RCT: Larval Debridement Therapy Versus Sharp Debridement to Remove Biofilm From Chronic Lower Extremity or Diabetic Foot Ulcers
Phase: NA
Conditions: Lower Extremity or Diabetic Foot Ulcers, Bacterial Infection
Sponsor: North Florida Foundation for Research and Education

 
 
 
Cover Page Required by NCT 
 
Official IRB Approved Revised Study Title: RCT: Larval Debridement Therapy versus Bedside Sharp 
Debridement to Remove Biofilm from Chronic Lower Extremity Ulcers or Diabetic Foot Ulcers 
 
NCT Study Title: Larval Debridement Therapy Versus Sharp Debridement to Remove Biofilm 
 
University of Florida IRB #: 201400590 
 
NCT number: NCT02294175 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Last Approved Revised Protocol (approved 8-3-2018) 
1. Project Title:  
RCT: Larval Debridement Therapy versus Sharp Debridement to Remove Biofilm from 
Chronic Lower Extremity or Diabetic Foot Ulcers 
 
2. Investigator(s):  
(Protocol revised only for change in PI of Record since original PI transferred stations) 
Matthew Morrow, Pharm D (PI of Record August 1, 2018 – 12/31/2018) 
Linda Cowan, PhD, ARNP, CWS (Co-I) 
Gregory Schultz, PhD (Consultant) 
 
3. Abstract:  
Drug resistant organisms and bacterial biofilm pose an increasing threat to the health of millions of 
individuals world-wide. These organisms are being identified with an alarming prevalence among 
persons with chronic wounds. The presence of necrotic tissue has been associated with the 
deterioration of open wounds and serves as a breeding ground and nutrient source for bacteria. The 
removal of necrotic tissue is widely accepted as required for optimal wound healing.     
The primary purpose of this study is to assess the efficacy of larval debridement therapy (LDT) with 
bagged, sterilized, live, medicinal blow fly (Lucilia sericata) larvae (or " BioBags") versus bedside sharp 
debridement in removing harmful bacteria, biofilm and necrotic tissue from chronic wounds to promote 
wound healing. Characteristics associated with chronic wound environments will be evaluated through 
analysis of samples of tissue taken from wound beds before and after both types of debridement.  One 
hundred and forty patients ≥ 21 years of age with an open, full thickness wound which is healing by 
secondary intention (of greater than 8 weeks duration and requires debridement) will be invited to 
participate. This recruitment number accounts for estimated 10% attrition rate, so final sample number 
is anticipated to be 128 subjects (64 in each arm). Samples of wound bed tissue and slough tissue (if 
present) will be collected on Days 0, 4 and 8 or prior to and after each larval debridement intervention 
or sharp debridement (control). Photos will be taken of the wound bed on Days 0, 4 and 8 or just prior 
to and after each debridement method. A randomized sampling procedure will place individuals into 
one of two groups: The intervention group will receive larval debridement therapy once every 4 days for 
2 applications (with saline moistened gauze as cover dressing changed daily) and the control group will 
receive sharp debridement therapy every 7 days for 2 debridements (with wound gel dressing changed 
daily).   
 
The wound and wound environment will be characterized by assessing wound Matrix 
Metalloproteinases (MMPs), inflammatory cytokines and the presence of total bacteria, planktonic (free 
floating) bacteria, and biofilm (sessile) bacteria. Clinicians will be asked to grade the amount of non-
viable tissue apparent in the wound bed, appearance of any signs of infection and overall visual 
appearance of the wound at each visit. Wound size will also be assessed. Subjects will be asked to rate 
their wound related pain level at each visit before and after the debridement method is applied. 
Subjects, caregivers and clinicians will be asked to complete a short survey to self-report their 
perceptions of each method of debridement. Data will be analyzed using bivariate and multivariate 
methods. 
 
4. Background: 
Chronic wounds are a significant health problem in the U.S.. Chronic wounds are defined as wounds 
that have “have failed to proceed through an orderly and timely reparative process to produce 
anatomic and functional integrity." Health care costs related to the management and treatment of 
chronic wounds in the U.S. exceeds $20 billion annually. For many health care providers, the 
treatment and management of non-healing wounds is challenging.  Traditionally, basic wound care has 
consisted of surgical debridement, manual irrigation, moisture retentive dressings, and topical and/or 
systemic antimicrobial therapy.  Although there has been tremendous progress in the science of 

 
wound healing, the prevalence and incidence of chronic wounds and their associated complications 
continues to escalate.  
 
The presence and complexity of bacterial biofilms in chronic wounds has recently been recognized as 
a key aspect of non-healing wounds. Bacterial biofilms are sessile colonies of polymicrobial organisms 
(bacterial, fungal, and possibly, viral) which are often symbiotic. These biofilm colonies produce a 
protective coating to protect the colonies from host defenses.  The character of this protective 
substance which is unique to biofilms is dynamic and production of its components seems to be 
triggered by hostile environments in the wound bed (such as the presence of topical antibiotics). 
Biofilms have been shown to have survival and defense mechanisms that: 1) inhibit the healing 
aspects of inflammatory cells; 2) resist antibiotics (topical and systemic) and other therapies; and 3) 
initiate cell to cell communication pathways (quorum sensing) which facilitate new biofilm growth, 
resulting in recalcitrant non-healing wounds.   
 
Wound larval debridement therapy (LDT) has been shown to have promise in healing chronic wounds 
by eradicating biofilms.  Recently (2012), the PI was part of a team of investigators who collaborated 
with the University of Florida Wound Research Laboratory and conducted several in vitro experiments 
to demonstrate the efficacy of larval exposure to biofilm (Cowan et al., 2013). These studies 
demonstrated bacterial biofilm (Pseudomonas aeruginosa (PA01) bacteria and Staphylococcus aureus 
(SA35556) bacteria) grown on pigskin explants were completely eradicated within 48 hours of 
exposure to loose medicinal maggots from Monarch Labs (larvae).  
 
