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 . 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 . 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 . 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) . 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 . 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 .
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.
PTB: Pulmonary tuberculosis is a commonailment of lungs results due to infectious agent mycobacterium tuberculosis, a small rod shaped bacilli causing diseases 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 .
Vitamin D Deficiency: Levels of serum 25(OH) D3 > 30 ng/ml are considered normal, levels > 20 and <30 ng/ml are insufficient and levels below < 20 ng/ml are labeled as deficiency .
2. 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.
2.1. 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.
2.2. 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.
2.3. Statistical Analysis
SPSS version 20.0 was used for collected data analysis. Frequencies & percentages 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.
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.
The average ages of patients were 37.80 years with SD of 15.05 years. The age ranged between 21 and 85 years. The mean age of healthy controls was 36.70 years with standard deviation was 14.14 years ranging between 20 and 78 years. Male to female ratio was 2.43/1 (273/112) in patients and in healthy control group it was 2.27/1 (100/44) out of 144. 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 (Table 1).
In Table 2 it is observed that 273 (70.9%) males and 112 (29.1%) were female. Out of all study subjects, 242 (62.9%) from young age, 101 (26.2%) from middle age and 42 (10.9%) were from old age group. There were 361 (93.8%) married patients while 24 (6.2%) were unmarried. Patients from rural setup were 209 (54.3%) while 176 (45.7%) belonged to urban areas. There were 202 (52.5%) subjects with primary to matriculation, 60 (15.6%) were intermediate to graduate and 123 (31.9%) uneducated. Socioeconomic class had shown dominant ratio of lower economic class 351 (91.2%), while 23 (6.0%), 11 (2.9%) belonged middle and upper class respectively. By occupation, 98 (25.5%) house wives, 146 (37.9%) manual workers, 55 (14.3%) were office workers and remaining 86 (22.3%) claimed no occupation. History of sun exposure < 01 hour was noted in 228 (59.2%), 2 hours in 68 (17.7%), 3 hours in 46 (11.9%), 4 hours in 21 (5.5%) and 5 hours in 22 (5.7%) subjects. Vitamin D levels were within normal or sufficient range in 134 (34.8%), below normal range in 251 (65.2%) cases, while insufficient in 09.56% (24/251) and deficient in 90.43% (227/251) cases. BCG vaccination history was negative 318 (82.6%), while positive in 67 (17.4%) cases. Family history of PTB was positive in 37 (09.6%) while negative in 348 (90.4%).
History of fever in 318 (82.6%) cases, cough in 348 (90.4%), heamoptysis 62 (16.1%), loss of appetite in 309 (80.3%), weight loss in 68 (17.7%), night sweats in 291 (75.6%), body ache 240 (62.3%) chest pain in 26 (6.8%), shortness of breath in 87 (22.6%), headache in 324 (84.2%) and joint pain in 47 (12.2%) was present respectively (Table 3).
Out of 273 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.
Table 1. Statistics of Vitamin D in patients and control Group.
Table 2. Frequency and percentage of different variables and non parametric test N = 385.
Table 3. Frequency and percentage of different symptoms of PTB and non parametric test N = 385.
Young age group males were 104 (38.6%), females 52 (46.4%), while middle age group males were 51 (18.7%), females 17 (15.2%) and old age group male were 15 (5.5%), old age females were 12 (10.7%) were vitamin D deficient.
The different statistical values for male and females like chi-square, df, asymp. Sig. (2-sided), Likelyhood ratio, Linear by linear association, Interval by interval pearsons R value, Ordinal by ordinal Spearman correlation and Approx. Sig. are described in Table 4.
A total of 251 different age group subjects were directly related to sun exposure in context of vitamin D deficiency, patients with sun exposure up-to 01 hour were 213 (93.4%), 2 hours 25 (36.8%), 3 hours 10 (21.7%), 4 hours 2 (9.5%) and 5 hours 1 (4.5%) were vitamin D deficient.
Out of 273 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 different statistical values for vitamin D, age group and sun exposure like chi-square, df, asymp. sig. (2-sided), Likelyhood ratio, Linear by linear association, Interval by interval pearsons R value, Ordinal by ordinal Spearman correlation and Approx. Sig. are described in Table 5(a) & Table 5(b).
Vitamin D deficiency was observed in 65.5% (230/351) belonging to lower socioeconomic class, 65.2% (15/23) from middle and 54.5% (906/11) belonging to upper socioeconomic class. Chi-square tests and Symmetric measures are shown in Table 6.
