M ILITARY M EDICINE, 180, 5:591, 2015
Airworthiness Testing of Medical Maggots George W. Peck, PhD*; Sandra M. Helgeson, E dD f; Maj Eric D. Powell, USAF B S C f; CPT Amanda L. Roth, MS USA§; Micah Flores, PhD§; MAJ Benjamin C. Kirkup Jr, MS USAII
ABSTRACT An investigation was conducted to test and certify medicinal maggots to facilitate rapid healing of traumatic and chronic wound infections in Wounded warriors being transported onboard military aircraft. Our specific aims included (1) to test the ability of medical grade larvae to withstand the rigors of U.S. Army aeromedical certifica tion. including tolerance to change in pressure, temperature, and humidity inside ground-based chambers; (2) to evaluate the efficacy of the medical grade larvae during a high-vibration rotary-wing medical transport flight; and (3) to gain U.S. Army aeromedical certification and U.S. Air Force safe-to-fly approval and begin the steps needed to deploy/ implement the use of medicinal maggots in patient care regimes for medical airlift standard operating procedures. This report outlines the ground-based and initial air-based tests performed during the study. Maggot mortality was very low during all tests, with a mortality rate of less than 1%. Maggot growth rates in wound arenas were mixed but generally depended on temperature. Overall, the results of these tests suggest that medicinal maggots can withstand the rigors of aeromedical evacuation flights in simulated flight environments and rotary- or fixed-wing aircraft.
INTRODUCTION M aggot debridem ent therapy (M D T) is an ancient w ound healing technology1 that has been recognized by m any clini cians as a potential adjunct to conventional therapy, and m any patients with nonhealing, chronic ulcers have been successfully treated. N um erous case reports, case series, and prospective clinical trials have described the effective use o f M D T in a variety o f ulcers.2 The use o f m edicinal m aggots (the green bottle fly, Lucilia sericata Meigen, Diptera: Calliphoridae) as a m edical treatm ent was cleared by the Food and D rug A dm inistration (FD A )3 and has been dem onstrated to be efficacious, w ell tolerated, and costeffective.1-4 In Europe, approxim ately 15,000 patients receive M D T for wound treatment annually.5 M any years o f clinical studies based on a com bination o f physical and biochemical actions have dem onstrated the success o f L. sericata larvae as debridem ent agents.5'6 L aboratory investigations have advanced our understanding o f the biochemical mechanisms underlying the beneficial effects o f m edicinal m aggots, including rem oval o f dead tissu e ,1-7 reduction o f the bacte rial b u rd en ,8-9 and prom otion o f tissue reg en eratio n .10-13 ♦Entomology Branch, Walter Reed Army Institute of Research, 503 Robert Grant Avenue. Building 503, Room 3w81, Silver Spring, MD 20910. tAirworthiness Certification & Evaluation Branch, United States Army Aeromedical Research Laboratory, Fort Rucker, AL 36362. tDepartment of Aeromedical Research, US Air Force School of Aero space Medicine, 2510 5th Street, Building 840 Area B, Room W 118.12, Wright-Patterson AFB, OH 45433. §Bacterial Diseases Branch, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910. || Department of Medicine, F. Edward Hebert School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814. This manuscript has been reviewed by the Walter Reed Army Institute of Research. The views, opinions, and/or findings in this article are those of the authors and should not to be construed as an official Department of the Army or the Department of Defense position, policy, or decision, doi: 10.7205/MILMED-D-14-00548
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M D T has been proven as a very safe, sim ple, and effective treatm ent m odality for chronic w ounds in am bulatory and hospitalized p a tie n ts.1-4-14 The logistics o f M D T in the U nited States relies on a single supplier, although there are other suppliers in Europe and Asia. Monarch Labs (ML)3 was incoiporated in September 2005, as a producer and distributor o f biotherapy medical prod ucts. Their first and lead product, Medical Maggots, was origi nally studied at the University o f California (Irvine, California) and at the D epartment o f Veterans Affairs (VA M edical Center, Long Beach, California).15 After receiving marketing clearance in 2004 by the FDA, the production o f Medical M aggots at the University o f California, Irvine M aggot Ther apy Laboratory was privatized by ML. M edical M aggots for M D T are available by prescription, and M L3 will ship the M edical M aggots overnight anyw here in the continental United States (CONUS), Hawaii, Puerto Rico, and Guam. In 2010, a fly rearing facility was established at the W alter Reed Arm y Institute o f Research (W RAIR) with the capacity to generate thousands o f maggots when needed. Initial studies included antim icrobial biocom patibility tests w ith m aggots (L. sericata) to ensure their usability and tolerance to m axim um clinical doses o f contem porary antibiotics, antileishmanials, and antifungals.16 A survey o f military physi cians in 2012 found a growing interest in the use o f m edicinal m aggots.17 The B acterial D isease B ranch at W R A IR is cu r rently testing the efficacy o f m edical grade m aggots to heal w ounds infected with m ultiple drug-resistant organism s (M D R O s) through the use o f a w ounded rat m odel. D espite advances in surgical m anag em en t o f com bat wounds, chronic infection remains a concerning and potentially preventable complication o f combat-related injuries across the various strata o f medical support for patients.18 W ounded U.S. military personnel are usually transported from Com bat Sup port Hospitals (Role 3) to Germany (Landstuhl) (Role 4) and then from Germany to the CONUS military medical centers
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on C-17 aircraft. This complex chain of care, by its nature, during an in-flight test. In collaboration with WRAIR, the may require multiple physical and care management handoffs U.S. Army Aeromedical Research Laboratory (USAARL) over a period of days to weeks from the point of injury conducted testing and evaluation of Medical Maggots in to care in a medical facility in the patient’s home country. accordance with an approved test plan (U.S. Army Aero Current recommendations to health care providers, to prevent medical Research Laboratory: Test plan for the use of Medi infectious complications of combat-injured patients with open cal Maggots [disinfected Phoenicia (Lucilia) sericata] during wounds, focus on care from point-of-injury until arrival at aeromedical care. Fort Rucker, 2012. U.S. Army Aeromedical tertiary care facilities outside the combat zone. Postinjury Research Laboratory and U.S. Air Force Aeromedical Test antimicrobials, early wound cleansing (irrigation) and sur Laboratory: Joint en route care equipment test standard gical debridement, delayed closure, and bony stabilization, (JECETS). Fort Rucker and Wright-Patterson AFB, 2012.). with emphasis on maintenance of infection control mea sures, are the essential components in reducing the inci S y s te m D e s c rip tio n dence of these infections. Each vial of Medical Maggots is shipped with approxi Emerging MDROs and the biofilms they create in wounds mately 600 L. sericata eggs that yield approximately 250 to present an ongoing challenge to normal wound healing.19'"0 500 maggots. Each vial contains a small amount of sterile Current recommendations of care may often fail when anti soy-based food on cotton gauze to keep newly hatched mag biotic resistance outpaces new treatment modalities for trau gots alive during shipment to a clinical site. When the vials matic and chronic wound infections. arrive at a clinical facility, their contents should be inspected En route care, medical treatment during evacuation between for viability and any evidence of contamination.3 ML’s rec levels of care from point of injury to a hospital in the CONUS, ommendations for the proper utilization of Medical Maggots is a critical component of casualty care and management include application within 24 hour of receipt and single within force health protection. Wounded warriors with acute dosing. “Used” or applied maggots become contaminated by traumatic wounds, including those with active MDRO infec the patient’s wound and must be handled as “contaminated tions, may benefit from cutting edge therapeutics during medi waste.” The maggots have a natural tendency to wander off cal evacuation in fixed-wing aircraft during transportation