A process for preparation of maggot egg dressing for the treatment of diabetic foot ulcer

Abstract

The invention discloses a process for preparation of maggot eggs as dressing for the treatment of diabetic foot ulcer. Maggot eggs are selected from Chrysomya megacephala or Chrysomya rufifacies. The process comprises the steps of collection of maggot eggs, pulverization, sterilization and finally the preparation of Blueggs. The prepared Blueggs is packed into a sterile packaging using copper tube which is further packed into a sachet, followed by further packing into a sterile cuvette of 2.5 cm as Blucop and in turn packed as Bluflask, which is useful as dressing for treating the wounds in patients with diabetic foot ulcer. Maggot egg therapy facilitates debridement, disinfection and healing thus making safe, effective and affordable therapeutic option in patients without riskin amputation of the limb.

Description

TITLE OF THE INVENTIONA process for preparation of Maggot egg dressing for the treatment of diabetic foot ulcerDESCRIPTION OF THE INVENTIONTechnical field of the invention

[0001] The present invention relates to a process for preparation of Maggot egg dressing for the treatment of diabetic foot ulcer. More particularly, the invention discloses a process for preparation of sterile maggot eggs as dressing for the treatment of diabetic foot ulcer in patients who were otherwise advised for amputation of limb due to non-healing diabetic foot ulcers.Background of the invention

[0002] Maggot therapy refers to a type of biotherapy involving the introduction of live or disinfected maggots (fly larvae) into non-healing skin and soft-tissue wounds of a human or other animal for the purpose of cleaning the necrotic or dead tissue within a wound for disinfection and debridement.

[0003] It is estimated that 2% of the population in developing countries suffer from a chronic wound, making it a hidden phenomenon that is increasing as populations age. The ease of access to maggot therapy has made it increasingly attractive for implementation. The practice of maggot therapy is based on observations that wounds naturally infested with maggots often are free of infection and debris.

[0004] A chronic wound is one that has failed to proceed through an orderly and timely reparative process to produce anatomic and functional integrity over aperiod of three months. The typical wound healing process is influenced by various extrinsic and intrinsic factors, which is systemic or local. These factors result in delayed healing and the development of a chronic wound.

[0005] Evidence exists that larvae have been used for the last thousand years by various ancient cultures, such as the aboriginal Ngemba tribe of New South Wales, the Hill people of Northern Myanma and the Mayan healers of Central America. Anthropological research suggests that the Mayans used-soaked dressings in the blood of cattle and exposed them to the sun before applying them to certain lesions, expecting the dressings to squirm with maggots. The French surgeon, Ambroise Pare (1510-1590), was the first doctor to note the beneficial effect of Bottle Green House fly larvae for wounds. The American Surgeon Forney Zacharias (1837-1901) officially documented the use of live maggot larvae during the American Civil War (1861-1865). In 1929, William Baer reports a case series of osteomyelitis treated with larval therapy based on his observations during World War I. Ronald Sherman and Edward Pechter rediscover and promote the use of larval therapy in the USA in the 1990s.

[0006] Maggot therapy is also known as maggot debridement therapy or larval therapy, which continues to attract interest in the treatment of chronic wounds.

[0007] Maggot therapy involves the deliberate utilization of live, medical-grade Bottle Green House fly larvae for the process of wound healing, disinfection, and the debridement of wounds. Maggot larvae are applied either in loose or confined form as dressings, which are clinician or surgeon dependent.

[0008] Maggots are effective against micro-organisms including bacteria, fungi etc. There are three midgut lysozymes of Green Bottle House Fly that have been confirmed to show antibacterial effects in maggot debridement therapy. It is noted that the majority of Gram-positive bacteria are destroyed in vivo within the particular section of the Green Bottle House Fly’s midgut where lysozymes are produced. During the passage through the intestine of the maggots, the ability ofbacteria to survive drastically decreased, implying the antibacterial action of the three midgut lysozymes.

[0009] Diabetic Foot Ulcers are the most common foot injuries leading to lower extremity amputation and also are much feared complications of diabetes and research estimates that the lifetime incidence of foot ulcers within the diabetic community is around 15% and may become as high as 25%.

[0010] Severe infections in the foot may lead to leg amputations. It is estimated that diabetes accounts for more than 50% of amputations, of which 85% of lower limb amputations in diabetes patients are preceded by foot ulcers. Approximately 34% of people with diabetes develop a foot ulcer in their lifetime. 18.6 million people worldwide develop foot ulcers each year. 15-20% of moderately to severely infected foot ulcers eventually lead to amputation. The mortality rate in amputated patients is above 70% due to depression and comorbidities.

