk,

Wejse, Gomes, Rabna, et al.: Vitamin D TB Trial

0.820.87), and for this we would need 134286 patients in each arm with
a power of 80% and a 5% significance level. For the mortality outcome
we stipulated case fatality to be 27% (24), and relative risk after
intervention to be 0.6, demanding a sample size of 286 patients in each
arm. We estimated enrollment of 300 patients per year, and the planned
and funded inclusion period was 2 years, but the incidence estimates we
had based this on proved to be too high. After 2 years we had enrolled
only 365 patients and the interim analysis showed no difference in
mortality or any trends for any of the arms being superior, and hence we
decided not to prolong the trial.

Outcome
The primary outcome was clinical improvement as assessed by TBscore
(23). The TBscore is a newly developed tool aimed at assessment of change
in clinical state in patients with TB. It is based on points assigned to signs
and symptoms, including cough, hemoptysis, dyspnea, chest pain, night
sweating, anemia, tachycardia, lung auscultation finding, fever, low body
mass index, and low midupper arm circumference, giving patients a TBscore
from 0 to 13. Change in TBscore has been shown to detect clinical change
well; a high TBscore correlates well with mortality and low TBscores
correlate with favorable outcomes, cure, and completed treatment.
The secondary outcome was all-cause mortality at 12 months of
follow-up. A verbal autopsy was conducted on all deaths, with a physician
using a standardized questionnaire to obtain information from the
nearest relative. No traumatic deaths were recorded; all died of causes
that may be related to TB or HIV.
We further assessed sputum conversion in smear-positive patients,
weight gain and changes in immunologic response by changes in CD41
T-lymphocyte count. The primary end point was available only for the
patients completing 8 months of treatment. Mortality was analyzed by
intention to treat, that is, for all included patients regardless of number
of follow-ups and study drug treatments.

Statistical Analysis
We expressed variables by their means or medians and standard deviations
or range. The Pearson chi-square (x2) was used to assess statistical
differences in proportions between groups (P , 0.05); the Student t test
to assess differences in means between two groups when a normal
distribution was present; and the Wilcoxon rank-sum test when nonparametric analysis was needed. Linear and logistic regression analyses
were used as multivariate models to adjust clinical outcomes for effects of
other factors. Cox regression and the Wilcoxon-Breslow-Gehan log-rank
test for equality of survivor functions were used to analyze mortality, and
Kaplan-Meier survival graphs were used to estimate the survival function.
A two-sided P , 0.05 was considered significant for the primary outcome,
for mortality P , 0.03 was considered significant because of interim
analyses. A false discovery rate was used to correct for multiple testing: test
P value 5 [(no. of tests 1 1)/(2 3 no. of tests)] 3 crude P value, hence for
exploratory subgroup analyses a P value of 0.03 were considered significant. Therefore 97% confidence intervals were used in estimates in all
subgroup analysis. Statistical analyses were performed with STATA
version 9 software (StataCorp, College Station, TX).

Interim Analysis
A data and safety monitoring board was established to monitor the study.
Interim safety analyses were done at 5 and 16 months for calcium
concentrations and mortality, and were blinded to treatment group. No
clinical outcomes were analyzed, only safety; the predefined stopping rule
was a difference in mortality between the two treatment arms at the 5%
significance level.

Ethics
Pretest and posttest counseling was provided for HIV testing; HIVinfected patients with TB were referred for cotrimoxazole treatment,
social support and retesting at a nongovernmental organization voluntary counseling and testing center in Bissau. No antiretroviral treatment
was available in Bissau during the study period. Written informed
consent was obtained before enrollment. The study was permitted by
the Health Ministry of Guinea-Bissau and approved by the National
Science and Ethics Committee as well as the Danish National Committee
on Biomedical Research Ethics.

