Introduction


Introduction:
The incidence of tuberculosis (TB) is increasing in Egypt, as well as other developing countries, especially among the immune compromised population 1. The global incidence of TB is estimated to be 10.4 million (90% adults; 65% males) new cases per year 2. The standard, antituberculosis therapy (ATT) shows efficacy for treatment of TB; it consists of isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB). However, the rate of therapeutic failure among the Egyptian patients with pulmonary TB ranges between 23% and 29% 3, 4. Vitamin D is one of the potential factors that may be involved in TB 5.
Vitamin D deficiency is estimated to affect between 24% and 77% of the general population in Egypt 6, 7. Studies exploring the effect of vitamin D on the therapeutic outcome of ATT revealed conflicting results 8. While it was reported that supplementation of vitamin D accelerated both clinical and radiographic improvement in all patients with TB 9, such supplementation was not successful in improving the rate of sputum clearance from Mycobacterium tuberculosis (Mtb) 10. Vitamin D binds to vitamin D receptor (VDR), activates VDR signaling, and induces a series of antimicrobial responses including induction of autophagy, phagolysosomal fusion, release and activation of the antimicrobial peptide “cathelicidin”, and killing of intracellular Mtb 11, 12.
Our research hypothesis is that vitamin D deficiency is prevalent among the general population in our locality. Vitamin D deficiency may contribute to the therapeutic failure of ATT; consequently, augmenting the standard ATT with cholecalciferol (vitamin D3) supplementation may lead to an improved therapeutic response of the patients with pulmonary TB compared to the standard ATT. We explored the role of adding cholecalciferol to the standard ATT in improving the therapeutic outcome among the naïve patients with pulmonary TB.

Patients and methods:
Study design:
A randomized, controlled, clinical study was conducted.
Study location:
The study population was recruited from the patients attending the outpatient clinics and admitted to the inpatient sectors of the department of Chest Diseases and the department of Tropical Medicine & Gastroenterology (Fever Unit), Assiut University, Egypt.
Study duration:
The study population was recruited during the period from October 2014 to August 2017.
Inclusion criteria:
The study included 500 naïve, patients with pulmonary TB eligible for ATT who accepted to be enrolled, consecutively. The patients were randomly allocated, by a computer-generated stratified, random assignments list (block randomization), to two groups. Group-A included 250 patients who received only ATT, while group-B included another 250 patients who received ATT with cholecalciferol supplementation (cholecalciferol-augmented ATT).
Pulmonary TB was diagnosed based on positive sputum culture on Lowenstein-Jensen medium performed by an experienced microbiologist, with or without the radiological evidence on chest radiography 13.

