Chief Complaint and Presenting Illness
A 64-year-old homemaker woman presented with a history of uncontrolled type 2 diabetes (A1C>7%) and painful foot wounds on the left leg for a year on 17 December 2019. During daily debridement, there was no bleeding in the wound that was washed with physiological serum and dressed with fibrinolysin and phenytoin creams. In terms of Wagner’s classification system, the DFU was of grade 1 (superficial ulcer of the skin or subcutaneous tissue) (9). The left first toe and the middle toe were involved, and the size of the wound was 3.5×2 cm. The patient had pain with a visual analog scale (VAS) score of 80 mm on 100 mm (10).
 The patient's objective findings were tenderness and coldness around the wound, hairless limb, decreased dorsalis pedis arterial pulse, and tactile sensation relative to the right foot. There was no vascular claudication.
As for the past medical history, she had uncontrolled type 2 diabetes for the past year, and hypertension for two years ago. Moreover, she had a heart attack and a cerebrovascular accident several years ago.
 She took losartan 50 mg twice daily, Glucophage 500 mg twice daily, Nitrocontin 2.6 mg twice daily, Gabapentin 300 mg every night after dinner, Praglinide 1 mg twice before meals, Concor 5 mg twice daily, atorvastatin 20 mg/day, Lanoxin 0.05 mg/day except for Fridays and Mondays, acetylsalicylic acid (ASA) 80 mg/day, Nephrovit daily, and amlodipine 5 mg/day. These drugs were continued during the patient's wound-specific treatment.
 On physical examination, the patient was alert, with BP: 140/90 mmHg, heart rate: 105/min, respiratory rate: 16/min, and venous filling time: 10 seconds.
 Laboratory tests were as follows: fasting blood sugar: 350 mg/dL (normal range: 70-110 mg/dL), 2-hour postprandial blood sugar: 370 mg/dL (normal range: <145mg/dL), Hb A1C: 6.9 g/dL (normal range: <5.7%), atrogen factor 1.9 (normal range: up to 4), hemoglobin (Hb): 9.60 g/dl (normal range: 12.3-15.3 g/dL), hematocrit (Hct): 30.80% (normal range: 35.9-44.5%), mean corpuscular volume (MCV):  90.30 fL(normal range: 77-96 FL), white blood cells (WBC): 4.60 m/mL (normal range: 4.4-11.3 m/mL), neutrophils: 54%, lymphocytes: 44%, eosinophils: 1%, red blood cells (RBC): 3.41 m/mL (normal range: 4.1-5.1 m/ml), platelets: 193 m/mL (normal range: 150-450 m/ml), blood sugar (PP): 400 mg%, serum bilirubin: 0⋅6 mg/dl, serum glutamic oxaloacetic transaminase (SGOT): 17 IU/L, serum glutamic-pyruvic transaminase (SGPT): 29 IU/L, serum alkaline phosphatase: 226 IU/l, total cholesterol: 148 mg/dl, serum triglyceride: 233 mg/dl, blood urea: 35 mg/dl, serum creatinine: 1.32 mg/dl, serum uric acid: 5.2 mg/dl, bleeding time: 1:20 minutes, and clotting time: 5:20 minutes.
 The patient was treated with 5% leech cream prepared using lyophilized leech at −80°C and formulated in the pharmaceutical form of oil/water. In step one, wounds were washed and debrided without bleeding (Figure 1).
 The cream was applied twice a day for a month from the knee to the tail of the toes. Every three or four days, and on the follow-up visit, a photo was taken of the wound (before applying the cream). A week after the application of the cream, bleeding was observed during the dress changing. The diameter of the wound was reduced and, after a month, the wound was completely healed, as displayed in Figure 1. During this time, the patient took no antibiotics and only kept the wound clean.

Figure 1. Diabetic foot ulcer a) baseline; b) after 2 weeks of treatment; c) after 4 weeks of treatment and d) follow-up four weeks after cessation of treatment.