845

RESULTS
Recruitment began in November 2003 and ended in December
2005. Follow-up was completed in December 2006. Three hundred and sixty-seven patients were enrolled and received study
drug at inclusion. Two patients were excluded as a revision of
chest X-rays showed no infiltrations, they were smear negative,
and therefore did not meet the WHO criteria for tuberculosis.
Hence 365 patients were analyzed by intention to treat. One
patient was mistaken for another patient and wrongly received
placebo instead of the study drug at 2 months of follow-up. He was
censored at that date. Two hundred and thirty-three patients
came for 8-month follow-up; 9 of these had not received the
second dose at 5 months but all were analyzed for the primary
outcome. Fifty-seven patients were monitored (found alive at
home visit) but were not seen at the final follow-up, 47 patients
died during the first 8 months, and 24 patients abandoned
treatment or transferred out (they were censored). The flow of
participants is outlined in Figure 1.
Baseline characteristics, including median age, body mass
index, type of TB, prevalence of HIV, baseline CD41 T-lymphocyte (CD4) counts, baseline vitamin D concentrations, and TB
severity score were similar between the vitamin D supplementation and placebo groups (Table 1). Baseline characteristics for
dropouts were analyzed separately and did not differ significantly
from those with complete follow-up (data not shown).
Adverse Events

The symptoms asked for as adverse effects were reported most

frequently at inclusion, at which time 103 of 365 reported any of
the symptoms before receiving the study drug. At 2 months only
24 reported any symptom, most frequently excessive thirst: 10 of
157 (6%) reporting adverse effects in the vitamin D group and 14
of 147 (9%) in the placebo group (P 5 0.31).
At 2 months of follow-up mean albumin-corrected serum
calcium in the first 120 patients was 2.05 mmol/L (95% confidence
interval [CI], 1.982.12) for the vitamin Dtreated group and 2.09
(95% CI, 2.012.17) for the placebo group. One patient exceeded
the reference range in the vitamin D group with an albumincorrected serum calcium of 2.93 mmol/L and two patients in the
placebo group with serum calcium at 2.923.02 mmol/L. At
8 months the mean serum calcium values were slightly higher
(2.17 vs. 2.19 mmol/L), but no cases of hypercalcemia were found.
Primary Outcome

Changes in TBscore and time to clinical improvement (progression to low-severity class) were similar in the two groups (Figure 2).
Sputum samples were not obtained from all patients every second
week, but sputum conversion rates were not different in the two
groups among the 247 initially smear-positive patients (data not
shown); smear positivity over time is presented in Figure 3.
Additional Outcomes

We further analyzed weight gain and change in CD4 counts as

additional outcomes. Median weight gain at 8 months was 5.9 kg or
11.1% of body weight in the vitamin Dtreated group and 5.7 kg or
11.8% in the placebo group (P 5 0.9). In the vitamin D group 56%
(70 of 124) gained more than 10% in weight, and in the placebo
group 52% (56 of 108) gained more than 10% in weight (P 5 0.48).
Weight gain did not differ by HIV status (data not shown).
The mean CD4 count at 8 months was higher than at
inclusion, but did not differ in the two treatment arms. In the
vitamin Dtreated group 97 patients had a mean CD4 count of
592 cells/ml (95% CI, 513671) and in the placebo group
90 patients had a mean CD4 count of 635 cells/ml (95% CI,
556714). Paired CD4 counts at both inclusion and 8 months

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2009

Figure 1. Flowchart of participants.

were available from 133 patients. Among the 70 vitamin D

treated patients there was a mean rise in CD4 count of 50 cells/ml
(95% CI, 257 to 158) and in the placebo group a mean fall of
7 cells/ml (95% CI, 2109 to 95). Results differed when stratified
by HIV status: Among 41 HIV-infected individuals the vitamin
Dtreated patients had a fall in CD4 count of 3 cells/ml (95%
CI, 282 to 76) versus a mean rise of 19 cells/ml (95% CI, 278 to
117) in placebo-treated patients (P 5 0.71). In contrast, 92 HIVuninfected individuals had a mean rise in CD4 count of 78 cells/ml
(95% CI, 282 to 238) in the vitamin Dtreated group and
a mean fall of 17 cells/ml (95% CI, 2153 to 120) in the placebo
group (P 5 0.37). However, this shift in change in CD4 count
was not significant (test of homogeneity, P 5 0.47).
The Secondary Outcome: Mortality