Maggot or larval debridement therapy (MDT or LDT) has been utilized for medical purposes for 
hundreds, if not thousands of years. Mayan Native Americans and other ancient cultures have 
documented reports of maggots being used in certain medical treatments. It was Napoleon's general 
surgeon, Baron Dominique-Jean Larrey (1766-1842), who documented observations that larvae of 
certain fly species, such as Phaenicia (Lucilia) sericata (blow fly), removed only dead tissue while 
promoting healthy tissue in the wound bed, helping wounds heal faster. 
 
Dr. John Forney Zacharias (1837-1901), a Confederate American Civil War surgeon, is recognized as 
the first healthcare provider in the United States (U.S.) who intentionally applied maggots for wound 
care/debridement purposes. He noted that "maggots could clean a wound better in one day" than any 
other agent they had at their disposal. He also accredited maggots with saving many soldier's lives. In 
World War I, an orthopedic surgeon named Dr. William S. Baer recognized the efficacy of maggots on 
the battlefield to "clean up" compound fractures and large flesh wounds, even crediting maggots with 
preventing sepsis in two battlefield cases. He was impressed with the application of maggots as a 
medical treatment, so he conducted research at Johns Hopkins in 1929 using maggots that he found in 
the neighborhood or grew on a windowsill. Two patients contracted tetanus from contaminated maggots 
(one died), so he developed sterile maggot growing procedures. He used maggot therapy in 21 patients 
with chronic osteomyelitis which did not respond to other treatments. He demonstrated rapid wound 
debridement of necrotic tissue, a return of the wound bed to an alkaline pH environment, the reduction 
of bacteria, reduced odor levels, and complete healing of the osteomyelitis infections within six weeks. 
 
With the development of antibiotics in the 1940's and various skin and wound antiseptics, the use of 
LDT declined. Arguably, one of the biggest reasons LDT may have lost favor in clinician's eyes was not 
ineffectiveness but was the "yuck factor"; patients, their caregivers and clinicians found it distasteful to 
apply small squirming worms that could crawl out of a wound. In fact, as much as Dr. Baer promoted 
the use of medicinal maggots, he himself said, "The sight [of maggots in an open wound] was very 
disgusting and measures were taken hurriedly to wash out these abominable looking creatures."   
 
With the advent of antibiotic resistant organisms and increasing drug sensitivities, there was a renewed 
interest in Maggot therapy in the 1980's. The Food and Drug Administration (FDA) approved the use of 
medicinal maggots (Phaenicia (Lucilia) sericata) for debriding non-healing necrotic skin and soft tissue 
wounds including lower extremity or diabetic foot ulcers, pressure ulcers, non-healing surgical or 

 
traumatic wounds and venous stasis ulcers. In the U.S., larval therapy with maggots is classified as a 
medical device. However, in Europe, Canada and Japan maggots are classified as medicinal drugs. 
  
Although anecdotal clinical evidence suggests that larvae therapy is successful in removing necrotic 
tissue and bacterial biofilm, there are limited data from randomized clinical trials to support this claim.  
A new (to the United States) method of larval debridement therapy delivery is a prepackaged mesh bag 
(similar to a tea bag) which contains a premeasured amount of sterilized, live, medicinal blow fly (Lucilia 
sericata) larvae. This bag is self-contained and increases the ease of use, at the same time, reassures 
patients that all the larvae are contained within the dressing and will not "escape" during therapy. This 
proposed study seeks to test this dressing (called a "Biobag") to investigate its effectiveness as a 
debridement therapy to remove necrotic tissue and biofilm, in comparison with a standard debridement 
technique   (bedside sharp debridement).  
 
Further studies validate findings that green bottle fly larvae or maggots (Lucilia sericata) used for 
wound debridement do not ingest healthy tissue of certain species such as humans, cows, horses, or 
dogs because of a genetic marker in these species as opposed to healthy tissue of sheep, rabbits, or 
hedgehogs, which the larvae will ingest because they are missing this specific DNA marker. This is in 
contrast to other fly maggots NOT used for wound care such as the New World Arm Screw Worm Fly 
larvae, 
which 
have 
been 
known 
to 
ingest 
healthy 
human 
tissue. 
(REFs   
http://www.dailymail.co.uk/health/article-2365158/Horrified-woman-27-discovers-headaches-scratching-
sounds-inside-head-FLESH-EATING-MAGGOTS.html ).   
 
Maggots used in the U.S. for larval debridement therapy are all processed under controlled laboratory 
conditions and are sterile (both free of disease as well as unable to reproduce).  Larval debridement of 
non-viable tissue within chronic wounds results partly from the proteolytic digestive enzymes liquefying 
the necrotic tissue, which the larvae then "suck up" (along with bacteria and biofilm) and remove from 
the wound bed. As such, they are a most efficient way to debride a wound without the pain, bleeding, or 
inflammatory response associated with sharp debridement.  
 
Unfortunately, current larval debridement methods available in the U.S. have not really addressed the 
"yuck factor" of loose or free-roaming maggots in open wounds. This may explain why, despite the 
clinically proven effectiveness of larval therapy to aid in the healing process, many U.S. clinicians do 
not select this method of wound treatment. Many nurses, doctors, caregivers, and patients alike have 
voiced an aversion to handling maggots or having to "count the number that go into a wound or come 
out of a wound" (Cowan et al., 2013). Having to count them is complicated by the fact that when the 
wound is sufficiently debrided, larger maggots may turn and eat smaller maggots. 
 
