Study design and population
This was a cross-sectional study conducted at the medical out-patient department of the Enugu State University Teaching hospital between June 2017 and August 2018. The inclusion criteria were subjects aged 30–80 years, who had been diagnosed with T2DM according to the 1999 World Health Organization criteria [18], and who have clinical suspicion of lower extremity PAD based on history of intermittent claudication (recurrent exertional calf pains that are relieved by rest) and/or diminished/absent peripheral pulses (dorsalis pedis or posterior tibial) on manual palpation. Subjects with active DFU and those with pedal edema that extended above the ankle joint were excluded from the study. The Research and Ethics Committee of Enugu State University Teaching Hospital approved the study protocol while informed consent was obtained from each patient prior to recruitment.
Sample size determination
The minimum sample size for the study was determined by Fisher’s formula: N = Z2PQ/d2 where N = minimum sample size, Z = the standard normal deviate corresponding to a 95% confidence level (I.e. 1.96). P = prevalence of peripheral artery disease in subjects with T2DM i, e, 12% [19], Q = complementary probability i.e., 1 − P = 1 − 0.12 = 0.88, and d = absolute precision limit desired (5%) = 0.05. Thus N = (1.96)2 (0.12) (0.88)/(0.05)2 ≈ 162. Subjects were consecutively recruited on each diabetes out-patient clinic day until a sample size of 170 was attained.
Initial screening and clinical examinations
After obtaining demographic and diabetes related history, history of intermittent claudication was sought. This was followed by manual palpation of the dorsalis pedis and posterior tibial artery pulsations in both lower limbs. Subjects were recruited for further vascular assessments (ABI and DUS) if they admitted to positive history of intermittent claudication and/or were adjudged to have diminished or absent peripheral pulses in at least one lower limb.
Ankle brachial index measurements
Oscillometric method using digital automatic blood pressure apparatus (OmronMX2,Omron Healthcare Europe B.V. Hoofddorp, The Netherlands) was used to obtain brachial and ankle (posterior tibial) blood pressures in both upper and lower extremities. Vegas et al. [16] had demonstrated that this method was more accurate than using manual blood pressure device and a Doppler probe. Examination was done with the patient lying supine after at least a 15-min rest. The ABI for each leg was calculated as a ratio of ankle blood pressure to that of the mean arm pressure. A ratio in the range 0.9–1.3 was regarded as normal according to international recommendations [13,14,15]. An ABI < 0.9 was diagnostic of PAD and values were stratified according to severity as follows: 0.7–0.89 = mild arterial obstruction, 0.5–0.69 = moderate arterial obstruction, ≤ 0.5 = severe arterial obstruction [14]. Subjects with ABI > 1.3 were adjudged to have incompressible lower limb arteries and excluded from analysis.
Duplex ultrasonography assessment
Duplex ultrasonography of the lower extremity arteries, from common femoral to pedal arteries was performed for each patient not later than two weeks following ABI measurements using a DC 60 Colour Duplex Scanner and a 7.5 MHz linear-array transducer (Mindray, China). The test was conducted by a certified radiologist and followed standard imaging protocols. Examination was done with the patient lying supine after resting for at least 15 min. The common femoral and anterior tibial arteries were imaged with the patient in the supine position while those of the popliteal, peroneal and posterior tibial arteries were done in lateral position. Imaging views were taken parallel to the vessel walls and within the center of laminar flow while maintaining the Doppler at or below angle 60°. Identification of arterial segments was based on the appearance of color signals, and where the artery is occluded, by the detection of a vessel wall accompanied by a vein. When turbulence or velocity increase was detected in the color image or the B-mode image suggested a change in vessel caliber, then flow velocity measurements were performed by means of spectral analysis and peak systolic velocities (PSV) recorded in meters per second (m/s).
Peripheral artery disease was diagnosed by the presence of at least a 50% reduction in luminal diameter, corresponding to PSV ≥ 150 cm/s. The severity of stenosis was graded as follows: 50–75% (PSV 200–300 cm/s) = mild stenosis; 76–99% (PSV > 300 cm/s) = moderate stenosis; complete occlusion = severe stenosis [20]. Complete luminal occlusion was diagnosed by the following criteria: loss of signal in the vessel segment, attenuated distal signal relative to that of the proximal, or detection of proximal exit or distal re-entry collaterals.
Data analysis
Data were analyzed with the Statistical Package for Social Sciences software (IBM version 23.0; SPSS Inc., Chicago, IL, USA). Categorical variables were presented as numbers and percentages while continuous variables were presented as means and standard deviations. Data were presented in tables and charts as appropriate. The sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy of ABI for diagnosis of PAD based on DUS as the gold standard were computed. Analysis was done at limb level rather than participants since ABI was calculated separately for each limb in line with international guidelines [13, 15]. Agreement between ABI and DUS was tested by Cohen’s Kappa (κ) statistics. The following stratification of κ was used: κ value of 1 = perfect agreement, κ 0.8–0.99 = excellent agreement, κ 0.6–0.79 = good agreement, κ 0.4–0.59 = moderate agreement, κ < 0.4 = poor agreement and κ value of zero signifies no agreement beyond chance.
