Volume 27 Number 2

Introduction

Diabetes mellitus (DM) is a global health challenge encountered in the public health care system. The International Diabetes Federation (IDF) diabetes atlas reported that in 2021, worldwide, an estimated 537 million adults aged 20–79 years were living with diabetes. It is projected to escalate to 643 million by 2030.¹ The global annual incidence of diabetes-related minor and major amputations during the 2010–2020 period was estimated to be 139.97 and 94.82 cases/100,000 people with diabetes, respectively.² The significance of diabetes stems from its intricate vascular, neuropathic and metabolic implications.³ Approximately 15% of diabetic patients are expected to experience diabetic foot ulcers (DFUs) at some point in their lives.⁴ If not expeditiously addressed, it can precipitate complications like infection, gangrene and the need for limb amputation.^5-6 Neuro-ischemic ulcers are the most frequently observed type of DFU.⁷ DFUs occur due to trauma and chronicity may be due to altered phases of healing.⁸ Chronic wounds may remain in a prolonged proliferative phase or revert to an inflammatory state.^9-10

Clinic presentation of DFU

Diabetic foot ulcers can cause discomfort, edema, erythema, induration, ulceration and sinus tract development in addition to other local foot issues impacting quality of life of patient. The classical signs and symptoms of infection in DFU includes increasing pain, erythema, edema, heat and purulent exudate.¹¹ Others may include fever, chills, nausea, vomiting, tachycardia and fatigue.

One of the essential components of DFU care is regular wound dressing after achieving glycemic and infection control. To improve healing in chronic ulcers, a variety of dressing materials have been developed over recent decades, ranging from basic to advanced types. Basic dressings serve primarily as physical barriers and help maintain a moist ulcer bed. However, they do not promote antibacterial activity or stimulate growth factors necessary for rapid healing.

Advanced dressings, including hydrogels, silicone meshes, hydrocolloids, alginates, vapor permeable adhesive films and those containing silver or collagen, may offer antibacterial properties and promote healing. Bioactive dressings, derived from synthetic materials or natural tissues like chitosan, alginate, hyaluronic acid, collagen and elastin, actively support tissue regeneration. Tissue engineered human skin equivalents (HSE) replicate the skin’s structure and activate wound healing by promoting epithelialisation. Despite their benefits, advanced and bioactive dressings especially HSE are costly and often require specialised storage, placing a financial and logistical burden.¹²

Diperoxochloric acid (DPOCL) is a novel therapeutic agent for severe wound-healing disorders. However, few studies have compared its efficacy with currently available dressing materials, leaving limited comparative data. This gap provided the rationale for the present study.

Mechanism of action of DPOCL

DPOCL exerts its functional effects by releasing chlorine dioxide (ClO₂), a reactive oxygen species (ROS), upon decomposition DPOCL exerts functional effects similar to ROS by releasing ClO₂ upon decomposition, which provides antibacterial activity. In studies with MRC-5 fibroblast cells, DPOCL has been shown to stimulate fibroblast proliferation.¹³

ClO₂ has the following benefits:

• It directly interacts with microbial cell walls
• It is the sole biocide that functions as a molecular free radical
• It eliminates even inactive bacteria, virus, fungi and certain spores by penetrating their cell walls and acting as a powerful oxidising agent that disrupts essential cellular components
• Microorganisms cannot develop resistance to ClO₂
• It can penetrate the slime layer of bacteria as it does not react significantly with the inert sugars that make up the extracellular polymeric substances (EPS), allowing it to penetrate deeper into the matrix

A multicenter, randomised, active-controlled, parallel-group study by Bal et al¹³ reported that DPOCL can be considered an effective and safe treatment option for DFU compared to isotonic normal saline. Similarly, a case series by Murthy et al¹⁴ reported positive wound contraction and reduction in ulcer size during the follow-up period, which ranged from 3 to 10 weeks.

Although DPOCL dressings have been shown to have several advantages in DFU, there is little research on its application to diabetic wounds. Thus, the purpose of this study was to evaluate the efficacy of DPOCL dressings compared to conventional normal saline dressings in DFUs aiming to provide a more affordable alternative to expensive novel dressings.

Objective To compare the percentage and rate of absolute wound surface area reduction among DPOCL and normal saline groups.

