vit a dan zink

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The effect of vitamin A and zinc supplementation on treatment

outcomes in pulmonary tuberculosis: a randomized controlled trial13

Marianne E Visser, Harleen MS Grewal, Elizabeth C Swart, Muhammad A Dhansay, Gerhard Walzl, Sonja Swanevelder,Carl Lombard, and Gary Maartens

ABSTRACT

Background: Low serum concentrations of vitamin A and zinc are

common in tuberculosis and may have an adverse effect on host

cellmediated responses. The role of adjunctive micronutrient sup-

plementation on treatment outcomes is uncertain.

Objective: The objective was to assess the efficacy of vitamin A

and zinc supplementation on sputum smear and culture conversion

and time to culture detection in adults with sputum smearpositivepulmonary tuberculosis.

Design: Participants attending a primary care tuberculosis clinic in

Cape Town, South Africa, were randomly assigned to receive micro-

nutrients (single dose of 200,000 IU retinyl palmitate plus 15 mg

Zn/d for 8 wk) or matching placebo. Sputum was collected weekly

for 8 wk for auramine staining and culture on liquid media (BACTEC

MGIT 960; Becton Dickinson, Sparks, MD). Performance status,

chest radiographs, and anthropometric measures were assessed at

baseline and again at 8 wk.

Results: The participants (n = 154) were randomly assigned to the

micronutrient (n = 77) or placebo (n = 77) group. Twenty partic-

ipants were HIV infected (13%), and 12 participants had an un-

known HIV status (8%). No differences in time to smear or

culture conversion were observed between the treatment groups

by Kaplan-Meier analysis (P = 0.15 and P = 0.38, respectively;

log-rank test). Log-logistic regression analysis found no significant

group interaction effect in time to culture detection over the 8-wk

period (P = 0.32). No significant differences in weight gain (2.3 6

3.5 compared with 2.2 6 2.4 kg, P = 0.68) or radiologic resolution

were observed between the treatment groups.

Conclusion: Supplementation with vitamin A and zinc did not

affect treatment outcomes in participants with pulmonary tubercu-

losis at 8 wk. This trial was registered at controlled-trials.com as

ISRCTN80852505. Am J Clin Nutr 2011;93:93100.

INTRODUCTION

South Africa has one of the highest incidence rates of tu-

berculosis globally, with 948 cases per 100,000 population in

2007 (1). Protein-energy malnutrition frequently occurs in pa-

tients with tuberculosis with or without HIV infection, as in-

dicated by reductions in anthropometric variables and serum

concentrations of visceral proteins and micronutrients (2).

Micronutrient deficiencies in particular may have an adverse

effect on components of the immune system for the control of

mycobacteria. Several observational studies have shown low

concentrations of vitamin A and zinc in adults and children with

pulmonary tuberculosis (38). Vitamin A is important for the

maintenance of mucosal immunity (9), and supplementation

thereof has been shown to modulate T helper (Th) 2 lymphocyte

responses in childhood tuberculosis (10); however, no effect on

clinical outcome was reported (3). Although high-dose vitamin A

reduces the mortality associated with respiratory infections of

children with measles (11), no beneficial effect on the incidence

(12) or clinical recovery of children with acute lower respiratoryinfections has been shown (13). Zinc deficiency causes thymic

atrophy, impairs the generation and proliferation of T lympho-

cytes (14), and is associated with Th1/Th2 T lymphocyte cyto-

kine imbalances, all of which may reduce resistance to disease

(15). All of these effects have been shown to reverse after ex-

perimental supplementation (14, 15). Zinc supplementation has

been shown to reduce the incidence and severity of diarrhea and

pneumonia in children (16). Furthermore, zinc supplementation

of children exposed to adults with smear-positive pulmonary

tuberculosis resulted in an increase in the size of the induration of

tuberculin skin tests (17).

