RISK OF NON-HEALING IN ISCHEMIC VERSUS NEUROPATHIC DIABETIC FOOT ULCERS IN RELATION TO GRADE, STAGE OF INFECTION AND TREATMENT PROTOCOL: A FOLLOW-UP STUDY

Background & Objectives: Diabetic foot ulcer (DFU) is a potentially crippling consequence of diabetic foot disease. Aim of this study was to determine the risk of non-healing in ischemic as compared to neuropathic diabetic foot ulcers in relation to its grade and stage of infection. Methods: This prospective non-interventional study was conducted from July 2019 to February 2020 in Diabetes Management Center, Services Hospital Lahore Pakistan. Patients presenting with DFU were assessed for neurological and vascular status in the lower limbs. Ulcer grading was determined by Wagner's and Texas classification. Patients were followed up to 2-6 months for healing status of the diabetic foot ulcer. Results: Of 132 patients, 97 (73%) patients presented with neuropathic ulcer and 35 (27%) were having ischemic ulcers. Most participants were aged between 40-59 years. Based on Wagner's ulcer classification, it was observed that patient with score 2 had three times more likely to have their ulcer healed compared to those with score 1 and 3 [OR =3.09(95% CI:0.62-15.38, P=0.17)]. Kaplan-Meier survival curves showed that healing pattern among ischemic ulcer is considerably better compared to neuropathic foot ulcers. The evidence of equal survival hypothesis using Log-rank (Mantel Cox) test was statistically significant (p<0.001). No statistically significant difference in healing pattern through time was found across Wagner's scoring categories. Conclusion: Peripheral neuropathy was the commonest pathology underlying DFU presenting at our tertiary level diabetes clinic. Early detection of neuropathy and timely foot care may help prevent ulceration with its often-grave consequences.

life, loss of income, and cost of treatment is huge. 3 Prevalence of DFU is 6.3% globally, whereas lifetime incidence of foot ulcer in a diabetic person can reach 4 up to 25%. In Pakistan, diabetic foot disease prevalence ranges from 4% to 10%, and the amputation rate after developing foot ulcers is between 8% and 5 21%. The cost of care of diabetic foot syndrome (DFS) increases significantly following the appearance of foot ulcers. In the first year, it is 5.4 times higher than for diabetic patients without foot ulcers, and in the 6 second year, it is 2.8 times higher. The management of diabetic foot disease costs between 9 to 13$ billion in USA due to lost earning potential and treatment 7 costs. Although hospital stays have become shorter due to better surgical treatment and the use of improved glycemic control and multidisciplinary strategies; the number of major lower extremity amputations has not 8 decreased.
The development of a diabetic foot ulcer is the outcome of various pathological processes, including underlying diseases like peripheral vascular and neuropathy and poor glycemic control; each of which contributes to its genesis and progression. When someone has chronic high blood sugar levels, it triggers a series of metabolic pathways downstream. These pathways include excessive release of cytokines, formation of more advanced glycation end products, increased activity in the polyol pathway, activation of protein kinase C, and a higher level of oxidative stress. Over time, these deranged metabolic processes lead to the develop-9 ment of vascular insufficiency and nerve damage. Furthermore, when the blood flow to the skin is inadequate due to micro vascular insufficiency, it causes arte- 10 ries and arterioles of the skin to contract abnormally.
Diabetic foot ulcers usually begin at vulnerable spots which are abnormal plantar pressure points occurring as a result of foot deformity, which is a long-term [11][12][13] sequel of diabetic peripheral neuropathy. Direct consequences of foot ulcers include chronic non-healing ulcers, osteomyelitis, systemic sepsis, amputation and even death. In addition, worsening of glycemic control occurs as a consequence of mobility restriction and wound infection, which in turn leads to progression 14 of other diabetic complications.
Optimum management of foot ulcer requires an accurate ulcer evaluation. This involves assessment of ulcer severity, presence of infection and the underlying 15 patho-physiological condition. Many systems for ulcer severity grading have been described, but the most widely used grading systems are the Wagner's classification and the University of Texas ulcer grading systems. The former system grades ulcer by its depth and presence of gangrene, while the latter also considers the 16 presence of infection and underlying ischemia. The presence of infection can be assessed clinically and confirmed on wound cultures. The patho-physiological categorization is based upon assessment for the presence of peripheral vascular ischemia and peripheral neuropathy. The former may be tested clinically by checking the ankle brachial pressure index or by peripheral arterial Doppler ultrasound, while the latter evaluation is usually done by peripheral sensory examination using at least a 10g monofilament and tuning fork, and more formally by biothesiometry or nerve 17,18 conduction studies. Previous studies have shown that the vast majority of diabetic foot ulcers occur in neuropathic and neuro-ischemic feet, while purely 19 ischemic ulcers are less common. Some authors have indicated that neuropathic ulcers tend to have a higher grade of ulcer severity as well as a higher rate of infection due to continued weight bearing on insensate feet compared to ulcers occurring in feet with intact 17 sensations. On the other hand, among ischemic ulcers, delayed ulcer healing has been observed compared 20 to non-ischemic ulcers.
Clinical characterization of diabetic foot ulcers, including assessment of ulcer severity, presence of infection and an assessment of the underlying etiology is essential for risk stratification, optimizing management, and reducing debilitating complications. This prospective study aimed to determine the risk of nonhealing of diabetic foot ulcers in relation to its grade, stage of infection and treatment protocol while comparing ischemic versus neuropathic ulcers and to correlate these with ulcer severity, grading, presence of infection and short term outcomes (complete healing, non-healing ulcers, amputation and/or death).

