Human Immunodeficiency Virus (HIV) disease is characterised by a progressive deterioration in immune function. 

It is a global health problem that has infected 60 million people and caused 25 million deaths worldwide. Currently, there are an estimated 33 million people living with HIV including 2 million children.One-third of HIV-infected individuals are co-infected with Mycobacterium tuberculosis, a leading cause of death among people living with HIV. It has been proposed that the increase in M. tuberculosis

Pathology associated with HIV infection is caused by the disruption of the local immune response within the tuberculosis granulomas, decreasing their ability to contain M. tuberculosis leading to increased mycobacterial replication, dissemination and clinical disease. Tuberculosis (TB) remains a major challenge to global public health. The global burden of Tuberculosis (TB) is huge, with an estimated one-third of the world population latently infected^1,2, 9 million new cases and 1.5 million deaths per year³. In a life-time, 10% of infected individuals progress to active TB disease in immune competent individuals increasing to 10% per year in HIV co-infected individuals⁴. The major contributing factors to disease progression in HIV uninfected individuals is malnutrition⁵ with micronutrient deficiencies such as vitamins A, B6, E, thiamine, folate, and zinc reported in patients with active TB disease ⁶. 25-hydroxyvitamin D (25(OH) D) has been implicated in the host defence against TB as an immunomodulator ^1,7,8. 25(OH) D has been shown to down-regulate the pro-inflammatory response and therefore may help to protect the host against increased lung pathology. Vitamin D status is influenced by a variety of factors including diet, sunlight exposure and underlying health conditions¹. In HIV-positive individuals, a high frequency of hypovitaminosis D has been reported, not only among populations at high latitudes but also among people living closer to the equator^{2- 6}.Vitamin D is an immunoregulatory hormone, and vitamin D deficiency and insufficiency in HIV-positive patients have been associated with clinical disease progression, treatment failure and mortality ^7–9. Only a few studies have compared the vitamin D status of HIV-positive with HIV-negative individuals ^4,10,11. This study will establish the association between serum vitamin D level and active pulmonary TB in HIV patients and will help in better understanding of vitamin D level in HIV patients.

Aim

The main objective of this study is the determination of serum 25-hydroxyvitamin D level in HIV patients with active tuberculosis and to determine difference in mean serum vitamin D level in both PLHIV with TB and PLHIV without TB

This hospital-based observational study was conducted at the Department of Medicine, SMS Hospital Jaipur, from May 2020 for one year or until the sample size was achieved, with an additional two months for data compilation and analysis. The sample included 30 cases in each group, with a total of 60 consecutive HIV patients, 30 with TB coinfection and 30 without, selected based on a 95% confidence level and 80% power to detect a mean difference of 4.9 (±2.8) in vitamin D levels. Inclusion criteria for the cases were HIV patients over 18 years old with TB coinfection confirmed by WHO 4 S screening and CBNAAT positivity, who consented to participate. Controls were age- and sex-matched HIV patients without TB. Exclusion criteria included known vitamin D deficiency, chronic renal disease, malabsorption disorders, and obesity.

Vitamin D

Vitamin D has a vital role in host immune defense against Mycobacterium tuberculosis (MTB). It has been observed that vitamin D induces antimicrobial peptide cathelicidin which inhibits mycobacterium multiplication in the macrophages^2,3.  High serum vitamin D concentrations and hypercalcemia have been described in patients with tuberculosis. Activated macrophages in granulomas are the most likely source of the high calcitriol levels⁴. But many studies worldwide have suggested that pulmonary tuberculosis (PTB) is associated with lower levels of vitamin D^6–8. Also, few studies showed no significant difference between the mean serum calcium and vitamin D level between the PTB patients and the controls^9. Present study was therefore undertaken to identify the relation between vitamin D levels and PTB. 

Serum 25-hydroxyvitamin D level in HIV patients with tuberculosis:

In vitro studies suggest vitamin D supplementation minimizes progression to active TB given the role of vitamin D in immune regulation^3,4. HIV infection itself has been shown to increase metabolism of 25(OH) D into the active form, further driving vitamin D deficiency, due to gp120 induced CYP27B1 production⁷. The Prospective Evaluation of Antiretrovirals in Resource Limited Settings (PEARLS) trial provided an opportunity to investigate the association between low 25(OH)D and cART outcomes in diverse settings. We used a case-cohort design in the PEARLS trial to assess risk factors for baseline low 25(OH)D in a cohort of human immunodeficiency virus type 1 (HIV-1) infected individuals at cART initiation and prospectively examined its association with HIV disease progression, virologic failure, and immunologic failure post-cART initiation in 8 low- and middle-income countries and the United States. As a result, vitamin D (Vit-D) supplementation can overcome the challenge and inhibit HIV and replication and growth.