1From the School of Public Health, University of the Western Cape,

Bellville, Cape Town, South Africa (MEV); The Gade Institute, Section of

Microbiology and Immunology; University of Bergen and Haukeland Uni-

versity Hospital, Bergen, Norway (HMSG); the Division of Dietetics, Uni-

versity of the Western Cape, Bellville, Cape Town, South Africa (ECS); the

Medical Research Council, Cape Town, South Africa (MAD); the Medical

Research Council Centre for Molecular and Cellular Biology, University of

Stellenbosch, Stellenbosch, South Africa (GW); the Biostatistics Unit, Med-

ical Research Council, Cape Town, South Africa (SS and CL); and the Di-

vision of Clinical Pharmacology, Department of Medicine, University of

Cape Town, South Africa (GM).2

Supported by research grants from the National Research Foundation

South Africa (2067444); the Norwegian Programme for Development, Re-search and Higher Education (NUFUPRO-2007/10183); the Research Coun-

cil of Norway (183694/S50)]; the National Research Foundation (South

Africa)/Research Council of Norway 180353/S50; Helse Vest (2007-2009-

Grewal); and the South African Sugar Association (200). GM was supported

in part by grant U2RTW007370 from the Fogarty International Center. The

South African Department of Health and Pharma Natura Pty (Ltd) donated

the vitamin A and placebo capsules, respectively, for the study.3 Address correspondence to ME Visser, School of Public Health, Univer-

sity of the Western Cape, Private Bag X17, Bellville, 7535, Cape Town,

South Africa. E-mail: [email protected].

Received August 10, 2010. Accepted for publication October 15, 2010.

First published online November 10, 2010; doi: 10.3945/ajcn.110.001784.

Am J Clin Nutr 2011;93:93100. Printed in USA. 2011 American Society for Nutrition 93

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An Indonesian randomized trial reported that vitamin A and

zinc supplementation resulted in earlier sputum smear conversion

and improved the resolution of chest radiographic lesion areas in

participants with pulmonary tuberculosis (18). One of the lim-

itations of their trial was that sputum culture data were not

evaluated. Although sputum smear is widely used as a marker of

treatment response in tuberculosis programs, it is well known that

the sensitivity thereof is affected by significant observer vari-

ability (19). On the other hand, a meta-analysis of trials withfollow-up periods of up to 2 y showed that culture positivity after

8 wk is strongly associated with relapse (20). Another micro-

nutrient trialfrom Mwanza, Tanzania, with multimicro-

nutrients or zincreported no effect on the proportion of

participants with a positive sputum culture at 2 mo (21). Time to

culture detection (TTD) has been shown to correlate with sputum

colony countsa well-recognized measure of the efficacy of

antitubercular therapy in clinical trials (19). The main aim of our

study was to evaluate the administration of vitamin A and zinc on

3 major bacteriological markers of treatment outcome: sputum

smear and culture conversion and TTD.

SUBJECTS AND METHODS

The study population consisted of adults attending the Delft

Community Health Centre, a primary care center in Cape Town,

South Africa, between May 2005 and August 2008. Participants

were eligible for inclusion if they were 1) 1860 y of age and 2)

had a positive sputum smear for acid-fast bacilli in 2 routine

spontaneous sputum specimens taken by clinic staff or 1 positive

sputum smear plus suggestive chest radiograph findings. Ex-

clusion criteria included 1) previous treatment of tuberculosis;

2) known or suspected multidrug resistance tuberculosis; 3)

clinical evidence of extrapulmonary tuberculosis or liver disease

(.5-fold increase in alanine aminotransaminase concentrations

above the normal range), renal failure, congestive heart failure,or neoplasm; 4) received any supplement containing vitamin A,

zinc, or iron 1 mo prior; 5) corticosteroid use; or 6) having given

birth within 6 mo of study entry. Because a large dose of vitamin

A may be teratogenic, all eligible women were interviewed re-

garding their method of contraception (regular use of birth

control pills, medroxyprogesterone acetate (Bodene Pty Ltd,

Port Elizabeth, South Africa), sterilization, or sexual abstinence

with regular menstrual periods) in addition to having a negative

urine pregnancy test result (Visitect pregnancy test; Omega

Diagnostics Ltd, Hillfoots, United Kingdom) on the day before

the vitamin A capsule was administered. The study protocol was

approved by the Ethics and Research Committee of the Uni-

versity of Cape Town. All participants gave written, informedconsent, and those who were willing to undergo voluntary testing

and counseling for HIV infection during the study period were

included. HIV infection was diagnosed on the basis of a positive

rapid test (Sensa; Seyama Solutions, Johannesburg, South Africa)

and was confirmed by a positive enzyme-linked immunosorbent

assay result for HIV-1 antibodies. Participants received coun-

seling by qualified counselors before and after the test.

A log-rank test simulating the smear conversion data in weekly

intervals from Karyadi et al (18) for both treatment groups was

used to estimate our sample size. Seventy participants in each

group were required to detect a significant difference between the

estimated survival curves at a 5% significance level with 90%

power, during an 8-wk follow-up period. We allowed for a 10%

loss to follow-up, giving a total number of 154 participants.

