Pioneering Biotherapy
(Maggot Debridement Therapy)
in the Philippines Using Materials Sustainable
in a Government Hospital
Derrick Chua, MD
Reynaldo joson, md, mha, mhped, ms surg
Ospital ng Maynila
Objectives: We describe a cost-effective and efficient way of rearing maggots and document its use for maggot debridement therapy (MDT). Methodology: Constructing cages, catching flies, harvesting and sterilizing eggs, culturing maggots, selecting patients, obtaining informed consent, applying maggots and monitoring response. Results: Blowflies are readily caught from the environment. A 1x1 foot cage houses 50 flies, which live for a month on water, dry sugar, and occasional meat. Every 3 days, eggs are collected from the underside of meat. Clusters are separated in 0.5% sodium hypochlorite, sterilized in 1% Lysol for 5 minutes, and hatch on chicken egg media. 1-2 day old maggots thrive on necrotic wounds. Designing dressing material is challenging. Patients accept MDT when properly explained. No adverse reactions to MDT were noted (single patient, 3rd maggot application). Conclusion: MDT can quickly be initiated in a government hospital, with minimal monetary cost and no specialized training. Patient acceptability can easily be attained. No adverse reactions were noted.
Introduction
Various methods of debridement are available1 for wounds with necrotic tissue. Depending on the amount of non-viable tissue, the urgency of its removal, the location of the wound, the patient’s ability to tolerate the procedure, the logistics and local expertise, one may elect to use one or a combination of these. Specific debridement techniques include sharp/ surgical (scalpel, nipper, curette), “wet to dry”, hydrotherapy, laser, ultrasound, biotherapy, chemical, enzymatic/ proteolytic, osmotic, and autolytic methods.
Biotherapy is the use of living animals as an aid to medical diagnosis and/or treatment. Biosurgery, or more specifically, maggot debridement therapy (MDT) is the topical application of live maggots (often sterilized) for treating wounds. Other synonyms include larva or larval therapy, larva debridement therapy, maggot therapy, and biodebridement.
It has been observed for centuries that wounds infested with maggots conferred a lower mortality to patients who were otherwise far-removed from medical care2,3. Confederate surgeon J. Zacharias during the American Civil War is the first western physician to use MDT2-4. W. Baer, the founder of modern maggot therapy, popularized MDT after WWI5 in the USA, and developed a sterilization process that was variously improved upon2,6-9. Many papers on the methodology for raising of flies, sterilizing and applying maggots were published since then10-16. MDT was and is used for abscesses17, leg ulcers18-24 and gangrene24-27, pressure ulcers28-30, skin infections31, necrotic skin flaps32, infected stumps33, burns34-36, inoperable malignancies17,37-40, empyema thoracis41, tuberculous abscesses42, mastoiditis43, compound fractures44,45, gas gangrene46, exposed prostheses47, and osteomyelitis2,48,49. With the introduction of sulfonamides and penicillin, and with the improvement of surgical techniques, MDT was relegated into the background50,51 after WWII. Since the 1990s, there has been resurgence in MDT, in part because of increasing antibiotic resistance. In January, 2004, the U.S. Food and Drug Administration (FDA) issued 510(k) #33391, allowing the production and marketing of maggots as a medical device. Some proponents of MDT now advocate it as 1st line treatment of certain types of wounds52,53.
In spite of the increasing number of people in various countries practicing MDT, its acceptability by the majority of physicians52 and patients is slow in coming. Although popular media may have exposed many to the concept of MDT (movie Gladiator, Times Magazine54, National Geographic Ultimate Explorer program55), maggots still traditionally signify the worst in dirtiness56,57.
Only few countries produce medicinal maggots commercially, and purchasing such can be expensive. Techniques for culturing sterile maggots are not standardized. Published protocols are neither easily found nor detailed enough for simple replication. Many of the materials needed are either expensive or not readily available.
It is ironic that this “low technology”58 ancient but effective debridement technique is more accessible in developed countries. The current resurgence includes various ongoing studies aimed at obtaining level-one evidence supporting the widespread use of MDT. The importance of bringing this technique into our country, where specialist medical care is deprived from most of the population is more important59,60 than ever.
Objective
To describe a cost-effective and efficient way of rearing sterile maggots for use in debridement therapy.
To document the application of MDT in a patient.