Methods

This is a randomised single blinded (patient) prospective study conducted among patients suffering from DFU, who attended the General Surgery OPD of a tertiary care hospital in North India and met the inclusion criteria and none of the exclusion criteria.

Inclusion criteria

1. Age >18 years
2. Non-healing diabetic ulcers of ≥6 weeks’ duration
3. Random blood sugar (RBS) <250mg/dl on anti-diabetic treatment
4. HbA1c <12%
5. Hemoglobin ≥10g/dl
6. Wagner’s grade¹⁵ 1 or 2 ulcers
7. Wound surface area between 1 and 15 cm-sq
8. Ankle-brachial pressure index (ABPI) >0.7
9. Willingness to attend follow-up visits

Exclusion criteria

1. Ulcers caused by arterial or venous insufficiency, or electrical or chemical burns
2. Ulcers with necrosis, purulent material or sinus tracts that could not be removed by debridement, as these will increase bioburden and delay healing
3. Ongoing treatment with corticosteroids, immunosuppressive agents or chemotherapeutic agents or radiotherapy
4. Poor nutritional status (serum albumin <3g/dl)
5. Wagner’s grade¹⁵ 3, 4 or 5 ulcers
6. Wound surface area more than 15cm-sq
7. Not willing to attend follow-up visits

After institutional ethical board approval, written informed consent was obtained from all subjects for the study and publication of data before starting the study. A total of 74 patients were enrolled in the study. Participants were divided into two groups, A and B, by permuted block randomisation. Patients were asked to pick-up folded chits containing random numbers generated from https://www.random.org/ and were predefined into either of the two groups. Patients in Group A were treated with daily DPOCL reconstituted solution and patients in Group B received daily conventional normal saline dressing. The study was carried out for 12 months from 30 June 2022 to 30 June 2023 and the treatment period was equal to follow-up period and lasted 30 days for each patient.

Colour doppler flow imaging (CDFI) of all patients demonstrated good vascular flow without luminal obstruction, with occasional vessel wall calcification in some cases. All patients had neuropathic DFUs; therefore, no revascularisation procedures were performed. Offloading shoes were provided during therapy and at discharge from the hospital’s artificial limb centre. Patients also received counselling on proper foot care to prevent recurrence. No systemic or topical antibiotics were used in either group, except in five cases where surgical debridement was done once in beginning of therapy in patients with infected diabetic foot ulcers with initial polymorphonuclear leucocytosis to reduce bacterial burden, remove necrotic tissue, and mitigate infection severity (Group A, n=2; Group B, n=3). These patients were given an injection of Augmentin 1.2g (875mg amoxicillin + 125mg clavulanic acid) 12 hourly for one week post debridement to cover the peak bacteraemia window and prevent septicaemia as per International Working Group on the Diabetic Foot (IWGDF) guidelines on diagnosis and treatment of diabetes related foot infection¹⁶ and institutional protocol.

Method of reconstitution of DPOCL solution and dressing

Diperoxochloric acid, the active ingredient, was supplied in 10mL bottles (Bottle A) containing 7.5mL of concentrate. This was then diluted with 22.5mL of sterile 0.9% w/v sodium chloride solution in a 30mL bottles (Bottle B) [Figure 1]. The reconstituted solution bottles were labeled with the date of reconstitution. They were stored in a cool, dark place and used within 14 days.

 

Figure 1. Materials used for dressing: Bottle-A containing DPOCL, Bottle-B containing sterile sodium chloride solution 0.9% w/v, dropper, inner gauze piece, outer gauze piece and roller gauze.

 

In Group A, as per the instruction manual of the product (DPOCL), the wound was washed with normal saline and 3.5mL of the reconstituted solution was spread over the inner gauze (3x3cm) in zig-zag fashion. Care was taken not to touch the gauze piece with dropper tip. This gauze was applied to the wound. It was covered by outer gauze (3x3cm) to prevent dryness at wound site and rolled gauze was applied over outer gauze to keep it secure [Figure 2]. On the next day, the reconstituted solution was applied over the previous inner gauze and closed with new outer gauze and roller gauze. The inner gauze was changed on alternate days and the outer and rolled gauze was changed daily. Photographs and tracing of the wound were taken every week. In Group B the wounds were washed with normal saline and gauze pieces soaked with 5mL normal saline was used for daily dressing of the wound and both inner and outer dressing was changed daily.