Participants were randomly assigned to the micronutrient or

placebo groups in computer-generated permuted blocks of 8,

generated by an independent epidemiologist. Treatment alloca-

tion was concealed by prepackaging supplements in sequentially

numbered packets according to the allocation schedule by the

Department of Pharmacy, University of the Western Cape. Active

and placebo capsules and tablets for both treatment groups wereidentical in size, shape, and color. All research team members as

well as the laboratory staff involved in the trial were blinded.

Participants received a single capsule containing 200,000 IU

vitamin A (retinyl palmitate) or placebo (sunflower oil) (Pharma

Natura Pty Ltd, Johannesburg, South Africa) from a research nurse

within 24 h after the start of tuberculosis therapy. This dosage

corresponded to a daily dosage of 5000 IU retinol over the first 2

mo, as used by Karyadi et al (18). Participants also received one

tablet containing 15 mg Zn (as zinc gluconate in a starch/gelatin

base) or placebo (starch/gelatin base) (Vitalfarm Pty Ltd, Cape

Town, South Africa) daily for 5 d/wk for 2 mo together with their

antitubercular therapy, as part of directly observed treatment. The

daily dietary zinc intake of urban black South Africans waspreviously estimated at 11 and 9 mg for men and women, re-

spectively (22). Therefore, the daily consumption of an additional

15 mg Zn was considered safe for our study participants (23).

Both treatment groups received tuberculosis therapy for 5 d/wk,

consisting of combination tablets contributing 600 mg rifampi-

cin, 300 mg isoniazid, 1.6 g pyrazinamide, and 1.1 g ethambutol

for participants weighing 3855 kg (Rifafour; Aventis Pharma

Pty Ltd, Johannesburg, South Africa). Doses were adjusted for

participants weighing ,38 or .55 kg. All participants also re-

ceived 25 mg pyridoxine/d. Most of our participants received

their tuberculosis treatment and supplements daily with directly

observed therapy at the clinic for the first 2 wk and thereafter

daily from trained community-based treatment supporters, whowere instructed to store the trial supplements in a cool, dark

place. Because of a change in national policy in April 2008, 15 of

our trial participants received tuberculosis treatment for 7 d/wk

with unsupervised weekend doses. The trial protocol for the

supplementation of zinc only on weekdays remained unchanged.

Adherence to the trial supplements was assessed by pill counts.

For participants receiving their tuberculosis therapy at the clinic,

trial tablets were counted weekly. For those receiving their

treatment in the community, the treatment supporter was visited

weekly or contacted by telephone to obtain a pill count. The mean

adherence rate to the trial supplements was 94 6 16% and was

independent of treatment arm. Adherence was calculated as the

number of doses received by each participant, divided by thenumber of treatment days that a particular participant was fol-

lowed up on the study.

Standardized operating procedures were followed for the

collection of all participant data. Vital signs (blood pressure,

pulse, and temperature), clinical symptoms, medical history, any

concomitant medication use, and Karnofsky performance status

were recorded for each participant. All participants were

screened with regard to the potential misuse of alcohol with

the Cut Down, Annoyance, Guilt and Eye-opener (CAGE)

questionnaire, which was previously validated locally (24).

Sociodemographic characteristics were documented for each

participant (eg, type of housing, housing density, household

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income, and smoking habits). Anthropometric measurements

were conducted at study entry and again at 8 wk. Body weight

was measured to the nearest 0.1 kg (A&D Personal Precision

Scale, Tokyo, Japan), height on a portable stadiometer to the

nearest 1 mm, midupper arm circumference to the nearest 1 mm

on the left arm with the use of a standardized measuring tape, and

skinfold thickness (triceps, biceps, subscapular, and supra-iliac)

measurements on the left arm to the nearest 0.2 mm with

a skinfold caliper (Scales 2000, Durban, South Africa). Bodydensity and percentage body fat were calculated with the use of

the Durnin and Womersley equations (25). Dietary intakes of the

participants were assessed by using the 24-h recall method at

baseline and at weeks 2 and 8 by a dietitian or trained dietetic

students using a standardized record form. Documentation was

made of any participant who received nutritional support from the

community health center as part of the health facilitybased

nutrition program.

