Vitamin D: Level of Vitamin D3 in AFB Positive PTB Patients in Initial Diagnostic Phase

Background: A relationship among vitamin D and pulmonary tuberculosis. Objective: To conclude the occurrence of Vitamin D insufficiency in subjects suffering from pulmonary TB. Design: This was a cross sectional study. Setting: Current study was performed at department of medicine, Peoples Medical College Hospital Nawabshah starting from January 2017 to December 2017. Samples: 385 AFB positive subjects of pulmonary tuberculosis after achieving the criteria of selection were recruited. Material and Methods: After a brief discussion, the patients were characterized for variable evaluation such as age, gender, sputum AFB and duration of disease and presence of low vitamin D levels. Sputum AFB criterion was used for Data collection. Fasting blood samples were collected for analysis of vitamin D levels in all subjects of AFB positive pulmonary tuberculosis. Results: In 385 diagnosed patients with smear positive PTB, 273 (70.9%) males and 112 (29.1%) were female. Vitamin D levels were within normal or sufficient range in 134 (34.8%), below normal range in 251 (65.2%) cases (insufficient in 09.56% (24/251) and deficient in 90.43% (227/251) cases). Conclusion: Decreased levels of vitamin D were common in subjects with AFB smear Positive PTB cases.


Introduction
Tuberculosis (TB) is the most important community health issue worldwide.
In 2011 it was assessed that about 8.7 million new cases of tuberculosis were reported and about 1.4 million deaths was related to this disease [1].Association of vitamin D deficiency/insufficiency as a cause of Tuberculosis or its manifestations as malnutrition is still undetermined.Incidence and progression of tuberculosis had been related with many factors and vitamin D deficiency is considered as one of them [2].Low levels of vitamin D were noted in subjects with Tuberculosis in comparison to controls.Vitamin D deficiency had been related with higher risk of Tuberculosis, also increased risk of developing active tuberculosis in subjects with latent tuberculosis infection had been noted [3].The chief source of vitamin D in humans remains the sun exposure.The conversion of 7-dehydrocholesterol to vitamin D3 via pre-vitamin D3 in human derma is induced by sun exposure.Liver and kidneys play essential role in the activation of vitamin D3 from 25-hydroxyvitamin D (25(OH)D) to bioactive form 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [4].In a meta-analysis on relationship among vitamin D deficiency and tuberculosis, increased risk of active tuberculosis was observed in subjects with decreased levels of this vitamin [5].The bioactive form of vitamin D3 binds with receptors of vitamin D (VDR), activation of VDR signaling and induction of mitochondrial response series leads to autophagy, fusion of phagolysosome, cathelicidin release and activation, and intracellular death of Mycobacterium Tuberculosis bacilli [4] [6] [7].
Studies had been conducted throughout the entire world on tuberculosis and vitamin D levels but no study had been carried out here in our setup.Vitamin D deficiency and pulmonary tuberculosis are the major issues of our population.This study will elaborate the association of vitamin D in subjects with AFB positive tuberculosis because both of these issues are linked with morbidity and mortality as reported all over the globe.This study will help in making the public health policies keeping in view with vitamin D levels and tuberculosis.This study will determine the frequency of decrease levels of vitamin D in subjects with different gender groups in tuberculosis and to find out whether vitamin D has any protective role in the tuberculosis.
The rationale behind current study was that there is paucity of such type of researches in our population and on other hand most of the previous studies were done on retrospective data.Further research on association of vitamin D with pulmonary tuberculosis in Pakistan is needed.This study estimates the risk of acquiring tuberculosis in relation to different levels of vitamin D in Pakistani population.

Operational Definitions
PTB: Pulmonary tuberculosis is a commonailment of lungs results due to infectious agent mycobacterium tuberculosis, a small rod shaped bacilli causing dis-eases in mankind.
Sputum AFB: This is a laboratory investigation also known as acid-fast bacillus (AFB) stain or a tuberculosis (TB) smear and accomplished on a sample of sputum from subjects suspected for tuberculosis to conclude if a subject has tuberculosis or other category of mycobacterial infection [8].

Material and Methods
Inclusion criteria: All male and female patients with clinical history of pulmonary tuberculosis and smear positive sputum for AFB were included.
Exclusion Criteria: Patients not willing for taking part in study, known cases of respiratory diseases other than PTB.