 

Figure 2. Dressing method

 

Day 1 and Day 30 or on the day on which the wound closed completely (where complete wound closure was achieved before 30 days) photographs and tracings were used for comparison of reduction of wound surface area. The tracings taken on a transparent sterilised (ETO) film were placed over a graph sheet with smallest square of 1x1mm that was used for calculation of surface area. One small square was equal to 1mm-sq. If the tracing line was covering more than or equal to half of small square, it was counted as one and if less than half area was covered it was counted as zero.

Sample size calculation

The sample size in each group of the study was calculated according to the formula given by Sahai & Kurshid (1996).¹⁷

Sample size estimation was based on the study by Bal et al¹³ which reported the following proportions of wound closure in each of the group:

P1 (Proportion in First Group)            =  0.7103 (71.03%)

P2 (Proportion in Second Group)      = 0.5753 (57.53%)

Type I error (α)=5%, Z_α (Value of Standard Normal Distribution for α =5%) = 1.96. Type II error (β)=20%, Power (1–β) = 80%, Z_1-β=0.842 Based on the above-mentioned formula, using the mentioned values, assuming 80% Power and 95% Confidence interval, the minimum calculated sample size for each arm was 197≈200 (Total 400).

Since a total of about 90 patients were available for inclusion into the study during the study period, applying finite population correction, we get a sample size of

400/ (1+(400/90)) ≈ 74 (37 in each group).

A total of 74 patients (both male & female) were included in the study.

Study flow

Statistical analysis

Data was coded and entered in MS Excel spreadsheet. SPSS v23 (IBM Corp.) was used for data analysis. Descriptive statistics was given as means/standard deviations and medians/IQRs for continuous variables, and frequencies and percentages for categorical variables. Data was presented graphically wherever needed for data visualisation. If data was normally distributed, appropriate parametric tests were used, for example continuously distributed data were compared using the independent sample t-tests when comparing two groups, while for non-normally distributed data, Mann-Whitney U test were used when comparing two groups and Friedmann test when comparing more than two dependent groups. Post-hoc- wise analysis was performed using the Wilcoxon signed-rank test. Chi-squared test was used for group comparisons of categorical data. Statistical significance was set at p<0.05.

Results

The characteristics of both the groups are given in the table below:

• At baseline WSA (mm-sq) of patients in Group A and Group B was 494.568±257.686 and 448.86±250.05 respectively. It was comparable at baseline. P value=0.441 (statistically insignificant).
• After seven days of treatment, WSA (mm-sq) of patients in Group A and Group B was 339.81±183.37 and 344.162±201.01. P value=0.923 (statistically insignificant).
• After 14 days of treatment WSA (mm-sq) of patients in Group A and Group B was 209.22±118.23 and 245.35±153.50. P value=0.260 (statistically insignificant).
• After 21 days of treatment, WSA (mm-sq) of patients in Group A and Group B was 102.13±54.27 and 166.56±113.30. It was statistically significantly less in Group A than Group B. P value=0.003.
• After 30 days of treatment WSA (mm-sq) of patients in Group A and Group B was 36.54±26.84 and 99.35±77.26. It was statistically significantly less in Group A than Group B. P value = 0.001. (Figure 3, Table 1).

 

Figure 3. Comparison of reduction in WSA (mm-sq) in the two study groups
WSA=Wound surface area, Blue= Group A (DPOCL group), Red=Group B (Normal saline group).

 

Table 1. Comparison of change in absolute WSA (mm2) of the two study groups

 

• After 30 days of treatment the percentage reduction in WSA (mm-sq) of patients in Group A and Group B was 93.02±3.39 and 79.64±6.106. Reduction in WSA was significantly higher in Group A than Group B. P value=0.001 (Table 2).
• Comorbidities distribution was comparable across both groups (Figure 4)
• Wound location distribution was comparable across both groups (Table 3)
• The healing rate of wounds as absolute area healed per day was 16.5mm-sq/day in Group A compared to 14.9mm-sq/day of Group B.
• None of the patient’s had any adverse reaction to DPOCL during treatment.
• Only one patient in each group achieved complete wound closure within 30 days.

 

Table 2. Comparison of change in WSA (mm2) and percentage reduction between the two study groups

 

Figure 4. Distribution of comorbidities among the two study groups
Blue= Group A (DPOCL), Red= Group B (normal saline).

