High-Dose Vitamin D3 Supplementation in the Treatment of Human Immune Deficiency Virus Patients: A Double-Blind Randomized Control Trial
Phase: PHASE3
Conditions: HIV-1-infection, Hypovitaminosis D
Sponsor: University of the Punjab

 
HDVDS-HIVT_Protocol_V 1.0_August 15, 2019 
 
Page 1 of 43  
Protocol Title 
High-Dose Vitamin D3 in the Treatment of Human 
Immune Deficiency Virus Patients, A Double-Blind 
Randomized Control Trial 
 
 
 
 
 
 
Protocol Version: 1.0 
Date: August 15, 2019 
 
 

 
HDVDS-HIVT_Protocol_V 1.0_August 15, 2019 
 
Page 1 of 43  
 
 
 
 
Institute of Social &Cultural Studies 
University of the Punjab, 
Lahore 
 
 

 
HDVDS-HIVT_Protocol_V 1.0_August 15, 2019 
 
Page 1 of 43  
 

 
1 
 
Abstract 
Human immunodeficiency virus is a key challenge for global health. Vitamin D 
deficiency is common in people living with HIV infection. Antiretroviral therapy may 
create unique risk factors for vitamin D insufficiency, including alterations of vitamin 
D metabolism by ART.  
The objective of the study is, to know the effect of high doseVitamin D, in the treatment 
of HIV patients with antiretroviral therapy. It will be a randomized controlled trial,in 
which 104patients visiting Said Mitha Teaching Hospital Lahore will be included.  
The patients will be divided into two groups. Group-A patients will receive ARV + 
Vitamin D supplements and patients in Group-B will receive only antiretroviral therapy 
and placebo. 
Methods/design: A randomized placebo double blind control trails ofpatients with age 
19-50 years will be the participants of our study at VCCT center of said MithaTeaching 
Hospital, located in walled city of Lahore,Pakistan. Participants will be randomly 
allocated to receive 25 hydroxyvitamin D supplementation (n=) or placebo (n=) with 
the start of ART.  
The primary outcome will be achieved with high doseofserum 25-hydroxyvitamin D 
level at 24weeks. Secondary outcome will include decrease in viral load, increase mean 
CD4 count to reduce prevalence of clinical symptoms (elevated levels of LFTs,decrease 
HB level& serum calcium level).Purposive sampling will be carried out. 
Data will be collected through Performa, which will be entered and statistically 
analyzed, using SPSS version 24.0, This Performa is designed by the Punjab AIDS 
Control 
Programme 
including 
socio-demographic 
information, 
Pretest 

 
2 
 
information,posttest information & follow up chart. Chi-square test will be used to 
estimate the association among qualitative variables. 
Discussion: High dose Vitamin D supplementation in HIV-infected patients has not 
previously been investigated in Pakistan and it is unknown whether increasing levels is 
associated with improved clinical outcome or not. Therefore, it is significant to conduct 
a study to know the effect of vitamin D in the treatment of HIV patients with 
antiretroviral therapy. 
From the public health point of view, it is also important to know the effectiveness of 
high dose of 25-hydroxy Vitamin-D level in positive human immune deficiency 
patients. 
Trial Registration: The trial will be registered with clinical trials.. 
Keywords: AIDS, HIV, anti-retroviral therapy, high dose 25-hydroxy Vitamin-D level, 
CD4 count, viral load. 
 
 