Fifty-four all-cause deaths were recorded during the 12-month

follow-up period, 30 of 187 (16%) in the vitamin D group and 24

of 178 (13%) in the placebo group (Table 2) (Kaplan-Meier

estimate shown in Figure 4, log-rank test P 5 0.45). In univariate
analyses, the only variables affecting the estimate were the
presence of VDI (increasing the hazard ratio [HZ] by 14%) and
HIV-1 infection (decreasing the HZ by 8%), and for HIV-1 we
noted a tendency for interaction (test of interaction, P 5 0.08).
When adjusting for both HIV-1 and VDI in a logistic regression
model, the estimate was only moderately increased, HZ 5 1.3
(95% CI, 0.72.3).
Stratified Analysis 1: Vitamin D Insufficiency

Because the effect of vitamin D supplementation may be seen

only among patients with vitamin D deficiency or insufficiency,
we assessed the outcomes in these two groups separately in an
exploratory, not prespecified, analysis. At treatment initiation,
mean TBscores differed according to vitamin D status, although
not significantly. The 181 patients with pulmonary TB with

Wejse, Gomes, Rabna, et al.: Vitamin D TB Trial

847

TABLE 1. BASELINE CHARACTERISTICS AT ENROLLMENT

Parameter
Age, years, mean (SD)
Men
Ethnicity
Balanta
Fula
Mandinga
Manjaco
Pepel
Others
Smear positive
Extrapulmonary TB, no.
HIV distribution
HIV-1
HIV-2
HIV-112
HIV negative
Missing
Mean CD41 T-lymphocyte
count, cells/ml (range)
CD41 cell count
, 200/ml, no. (%)
Weight, kg, mean (SD)
Mean BMI (range)
MUAC, cm, mean (range)
TBscore* (95% CI)
Distribution of severity class
I
II
III
Mean s-25(OH)D3 (SD)
No. with s-25(OH)D3
, 50 nmol/L (%)
No. with s-25(OH)D3
, 75 nmol/L (%)
Albumin-corrected serum
calcium, mmol/L, mean (SD)

Vitamin D
(n 5 187)

Placebo
(n 5 178)

37 (13)
116 (62%)

38 (14)
106 (60%)

28
29
20
36
41
33
123

29
25
8
32
37
47
124

42/187
18/187
12/187
111/187
4/187
505

(15%)
(16%)
(10%)
(19%)
(22%)
(17%)
(66%)
7
(23%)
(10%)
(6%)
(59%)
(2%)
(161733)

30/138 (22%)
51.9
18.8
237.1
6.7

(9.4)
(1233)
(154358)
(6.47.0)

60 (32%)
69 (37%)
58 (31%)
77.5 (23.8)
17 (10%)

34/178
18/178
7/178
117/178
2/178
561

(16%)
(14%)
(5%)
(18%)
(21%)
(26%)
(70%)
4
(19%)
(10%)
(4%)
(66%)
(1%)
(62328)

23/138 (17%)
51.1
18.5
236.7
6.8
44
78
56
79.1
13

(8.7)
(1227)
(144328)
(6.57.1)
(25%)
(44%)
(32%)
(21.8)
(8%)

86 (48%)

77 (45%)

2.03 (0.26)

2.03 (0.24)

Definition of abbreviations: BMI 5 body mass index; CI 5 confidence interval;

MUAC 5 midupper arm circumference; s-25(OH)D3 5 serum 25-hydroxyvitamin
D3; TB 5 tuberculosis; TBscore 5 clinical scoring system for tuberculosis.
Data are presented as number (%) unless otherwise stated.
* Only patients with pulmonary TB.

normal vitamin D status had a mean TBscore of 6.5 (95% CI, 6.2
6.8) compared with 157 patients with pulmonary TB with VDI
having a mean TBscore of 6.8 (95% CI, 6.57.1), and the
29 patients with pulmonary TB with VDD had a mean TBscore
of 7.1 (95% CI, 6.37.9). The TBscore at end of treatment differed

Figure 2. TBscore (clinical scoring system for tuberculosis) at various

time points for the two treatment arms.