Clinical examination

A detailed history was taken from the selected subjects about age, sex, residential address, economic status, occupation, any drug history, bleeding disorder and febrile illness. The subjects were examined clinically comprising of general physical examination, assessment of vital parameters and systemic examination. Standing height and weight measured .Body mass index (BMI) was calculated. Blood pressure as measured manually by standardized mercury sphygmomanometer by following the guidelines given by the British and Irish Hypertension Society 2017. Participants were advised to avoid alcohol, cigarettes, coffee/tea and exercise for at least 30 minutes before blood pressure measurement. Blood pressure was measured twice for each subject.

Statistical Analysis

The data was collected and complied on M.S. Excel 2020 and data was analysed using SPSS 20.0 version. Data were analysed and statistically evaluated using Statistical Package for Social sciences (SPSS)-PC-20 software (version 20, SPSS, Inc, Chicago, IL, USA). Data were presented as mean and standard deviation (SD) for normally distributed continuous variables and as frequencies for categorical variables. Comparisons were made for means of two sample using Student‗s t test for continuous variables and by x2 analysis for categorical variables. ANOVA statistical analysis was performed to evaluate intergroup comparison between three groups. Pearson correlation coefficient were performed with Vitamin D ESR, CRP, BMI and WHO clinical staging. All statistical analyses were performed taking level of significance at p-value<0.05.

FINANCIAL SUPPORT - Nil 

CONFLICT OF INTEREST - Nil.

Table 1: Distribution of patients according to age and gender.

In our study the majority (38%) patients were in age group 31-40 years followed by 28% in the age 41-50 years in group A. In group B majority (52%) patients were in the age group 31-40 years followed by 20% patients in the 41-50 years age group. The mean age for group A was 36.68 years and for group B it was 38.42 years ( p value 0.17 i.e. >0.05.Also 66% patients in group A and 68% patients in group B were male. p value  0.83 I.e. >0.05.

Table 2: Distribution of patients according to ART regimen

We found that 80% of patients in both groups were on TLD regimen. The p value was 1. There was no significant difference between these groups as regard to art regimen  as p value was >0.05.

Table 3: Distribution of patients according to BMI

In our study we found that 76% patients in group A were of normal BMI followed by 16% patients with underweight BMI. In group B, 58% of patients were of normal BMI followed by 20% of overweight patients. The mean BMI for group A was 20.58 kg/m2 and for group B it was 21.4 kg/m2. The p value was 0.04. There was a significant difference between these groups as p value was <0.05.

Table 4: Distribution of patients according to laboratory parameters

In our study we found that mean ESR for group A was 47.68 mm\brand for group B it was 51.48mm\hr. The mean ALK PO4 for group A was 142.46IU\L and for group B it was 104.04IU\L. Mean CRP for group A was 3.9mg\l and for group B it was 2.02mg\l. The p value for ESR was 0.42, for ALK PO4 was 0.002 and for CRP it was <0.0001.There was no significant difference found in mean SGOT, SGPT, S.Bilirubin as p value was >0.05.  Calcium was found in both groups but a significant difference was not present. There was no significant difference found in Blood Urea, S. Creatinine, Calcium and albumin between these groups as p value was >0.05.

Table 5:Distribution of patients according to Vitamin D level.

In our study we found that over all study was Vitamin D deficient but mean significant difference was present in both groups.  Mean Vitamin D in group A was 20.33 ng/dl and for group B it was 17.57 ng/dl. The p value was 0.04. There was a significant difference between these groups as p value was <0.05.

Table 6: Correlation of CRP, ESR, and BMI with Vitamin D level

In our study we found direct correlation between CRP and Vitamin D in both the groups. The r value and p value of CRP and Vit D for group A was 0.12 and 0.26 respectively. For group B it r value was 0.09 and p value was 0.53. We found indirect or inverse correlation between ESR and Vitamin D in both the groups. The r value and p value of ESR and Vit D for group A was -0.09 and 0.53 respectively. For group B its r value was 0.07 and p value was 0.62. we found inverse correlation between BMI and Vitamin D in both the groups. The r value and p value of BMI and Vit D for group A was -0.3 and 0.03 respectively. For group B it r value was -0.33 and p value was 0.19.