After fulfilling the study entry criteria, the participants sup-

plied one spontaneous sputum specimen to the research nurse for

microscopic examination by means of fluorescent microscopy

(auramine stain) in the National Health Laboratory Service

Laboratory, Groote Schuur Hospital, Cape Town, South Africa.Sputum specimens were also cultured on liquid media by using

the BACTEC MGIT 960 system (Becton Dickinson, Sparks,

MD). Positive culture results were stained with a Ziehl Neelsen

stain, and cultures with acid fast bacilli were confirmed as

Mycobacterium tuberculosis complex with an in-house poly-

merase chain reaction assay (26). Sputum smears were graded

according to international standards (27). The baseline specimen

with the highest smear grading was used to compare both

treatment groups at baseline. One early-morning unassisted spu-

tum specimen was collected from each participant every week, up

to 8 wk. The date of culture or smear conversion was taken as the

date of the first negative culture or smear, provided that there were

no subsequent positive cultures or smears. Participants whose firstnegative culture or smear occurred at week 8 were regarded as

converters. Routine drug susceptibility testing (isoniazid and ri-

fampicin only) of isolates from the first 2 wk was carried out by

using the MGIT 960 system from January 2008; isolates that were

cultured before that date were tested retrospectively.

The extent and size of the lung cavities were assessed in-

dependently by 2 pulmonologists experienced in the use of the

Chest Radiograph Reading and Recording System (28). If there

were discrepancies, consensus was reached. Venipuncture sites

were cleaned with trace elementfree alcohol, and blood for trace

element analysis was collected in a trace elementfree tube

(Vacutainer; Becton-Dickinson, Franklin Lakes, NJ), protected

from light, and stored at 70C after centrifugation. Venipuncturewas performed at study entry and after 2 and 8 wk for full blood

count, serum retinol (measured by HPLC), C-reactive protein

(CRP) (DRG International Inc, Mountainside, NJ), serum zinc

and copper (measured with a Pye Unicam SP9 atomic absorption

spectrometer), and serum albumin (measured with a colorimetric

method; Diagnostics Worldwide, Wiesbaden, Germany).

All data were captured in duplicate in Microsoft Office Excel

2003 and validated with SAS version 9.2 software (SAS Institute

Inc, Cary, NC). The dietary data of the study participants were

analyzed with the Foodfinder food-composition database (29).

Missing values for dietary zinc were replaced with values from

other food-composition databases (30).

Statistical analyses were carried out by using SAS version 9.2

and Stata 9.0 (StataCorp, College Station, TX). The Kolmogorov-

Smirnov test was used to investigate whether variables were

normally distributed and, if necessary, variables were trans-

formed. Baseline comparability of the treatment groups was

assessed by Students 2-tailed ttest or 2-tailed chi-square analysis,

where appropriate. Study outcomes were analyzed on an intent-

to-treat basis. An analysis of covariance regression model was

used with the group indicator, time of measurement, and thebaseline response as covariates to analyze changes in anthropo-

metric, dietary, and biochemical variables. The covariance structure

of the repeated measurement residuals within each participant was

modeled by an unstructured process. Logistic regression was used

to examine any changes in radiological variables. Time to sputum

smear and culture conversionthe time point after which all

smears or cultures were negativewas estimated by using the

Kaplan-Meier method. The log-rank test was used to evaluate any

treatment effect between the 2 groups. TTD data from baseline to

week 8 for each group was compared by means of a log-logistic

regression model, a frailty regression model that accounts for

nonproportional hazards present at each time point (31).

RESULTS

One hundred fifty-four participants were randomly assigned to

the micronutrient and placebo groups (Figure 1). No significant

differences in baseline characteristics were observed between

the treatment groups (Table 1). In total, 15 women and 5 men

were HIV-infected (13%), and the HIV status was unknown for

12 participants who were lost to follow-up before counseling

FIGURE 1. Trial profile.

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and testing could be performed (8%). Two participants were

included with a known diagnosis of HIV infection (one pergroup), both of whom were stable on antiretroviral therapy for

.6 mo. All participants with newly diagnosed HIV infection

were referred to the antiretroviral clinic for assessment. At the

time the study was done, the local policy was to initiate anti-

retroviral therapy after the intensive phase of antitubercular

therapy, which was after our study period. All HIV-infected

participants were given cotrimoxazole prophylaxis.

