Methods
TB, alcoholism malabsorption, cancer, autoimmune liver, chronic disease, diabetes
PULMONARY TUBERCULOSIS, CANCER, RENAL FAILURE, CHRONIC DISEASE, BLOOD, EXTRAPULMONARY TUBERCULOSIS, HYPERTENSION, DIABETES
The research design was the true Experiment Desain Pretest–Posttest dengan Grup Kontrol (Pretest–Posttest with Control Group) which examined the effect of vitamin D supplementation on TLR2 and TLR4 as well as clinical outcomes measured before and after administration of vitamin D supplementation. This research was conducted from July 2022 to December 2022. Subjects were selected by a convenience random sampling method. The total number of subjects found was 53 participants who were assessed for eligibility in this study. However, 11 participants did not meet the inclusion criteria, and one participant declined to participate in this study because of refusal to have blood samples collected. The inclusion criteria were TB patients aged 19–50 years, who were diagnosed with hematology, MRI, and GenExpert. Subjects had to sign a consent form. The exclusion criteria were pulmonary tuberculosis, other extrapulmonary tuberculosis, and any chronic disease (renal failure, diabetes, hypertension, autoimmune liver, cancer, alcoholism malabsorption syndrome). (Fig. FlowchartBefore supplementation, 41 subjects met inclusion criteria and agreed to involve in this study. Three participants were excluded because they died in the middle of the study and two participants were excluded due to loss to follow-up. All the subjects were divided into three groups. The study group received vitamin D supplementation at 10,000 IU/day and 5,000 IU/day, and the control group received 400 IU/day. Oral vitamin D supplementation was started concurrently with anti-TB drugs. A total of 36 participants were treated and completed with supplementation and blood sampling; 12 participants were placed in the vitamin D dose group of 10,000; 12 participants were included in the vitamin D dose group of 5000 as a study group; and 12 participants were placed in the 400-dose vitamin D group as a control group. (Fig. Blood samples were collected after 4 and 8 weeks of supplementation. Three milliliter of venous blood samples were collected from the subjects. The sample was immediately centrifuged for 10–15 min to separate the blood serum. The blood serum was put into Eppendorf tubes and stored in a freezer at –20◦C and taken to the laboratory. The samples were processed for ELISA analysis. TLR2 and vitamin D concentrations were recorded in ng/ml, and TLR4 was recorded in pg/ml.All reagents were incubated at room temperature (18–25 °C) before use. After that, 30 mL of concentrated wash buffer was diluted with 720 mL of deionized or distilled water to prepare 750 mL of wash buffer. The standard will be centrifuged at 10,000 × g for 1 min. The sample diluent and 1.0 mL of reference standard were allowed to stand for 10 min and inverted gently several times. Each well was filled with 100 μL standard or sample and incubated for 90 min at 37 °C. The liquid was removed, and 100 μL of biotinylated detection was added. The sample was incubated for 1 h at 37 °C and washed three times. HRP Conjugate 100 μL was added. The samples will be incubated for 30 min at 37 °C. Then, the sample was washed five times. Substrate reagent (90 μL) was added and incubated for 15 min at 37 °C. The stop solution (50 μL) was added. Then, all the samples will be read at 450 nm in an ELISA microplate reader.Statistical analysis was performed with SPSS v.23 using a descriptive data normalization test and bivariate analysis of numerical data. Kolmogorov–Smirnov ANOVA test, repeated ANOVA test, and Pearson test. The statistical test is significant if the 