Fig. 1 Correlation of CRP with vitamin D

Fig. 2:  Co-relation of ESR with vitamin D

Fig.3 Co-relation of BMI with vitamin D

In our study, 38% of patients in Group A were aged 31-40 years, followed by 28% aged 41-50 years. In Group B, 52% of patients were aged 31-40 years, followed by 20% aged 41-50 years. The mean age was 36.68 years for Group A and 38.42 years for Group B, with a p-value of 0.17, indicating no significant difference between the groups (p > 0.05). Additionally, 66% of patients in Group A and 68% in Group B were male, with a p-value of 0.83, again showing no significant difference between the groups (p > 0.05).As in Jaimni V et al¹² found that in both groups, the majority were men (88%). There was no significant variation in age and sex between the two groups.

In our study, 80% of patients in both groups were on the TLD regimen, with a p-value of 1. This indicates no significant difference between the groups (p > 0.05).Also in Musarurwa et al¹³ found that of 140 patients on cART, 109 (77.9%) were on an efavirenz (EFV)-based cART regimen. There was however, no significant difference in proportions of patients on EFV-based regimen between the two groups (p = 0.061).ART has been associated with vitamin D deficiency in HIV patients^2,6,14-17.

In our study, the mean ESR was 47.68 for Group A and 51.48 for Group B (p = 0.42). The mean ALK PO4 was 142.46 for Group A and 104.04 for Group B (p = 0.002). The mean CRP was 3.9 for Group A and 2.02 for Group B (p < 0.0001). These results indicate no significant difference in ESR (p > 0.05), but significant differences in ALK PO4 and CRP (p < 0.05). Similarly,Yilma D et al¹⁶ found that HIV-positive patients had 17 % (95 % CI 6, 33 %) higher serum 25(OH)D than HIV-negative persons. The age groups 35–45 and >45 years had 12 % (95 % CI 1, 22 %) and 18 % (95 % CI 4, 30 %) lower serum 25(OH)D, respectively, than the age group 18–25 years. A number of studies have reported high proportion of vitamin D deficiency and insufficiency in HIV-positive patients^2,5,18-19.

In our study, 76% of patients in Group A had a normal BMI, followed by 16% with underweight BMI. In Group B, 58% had a normal BMI, followed by 20% with overweight BMI. The mean BMI was 20.58 kg/m² for Group A and 21.4 kg/m² for Group B, with a p-value of 0.04, indicating a significant difference between the groups (p < 0.05). We found a correlation between BMI and Vitamin D in both groups. For Group A, the r value was -0.3 and the p value was 0.03. For Group B, the r value was -0.33 and the p value was 0.19.Similarly, Sudfeld et al.²⁰ explored the relationship between vitamin D levels and significant weight loss (>10% from baseline) in their study. They discovered that individuals with a deficiency in vitamin D had a notably higher hazard (2.10 times) of experiencing such weight loss compared to those with sufficient levels of vitamin D, after adjusting for various factors. However, they did not find a significant association for individuals with vitamin D insufficiency. Their analysis also didn't reveal any clear linear or nonlinear relationship between continuous 25 (OH)D levels and the incidence of >10% weight loss. They conducted sensitivity analyses, which showed that the elevated risk estimates for weight loss in individuals with vitamin D deficiency were not statistically significant when certain factors were excluded from the analysis, such as events occurring before 2 months of follow-up or individuals with pulmonary tuberculosis.

In our study, we found a direct correlation between CRP and Vitamin D in both groups. For Group A, the r value was 0.12 and the p value was 0.26. For Group B, the r value was 0.09 and the p value was 0.53. We also found an inverse correlation between ESR and Vitamin D. For Group A, the r value was -0.09 and the p value was 0.53. For Group B, the r value was 0.07 and the p value was 0.62.Haver F et al¹⁷ found that low 25(OH)D was independently associated with higher rates of clinical progression and death is biologically plausible given the wealth of studies now emerging on the diverse roles of vitamin D in the immune system.

 Limitations

Further prospective studies with larger sample size are required to understand the relation between vitamin D and tuberculosis. There may be differences in UV exposure among study participants and its possible role in serum vitamin D deficiency. Further studies are required to study vitamin D‘s role in the prognosis and outcome of pulmonary tuberculosis. Association of Vitamin D level with viral load was not done due to lack of sufficient data

Conclusions Our findings, though at variance with the generally accepted association between serum vitamin D status and PTB, are in concordance with findings from a few other studies that report an association between high serum 25(OH) D concentrations and risk of PTB. It is possible that the reason for this observed variation could be genetic given the complex and diverse actions of vitamin D. Thus, genetic polymorphisms in the vitamin D receptor, or in the multiple enzymes involved in vitamin D metabolism, remain attractive candidates for further study.

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