 
3 
 
1. INTRODUCTION 
 
1.1 Background 
 
Human immunodeficiency virus (HIV) is retrovirus; it infects immune system cell and 
impairs their function. This virus causes infection that leads to worsening the immune 
system, resulting in immune deficiency. There are two types of HIV i.e. HIV 1 and HIV 
2. HIV 1 is more virulent easily transmit. HIV in majority is caused by HIV 1 while 
HIV 2 is less easily transmitted and it is mostly confined to West Africa. HIV 2 is also 
called non-progresive, which means the chance of progression into AIDS is less 
(Nyamweya et al., 2013; Abbas, Naveed, Qamar, Zehra& Jawed, 2016). 
Human immunodeficiency virus is a global health problem (Mansueto, Seidita, Vitale, 
Gangemi, Iaria&Cascio, 2015)and a major health concern which remains incurable 
(Imran, Nasi&Riaz, 2018). Globally, about 36.7 million people are living with HIV. 
HIV infection causes AIDS and is epidemic throughout the world. In 2016; about 1.8 
million were reported as newly infected and 1 million died of AIDS-related illnesses in 
the same period (Khan, Wali, Fatima, Yaqoob& Aziz, 2019). 
The HIV infection is becoming a prevalent disease in Pakistan, and its death toll has 
been steadily increasing each year since 1987 (Hussain et al., 2018).In Pakistan, HIV 
epidemic trend has increased from “low prevalence, high risk” to “concentrated” 
epidemic particularly in high risk populations. According to World Health Organization 
and National AIDS Control Program, an estimated 91,340 people were living with HIV 
in 2014. Majority of HIV-infected population belonged to high risk groups including 

 
4 
 
injection drug users (IUD), transgender, males and female sex workers. Also, the 
previous studies have shown prevalence rate of 27.2%, 5.2%, 1.6% and 0.6% 
respectively in these high risk groups (Shaukat et al., 2018). 
1.2 Study Medication 
The introduction of antiretroviral therapy (ART) has not only improved the prognosis 
of HIV patients by reducing their mortality and morbidity, but also has revealed some 
complications related to the therapy (Grana et al., 2019).Vitamin D deficiency is 
common in people living with HIV infection (Hileman, Overton&McComsey, 2016; 
Hsieh & Yin, 2018).HIV infection and exposure to certain anti-retroviral might 
contribute to altered levels of25-hydroxy vitamin D 25(OH)D).The preferred and most 
commonly used parameter for assessment of vitamin D status is serum 25(OH)D 
concentration. 25(OH)D, which is the major circulating metabolite of vitamin D and 
reflects the vitamin D inputs from cutaneous synthesis and dietary intake(Lips, 2010; 
Deshwal&Arora, 2019). Most current guidelines define vitamin D deficiency and 
insufficiency as a serum vitamin D value 25 (OH) D) <30 nmol/L (<12 ng/ml) and 
between 30–50 nmol/L respectively (12–20 ng/ml) (Penner et al., 2018).The prevalence 
of vitamin D deficiency in HIV infected individuals ranges from 60% to 90% and is 
associated with female gender, black ethnicity and antiretroviral use (Tiraboschi et al., 
2016). 
HIV infection may create unique risk factors for vitamin D insufficiency such as 
chronic inflammation, and both the protease inhibitor and non-nucleoside reverse 
transcriptase inhibitor classes of antiretroviral agents may enhance vitamin D 
metabolism via modulation of the cytochrome P450 system and vitamin D 
hydroxylation. Additionally, in HIV-infected persons, vitamin D insufficiency has been 

 
5 
 
associated with lower CD4+ T lymphocyte counts (Aziz et al., 2013; Coelho et al., 
2015). 
The main role of vitamin D is to maintain the function of monocytes and macrophages 
which are linked to human innate immunity to certain infectious agents. This role is 
very important in body’s natural defense against infection in which macrophagesplays 
a vital role in pathogenesis. Vitamin D acts by combining the nuclei receptor on affected 
cells so both abnormality in receptor function and structure or low vitamin D level alter 
the immunity against HIV (Nnoaham& Clarke, 2008; Afzal, Rathore, Butt 
&Randhawa, 2018). 
Vitamin D is obtained in its natural form by exposing the skin to sun rays that converts 
7-dehydrocholesterol (pro-vitamin D3) into Cholicalceferol (vitamin D3) through 
which the bloodstream passes, where it joins the D-binding protein (VDBP) and is 
transported to the liver. The second source of vitamin D is via oral intake, through the 
transformation of ergo sterol (vitamin D2) in plants and fungi. Vitamins D3 and D2 are 
both metabolized in the liver to 25-hydroxicholecalciferol and are transformed in 
kidneys into its active form, 1,25dihydroxyvitamin D, which is a steroid hormone vital 
forcalcium and bone metabolism (Conrado, de Barros Miranda-Filho & Bandeira, 
2010). 
1.3 Rationale for the Proposed Study 
Human immunodeficiency virus is a leading health problem that exacerbates the quality 
of life and a major cause of mortality among patients. In Pakistan, HIV infection is also 
increasing rapidly while injection drug users, transgender and sex workers are high risk 
groups. Vitamin D deficiency is common in people living with HIV infection. It has 
recently been confirmed that Vitamin D supplementation reduces the progression of 