Exclusion criteria:
Pregnant or lactating patients, patients less than 18 years old, those with previous ATT, and those with extrapulmonary TB were excluded from being enrolled. In addition, we excluded patients with hypercalcemia, chronic liver disease and/or elevated serum levels of aminotransferases, chronic kidney disease and/or elevated serum level of creatinine, any alcohol intake, and those receiving hepatotoxic drugs other than ATT, as well as those receiving corticosteroids or antimetabolites for any other indication. Patients with human immunodeficiency virus (HIV) infection were not included in the study.
Methods:
All the participating patients had pretreatment evaluation including clinical evaluation (medical history and physical examination with estimation of weight and height), imaging studies (chest radiography and abdominal ultrasonography), laboratory investigations (serum level of calcium, fasting serum level of glucose, liver chemistry panel, virology panel, kidney chemistry panel, and complete blood count). Body mass index (BMI) was calculated according to the following: body weight (kg) / height (m)2. Diabetes mellitus (DM) was defined as serum level of glucose of 7 mmol/L or more. Liver chemistry panel included estimation of serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, bilirubin, and albumin. Virology panel included testing for serum antibody to hepatitis C virus (anti-HCV Ab), hepatitis B surface antigen (HBsAg), antibody to hepatitis B core (anti-HBc Ab), and antibody to human immunodeficiency virus (Anti-HIV Ab).
All the enrolled patients received the World Health Organization (WHO)-recommended, standard ATT therapy: INH (5 mg/kg/day; the maximum dose was 300 mg/day), RIF (10 mg/kg/day; the maximum dose was 600 mg/day), PZA (30 mg/kg/day; the maximum dose was 2000 mg/day), and (EMB) (20 mg/kg/day; the maximum dose was 1600 mg/day) for two months, followed by INH and RIF for extra four months 14. All patients received pyridoxine (vitamin B6, 50 mg/day) during the whole treatment period for prophylaxis against isoniazid-related peripheral neuropathy 15. Group-B patients, in addition to ATT, received cholecalciferol supplementation during the whole treatment period (600 IU/day, orally, immediately after lunch). The dose of cholecalciferol was determined according to the recommended dietary allowances for persons 19-70 years old 16. Higher doses of cholecalciferol were avoided to decrease the risk of hypercalcemia. Vitamin D3 was administered with the largest meal to improve its absorption 17. Cholecalciferol supplementation was produced by the same pharmaceutical company.
Follow up included clinical evaluation and laboratory investigations. Daily clinical evaluation during the period of hospital admission, followed by clinical evaluation and laboratory investigations (liver chemistry panel) at the outpatient clinic on weekly basis for the first two months of treatment period, and then monthly for rest of the period, were carried out. Sputum culture on Lowenstein-Jensen medium was performed twice, at the third month and at the fifth month of therapy, for all the patients. Early therapeutic response to ATT was defined as negative sputum culture at the third month during the treatment period, while therapeutic failure of ATT was de?ned as positive sputum culture at the fifth month 18.
For the patients who had elevated serum levels of ALT and/or AST (three times the upper limit of normal (ULN) or more with symptoms or five times the ULN with or without symptoms) during the first two months of ATT, RIF and PZA were discontinued immediately. Less hepatotoxic drug (streptomycin (SM)) was used in addition to INH and EMB until RIF and PZA were resumed after decline of ALT and/or AST serum levels to less than two times the ULN. The dose of SM was 15 mg/kg/day by intramuscular injection with a maximum daily dose of 1000 mg. For patients with the same criteria but after the first two months of ATT, RIF was replaced by EMB temporarily until it was resumed according to the previously mentioned rules 14.
Statistical analysis:
Data were analyzed using the Statistical Package for Social Sciences (IBM SPSS Statistics, version 22.0, release 22.0.0.0; IBM Corp, Armonk, New York, US) for Microsoft Windows® (64-bit version). Results were expressed as mean ± standard deviation or frequency (percentage) as appropriate. Student’s t-test or Mann-Whitney U test, and Yates’ corrected chi-squared test or Fischer’s exact test as appropriate were used to compare the variables between the study groups. A p value less than 0.05 was considered statistically significant.
The software G*Power version 3.1.9.2 was used for a post hoc power analysis of the performed chi-square tests. An arbitrary effect size was chosen for the power analysis, which precisely was a Cohen’s w statistic of 0.3. This value conventionally corresponds to a medium sized effect. The power achieved was 0.84.
Ethical considerations:
The study was conducted after approval of the Clinical Research Ethical Committee of Assiut Faculty of Medicine, and was carried out according to the code of ethics of the World Medical Association (Declaration of Helsinki). All the participants signed a consent certi?cate after discussing in detail with the investigators the certi?cate subjects and the study aim. Participants were clearly informed that refusing to participate in the study will not affect having full bene?t of the available medical service. Data con?dentiality was respected.

Results:
After exclusion of three patients of group-A (due to development of severe ATT-related fulminant hepatopathy leading to death of one patient and acute liver failure of two more) and a single patient of group-B (died because of motor vehicle accident), the study included 496 patients (247 of group-A and 249 of group-B). Pretreatment demographic, clinical, radiological, and laboratory characteristics of the study population are shown in table-1. The two groups were matching. The mean age of the study population 32.5±10.8; the mean age of group-A patients was 31.9±10.4, while it was 33.2±11.1 for those of group-B. The female patients represented 55.6% of the study population; 53% of patients of group-A, and 58.2% of those of group-B. Among 5.8% of the study population, DM was detected (7% of patients of group-B and 5% of those of group-A. Fever was present among 66.1% of the study population (69.6% of group-A patients and 62.7% of those of group-B).
Table-2 shows the therapeutic outcome of ATT among the study population. The overall rate of therapeutic failure among the study population was 29.4%; the rate was significantly lower among patients of group-B compared to those of group-A (22.1% (95% CI 14.7-26.2) vs 38.1% (95% CI 31.5-46.1), p 0.036). The rate of early therapeutic response was significantly higher among patients of group-B compared to those of group-A (35.3% (95% CI 29.6-42.3) vs 19.4% (95% CI 15.1-24.6), p 0.041). Comparing the patients of group-B to those of group-A, the late therapeutic response was nearly equal among the patients of both groups. The therapeutic outcome of the study population is presented in figure-1. All the patients with therapeutic failure (positive sputum culture at the fifth month of ATT) had a positive sputum culture at the seventh month of ATT.
Regarding the adverse effects of ATT among our study population, the overall incidence rate was 19.3%; it was higher among patients of group-A compared to those of group-B (21.9% vs 16.9%). However, this difference was not statistically significant. The most frequent adverse effect among the study population was elevated liver chemistry (9.9%). Patients of group-A compared to those of group-B, had significantly higher rates of elevated liver chemistry (15% vs 4.8%, p 0.001), jaundice (2% vs 0.8%, p 0.008), and myalgia (4.1% vs 2.4%, p 0.047).

