Assessment of peripheral nerve affection of the lower limbs in type 2 diabetic patients using neuromuscular ultrasound and electrodiagnostic studies

Type 2 diabetes mellitus (T2DM) presents as one of the major health problems due to its considerable influence on man’s life as well as its high medical costs [1]. One of the most common microvascular sequalae of diabetes mellitus is diabetic peripheral neuropathy (DPN). DPN is considered a main predisposing factor for diabetes related lower limb problems [2]. Neuropathic effect of diabetes on the lower limbs could be classified into sensory, motor and/or autonomic peripheral neuropathy (PN). Nearly half of the patients with diabetes mellitus would subsequently present with symptomatic DPN within 25 years of diabetes onset [3].

Huge efforts were carried out recently to enable physicians to early detect DPN in the subclinical stage. Despite the advantages of nerve conduction studies (NCS) being widely known, non-invasive, and objective methods for diagnosis of DPN, the absence of symptoms in half of diabetic patients and the low sensitivity of electro-neurophysiology to small fibers usually result in frequently missed diagnosis [4].

Lately, with the improved technology, neuromuscular ultrasound (NMUS) is considered a relatively cheap, replicable, and a more convenient technique that could be used for diagnosing neuropathies [5]. The expected anatomical location and the peculiar appearance of the peripheral nerves, in addition to the superficial layout in part of their course in most of them, allow them to be scanned easily for evaluation by sonography [6]. Narayan et al. [7] found that cross-sectional area (CSA) measurements of peripheral nerves using high-resolution ultrasound were increased in cases of DPN as compared to healthy controls. Moreover, axonal affection was the predominant neuropathic pattern in lower limb peripheral nerves.

Since the pathogenesis and mechanism of occurrence of DPN are not fully understood, there are no efficient treatment measures to reverse the pathology and abolish the symptoms. Early detection is, therefore, crucial for improving prognosis and quality of life in patients with DPN. High-resolution ultrasound, therefore, shows a great promising tool for screening, diagnosing, and follow-up of DPN [4].

Although there were studies on the value of assessment of nerves in patients with DPN using ultrasound, its potential role in subclinical assessment has remained largely unmet [8]. Further research work would, therefore, enable us to better identify peripheral nerves affection occurring in the lower limbs of type 2 diabetic patients using NMUS to decide if ultrasound might be a useful tool for the diagnosis of DPN in asymptomatic patients. Moreover, additional studies are crucial to determine the presence or absence of any correlation between NMUS and NCS studies results aiming to determine if NMUS could complement or replace electrodiagnostic studies in early detection of DPN.

This diagnostic accuracy testing (an observational analytic case control study) included 40 patients diagnosed with type 2 diabetes mellitus (according to the American diabetes association diagnostic criteria [9]) and aged from 30 to 60 years who were recruited from Ain Shams University diabetic outpatient clinic through a period of 9 months. All subjects who were presented with a history of traumatic peripheral nerves injury, lumbar radiculopathy, foot/ankle fractures or deformities, foot ulceration, smoking/alcohol intake or common systemic disorders causing polyneuropathy were excluded from the study. Twenty healthy subjects, matched to cases in age and sex, who did not have any neuropathic symptoms or risk factors for neuropathy based on neurological examination were selected as controls. Ethical approval was obtained from the ethical committee at the Faculty of Medicine, Ain Shams University research board FMASU MD 41/2022. A written informed consent was taken from all participants.

All patients underwent full medical history taking with emphasis on duration of diabetes since date of diagnosis, presence of hypertension and symptoms suggestive of PN. Clinical examination was performed for all patients including general and full neurological examination; stressing on PN signs including but not limited to reduced muscle power, muscle atrophy, reduced deep tendon reflexes, orthopedic deformities (pes cavus and hammer toes), glove and stock hypothesia, decreased proprioception, decreased vibration, sensory ataxia and trophic changes. Monofilament testing was done for all patients as a quantitative sensory test using DARCO foot filament 5.07 (10 g) Semmes Weinstein monofilament. Ten points in each foot were examined including nine plantar sites (distal great toe, third and fifth toes; first, third, and fifth metatarsal heads, medial foot, lateral foot and heel) and one dorsal site [10]. Anthropometric measurements including weight (Wt), height (Ht), body mass index (BMI), waist circumference (WC), hip circumference (HC), and waist-to-hip ratio (WHR) were measured in all cases and controls. Laboratory investigations were performed including glycated hemoglobin (HbA1C) and serum cholesterol level.