[0011] While technological advances have led to significant improvements in medical care, including wound care, non-healing wounds still remain a significant problem.1Several studies reported that Maggot therapy was more effective in debriding non-healing foot and leg ulcers, however, it is not so much popularised India, though it is proved to be effective in non-healing ulcers.

[0012] Over the past few years, there has been a resurgence in the use of maggot therapy, even though its optimal role has not been clearly defined. Large prospective clinical trials have not been conducted for maggot therapy, and there are no commercial backers to support such studies.

[0013] There are many conventional and advanced methods for managing diabetic foot ulcers and preventing amputation, including antibiotic therapy, necrotic tissue debridement, wound dressing, Negative Pressure Wound Therapy (NPWT), Hyperbaric Oxygen Therapy (HBOT), stem cell-based therapy, growth factor therapy. The single use of different methods may not be considerably efficient and cause pain and mechanical damage to healthy underlying tissues.

[0014] The patent application “W02010011611A2” entitled “Antimicrobially- primed medicinal maggot therapy ' discloses a elates in part to medicinal maggot therapy methods and compositions for treatment of a variety of indications, including, without limitation, wound care, infection and septic wound care.

[0015] The patent application “US20070022960A1” entitled “Method for the production of an agent from larvae in flies for treating wounds and agent produced according to said method" discloses treatment of wounds by means of larvae from flies is improved by virtue of the fact that the larvae are contaminated with bacteriophages.

[0016] The patent application “US20030124199A1” entitled “Use of fly larval extracts for wound treatment” discloses topical application of fly larval extracts obtainable from fly larvae which are killed and extracted with cooling in aqueous medium or in solvents and are freed of undissolved constituents. The fly larval extracts of various species are suitable for the treatment of superficial or deep chronic and acute wounds of any etiology. The fly larval extracts with a woundhealing effect are obtainable for example from fly larvae of the genera Sarcophaga or Lucilia.

[0017] The patent application “US2018265892A1” entitled “Genetically engineered larvae for wound healing ” discloses methods and compositions for maggot debridement therapy. The invention relates to recombinant nucleic acid constructs, transgenic maggots comprising the recombinant nucleic acids, methods for making the maggots, and methods for the use of maggots, including debridement and promoting wound healing.

[0018] Although, there are various studies or applications related to maggot therapy for diabetic foot ulcer, the existing methods or treatment approaches utilize the live larva for treatment, which has the technical problem of handling the live larva and the crawling of live larva consume long time for fortification, Hence, there is a requirement for a method for production of maggot egg dressing with maggot eggs for the treatment of diabetic foot ulcer.Summary of the invention

[0019] The present invention specifically discloses a process for preparation of sterile maggot eggs dressing for the treatment of diabetic foot ulcer in patients who were otherwise advised for amputation of limb due to non-healing diabetic foot ulcers.

[0020] The process of the present invention comprises multiple steps such as collection of maggot eggs, pulverization, sterilization and finally the preparation of ‘Blueggs’. The prepared ‘Blueggs’ is packed into a sterile packaging using copper tube as ‘Blucop’, which is further packed with methylene blue ice cubes in 500 ml thermal flask as ‘Bluflask’, which is useful as dressing for treating the wounds in patients with diabetic foot ulcer.

[0021] The process of the present invention comprises the steps of collection of maggot eggs from a rearing cage. A putrefied minced meat is placed in the rearing cage, which results in collection of maggot eggs. The maggot eggs are gently removed from the meat cup from the rearing cage. Maggot Egg Clumps (MEC) are carefully separated using a spatula. MEC is transferred into a sterile petri dish using stainless- steel forceps. MEC is subjected to pulverization to separate the clumps into mass. A stainless-steel bin of 3-liter capacity is filled with 2 liters of soapnut (Sa tWnsolution and approximately 450 mg to 600 mg of MEC is placed between two sponges of approximate size of 6’-7’ in diameter. MEC is gently massaged using a surgical glove and pulverized in between the sponge and a thin skin plaster. The mixture is filtered using a Polyurethane Mesh Milk Filter (PMMF) and the eggs collected after filtration are considered for the next step. The eggs collected from this step i.e. after pulverization is termed Maggot Egg Mass (MEM).