before and after they have finished feeding. Thus, they must from theater hospitals to established centers of care in Europe be kept in place by dressings that confine them to the and the CONUS. wound, but allow air circulation, liquefied necrotic tissue, New paradigms of wound treatment are needed to meet and other wound discharge to drain out. The conventional the challenge of MDROs and their associated biofilms in way to accomplish this is to build a maggot “cage-dressing” wounded warriors. This is true for ground-based care as well over and around the wound containing the maggots. M L’ as medical airlift operations. Recruiting the power of medici has designed “preassembled” maggot dressings that confine nal maggots can be an important step in meeting this chal the maggots to the wound. Two of these dressings, Le Soc lenge by expanding the range of tools within the aeromedical (1.75 in by 2.5 in) and LeFlap Dujour (4 in by 4 in) were wound treatment toolbox. Military physicians will benefit by tested in simulated wounds. having MDT for use alone or in combination with other proven wound disinfection/healing regimes. To facilitate the adoption of medicinal maggots for aero G e n e ra l T e s t D e s ig n medical wound treatment, the Wound Infections Department, Following guidelines in the Joint Enroute Care Equipment Bacterial Diseases Branch at WRAIR, initiated a project that Test Standards, the USAARL tested baseline performance as used the U.S. Army aeromedical certification process to test well as climactic and altitude stresses related to certification and certify Medical Maggots for use in medical evacuation of medical devices for use in military medical airlift opera airlift operations. Potential failure of medical devices during tions. During all phases of testing, the Airworthiness Certifi in-flight medical care may expose patients and aircrew to cation and Evaluation Branch's safety precautions, quality hazardous conditions. Consequently, medical devices undergo assurance standards, and standard operating procedures were rigorous testing before use on military aircraft. The U.S. followed, including those set forth by the off-site laborato Army aeromedical certification process utilizes military ries involved in testing. Baseline testing was conducted at and civilian standards, regulations, and specifications, as ML (Irvine, California), and Environment Associates (EA, well as professional experience and expertise for guidance Santa Ana, California). During all tests, the maggots were monitored for mortality, escape from the dressing, and weight throughout the testing and evaluation process. gain. Weight gain was assumed to be the best metric of medi cal device efficacy as it is directly correlated with wound MATERIALS AND METHODS The U.S. Army airworthiness test and evaluation process debridement success.1 To simulate a wound, raw pork (belly has two phases. The first involves laboratory testing that cut) was used as a substrate for the maggots. The pork was simulates the military aeromedical operational environment. left at room temperature for 14 hours before cutting into 1-cm The second phase involves evaluation of medical equipment cubes. A 10-cm plastic petri plate served as the containment
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area for the maggots and the pork cubes (wound arena). Mag gots were removed from a ML shipping vial and manipulated throughout the experiments with a moistened No. 2 camel’s hair paint brush. Groups of maggots were weighed before and after placement in wound arenas to ±0.001 g (Model XE-410D, Denver Instruments, Bohemia, New York). The one exception to this was during flight testing, when mag gots were weighed to ±0.01 g. Approximately 19 g (range 18.88-19.94 g for the baseline, temperature, and altitude tests; 26.27 [1.42] [mean (1SD, n = 4)] g for the rotary wing aircraft test) of pork cubes were used in each wound arena along with 35 maggots. Second instar maggots (24- to 48-hour posthatch) were used for all tests. This design was used to approximate the directed dosage for Medical Maggots in open wounds (5-10 maggots/cm2).'1 Lids were placed onto the wound arenas and each arena was placed into a LeSoc dressing sealed with heat (baseline ML below) or medical tape (all other tests) for maggot containment in case of escape from the arena. Four replicate arenas were run simulta neously during each test type. In clinical situations, a LeSoc dressing would be placed around an appendage with a wound and sealed with medical tape. A fifth petri dish with similar amounts of pork and maggots was used to test the LeFlap Dujour for its ability to restrain maggots within a sim ulated wound. The LeFlap Dujour dressing contains a strong adhesive that prevents maggots from escaping from a wound and is designed to be placed directly on the skin around the margin of a wound. In this study, a 5-cm opening was cut into the lid of wound arena five, and the LeFlap Dujour was applied to just cover the hole. The lid of wound arena five was sealed with tape to prevent maggot escape. Baseline Performance Assessment The initial baseline evaluation occurred at ML at ambient temperatures, and additional baseline testing occurred at EA. During this baseline testing at EA, maggots and wound arenas were treated as stated previously, with the additional factors of elevated temperature and humidity within a con trolled environmental chamber (Cincinnati Sub-zero humid ity chamber, model No. Zh-32-2-2-H/AC). The elevated temperature for this baseline test was chosen to simulate a human wound and the low humidity to simulate the typi cal conditions of high altitude medical evacuation flights in C-17 aircraft. High and Low Temperature Tests Extreme temperature evaluations took place at EA in con trolled environmental chambers. Maggots and wound arenas were prepared as previously described. The high temperature test had two phases. Phase 1 was a test to simulate overnight storage of the maggots in a high temperature and a low humidity environment. The maggots were left in their ship ping vial for this portion of the test. Since the maggots were not exposed to a wound arena for Phase 1 of the high
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temperature test, no attempt was made to take their weight. Phase 2 was a test to simulate transportation of the maggots in a high temperature and low humidity environment. Since Phase 2 was simulating the treatment of a wound, maggots were placed in pork-filled wound arenas. Low temperature tests also had two phases. Phase 1 was a test to simulate stor age overnight in a cold shipping container and a relatively high humidity environment. Again, the maggots were left in their shipping vial for this storage simulation test, and their weight was not measured. Phase 2 was a test to simulate transportation of the maggots while placed in pork-filled wound arenas in a moderately low temperature and moderate humidity environment. Altitude Tests Altitude evaluations took place at EA. Maggots and wound arenas were prepared as above. Humidity could not be con trolled in the chamber (Thermotron altitude chamber, model F-27CHA-5-5); however, humidity and temperature readings were taken inside the chamber (Fluke thermometer, model 51K/J). Maggots were subjected to comparatively low pres sures, simulating an altitude of 3,048 m (10,000 ft); test chamber pressure was held at 520 Torr, and ambient pres sure at EA was 747 Torr. Depressurization at test start occurred in a 2-minute interval, whereas repressurization at test completion occurred in a 15-minute interval. This pres surization schedule simulates current practice in a C-17 fixed-wing aircraft. Rotary-Wing Aircraft Testing Rotary-wing aircraft testing occurred at Foil Rucker, Alabama. Maggots and wound arenas were prepared as stated previ ously. Testing was conducted onboard USAARL’s JUH-60A Black Hawk (serial number 88-26069) research helicopter. The flight test introduced the maggots to aircraft vibration. Wound arenas were placed into a cardboard box, with box lid flaps folded in a pinwheel fashion to allow air circulation. The box was secured in the rear center seat using helicopter seatbelts. Ambient temperature was taken at start and end of the flight (Fluke thermometer, model 54 II) with no change observed (initial temperature = final temperature); the flight duration was 4 hours. The flight profile included hovering, high vibration acceleration, high-speed takeoff and landing, 60 degree left and right bank at 120 knots, and level flight at maximum speed. Maximum altitude was 1,500 ft. Fixed-Wing Aircraft Testing Fixed-wing aircraft testing occurred on board a C-17 air craft, flying from Andrews AFB, Maryland, to Ramstein AB, Germany, with a 2.3-hour layover at McGuire AFB, New Jersey. Baseline for fixed-wing testing occurred at WRA1R, Silver Spring, Maryland. Ambient temperature and relative humidity were taken with a HOBO monitor (model U12; Onset Computer, Bourne, Massachusetts). The four petri