NCS were performed for all patients following the electrodiagnostic protocol of PN proposed by Preston and Shapiro [11]. NCS were done using EMG machine model Nihon Kohden MEB-9400 K Neuropack S1 in Physical Medicine, Rheumatology and Rehabilitation department in Ain Shams University hospital. Sensory NCS were performed for the sural, median and ulnar nerves bilaterally. Motor NCS were performed for the common peroneal, posterior tibial, median, and ulnar nerves on both sides as well. Skin temperature was maintained at 36 °C during testing. Values obtained were compared against reference values proposed by Preston and Shapiro [11]. Patients were classified to either having electrophysiological diabetic peripheral neuropathy (EDPN) or not according to England et al. [12] minimum case definition criterion. EDPN pattern was identified as axonal, demyelinating, or mixed axonal and demyelinating according to Grimm et al. criteria [13]. Severity of EDPN in patients was determined where any parameter abnormality involving two, three to four or five nerves was graded as mild, moderate & severe neuropathy, respectively, as stated by Joy et al. [14].

All cases and controls underwent NMUS for lower limbs selected nerves using LOGIQ P5 Ultrasound Machine (General Electric Company, Milwaukee, USA) with a 7–12 MHZ linear array probe. Frequency, focus, depth and gain were adjusted according to the examined nerve. CSA measurements were recorded in the transverse plane where the transducer was placed perpendicular to the nerve with minimal probe pressure to avoid false distortion of the nerve. Measurement was performed from the inner border of the echogenic epineurium using the tracing method [15]. For consistency, three tracing trials were recorded with the mean value used. In addition, assessment of echogenicity (subjective assessment) was done and stated as normal or hypoechoic [8]. The CSA measurements were measured bilaterally in the following nerves at standardized anatomical sites as follows: tibial nerve CSA measurement was done proximally in the popliteal fossa [8] and measured distally at the proximal tunnel then a second axial image was obtained beneath the flexor retinaculum (within tunnel level) [16]. Fibular nerve measurement was recorded just distal to the bifurcation of the Sciatic nerve in the popliteal fossa and at the posterior fibular head proximal to the fibular tunnel [17]. Sural nerve measurement was recorded at the calf [18] with another measurement recorded at the level of the lateral malleolus at the ankle [15]. The sonographic measurements were done by a sonographer who was blinded to NCS results.

No statistically significant differences were detected on comparison between cases and controls regarding age and sex. Cases were found to have statistically significant higher BMI and waist-to-hip ratio when compared to controls, as shown in Table 1.

Six out of the forty cases (15%) had hypertension. At time of recruitment, cases showed a mean diabetes duration of 8.4 ± 6.8 years with mean Glycated Hb (HbA1C) level of 8.34 ± 1.85 gm/dl ranging from 5.1 to 12.5 gm/dl. Serum cholesterol levels in diabetic cases were 191.90 ± 36.30(mg/dl). As regards diabetes duration classification, 14 cases were found to have a diabetes duration of 5–10 years which represented 35% of all cases, while in diabetes durations of less than 5 years and more than 10 years, 13 patients representing 32.5% of all cases were found in each duration.

At time of recruitment, only 19 (47.5%) out of the 40 cases were symptomatic, while 21 (52.5%) patients were asymptomatic. On clinical examination, 31 (77.5%) patients were presented with symptoms and/or signs suggestive of PN with glove and stock hypothesia being the most prominent sign.