Fortunately, there is a brilliant new alternative. A European company, BioMonde, has successfully 
addressed this issue by manufacturing sterile maggots in a mesh bag called a BioBag. This allows for 
an aesthetically and psychologically acceptable method of introducing the larval therapy to the wound 
and removing them all in "one neat package." BioMonde has recently obtained FDA approval for this 
medical device in the U.S. In addition, several studies validate the comparative effectiveness of these 
maggots in BioBags versus currently "free range" or loose maggots (Monarch Labs) (Blake et al., 
2007). Now that this research has been completed, a randomized controlled trial (RCT) testing larval 
debridement therapy, also known as “biosurgery” using BioBags versus traditional bedside sharp 
debridement methods in human subjects with chronic wounds is warranted.  
 
Purpose Statement 
Our proposal includes the testing of the BioBag device in a randomized control trial (RCT) in a sample 
of subjects with chronic open, non-healing lower extremity or diabetic foot ulcers. While this would be 
the first clinical test in the U.S., the product has been widely tested elsewhere and is successfully used 
in over 40,000 British and European human wound treatments per year. However, there has been no 
quantitative measure of the comparative effectiveness of this product versus sharp debridement in 
regards to bacterial biofilm measures. The purpose of this proposed RCT research study is to 

 
investigate the efficacy and acceptability of larval debridement therapy using "BioBags" versus bedside 
sharp debridement therapy in humans with chronic wounds.  
 
Significance   
This study has an enormous opportunity to contribute to wound care practice within the VA as well as 
all of the United States. The potential for improved quality of life for Veterans (due to avoidance of 
amputation and healing of chronic wounds) is priceless.  This research is the first step in a program of 
research aimed at improving effectiveness of wound care at the bedside. This proposed research aims 
to provide clinicians with more effective tools in wound debridement and in particular, in the fight 
against biofilm (such as the removal of biofilm in wounds by larval therapies). Using the knowledge 
gained in this study, we will also be able to answer questions related to the comparative effectiveness 
of antibiofilm strategies such as larval debridement therapy versus sharp debridement therapy – and 
apply this knowledge to clinical practice. For instance, if larval therapy is demonstrated to be more 
effective at removing biofilm, and may also be appropriately applied by a nurse or informal caregiver (in 
the patient’s own home) rather than a physician/ARNP/PA in a clinical setting, this could reduce health 
care costs and improve access to care.  Furthermore, decreasing wound infection and improving 
wound healing has the potential for decreasing complications and improving the quality of life for 
Veterans, which is priceless.   
 
5. Specific Aims: 
 
Specific Aim 1 
 
To compare the clinical effectiveness of a self-contained bagged larval debridement therapy 
(intervention) (BioBag) with bedside sharp debridement (control). The following comparisons will be 
made: 1) The visual reduction of non-viable tissue in the wound bed of chronic full-thickness lower 
extremity or diabetic foot ulcers, and 2) The reduction in colony forming units (CFU) of bacterial biofilm 
in samples taken from the wound bed. 
 
Specific Aim 2 
 
To compare wound characteristics (e.g., inflammatory markers, wound size) in the intervention group 
(BioBag therapy) and control group (Sharp debridement) over time. 
 
Specific Aim 3 
 
To investigate patient, caregiver and clinician perceptions of larval therapy dressings and bedside 
sharp debridement using measures of self-reported aesthetics, ease of use, and wound pain.    
 
6. Research Plan: 
 
Research design and methods  
Design:  A repeated measures, randomized control trial experimental design will be used for this study. 
Subjects will serve as their own control, in addition to a control group. Initial wound samples will be 
followed by two subsequent repeated wound samplings approximately four days apart. Informed consent 
will be obtained prior to all procedures, photo consents will also be obtained for wound photos.   
 
Veteran Site: North Florida South Georgia Veterans Health System 
Setting, Sample:  A target goal final sample size of one hundred twenty eight adults from the North 
Florida South Georgia VHS will be recruited (total recruited number anticipated to be ~140 allowing for 
10% attrition). Including target of 128 Caregivers and 6 Providers, and accounting for 12 caregivers lost 
to follow up or 10 subjects who fail screening, the total study recruitment population is 296. Wound 
providers and clinicians who care for lower extremity or diabetic foot ulcers will be provided with a 
“Provider Study Flyer” which gives a brief description of the study with inclusion/exclusion criteria listed 
(to remind the Provider to approach potentially eligible patients with Research Team Contact 

 
information). The clinicians will also be asked to make a Recruitment Brochure available to potentially 
eligible patients (those with lower extremity or diabetic foot ulcers over 8 weeks duration). In addition, 
study flyers will be sent to select VA wound and podiatry offices in the NF/SG VHS. Providers will be 
asked to contact study team if any patient is willing to hear about the study. If the patient is willing to 
hear about the study, they may contact a study team member from the contact number on the 
recruitment brochure or the provider can let the patient know when the research team member is 
scheduled to be in the provider clinic office and the patient may talk directly to the research study team 
member for more information. If patients call a research team member, they will have their questions 
answered but will not be screened over the phone. They will be scheduled to come into the provider 
office to discuss the study with a research team member and review informed consent. Members of the 
research study team will review the informed consent with the patient and enroll the subject if they 
agree to participate. Wound clinicians will not be asked to consent subjects, or to provide a list of 
patients or appointment times to research team. However, the subject’s wound providers (clinicians) will 
be the ones to perform the sample collection and apply the LDT or bedside sharp debridement. The 
sample also includes one available caregiver of the patient (non-professional) to answer the caregiver 
surveys if willing, and the wound care providers, who will be asked to answer the clinician survey 
regarding perceptions of debridement methods.  
 