 
6 
 
disease and prevents HIV-infected individuals from mortality (Anderson et al., 2010; 
Mehta et al., 2010; Barbosa et al., 2014). Present study evaluates the effect of vitamin 
D in the treatment of HIV patients with antiretroviral therapy.  
 
 
 
 
 
 
2. RESEARCH OBJECTIVE 
2.1 Primary objectives: 
1. To determine whether proposed oral high dose vitamin D3 is sufficient for achieving 
the physiological concentration in HIV positive patients on ART. (primary outcome) 
2.2 Secondary objectives: 
1. To determine whether this intervention also improves CD4 count, viral load count, 
liver enzyme in study participants. (Efficacy outcome). 
2. To discover whether this interventional regimen is safe and well tolerated by study 
participants. (Safety outcome). 
3. To ascertain socio-demographics factors responsible for decreased level of vitamin 
D3 in HIV positive patients on ART.  
 
2.3 Tertiary objectives: 
 

 
7 
 
1. To conclude the effects of total leucocytes count in HIV patients with vitamain D 
supplementation.  
2. To determine the effects of total neutrophil count in HIV patients with vitamain-D 
supplementation.  
3. To investigate the effects of total basophil count in HIV patients with vitamain-D 
supplementation.  
4. To determine the effects of total eosinophil count in HIV patients with vitamain-
D supplementation.  
5. To conclude the effects of total monocyte count in HIV patients with vitamain-D 
supplementation.  
6. To establish the effects of total platelets count in HIV patients with vitamin-D 
supplementation.  
7. To find out the effects of total hemoglobin count in HIV patients with vitamin-D 
supplementation.  
8. To determine the effects of total lymphocytes count in HIV patients with vitamin-
D supplementation.  
 
 
 
 
 
 

 
8 
 
3. Study Population  
Patients who visited the VCCT center of GOVT Said Mitha Teaching Hospital Lahore 
and the treatment center of Jinnah hospital HIV clinic were the study population and they 
were HIV positive and on ART. The study population consisted of HIV patients ranging 
in age from 19 to 50 years. Patients with severe vitamin D3 deficiency were seen at the 
multi-centric outpatient HIV department. 
 
 
3.1 Eligibility Criteria  
Inclusion criteria:  
 
Age at enrolment between 19-50 years old  
 
Vitamin D deficiency level>20ng/ml  
 
Had not been taking any kind of vitamin D supplementation mega doses for since 
six months  
 
Written consent was signed by participants voluntarily  
 
Exclusion criteria  
 
Pregnant women  
 
Vitamin D level<20ng/ml or taking vitamin D supplementation mega doses since 
last three months were not included  

 
9 
 
4. STUDY PROCEDURES 
4.1 Study Population 
Patients visiting Said Mitha Teaching Hospital Lahore will be the study population and 
having diagnosed with HIV positive patients. 
4.2 Sampling technique: 
Random list was generated for the double blind purpose. This list was generated 
through Excel sheet.  
4.3 Ethical Consideration: 
The study will be conducted after approval from the Punjab University Ethical Review 
Board. Permission will also be obtained from Punjab AIDS Control Programme to use 
health facilities and this study will be registered atClinical Trail.gov, to use the health 
facilities and services of paramedics staff for data collection. Written consent of parents 
and guardians will be taken before starting the study. 
4.4 Data Collection: 
All staff involved in the study will be trained by the principal investigator before start 
of data collection. Trained  laboratory technician will screen the  patients  aged 19  years 
to 50 years  of age  in(VDT) diagnostic center of  hospital  visiting outpatients 
department in their community and will refer the  positive patients to  treatment center 
of  AIDS Control Programme clinic of Mayo Hospital Lahore, After receiving their 
prescribed ART, the two positive groups would be divided into two equal groups, one 
group will receive 25hydroxy vitamin –D and other halves will receive placebo at the 
same time once weekly for 12 weeks. Patients will be called for follow-up after every 