METHODS
This prospective, non-interventional study was conducted at a tertiary care teaching hospital of Lahore city in Pakistan. Ethical approval for this study was granted by institutional review board Services Institute of Medical Sciences (SIMS) Lahore (IRB/ 2019/562/SIMS). Participants were recruited between July 2019 and December 2020 and then followed up to June 2021. Sample size of 132 was found to be suitable to detect the effect size. This sample size was calculated using WinPEPI software, taking prevalence of diabetic foot ulcer as 5 % and at 5 % precision around the prevalence, using smoking status. The proportional hazard assumption was examined and there was no significant violation of this assumption. Non-healing was coded 1 as our primary outcome and P value < 0.05 was considered significant.

RESULTS
The aim of this prospective study was to determine risk of non-healing of diabetic foot ulcers in relation to its grade, stage of infection and treatment protocol while comparing ischemic against neuropathic ulcers. Table 2 describes the baseline characteristic of participants with DFU. Of 132 participants 97(73%) patients had neuropathic ulcers and 35(27%) were diagnosed with ischemic ulcers respectively. Most participants were males with average age of those with ischemic ulcers was two years higher than those with neuropathic ulcers; with majority were between aged 40-59 years; yet no statistically significant difference was observed in these individuals. About 68% patients with neuropathic ulcers were males compared to 74% with ischemic ulcers. As regards smoking status, no statistically significant difference was observed among those with neuropathic versus ischemic ulcers patients (p=0.66). Most participants had type 2 Diabetes Mellitus and more than fifty percent patients had DM for ten years or less (Table 2). Regarding diabetic control of these patients, we found that more than 90% of both neuropathic and ischemic ulcer patients have poor diabetic control with HBAIc level more than 7%. Both neuropathic and ischemic ulcers patients did not give history of myocardial infarction or cardiovascular accidents, however, the difference of reporting myocardial infarction between those with neuropathic ulcer and ischemic ulcers was statistically significant (p=0.007). We found that a total of 33 patients (34%) with neuropathic ulcers and 18 patients (51.4%) with ischemic ulcers had infected wound, but this difference was not found to be statistically significant (p=0.07). Regarding the ulcer outcome in terms of healing, 82 (85%) of the patients with neuropathic ulcers and 26 (74%) patients with ischemic ulcers reported complete healing of ulcer, but there was no significant difference in healing versus non-healing found among these two types of ulcers (p=0.18). Table 3 represents Pathophysiological characterization and severity/grading of diabetic foot ulcers based on Wagner and Texas classification systems. Using Wagner's ulcer score, categorizing the ulcer based on its depth and presence of gangrene, it was found that 55 patients with neuropathic ulcers (56.7%) where having superficial ulcers without infection (Score 1), whereas 23 patients (24%) having deep ulcer with or without infection (score 2). Likelihood of healing DFU based on Wagner ulcer score, it was observed that patient with score 2 had three times more likely to have their ulcer healed compared to those with score 1 and 3; OR =3.09 (95% CI:0.62-15.38, P= 0.17) (Table 4). Comparatively, of 35 patients with ischemic ulcers, 13 (37%) were categorized as score-1 and 10 (28.6%) were given score 3 (deep ulcer with bone involvement). The likelihood of healing of ischemic ulcers of those patients with score 3 and above was higher [OR =1.09 (95% CI 0.12 -9.58, P=0.94)] compared to patients at score 1 and 2 ( Table 4).
The risk of non-healing of DFU, hazard ratios comparing healing of DFU among patients with neuropathic against ischemic ulcers, in relation to Wagner   (Table 5). On the other hand, the estimates for patients with ischemic ulcers for healing pattern using Wagner's ulcer scoring were insignificant and unremarkable (p=0.68). (Table 5). Figure 1 depicts Kaplan-Meier survival curves  and diabetic ischemic ulcers (Mantel Cox p =0.07) based on Wagner ulcer scores respectively. We found no statistically significant difference in healing pattern through time across the scoring categories. Finally, we did not find any statistically significant difference in healing through time using Texas staging and grading (data not shown).