Figure 3. Proportion of patients with positive acid-fast bacteria sputum

examination.

significantly by vitamin D status at treatment start. Among the

220 patients with pulmonary TB seen at 8 months with an initial
vitamin D measurement, the 117 with normal vitamin D status
had a mean TBscore of 0.7 (95% CI, 0.50.9) compared with 1.3
(95% CI, 1.01.6) among 103 patients with initial VDI. Among
these VDI patients, the TBscore was insignificantly lower at
8 months: among 55 vitamin Dtreated the mean score was 1.1
(95% CI, 0.81.4) compared with 1.4 (95% CI, 1.01.9) among 48
placebo-treated (P 5 0.21).
For the secondary outcome, mortality, the crude mortality
ratio of 1.2 changed moderately when looking at the 163
patients with initial VDI, HZ 5 1.4 (95% CI, 0.53.7). But
when looking at the 30 patients with VDD, HZ for death among
vitamin Dtreated patients was 0.7 (95% CI, 0.16.4). Adjusting
for HIV and background factors did not change these estimates.
Stratified Analysis 2: The Role of HIV

Because HIV infection may modify the effect of health interventions, for example, vitamin A, we examined the impact of
vitamin D among both HIV-infected and HIV-uninfected individuals in an exploratory, not prespecified, analysis. As demonstrated in Table 3, the data suggest a tendency after the first dose
of vitamin D toward a more rapid decrease in the TBscore among
HIV-uninfected patients treated with vitamin D and a more slow
decrease in HIV-infected patients treated with vitamin D,
TBscores being significantly different by HIV status in the
vitamin Dtreated group (P 5 0.008) after 2 months of followup but not in the placebo group (P 5 0.45). This pattern was
similar at 5 and 8 months with significant differences by HIV
status in the vitamin Dtreated group, although TBscores at
inclusion did not differ significantly. Table 3 also shows a lower
TBscore at the end of treatment in the HIV-uninfected group;
however, this difference was not significant.
In addition, we analyzed whether mortality differed when
stratified for HIV status. Among the 131 HIV-infected patients,
12-month mortality was high, as shown in Table 2. In a Cox
regression model we examined mortality separately for both
HIV-1infected patients and for all HIV-infected patients. HZ
estimates were not changed when looking at all HIV-infected
patients, and among the 95 HIV-1infected patients the mortality
risk was not significantly higher in the vitamin Dtreated arm,
HZ 5 1.8 (95% CI, 0.84.1) or significantly lower among the 228
HIV-uninfected patients, HZ 5 0.9 (95% CI, 0.32.8). This

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2009

TABLE 3. TBscore IN TREATMENT GROUPS BY TIME

AND HIV STATUS
Mean TBscore (NVitamin D/NPlacebo)

Vitamin D (SD)

Placebo (SD)

P Value

Inclusion
HIV1* (72/58)
HIV2 (104/114)

6.9 (2.1)
6.5 (1.9)

6.8 (2.1)
6.7 (1.9)

0.79
0.54

Two months
HIV1 (57/49)
HIV2 (94/97)

2.8 (2.3)
1.9 (1.7)

2.4 (2.0)
2.1 (2.0)

0.34
0.35

Five months
HIV1 (49/43)
HIV2 (88/91)

1.6 (1.6)
1.0 (1.4)

1.6 (2.2)
1.2 (1.3)

0.89
0.35

Eight months
HIV1 (39/32)
HIV2 (81/74)

1.3 (1.4)
0.7 (1.0)

1.2 (1.3)
1.0 (1.4)

0.91
0.09

Figure 4. Kaplan-Meier plot of cumulative incidence proportion (1

survival) for the two groups.

twofold difference in HZ ratio estimates, although not significant,

showed indications of effect modification of HIV on vitamin D
treatment (test of interaction, P 5 0.08). By adjusting for the
presence of VDI the mortality risk for HIV-1infected individuals was 2.4 (95% CI, 0.956.5).
Vitamin D Status at 2 and 8 Months