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dishes containing 35 medicinal maggots each were placed in a cardboard box, placed in the main cargo area, and secured to a passenger seat toward the front of the aircraft. Altitude was stepped between 33,000 and 37,000 ft, with cabin pressure maintained at 8,000 ft equivalent. No abnor mal barometric perturbations were observed during the course of the flight. Data Analysis Standard formulas were used to calculate measures of central tendency, including mean and standard deviation. Analysis of variance (ANOVA) was used to compare the entire range of growth rates for all six test types; /-tests were used to compare means. RESULTS
Differences existed among the eight testing regimes, includ ing variation in time inside the test chambers and time exposed to the pork, temperature, relative humidity, and weight gain (Table I). A key finding with clinical implications was sig nificant differences in maggot growth rate (pg/h) among the various testing regimes (ANOVA, F = 38.26, d f = 0 7, p = 1.65 x 1CT11). All tests at ML and EA occurred during July 2012, whereas the rotary-wing test occurred on Sep tember 6, 2012, and the fixed-wing test occurred on June 20 and 21, 2014. Baseline Tests Baseline tests were conducted at ML and EA. Maggots gained significant weight during baseline tests (Table I), averaging 3.60 times their starting weight (jag) for baseline ML and 2.83 times their starting weight for baseline EA. Maggot survival rates were 100% for baseline ML and 99.4% for baseline EA testing. Maggot growth rate for the EA baseline was significantly higher than the growth rate for the ML baseline (20.6 vs. 12.2 pg/h; t = 4.38, d f = 3,
p = 0.011). The LeFlap Dujour was able to restrain maggots
within a simulated wound, for example, none escaped during baseline tests. High and Low Temperature Tests Maggot growth rate for the high temperature transport test was significantly lower than the baseline ML (Table I; t = 4.19, d f = 3, p = 0.012), and significantly lower than the baseline EA (t = 4.87, d f = 3, p = 0.008). Maggot growth for the low temperature test was significantly lower than the baseline ML growth rate (t = 12.47, d f = 3, p = 0.001), and significantly lower than the baseline EA growth rate (t = 8.58, d f = 3, p ~ 0.001). This depressed growth rate, when compared to baseline test growth rate, was an expected outcome for testing at lower temperatures. Maggot survival during the high and low temperature transport tests were 98.7% and 100%, respectively. Altitude Test Maggot growth rate for the altitude test was significantly lower than the baseline ML (Table I, t = 5.80, d f = 3, p = 0.005), and significantly lower that the baseline EA (/ = 7.77, d f = 3, p = 0.001). Interestingly, the maggot growth for the low temperature test was statistically identical to the altitude test growth rate (t = 0.73, d f= 3, p = 0.26). This equivalence of growth rate may be attributed to dissimilar stress (cold vs. low oxygen) affecting maggot growth in similar ways. Maggot survival for altitude chamber testing was 100%. Rotary-Wing Aircraft Testing Maggot growth rate for the rotary-wing aircraft test was significantly lower than the baseline ML (Table I; t = 11.77, d f= 3, p = 0.001), and significantly lower than the baseline EA (/ = 6.26, d f= 3, p = 0.002). Maggot survival for rotary wing testing was 100%.
TABLE I. Environmental Factors and Growth Characteristics of Maggots Across Test Types. Means Are for Four Replicate Wound Arenas That Were Used in Each Test, With 35 Maggots Per Arena. Mean Time to Feed Is the Total Time the Maggots Were in Contact With the Pork. Maggots Were Kept Inside Their Shipping Vial for Storage Tests (High and Low Temperature)
Test Type
Mean Temperature °C (Range)
Baseline ML“ Baseline EA Baseline WRAIR* High T (Storage) High T (Transport) Low T (Storage) Low T (Transport) Altitude Rotary-Wing Fixed-Wing
26.5 (25-28) 39 (35-43) 21.9 (21.5-23.0) 40 (37—13) 39 (36-42) 5 (2-8) 15 (12-18) 20.0 (18.6-21.4) 29.39 22.2 (18.3-28.2)
Mean Percent RH (Range) 45 30 54 40 29 84 81 37 78 43
(40-50) (27-33) (49-56) (37-43) (22-36) (79-89) (78-84) (28-44) (76-80) (37-66)
Time in Chamber (Hours)
Mean Change in Weight (pg) (1SD)
Mean Time to Feed (Hours) (ISD)
Mean Growth Rate (pg/h) (ISD)
10.50 10.07 20.77 17.17 10.00 12.25 10.00 8.08 4.00“ 20.54
128.0 (19.7) 273.0 (57.6) 101.3 (32.5)
10.5 (0.3) 13.2 (0.1) 20.8 (0.4) — 11.6 (0.2)
12.2 (1.6) 20.6 (4.2) 4.88 (1.55) — 8.21 (1.33) — 2.12 (0.81) 2.95 (1.67) 6.43 (1.62) 2.95 (1.38)
—
95.5 (15.2) —
22.0 32.8 47.5 60.7
—
(8.4) (18.1) (12.6) (28.5)
10.4 11.2 7.4 20.5
(0.2) (0.1) (0.2) (0.3)
T, temperature; SD, standard deviation; — , not measured. “Wound arenas were placed on the laboratory counter at ML, with ambient laboratory conditions acting as a “chamber.” ^Baseline growth treatment for Fixed-Wing study. ““Chamber” in this case was the helicopter passenger area.
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Fixed-Wing Aircraft Testing Maggot growth rate for the fixed-wing aircraft test was equal to the baseline data recorded at WRAIR (Table I; t = 1.72, d f = 3, p = 0.092). However, the baseline (WRAIR) was significantly lower than the baseline ML (t = 6.97, d f = 3, p = 0.003) and EA (t = 5.71, d f= 3, p = 0.005).
DISCUSSION This study modeled the wound debridement ability of Medical Maggots under various testing regimes measured as weight gain. Maggot growth was generally a function of temperature, with higher temperatures contributing to the largest growth rates. The comparatively higher maggot growth rate observed for the baseline EA is most likely attributable to the increased testing temperature (39 vs. 26°C). However, expected growth trends can be impacted by adverse events, including suboptimal food substrates.21 Thus, the depressed high temperature transport test maggot growth rate may be attributed starvation stress (sole food source was transport vial medium [ML sterile soy broth mix]) during the 17-hour high temperature storage test. Similarly, differences in pork quality may be one possible explanation for the lower weight gain for the WRAIR baseline when compared to the ML and EA base lines. Variation in temperature and humidity are known to affect L. sericcita growth rate22-24 and survival.25-27 One reason for the lower rate of growth observed during the altitude tests may have been the slight decrease in tem perature between the baseline and altitude testing regimes (Table I). However, a more probable reason was the lower partial pressure of oxygen in the altitude testing chamber. Unfortunately, the primary entomological literature lacks information on how a low oxygen environment may affect maggot growth rates. Interestingly, the fixed-wing growth rate was identical to the altitude test growth rate (Table I). This may be because of the similar conditions of lower pressure and its effect on maggot growth. The vibration of the aircraft is one possible explanation for the lower weight gain during flight testing as compared to the base line weight gain at ML. Maggots may have had difficulty feeding because of vibration of their food substrate during the test. As with low oxygen effects, the primary entomo logical literature lacks information on how aircraft vibration may affect maggot growth rates. Nevertheless, it seems plau sible that low oxygen and vibration could potentially impact maggot growth, depending on their magnitude and duration. Thus, starvation stress during high temperature storage testing before high temperature transport testing, delayed recovery from low temperature storage testing before low temperature transport testing, the lack of sufficient oxygen during high altitude testing, and mechanical vibrations during rotary-wing aircraft testing are all factors likely impacting the observed deviation of growth rates from expected growth rates. The data presented here demonstrate the effects of low pressure, high vibration, and extreme climatic environments