After applying the cream for a week, bleeding occurred when changing the dressing of the wound, which clearly indicated the improvement of the venous blood supply; this was not observed with other parallel treatments. Another important feature of this treatment compared to other methods of treatment is the trypsin inhibitor enzyme, inhibiting the trypsin and helping protect the wound and accelerate the healing process. Also, pain severity was significantly reduced (VAS: 4) after two days. At the end of the 3^rd day, the pain was completely disappeared. New tissue was found in the wound after a week. The wound size was reduced by ~50% during the 3^rd week. The diameter of the wound was decreased and, after one month, the wound was completely healed. Then, after four weeks, the ulcers were completely healed (Figure 1c). The follow up was done for two months and no recurrence was observed (Figure 1d).

Table 1. Laboratory tests

Discussion

The wound-healing effect of this product is visible in this study. This treatment seems more rational compared to the previous treatments of DFUs which commonly include debridement. Improving blood flow is a therapeutic principle in DFUs. In a study conducted on 564 diabetic patients with limb-threatening ischemia, PTA (percutaneous transluminal angioplasty) or BPG (bypass grafting) reduced member amputation. This feature is powered by hirudin and other anticoagulant agents present in the leech saliva. The salivary gland secretion of leeches contains proteins and peptides with different pharmacological activities. The mechanism of action of only eight known proteins from the leech salivary gland secretion that are biologically active includes: saratin protein with 12 kD (molecular weight) and the mechanism of platelet adhesion to the damaged vascular wall, calin protein with 65 kD, destabilase-lysozyme with 12.6 kD and the mechanism of antibacterial activity, hyaluronidase with 28.5 kD and the mechanism of smiting hyaluronic acid in the cellular matrix (8), collagenase with 100 kD and the cleaving and digesting activity of collagen (11), apyrase with 45 kD and the prevention of platelet initiation, antistasin with 15 kD and inhibition of tissue damage or inflammation, and hirudin with 8 kD and the mechanism of anticoagulation activity (11).
 Early diagnosis and effective treatment are considered essential for wound healing because they can prevent amputation and preserve the quality of life of the patient. Therefore, specific treatments should be considered based on the size, depth, and location of the wound (12). Basic and standard principles for DFUs include nutrition and control of blood sugar, infectious control, proper debridement, vascular insufficiency and decreased blood flow, foot pressure, and moisture retention (13, 14). In recent decades, leech therapy has attracted surgeons' attention for organ transplants and is used to prevent rejection. Because of the possibility of infection by the microbial flora of the digestive tract of leeches, despite the positive effects of leech therapy, , their use has been associated with risks. Reports on the side effect of leeching indicates the incidence to range from 4.1% to 36.2%. Ischemia, neuropathy, and infection are three important pathological factors leading to complications of the diabetic foot. Although not all wounds can be prevented, they can be reduced (12). Sig et al. showed that leech enzymatic activity reduced infection due to its anti-inflammatory and antimicrobial effects, and also reduced ischemia due to anticoagulant effects, blood flow increase and regulation, and platelet function inhibition. Also, they mitigated wound infection and accelerated the state of wound healing (15).
 
 

Conclusion

This study demonstrated that the daily application of topical leech cream can effectively enhance wound healing in diabetic wounds in a mean period of four weeks without any adverse side effects while also contributing to patient outcome. Several treatments are employed for DFUs, and their goal is to preserve the organ. In previous treatments, the probability of treatment failure was high and treatment often led to amputation. These treatments only include the debridement and disinfection of necrotic tissue. In this study, the main goal was to improve blood supply and prevent tissue damage; with improved blood supply, tissue repair can also be achieved. Further investigation is required with more clinical trials. It should be notes that treatment should be tailored to each patient's condition, health policy, and economic status.

Acknowledgements

The authors thank all those who helped them in writing and reviewing this manuscript.

 

Conflicts of Interest

Authors declared no conflict of interests.