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Background: Low vitamin D levels may increase the risk of tuberculosis disease; however, previous observational cohort studies showed variable results. We investigated the relationship between vitamin D levels in infancy and subsequent development of tuberculosis disease throughout childhood. Methods: We enrolled pregnant women at 20-28 weeks’ gestation attending antenatal care in a periurban South African setting in the Drakenstein Child Health Study. Serum 25(OH)D concentrations were measured in newborn infants aged 6-10 weeks. Children were followed prospectively for tuberculosis infection and disease using annual tuberculin skin testing, radiographic examinations, and microbiological diagnosis with GeneXpert, culture, and smear testing. Univariable and multivariable Cox regression was performed and HRs with 95% CIs were calculated. Results: Children were followed for tuberculosis disease for a median of 7.2 years (IQR, 6.2-7.9). Among 744 children (<1% with human immunodeficiency virus (HIV), 21% HIV-exposed without HIV), those who were vitamin D deficient in early infancy were not at increased risk of developing tuberculosis disease (adjusted HR,. 8; 95% CI,. 4-1.6). Infants in the lowest vitamin D concentration tertile were at similar risk of tuberculosis as the highest tertile (adjusted HR,. 7; 95% CI,. 4-1.4). Vitamin D deficiency was associated with tuberculin conversion ≤2 years of age at a <30-nmol/L (adjusted OR, 1.9; 95% CI, 1.2-3.2), but not <50-nmol/L (adjusted OR, 1.5; 95% CI,. 8-2.9), cutoff. Conclusions: In a setting with hyperendemic rates of tuberculosis, vitamin D concentrations in infancy did not predict tuberculosis disease at any point in childhood. However, very low vitamin D levels were associated with tuberculin conversion in young children.
The study cohort as well as the subcohort tested for vitamin D has been described previously [3, 14, 15]. Briefly, we enrolled pregnant women between 20 and 28 weeks’ gestation attending antenatal care in Paarl, a periurban setting outside of Cape Town, South Africa. Participants were recruited from 2 neighboring community clinics, TC Newman and Mbekweni, serving impoverished communities. Infants received the bacillus Calmette–Guérin (BCG) vaccination at birth. All mothers accessed care in the public sector, which has a strong primary healthcare program, including an effective mother-to-child human immunodeficiency virus (HIV) prevention and antiretroviral therapy program. Women were followed through pregnancy and childbirth, and newborn infants were followed into childhood. Vitamin D supplementation was not routinely provided to infants in the national health services, unless born premature. Exclusion criteria for pregnant women were being younger than 18 years and intending to leave the area within 1 year. We obtained ethics approval from the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee (reference numbers 401/2009 and 651/2013) and the Provincial Child Health Research Committee. Mothers provided written informed consent at enrollment, which was renewed annually. Surveys focusing on maternal health were administered at enrollment and antenatal data were concurrently collected. Detailed birth information was obtained at delivery. Obstetric care and all births took place at the regional hospital in Paarl. Follow-up visits, including clinical examinations, were done at 6, 10, and 14 weeks; 6 and 12 months; and then annually until the end of follow-up. Data for environmental exposures, household characteristics, respiratory risk factors, anthropometry, and child symptoms were obtained at scheduled visits. Missed visits were rebooked with a study mobile phone network system or by study community-based fieldworkers. Mothers were counselled about respiratory symptoms at every visit and advised to attend the study site or contact study staff between scheduled study visits whenever the child developed cough or difficulty breathing. All mothers were tested for HIV during pregnancy with Abbott Determine HIV 1/2 rapid HIV antibody test (Abbott Laboratories, North Chicago, IL, USA). If positive, a confirmatory enzyme-linked immunosorbent assay was done. All mothers living with HIV received ART as per national guidelines. Infants of mothers living with HIV were tested with DNA polymerase chain reaction (PCR; Cobas Ampliprep System; Roche Molecular Systems, Branchburg, NJ, USA) at age 6 weeks and 6 weeks after the end of breastfeeding as per national guidelines. Children were re-tested at 18 months with the rapid antibody test [16, 17]. Serum samples were taken from infants between 6 and 10 weeks of age. Vitamin D status was assessed through serum 25-hydroxyvitamin D [25(OH)D] concentration (nmol/L) and measured at Vitas AS (Oslo, Norway; a reference laboratory in Europe with a Vitamin D External Quality Assessment Scheme certificate) from specimens stored at –80°C using liquid chromatography–tandem mass