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Efficacy of Maggot Therapy on Staphylococcus aureus and Pseudomonas aeruginosa in Diabetic Foot Ulcers

A Randomized Controlled Trial

Malekian, Azam; Esmaeeli Djavid, Gholamreza; Akbarzadeh, Kamran; Soltandallal, Mehdi; Rassi, Yavar; Rafinejad, Javad; Rahimi Foroushani, Abbas; Farhoud, Amir Reza; Bakhtiary, Ronak; Totonchi, Mehrangiz

Journal of Wound Ostomy & Continence Nursing: January/February 2019 - Volume 46 - Issue 1 - p 25–29
doi: 10.1097/WON.0000000000000496
Wound Care
Free

PURPOSE: This study was conducted to evaluate the antimicrobial effects of medicinal maggots of Lucilia sericata on Staphylococcus aureus and Pseudomonas aeruginosa on diabetic foot ulcers (DFUs).

DESIGN: Randomized controlled trial.

SUBJECTS AND SETTING: The sample comprised 50 adult patients from the clinic of the Academic Center for Education, Culture and Research of Tehran University of Medical Sciences, Iran. All participants who had at least 1 DFU present for at least 12 weeks, an arterial brachial index value of more than 0.6, and a hemoglobin A1c value of less than 8% were included in this study.

METHODS: Subjects were randomly selected for the maggot-treated (treatment) or conventional treatment (control) group. Conventional treatments such as antibiotic therapy, debridement, and offloading were done for both groups, but maggot therapy (MT) was added to the protocol of the treatment group. Bacterial burden was monitored and compared for both groups using cultures collected using swab technique. Wound secretions were measured and compared in both groups.

RESULTS: The number of infected cases with S aureus in the treatment group was significantly reduced after 48 hours in comparison with the control group (P = .047). The number of infected cases with P aeruginosa was significantly reduced after 96 hours (P = .002). We also found that wound secretions in the treatment group were significantly higher than in the control group (P < .00).

CONCLUSIONS: Our findings indicate that MT is a safe and efficacious treatment of DFUs.

Azam Malekiam, MSc, Maggot Debridement Therapy Clinic, Iranian Center for Wound Healing, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran; and Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Gholamreza Esmaeeli Djavid, MD, Maggot Debridement Therapy Clinic, Iranian Center for Wound Healing, ACECR, Tehran, Iran; and Medical Laser Research Center, ACECR, Tehran, Iran.

Kamran Akbarzadeh, PhD, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Mehdi Soltandallal, PhD, Department of Medical Pathology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Yavar Rassi, PhD, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Javad Rafinejad, PhD, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Abbas Rahimi Forooshani, PhD, Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Amir Reza Farhoud, MD, Joint Reconstruction Research Center, Department of Orthopedic Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran.

Ronak Bakhtiary, PhD, Department of Medical Pathology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Correspondence: Kamran Akbarzadeh, PhD, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (kakbarzadeh@tums.ac.ir).

The authors declare no conflicts of interest.

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INTRODUCTION

Long-term complications of diabetes mellitus include diabetic foot ulcers (DFUs), heart disease, stroke, chronic kidney failure, and damage to the eyes.1 Diabetic foot ulcers are the most common foot injuries leading to lower extremity amputation.2 A DFU does not heal in an orderly fashion, nor in a predictable amount of time.3 Wound healing in DFUs may be delayed by multiple factors including infection with various species of bacteria.4 For example, infection with antibiotic-resistant organisms and development of biofilms are known to delay wound healing.5

The main bacteria associated with DFUs are Staphylococcus aureus and Pseudomonas aeruginosa.6 Staphylococcus aureus, a gram-positive bacterium, is common in the flora of human skin. Pathogenic strains create potent protein toxins and promote cell-surface proteins that bind and inactivate antibodies. The advent of antibiotic-resistant strains of S aureus such as methicillin-resistant S aureus is a global problem in clinical medicine.7 Pseudomonas aeruginosa is a gram-negative, rod-shaped bacterium that causes disease in humans.8 This species is a typical “multidrug-resistant pathogen” recognized for its ubiquity, intrinsically advanced antibiotic resistance mechanisms, and association with serious illnesses (especially nosocomial infections). In all infections colonized by P aeruginosa, treatment is complicated by the organism's resistance profile, which may fail treatment.9

Maggot therapy (MT), also referred to as larva therapy, has been used since the 1930s for cleaning soft-tissue lesions.10 Maggot therapy has been shown to be effective for various kinds of wounds with gangrenous or necrotic tissues.11 One of the main healing properties of MT is its ability to reduce bacteria from the wound through digestion,12 , 13 production of antibacterial excreta/secreta,bacteria of a <500-Da fraction from maggot excretions/secretions of Lucilia sericata (Diptera: Calliphoridae). Microb Infect. 2008;10(4):325–333.','400');" onMouseOut="javascript:ImageWrapperControl_ImageMouseOut();">14 and destruction of biofilms.15 The efficacy of MT for reducing bacterial infections has been demonstrated in multiple studies.16–21 We searched the Iranian medical literature and did not find any adequate studies of MT. This study was conducted to evaluate antimicrobial effects of MT on S aureus and P aeruginosa in Iranian subjects with DFUs.