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 significant association between gender and address of patients. Marital status and BCG vaccine status were statistically significant. Sun exposure was significantly related to education, occupation and family history of tuberculosis. Educational status was statistically related to vitamin D deficiency and sun exposure. Economical class was statistically related to marital status, BCG vaccination status and family history of PTB. Occupation of patients was directly related to vitamin D deficiency and sun exposure. While the relationship of vitamin D deficiency with age group, gender, marital status, address, economical class and BCG vaccination was not significant statistically (Table 7).
Vitamin D deficiency and different symptoms of tuberculosis were related statistically like cough, loss of appetite, chest pain and headache. While fever, heamoptysis, weight loss, night sweats, bodyache, shortness of breath and joint pain did not show significant statistical relation. Different symptoms were interrelated and shown significant relation like fever, cough, heamoptysis, chest pain, and shortness of breath, headache and joint pain were interrelated with significant statistical relation (Table 8).
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 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).
Table 7. Correlations of vitamin D and different variables of study N = 385.
*Correlation is significant at the 0.05 level (2-tailed); **Correlation is significant at the 0.01 level (2-tailed).
Table 8. Correlations of vitamin D and different symptoms of PTB, N = 385.
*Correlation is significant at the 0.05 level (2-tailed), **Correlation is significant at the 0.01 level (2-tailed).
Table 9. Vitamin D and demographic variables (paired statistic and correlations).
Table 10. Vitamin D and different parameters (paired sample tests).
Pulmonary tuberculosis including extra-pulmonary tuberculosis is general health issue in Pakistan. List of factors are accountable to the development of tuberculosis. Vitamin D is also labeled as one of the risk agents for the occurrence and progress of tuberculosis. Worldwide studies are available on different health issues and role of vitamin D, but our setup lacks that. Nawabshah is located in Sindh Pakistan a hot area in summer the temperature reaches at the world record temperatures. Inspite of sunny environment our controls and patients are vitamin D deficient. Nowadays vitamin D is blamed for a list of problems all over the world as reported. Here our setup is also one of the victims out of them.
The hazard of tuberculosis (TB) infection is related to the decreased levels of vitamin D . 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 and worse progression of disease in tuberculosis subjects . 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 controls 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 . The role of vitamin D after development of Pulmonary TB needs randomized control trials to check out the effect after therapy.
Vitamin D deficiency is associated with list of problems as reported and Vitamin D deficiency had been labeled for many diseases. Naveed and Anwar concluded that chronic kidney disease is a chief contributing cause of vitamin D deficiency which was observed in about 83% of subjects .
Jamali A.A. et al. found that considerably low levels of vitamin D3 were seen in (62.1%) of patients suffering from parkinsons disease and in second study they found low levels were associated with primary infertility as compared to controlled population .
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 . 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. 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 , 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 . The needs of retrograde studies are essential here to look in this regard.
Significant decreased levels of vitamin D were observed in subjects with tuberculosis in comparison to healthy controls and lack of vitamin D was related with amplified hazard of tuberculosis but subjects of the African populations with tuberculosis and HIV infected were lacking such association. Decreased levels of vitamin D were analyzed in subjects co-infected with tuberculosis and HIV on antiretroviral treatment with immune reconstitution inflammatory syndrome vs. those co-infected subjects who did not develop Tuberculosis related immune reconstitution syndrome. Subjects with latent tuberculosis infection with decreased vitamin D levels had increased risk to develop the active tuberculosis vs. those subjects with sufficient levels of vitamin D, also high risk of conversion of tuberculin skin test/tuberculosis infection, decreased levels of vitamin D in active tuberculosis subjects vs. latent tuberculosis infection did not 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 prevention and treatment of tuberculosis .
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 . 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 .
Levels of bioactive form of vitamin D (1,25(OH)D3) were higher in tuberculosis subjects in comparison to controls without tuberculosis .
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 .
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.
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.
Current study had shown an association between serum levels of vitamin D and tuberculosis. Vitamin D deficiency is probably a risk factor for tuberculosis than its consequence. Further studies are required to conclude about the beneficial effects with vitamin D supplementations in treatment and prevention of tuberculosis.
We gratefully acknowledge Dr Bhojo Mal Tanwaniand Dr Ghulam Mustafa Jamaliand for their excellent support.
We also thank Mr Parkashkumar and staff of Jholylal Diagnostic Lab for their positive role and cooperation in this research.
This study was not supported financially by any institutional/governmental and nongovernmental organization.
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