Overall, menhad a poorer nutritional status than did women, as

reflected by lower body mass index (BMI) values in men than in

women at study entry in both groups (P = 0.012), despite higher

reported dietary energy intakes. Men reported higher intakes of

macronutrients and zinc than did women, but the intakes of the

2 treatment groups at baseline were similar (Table 2).Baseline drug susceptibility data were available for 118 par-

ticipants (76%). Monoresistance to isoniazid was observed in

6 participants (3 per group), whereas 1 participant in the mi-

cronutrient group was monoresistant to rifampicin. Multidrug

resistance (to both isoniazid and rifampicin) was observed in

3 participants (4%) (2 in the micronutrient group and 1 in the

placebo group). Sixty-six of 77 participants (86%) in the micro-

nutrient group and 58 of 77 participants (75%) in the placebo group

completed the trial (P = 0.104) (Figure 1). One HIV-infected

participant in the micronutrient group died during the study pe-

riod. The gastrointestinal side effects reported included nausea

and vomiting in 3 participants in the micronutrient group (nausea

and vomiting) and in 2 participants in the placebo group (epi-

gastric discomfort and diarrhea).

Primary analyses

At study entry, 91% of participants were culture-positive, and

the TTD was similar in both groups (Table 1). In the placebo

group, one baseline sputum culture result was unavailable be-

cause of contamination. Kaplan-Meier analysis showed no sig-

nificant difference in the time to sputum smear or culture

conversion between the treatment groups during the 8-wk period

(Figure 2). After 8 wk, 73% and 60% of participants in the

micronutrient group compared with 65% and 51% of partic-

ipants in the placebo group had undergone smear and culture

conversion, respectively. No significant group interaction effectin TTD were found over the 8-wk period in the regression model

(Table 3). For both groups, TTD at baseline was a significant

contributor of TTD at week 8.

Secondary analyses

Most participants underwent chest radiograph examination at

baseline (n = 125) and after 2 mo (n = 109). HIV-infected

participants were less likely to have lung cavities at baseline

(P = 0.005). After 2 mo, a significant reduction in the total

number of participants with lung cavities was found [odds ratio

(OR): 0.56; 95% CI: 0.32, 0.55), but no significant effect of

micronutrient supplementation on cavity resolution was found.

TABLE 1

Characteristics of adults with smear-positive pulmonary tuberculosis by treatment group at study entry

Micronutrient group Placebo group

Characteristics n Values n Values P

Age (y) 77 30 (2342)1

77 27 (2143) 0.125

Sex (% male) 77 67.5 77 63.6 0.611

HIV status [n (%)] 77 77

Negative 63 (81.9) 59 (76.7) 0.717

Positive 9 (11.6) 11 (14.3)

Unknown 5 (6.5) 7 (9.0)

History of diabetes mellitus [n (%)] 77 4 (5.2) 77 2 (2.6) 0.681

Sputum smear grade [n (%)] 77 77

Scanty positive 3 (3.9) 3 (3.9) 0.944

1+ 10 (13.0) 9 (11.7)

2+ 9 (11.7) 7 (9.1)

3+ 55 (71.4) 58 (75.3)

Culture-positive [n (%)] 77 70 (90.9) 76 69 (90.8) 0.98

Time to detection (d) 7.0 (6.011.0) 7.0 (6.012.0) 0.35

Lung cavities [n (%)] 61 49 (80.3) 64 50 (78.1) 0.762

No. of lung zones affected 2 (12) 1 (12) 0.609

Karnofsky score 77 90 (8090) 77 90 (8090) 0.878

BMI (kg/m

2

)Male 52 18.9 6 2.7

249 19.0 6 2 0.726

Female 24 23.0 6 4.3 27 21.6 6 4.8 0.279

Arm muscle circumference (cm)

Male 51 21.5 6 2.6 49 22.1 6 2.3 0.252

Female 24 21.4 6 2.9 27 20.9 6 2.9 0.539

History of cigarette smoking [n (%)] 77 54 (70.1) 77 55 (71.4) 0.859

Current cigarette smoking [n (%)] 77 35 (45.5) 77 40 (51.9) 0.420

Alcohol misuse [n (%)] 77 27 (35.1) 77 31 (40.3) 0.506

1Median; interquartile range in parentheses (all such values).

2Mean 6 SD (all such values).