 

Table 3. Comparison of wound location distribution in the two study groups

 

Discussion

An increased risk of readmission, amputations and death is associated with DFU. DFUs are debilitating and painful, drastically lowering a patient’s quality of life.^18-21 Until the nineteenth century, DFUs were treated with lengthy bed rest; nevertheless, the ulcers resurfaced as the patient began to move.²² Dressing is an important part of DFU treatment after achieving glycemic and infection control. Modern dressings have the limitation of special storage, like for growth stimulators, and most of them are not cost-effective. The current study intended to assess the effectiveness of DPOCL in the treatment of chronic non-healing DFU.

When we compared the WSA (mm-sq) across two research groups, we discovered that Group A had a considerably greater reduction in WSA after three weeks (p=0.003) and 30 days (p=0.001) than Group B. This is consistent with the findings of Bal et al,¹³ who compared the effectiveness and safety of a topical solution of DPOCL to isotonic sodium chloride solution (ISCL). The mean WSA in the DPOCL arm at the end of their study (0.639cm-sq) was substantially lower (P=0.0209) than the ISCL arm (0.818 cm-sq). These data demonstrated that DPOCL may be regarded as an effective and safe therapy for DFU.

In the current study, the percentage decrease in WSA (baseline to 30 days) was shown to be considerably larger in Group A than in Group B (p=0.001). This is also consistent with the findings of another case series published by Murthy et al,¹⁴ where diperoxochloric acid was shown to be beneficial in treating infected diabetic foot ulcers associated with osteomyelitis. DPOCL releases chlorine-dioxide and may have cytotoxic effects. However, during our study no increase in skin inflammation or any other sign of toxicity was noticed.

 

Figure 5. Wound healing in a DFU with DPOCL dressing achieved during this study: A) Initial DFU. B) After 7 days of dressing with DPOCL. C) After 14 days of dressing with DPOCL. D) After 21 days of dressing with DPOCL. E) After 30 days of dressing with DPOCL. F) Graphical tracing of wound.

 

Limitations

Our study has several limitations firstly; it is a single-center study done at a tertiary care hospital. As a result, the findings of our study may not apply to small hospitals or primary care clinics.

• Only a small sample size was available.
• A longer duration of follow-up (3 months or more) is required to calculate the time required for complete closure of the wound/ healing rate.
• To rule out cytotoxicity arising out of DPOCL treatment further pathological evaluation is needed.

Potential bias

This study may be influenced by selection bias due to the small sample size and limited population, which may reduce generalisability. Observer bias is also possible, since wound healing outcomes were partly assessed through visual inspection, despite efforts to maintain objective measurement standards. Finally, confounding factors, such as variations in comorbid conditions and nutritional status, may have contributed to differences in healing outcomes, despite randomisation and attempts to standardise treatment protocols.

Conclusion

Diperoxochloric acid is a recently approved agent for the treatment of diabetic foot ulcers. The study findings suggest that DPOCL is a safe and effective option and may offer advantages over conventional normal saline dressings. Compared to normal saline, DPOCL was associated with a greater reduction in wound surface area, indicating its potential to promote faster healing and improve clinical outcomes in patients with diabetic foot ulcers.

Axknowledgements

Thanks to Dr K J Singh, former HOD Surgery for his support of this research. Thanks also to Dr VC Jha, Dr Sanjay Kumar, Dr Vikram Trehan, for encouragement and support, and to all the nursing staff and technicians for their cooperation during the study. Thanks also to the patients and their parents for giving informed consent and participating.

Conflicts of interest/Competing interests

All authors certify that they have no affiliations with or involvement in any organisation or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Ethics Committee (Date: 29 June 2022/No. 09/29/Jun/CHWC/2022).

Funding

No funds, grants or other support was received by the authors for the submitted work.

Consent for publication

The participants have consented to the submission of data to the journal.

Author(s)

Pradeep Kumar Singh¹* MS General Surgery, Gurpreet Singh Bhatia¹ MS General Surgery, Amandeep Singh Kalra¹* MS General Surgery, Aman Arora² MS General Surgery, Gagandeep Singh¹ MS General Surgery
¹Department of Surgery, Command Hospital (Western) Chandimandir, Panchkula, India
²Military Hospital Jamnagar, India

*Corresponding authors email pradeepthename@gmail.com  dr.askalra@gmail.com

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