Out of the EDPN group (21 patients), 10 patients were asymptomatic at time of recruitment.

Regarding NMUS, CSA values of all examined nerves obtained of both sides were compared in the whole study group and no statistically significant side-to-side difference was found at all the measured sites. Thus, mean CSA value for each site of all examined nerves were calculated and used in statistical analysis using the formula [Mean CSA = (CSA of right limb + CSA of left limb)/2]. All cases showed normal nerve echogenicity on subjective assessment of all examined nerves at the measured sites.

Mean CSA values for all cases showed statistically significant higher values when compared to controls at all sites of all examined nerves except at the proximal tunnel of the tibial nerve, as shown in Table 2 and Fig. 1. Moreover, on comparing CSA values of controls, asymptomatic and symptomatic diabetic cases, again asymptomatic diabetic cases showed statistically significant higher values than controls at the following sites: tibial nerve both at Popliteal fossa and within tarsal tunnel and sural nerve at the ankle. Comparing mean CSA values between symptomatic and asymptomatic cases at all measured sites of examined nerves did not show any significant difference. In addition, symptomatic cases showed statistically significant higher CSA values than controls in the popliteal fossa site of the tibial nerve, as shown in Table 3.

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In asymptomatic diabetic cases comparison with controls, tibial nerve both at Popliteal fossa and within tarsal tunnel sites and sural nerve at the ankle site were of the highest significance (P values > 0.001,0.003, 0.001, respectively) with AUC values of 0.746, 0.739 and 0.754, respectively. The cutoff values were > 27, > 18 and > 4, respectively, Table 4.

Meanwhile, when patients of both groups were compared together, patients with EDPN were found to have higher mean CSA values than patients without EDPN at almost all sites measured. However, this difference did not reach a statistically significant limit except for the sural nerve at the ankle site which showed a statistically significant difference (P value = 0.009), as shown in Table 5.

Neither the pattern of affection whether axonal or mixed nor the severity of affection whether mild, moderate or severe showed a statistically significant difference in mean CSA values when comparing patients of group 1 with EDPN, as shown in Tables 6 and 7.

Correlation of diabetes duration with mean CSA among cases, did not show any significance except only at the popliteal site of posterior tibial nerve which showed statistically significantly higher values in patients with diabetes duration more than 10 years when compared to patients of other groups.

ROC test was used to explore the potential diagnostic value of mean sural nerve CSA at ankle site. For discrimination of diabetic patients with EDPN from diabetic patients without EDPN, the cut off value was > 5 mm² with a sensitivity of 47.6% and specificity of 89.5%. The positive predictive value was 83.3%, and the negative predictive value was of 60.7%, as shown in Table 8 and Fig. 2.

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In this study, we aimed to define the pattern of affection of lower limb peripheral nerves in type 2 diabetic patients using neuromuscular ultrasound and to assess its utility in evaluation of diabetic peripheral neuropathy in asymptomatic diabetic patients.

DPN commonly occurs in absence of obvious symptoms [19]. Up to 50% of DPN cases may be asymptomatic. Thus, regular screening for signs of neuropathy in diabetic patients is crucial, as it may detect the earliest stages of neuropathy [20] and this explains the higher percentage of diabetic patients detected with DPN based on signs (77% of patients) through clinical examination for DPN rather than that solely based on symptoms (47.5% of patients) and history taking, therefore, detecting DPN early in the subclinical stage.

In this study, sensory signs of PN in the form of glove and stock hypothesia were the most commonly detected sign and this abides with the nature of the disease where patients usually complain of symptoms of sensory loss before motor symptoms of DPN are manifested [21, 22].

Shabeeb et al. [23] concluded that the best quantitative tool for DPN diagnosis was electrophysiological studies. Bably et al. [24] study on type 2 diabetic patients found that 44.4% of patients had polyneuropathy by NCS. This was in accordance with our results, since nearly half of our patients were found to show EDPN on classifying them according to their electrodiagnostic findings. On further classification of EDPN patients according to the pattern, the majority of our patients showed mixed axonal and demyelinating pattern of EDPN. Hobson Webb et al. [17] confirms our findings denoting that electrodiagnostic studies often reveal a mixed axonal and demyelinating process.