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Significant weight gain occurred after 2 mo in the micronutrient

and placebo groups (2.3 6 3.5 and 2.2 6 2.4 kg respectively;

P = 0.68). Overall, the mean weight gain for men was 3.18 (95%

CI: 1.84, 4.52) kg and 2.28 (95% CI: 1.22, 3.34) kg among HIV-

uninfected women, whereas no significant weight change among

HIV-infected women was observed. Significant increases in

other anthropometric indicators, such as arm muscle circum-

ference, were shown in the micronutrient (0.75 cm; 95% CI:

0.43, 1.08 cm) and placebo (0.71 cm; 95% CI: 0.36, 1.06 cm)

groups. However, the reported dietary intakes of men and

women did not change significantly during the study period.

Forty-seven percent of participants in both groups had low

serum concentrations of retinol (, 20 lg/dL) at baseline, and

75% and 83% of participants in the micronutrient and placebo

groups had low zinc concentrations (,70 lg/dL), respectively

(P = 0.156). Regression analysis showed a significant increase in

serum retinol and albumin concentrations over time in both

groups, whereas serum CRP and copper concentrations de-

creased (Table 4). However, no significant change in serum zinc

was observed during the study period in either group. Hemoglo-

bin increased significantly over the 8-wk period, whereas reduc-

tions were noted for total white blood cell count and neutrophil

count; differences between groups were not significant.

DISCUSSION

We observed no significant effect of supplementation with

a single dose of vitamin A and daily zinc on the rate of sputum

smear or culture conversion by 8 wk or on TTD. Participants in

both groups with a high baseline bacterial load, as reflected by

a short TTD, were likely to have a shorter TTD at week 8. TTD

during the first 2 wk of treatment has been shown to predict

TABLE 2

Reported daily dietary intake (24-h recall) of participants at study entry1

Nutrient

Placebo group

(49 M, 24 F)

Micronutrient group

(47 M, 26 F) P

Energy (kJ)

Male 7520 (56379213) 7187 (47169222) 0.593

Female 4709 (34107301) 5006 (40065808) 1.000

Protein (g)

Male 59.7 (38.090.1) 59.3 (36.086.7) 0.753

Female 31.5 (22.153.3) 41.3 (23.554.6) 0.763

Carbohydrate (g)

Male 219.5 (189.2298.0) 216.0 (153.0304.6) 0.337

Female 158.8 (115.9218.3) 163.5 (131.9201.6) 0.823

Fat (g)

Male 59.4 (30.677.2) 54.8 (28.578.5) 0.832

Female 33.1 (24.159.6) 34.4 (21.642.4) 0.593

Dietary fiber (g)

Male 16.2 (11.924.3) 15.3 (7.921.9) 0.281

Female 10.0 (6.314.5) 10.0 (5.516.5) 0.946

Vitamin A (RE)

Male 451 (288552) 417 (166691) 0.500

Female 295 (198583) 335 (235541) 0.676

Zinc (mg)Male 12.2 (6.015.1) 9.1 (5.513.6) 0.164

Female 5.5 (3.79.2) 6.6 (4.79.6) 0.479

1All values are medians; interquartile ranges in parentheses. RE, retinol equivalents.

FIGURE 2. Kaplan-Meier graphs showing the rate of sputum smear conversion (A) and sputum culture conversion (B) between the treatment groups. Thetables under each figure represent the number of participants at risk. Follow-up data were unavailable for 6 participants (3 per group). Seven participants (2 inthe micronutrient group and 5 in the placebo group) were culture-negative throughout the 8-wk period.



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treatment responses in patients with first-time pulmonary tu-

berculosis (32).

Our results contrast with those by Karyadi et al (18), who

reported an earlier sputum smear conversion among Indonesian

patients supplemented with zinc and vitamin A. One explanation

for the different findings in our study could be that our patients

had more advanced tuberculosis. Compared with the participants

in the Karyadi et al study, our study participants were more likely

at baseline to have higher-grade sputum smear positivity (36% 3+

compared with 7.5% 3+) and to have pulmonary cavities (73%

compared with 37.5%) (18). Both of these factors are known to be

associated with longer sputum smear and culture conversion times(33). However, our failure to find a benefit for the micronutrient

intervention is in keeping with 2 other studies: a second Indonesian

trial, as yet unpublished, found no improvement in sputum smear

conversion with zinc and vitamin A (34), and a Tanzanian trial

reported no effect of a multimicronutrient intervention or zinc on

sputum culture conversion at 2 mo (21).