Sample Size Power Analysis: Power analysis is based on the patient sample and the primary study aim 
of comparing debridement methods on clinical effectiveness. The POWER procedure in SAS version 
9.2 (Cary, N.C.) was used to conduct the power analysis. Assuming a Type I error rate of .05 and two-
sided testing, a sample of 64 is needed in each group to detect a medium effect size of .50 with at least 
80% power. This magnitude of effect is considered clinically significant. 
 
Patient characteristics to be recorded: Age (in years), gender (Male/Female), presence of diabetes 
diagnosis (any “no” would be excluded from study), smoking behavior (Yes/No), presence or absence 
of pedal pulses (Yes/No) and Ankle Brachial Index (ABI), relevant laboratory values, culture reports and 
other pertinent medical history (co-morbid conditions). Additionally, telephone numbers will be obtained, 
with the participants consent, in order to call and remind the participant the day before there next 
upcoming visit.      
 
Inclusion criteria:  (1) Veterans over 21 years of age; (2) with chronic lower extremity or diabetic foot 
ulcers (wound duration over 8 weeks); (3) who at the clinician’s judgment requires wound debridement 
(25% or more of wound bed covered with non-viable tissue); (4) wound size 1.5 cm (roughly the size of 
a quarter) or larger in diameter.  
 
Exclusion criteria:  (1) Cognitive impairment that would interfere with patient signing own Informed 
Consent; (2) Veterans on active anticoagulant therapy with most recent (within last week) PT/INR 
(international normalized ratio of prothrombin time) > 3.0, or other significant bleeding risk; (3) Active 
immune suppression just prior to or during study (on systemic corticosteroids* within 7 days prior, or 
chemotherapy for cancer or RA treatment within 4 weeks prior to study, or with diagnosis of HIV/AIDS) 
- *Nasal steroid sprays will not be excluded; (4) Active systemic antibiotics is an exclusion; (5) Absent 
dorsalis pedis pulses and Ankle Brachial Index (ABI) < 0.5 is an exclusion (indicates critical limb 
ischemia).   Other possible reasons participants could be removed from this study include: transfers to 
other non-VA facilities, participant is unable to tolerate tissue sampling even with local anesthesia, at 
the discretion of the provider for clinical reasons, and/or inability to comply with scheduled research 
visits. Furthermore, if the participant has significant wound healing so that sampling is not possible after 
the initial sampling, they will be removed from the study.  
 
Human Subjects Involvement and Characteristics: One hundred and forty consented veterans and their 
(non-professional) caregivers will be randomly assigned to one of two study arms:  (1) Larval 
Debridement Therapy (LDT) or (2) Bedside Sharp Debridement (control).  The intervention group, or 
LDT participants, will undergo LDT every 4 days for 2 applications as the main method of debridement 
strategy. They will have wound samples collected before the initial larvae are applied after 4 days of 
larval therapy and after 8 days of larval therapy. They will also both be asked to complete a short paper 

 
survey on Days 0, 4 and 8. The subject’s wound clinician will also be asked to complete a similar 
survey (only one round of surveys per consented wound clinician) at Day 0 and Day 8 and 3 months 
later or at completion of study regarding perceptions of wound debridement therapy.   
    
The control group (bedside sharp debridement) will receive sharp debridement of their wounds 7 days 
apart x 2 sharp debridements and also have wound samples collected before the initial sharp 
debridement (Day 0), 4 days after the initial sharp debridement (Day 4), then eight days after initial 
sharp debridement, which will be 24 hours after the second sharp debridement (Day 8).   
 
Both groups will also receive standard care and therapies ordered by their VA health care providers for 
their chronic wound care (excluding systemic antibiotics and topical wound antimicrobials), and 
standard emergency contact numbers. The study duration for the participants is 8 days, 
 
IRB, Safety Committee, Information Security Officer (ISO) and VA R&D committee approval will be 
obtained prior to beginning this research study. The sample size is anticipated to be 140 total subjects 
(desired n=128 + 10% attrition). The desired sample size for data analysis is 128 to achieve adequate 
sample size for statistical power.  
 
Data collection from human subject’s electronic health record will include characteristics such as wound 
etiology, wound location and duration, wound treatments, wound photos, wound progress notes, age, 
gender, telephone number, comorbid conditions, pertinent laboratory values and medical history. This 
data will be conducted only by trained research team members designated in the IRB approved study 
protocol over the duration of the study. Data collection will only occur in compliance with VA Information 
Security requirements. The Veterans selected for this study will be adults and may involve any age over 
18, race/ethnicity, gender, and medical comorbid conditions. No special classes of subjects will be 
sought out.  
 
Recruitment and informed consent: Wound care providers such as in podiatry and lower extremity or 
diabetic limb preservation program (DPM) and Wound Team Clinicians will be approached and the 
study will be described. They will be asked to notify study investigators of potential subjects who are 
likely to have chronic lower extremity or diabetic foot ulcers (> 8 weeks duration) of 1.5cm size or 
greater -and who may be interested in participating in the study. If Veteran says they are willing to 
participate, wound provider will notify investigator and one of research team will meet Veteran and 
wound provider in clinic. Study will be explained to Veteran and the Veteran will sign informed consent 
if they agree. 
 
