The effect of vitamin A and zinc supplementation on treatment outcomes in pulmonary tuberculosis: a randomized controlled trial1–3 Marianne E Visser, Harleen MS Grewal, Elizabeth C Swart, Muhammad A Dhansay, Gerhard Walzl, Sonja Swanevelder, Carl Lombard, and Gary Maartens

INTRODUCTION

South Africa has one of the highest incidence rates of tuberculosis globally, with 948 cases per 100,000 population in 2007 (1). Protein-energy malnutrition frequently occurs in patients with tuberculosis with or without HIV infection, as indicated 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 (3–8). 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 respiratory infections has been shown (13). Zinc deficiency causes thymic atrophy, impairs the generation and proliferation of T lymphocytes (14), and is associated with Th1/Th2 T lymphocyte cytokine imbalances, all of which may reduce resistance to disease (15). All of these effects have been shown to reverse after experimental 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).

1

From 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 University Hospital, Bergen, Norway (HMSG); the Division of Dietetics, University 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, Medical Research Council, Cape Town, South Africa (SS and CL); and the Division 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, Research and Higher Education (NUFUPRO-2007/10183); the Research Council of Norway (183694/S50)]; the National Research Foundation (South Africa)/Research Council of Norway 180353/S50; Helse Vest (2007-2009Grewal); 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, University 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;93:93–100. Printed in USA. Ó 2011 American Society for Nutrition

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ABSTRACT Background: Low serum concentrations of vitamin A and zinc are common in tuberculosis and may have an adverse effect on host cell–mediated responses. The role of adjunctive micronutrient supplementation 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 smear–positive pulmonary tuberculosis. Design: Participants attending a primary care tuberculosis clinic in Cape Town, South Africa, were randomly assigned to receive micronutrients (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 participants were HIV infected (13%), and 12 participants had an unknown 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 tuberculosis at 8 wk. This trial was registered at controlled-trials.com as ISRCTN80852505. Am J Clin Nutr 2011;93:93–100.

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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 limitations 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 variability (19). On the other hand, a meta-analysis of trials with follow-up periods of up to 2 y showed that culture positivity after 8 wk is strongly associated with relapse (20). Another micronutrient trial—from Mwanza, Tanzania, with multimicronutrients or zinc—reported 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 counts—a 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.

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) 18–60 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. Exclusion 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 regarding 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 University of Cape Town. All participants gave written, informed consent, 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 counseling 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%

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SUBJECTS AND METHODS

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 allocation 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 were identical 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 was previously estimated at 11 and 9 mg for men and women, respectively (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 rifampicin, 300 mg isoniazid, 1.6 g pyrazinamide, and 1.1 g ethambutol for participants weighing 38–55 kg (Rifafour; Aventis Pharma Pty Ltd, Johannesburg, South Africa). Doses were adjusted for participants weighing ,38 or .55 kg. All participants also received 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, who were 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 the number of treatment days that a particular participant was followed 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

VITAMIN A AND ZINC IN PULMONARY TUBERCULOSIS

Statistical analyses were carried out by using SAS version 9.2 and Stata 9.0 (StataCorp, College Station, TX). The KolmogorovSmirnov test was used to investigate whether variables were normally distributed and, if necessary, variables were transformed. Baseline comparability of the treatment groups was assessed by Student’s 2-tailed t test or 2-tailed chi-square analysis, where appropriate. Study outcomes were analyzed on an intentto-treat basis. An analysis of covariance regression model was used with the group indicator, time of measurement, and the baseline response as covariates to analyze changes in anthropometric, 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 conversion—the time point after which all smears or cultures were negative—was 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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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). Body density 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 facility–based nutrition program. After fulfilling the study entry criteria, the participants supplied 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 polymerase 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 sputum 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 first negative culture or smear occurred at week 8 were regarded as converters. Routine drug susceptibility testing (isoniazid and rifampicin 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 independently 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 element–free alcohol, and blood for trace element analysis was collected in a trace element–free tube (Vacutainer; Becton-Dickinson, Franklin Lakes, NJ), protected from light, and stored at –70°C after centrifugation. Venipuncture was 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).