Methodology
• Materials preparation
– Fly cage frame options:
• G16 wire (<1/4 kg) plus G18 wire (<1/4 kg) for cage suspension
• Wood (10-16 inch long slender sticks)
– Fly cage wall options:
• Fine nylon mesh/ net (1-2 yards)
• Cheesecloth (cannot see inside cage)
• Fine wire screen (cannot launder, fly agitation with nearby motion)
– Garter or rope to fit cage net sleeve edge
– Maggot dressing/ containment material (<0.4mm pore size mesh or >60 threads per inch) options:
• Fine nylon11 net
• Not stockings (stretchable enables maggots to squeeze thru easily)
• Metal (stainless, brass, copper) mesh net 61
– Gauze 8x16 or larger as top wound dressing material (daily and as needed)
– Wide-mouthed liter plastic bottle (like for pasteurized milk)
– Pest-control measures for ants, options:
• Insecticide chalk (deltamethrin 0.2% in Bayer Chalk Roach & Ant Killer from S.C. Johnson & Son, Inc. at Makati) mixed into insecticide gel (imidacloprid 2.15% in Bayer Blattanex Cockroach Gel from Calamba, Laguna) applied on suspension wires
• Water moat around wooden cage
– Plastic cup (~6-8oz) inverted on plastic plate (to fit cage)
– Maggot sterile food options:
• Chicken egg (1, small)
• Agar (unflavored, unsweetened gelatin) with meat
– Sugar (teaspoonfuls), may use brown
– Small (1-2cm) cubes of rotting lean meat (100g), e.g. pork
– Disposable glass vials (1 per 3d)
– Disposable plastic film canisters (1 per 3d), preferably black
– All laboratory glassware can be inexpensive variety made in China, not necessary thicker material made in Germany.
– Autoclave or pressure cooker.
– Glass funnel (60ml, 2 per 3d)
– Sterilizing solution container options:
• Small glass bottle (solution stock)
• Smallest Erlenmeyer flask (2 per 3d, new solution every time)
– Egg cluster separator options:
• Toothpicks (preferably flat type) or wooden applicators
• Glass beads (handful; or marbles smaller than standard; metal bearings unsuitable; used for gentle agitation)
– Glass pipette (2, to measure <1/100th of bottle/ flask capacity)
– Petri dish (1 set per 3d)
– Filter for sterilized eggs, options:
• Gauze 4x4 (2 per 3d, 2-ply or thicker) for Lysol rinsing (also for wound lining, and top dressing)
• Cheesecloth
• Filter paper (1 per 3d, optional, cut circle to fit funnel, folded to corrugate, for hypochlorite pre-treatment)
– Rinsing solution for sterilized eggs, options:
• Commercial NSS/ LR (place a tuberculin syringe shaft thru the IV bottle and use the plunger as a cork)
• Sterilized normal saline solution
• Distilled water
– Sterile gloves (can autoclave a box of latex gloves)
– Sterilizing solution options:
• Lysol® disinfectant concentrate (o-phenlyphenol, o-benzyl-p-chlorophenol & xylenols from Reckitt Benckiser, Pasig) 1% for 5-6min
• Various Lysol ® preparations may have differing active ingredients
• Lysol® 3%11 for 5-6min
• Mercuric bichloride 1/2000, with 25% alcohol & HCl for 15-30min2
• Formalin 4-5% for 3 min
• Formalin 1%62
• Sodium hypochlorite 0.5%62
– Pretreatment (removes albuminous coating) options:
• Bleach (small bottle of 5.25% sodium hypochlorite, Purex®)
• None
• Dakin’s solution
– Wound edge skin protector options:
• Zinc oxide cream (e.g. Desitin ®, Little Ones ® Diaper Rash Ointment)
• Petrolatum
• Hydrocolloid dressing (e.g. Duoderm ®)
– Plaster tape (Leukoplast ® tape from BSN Medical Ltd, South Africa, 1 inch width)
– Pupation jar (2, 1 gallon containers with cover)
– Pupation can (tin can with mesh/ net cover)
– Sawdust (1 pint; from sawmill, not insecticide-treated lumber mill; sun dried)
– Fine-meshed sieve (large enough to easily sift sawdust between gallon containers)
– Rubber-band (1 per vial maggots and per pupation cycle)
– Trash bag (to fit pupation jar with air reservoir space)
• Locations/ facilities
– Refrigerator (maggots & egg media far from freezer compartment, meats in freezer) if materials not used immediately.
– Isolated insectiary area (not necessarily enclosed, but isolable from ants, cats, rats, birds, lizards, other flies, curious people) whose smell15 would not disturb other people, with relatively high ambient temperature (30ºC15).
– Workbench for sterilization (laminar flow hood not necessary)
– Incubators made of glass or cabinets and rooms, containing tiers of cages, uses exhaust fans and humidified air, may be needed for commercial high volume production
• Fly cage & food (Figure 1)
– 1x1 foot square wire frames, tie together 4 of these to form a cube frame.
– Use fine nylon mesh (prevents Drosophila entrance). Fashion sleeve encompassing cube frame. If sleeve protruding beyond frame is not long enough for easy knotting, garter or string should be weaved into edge of sleeve.