Non-Veteran site: adding North Florida Regional Medical Center as a second site to meet 
recruitment 
Setting, Sample:  A target goal final sample size of forty adults (plus willing caregivers) from the North 
Florida Regional Medical Center will be recruited. Including the initial total target of 128 Caregivers and 
6 Providers, and accounting for 12 caregivers lost to follow up or 10 subjects who fail screening, the 
total study recruitment population is still 296 for all recruitment sites. Wound providers and clinicians 
who care for lower extremity or diabetic foot ulcers will be provided with a “Provider Study Flyer” which 
gives a brief description of the study with inclusion/exclusion criteria listed (to remind the Provider to 
approach potentially eligible patients with Research Team Contact information). The clinicians will also 
be asked to give a Recruitment Brochure to potentially eligible patients (those with lower extremity or 
diabetic foot ulcers over 8 weeks duration). In addition, study flyers will be sent to select NFRMC 
wound and podiatry clinics. Providers will be asked to contact study team if any patient is willing to hear 
about the study. If the patient is willing to hear about the study, they may contact a study team member 
from the contact number on the recruitment brochure or the provider can let the patient know when the 
research team member is scheduled to be in the provider clinic office and the patient may talk directly 
to the research study team member for more information. If patients call a research team member, they 
will have their questions answered but will not be screened or consented over the phone. They will be 
scheduled to come into the provider office to discuss the study with a research team member and 
review informed consent. Members of the research study team will review the informed consent with 

 
the patient and enroll the subject if they agree to participate. Wound clinicians will not be asked to 
consent subjects, or to provide a list of patients (PHI) to the research team. The subject’s wound 
providers (clinicians) will be the ones to perform the sample collection and apply the LDT or bedside 
sharp debridement. The study sample also includes one available caregiver of the patient (non-
professional) to answer the caregiver surveys if they are willing, and any willing  wound care providers, 
who will be asked to answer the clinician survey regarding perceptions of debridement methods.  
 
Sample Size Power Analysis: Power analysis for the entire study is based on the patient sample and 
the primary study aim of comparing debridement methods on clinical effectiveness. The POWER 
procedure in SAS version 9.2 (Cary, N.C.) was used to conduct the power analysis. Assuming a Type I 
error rate of .05 and two-sided testing, a sample of 64 is needed in each group to detect a medium 
effect size of .50 with at least 80% power. This magnitude of effect is considered clinically significant. 
 
Patient characteristics to be recorded: Age (in years), gender (Male/Female), presence of diabetes 
diagnosis (any “no” would be excluded from study), smoking behavior (Yes/No), presence or absence 
of pedal pulses (Yes/No) and Ankle Brachial Index (ABI), relevant laboratory values, culture reports and 
other pertinent medical history (co-morbid conditions). Additionally, telephone numbers will be obtained, 
with the participants consent, in order to call and remind the participant the day before there next 
upcoming visit.      
 
Inclusion criteria:  (1) Individuals over 21 years of age; (2) with chronic lower extremity or diabetic foot 
ulcers (wound duration over 8 weeks); (3) who at the clinician’s judgment requires wound debridement 
(25% or more of wound bed covered with non-viable tissue); (4) wound size 1.5 cm (roughly the size of 
a quarter) or larger in diameter.  
 
Exclusion criteria:  (1) Cognitive impairment that would interfere with patient signing their own Informed 
Consent; (2) Individuals on active anticoagulant therapy with most recent (within last week) PT/INR 
(international normalized ratio of prothrombin time) > 3.0, or other significant bleeding risk; (3) Active 
immune suppression just prior to or during study (on systemic corticosteroids* within 7 days prior, or 
chemotherapy for cancer or RA treatment within 4 weeks prior to study, or with diagnosis of HIV/AIDS) 
- *Nasal steroid sprays will not be excluded; (4) Active systemic antibiotics is an exclusion; (5) Absent 
dorsalis pedis pulses and Ankle Brachial Index (ABI) < 0.5 is an exclusion (indicates critical limb 
ischemia).   Other possible reasons participants could be removed from this study include: transfers to 
other non-study participating facilities, participant is unable to tolerate tissue sampling even with local 
anesthesia, at the discretion of the provider for clinical reasons, and/or inability to comply with 
scheduled research visits. Furthermore, if the participant has significant wound healing so that sampling 
is not possible after the initial sampling, they will be removed from the study.  
 
Human Subjects Involvement and Characteristics: After signing informed consent, participants and their 
(non-professional) caregivers will be randomly assigned to one of two study arms:  (1) Larval 
Debridement Therapy (LDT) or (2) Bedside Sharp Debridement (control).  The intervention group, or 
LDT participants, will undergo LDT every 4 days for 2 applications as the main method of debridement 
strategy. They will have wound samples collected before the initial larvae are applied after 4 days of 
larval therapy and after 8 days of larval therapy. They will also both be asked to complete a short paper 
survey on Days 0, 4 and 8. The subject’s wound clinician will also be asked to complete (if willing) a 
similar survey (only one round of surveys per consented wound clinician) at Day 0 and Day 8 and 3 
months later or at completion of study regarding perceptions of wound debridement therapy.   
    
The control group (bedside sharp debridement) will receive sharp debridement of their wounds 7 days 
apart x 2 sharp debridements and also have wound samples collected before the initial sharp 
debridement (Day 0), 4 days after the initial sharp debridement (Day 4), then eight days after initial 
sharp debridement, which will be 24 hours after the second sharp debridement (Day 8).   
 

 
Both groups will also receive standard care and therapies ordered by their health care providers for 
their chronic wound care (excluding systemic antibiotics and topical wound antimicrobials), and 
standard emergency contact numbers. The study duration for the participants is 8 days. 
 
UF IRB and designated NFRMC and VA (as parent study site) approval(s) will be obtained as needed 
prior to beginning this research study. The total sample size (for all combined sites) is anticipated to be 
140 total subjects (desired n=128 + 10% attrition).   
 