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METHODS

This randomized clinical trial was conducted in the clinic of Academic Center for Education, Culture and Research of Tehran University of Medical Sciences (registered in IRCT as code no. IRCT2016060328232N1). The study was reviewed and approved by the Medical Ethics Board of Tehran University of Medical Sciences, Tehran, Iran, with code no. IR.TUMS.REC.1395.2626, 24/05/2016.

A cohort of 50 adult patients, who each had at least 1 DFU, classified as Wagner22 grades 2 and 3, and present at least 12 weeks, was selected and randomly categorized into 2 groups (treatment or control). Data were collected from December 2015 to March 2016. Additional inclusion criteria were an arterial brachial index of more than 0.6 and a hemoglobin A1c value of less than 8% on study enrollment and randomization. Exclusion criteria were presence of severe infection requiring hospitalization, gangrene, systemic diseases such as collagen-vascular diseases, evidence of ischemia, entomophobia (fear of ≥1 classes of insect), and refractory and severe pain. The treatment group received MT in addition to conventional treatment (sharp debridement, antibiotic therapy, offloading). The control group received conventional treatment (sharp debridement, antibiotic therapy, offloading).

Sterile larvae of Lucilia sericata (Diptera: Calliphoridae) were used as the agent for MT. The larvae were prepared and disinfected at the Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences. The process of disinfection included washing out of the eggs with distilled water, submerging them in the chlorhexidine 5% for 8 minutes, submerging them in EtOH 70% for 3 minutes, washing again in sterile distilled water as a final step, and release onto sterile blood agar media in the filter top flask. The flask was left in an incubator at 28°C to 30°C for hatching. After 24 hours, the larvae were ready to use.

To ensure a consistent and standard dressing procedure for both the control and treatment groups, topical dressings were managed by an expert nurse who had been trained for this study. The control group received conventional treatment including sharp debridement, antibiotic therapy, and offloading as indicated. The treatment group received conventional treatment and MT. The skin within a circumference of approximately 2 cm of the wound was covered with a zinc oxide or similar skin protectant to prevent contact of healthy tissues with excretions of the larvae. Preparation of the treatment dressing included cutting an opening in the sterile gauze as the shape of the wound. The sterile gauze was then applied over the barrier protected periwound skin. This was done for 2 reasons: it restricted the larvae to the wound bed and created a space for their activity. Maggots were applied directly onto the wound at a dose of 5 to 7 maggots/1 cm2 of wound surface. Maggot therapy was applied at 48 hours' intervals. A special soft mesh net of polyvinyl alcohol, prepared beforehand and sterilized, was used for covering of the cage-like space and maggots on the wound. We placed 2 to 3 sterile gauze pads over the wound bed to absorb drainage. These gauze pads were replaced if saturated with wound drainage and a new one applied. The dressing was secured with soft roll bandage.

A culture using the swab technique was collected before and immediately after each maggot application. Wound drainage was calculated by weighing dressings before and after use. Synchronized sampling was done in wounds of the control group. The samples were transferred to the laboratory by dipping in transport media, MacConkey Agar for gram-negative bacteria and Blood Agar for all species of bacteria. Both media were maintained at 37°C in an incubator for a 48-hour period to allow adequate growth needed to exert a therapeutic response when placed in the wound bed. MacConkey Agar was transferred to a triple sugar iron media for 24 hours for identification of P aeruginosa, which was confirmed with lysine decarboxylase,23 while Blood Agar media was transferred to Mannitol Salt Agar for 24 hours for identification of S aureus, which was confirmed with DNAse and CoAgulase.23

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DATA ANALYSIS

All data were imported to SPSS software for analyzing (Statistical Package for the Social Sciences, Chicago, Illinois). The χ2 test was used to compare outcomes between groups; the McNemar test was used to analyze data among sampling intervals of the treatment group.

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RESULTS

The sample population comprised 50 individuals with DFUs; 25 were randomly allocated to the treatment and control groups. The mean age of the treatment and control groups was 59.4 and 61.9 years, respectively; there were no significant differences between groups based on age (P = .463). The sex ratio (proportion of men to women) for the treatment group was 3.2 and for the control group was 2.6, with no significant difference between groups (P = .747).

Forty-four out of 50 DFUs were infected with bacteria, 18 were infected with S aureus, and 16 with P aeruginosa (Figure 1). The average depth and size of the wounds were 2 cm and 7 cm2, respectively. Maggot therapy on the wounds of the 9 cases infected with S aureus resulted in a significant reduction after 48 hours in the treatment group as compared to the 9 cases in the control group (P = .047). We found no reduction in bacterial infection of the wounds in control group participants (Figure 2). Using the McNemar test and excluding control group data, we found that removing S aureus among cases of the treatment group was significant after the first application of MT at 2 days (P = .031) and after the second application at 4 days (P = .0).