[0022] MEM thus obtained is subjected to sterilization and the process is carried out in a sterile room. A 7” plastic blender blade with a blunt edge is coated with the impression material and dried for approximately 6 hours to form a soft coating. MEM is transferred into a 8” plastic blender bin containing 100 ml of 5%povidone iodine solution using a stainless-steel spatula and blended at a speed of 100 RPM for 3 to 5 minutes. After blending, MEM is washed with 500 ml of sterile water. The blending is repeated for one more time and MEM is filtered using PMMF filter under sterile tap water using a sterile brush. MEM obtained is used to prepare Blueggs using 100 ml of 0.1% methylene blue solution. MEM is transferred into a 8” plastic blender bin containing 100 ml of 0.1% methylene iodine solution using a stainless-steel spatula and blended at a speed of 100 RPM for 3 to 5 minutes. The blended mixture is further subjected to filtration using the PMMF filter of No 100 and a pore size of 150 microns, thus collecting the ‘Blueggs’.

[0023] The collected ‘Blueggs’ is packed in a completely sterile mode. Approximately 90 mg to 120 mg of ‘Blueggs’ are fdled in a sterile copper tube, which is further packed into a sachet, followed by further packing into a sterile cuvette of 2.5 cm and is termed as ‘Blucop’. ‘Blucop’ thus packed in said manner exhibits a shelf life of 8 hours and is used to prepare “Bluflask” using methylene blue ice cubes in a 500 ml thermal flask and ‘Blucop’ prepared in earlier step is placed in a zip lock plastic cover, which consists of 3 ml of 5% povidone iodine solution.

[0024] “Bluflask” exhibits a shelf life of 18 hours and is ready to be used as dressing in the treatment of patients with diabetic foot ulcer.Brief description of the drawings

[0025] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings.

[0026] Figure 1 illustrates a flowchart for a process of preparation of the maggot egg dressing for the treatment of patient with diabetic foot ulcer according to an embodiment of the invention.

[0027] Detailed description of the invention

[0028] In order to make the matter of the invention clear and concise, the following definitions are provided for specific terms used in the following description.

[0029] The term “Maggot” refers to a soft-bodied legless larva of a fly or other insect, found in decaying matter.

[0030] The term “Diabetic Foot Ulcer” refers to an open sore or wound that occurs in approximately 15% of patients with diabetes and is commonly located on the bottom of the foot.

[0031] The term “Blueggs” refers to the Maggot eggs treated with methylene blue.

[0032] The term “Blucop” refers to the Blueggs packed in a sterile copper tube which is further packed into a sachet, followed by packing into a 2.5 cm sterile cuvette.

[0033] The term “Bluflask” refers to a sterile packed “Blucop” in a thermal flask containing methylene blue ice cubes.

[0034] The term “Dressing” refers to a piece of material applied to a wound to promote healing and protect the wound from further harm.

[0035] The present invention specifically discloses a process for preparation of sterile maggot egg dressing for the treatment of diabetic foot ulcer in patients who were otherwise advised for amputation of limb due to non-healing diabetic ulcers.

[0036] Maggot therapy is achieved using surgical grade maggot eggs of particular Green Bottle House Fly (Chrysomya) species, which is cultured by integrating quality control measure throughout the process. The maggot eggs are selected from Chrysomya megacephala or Chrysomya rufifacies.

[0037] The process of the present invention is unique and results in the processing of maggot egg dressing, which is useful for treatment of non-healing wounds in patients with diabetes.

[0038] The process of the present invention comprises series of steps such as collection of maggot eggs, pulverization, sterilization and finally the preparation of surgical grade ‘Blueggs’. The prepared sterile ‘Blueggs’ are packed as ‘Blucop’ and subsequently as ‘Bluflask’, which is used as dressing for treating the wounds in patients with diabetic foot ulcer.

[0039] The maggot egg dressing prepared by the process of the present invention is effective and affordable therapeutic option for patients who were otherwise advised for amputation of limb due to non-healing diabetic ulcers.

[0040] Figure 1 illustrates a flowchart for a process of preparation of the maggot eggs dressing for the treatment of patient with diabetic foot ulcer according to an embodiment of the invention. The process (100) comprises a step (101) of collection of maggot eggs from a rearing cage of Green Bottle House Fly (Chrysomya) species. The rearing cage is used to collect the maggot eggs in which a putrefied minced meat is placed, which aids in collection of maggot eggs. The Maggot Egg Clumps (MEC) are gently removed from the meat cup from the rearing cage. It is required to ensure that the flies should not escape from the rearing cage. Maggot Egg Clumps are carefully separated the using a spatula. The spatula is made up of either plastic or wood and coated with soft impression material to make sure that eggs are not damaged during transfer. The egg mass is transferred into a sterile petri dish using a stainless-steel forceps.