Data collection from human subject’s electronic health record will include characteristics such as wound 
etiology, wound location and duration, wound treatments, wound photos, wound progress notes, age, 
gender, telephone number, comorbid conditions, pertinent laboratory values and medical history. This 
data will be conducted only by trained research team members designated in the IRB approved study 
protocol over the duration of the study. Data collection will only occur in compliance with UF IRB and 
study parent site (VA) Information Security requirements. The participants selected for this study will be 
adults and may involve any age over 21, race/ethnicity, gender, and medical comorbid conditions. No 
special classes of subjects will be sought out.  
 
Recruitment and informed consent: Wound care providers such as in podiatry, and wound and 
hyperbaric clinics will be approached and the study will be described. They will be asked to notify study 
investigators of potential subjects who are likely to have chronic lower extremity or diabetic foot ulcers 
(> 8 weeks duration) of 1.5cm size or greater -and who may be interested in participating in the study. If 
patients say they are willing to participate, their wound provider will notify the study investigator(s) and 
one of the research team members will meet with the patient and wound provider in clinic. The study 
will be explained to the patient and if they agree to participate, the participants will sign informed 
consent. 
 
 
Both Research Recruitment Sites: 
Confidentiality: All information collected from participants will be kept strictly confidential and used for 
research purposes only.  All samples will be marked with de-identified information (an assigned subject 
number for each patient, date of collection, visit number, location of wound and duration of wound). 
Information regarding personal identifiers will be kept by the PI/project manager and stored separately 
in a locked cabinet.  Participants will be assured that only the research study team and the Human 
Subjects Review Committees will have access to the study materials.  All the study materials will be 
maintained in a locked/protected file within the VA Research Office until such time as the VA Research 
Compliance Officer gives the directive to destroy the files. 
 
Study procedures (Sample Collection) 
Larval Debridement Therapy (intervention) group: Briefly, wounds may be rinsed with saline and 
blotted with sterile gauze prior to collecting baseline wound samples at Day 0. Wound measurements 
and wound photos will be taken at baseline and Day 8. Wound samples will be collected on Day 0, Day 
4, and Day 8. One BioBag (larval debridement therapy) will be applied to wound on Day 0 after 
baseline wound samples are obtained, and this will be removed on Day 4, at which time 2rd wound 
sample is obtained. Immediately after 2nd wound sample obtained, the 2nd BioBag will be applied. The 
dead larvae from the Biobags after removal from the subjects in this study will be placed in a sterile 
container and labeled with study subject number and “1st” or “2nd application.” These specimens will be 
frozen in our VA research laboratory for a future secondary analysis. This will be the subject of a new 
grant proposal being submitted by the current investigators in 2015 with the aim of examining the 
digestive track and body of the larvae for the presence of bacteria and correlating this with the bacteria 
identified in the bacterial analysis of the subject wounds.   Size of the Biobag is determined by the size 
of the wound (typically 5 to 8 larvae per cm2 or 50 to 400 larvae per single Biobag). Only cover dressing 
(saline moistened gauze + dry gauze/secondary cover) will be changed over BioBag on days 1,2,3,5,6, 
and 7. Last wound sampling (3rd) will be done on Day 8, after 2nd BioBag is removed. Clinicians as well 
as Veteran participants and non-Veteran participants (at NFRMC) and their caregivers will be given 
copies of LDT instructions for use (Appendix A/Appendix B).  Wound samples will be obtained at each 

 
sampling date by sterile swab(s) and 3-4mm curette scraping of wound bed using a sterile curette 
sweeping z track method across wound bed. Sterile filter paper or micropipette/capillary tube may also 
be used to collect wound fluid. Wound samples will be collected at wound provider’s office/clinic and 
placed in appropriate receptacle (sterile transport tubes with caps or vials with caps) for transport. The 
sample receptacles will be labeled with assigned subject number, number of visit (1, 2, 3, etc.), date of 
sample collection, duration and location of wound. Labeled samples will be placed in sealed biohazard 
bag, then in a biohazard marked cooler with a cold pack, and this container will be placed in a larger 
transport container for transport to the VA laboratory.  Clinicians and clinic staff will be asked to 
complete sample collection training and biohazard shipping (if shipping is deemed necessary) training 
before collecting or shipping any wound samples to standardize the procedures and be in compliance 
with research, safety and shipping regulations. Clinicians will be asked to view a LDT training video 
before performing application of Larvae in order to further standardize the application. 
 
Sharp Debridement Therapy (control) group: Briefly, wounds may be rinsed with saline and blotted 
with sterile gauze prior to collecting baseline wound samples at Day 0. Wound measurements and 
wound photos will be taken on Day 0, Day 4, Day 7 (before and after sharp debridement) and on Day 8. 
Wound samples will be obtained on Day 0, Day 4, and Day 8. Sharp debridement will be performed by 
patient’s wound clinician using standard technique on day 0 after baseline wound measures are 
obtained and again on day 7.  Wound samples will be obtained by sterile swab(s) and 3-4mm curette 
scraping of wound bed using a sterile curette sweeping z track method across wound bed. Sterile filter 
paper or micropipette/capillary tube may also be used to collect wound fluid. Wound samples will be 
placed in appropriate receptacle for transport as described above. Wound gel dressing (with gauze as 
secondary cover) will be applied to wound after debridement and changed daily as a control dressing. 
The following depicts the anticipated sampling timeframe for each participant. 
 