In this study, mean CSA values of lower limbs peripheral nerves of diabetic cases were found to be statistically higher than controls at all sites of the examined nerves except at the proximal tunnel of the tibial nerve. Similar findings were revealed by Tóth et al. [25] who performed nerve ultrasonography at multiple sites on upper and lower nerves in 67 patients with T2DM and 25 healthy controls. Statistically significant higher CSA values were found at most of the sites performed in the lower limbs of diabetic patients than of controls. In this study, the only site that had higher mean CSA values in diabetic cases than controls yet did not reach a statistically significant result was the tibial nerve at proximal Tarsal tunnel.

In this work, patients with EDPN were found to have higher mean CSA values than patients without EDPN at almost all sites measured in all examined nerves; however, it was statistically significant only at the ankle site of the sural nerve. This would agree with the well-known fact that sural nerve is one of the earliest nerves to be affected in diabetic neuropathy [26, 27]. Goyal et al. [15] had similar results where sural nerve CSA value at ankle was statistically higher in diabetic patients with EDPN than in patients without EDPN. The cutoff value of sural nerve CSA to predict EDPN in that study was 4.41 mm², while in this study, it was 5 mm². This slight difference could be due to the smaller and the different population. However, our findings were not in line with Breiner et al. [28] as they did not record statistically significant differences in CSA measurements of the lower limbs between diabetic patients diagnosed with DPN and those who did not demonstrate DPN and only the Ulnar nerve at the Ulnar groove showed statistically significant difference in CSA measurements. These contradicting results to ours could be due different sample population examined by Breiner et al. [28] where type 1 diabetics were also included in addition to type 2 diabetic patients.

Our study showed that there was no statistical difference between symptomatic and asymptomatic cases regarding mean CSA values. Furthermore, asymptomatic diabetic patients had statistically higher CSA values than controls at almost all sites which supports the hypothesis that subclinical diabetes results in larger CSA than normal healthy population. These findings correspond with Breiner et al. [28] study findings which was performed on 67 patients with type 2 diabetes, with and without DPN, and 100 healthy volunteers that underwent NMUS measurements for selected sites of upper and lower limb nerves. Their study showed that all diabetic patients had higher CSA values in NMUS with no statistically significant correlation found between CSA values and clinical parameters such as presence of neuropathic symptoms and signs. In addition, Pitarokoili et al. [29] found no correlation in their study between sonographic measures and the clinical data of 44 diabetic patients proving our results that all diabetic patients will show changes in CSA regardless presence or absence of any peripheral neuropathic symptomatology. On the contrary, Yavuz et al. [30] stated that diabetic patients not having any clinical symptoms of polyneuropathy did not exhibit any CSA changes when compared with symptomatic group thus, they excluded the hypothesis that subclinical diabetes had an effect on CSA values.

Furthermore, comparison of mean CSA values between different patterns of EDPN among cases showed a non-statistically significant result at all measured sites of examined nerves. These findings were in agreement with Tóth et al. [25] where 102 diabetic patients underwent NCS. The diabetic subgroup with PN (n = 68) was divided into subgroups with (23 patients) or without (45 patients) signs of demyelination. Nineteen patients out of the 23 patients had mixed (demyelination and axon loss) electrophysiological abnormalities, while the majority of patients were found with axonal pattern. No significant difference in nerve size was found between the subgroups whether with some degree of demyelination or purely axonal PN subgroup. In addition, these patterns found in Tóth et al. [25] were similar to ours except that the mixed pattern was constituting our greater percentage unlike Toth et al. [25] which could be due to longer duration of diabetes in our patients, since early axonal changes are subsequently followed by added demyelination as diabetes duration increases, resulting in a mixed pattern due to progressive loss of the fastest and largest axons [31]. No pure demyelinating cases of DPN were detected in this study and it is well known that purely demyelinative neuropathy is rare in diabetic patients, and suggests more a demyelinative neuropathy of inflammatory cause [32].