 
10 
 
4 weeks. At the end, we will perform posttest and check the level of 25 hydroxy in both 
groups and other parameters and by using logistic regression we will evaluates the 
results of effectiveness of 25 hydroxyin blood levels of HIV patients 
4.5 Randomization: 
The random allocation sequence will be generated in an excel spread sheet by a 
statistician who will be independent of the study. Consecutive numbers will be assigned 
to active vs. placebo in equal numbers. No restrictions (e.g. stratification, block size) 
will be applied. This sequence will be used by the study pharmacy to label pairs of 
syringes containing active and placebo medication with a study number assigned to 
active and placebo arms, respectively. These staff will assign the consecutive ID 
numbers to participants according to the sequence in which they are enrolled, and the 
hospital pharmacy then supply syringes of placebo active medication bearing this ID 
number. 
4.6 Blinding: 
Patients of all study participants will be blinded to allocation, as will be the doctors and 
the staff nurse who will enrol participants and perform study assessments. Active and 
placebo medication will be presented identically in 1 ml syringes, and will have 
identical appearance and texture. 
4.7 Interventions: 
All participants will be treated with vitamin D supplements (Annexure attached) 
provided by AIDS Control Programme at outpatients department according to 
WHOguidelines.Vitamin D and calcium will be supplied to HIV positive patients 
according to the study (Annexure attached) on a weekly basis by suitably trained staff 

 
11 
 
who will provide information regarding benefits of vitaminD advise as to how it should 
be taken. Participants who will be randomised to the intervention arm of the trial will 
receive one oral dose of 100,000 IU (10 mg) vitamin D3 (cholecalciferol) in 1 ml olive 
oil, will administer via a syringe at 5 and 12 weeks’ post-initiation of vitamin D3 
supplementation. Participants who will randomised to the control arm of the trial will 
receive one oral dose of placebo (1 ml extra virgin) via a syringe at 5 and 12 week’s 
post-initiation of vitamin D3 supplementation. After screening further dose of RDA 
Vitamin-D  will be provided for next 5 weeks to complete 24 weeks of study. 
 
5. LAB ANALYSIS AND COLLECTION OF 
BLOOD SAMPLE: 
25-hydroxyvitaman D concentration will be measured 0 and 60 days to assess baseline 
deficiency and then effect of supplementation,3ml blood sample will be taken in gel 
tube after clot formation will be centrifuged. Serum or plasma may be stored for up to 
3 days at 2-8 centigrade. Initially Alere HIV combo strips will be used for screening of 
HIV and syphilis, then on positive result we will use second screening on uni-gold HIV 
strip. Then on third step we will use SD HIV ½ 3.0 strip for final confirmation of 
screening step and finally we perform RT-PCR at last. 
The haemoglobin level will be determined at 0 days and 60 days to assess the anaemia 
and effect of supplementation in all selected participants. Blood sample will be drawn 
from patients who will give their consent voluntarily. 
 

 
12 
 
6. STUDY SCHEDULE 
Schedule of clinical assessment and procedure: 
 
6.1 STUDY PROCEDURES AND ASSESSMENTS:  
Visit Schedule:  
Visit 1 (Screening, Days -3 to 1)  
Subjects who were informed about the study and voluntarily gave written consent to 
participation had the following tests.  
a. Informed consent and screening number assignment  
b. Demographics  
 
Screening 
Treatments 
 
V 1 
Screening & 
Assessment 
V 2 
Randomization 
& 1st Dose 
V 3 
 2nd dose 
V 4 
3rd dose  
V 5 
Post 
Assesments 
 
-7~1 
1 Day 
5 week 
12 week 
24 week 
ICF 
× 
 
 
 
 
Eligibility Criteria 
× 
× 
 
 
 
Baseline 
Questionnaire 
× 
 
 
 