DISCUSSION
Worldwide, diabetic foot problems have become most prevalent issues which lead to severe economic 7 crises for the patients, their families, and society. The main underlying reasons of diabetic foot ulcers are diabetic peripheral sensory neuropathies, peripheral vascular diseases, and resulting deformities. Additionally, callus formation, edema, and trauma are commonly identified as the factors that precede the development of diabetic foot ulcers.
Our study showed that there are certain risk factors that are associated with foot ulcers, including having diabetes for a longer period of time and having higher HbA1C levels. Males had a higher frequency of foot ulcers, and several studies have shown that advancing age is also a contributing factor to foot ulceration in 22 diabetic patients. We also found that the frequency of foot ulcers was particularly high among those over the age of 50 which is consistent with the findings of 25 a previous study by Khan et al. Neuropathy plays a significant role in the development of diabetic foot ulcers (DFU) because it causes the feet to become insensitive and lose their position sense. As a result, patients may injure their feet without realizing it and are unable to take steps to prevent further 25 damage, which impairs the healing process. A decrease in foot pulses has been linked to an increased risk of foot ulcers, and this may be a practical clinical alternative to more complex peripheral vascular assessments. However, the ankle/brachial pressure index has also 26 been identified as an independent risk factor.
Younis et al. found that 74% of ulcers were neuropathic, 19% were neuro-ischemic, and 7% were ische-19 mic. Our study also found that 73.48% of ulcers were neuropathic, and 26.51% were ischemic, which is similar to study by Younis et al. Other studies have reported that around 45-60% of diabetic foot ulcers are purely neuropathic, while approximately 45% have 27 both neuropathic and ischemic components. Similarly, 28 Shahbazian et al. found that 33.3% of the patients suffering from grade 1 or higher DFU had co-morbidities. In our study, we found that 57% of cases had comorbidities, including 13.6% with retinopathy, 15.9% with an eGFR of 59 ml/min or lower, and 19.7% with macrovascular complications. These co-morbidities contribute to the development of foot ulcers probably due to factors like generalized ischemia, chronic eczema, oozing ulcers in edematous feet as well as 25 immobility.
Jia et al. Found that 37% of cases had neuropathic ulcers, 28.4% had neuro-ischemic ulcers; ischemic ulcers were presented in 6.2%, and the remaining 28.4% reported having other types of ulcers. The overall infection rate of foot ulcers was 40.1% with the highest rate of 42.1% among neuropathic ulcers, 26.4% of these were ischemic ulcers, and 43.8% neuro-ischemic ulcers were infected (p = 0.11). Deep ulcers were found in 6.5% of neuropathic ulcers, 3.8% of ischemic ulcers, 29 and 7.5% of neuro-ischemic ulcers (p = 0.003%). Furthermore, past history of amputation was reported in 28.4% of the cases. In our study, we found that 38.6% of patients had wound infections. Neuropathic ulcers accounted for 73% of cases, while ischemic ulcers accounted for 27% of cases. On the other hand, about 34% of the neuropathic ulcers, 51.4% of the ischemic ulcers were infected (p = 0.07). Furthermore, 23.7% of neuropathic ulcers and 25.7% of ischemic ulcers had Wagner's grade 2 indicating a deep ulcer without bone involvement.
The most frequent reason for hospitalization related to diabetes is foot ulcer infection, which also remains a significant cause of amputation of the lower limbs. In the presence of neuropathy or peripheral arterial disease, the typical signs of local infection and the local inflammatory response are masked or reduced. Despite appropriate care, diabetic foot ulcers can progress to grave complications like infections, amputations, or even death. Previous studies indicate that peripheral arterial disease (PAD) is a more significant risk factor for diabetic foot ulcers compared to infection, possibly due to the participants from diverse populations. If a diabetic foot ulcer becomes infected, underlying PAD can speed up the infection's progression, leading to greater risk of hospitalization and ultimately amputa- 30 tion. Our study shows that there was a greater infection rate among ischemic type of DFUs; Regarding healing Hafiza Ammarah Sadiq pattern, it was found that comparing neuropathic diabetic foot with ischemic ulcer, healing pattern among ischemic ulcer is considerably better which may depend upon the level of care such patients are getting over there; lack of diagnostic facilities for neuropathy at primary health care centers can also lead to a delay in diagnosis. Hence, our suggestion is for patients to receive a thorough evaluation, preferably through noninvasive testing methods such as ABI measurements. This will also create an opportunity for conducting larger-scale studies with a more extensive sample size.
Results of this study should be interpreted after considering few limitations. Participants were recruited using convenient sampling and size of sample was relatively small for precise estimates. These issues might limit the generalization of findings. Follow-up was conducted by telephone (due to COVID-19 lockdown) with images of wound were virtually examined. This indirect examination might affect the inconsistency of grading of these wounds. We analyzed the data using both descriptive and inferential statistics especially examined the time to event analysis, which is unique for such data and highlight different aspects of exposure and outcome relationship.

CONCLUSION
Risk of non-healing DFU is significantly higher among those with ischemic diabetic ulcers compared to those with neuropathic ulcers. Infected wound at presentation is an important determinant of its subsequent healing. Early detection of neuropathy and preventive foot care may help prevent ulceration with its resultant disability. In most cases, neuropathy was the primary contributing factor to diabetic foot ulcers rather than peripheral arterial disease.