Vitamin D status was remeasured at 2 and 8 months, which were 2

and 3 months after the first and second doses were given at inclusion and at 5 months. At 2 months 270 samples were available
for analysis, and at 8 months 226 samples were available. A general
rise was seen in serum 25(OH)D3 concentrations, in 203 patients
with paired samples at inclusion, 2 months, and 8 months (Figure
5). In this group mean serum 25(OH)D3 increased from 78 nmol/L
(95% CI, 7581) to 103 nmol/L (95% CI, 99108) at 2 months of
follow-up and 98 nmol/L (95% CI, 94102) at 8 months. The
increase was also present in the placebo group: Mean 25(OH)D3
increased to 105 nmol/L (95% CI, 99110) in the treatment arm
and 103 nmol/L (95% CI, 96110) in the placebo arm at 2 months
and to 102 nmol/L (95% CI, 96107) in the treatment arm and
95 nmol/L (95% CI, 89102) in the placebo arm at 8 months.
Fractions of patients with VDD/VDI were not lower to at inclusion but were lower in the treatment arm at 2 and 8 months,
although not significantly different (data not shown); crude data
are displayed in Figure 5. A nonsignificant effect on serum vitamin D
levels was noted among vitamin Dinsufficient patients at baseline:
Patients with 25(OH)D3 concentrations less than 75 nmol/L increased from 60 nmol/L (95% CI, 5763) to 102 nmol/L (95% CI,
93110) whereas placebo-treated patients increased from 60 nmol/L
(95% CI, 5763) to only 95 nmol/L (95% CI, 85106).

DISCUSSION
Vitamin D supplementation of patients with TB in GuineaBissau did not result in serious adverse events, and we showed
no effect on the chosen outcomes in the entire group of patients.
The data show no significant differential effects of vitamin D
TABLE 2. MORTALITY BY HIV STATUS
Infection
HIV-1 and HIV-112 infected
HIV-2 only infected
HIV-1 and HIV-2 uninfected
HIV status unknown
Total

Vitamin D

Placebo

20/54
1/18
7/111
2/4
30/187

10/41
5/18
8/117
1/2
24/178

Mortality Ratio
1.52
0.2
0.92
1
1.19

(0.802.88)
(0.031.55)
(0.352.46)
(0.185.5)
(0.721.95)

* HIV infected (HIV-1 and HIV-2 and HIV-112 combined).

HIV uninfected.

supplementation in either HIV-infected or vitamin Dinsufficient

subjects. We may, however, speculate that there is a trend
toward positive effects for HIV-uninfected and vitamin D
insufficient subjects and a trend toward higher mortality among
HIV-infected patients treated with vitamin D.
None of the HIV-positive patients were receiving antiretroviral
treatment although 53 of 273 (19%) had CD41 T-lymphocyte
counts indicating a need for such treatment, which unfortunately
was not available in Bissau at the time of the study. Therefore the
findings cannot be generalized to HIV-positive patients treated
with antiretroviral therapy.
As reported elsewhere (28), VDI was common among patients
with TB in the study area, but severe vitamin D deficiency was
rare. Our findings indicate a slightly beneficial effect among
patients with TB with low vitamin D, but the study was not
sufficiently powered or designed to determine this. Furthermore,
because measuring vitamin D is costly and requires a certain level
of laboratory facility, it is unlikely that measuring vitamin D in all
patients with TB will be routine in resource-poor settings. Our
aim was to test vitamin D treatment in such a setting to determine
whether vitamin D is of value for patients with TB in resourcepoor areas.
Our study addresses the controversy of hypercalcemia in
patients with TB (29). We found no evidence of TB-associated
risk of hypercalcemia in this population, but we report a slight rise
in calcium concentrations during antituberculosis treatment
irrespective of vitamin D treatment arm. This observation is in
line with Fuss and colleagues, who did not find that vitamin D
supplementation of patients with TB resulted in hypercalcemia
(30). In 1969 Brincourt reported the result of repeated oral
supplementation with 15 mg (600,000 IU) of vitamin D2 (3) as
supplementary treatment to antibiotics in 150 patients in a study
with no control group and noted an effect on dissolving cavities;
Brincourt observed no hypercalcemia.
Our findings of lack of clinical effect are contradictory to what
Brincourt, as well as numerous studies from the preantibiotic era,
have reported. However, they all used much higher dosages than
in this trial, and all of these studies were uncontrolled (3133). In
modern times we know of only two small randomized trials
enrolling 24 children with TB in Egypt (15) and 67 adults in
Indonesia (14). They used oral vitamin D at 1,000 and 10,000 IU/
day, respectively, or placebo. They reported a more evident
clinical and radiographic improvement in the treated group in
Egypt (although not statistically significant), and improved
sputum conversion rates in the Indonesian study. These studies
were small and HIV status was not reported; however, it is
unlikely that these studies included many HIV-infected individ-