Ethical Consideration
Approval of study was sought from the hospital ethics committee PMCH Nawabshah.Permission for data collection will be taken from the head of department of the Medicine.Written informed consent will be obtained from adult subjects while ensuring that the data will be kept confidential.People will be thoroughly informed about the objectives and methods of the study.

Data Collection
After a brief discussion, the patients were characterized for variable evaluation such as age, gender, sputum AFB and duration of disease and presence of low vitamin D levels.Sputum AFB criterion was used for Data collection.Fasting blood samples were collected for analysis of vitamin D levels in all subjects of AFB positive pulmonary tuberculosis.Data were collected through interview based questionnaire.Sample size was calculated used 95% level of confidence and margin error of 5%, from 1.6 million populations with 50% distribution response rate through the Rao-software.Sample size included 385 participants.Subjects grouping were done with positive sputum AFB for tuberculosis to analyze the frequency of low levels of vitamin D levels and AFB positivity.
Sputum examination was done after preparation of the sputum slide for ZN staining for proper microscopic examination of AFB.
Collected blood samples were stored before to examination.Total serum levels of vitamin D were analyzed by automatic analyzer.Convenience sample procedure was performed.This Current study was hospital based, subjects aged more than 20 years and diagnosed cases of PTB with sputum AFB positive were included.

Statistical Analysis
SPSS version 20.0 was used for collected data analysis.Frequencies & percentag-es were calculated for variables such as sex, Sputum AFB & vitamin D levels.
Quantitative variables as age were calculated for mean and standard deviation.Importance of vitamin D was observed in subjects with pulmonary TB with relation to age, sex, and sputum positive for AFB and PTB duration to conclude the effect on consequences.

Results
A total of 1245 subjects suspected of Pulmonary Tuberculosis who presented with different symptoms like cough, fever, and weight loss, loss of appetite, body ache, and difficulty in breath were included.They were advised sputum for AFB and 385 cases with AFB positive were included.In parallel to this 144 healthy controls were selected in respect to age and gender for vitamin D levels.9).
In relation to tuberculosis parameters and vitamin D levels paired sample test was performed with mean and SD, upper and lower limits, with 95% confidence interval as shown in Table 10, the p-value was statistically significant vitamin D level with pair of vitamin D and age group < 0.002, address < 0.002, economical status < 0.000, occupation < 0.000, sun exposure < 0.000, BCG vaccination < 0.000 and family history of PTB < 0.000.While there was insignificant statistical relation of gender 0.105 and education 0.629 (Table 10).The hazard of tuberculosis (TB) infection is related to the decreased levels of vitamin D [10] [11].Current research also declares the low levels of vitamin D in subjects who were suffering from the pulmonary tuberculosis.
Subjects with vitamin D deficiency had increased vulnerability to acquire the tuberculosis [12] and worse progression of disease in tuberculosis subjects [12] [13].Although the current study was not focusing the before analysis of vitamin D levels in subjects who are at increased risk level for the development of pulmonary tuberculosis.
Receptor for vitamin D (VDR) is polymorphic nuclear receptor which con- tion and treatment of tuberculosis [3].In present study male patients of different age groups 170 (62.3%) were vitamin D deficient, while out of 112 female patients 81 (72.3%) were vitamin D deficient.The majority of population belonged to young age group and HIV is not a common risk factor here for tuberculosis in our setup.
Nnoaham et al. in a meta-analysis on the relationship among vitamin D and tuberculosis had observed decreased levels of vitamin D in subjects with tuberculosis vs. controls and vitamin D deficiency confidently related with high risk of tuberculosis [5].In current research the mean serum vitamin D3 level of patients was 26.98 with SD 22.10 ng/ml.The mean vitamin D3 levels of healthy controls were 34.46 with SD 44.4 ng/ml these results are matchable with the meta-analysis by Nnoaham et al.
Supplements of vitamin D were not significantly beneficial in tuberculosis subjects on treatment as denoted by Xia et al. in 2012, their analysis did not discourse the query whether it could be beneficial in prevention of tuberculosis.More controlled studies are further required to conclude about the beneficial effects of supplement vitamin D in tuberculosis patients [22].
Levels of bioactive form of vitamin D (1,25(OH)D3) were higher in tuberculosis subjects in comparison to controls without tuberculosis [4].Selvaraj et al. suggested that such rise in 1,25(OH)2D3 may be due to CYP27B1 expression up regulation that led to augmented transformation of 25(OH)D to 1,25(OH)2D3, that can be the reason for 25(OH)D lack since of its amplified use [23].
The role of vitamin D in prevention and treatment of pulmonary tuberculosis still needs much more clarifications in future.The test availability at government setup is not available at large scale.There should be vitamin D screening in general population throughout the nation for to address the exact levels of deficiency and future plan to manage it properly.Early identification and diagnosis of vitamin D deficiency may prevent the burden of some morbid diseases which may result in morbidity and mortality.