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on the short term growth rate of the Medical Maggots. Although most variation in growth rate occurred between test types, it should be noted that intratest variation was also observed (SD values, Table I). Heterogeneity in pork com position among wound arenas in a test type may have con tributed to the large intratest variation. The pork was a mix of lean and fatty tissue, and some wound arenas may have contained greater amounts of fatty pork, which is of lower nutritional value to maggots, impacting their growth rates. The impact of different tissue composition on L. sericata growth rate has been elucidated previously.26 Earlier studies have used maggot length as a primary factor of growth rate models.23,25 However, these studies measured maggot growth over many days. For the purpose of determining environmental effects on wound debridement efficacy over just a few hours, weight gain was considered the best metric of device efficacy as it is directly correlated with wound debridement success.1 Although individual weights of maggots would have been ideal, weights of pooled cohorts of early instar maggots has previously yielded accu rate linear models of growth.27 Because American Medical Association and Centers for Medicare and Medicaid recently released reimbursement coding guidelines with regards to maggot therapy, there is a potential for wider use of maggot therapy in the United States in the near future. However, comparative clinical trials (in particular randomized controlled trials) investigating the efficacy of MDT are needed." 6 A systematic search in the lit erature evaluating the efficacy of sterile L. sericata applied on ulcers with various etiologies report MDT as being signifi cantly more effective than hydrogel or a mixture of conven tional therapy modalities, including hydrocolloid, hydrogel, and saline moistened gauze.4 Further, the ability of medicinal maggots to tolerate a wide variety of antimicrobial classes suggests that MDs may find a use for them in a wide variety of clinical situations, including topical treatments of highly concentrated, broad-spectrum antibiotics.18 MDT, despite its long history and safety profile, finds limited use in the military health care system. Although new methods are continually being investigated to debride wounds more quickly and effectively, MDT largely remains a therapy of last resort. Recently, the frequency of MDT used in the Army sector of the Military Health System and the decision making process surrounding its use was evaluated.I7A 22-question survey was prepared and distributed to Army physicians through select Medical Corps Consultants in specialties likely to practice debridement. Eighty-three per cent of respondents were familiar with MDT, and of those familiar, 63% were aware of FDA approval for the product and 10% had used the Medical Maggots themselves. The three most frequently cited reasons for not using the therapy were no need (52%), no access (23%), and insufficient expe rience (19%). Military physicians faced with nonhealing wounds that are infected with MDROs will benefit by having medicinal maggots in their toolbox, to be used alone
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or in combination with other proven wound disinfection/ healing regimes. CONCLUSION Wounded warriors with nonhealing wounds that are treated with medicinal maggots will likely have better outcomes, including avoidance of amputations and shorter recovery times. This will translate to reduced treatment costs to the military medical system. The overall results of these tests suggest that medicinal maggots can withstand the rigors of aeromedical evacuation flights in simulated flight environments and rotary wing aircraft, and fixed-wing aircraft. Since direct electrome chanical testing is not possible with this type of medical device (Medical Maggots), future work will focus on obtaining some sort of alternate document, likely termed an “approval for use” aboard MEDEVAC aircraft, through a USAARL aero medical certification memorandum. ACKNOWLEDGMENTS The authors would like to thank the Airworthiness Certification and Evalua tion Branch of USAARL for their test guidance, airworthiness testing, and data reporting. This study was funded by Defense Medical Research and Development Program (DMRDP), DHP 6.7—Medical Products and Capabili ties Enhancements No. 2011053 Airworthiness Testing of Medical Maggots.
REFERENCES 1. Sherman R, Hall M, Thomas S: Medicinal maggots: an ancient remedy for some contemporary afflictions. Ann Rev Entomol 2000; 45(1): 55-81. 2. Davydov L: Maggot therapy in wound management in modem era and a review of published literature. J Pharm Pract 2011; 24: 89-93. 3. Monarch Labs, Inc: Clinical protocols and device details. Available at http://www.monarchlabs.com/; accessed December 10, 2013. 4. Zarchi K. Jemec G: The efficacy of maggot debridement therapy: a review of comparative clinical trials. Int Wound J 2012; 9: 469-77. 5. Cazander G. Pritchard D. Nigam Y, Jung W, Nibbering P: Multiple actions of Lucilia sericata larvae in hard-to-heal wounds: larval secretions contain molecules that accelerate wound healing, reduce chronic inflam mation and inhibit bacterial infection. BioEssays 2013; 35: 1083-92. 6. Mudge E, Price P, Neal W, Harding K: A randomized controlled trial of larval therapy for the debridement of leg ulcers: results of a multi center, randomized, controlled, open, observer blind, parallel group study. Wound Repair Regen 2014; 22(1): 43-51. 7. Barnard D: Skeletal-muscular mechanisms of the larva of Lucilia sericata (Meigen) in relation to feeding habit. Pan-Pac Entomol 1977; 53: 223-9. 8. Jiang K, Sun X, Wang W, et al: Excretions/secretions from bacteriapretreated maggot are more effective against Pseudomonas aeruginosa biofilms. PLoS One 2012; 7: e49815.
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Cerovsky V, Zd’arek J, Fucfk V, Monincova L, Voburka Z, Bern R: Lucifensin, the long-sought antimicrobial factor of medicinal maggots of the blowfly Lucilia sericata. Cell Mol Life Sci 2010; 67: 455-66. 10. Bexfield A, Bond A, Morgan C, et al: Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis. Brit J Dermatol 2010; 162: 554-62. 11. Wang S, Wang K, XinY Lv D: Maggot excretions/secretions induces human microvascular endothelial cell migration through AKT1. Mol Biol Rep 2010; 37: 2719-25. 12. Zhang Z, Wang S, Diao Y, Zhang J. Lv D: Fatty acid extracts from Lucilia sericata larvae promote murine cutaneous wound healing by angiogenic activity. Lipids Health Dis 2010; 9: 24. 13. Zhang Z, Wang S, Tian X, Zhao Z, Zhang J, Lu D: Maggot homoge nate is associated with neural regeneration and wound healing in the rat. Neural Regen Res 2010; 5: 623-9. 14. Gilead L, Mumcuoglu K, Ingber A: The use of maggot debridement therapy in the treatment of chronic wounds in hospitalized and ambula tory patients. J Wound Care 2012; 21: 78-85. 15. Sherman R. Wyle F: Low-cost, low-maintenance rearing of maggots in hospitals, clinics, and schools. Am J Trap Med Hyg 1996; 54: 38-41. 16. Peck G, Kirkup B: Biocompatibility of antimicrobials to maggot debride ment therapy: medical maggots Lucilia sericata (Diptera: Calliphoridae) exhibit tolerance to clinical maximum doses of antimicrobials. J Med Entomol 2012; 49(5): 1137-43. 17. Heitkamp R, Peck G, Kirkup B: Maggot debridement therapy in the US Army Medical Command: perceptions and prevalence. Mil Med 2012; 177(11): 1411-6. 18. Hospenthal D. Murray C, Andersen R: Guidelines for the prevention of infections associated with combat-related injuries. J Trauma 2011; 71(2): S210-34. 19. James G, Swogger E, Wolcott R: Biofilms in chronic wounds. Wound Repair Regen 2008; 16(1): 37-44. 20. Hospenthal D, Murray C: Preface: guidelines for the prevention of infections associated with combat-related injuries. J Trauma 2011; 71(2): S197—201. 21. Tarone A, Foran D: Components of developmental plasticity in a Michigan population of Lucilia sericata (Diptera: Calliphoridae). J Med Entomol 2006; 43(5): 1023-33. 22. Greenburg B: Flies as forensic indicators. J Med Entomol 1991; 20: 565-77. 23. Anderson G: Minimum and maximum development rates for some forensically important Calliphoridae (Diptera). J Forensic Sci 2000; 45: 824-32. 24. Grassberger M, Reiter C: Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram. Forensic Sci Int 2001; 120: 32-6. 25. Wall R, Pitts K, Smith K: Pre-adult mortality in the blowfly Lucilia sericata. Med Vet Entomol 2001; 15: 328-34. 26. Clark K, Evans L, Wall R: Growth rates of the blowfly, Lucilia sericata, on different body tissues. Forensic Sci Int 2006; 156: 145-9. 27. Tarone M, Foran D: Generalized additive models and Lucilia sericata growth: assessing confidence intervals and error rates in forensic ento mology. J Forensic Sci 2008; 53: 942-8.
