Risk factors for active tuberculosis in 938 QuantiFERON-positive schoolchildren in Mongolia: a community-based cross-sectional study

Mongolia is a land -locked country with Russia to its north, and China to its east, south and west. Mongolia itself is thinly populated with only 3.1 million people. The capital, Ulaanbaatar, however, is densely populated, containing 46% (1.3 million) of the population. Secondary education is compulsory for children between the ages of 6 and 16.

TB is common in Mongolia. The World Health Organization (WHO) has estimated the incidence of active TB to be 428 per 100,000 people per year [9]. The prevalence of HIV infection is low, estimated at 0.02% of the population [10].

The study reported herein was approved by the Ethical Review Boards of the Mongolian Ministry of Health and the Mongolian National University, and by the Office of Human Research Administration at the Harvard T.H. Chan School of Public Health in Boston, MA, USA (IRB ref. no. 14–0513).

These children were enrolled as part of the clinical trial of vitamin D supplementation in TB prevention with broad eligibility criteria. Children between the ages of 6 and 13, whose parents gave written consent, and who themselves gave written assent, were enrolled, unless: 1) the family planned to move away from UB within the next four years; 2) they were taking over 10 micrograms per day of any form of vitamin D; 3) they showed evidence of rickets on physical exam (leg bowing, knock knees, pectus carinatum, thickened wrists or ankles; 4) they were known to be seropositive for Human Immunodeficiency Virus (HIV) or to have hyperparathyroidism, sarcoidosis, or previous latent or active TB;, 5) they were taking cytotoxic or immunosuppressant medication, enzyme-inducing anticonvulsants or cardiac glycosides.

We collected the following information from parents or legal guardians, using an electronic questionnaire connected to the REDCap database: age, gender, monthly household income, type and ownership of residence, highest education level attained by either parent, number of people sleeping per room, smoking by household members and the children themselves, and exposure to a case of pulmonary TB present in the household at any time during the child’s lifetime.

Height and weight were measured with light clothing, but no shoes, hair ornaments or hats. A portable stadiometer (SECA, Hamburg, Germany) was used to measure height to the nearest 0.1 cm. A digital floor scale (SECA) was used to measure weight to the nearest 0.1 kg. Body Mass Index (BMI) was calculated as weight (kg) divided by the square of the height (m). Parameters of body composition; impedance, % body fat and fat-free mass, were measured using a body composition analyzer (SC-331S, Tanita, Tokyo, Japan). Children were examined for evidence of a BCG scar.

We used QuantiFERON-TB Gold High Altitude tubes (Qiagen, Hilden, Germany) to collect 3 ml of venous blood, which we processed according to the manufacturer’s instructions by the Global Laboratory (G-Lab) in UB, which participates in the QuantiFERON Quality Assurance Program of the Royal College of Pathologists in Australia. Children with positive test results were sent to physicians at the Mongolian National Centre for Communicable Diseases (NCCD) for clinical and radiological assessment for active TB, which was diagnosed using published criteria [11]. Facilities for mycobacterial culture were not available. Those diagnosed with active TB were treated at NCCD clinics, or District TB Dispensaries with 2 months of isoniazid, rifampicin, pyrazinamide and ethambutol, followed by 4 months of isoniazid and rifampicin. Response to treatment was based on a clinical and radiological evaluation over the course of anti-TB treatment. Specifically, a ‘good response’ was evidenced by a) reduced cough, b) resolution of pyrexia, c) increased weight, d) increased playfulness /activity reported by parents or caregivers, and/or e) improved chest radiograph appearance (in cases where this was abnormal at baseline).QuantiFERON-positive children in whom active TB was excluded were not preventively treated for latent tuberculosis infection, in line with Mongolian National TB Program (NTP) (Minister order #306) and WHO recommendations [12].

25-hydroxyvitamin D levels were measured using an enzyme linked fluorescent assay (VIDAS 250H Vitamin D total; Biomerieux, Marcy-l’Etoile, France). The assay was accredited by the Vitamin D External Quality Assessment Scheme (DEQAS). The total Coefficient of Variation (CV) was 7.9%, mean bias was 7.7% and the limit of quantitation (LOQ) was 8.1 ng/ml. We standardized non-zero 25(OH) D values using a set of 40 DEQAS serum samples as previously described [13]. Values below the LOQ were imputed as 5.7 ng/ml (i.e. the limit of quantitation divided by the square root of two). We then calculated deseasonalized (season-adjusted) values for each participant from their individual standardized 25(OH) D concentration and date of blood sample collection as described elsewhere [14].

SAS software (version 9.4; SAS Institute, Cary, NC USA), STATA (version 15; StataCorp, College Station, TX USA) and Prism (version 7.03; GraphPad Software, San Diego, CA USA) were used to analyze data. We used a Wilson procedure without correction for continuity [15] to calculate 95% confidence intervals (CI) for the estimate of disease prevalence in QFT-G-positive children. Two multivariable analyses were conducted to identify factors that were independently associated with risk of active TB. Models were first adjusted for age and sex only. In the other models we additionally adjusted for all covariates that were associated with risk of active tuberculosis with P < 0.20 in the age- and sex-adjusted analysis, namely type of residence (centrally heated; not centrally heated; yurt), parental education (university/polytechnic; secondary school or lower), monthly household income, number of people per room, month of sampling, presence vs. absence of indoor smokers in the household, active smoking by the child, body mass index, % body fat, household exposure to an index case of pulmonary tuberculosis (PTB) and vitamin D deficiency, defined as serum 25(OH) D concentration < 10 ng/ml. We pre-specified this threshold based on findings of a meta-analysis reporting susceptibility to tuberculosis to be increased below this cut-off [16]. Risk ratios for the association between these independent variables and risk of active tuberculosis were estimated using generalized estimating equations (GEE) with the binary distribution, log link function and exchangeable working correlation structure [17]. We used Log-Poisson models when the log-binomial model failed to converge, which provide consistent but not fully efficient estimates of the risk ratio and its confidence intervals, [18]. We calculated Population Attributable Fraction (PAFs) and their 95% confidence intervals for potentially modifiable independent risk factors for active TB using STATA as previously described [19].