Limitations
In current study there are certain restrictions and strengths.Current study demonstrates completely most features of association of vitamin D and tuberculosis, and solved the query about vitamin D deficiency was cause or sequel of tuberculosis.Current analysis was based on most of the available quality data to ensure the outcomes.Certain limits are also concerned with this study such as number of related studies was restricted with small sample size that indeed may have certain effects on study outcomes.Moreover inconsistency in VDD definition was seen due to the different standards for VDD in different studies that could affect our study outcomes.

Conclusion
Current study had shown an association between serum levels of vitamin D and

Table 1 .
Statistics of Vitamin D in patients and control Group.

Table 3 .
Frequency and percentage of different symptoms of PTB and non parametric test N = 385.

Table 4 .
Vitamin D, Age Group and Gender.

Table 5 .
Vitamin D, Age Group and sun exposure.

Table 6 .
Vitamin D, Age Group and economical Class.
Vitamin D deficiency was significantly associated with education, occupation, sun exposure and family history of pulmonary tuberculosis.There was signifi-DOI: 10.4236/jtr.2018.The paired sample testing and paired samples correlation were analyzed and found statistically significant.Various means and standard deviations with standard error of mean were checked in parallel to correlations and significance as shown in the p-value that was statistically significant vitamin D level with occupation (<0.000), sun exposure (<0.000) and family history of tuberculosis (<0.003).While age group (<0.828), gender (<0.060), address (<0.874), education (<0.032), economical class (<0.533) and BCG vaccination was statistically insignificant in paired sample testing and paired correlations (Table

Table 7 .
Correlations of vitamin D and different variables of study N = 385.

Table 8 .
Correlations of vitamin D and different symptoms of PTB, N = 385.

Table 9 .
Vitamin D and demographic variables (paired statistic and correlations).

Table 10 .
Vitamin D and different parameters (paired sample tests).
[20]s the expression of genes responsible for immune function.The process by which tuberculosis infection may be limited or prevented is through the binding of bioactive form of vitamin D binds with VDR[14][15][16].The role of vitamin D after development of Pulmonary TB needs randomized control trials to check out the effect after therapy.In Pakistan Najeeha Talat et al. determined the serum levels of vitamin D on subjects with tuberculosis and their contacts; they observed that 79% subjects were vitamin D deficient (<20 ng/ml), 14% with insufficient and 07% had sufficient vitamin D levels[12].The results of current research are also at match able position to Najeeha study as low serum vitamin D levels were seen in 60.00% AFB positive PTB patients in our study.There is 5 fold augmented risk for tuberculosis progression in subjects with vitamin D deficiency.Sutaria et al.[20]observed the association among deficient vitamin D status and tuberculosis, relationship among VDR polymorphism and vulnerability of tuberculosis and also evaluated part of vitamin D supplements in the prevention and treatment of tuberculosis.They concluded that subjects with tuberculosis had low vitamin D in comparison to healthy age and sex matched controls[21], subjects with convinced VDR polymorphisms (BsmI and FokI) were found to have high vulnerability for tuberculosis and subjects who were given supplementary vitamin D showed the enhanced results in most of studies[20].The needs of retrograde studies are essential here to look in this regard.
achieve the statistical significance, thus representing that vitamin D deficiency expected as a risk agent rather than a result of tuberculosis.Further researches are required to analyse that either vitamin D supplements are helpful in preven-A.A.Jamali et al.DOI: 10.4236/jtr.2018.64023267 Journal of Tuberculosis Research