 
Weight 
× 
 
× 
× 
× 
Height 
× 
 
× 
× 
× 
Vital Signs 
× 
× 
× 
× 
× 
Vit-D 
× 
 
 
× 
V4+1 Day 
 
CD4 count 
× 
 
 
× 
V4+1 Day 
 
Viral load 
× 
 
 
× 
V4+1 Day 
 
PCR 
× 
 
 
× 
V4+1 Day 
 
CBC 
× 
 
 
× 
V4+1 Day 
 
Study Medication  
 
× 
× 
× 
 
AE Monitoring 
 
× 
× 
× 
× 
RDA 
 
 
 
 
× 
End of Study 
 
 
 
 
× 

 
13 
 
c. Information on exposure to HIV infection  
d. Medical history  
e. Previous HIV lab test if any  
f. RT-PCR  
g. Laboratory tests  
h. Prior medications and therapies  
i. Physical examination (investigator questioning)  
j. Symptoms  
k. Body weight measurement  
l. Assessment of the inclusion/exclusion criteria  
Visit 2 (Randomization, Baseline, Day 1):  
a. Medical history  
b. Prior medications and therapies  
c. Physical examination (investigator questioning)  
d. RT-PCR  
e. Laboratory tests  
f. Symptoms  
g. Assessment of the inclusion/exclusion criteria  
h. Randomization  
i. Prescription of test drug or placebo  
Visit 3 (Day 4)  
a. Concomitant medications and therapies  
b. Physical examination (investigator questioning)  
c. RT-PCR  
d. Symptoms  

 
14 
 
e. Treatment adherence.  
Visit 4 (Day 7):  
a. Concomitant medications and therapies  
b. Physical examination (investigator questioning)  
c. RT-PCR  
d. Laboratory tests  
e. Symptoms  
Visit 5 (Day 14)  
a. Concomitant medications and therapies  
b. Physical examination (investigator questioning)  
c. RT-PCR  
d. Laboratory tests  
e. Symptoms  
f. Body weight measurement  
g. Adverse events  
Visit 6 (Day 28):  
a. Physical examination (investigator questioning)  
b. Laboratory tests  
c. AEs  
d. Collection of clinical treatment information  
Even if a subject was withdrawn, unless the subject withdrew the consent for safety 
follow-up, the safety follow-up was conducted at the scheduled visits (Visits 3, 4, 5, 
and 6) to collect the following clinical treatment information.  
e. Treatment adherence  
 

 
15 
 
Unscheduled Visit:  
If a subject arrived at the site for medical treatment on an unscheduled date due to an 
AE, a change in concomitant drugs, withdrawal status, or measurement data acquired 
during the research, all relevant facts were documented as thoroughly as possible in the 
SD. Unplanned visits did not interfere with a planned study schedule. Unscheduled 
visits may had happen at the subject's solicitation or when the examiner considered it 
fundamental. There had been no limitations on the tests done during impromptu visits, 
and the specialist embraced tests that were reasonable and needed for the subject.  
 
7. STATISTICAL CONSIDERATION  
Statistical analyses will be conducted using SPSS VERSION 23.Qualitative data will 
be presented in form of frequency and percentage and quantitative data will be 
presented in form of mean ± S.D. For the comparison of both treatments (randomly 
allocated to patients) Wilcoxon test will be applied to compare effectiveness of both 
treatment plans. P-value ≤ 0.05 will be considered as significant, Baseline 
characteristics of trial participants will be summarised separately for both randomised 
group. For each group dichotomous and categorical data of participants will be 
presented as number and percentages. 
The primary outcome, effect of allocation on continuous outcomes that will assessed 
both at baseline and at the end of study(e.gVitamin-D supplementation at24 weeks will 
be assessed using linear regression, adjusting for the baseline values. The effect of 
allocation of categorical outcomes variables that will be assessed both at baseline and 
at the end of study (clinical outcome)will be analysed with generalized linear regression 
with  a log link and binomial distribution to yield a risk ratio adjusted for the baseline 