Moreover, comparison of mean CSA values between different grades of severity of EDPN in patients showed a non-statistically significant result at all measured sites of examined nerves. This comes in contrary to Arumugam et al. [33] study which was conducted on 100 subjects with T2DM that underwent NCS and ultrasonographic CSA measurement for selected sites of the tibial, peroneal and sural nerves similar to our measured sites. Statistically significant differences among different grades of severity were found in the tibial and sural nerve CSAs where both nerves showed progressively larger nerve CSAs with worsening severity. This difference could be due to use of different criteria for classifying severity of DSPN, since Arumugam et al. [33] study used Toronto clinical scoring system which could be less accurate in severity assessment than using severity classification criteria based on electrophysiological data. Previous studies supported the use of NCS for determining the severity of DPN objectively unlike clinical psychophysical examinations [34, 35].

Regarding correlation of diabetes duration with mean CSA among cases, non-statistically significant difference was found at all sites measured except only at the popliteal site of posterior tibial nerve which showed statistically significantly higher values in patients with diabetes duration more than 10 years when compared to patients of other groups. Dhanapalaratnam et al. [36] study supports our results as mentioned previously regarding the high sensitivity of tibial nerve measurements for detecting neuropathy consistent with the length-dependent nature of neuropathy in diabetes. It is well known that in distal symmetrical peripheral neuropathy of diabetes, the neuropathic pattern starts and manifests early in the feet then proceeds to affect the more proximal parts of the lower limb nerves [32].

In this study, ROC test used to explore the potential diagnostic value of mean sural nerve CSA at ankle site revealed an AUC of 0.722. For discrimination of diabetic patients with EDPN from diabetic patients without EDPN, the cut off value was > 5 mm² with a sensitivity of 47.6% and specificity of 89.5%. The positive predictive value was 83.3%, and the negative predictive value was of 60.7%. Breiner et al. 28 ROC analysis showed that CSA of the sural nerve had acceptable performance with AUC 0.70–0.79 which was same as our AUC. Kang et al. [37] results were close to our results where the optimal cutoff values of CSA for diagnosis of DPN was 4.15 mm2 for the sural nerve. The optimal cutoff values of these nerves yielded sensitivity of 65–80% and specificity of 60–70%.

One of our study limitations was the small sample size used; therefore, further future studies on larger scale of patients are recommended with performing prospective studies on recently diagnosed patients with DM without clinical/ electrophysiological DPN, who will be followed with multiple NCS and NMUS measurements of CSA with parallel evaluation of HbA1c levels to be able to enhance our understanding to early morphological alterations in DPN. More studies are needed to show the value of NMUS in better understanding of the changes that happen in upper limb DPN.

Also, the use of more advanced ultrasound techniques with higher frequency for better resolution is needed that would enable us to visualize structures in more details and adding the use of color Doppler imaging to the study might also provide further insight. In addition, one of our limitations was using a subjective method for echogenicity assessment; accordingly, in future studies we are aiming at use of software systems objectively assessing nerve echogenicity.

This study concluded that diabetic cases had statistically significant higher mean CSA values of lower limbs nerves than controls. Moreover, mean CSA values of the tibial nerve at the popliteal fossa and within the tarsal tunnel in addition to sural nerve at ankle were statistically significant higher in asymptomatic diabetic cases than in controls. Therefore, CSA measurements at these sites will be helpful to screen for PN early in asymptomatic diabetic patients. This study also revealed that patients with EDPN were found to have higher statistically significant mean CSA values of the sural nerve at the ankle site than patients without EDPN. Thus, NMUS using sural nerve CSA measurement at the lateral malleolus could be used as a screening tool for detection of DPN in diabetic patients being less painful for the patients and less time consuming for the physicians to be followed by NCS as a confirmatory tool for DPN diagnosis.