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Airworthiness Testing of Medical Maggots George W. Peck, PhD*; Sandra M. Helgeson, E dD f; Maj Eric D. Powell, USAF B S C f; CPT Amanda L. Roth, MS USA§; Micah Flores, PhD§; MAJ Benjamin C. Kirkup Jr, MS USAII
ABSTRACT An investigation was conducted to test and certify medicinal maggots to facilitate rapid healing of traumatic and chronic wound infections in Wounded warriors being transported onboard military aircraft. Our specific aims included (1) to test the ability of medical grade larvae to withstand the rigors of U.S. Army aeromedical certifica tion. including tolerance to change in pressure, temperature, and humidity inside ground-based chambers; (2) to evaluate the efficacy of the medical grade larvae during a high-vibration rotary-wing medical transport flight; and (3) to gain U.S. Army aeromedical certification and U.S. Air Force safe-to-fly approval and begin the steps needed to deploy/ implement the use of medicinal maggots in patient care regimes for medical airlift standard operating procedures. This report outlines the ground-based and initial air-based tests performed during the study. Maggot mortality was very low during all tests, with a mortality rate of less than 1%. Maggot growth rates in wound arenas were mixed but generally depended on temperature. Overall, the results of these tests suggest that medicinal maggots can withstand the rigors of aeromedical evacuation flights in simulated flight environments and rotary- or fixed-wing aircraft.
INTRODUCTION M aggot debridem ent therapy (M D T) is an ancient w ound healing technology1 that has been recognized by m any clini cians as a potential adjunct to conventional therapy, and m any patients with nonhealing, chronic ulcers have been successfully treated. N um erous case reports, case series, and prospective clinical trials have described the effective use o f M D T in a variety o f ulcers.2 The use o f m edicinal m aggots (the green bottle fly, Lucilia sericata Meigen, Diptera: Calliphoridae) as a m edical treatm ent was cleared by the Food and D rug A dm inistration (FD A )3 and has been dem onstrated to be efficacious, w ell tolerated, and costeffective.1-4 In Europe, approxim ately 15,000 patients receive M D T for wound treatment annually.5 M any years o f clinical studies based on a com bination o f physical and biochemical actions have dem onstrated the success o f L. sericata larvae as debridem ent agents.5'6 L aboratory investigations have advanced our understanding o f the biochemical mechanisms underlying the beneficial effects o f m edicinal m aggots, including rem oval o f dead tissu e ,1-7 reduction o f the bacte rial b u rd en ,8-9 and prom otion o f tissue reg en eratio n .10-13 ♦Entomology Branch, Walter Reed Army Institute of Research, 503 Robert Grant Avenue. Building 503, Room 3w81, Silver Spring, MD 20910. tAirworthiness Certification & Evaluation Branch, United States Army Aeromedical Research Laboratory, Fort Rucker, AL 36362. tDepartment of Aeromedical Research, US Air Force School of Aero space Medicine, 2510 5th Street, Building 840 Area B, Room W 118.12, Wright-Patterson AFB, OH 45433. §Bacterial Diseases Branch, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910. || Department of Medicine, F. Edward Hebert School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814. This manuscript has been reviewed by the Walter Reed Army Institute of Research. The views, opinions, and/or findings in this article are those of the authors and should not to be construed as an official Department of the Army or the Department of Defense position, policy, or decision, doi: 10.7205/MILMED-D-14-00548
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M D T has been proven as a very safe, sim ple, and effective treatm ent m odality for chronic w ounds in am bulatory and hospitalized p a tie n ts.1-4-14 The logistics o f M D T in the U nited States relies on a single supplier, although there are other suppliers in Europe and Asia. Monarch Labs (ML)3 was incoiporated in September 2005, as a producer and distributor o f biotherapy medical prod ucts. Their first and lead product, Medical Maggots, was origi nally studied at the University o f California (Irvine, California) and at the D epartment o f Veterans Affairs (VA M edical Center, Long Beach, California).15 After receiving marketing clearance in 2004 by the FDA, the production o f Medical M aggots at the University o f California, Irvine M aggot Ther apy Laboratory was privatized by ML. M edical M aggots for M D T are available by prescription, and M L3 will ship the M edical M aggots overnight anyw here in the continental United States (CONUS), Hawaii, Puerto Rico, and Guam. In 2010, a fly rearing facility was established at the W alter Reed Arm y Institute o f Research (W RAIR) with the capacity to generate thousands o f maggots when needed. Initial studies included antim icrobial biocom patibility tests w ith m aggots (L. sericata) to ensure their usability and tolerance to m axim um clinical doses o f contem porary antibiotics, antileishmanials, and antifungals.16 A survey o f military physi cians in 2012 found a growing interest in the use o f m edicinal m aggots.17 The B acterial D isease B ranch at W R A IR is cu r rently testing the efficacy o f m edical grade m aggots to heal w ounds infected with m ultiple drug-resistant organism s (M D R O s) through the use o f a w ounded rat m odel. D espite advances in surgical m anag em en t o f com bat wounds, chronic infection remains a concerning and potentially preventable complication o f combat-related injuries across the various strata o f medical support for patients.18 W ounded U.S. military personnel are usually transported from Com bat Sup port Hospitals (Role 3) to Germany (Landstuhl) (Role 4) and then from Germany to the CONUS military medical centers
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on C-17 aircraft. This complex chain of care, by its nature, during an in-flight test. In collaboration with WRAIR, the may require multiple physical and care management handoffs U.S. Army Aeromedical Research Laboratory (USAARL) over a period of days to weeks from the point of injury conducted testing and evaluation of Medical Maggots in to care in a medical facility in the patient’s home country. accordance with an approved test plan (U.S. Army Aero Current recommendations to health care providers, to prevent medical Research Laboratory: Test plan for the use of Medi infectious complications of combat-injured patients with open cal Maggots [disinfected Phoenicia (Lucilia) sericata] during wounds, focus on care from point-of-injury until arrival at aeromedical care. Fort Rucker, 2012. U.S. Army Aeromedical tertiary care facilities outside the combat zone. Postinjury Research Laboratory and U.S. Air Force Aeromedical Test antimicrobials, early wound cleansing (irrigation) and sur Laboratory: Joint en route care equipment test standard gical debridement, delayed closure, and bony stabilization, (JECETS). Fort Rucker and Wright-Patterson AFB, 2012.). with emphasis on maintenance of infection control mea sures, are the essential components in reducing the inci S y s te m D e s c rip tio n dence of these infections. Each vial of Medical Maggots is shipped with approxi Emerging MDROs and the biofilms they create in wounds mately 600 L. sericata eggs that yield approximately 250 to present an ongoing challenge to normal wound healing.19'"0 500 maggots. Each vial contains a small amount of sterile Current recommendations of care may often fail when anti soy-based food on cotton gauze to keep newly hatched mag biotic resistance outpaces new treatment modalities for trau gots alive during shipment to a clinical site. When the vials matic and chronic wound infections. arrive at a clinical facility, their contents should be inspected En route care, medical treatment during evacuation between for viability and any evidence of contamination.3 ML’s rec levels of care from point of injury to a hospital in the CONUS, ommendations for the proper utilization of Medical Maggots is a critical component of casualty care and management include application within 24 hour of receipt and single within force health protection. Wounded warriors with acute dosing. “Used” or applied maggots become contaminated by traumatic wounds, including those with active MDRO infec the patient’s wound and must be handled as “contaminated tions, may benefit from cutting edge therapeutics during medi waste.” The maggots have a natural tendency to wander off cal evacuation in fixed-wing aircraft during transportation before and after they