 
16 
 
values with 95% CI and p value(cummings,2009).. Serum concentration of 25{OH} D 
will be measured at baseline,05 03 months at baseline, Statistical significance will be 
inferred where p< 0.05. 
Allocated to patients) Wilcoxon test will be applied to compare effectiveness of both 
treatment plans. P-value ≤ 0.05 will be considered as significant. 
8. ETHICAL CONSIDERATION 
The study will be conducted after approval from the Punjab University Ethical review 
board. Permission will also be obtained from Punjab  AIDS control programme to use 
health facilities and this study will be registered  at  CLICAL TRAIL.gov, to use the 
health facilities and services of  paramedics staff for data collection. Written consent of 
parent and guardian will be taken before starting the study. 
8.1 Data Collection 
A Performa will be prepared by  researcher and finalized after pre-testing. Each 
patientvisiting Said Mitha Teaching Hospital Lahore will be assessed by the researchers 
and their demographic information will be noted on the pre-designed Performainitially. 
Data is collected from all those patients whom would be registered in our 03 months of 
study after approval of synopsis. 
8.2 Human Immunodeficiency Virus 
The human immunodeficiency virus (HIV) infects cells of the immune system, 
destroying or impairing their function. Infection with the virus results in progressive 
deterioration of the immune system, leading to "immune deficiency."  

 
17 
 
8.3 Standard Antiretroviral Therapy 
Standard antiretroviral therapy (ART) consists of the combination of at least three 
antiretroviral (ARV) drugs to maximally suppress the HIV virus and stop the 
progression of HIV disease. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
18 
 
9. ANNEXURE 1: 
 
ABBREVIATIONS 
AIDS:                      
Acquired immunodeficiency syndrome 
25(OH)vitamin-D:   25 Hydroxy Vitamin-D 
RDA:                       
Recommended daily allowances. 
ART:                        Anti-retroviral therapy. 
HIV:                         Human-immune deficiency virus 
WHO: 
 World health organization. 
PACP:                       Punjab AIDS control programme. 
CD4 count:                Cluster of differentiation 
10. 
WORK PLAN 
Weeks 
Months 
1 
2 
3 
4 
5 
6 
Synopsis writing and approval 
 
 
 
 
 
 
Data collection  
 
 
 
 
 
 
Data analysis 
 
 
 
 
 
 
Thesis writing  
 
 
 
 
 
 
Thesis submission  
 
 
 
 
 
 
 
 

 
19 
 
11. 
References 
 Abbas, S.S., Naveed, S., Qamar, F., Zehra, S., & Jawed, S.H. (2016). 
Comprehensive delineation on HIV; latest encroachment in clinical findings, 
future 
trends 
and 
pharmacotherapy.Journal 
of 
Bioequivalence 
& 
Bioavailability, 8(1), 37-36. 
 Afzal, A., Rathore, R., Butt, N.F., &Randhawa, F.A. (2018).Efficacy of vitamin 
D supplementation in achieving an early sputum conversion in smear positive 
pulmonary tuberculosis.Pakistan Journal of Medical Sciences, 34(4), 849-854. 
 Anderson, J.L., May, H.T., Horne, B.D., Bair, T.L., Hall, N.L., Carlquist, J.F., 
… Muhlestein, J.B. (2010). Relation of vitamin D deficiency to cardiovascular 
risk factors, disease status, and incident events in a general healthcare 
population.American Journal of Cardiology,106, 963-968. 
 Ashenafi, S., Amogne, W., Kassa, E., Gebreselassie, N., Bekele, A., Aseffa, G., 
…Brighenti, S. (2019). Daily nutritional supplementation with vitamin D3 and 
phenylbutyrate to treatment-naïve HIV patients tested in a randomized placebo-
controlled trial. Nutrients, 11, 133. 
 Aziz, M., Livak, B., Burke-Miller, J., French, A.L., Glesby, M.J., Sharma, A. 
Adeyemi, O.M. (2013). Vitamin D insufficiency may impair CD4 recovery 
among women’s interagency HIV Study participants with advanced disease on 
HAART. AIDS, 27, 573-578. 
 Barbosa, N., Costa, L., Pinto, M., Rosinha, P., Rosinha, I., &Couto, M. (2014). 
Vitamin D and HIV infection: a systematic review. Journal of 
Immunodeficiency & Disorders, 3, 1-11. 