– Water supply- fill plastic cup with tap water, invert onto gauze pad (instead of paper) on plastic plate. This provides continuous water supply without drowning flies. With such, insectiary humidity is not important11 for fly survival. Replace cup-plate system as needed just prior to it drying up. Open the mouth of the nylon sleeve carefully just enough to fit your gloved hand as it brings items into or out of the cage. Expect some escapes during the first few tries.
– Food supply- place dry sugar in film canister inside cage
– Protein supply- place a piece of meat (see egg harvesting) every 2-3days
– (Water-protein supply- optional suggestion of milk instead of water in cup inverted onto gauze on plate)
– Nylon sleeve can be removed from wire frame and laundered as needed.
• Fly catching
– Using bait is slow and flies caught are of differing species
– Bring a wide-mouthed liter plastic bottle to a dump site around noontime. Catch green metallic scavenger flies by covering them with the bottle, capping the bottle once fly flies in, and opening the bottle into a nylon cage.
– One square foot cage can sustain 200-250 flies11. This concentration of insects may require a ventilation system like an exhaust fan beside. My insectiary contains only one-tenth the population and did not need artificial ventilation systems.
– Once cage is filled, suspend cage onto wires on four corners. Insecticide gel/chalk mixture is applied onto wires to prevent ant infestation.
– The author’s insectiary is located beneath a window sill outside an air-conditioned room on the second floor roof. This provided some shelter from rain, direct sunlight, and scavenger/predatory animals.
• Egg harvesting (Figure 2)
– Either purchase new meat from a supermarket (allows small portions to be bought) or ask for meat scraps (unfresh) at wet market while buying other items
– Studies show that liver and egg are inferior to lean meat12. Pork is cheaper than beef and easier to purchase in small cubes than chicken.
– Leave meat (inside plastic bag to reduce smell and prevent scavenging) at room temperature for a day or so.
– Place each cube inside a film canister (which photo shops throw away), wrap in multiple layers of plastic (prevents odor from escaping), then store inside freezer (prevents odors).
– May defrost each canister prior to use.
– Schedule placing meat every 2 to 3 days (also as part of protein source for adult flies). Place in the morning and remove in the afternoon, or leave and inspect for eggs every evening.
– Place canister of meat inside cage using gloved hand snuggly fitting nylon sleeve of cage to prevent escape of flies.
– Remove canister similarly, shaking and tapping canister to remove flies still inside.
– Separate clusters of white eggs from meat onto Petri dish
– Meat may be washed in tap water, refrozen, and reused.
– When trying to achieve aseptic insectiary (e.g. 7th generation flies in captivity without sterilization of eggs), can use char-broiled meat cube in Petri dish of agar/ gelatin
• Egg sterilization (Figure 3)
– Non-sterile larvae carry risks63-65 of botulism, tetanus, gas gangrene66, and sepsis67 from bacteria carried in their gut and on the skin. Hence sterilization is recommended before the eggs hatch. Eggs also have better tolerance to the chemicals used than maggots themselves.
– Glassware and other materials for sterilization may be autoclaved or pressure-cooked (15min).
– Prepare stocks of 1/100 bleach (5% into 0.05% sodium hypochlorite) and Lysol (as per manufacturer’s recommendation. 3% Lysol recommended in literature11 seemed toxic to eggs in author’s experience) using sterile NSS or LRS inside sterilized, capped glass bottles. Or fresh small batches could be prepared just prior to use inside small flasks.
– Separate egg clusters in small amount of bleach solution using toothpick on Petri dish or agitation with beads in flask. Eggs can survive even quite robust handling61 but macerates with significant direct pressure by wooden applicator against gauze surface).
– Wash bleach off. Decanting or pipetting solution off is impractical. Filter thru filter paper (drains slow) or gauze (eggs enter onto holes, causing more difficult retrieval) on funnel.
– Return eggs into Lysol solution. Stopwatch time for 5 minutes. Rinse off Lysol within a minute after 5 minutes soak. Gauze on funnel enables quick repetitive rinsing.
• Maggot culturing (Figure 4)
– Create media by placing small amount of chicken egg (50%)-water (50%)-sugar (pinch of) mixture into disposable glass vials. Vials not stoppered, but caps placed under vial, both wrapped with paper and placed upright for autoclaving. Vials capped after autoclaving, observing aseptic technique, and refrigerated until ready for use.
– Place sterilized eggs into prepared vial of media. Incubate at room temperature with loose cap. Eggs will hatch into similar-sized maggots within 12 hrs.
• Maggot storage
– Let maggots feed on media for 1-2 days (higher survival in wound & better storage cold tolerance11,68), change occlusive vial covering with net to let fresh air in within 12hrs of hatching.
– Store capped maggot vial inside refrigerator (bottom of a fridge not near the freezer, 8-10ºC) and use within (optimal 60hrs68) 14-18 days, allowing return to room temperature before use.