Figure 1

Figure 1

Figure 2

Figure 2

The number of infected cases with P aeruginosa was reduced from 9 to 5 after the first application of MT in the treatment group. Treatment group subjects were more likely to be infection free than were controls (P = .508). A second application of MT did not remove P aeruginosa from all patients, but the reduction was significant in comparison with the control group (χ2 test, P = .048; Figure 3). Using the McNemar test and excluding control group data, we found that removing P aeruginosa among cases in the treatment group was significant after the first application of MT at 2 days (P = .03), as well as after the second application at 4 days (P = .002).

Figure 3

Figure 3

We compared drainage from the DFU in the treatment and control groups. The average volume of drainage was 152.2 ± 41.4 and 50.6 ± 18.8 g in the treatment and control groups, respectively, a statistically significant difference (Mann-Whitney U test, P < .00). Figures 4 and 5 illustrate 2 DFUs infected with P aeruginosa and S aureus, respectively.

Figure 4

Figure 4

Figure 5

Figure 5

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DISCUSSION

Several mechanisms have been mentioned for the antimicrobial activity and effectiveness of MT.24 The effect of MT depends on the type of larvae.21 Findings from our study demonstrate the ability of an Iranian strain of L sericata in removing single-organism infection of P aeruginosa or S aureus or infection with both pathogens. Other studies using in vitro methods demonstrated the ability of excretions/secretions (E/S) of L sericata larvae in reducing the density of S aureus.bacteria of a <500-Da fraction from maggot excretions/secretions of Lucilia sericata (Diptera: Calliphoridae). Microb Infect. 2008;10(4):325–333.','400');" onMouseOut="javascript:ImageWrapperControl_ImageMouseOut();">14 In vivo methods on human cases showed similar findings in a before/after study by Jaklic and colleagues,21 who found that MT eradicated S aureus colonies from the wound bed.

Study findings also demonstrated the antimicrobial effects of L sericata larvae on P aeruginosa. Our results are similar to Cazander and colleagues,15 who studied the effects of maggot excretions on PAO1 biofilm formation in different biomaterials.15 Our results are similar to Jaklic and colleagues,21 whose in vitro assays demonstrated different mechanisms of L sericata excretions and secretions on S aureus and P aeruginosa.

We found that higher doses (∼20-fold higher) of larvae E/S were needed for reducing P aeruginosa as compared to S aureus biofilms. Jaklic and colleagues21 added the larval E/S once in the beginning of the test, but in our study, the live larvae produced these effluents continuously. Similarly, Steenvoorde and Jukema17 found negative effects of larvae on removing P aeruginosa. Results of our study indicate that the ongoing use of larvae in parallel with other medical interventions can be effective for removing this bacterium. Our investigation also suggests that the number of larvae applied to each wound is important. The number of larvae we used was based on the wound depth and surface in order to minimize any deleterious effects. It seems that the continued presence of an adequate number of larvae inside the wounds and coordinated clinical care of patients were necessary for overcoming the bacterial infections, especially for the cases of P aeruginosa.

Others have found that the combination of excretions and secretions of L sericata in conjunction with systemic antibiotics improves eradication of S aureus and P aeruginosa and the biofilms of chronic wounds.24 In our study, ciprofloxacin and clindamycin were used in conjunction with MT in the treatment group.

Some evidence suggests that P aeruginosa may impair the life span of larvae introduced during MT.25 Maggot therapy is a controlled myiasis accomplished by introducing live creatures to a human body to feed on selected tissue. The activity of larvae in the dressed wound is related to their ability to adapt to the environment surroundings. The bacterial and physiochemical environment, the number of larvae per square centimeter, their preparation, and sterilizing processes prior to introduction into the wound bed affect their ability to create the intended therapeutic effect. Nevertheless, we found no observable changes in development of larvae in the wounds that were infected with either P aeruginosa or S aureus.

Deep abscess is a common characteristic of DFUs and is considered when classifying the severity of the wound.26 Abscesses increase the likelihood of osteomyelitis.27 Maggot therapy has demonstrated a positive effect on the treatment of abscesses in animal studies,28 , 29 but there is limited evidence regarding the ability of MT to treat abscesses in humans. Our study suggests a benefit when MT is added to conventional treatment.

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LIMITATIONS

We found that patient acceptance was a limitation for using MT. This behavior has been reported from some other previous studies.30 In addition, the relatively small sample size and setting homogeneity limit generalizability of our findings.

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CONCLUSIONS

Study findings suggest that MT is a safe and effective treatment for persons with DFU colonized or infected with S aureus and P aeruginosa.

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ACKNOWLEDGMENT

The authors thank all staff and personnel of the clinic and pathobiology lab for their kind help with this project. This project was financially supported by Vice Chancellor of Research of Tehran University of Medial Sciences (code no. 95-01-27-30903).

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Keywords:

Bacteria; Biofilm; Diabetic foot ulcer; Disinfection; Infection; Larva; Larval therapy; Lucilia sericata; Maggot therapy; Pseudomonas aeruginosa; Randomized controlled trial; Staphylococcus aureus

© 2019 by the Wound, Ostomy and Continence Nurses Society.