 
20 
 
 Coelho, L., Cardoso, S.W., Luz, P.M., Hoffman, R.M., Mendonça, L., Veloso, 
V.G. Lake, J.E. (2015). Vitamin D3 supplementation in HIV infection: 
effectiveness and associations with antiretroviral therapy. Nutrition Journal, 14, 
81.  
 Conrado, T., de Barros Miranda-Filho, D., &Bandeira, F. (2010).Vitamin D 
deficiency in HIV-infected individuals: one more risk factor for bone loss and 
cardiovascular disease?ArquivosBrasileiros de Endocrinologia&Metabologia, 
54(2), 118-122. 
 Conrado, T., Miranda-Filho, D.D.B., de AlencarXimenes, R.A., Albuquerque, 
M.D.F., Lacerda, H.R., Ramos, R.C.F., …Bandeira, F. (2011). Vitamin D 
deficiency in HIV-infected women on antiretroviral therapy living in the 
tropics.Journal of the International Association of Physicians in AIDS Care, 
10(4), 239-245. 
 Deshwal, R., &Arora, S. (2019). High prevalence of vitamin D deficiency in 
HIV infected on antiretroviral therapy in a cohort of Indian patients.Journal of 
the Association of Physicians of India, 67, 42-45. 
 Grana, E.D., Rigo, F., Lanzafame, M., Lattuada, E., Suardi, S., Mottes, M., 
Carbonare, L.D. (2019). Relationship between vertebral fractures, bone mineral 
density, and osteometabolic profile in HIV and hepatitis B and C-infected 
patients treated with ART. Frontiers of Endocrinology, 10, 302. 
 Hileman, C.O., Overton, E.T., &McComsey, G.A. (2016). Vitamin D and bone 
loss in HIV. Current Opinion in HIV and AIDS, 11(3), 277–284. 
 Hsieh, E., & Yin, M.T. (2018).Continued interest and controversy: vitamin D 
in HIV.Current HIV/AIDS Reports, 15(3), 199-211. 

 
21 
 
 Hussain, A., Hussain, S., Ali, S.M., Ali, E., Mehmood, A., & Ali, F. (2018). 
HIV/AIDS- a growing epidemic in Pakistan. Journal of Evolution of Medical 
and Dental Sciences, 7(08), 1057-1062. 
 Imran, M., Nasi, J.A., &Riaz, S. (2018). Regional pattern of HIV cases in 
Pakistan. Journal of Postgraduate Medical Institute, 32(1), 9-13. 
 Khan, M.D., Wali, A., Fatima, R., Yaqoob, A., & Aziz, S. (2019). Prevalence 
and associated risk factors of HIV in prisons in Balochistan, Pakistan: a cross-
sectional study. F1000Research, 7, 1821. 
 Lerma-Chippirraz, E., Güerri-Fernández, R., García, J.V., Mena, A.G., 
Grinberg, A.G., Montero, M.M., … Freud, H.K. (2016). Validation protocol of 
vitamin D supplementation in patients with HIV-infection.AIDS Research and 
Treatment, 2016, 8. 
 Lips, P. (2010).Worldwide status of vitamin D nutrition.Journal of Steroid 
Biochemistry and Molecular Biology,121, 297-300. 
 Mansueto, P., Seidita, A., Vitale, G., Gangemi, S., Iaria, C., &Cascio, A. (2015). 
Vitamin D deficiency in HIV infection: not only a bone disorder. BioMed 
Research International, 2015, 18. 
 Mehta, S., Giovannucci, E., Mugusi, F.M., Spiegelman, D., Aboud, 
S., Hertzmark, E., … Fawzi, W.W. (2010). Vitamin D status of HIV-infected 
women and its association with HIV disease progression, anemia, and 
mortality.PLoS One, 5(1), e8770. 
 Nnoaham, K.E., & Clarke, A. (2008). Low serum vitamin D levels and 
tuberculosis: a systematic review and meta-analysis. International Journal of 
Epidemiology, 37(1), 113-119. 