Almost half of our participants were still culture-positive for

M. tuberculosis on liquid culture media at the end of 8 wk,

which was higher than we anticipated. However, similar high

rates of culture positivity have been observed in liquid culture

media at 2 mo at African sites (3537). It has been suggested

that the use of a less sensitive method, such as solid media, may

prove to be more useful in a clinical trial, because patients withstrongly positive results at 8 wk are likely to relapse, whereas

patients with scanty positive cultures are most likely to convert

during the continuation phase; the inclusion of the latter only

weakens the association with relapse (35).

We showed a significant increase in serum retinol concen-

trations during the study period in both treatment groups. Most of

our participants had serum concentrations consistent with a de-

ficiency of retinol (,20 lg/dL) at baseline. An important factor

that contributes to low retinol concentrations in active tubercu-

losis is the acute phase response, during which the hepatic

production of several proteins, such as CRP and ceruloplasmin,

increases, whereas that of others (eg, retinol-binding protein)

decrease (38). It has been shown that CRP concentrations de-crease within days of starting antitubercular therapy in pulmo-

nary tuberculosis (39). Acute fever is also associated with

significant urinary losses of retinol and retinol-binding proteins

(40). A study of Tanzanian patients receiving antitubercular treat-

ment without any supplementation of vitamin A showed a signifi-

cant increase in retinol concentrations after 2 mo (6). Our findings

confirm this observation and suggest, therefore, that the low

baseline retinol concentrations were most likely due to the pres-

ence of active disease rather than underlying deficiency.

Despite good adherence, no significant increase in serum zinc

concentrations occurred in the supplemented group in our study.

Our findings agree with data from 2 supplementation trials, both

TABLE 3

Log-logistic regression estimates for time-to-detection (TTD) data1

Coefficient 95% CI P

Rx 20.383 20.128, 0.514 0.403

Rx week 0.109 20.107, 0.033 0.322

Week 0.138 0.122, 0.154 ,0.0001

Baseline TTD 0.325 0.257, 0.392 ,0.0001

1

Rx, treatment group; Rx week, interaction term.

TABLE

4

Laboratoryvariablesofparticipantsatbaselineand2and8wk1

Baseline

Week2

Week

8

n

Micronutrie

ntgroup

n

Placebogroup

n

Micronutrientgroup

n

Placebog

roup

n

Micronutrientgroup

n

Placebogroup

Retinol(lg/dL)

76

21.1(15.1

27.8)2

76

21.2(15.728.9)

72

32.9(25.044.6)

63

32.7(24.041.1)

63

40.3(28.748.5)3

54

35.8(27.743.2)3

Zinc(lg/dL)

72

62(5371.8)

72

59(51.865.3)

70

67.5(57.076.0)

60

60.5(52.868.8)

61

62.5(55.570.0)

49

61.5(54.069.0)

Copper(lg/dL)

72

171(143.5

198.5)

72

176.8(153.5197.4)

70

172.8(154.0200.0)

60

164.0(149

.0196.5)

61

152.0(135.0169.0)4

49

155.0(123.5174.0)4

CRP(mg/L)

75

50.9(36.6

73.8)

75

50.1(31.271.5)

70

27.9(14.743.2)

63

16.9(8.730.5)

63

11.9(4.426.8)4

53

8.3(2.917.3)4

Albumin(g/L)

75

35.9

6

5.6

5

74

35.8

6

4.4

69

37.0

6

5.6

63

37.0

6

5.0

63

416

4.6

4

52

416

5.0

4

Hemoglobin(g/dL)

76

11.8

6

1.7

76

11.8

6

1.9

72

8.6

6

3.5

65

12.0

6

1.8

67

13.1

6

1.6

4

59

13.3

6

1.6

4

WBC(109/L)

76

10.3

6

3.6

76

106

3.6

72

5.8

6

3.2

65

8.5

6

3.3

67

7.4

6

2.6

4

59

7.0

6

2.8

4

Neutrophils(109/L)

76

7.6

6

3.3

76

7.5

6

3.3

72

5.8

6

3.2

65

5.7

6

2.9

67

4.6

6

2.3

4

59

4.4

6

2.4

4

1

CRP,C-reactiveprotein;WBC,whitebloodcell.

2

Median;interquartilerangeinparentheses(allsuchvalues).

3

Significantchangeoverthe8-wkperiodinbothgroups,P

=0.0003(ANCOVA).

4

Significantchangeoverthe8-wkperiodinbothgroups,P

,

0.0001(ANCOVA).

5

Mean6

SD(allsuchvalues).