have finished feeding. Thus, they must from theater hospitals to established centers of care in Europe be kept in place by dressings that confine them to the and the CONUS. wound, but allow air circulation, liquefied necrotic tissue, New paradigms of wound treatment are needed to meet and other wound discharge to drain out. The conventional the challenge of MDROs and their associated biofilms in way to accomplish this is to build a maggot “cage-dressing” wounded warriors. This is true for ground-based care as well over and around the wound containing the maggots. M L’ as medical airlift operations. Recruiting the power of medici has designed “preassembled” maggot dressings that confine nal maggots can be an important step in meeting this chal the maggots to the wound. Two of these dressings, Le Soc lenge by expanding the range of tools within the aeromedical (1.75 in by 2.5 in) and LeFlap Dujour (4 in by 4 in) were wound treatment toolbox. Military physicians will benefit by tested in simulated wounds. having MDT for use alone or in combination with other proven wound disinfection/healing regimes. To facilitate the adoption of medicinal maggots for aero G e n e ra l T e s t D e s ig n medical wound treatment, the Wound Infections Department, Following guidelines in the Joint Enroute Care Equipment Bacterial Diseases Branch at WRAIR, initiated a project that Test Standards, the USAARL tested baseline performance as used the U.S. Army aeromedical certification process to test well as climactic and altitude stresses related to certification and certify Medical Maggots for use in medical evacuation of medical devices for use in military medical airlift opera airlift operations. Potential failure of medical devices during tions. During all phases of testing, the Airworthiness Certifi in-flight medical care may expose patients and aircrew to cation and Evaluation Branch's safety precautions, quality hazardous conditions. Consequently, medical devices undergo assurance standards, and standard operating procedures were rigorous testing before use on military aircraft. The U.S. followed, including those set forth by the off-site laborato Army aeromedical certification process utilizes military ries involved in testing. Baseline testing was conducted at and civilian standards, regulations, and specifications, as ML (Irvine, California), and Environment Associates (EA, well as professional experience and expertise for guidance Santa Ana, California). During all tests, the maggots were monitored for mortality, escape from the dressing, and weight throughout the testing and evaluation process. gain. Weight gain was assumed to be the best metric of medi cal device efficacy as it is directly correlated with wound MATERIALS AND METHODS The U.S. Army airworthiness test and evaluation process debridement success.1 To simulate a wound, raw pork (belly has two phases. The first involves laboratory testing that cut) was used as a substrate for the maggots. The pork was simulates the military aeromedical operational environment. left at room temperature for 14 hours before cutting into 1-cm The second phase involves evaluation of medical equipment cubes. A 10-cm plastic petri plate served as the containment
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area for the maggots and the pork cubes (wound arena). Mag gots were removed from a ML shipping vial and manipulated throughout the experiments with a moistened No. 2 camel’s hair paint brush. Groups of maggots were weighed before and after placement in wound arenas to ±0.001 g (Model XE-410D, Denver Instruments, Bohemia, New York). The one exception to this was during flight testing, when mag gots were weighed to ±0.01 g. Approximately 19 g (range 18.88-19.94 g for the baseline, temperature, and altitude tests; 26.27 [1.42] [mean (1SD, n = 4)] g for the rotary wing aircraft test) of pork cubes were used in each wound arena along with 35 maggots. Second instar maggots (24- to 48-hour posthatch) were used for all tests. This design was used to approximate the directed dosage for Medical Maggots in open wounds (5-10 maggots/cm2).'1 Lids were placed onto the wound arenas and each arena was placed into a LeSoc dressing sealed with heat (baseline ML below) or medical tape (all other tests) for maggot containment in case of escape from the arena. Four replicate arenas were run simulta neously during each test type. In clinical situations, a LeSoc dressing would be placed around an appendage with a wound and sealed with medical tape. A fifth petri dish with similar amounts of pork and maggots was used to test the LeFlap Dujour for its ability to restrain maggots within a sim ulated wound. The LeFlap Dujour dressing contains a strong adhesive that prevents maggots from escaping from a wound and is designed to be placed directly on the skin around the margin of a wound. In this study, a 5-cm opening was cut into the lid of wound arena five, and the LeFlap Dujour was applied to just cover the hole. The lid of wound arena five was sealed with tape to prevent maggot escape. Baseline Performance Assessment The initial baseline evaluation occurred at ML at ambient temperatures, and additional baseline testing occurred at EA. During this baseline testing at EA, maggots and wound arenas were treated as stated previously, with the additional factors of elevated temperature and humidity within a con trolled environmental chamber (Cincinnati Sub-zero humid ity chamber, model No. Zh-32-2-2-H/AC). The elevated temperature for this baseline test was chosen to simulate a human wound and the low humidity to simulate the typi cal conditions of high altitude medical evacuation flights in C-17 aircraft. High and Low Temperature Tests Extreme temperature evaluations took place at EA in con trolled environmental chambers. Maggots and wound arenas were prepared as previously described. The high temperature test had two phases. Phase 1 was a test to simulate overnight storage of the maggots in a high temperature and a low humidity environment. The maggots were left in their ship ping vial for this portion of the test. Since the maggots were not exposed to a wound arena for Phase 1 of the high
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temperature test, no attempt was made to take their weight. Phase 2 was a test to simulate transportation of the maggots in a high temperature and low humidity environment. Since Phase 2 was simulating the treatment of a wound, maggots were placed in pork-filled wound arenas. Low temperature tests also had two phases. Phase 1 was a test to simulate stor age overnight in a cold shipping container and a relatively high humidity environment. Again, the maggots were left in their shipping vial for this storage simulation test, and their weight was not measured. Phase 2 was a test to simulate transportation of the maggots while placed in pork-filled wound arenas in a moderately low temperature and moderate humidity environment. Altitude Tests Altitude evaluations took place at EA. Maggots and wound arenas were prepared as above. Humidity could not be con trolled in the chamber (Thermotron altitude chamber, model F-27CHA-5-5); however, humidity and temperature readings were taken inside the chamber (Fluke thermometer, model 51K/J). Maggots were subjected to comparatively low pres sures, simulating an altitude of 3,048 m (10,000 ft); test chamber pressure was held at 520 Torr, and ambient pres sure at EA was 747 Torr. Depressurization at test start occurred in a 2-minute interval, whereas repressurization at test completion occurred in a 15-minute interval. This pres surization schedule simulates current practice in a C-17 fixed-wing aircraft. Rotary-Wing Aircraft Testing Rotary-wing aircraft testing occurred at Foil Rucker, Alabama. Maggots and wound arenas were prepared as stated previ ously. Testing was conducted onboard USAARL’s JUH-60A Black Hawk (serial number 88-26069) research helicopter. The flight test introduced the maggots to aircraft vibration. Wound arenas were placed into a cardboard box, with box lid flaps folded in a pinwheel fashion to allow air circulation. The box was secured in the rear center seat using helicopter seatbelts. Ambient temperature was taken at start and end of the flight (Fluke thermometer, model 54 II) with no change observed (initial temperature = final temperature); the flight duration was 4 hours. The flight profile included hovering, high vibration acceleration, high-speed takeoff and landing, 60 degree left and right bank at 120 knots, and level flight at maximum speed. Maximum altitude was 1,500 ft. Fixed-Wing Aircraft Testing Fixed-wing aircraft testing occurred on board a C-17 air craft, flying from Andrews AFB, Maryland, to Ramstein AB, Germany, with a 2.3-hour layover at McGuire AFB, New Jersey. Baseline for fixed-wing testing occurred at WRA1R, Silver Spring, Maryland. Ambient temperature and relative humidity were taken with a HOBO monitor (model U12; Onset Computer, Bourne, Massachusetts). The four petri