 
22 
 
 Noe, S., Heldwein, S., Pascucchi, R., Oldenbuttel, C., Wiese, C., von Krosigk, 
A. Wolf, E. (2017). Cholecalciferol 20 000 IU once weekly in HIV-positive 
patients with low vitamin D levels: result from a cohort study. Journal of the 
International Association of Providers of AIDS Care, 16(4), 315-320. 
 Nyamweya, S., Hegedus, A., Jaye, A., Rowland-Jones, S., Flanagan, K.L., 
&Macallan, D.C. (2013). Comparing HIV-1 and HIV-2 infection: lessons for 
viral immunopathogenesis. Reviews in Medical Virology, 23, 221-240.  
 Penner, J., Ferrand, R.A., Richards, C., Ward, K.A., Burns, J.E., &Gregson, 
C.L. (2018). The impact of vitamin D supplementation on musculoskeletal 
health outcomes in children, adolescents, and young adults living with HIV: a 
systematic review. PLoS ONE, 13(11), e0207022. 
 Roozbeh, F., &Ghajar, M. (2018). Systematic review and meta-analysis effect 
of vitamin D supplementation with anti-viral therapy on CD4 levels in HIV-
infected patients: systematic review and meta-analysis. International Journal of 
Medical Investigation, 7(3), 1-10. 
 Shaukat, S.N., Khan, S., Raza, A., Khanani, R., Ghayaz, A., &Kazmi, S.U. 
(2018). Prognostic markers in HIV mono-and co-infected individuals: a study 
from Karachi–Pakistan. Journal of Infection and Public Health, 11, 250-254. 
 Tiraboschi, J., Ibrahim, F., Teague, A., Mant, C., Dew, T., Cason, J., & Fox, J. 
(2016). No benefit of standard vitamin D/calcium supplementation in HIV 
infected individuals. Journal of Infectious Diseases and Therapy, 4, 301. 
 Yilma, D., Kæstel, P., Olsen, M.F., Abdissa, A., Tesfaye, M., Girma, T.Friis, 
H. (2016). Change in serum 25-hydroxyvitamin D with antiretroviral treatment 
initiation and nutritional intervention in HIV-positive adults. British Journal of 
Nutrition, 116, 1720-1727. 

 
23 
 
Research Performa 
1. Biographic Data 
Registration No.  ___________  
 
Date: __________ 
 
 
Group-A (ARV + Vit.-D) 
 
 
Group-B (ARV+ Placebo)  
Name_________________ 
 
 
 
Age _________________ (years) 
 
Gender 
 
Male   
 
 
 
Female  
 
Education  
 
Illiterate  
                                                               Under matric   
 
Matric and above   
 
Employed  
 
Unemployed    
Family monthly income (Rs.)  
 
 
 
Marital Status 
Married  
 
 
 
Unmarried  
 
 
 
Occupation 
BMI 
 
Underweight   
 
 
 
Overweight   
 
 
 
Obese   
 
 
 
2. Exposure to sunlight  
 
Yes  
 
 
 
 
 
No  
 
 
 
 

 
24 
 
3. Serum vitamin D level 
 
 
ng/ml 
4. Vitamin D deficiency 
Yes  
 
 
 
 
No  
 
 
 
 
5. Consent Form Taken       
Yes  
 
 
 
 
No  
 
 
6. Ethical committee minutes of meetings 
7.Presentingcomplaint 
_______________________________________________________________ 
8.Brief Physical Examination (Vital Sign & Brief History) 
BP 
 
 
Temp   
 
RR  
 
 
Pulse 
9. Inclusion Criteria 
(All patients which are not taking vitamin-D Supplementation from last 6 months & 
having deficiency) 
10.Exclusion Criteria 
1. 
pregnant women 
2. 
Patients on active TB medicines  
3. 
Patients do not sign consent form freely 
 
 Follow up visits:                     
_____________________________________________________________________ 
_____________________________________________________________________
_____________________________________________________________________ 
 
 

 
25 
 
 
11. Treatment obtained: 
 
 
Yes         No 
12.Adverse effects of medicine: 
 
Yes          No 
13. Compliance of medicine  
            Yes          No 
14. Outcome of achievement:: 
 
Yes          No 
 
 
 
Investigator signature: 
 
___________________