98 VISSER ET AL


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of which failed to show increases in plasma zinc concentrations

after 2 mo, but noted significant increases at the end of treatment

in both supplemented and nonsupplemented participants (18, 34).

Again, the effect of symptomatic disease on zinc homeostasis

should be considered. Because the concentration of zinc is much

higher in tissues such as the liver, than in serum, small differences

in the hepatic uptake of zinc may have a profound effect on serum

concentrations, such as an increased hepatic synthesis of met-

allothionein. The latter is an intracellular metal-binding proteinthat is activated by cytokines during the acute phase response

(41). During this trial, we measured serum copper concentrations

in an attempt to monitor any adverse effects of chronic ingestion

of zinc on copper absorption (42). We found a significant re-

duction in serum copper in both treatment groups, which sug-

gested an indirect effect of the reduction in the inflammatory

response, rather than a direct antagonistic effect of zinc sup-

plementation on serum copper concentrations.

Few trials have examined the effect of micronutrient supple-

mentation on other tuberculosis treatment outcomes. A trial from

Dar es Salaam, Tanzania, showed that multinutrient supplemen-

tation without zinc reduced the risk of tuberculosis recurrences

between 1 and 8 mo after the start of treatment in HIV-positiveparticipants, but not in those who were HIV-negative (43). Three

trials reported on the effect of supplementation of multimicro-

nutrients on the mortality of participants with pulmonary tuber-

culosis (4345), only one of which showed a significant reduction

in the mortality of HIV-positive participants receiving a multi-

micronutrient supplement, which included zinc (44).

Our study had several limitations. First, our follow-up rate was

lower than anticipated, which together with the observed low

conversion rates may have reduced the statistical power of the

study. Second, most of our participants were not in a fasting state

at the time of blood collection. Participants were encouraged to

eat before receiving their antitubercular medication to minimize

gastrointestinal side effects. Postprandial zinc concentrationstend to be marginally lower than fasting concentrations (46). It is

also possible that the bioavailability of zinc from the study

supplement may have been influenced by the presence of dietary

factors such as phytate, type and amount of protein, and dietary

zinc (47). Third, a single 24-h dietary recall provides an estimate

of the mean dietary intake of a group, provided that days in all

parts of the week are represented (46). For the purpose of this

trial, a single recall at each time point was deemed sufficient to

ascertain whether there were significant changes in the dietary

intake of the 2 treatment groups during the study period. Fourth,

we evaluated a surrogate marker, bacteriologic outcomes at 8 wk,

to evaluate treatment success rather than failure at the end of

treatment or relapse.In conclusion, we found that micronutrient supplementation had

no effect on bacteriologic outcomes at 8 wk in participants with

pulmonary tuberculosis. Additional clinical trials with longer

follow-up periods to assess the efficacy of such interventions on

treatment outcomes and with adequate power to assess the

interventions in those with and without HIV infection are needed.

We thank our field staff and the clinic staff at the Delft and Delft-South

Community Health Centres for their valuable contributions to this trial.

We also thank the following people and institutions: L Birch from PathCare;

R Bapoo from the Department of Pharmacy, University of the Western Cape;

C Morroni from the School of Public Health, University of Cape Town;

A Whitelaw, V January, and K Mentoor from t he National Health Laboratory

Service; Groote Schuur Microbiology Laboratory; D Marais, M Marais,

E Harmse, and K Rossouw from the Nutritional Intervention Unit, Medical

Research Council forthe biochemical analyses;A Menezes, N Chegou, andM

de Kock from theMedical ResearchCouncil Centreof Molecularand Cellular

Biology at the University of Stellenbosch for the processing and storing of all

blood samples; R Dawson and K Narunsky from the University of Cape Town

Lung Institute for evaluation of the chest radiographs; and R Laubscher from

the Statistics unit, Medical Research Council.

The authors responsibilities were as followsMEV, ECS, and GM:

developed the protocol; MEV: responsible for the conduct of the trial and

for data management; ECS, HMSG, GM, and GW supervised the conduct

of the trial and the data management; MAD: responsible for the biochemical

analyses; MEV: analyzed and interpreted the data; and SS and CL: assisted

with the analysis and interpretation of the data and wrote the first version of

the manuscript. All authors contributed to the final version of the manuscript.

There were no conflicts of interests. The funding sources had no influence on

the study design, interpretation of data, or the decision to submit the manu-

script for publication.

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