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dishes containing 35 medicinal maggots each were placed in a cardboard box, placed in the main cargo area, and secured to a passenger seat toward the front of the aircraft. Altitude was stepped between 33,000 and 37,000 ft, with cabin pressure maintained at 8,000 ft equivalent. No abnor mal barometric perturbations were observed during the course of the flight. Data Analysis Standard formulas were used to calculate measures of central tendency, including mean and standard deviation. Analysis of variance (ANOVA) was used to compare the entire range of growth rates for all six test types; /-tests were used to compare means. RESULTS
Differences existed among the eight testing regimes, includ ing variation in time inside the test chambers and time exposed to the pork, temperature, relative humidity, and weight gain (Table I). A key finding with clinical implications was sig nificant differences in maggot growth rate (pg/h) among the various testing regimes (ANOVA, F = 38.26, d f = 0 7, p = 1.65 x 1CT11). All tests at ML and EA occurred during July 2012, whereas the rotary-wing test occurred on Sep tember 6, 2012, and the fixed-wing test occurred on June 20 and 21, 2014. Baseline Tests Baseline tests were conducted at ML and EA. Maggots gained significant weight during baseline tests (Table I), averaging 3.60 times their starting weight (jag) for baseline ML and 2.83 times their starting weight for baseline EA. Maggot survival rates were 100% for baseline ML and 99.4% for baseline EA testing. Maggot growth rate for the EA baseline was significantly higher than the growth rate for the ML baseline (20.6 vs. 12.2 pg/h; t = 4.38, d f = 3,
p = 0.011). The LeFlap Dujour was able to restrain maggots
within a simulated wound, for example, none escaped during baseline tests. High and Low Temperature Tests Maggot growth rate for the high temperature transport test was significantly lower than the baseline ML (Table I; t = 4.19, d f = 3, p = 0.012), and significantly lower than the baseline EA (t = 4.87, d f = 3, p = 0.008). Maggot growth for the low temperature test was significantly lower than the baseline ML growth rate (t = 12.47, d f = 3, p = 0.001), and significantly lower than the baseline EA growth rate (t = 8.58, d f = 3, p ~ 0.001). This depressed growth rate, when compared to baseline test growth rate, was an expected outcome for testing at lower temperatures. Maggot survival during the high and low temperature transport tests were 98.7% and 100%, respectively. Altitude Test Maggot growth rate for the altitude test was significantly lower than the baseline ML (Table I, t = 5.80, d f = 3, p = 0.005), and significantly lower that the baseline EA (/ = 7.77, d f = 3, p = 0.001). Interestingly, the maggot growth for the low temperature test was statistically identical to the altitude test growth rate (t = 0.73, d f= 3, p = 0.26). This equivalence of growth rate may be attributed to dissimilar stress (cold vs. low oxygen) affecting maggot growth in similar ways. Maggot survival for altitude chamber testing was 100%. Rotary-Wing Aircraft Testing Maggot growth rate for the rotary-wing aircraft test was significantly lower than the baseline ML (Table I; t = 11.77, d f= 3, p = 0.001), and significantly lower than the baseline EA (/ = 6.26, d f= 3, p = 0.002). Maggot survival for rotary wing testing was 100%.
TABLE I. Environmental Factors and Growth Characteristics of Maggots Across Test Types. Means Are for Four Replicate Wound Arenas That Were Used in Each Test, With 35 Maggots Per Arena. Mean Time to Feed Is the Total Time the Maggots Were in Contact With the Pork. Maggots Were Kept Inside Their Shipping Vial for Storage Tests (High and Low Temperature)
Test Type
Mean Temperature °C (Range)
Baseline ML“ Baseline EA Baseline WRAIR* High T (Storage) High T (Transport) Low T (Storage) Low T (Transport) Altitude Rotary-Wing Fixed-Wing
26.5 (25-28) 39 (35-43) 21.9 (21.5-23.0) 40 (37—13) 39 (36-42) 5 (2-8) 15 (12-18) 20.0 (18.6-21.4) 29.39 22.2 (18.3-28.2)
Mean Percent RH (Range) 45 30 54 40 29 84 81 37 78 43
(40-50) (27-33) (49-56) (37-43) (22-36) (79-89) (78-84) (28-44) (76-80) (37-66)
Time in Chamber (Hours)
Mean Change in Weight (pg) (1SD)
Mean Time to Feed (Hours) (ISD)
Mean Growth Rate (pg/h) (ISD)
10.50 10.07 20.77 17.17 10.00 12.25 10.00 8.08 4.00“ 20.54
128.0 (19.7) 273.0 (57.6) 101.3 (32.5)
10.5 (0.3) 13.2 (0.1) 20.8 (0.4) — 11.6 (0.2)
12.2 (1.6) 20.6 (4.2) 4.88 (1.55) — 8.21 (1.33) — 2.12 (0.81) 2.95 (1.67) 6.43 (1.62) 2.95 (1.38)
—
95.5 (15.2) —
22.0 32.8 47.5 60.7
—
(8.4) (18.1) (12.6) (28.5)
10.4 11.2 7.4 20.5
(0.2) (0.1) (0.2) (0.3)
T, temperature; SD, standard deviation; — , not measured. “Wound arenas were placed on the laboratory counter at ML, with ambient laboratory conditions acting as a “chamber.” ^Baseline growth treatment for Fixed-Wing study. ““Chamber” in this case was the helicopter passenger area.
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Fixed-Wing Aircraft Testing Maggot growth rate for the fixed-wing aircraft test was equal to the baseline data recorded at WRAIR (Table I; t = 1.72, d f = 3, p = 0.092). However, the baseline (WRAIR) was significantly lower than the baseline ML (t = 6.97, d f = 3, p = 0.003) and EA (t = 5.71, d f= 3, p = 0.005).
DISCUSSION This study modeled the wound debridement ability of Medical Maggots under various testing regimes measured as weight gain. Maggot growth was generally a function of temperature, with higher temperatures contributing to the largest growth rates. The comparatively higher maggot growth rate observed for the baseline EA is most likely attributable to the increased testing temperature (39 vs. 26°C). However, expected growth trends can be impacted by adverse events, including suboptimal food substrates.21 Thus, the depressed high temperature transport test maggot growth rate may be attributed starvation stress (sole food source was transport vial medium [ML sterile soy broth mix]) during the 17-hour high temperature storage test. Similarly, differences in pork quality may be one possible explanation for the lower weight gain for the WRAIR baseline when compared to the ML and EA base lines. Variation in temperature and humidity are known to affect L. sericcita growth rate22-24 and survival.25-27 One reason for the lower rate of growth observed during the altitude tests may have been the slight decrease in tem perature between the baseline and altitude testing regimes (Table I). However, a more probable reason was the lower partial pressure of oxygen in the altitude testing chamber. Unfortunately, the primary entomological literature lacks information on how a low oxygen environment may affect maggot growth rates. Interestingly, the fixed-wing growth rate was identical to the altitude test growth rate (Table I). This may be because of the similar conditions of lower pressure and its effect on maggot growth. The vibration of the aircraft is one possible explanation for the lower weight gain during flight testing as compared to the base line weight gain at ML. Maggots may have had difficulty feeding because of vibration of their food substrate during the test. As with low oxygen effects, the primary entomo logical literature lacks information on how aircraft vibration may affect maggot growth rates. Nevertheless, it seems plau sible that low oxygen and vibration could potentially impact maggot growth, depending on their magnitude and duration. Thus, starvation stress during high temperature storage testing before high temperature transport testing, delayed recovery from low temperature storage testing before low temperature transport testing, the lack of sufficient oxygen during high altitude testing, and mechanical vibrations during rotary-wing aircraft testing are all factors likely impacting the observed deviation of growth rates from expected growth rates. The data presented here demonstrate the effects of low pressure, high vibration, and extreme climatic environments