Discussion:
Cholecalciferol-augmented ATT needs to be considered as the standard of care therapy for the naïve patients with pulmonary TB. According to the results of our study, the naïve patients with pulmonary TB who received vitamin D3 combined with ATT achieved significantly lower rates of therapeutic failure compared to those who received only the standard ATT. Moreover, the patients on cholecalciferol-augmented ATT compared to those on the standard ATT, had significantly higher rates of early therapeutic response; the earlier the therapeutic response, the less frequent the possibility of spreading the infection. The advantage of the therapeutic outcome gained by adding vitamin D3 to ATT was not on the expense of the safety. On the contrary, the patients who received cholecalciferol-augmented ATT had lower incidence rates of adverse effects compared to those who received the standard ATT, although this was not statistically significant except for the hepatic and muscular side effects, which were significantly lower among the patients with cholecalciferol-augmented ATT compared to those with the standard ATT.
Compared to the reported rates of therapeutic failure for ATT among the Egyptian, naïve patients with pulmonary TB, which ranges between 23% and 29% 3, 4, the overall rate of therapeutic failure among our study population with naïve pulmonary TB was 29.4% which is equal to the highest rate reported. A lower therapeutic failure rate of ATT when combined with vitamin D3 compared to the standard ATT (19% vs 27%) was reported among the Egyptian patients with naïve pulmonary TB who received vitamin D3 to decrease the ATT-related hepatopathy. However, this difference was not statistically significant. This might be attributed to the larger size of our study population (496 vs 300 patients). In addition, our study revealed significantly higher rate of early therapeutic response among the patients who received cholecalciferol-augmented ATT compared to those who received the standard ATT; the early therapeutic response was not evaluated by the study of Hasanain, et al 4.
Sufficient vitamin D3 levels in serum protects against tuberculin skin test conversion 19. Vitamin D insufficiency is a risk factor for tuberculosis in men 20 and is associated with progression to active TB among healthy subjects 21. The SUCCINCT Study showed that high doses of vitamin D accelerated the improvement of patients with pulmonary TB 10. Also, this accelerated improvement was previously evident among the pediatric patients with pulmonary TB 22. The clearance rate of sputum among patients with moderately advanced pulmonary TB is associated with vitamin D supplementation 23. In addition, among the patients with HIV infection, vitamin D3 supplementation reduces the incidence of pulmonary TB 24.
A single, large, oral dose of ergocalciferol significantly reduces the growth of Mtb 25. Vitamin D metabolites contribute to Mtb elimination via enhancement of macrophage phagocytosis, activation of monocytes, cytokines modulation, limitation of intracellular growth of Mtb, and suppression of enzymes involved in pulmonary cavitation 25, 29. The immunomodulatory role of vitamin D in Mtb infection depends on the activation of cathelicidin-mediated mycobacterial destruction through binding with vitamin D receptors (VDR) 26. Vitamin D-mediated enhanced innate immunity of monocytes among patients with Mtb infection is related to the transcriptional activation of the antimicrobial peptide LL-37 11. Vitamin D supplementation enhances Mtb-induced interferon-gamma secretion in patients with vitamin D deficiency, leading to improved cell mediated immunity against Mtb 10. In addition, gene polymorphism of VDR contributes to TB susceptibility 27.
Disagreeing with our findings, vitamin D supplementation did not alter the therapeutic response among patients with pulmonary TB 28, possibly due to the high prevalence of obesity, alcohol use, and HIV infection among those patients. Also, among patients undergoing hemodialysis, vitamin D supplements was not associated with lower incidence of TB 29. This can be explained by the defective renal conversion of vitamin D to its active form 30. Two further studies reported lack of enhanced therapeutic outcome among patients with pulmonary TB who received high doses of vitamin D 31, 32. Nielsen, et al, reported the association of TB with both low and high levels of vitamin D 33. High levels of vitamin D may lead to downregulation of VDR expression resulting in their defective signaling 34.
The lower rates of hepatic adverse effects were previously reported among the Egyptian, naïve patients with pulmonary TB receiving vitamin D3 supplementation with ATT compared to those receiving the standard ATT 4. Deficiency of vitamin D can aggravate the damage to hepatocytes 35, 36.
The serum levels of vitamin D were not measured among our study population which is considered as a major limitation of the study. Also, antibiotic susceptibility testing for Mtb cultures was not available. However, this study included a large sample size and is considered the first of its type among the Egyptian, naïve, patients with pulmonary TB.
In conclusion, cholecalciferol-augmented ATT can be more efficacious in treating naïve patients with pulmonary TB compared to the standard ATT. In addition, adding vitamin D3 to ATT provides extra protection against the hepatic and muscular adverse effects of ATT. Considering vitamin D3 as a safe and inexpensive medication, it is recommended to be combined with the standard ATT. Further studies are encouraged to explore the different mechanisms by which vitamin D3 contributes to the improved therapeutic outcome of ATT.