 
 
 
 
 
LDT group (X denotes sampling procedure) 
Day 0 - X pre-measures (sample 1);      
 surveys; photos 
 
 
 Apply larvae bag 1 
Day 4 - Remove larvae bag 1  
 X post-measure (sample 2); Pain   
 survey; photos  
 
 
 Apply larvae bag 2 
Day 8 - X final measures (sample 3);  
             surveys; photos  
SD group  
Day 0 – X pre-measures (sample 1); surveys;  
             photos 
 Sharp Debridement 1 
Day 4 - X post measures (sample 2); pain survey;   
             Wound photos 
Day 7 - Sharp debridement 2 & photos 
 
Day 8 - X final measures (sample 3); surveys;                
             photos  
 
 
Data Collection & Data Management 
Measurement of bacterial colonization.   A microbiological assessment of the wound samples will be 
performed.  Samples will be measured for both planktonic (single) bacteria and biofilm bacteria by 
standard clinical microbiology techniques with serial dilution and plating on complete agar medium 

 
plates. Colony forming units (CFUs) will be measured after approximately 24 hours of culture under 
standard laboratory conditions.  Levels of planktonic bacteria and biofilm bacteria will be compared 
within each sample and chronologically within each patient. Photos will be reviewed by blinded wound 
clinicians and rated on amount of necrotic tissue present visually in wound bed at each sampling time 
(reported on scale 0 – 10 based on 0%, 1 = 10% or less, etc.).  Data collected from human subject’s 
electronic health record will include characteristics such as wound etiology, wound location/duration, 
wound treatments, wound photos, wound progress notes, age, gender, comorbid conditions, pertinent 
laboratory values and medical history. This data will be conducted only by trained research team 
members designated in the IRB approved study protocol over the duration of the study.   
 
Only de-identified pertinent information per subject sample such as age of subject, visit number (Day 0 
,4, 8), location of wound, duration of wound, co-morbid conditions, medications and current wound 
treatments will be shared with the investigational laboratories or their personnel. Only the PI, site PI, 
Co-Investigators, and study coordinator(s) will have access to the subject PHI and study folders. UF lab 
has agreed to destroy any remaining samples at the conclusion of the current study.  
 
Measurement of MMPs.  Using techniques such as ELISA or florescence resonance energy transfer 
(FRET) assay methodology, the levels of active MMPs (or measures of MMP activity) will be calculated 
and expressed as pg/ml of wound fluid and pg/mg protein.  
 
Measurement of IL6.  Using techniques such as ELISA and florescence resonance energy transfer 
(FRET) assay methodology, the levels of active  IL6 will be calculated and reported. 
Methods for Data Collection & Data Management 
Variable 
Significance 
Sampling time 
Source 
Specific 
Aim 
Age 
Demographic 
Baseline 
EHR 
n/a 
ABI 
Exclusion criterion 
Baseline - 
enrollment 
ABI done after 
ICF 
n/a 
% nonviable wound 
tissue  
Appearance of 
wound bed 
Baseline Day 0 and 
Day 8 
Provider 
assessment 
Aim 1 
Quantitative  
Cultures (planktonic 
and biofilm) 
Microbial 
identification 
Day 0 (pre-test), + 
Day 4 and Day 8 
Wound 
samples 
Aim 1 
Wound Size in cm2 
(volume: L x W x D) 
Descriptive 
Baseline Day 0 and 
Day 8 
Wound 
measurements 
Aim 2 
Interleukin 6 
Marker of 
inflammation 
Day 0 (pre-test), + 
Day 4 and Day 8 
Wound sample 
(fluid and/or 
tissue) 
Aim 2 
Matrix 
metalloproteinases 
(MMP-2 and 9 
equivalent) 
Elevated MMPs   
associated with non-
healing wounds 
Day 0 (pre-test), + 
Day 4 and Day 8 
Wound sample 
(fluid and/or 
tissue) 
Aim 2 
Perceptions of 
therapy 
Perceptions of 
debridement 
Day 0 (pre-test) + 
Day 8  
Patient, 
Caregiver & 
Clinician 
Surveys 
Aim 3 
 
Survey Methods 
Subjects will be asked to complete a paper survey at Day 0, Day 4 and Day 8 of the study, querying the 
patient’s perceptions of: wound pain, satisfaction with most recent/current wound care method, ease of 

 
care, effectiveness of most recent wound treatment, aesthetics of current wound treatment and toward 
sharp debridement and maggots for wound care. 
 
Caregivers and wound clinical providers will be asked to complete a paper survey at Day 0 and Day 8 
of the study (if wound clinician is caring for more than one study subject, they will be asked to complete 
a survey at day 0 of first subject, day 8 of first subject and 3 months later or at completion of the study 
for each type of debridement), querying them about their perceptions of what the patient’s experience 
was regarding the most recent wound debridement methods as well as satisfaction with most 
recent/current wound care method, ease of care, effectiveness of most recent wound treatment, 
aesthetics of most current wound treatment and toward sharp debridement and maggots for wound 
care. 
 
Data Analysis 
Data will be summarized with descriptive statistics. Data will be checked for implausible values, 
missingness, and distributional form. Data transformations will be made as necessary to achieve 
normality. To address Specific Aim 1 (comparing the clinical effectiveness of a self-contained larval 
debridement therapy with a bedside sharp debridement therapy), the numeric outcome measures of 
changes (post minus pre) in percent of non-viable tissue and bacterial biofilm will be analyzed using 
independent samples t-tests. To address Specific Aim 2 (comparing wound characteristics of larval 
debridement and bedside sharp debridement therapies) over time, longitudinal generalized mixed 
models (GLMMs) will be used with the dependent repeated measures being wound characteristics 
(e.g., inflammatory markers, wound size) and the independent variable of group assignment (larval 
versus sharp debridement). To address Specific Aim 3 (investigating patient, caregiver, and clinician 
perceptions of larval therapy dressings and bedside sharp debridement using self-reported 
assessments, independent samples t-tests will be used to analyze data measured at the interval level, 
Wilcoxon rank sum tests will be used to analyze data measured at the ordinal level, and chi-square 
tests (or Fisher exact tests in the case of data sparseness) will be used to analyze categorical data. All 
hypotheses testing will be two-sided using a level of significance of .05. Data will be analyzed as “intent 
to treat.” SAS version 9.2 (Cary, N.C.) will be used for all analysis. 
 