Wejse, Gomes, Rabna, et al.: Vitamin D TB Trial

Figure 5. Vitamin D serum levels before and during study.

uals. It is worth noting that the Indonesian study showed benefit

with a combined dose 1 log higher than the present study; the
reason for this trial not showing effect may therefore be that the
dose was insufficient. We based our choice of dose on what has
been shown effective toward osteomalacia, but the optimal dose
for an antituberculous effect is not known and it may well be
higher. As shown in Figure 5, the doses of vitamin D used did not
have a lasting effect on vitamin D levels in the patients who
received vitamin D and the lack of effect may therefore be due to
a suboptimal dose concentration or a suboptimal dosage interval.
The finding that vitamin D levels were higher at 2 months of
follow-up in both treatment arm and placebo arm is curious and
unexpected. The half-life for a single large dose is 2 months and no
sample was available earlier, and therefore a difference may have
been present right after vitamin D supplementation. A large
proportion of the patients had normal vitamin D levels before the
intervention, and if the dose given was suboptimal even for
nondeficient patients no difference between groups would be
expected 2 months later. There are no previous data on repeated
vitamin D measurements during a course of standard tuberculosis
treatment, and therefore a rise in vitamin D levels at 2 months
may be a normal phenomenon. It is likely that patients with a TB
diagnosis in the intensive phase of treatment are allowed a better
share of food by the family to compensate for their illness; in our
experience the patients with TB are focused on obtaining a rich
diet with meat and eggs if possible and may therefore have
increased their dietary vitamin D intake. A change in sun
exposure may also explain the increased vitamin D levels, but
we have no knowledge of this being the case. Activated macrophages have been shown to hydroxylate 25(OH)D (34) to 1,25dihydroxyvitamin D [1,25(OH)2D]; active synthesis of 25(OH)D
from nonhydroxylated vitamin D by macrophages no longer
occupied by mycobacteria may therefore be a possible additional
mechanism for increased vitamin D in the placebo group.
Our findings also contrast with those of Range and colleagues
(35), who reported a 50% reduction in mortality among HIVinfected patients with TB treated with multivitamin supplementation including vitamin D in a randomized clinical trial in
Tanzania. This was a subgroup analysis result, currently being
tested in a new trial, and they used much lower vitamin D doses as

849

well as a variety of other micronutrients such as zinc, which was

shown to have a separate beneficial effect.
We observed trends in CD4 counts indicating an increase in
CD4 count in vitamin Dtreated subjects who were not HIV
infected and a decrease in HIV-infected patients. We therefore
speculate that vitamin D has differential effects depending on
whether the immune system is intact.
We may hypothesize that a small beneficial effect is present in
HIV-uninfected patients with TB; vitamin D has been shown to
exert an effect in vitro on host immune response to Mycobacterium tuberculosis (5). HIV-infected patients, on the other hand,
are known to suffer from further immune activation (36), which
may cause the slower clinical improvement and the nonsignificantly higher mortality present among vitamin Dtreated HIV-1
infected patients. We may later analyze additional nonplanned
outcomes such as HIV viral load in this cohort to furnish more
data on this matter.
Indeed, vitamin D supplementation may have only a minor
effect on the outcome of TB treatment. Future studies may
therefore instead aim to evaluate vitamin D as an agent to
improve immune function in order to prevent latent TB infection.
In conclusion, we found satisfactory safety when supplementing
patients with TB with vitamin D at high doses. In this trial vitamin
D supplementation showed no beneficial effect for patients with
TB in general, but this may have been due to suboptimal dosage.
Further trials investigating larger, perhaps daily, vitamin D doses
for prevention of latent TB infection appear warranted.
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgment: The authors thank the TB study team at the Bandim Health
Project; the staff at the Bandim, Belem, and Cuntum Health Centers and at Sant
DEgidio TB Hospital; Henrik Friis and Sren Johnsen for useful suggestions early
in the study; and Lars stergaard, Lars Pedersen, and Henrik Toft Srensen for
serving on the Data and Safety Monitoring Board and Jesper Eugen-Olsen and
Jens Nielsen for advice on interpreting the data. The authors thank Crinex
Pharmaceuticals for providing the cholecalciferol and placebo ampules.

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