Study design and setting
This was a cross sectional study that was conducted from January 2020 to March and May 2020. The study took place in the Rheumatology outpatient clinic of a 200-bed capacity national referral hospital in Kampala, Uganda. Uganda is an equatorial country that receives 12 h of sunshine daily. Majority of the people wear light clothing and spend 8–10 h outdoors [15]. The Rheumatology clinic assesses patients with joint, muscle and autoimmune diseases, and runs once a week attending to approximately 15–20 patients on each clinic day. Three quarters of these are patients with osteoarthritis. Determination of serum vitamin D levels was done at Mulago National Referral Hospital clinical chemistry laboratory. Permission was sought from the study site to transfer the blood samples to for analysis.
Participants’ enrolment and study definitions
For screening, files were reviewed and patients with a diagnosis of osteoarthritis were approached for informed consent. New patients with knee pain for at least 6 months were also approached for informed consent. Adult patients (aged ≥ 18 years) with knee osteoarthritis diagnosed using the American College of Rheumatology (ACR) clinical criteria for knee osteoarthritis [16] were consecutively enrolled into the study (Fig. 1). The ACR clinical criteria for knee osteoarthritis [16] includes patients with knee pain for at least 6 months and at least three of the following six items: age > 50 years, morning stiffness of short duration < 30 min, crepitus on knee range of motion (ROM), bony tenderness, no palpable warmth and bony enlargement.
We excluded patients with a known diagnosis of rheumatoid arthritis, systemic lupus erythematosus and gout.
Patients’ serum vitamin D levels were categorized as sufficient (≥ 30 ng/ml), insufficient (20–29 ng/ml), deficient (< 20 ng/ml) based on the Endocrine Society Clinical Practice Guidelines on evaluation, treatment and prevention of vitamin D deficiency [17]. The categories insufficient and deficient were operationally defined and categorised as suboptimal.
Data collection
After obtaining informed consent, we administered a pretested questionnaire to obtain socio-demographic characteristics, history of chronic illnesses, drugs including vitamin D and calcium supplementation, duration of daily sunshine exposure, prior or current involvement in sports, alcohol and smoking. Weight and height were taken using a calibrated weighing scale and stadiometer respectively for body mass index calculation.
Assessment of osteoarthritis symptom Severity (joint pain, stiffness and physical function)
Joint pain, stiffness and physical function were assessed by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) [18]. The WOMAC tool consists of 24 items divided into 3 subscales each with a score ranging from 0 to 4 where 0 = none, 1 = slight, 2 = moderate, 3 = severe, 4 = extreme [18]. The total WOMAC score ranges from 0 to 96 and was determined by the sum of the pain, stiffness and physical function scores. Higher scores indicate more severe disease.
The 3 subscales assessed:
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1.
Pain (5 items): during walking, climbing stairs, sleeping at night (in bed), resting (sitting or lying), and standing upright.
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2.
Stiffness (2 items): after first waking (morning) and later in the day (evening).
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3.
Physical Function (17 items): descending stairs, ascending stairs, rising from sitting, standing, bending to floor, walking on even floor, getting in/out of a car, shopping, putting on socks, taking off socks, rising from bed, lying in bed, getting in / out of bath, sitting, getting on/off toilet, doing light domestic duties, doing heavy domestic duties.
Laboratory analysis
Sample collection
We drew about 4 mls of venous blood from each consented study patient aseptically, into a plain blood sample tube (BD Vacutainer) and allowed the blood to coagulate at room temperature for not more than 4 h. The blood samples were then taken to a clinical chemistry laboratory at the study site, centrifuged immediately and stored at − 20 °C in a refrigerator. Upon collection of an ample number of blood samples, the stored serum was transported from the study site in a cooler box with ice packs to Mulago National Referral Hospital clinical chemistry laboratory within one and half hours for further storage at − 20 °C in a refrigerator until testing for up to 3 months.
Diagnostic testing
Serum Vitamin D concentrations were measured on thawed sera by electrochemilumniscence immunoassay using a Cobas-6000 machine supplied by Roche Diagnostics. It measures the serum vitamin D (25 (OH) D) concentrations in the range of 4–100 ng/ml.
Measurement of albumin, calcium concentration was also done. We corrected serum calcium levels for albumin for deranged albumin levels using the following formula: Corrected calcium (mmol/L) = measured serum calcium + [(normal albumin − patient’s albumin) × 0.02]. Hypocalcaemia was defined as serum calcium concentration corrected for albumin < 2.2 mmol/L.
Statistical analysis
Data was entered into entered into SPSS version 21 and excel and exported to STATA 15 software for analysis. Participant characteristics were summarized into means and standard deviations for continuous variables that were normally distributed and medians with their 25th and 75th percentiles for continuous variables that were not normally distributed. Patient demographic and clinical characteristic were summarized as frequencies and proportions. Using a mean pain, stiffness and physical function severity score (total WOMAC score) of 57.2 determined by the WOMAC tool with a standard deviation of 17.6 from a cross-sectional study carried out in patients with osteoarthritis in Turkey [9], sample size estimation for mean in one group was used for sample size calculation. A sample size of 107 patients was obtained and it powered the study to 80%.
The distribution of serum vitamin D levels was summarized as mean and its standard deviation because it was normally distributed. Joint pain, stiffness, physical function and total WOMAC scores were summarized into medians with their 25th and 75th percentiles because they were not normally distributed. The Spearman’s rank correlation was used to see the relationship between joint pain, stiffness, physical function, total WOMAC score and serum vitamin D levels. The coefficients were considered statistically significant, if they had a p value < 0.05.
Study approval
Ethical clearance to carry out the study was obtained from the Department of Medicine Makerere University College of Health Sciences and the School of Medicine Research and Ethics Committee; Ref No: 2020-015. All study participants provided written informed consent before enrolment into the study. The result was explained to the patient/caretaker by telephone and suboptimal serum vitamin D levels were replaced.