Data and Safety Monitoring Plan 
The main study PI will review the collected data monthly during sample collection to identify any 
possible breach of the study protocol, including incorrect consent procedure or data storage.  This 
study will be monitored by the University of Florida IRB and the NF/SG VHS R&D Committee as well as 
the Research Compliance Officer, who have institutional responsibility to oversee the progress of 
research studies and the safety of study participants. The PI has the responsibility to promptly report 
any adverse event or breach of the study protocol. Should any adverse event or breach of protocol 
occur, they will be reported in accordance with the IRB standard operating procedures enforced by the 
IRBs at University of Florida, North Florida Regional Medical center, and the NF/SG VHS research 
Offices.  All of the research team members will be required to pass the mandatory training programs for 
human subject research and be certified by the IRB for conducting this study.   
 
Inclusion of women, minorities and/or children: Efforts will be made to recruit women and minority 
patients. 
 
7. Possible Discomforts and Risks: 
 
Adequacy of Protection from Risk  
Potential Risks: The risks to human subjects associated with this study are minimal related to the 
collection of wound samples, since these samples will be collected by the PI or patient’s wound care 
clinicians. The risks related to the debridement techniques are also minimal since bedside sharp 
debridement is typically a standard of care in these patients with lower extremity or diabetic foot ulcers 
and the risk of larval debridement therapy is considered to be less than that of sharp debridement. 
Potential risks related to sharp debridement, and to a lesser extent, to larval debridement, may include: 
bleeding, pain, increased wound draining, infection, increased wound odor, and in the case of larval 

 
debridement, a “crawling” sensation in wound. Potential risks related to subject, caregiver and wound 
clinician participation with the study survey component are minimal, due to the fact that no sensitive 
information is being collected.   
 
Prior to study, it will be approved by the VA Subcommittee on Clinical Investigation (Research and 
Development committee) and the University of Florida Health Science Center Institutional Review 
Board (UF IRB). The UF IRB is the official review and approval process for all VA research studies at 
NF/SG VHS, and is the IRB of record for the additional North Florida Regional Medical Center Site.  
Despite the low risk of this study, we will take measures to safeguard all of the study participants 
(including caregiver and clinician) from inconvenience, unnecessary discomfort and Personal Health 
Information risk. First, we will meet with patient, caregiver and clinician at a convenient time for the 
participant, caregiver, and wound care provider/clinician.  The surveys will be brief and will not ask for 
personally identifying or sensitive information (see Appendixes). We will take measures to safeguard 
any data collected and keep all data collected in a secure locked file on the VA server and in locked file 
cabinet (copies of regulatory documents and ICFs pertinent to NFRMC will be kept on site at NFRMC 
by site PI, Dr. Ellis). We will ensure that the participants are fully informed about the procedures, risks 
and objectives of the wound sample collection. The informed consents and all data collected at the 
clinician’s office or secondary study site(s) will be transported in a locked file box by the research team 
member and then stored in the Research office in a locked file cabinet. For VA participants, the original 
is scanned into patient’s electronic health record/CPRS per VA regulatory protocol; for NFRMC 
participants, the original is copied and kept onsite at NFRMC in a locked file cabinet with the study 
binder.  
 
Potential Financial Risks: There are no anticipated financial risks to study subjects. The larval BioBags 
and lab tests performed on the wound samples collected from human participants for the sole purpose of 
this study will be the responsibility of the principal investigator.   
 
8. Possible Benefits: 
 
Benefits to Subjects 
Potential Health Benefits:  The human subjects in both arms of this study may benefit from the 
debridement therapies, as this has been demonstrated to improve wound healing trajectories. Information 
gained from this research may benefit future patients who suffer from chronic wounds. 
 
9. Conflict of Interest: 
Conflict of Interest Statements have been completed by all VA Investigators and are maintained on file 
in regulatory documentation. There is no conflict of interest identified with any of the Investigators. The 
Funding source/study Sponsor is Industry (Biomonde) and that is readily disclosed, but there are no 
financial obligations between this company and any of the investigators other than for study purposes 
as stated in the signed Cooperative Research and Development Agreement (CRADA) on file with the 
VA’s affiliated not-for profit entity, the North Florida Foundation for Research and Education (NFFRE).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Literature Cited 
 
Blake, F.A., Abromeit, N., Bubenheim, M., Li, L. & Schmelzle, R. (2007). The biosurgical wound 
debridement:  
experimental investigation of efficiency and practicability. Wound Repair Regen, 15 (5), 756-
761. 
Cowan, L.J., Stechmiller, J.K., Phillips, P., Yang, Q. & Schultz, G.S. (2013). Chronic wounds, biofilms, 
and use  
of medicinal larvae. Ulcers, 2013, Article ID 487024, 7 pages, 2013. Doi:10.1155/2013/487024  
Cowan, L.J., Phillips, P., Stechmiller, J., Yang, Q., Wolcott, R. & Schultz, G. (2013) Chapter 4: 
Antibiofilm  
Strategies and Antiseptics. Antiseptics in Surgery – Update 2013, edited by Christian Willy. 
Lindqvist  
Book Publishing: Berlin, Germany.