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on the short term growth rate of the Medical Maggots. Although most variation in growth rate occurred between test types, it should be noted that intratest variation was also observed (SD values, Table I). Heterogeneity in pork com position among wound arenas in a test type may have con tributed to the large intratest variation. The pork was a mix of lean and fatty tissue, and some wound arenas may have contained greater amounts of fatty pork, which is of lower nutritional value to maggots, impacting their growth rates. The impact of different tissue composition on L. sericata growth rate has been elucidated previously.26 Earlier studies have used maggot length as a primary factor of growth rate models.23,25 However, these studies measured maggot growth over many days. For the purpose of determining environmental effects on wound debridement efficacy over just a few hours, weight gain was considered the best metric of device efficacy as it is directly correlated with wound debridement success.1 Although individual weights of maggots would have been ideal, weights of pooled cohorts of early instar maggots has previously yielded accu rate linear models of growth.27 Because American Medical Association and Centers for Medicare and Medicaid recently released reimbursement coding guidelines with regards to maggot therapy, there is a potential for wider use of maggot therapy in the United States in the near future. However, comparative clinical trials (in particular randomized controlled trials) investigating the efficacy of MDT are needed." 6 A systematic search in the lit erature evaluating the efficacy of sterile L. sericata applied on ulcers with various etiologies report MDT as being signifi cantly more effective than hydrogel or a mixture of conven tional therapy modalities, including hydrocolloid, hydrogel, and saline moistened gauze.4 Further, the ability of medicinal maggots to tolerate a wide variety of antimicrobial classes suggests that MDs may find a use for them in a wide variety of clinical situations, including topical treatments of highly concentrated, broad-spectrum antibiotics.18 MDT, despite its long history and safety profile, finds limited use in the military health care system. Although new methods are continually being investigated to debride wounds more quickly and effectively, MDT largely remains a therapy of last resort. Recently, the frequency of MDT used in the Army sector of the Military Health System and the decision making process surrounding its use was evaluated.I7A 22-question survey was prepared and distributed to Army physicians through select Medical Corps Consultants in specialties likely to practice debridement. Eighty-three per cent of respondents were familiar with MDT, and of those familiar, 63% were aware of FDA approval for the product and 10% had used the Medical Maggots themselves. The three most frequently cited reasons for not using the therapy were no need (52%), no access (23%), and insufficient expe rience (19%). Military physicians faced with nonhealing wounds that are infected with MDROs will benefit by having medicinal maggots in their toolbox, to be used alone
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Maggot Airworthiness Testing
or in combination with other proven wound disinfection/ healing regimes. CONCLUSION Wounded warriors with nonhealing wounds that are treated with medicinal maggots will likely have better outcomes, including avoidance of amputations and shorter recovery times. This will translate to reduced treatment costs to the military medical system. The overall results of these tests suggest that medicinal maggots can withstand the rigors of aeromedical evacuation flights in simulated flight environments and rotary wing aircraft, and fixed-wing aircraft. Since direct electrome chanical testing is not possible with this type of medical device (Medical Maggots), future work will focus on obtaining some sort of alternate document, likely termed an “approval for use” aboard MEDEVAC aircraft, through a USAARL aero medical certification memorandum. ACKNOWLEDGMENTS The authors would like to thank the Airworthiness Certification and Evalua tion Branch of USAARL for their test guidance, airworthiness testing, and data reporting. This study was funded by Defense Medical Research and Development Program (DMRDP), DHP 6.7—Medical Products and Capabili ties Enhancements No. 2011053 Airworthiness Testing of Medical Maggots.
REFERENCES 1. Sherman R, Hall M, Thomas S: Medicinal maggots: an ancient remedy for some contemporary afflictions. Ann Rev Entomol 2000; 45(1): 55-81. 2. Davydov L: Maggot therapy in wound management in modem era and a review of published literature. J Pharm Pract 2011; 24: 89-93. 3. Monarch Labs, Inc: Clinical protocols and device details. Available at http://www.monarchlabs.com/; accessed December 10, 2013. 4. Zarchi K. Jemec G: The efficacy of maggot debridement therapy: a review of comparative clinical trials. Int Wound J 2012; 9: 469-77. 5. Cazander G. Pritchard D. Nigam Y, Jung W, Nibbering P: Multiple actions of Lucilia sericata larvae in hard-to-heal wounds: larval secretions contain molecules that accelerate wound healing, reduce chronic inflam mation and inhibit bacterial infection. BioEssays 2013; 35: 1083-92. 6. Mudge E, Price P, Neal W, Harding K: A randomized controlled trial of larval therapy for the debridement of leg ulcers: results of a multi center, randomized, controlled, open, observer blind, parallel group study. Wound Repair Regen 2014; 22(1): 43-51. 7. Barnard D: Skeletal-muscular mechanisms of the larva of Lucilia sericata (Meigen) in relation to feeding habit. Pan-Pac Entomol 1977; 53: 223-9. 8. Jiang K, Sun X, Wang W, et al: Excretions/secretions from bacteriapretreated maggot are more effective against Pseudomonas aeruginosa biofilms. PLoS One 2012; 7: e49815.
596
9.
Cerovsky V, Zd’arek J, Fucfk V, Monincova L, Voburka Z, Bern R: Lucifensin, the long-sought antimicrobial factor of medicinal maggots of the blowfly Lucilia sericata. Cell Mol Life Sci 2010; 67: 455-66. 10. Bexfield A, Bond A, Morgan C, et al: Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis. Brit J Dermatol 2010; 162: 554-62. 11. Wang S, Wang K, XinY Lv D: Maggot excretions/secretions induces human microvascular endothelial cell migration through AKT1. Mol Biol Rep 2010; 37: 2719-25. 12. Zhang Z, Wang S, Diao Y, Zhang J. Lv D: Fatty acid extracts from Lucilia sericata larvae promote murine cutaneous wound healing by angiogenic activity. Lipids Health Dis 2010; 9: 24. 13. Zhang Z, Wang S, Tian X, Zhao Z, Zhang J, Lu D: Maggot homoge nate is associated with neural regeneration and wound healing in the rat. Neural Regen Res 2010; 5: 623-9. 14. Gilead L, Mumcuoglu K, Ingber A: The use of maggot debridement therapy in the treatment of chronic wounds in hospitalized and ambula tory patients. J Wound Care 2012; 21: 78-85. 15. Sherman R. Wyle F: Low-cost, low-maintenance rearing of maggots in hospitals, clinics, and schools. Am J Trap Med Hyg 1996; 54: 38-41. 16. Peck G, Kirkup B: Biocompatibility of antimicrobials to maggot debride ment therapy: medical maggots Lucilia sericata (Diptera: Calliphoridae) exhibit tolerance to clinical maximum doses of antimicrobials. J Med Entomol 2012; 49(5): 1137-43. 17. Heitkamp R, Peck G, Kirkup B: Maggot debridement therapy in the US Army Medical Command: perceptions and prevalence. Mil Med 2012; 177(11): 1411-6. 18. Hospenthal D. Murray C, Andersen R: Guidelines for the prevention of infections associated with combat-related injuries. J Trauma 2011; 71(2): S210-34. 19. James G, Swogger E, Wolcott R: Biofilms in chronic wounds. Wound Repair Regen 2008; 16(1): 37-44. 20. Hospenthal D, Murray C: Preface: guidelines for the prevention of infections associated with combat-related injuries. J Trauma 2011; 71(2): S197—201. 21. Tarone A, Foran D: Components of developmental plasticity in a Michigan population of Lucilia sericata (Diptera: Calliphoridae). J Med Entomol 2006; 43(5): 1023-33. 22. Greenburg B: Flies as forensic indicators. J Med Entomol 1991; 20: 565-77. 23. Anderson G: Minimum and maximum development rates for some forensically important Calliphoridae (Diptera). J Forensic Sci 2000; 45: 824-32. 24. Grassberger M, Reiter C: Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram. Forensic Sci Int 2001; 120: 32-6. 25. Wall R, Pitts K, Smith K: Pre-adult mortality in the blowfly Lucilia sericata. Med Vet Entomol 2001; 15: 328-34. 26. Clark K, Evans L, Wall R: Growth rates of the blowfly, Lucilia sericata, on different body tissues. Forensic Sci Int 2006; 156: 145-9. 27. Tarone M, Foran D: Generalized additive models and Lucilia sericata growth: assessing confidence intervals and error rates in forensic ento mology. J Forensic Sci 2008; 53: 942-8.
MILITARY MEDICINE, Vol. 180, May 2015
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![[Infested sore by native maggots].](/assets/img/hold.png)