spectrometry. Clinicians did not have access to vitamin D results when making diagnoses, as measurements were done on biobanked samples several years after collection. Data were collected on infant factors relevant to vitamin D and tuberculosis disease risk based on prior studies with this cohort [3, 15] as well as the medical literature [18]. Infant variables included sex, height-for-age z score (HAZ), weight-for-age z score (WAZ), maternal HIV, gestational age, breastfeeding practices in the first year of life, and season of birth. Season of birth was categorized into summer (December–February), autumn (March–May), winter (June–August), and spring (September–November). We also collected maternal factors including age, smoking, educational level achieved, and various markers for household socioeconomic status. Socioeconomic status was assessed using a validated composite score comprising 4 variables including asset ownership, household income, employment, and education [14]. Tuberculin skin tests were done at the 6-month visit and then at 12, 24, 36, 48, and 60 months of age, and at the time of a lower respiratory tract infection (LRTI) [3]. Tuberculin skin test conversion was defined as an induration reaction greater than or equal to 10 mm, to minimize the risk of misclassification due to BCG vaccination or exposure to environmental mycobacteria [19, 20]. As tuberculin skin test boosting may occur after recurrent tests, children with reactive but negative skin test results (1–9 mm) were not given another test and were censored from conversion analysis at that time point. Because most children were censored by 2 years of age in our cohort, we only used tuberculin skin test results before this age in this analysis. Children with positive tuberculin skin tests were screened for tuberculosis disease and, if none, were referred to local tuberculosis clinics for isoniazid preventive therapy. Children were followed up for tuberculosis disease from birth at regular study visits as previously described [3]. Tuberculosis disease was diagnosed by experienced physicians and nurses in local tuberculosis community clinics or by study staff, and chest radiographs were read and reported by an experienced clinician. Trained staff collected induced sputum for microbiological confirmation using liquid culture and nucleic acid amplification (Xpert MTB/RIF; Cepheid, Sunnyvale, CA, USA) from children with a tuberculin skin test induration of 10 mm or greater, those presenting with an LRTI, and in participants in whom tuberculosis disease was considered presumptive. A chest radiograph was taken in all children with presumptive (or possible) tuberculosis disease. Children were included in this analysis if they had a vitamin D measurement at 6–10 weeks of age. We summarized continuous variables as medians with interquartile ranges (IQRs) and categorical variables using proportions. Our primary outcome was tuberculosis disease incidence after 10 weeks of age to the end of follow-up (30 January 2021). For tuberculosis disease incidence, time-to-event was constructed between birth and the date of tuberculosis. Follow-up was censored at death, development of tuberculosis disease, end of follow-up, or until 30 January 2021. We compared tuberculosis disease incidence in infants with and without vitamin D deficiency using hazard ratios (HRs) and 95% confidence intervals (CIs) obtained from Cox proportional hazards models. We completed this analysis independently for the entire follow-up and then conducted a landmark analysis (ie, analyzing only subjects at that time point still eligible for the analysis) prior to 1 year of age. Two-sample likelihood ratio tests were used. To assess whether there was a dose–response relationship between vitamin D concentration, we categorized vitamin D concentrations into tertiles, as used elsewhere and to enable comparison with such studies [11, 12, 15]. We compared incident tuberculosis disease among participants at each tertile level. We also compared our results using vitamin D cutoffs. Children were categorized into distinct categories based on their serum 25(OH)D concentration, including deficient (<50 nmol/L), insufficient (50–74 nmol/L), and sufficient (≥75 nmol/L). There is no consensus on the definition of vitamin D deficiency [18]. Due to this, we conducted separate analyses with <50-nmol/L and <30-nmol/L cutoffs for vitamin D deficiency in our cohort [18]. Last, we assessed the relationship between vitamin D levels and tuberculin conversion at 1 year of age or younger and 2 years of age using logistic regression models. We assessed the odds of a tuberculin conversion at 1 year of age or younger or 2 years of age independently for each vitamin D deficiency cutoff and in each vitamin D tertile. Multivariable models were built including all relevant variables related to tuberculin conversion in this cohort [3, 15]. All analyses were performed using Stata (version 14.1; StataCorp, College Station, TX, USA).