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TABLE 1 Characteristics of adults with smear-positive pulmonary tuberculosis by treatment group at study entry Micronutrient group Characteristics

1 2

77 77 77

77 77

Values

n 1

30 (23–42) 67.5 63 9 5 4

(81.9) (11.6) (6.5) (5.2)

77 77 77

77 77

Values

P

27 (21–43) 63.6

0.125 0.611

59 11 7 2

(76.7) (14.3) (9.0) (2.6)

0.717

3 9 7 58 69 7.0 50 1 90

(3.9) (11.7) (9.1) (75.3) (90.8) (6.0–12.0) (78.1) (1–2) (80–90)

0.681

77

3 (3.9) 10 (13.0) 9 (11.7) 55 (71.4) 70 (90.9) 7.0 (6.0–11.0) 49 (80.3) 2 (1–2) 90 (80–90)

77

52 24

18.9 6 2.72 23.0 6 4.3

49 27

19.0 6 2 21.6 6 4.8

0.726 0.279

51 24 77 77 77

21.5 6 2.6 21.4 6 2.9 54 (70.1) 35 (45.5) 27 (35.1)

49 27 77 77 77

22.1 6 2.3 20.9 6 2.9 55 (71.4) 40 (51.9) 31 (40.3)

0.252 0.539 0.859 0.420 0.506

77 61

76 64

0.944

0.98 0.35 0.762 0.609 0.878

Median; interquartile range in parentheses (all such values). Mean 6 SD (all such values).

and testing could be performed (8%). Two participants were included with a known diagnosis of HIV infection (one per group), 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 antiretroviral therapy after the intensive phase of antitubercular therapy, which was after our study period. All HIV-infected participants were given cotrimoxazole prophylaxis. Overall, men had 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 participants (76%). Monoresistance to isoniazid was observed in 6 participants (3 per group), whereas 1 participant in the micronutrient 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 micronutrient 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 period. 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 (epigastric 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 because of contamination. Kaplan-Meier analysis showed no significant 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 participants in the placebo group had undergone smear and culture conversion, respectively. No significant group interaction effect in 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.

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Age (y) Sex (% male) HIV status [n (%)] Negative Positive Unknown History of diabetes mellitus [n (%)] Sputum smear grade [n (%)] Scanty positive 1+ 2+ 3+ Culture-positive [n (%)] Time to detection (d) Lung cavities [n (%)] No. of lung zones affected Karnofsky score BMI (kg/m2) Male Female Arm muscle circumference (cm) Male Female History of cigarette smoking [n (%)] Current cigarette smoking [n (%)] Alcohol misuse [n (%)]

n

Placebo group

VITAMIN A AND ZINC IN PULMONARY TUBERCULOSIS

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TABLE 2 Reported daily dietary intake (24-h recall) of participants at study entry1 Nutrient

1

Micronutrient group (47 M, 26 F)

P

7520 (5637–9213) 4709 (3410–7301)

7187 (4716–9222) 5006 (4006–5808)

0.593 1.000

59.7 (38.0–90.1) 31.5 (22.1–53.3)

59.3 (36.0–86.7) 41.3 (23.5–54.6)

0.753 0.763

219.5 (189.2–298.0) 158.8 (115.9–218.3)

216.0 (153.0–304.6) 163.5 (131.9–201.6)

0.337 0.823

59.4 (30.6–77.2) 33.1 (24.1–59.6)

54.8 (28.5–78.5) 34.4 (21.6–42.4)

0.832 0.593

16.2 (11.9–24.3) 10.0 (6.3–14.5)

15.3 (7.9–21.9) 10.0 (5.5–16.5)

0.281 0.946

451 (288–552) 295 (198–583)

417 (166–691) 335 (235–541)

0.500 0.676

12.2 (6.0–15.1) 5.5 (3.7–9.2)

9.1 (5.5–13.6) 6.6 (4.7–9.6)

0.164 0.479

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

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 HIVuninfected women, whereas no significant weight change among HIV-infected women was observed. Significant increases in other anthropometric indicators, such as arm muscle circumference, 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 decreased (Table 4). However, no significant change in serum zinc was observed during the study period in either group. Hemoglobin increased significantly over the 8-wk period, whereas reductions 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

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

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Energy (kJ) Male Female Protein (g) Male Female Carbohydrate (g) Male Female Fat (g) Male Female Dietary fiber (g) Male Female Vitamin A (RE) Male Female Zinc (mg) Male Female

Placebo group (49 M, 24 F)

98

35.8 61.5 155.0 8.3

54 49 49 53 52 59 59 59 (28.7–48.5) (55.5–70.0) (135.0–169.0)4 (4.4–26.8)4 41 6 4.64 13.1 6 1.64 7.4 6 2.64 4.6 6 2.34 63 61 61 63 63 67 67 67

5

4

3

CRP, C-reactive protein; WBC, white blood cell. Median; interquartile range in parentheses (all such values). Significant change over the 8-wk period in both groups, P = 0.0003 (ANCOVA). Significant change over the 8-wk period in both groups, P , 0.0001 (ANCOVA). Mean 6 SD (all such values). 1

76 72 72 75 75 76 76 76 Retinol (lg/dL) Zinc (lg/dL) Copper (lg/dL) CRP (mg/L) Albumin (g/L) Hemoglobin (g/dL) WBC (· 109/L) Neutrophils (· 109/L)

2

21.1 (15.1–27.8) 62 (53–71.8) 171 (143.5–198.5) 50.9 (36.6–73.8) 35.9 6 5.65 11.8 6 1.7 10.3 6 3.6 7.6 6 3.3

76 72 72 75 74 76 76 76

21.2 (15.7–28.9) 59 (51.8–65.3) 176.8 (153.5–197.4) 50.1 (31.2–71.5) 35.8 6 4.4 11.8 6 1.9 10 6 3.6 7.5 6 3.3

72 70 70 70 69 72 72 72

32.9 (25.0–44.6) 67.5 (57.0–76.0) 172.8 (154.0–200.0) 27.9 (14.7–43.2) 37.0 6 5.6 8.6 6 3.5 5.8 6 3.2 5.8 6 3.2

63 60 60 63 63 65 65 65

32.7 (24.0–41.1) 60.5 (52.8–68.8) 164.0 (149.0–196.5) 16.9 (8.7–30.5) 37.0 6 5.0 12.0 6 1.8 8.5 6 3.3 5.7 6 2.9

40.3 62.5 152.0 11.9

Placebo group n Micronutrient group n Placebo group n Micronutrient group n Placebo group n

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treatment responses in patients with first-time pulmonary tuberculosis (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 (35–37). 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 with strongly 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 concentrations during the study period in both treatment groups. Most of our participants had serum concentrations consistent with a deficiency of retinol (,20 lg/dL) at baseline. An important factor that contributes to low retinol concentrations in active tuberculosis 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 decrease within days of starting antitubercular therapy in pulmonary 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 treatment without any supplementation of vitamin A showed a significant 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 presence 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

Week 2

Rx, treatment group; Rx week, interaction term.

2

0.403 0.322 ,0.0001 ,0.0001

Micronutrient group

0.514 0.033 0.154 0.392

n

20.128, 20.107, 0.122, 0.257,

TABLE 4 Laboratory variables of participants at baseline and 2 and 8 wk1

1

20.383 0.109 0.138 0.325

P

Week 8

Rx Rx week Week Baseline TTD

95% CI

Baseline

Coefficient

3

TABLE 3 Log-logistic regression estimates for time-to-detection (TTD) data1

(27.7–43.2)3 (54.0–69.0) (123.5–174.0)4 (2.9–17.3)4 41 6 5.04 13.3 6 1.64 7.0 6 2.84 4.4 6 2.44

VISSER ET AL

VITAMIN A AND ZINC IN PULMONARY TUBERCULOSIS

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 the 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 for the biochemical analyses; A Menezes, N Chegou, and M de Kock from the Medical Research Council Centre of Molecular and 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 follows—MEV, 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 manuscript for publication.

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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 metallothionein. The latter is an intracellular metal-binding protein that 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 reduction in serum copper in both treatment groups, which suggested an indirect effect of the reduction in the inflammatory response, rather than a direct antagonistic effect of zinc supplementation on serum copper concentrations. Few trials have examined the effect of micronutrient supplementation on other tuberculosis treatment outcomes. A trial from Dar es Salaam, Tanzania, showed that multinutrient supplementation without zinc reduced the risk of tuberculosis recurrences between 1 and 8 mo after the start of treatment in HIV-positive participants, but not in those who were HIV-negative (43). Three trials reported on the effect of supplementation of multimicronutrients on the mortality of participants with pulmonary tuberculosis (43–45), only one of which showed a significant reduction in the mortality of HIV-positive participants receiving a multimicronutrient 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 concentrations tend 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.

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