[0041] MEC thus collected is subjected to pulverization in step (102). At step (102), Maggot Egg Clumps are subjected to pulverization to separate the clumps into mass. A stainless-steel bin of 3 litre capacity is filled with 2 litres of soapnut solution ( / oharas) and 450 mg to 600 mg of MEC is placed between two sponges of size in a range between 6” to 7” MEC is gentle massaged using asurgical glove and pulverized in between the sponge and a thin skin plaster. This activity results in separation of the eggs. The separated eggs are left in the stainless-steel bin. Further, the entire mixture is filtered using a Polyurethane Mesh Milk Filter (PMMF) and the eggs collected after filtration is considered for the next step. The PMMF filter used in the present invention is of No. 100 and with a pore size of 150 microns. The eggs collected from this step i.e. after pulverization is termed as Maggot Egg Mass (MEM).

[0042] At step (103), MEM obtained in the previous step is subjected to sterilization and the process is carried out in a sterile room. A 7” plastic blender blade with a blunt edge is coated with the impression material and dried for approximately 6 hours to form a soft coating. The coating with the impression material prevents any damage of the eggs during sterilization or blending. MEM is transferred into a 8” plastic blender bin consisting of 100 ml 5% to 10% povidone iodine solution using a stainless-steel spatula and blended at a speed of 100 RPM for 3 to 5 minutes. After blending, MEM is washed with 500 ml of sterile water. The blending is repeated for one more time and MEM is filtered using PMMF filter of No. 100 and pore size of 150 microns under sterile tap water using a sterile brush.

[0043] The MEM obtained at step (103) is used to prepare ‘Blueggs’ in step (104). A 7” plastic blender blade with a blunt edge is coated with the impression material and dried for 6 hours to form a soft coating. MEM is transferred to 8” blender bin using a sterile stainless-steel spatula and MEM is blended at a speed of 100 rpm using 100 ml of 0.1% methylene blue solution. Further, MEM is filtered using PMMF filter of No. 100 and pore size of 150 microns and MEM, which is blue in colour, imparted by methylene blue is ready for packaging and hence termed as ‘Blueggs’.

[0044] The coating with methylene blue makes the maggot eggs resistant to microbial contamination thus ensuring effective sterilization. This step of coating with methylene blue prevents bacterial growth during transportation.

[0045] ‘Blueggs’ thus prepared by said process is packed in a unique manner to retain the stability of ‘Blueggs’. The packing of ‘Blueggs’ is completed in a sterile mode. 90 mg to 120 mg of ‘Blueggs’ are filled in a sterile copper tube using a sterile stainless- steel spatula. The copper tube used is of 1 mm thickness and 1 cm length with an outer diameter of 6 mm and inner diameter of 4 mm. The copper tube containing ‘Blueggs’ is packed into a sachet of 15 mm X 8 mm and is made up of PMMF No. 100 of 150 microns and sealed. The sealed sachet is packed in a sterile cuvette of 2.5 cm and the packaging is termed as “Blucop”.

[0046] “Blucop” is placed in a zip lock plastic sachet of 3 cm X 2 cm, containing 3 ml of 5% povidone iodine solution. The zip lock plastic sachet containing “Blucop” is further placed in 500 ml thermal flask, containing methylene blue ice cubes and is termed as “Bluflask”.

[0047] “Bluflask” exhibits a shelf life of 18 hours at a temperature of 5°C to 10°C and is ready to be used as dressing in the treatment of patients with diabetic foot ulcer.

[0048] The morphology of each Maggot egg prepared by the process of the present invention exhibits a width of 300-350 microns, length of 1400-1500 microns, weight of 0.10 mg to 0.13 mg thus facilitating a unique and effective treatment option in patients with diabetic foot ulcer.

[0049] The soapnut used in the present invention is purchased from the vendor and the vendor details are as follows:Vendor Name: Vasavi Grandhige AngadiAddress: Vasavi Road , Doddapete,Kolar -563101, KarnatakaIndia

[0050] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.

[0051] Example 1: Treatment of Maggot therapy in patients with diabetic foot ulcer.

[0052] The effect of maggot therapy using the dressing of the present invention is analysed in patients with diabetic foot ulcer, who were advised knee amputation. Open wounds in large body cavities and wounds in close proximity to large blood vessels were excluded from the treatment. In addition, the treatment group also excluded the patients allergic to eggs, soybeans or fly larvae.

[0053] The patients, who were treated with maggot therapy had diabetes in addition to multiple other co-morbidities, having Haemoglobin Ale > 10 (range 6.1 to 17.3, mean of 10.2). These diabetic patients had the most challenging wounds for debridement due to compromised vascularity, not amenable to sharp debridement and were advised amputation.

[0054] The ulcer was prepared initially by normal saline wash. The surroundings of the ulcer were given soap wash followed by hydrogen peroxide and Iodine lotion wash. The surrounding area was dried for five to ten minutes. The remaining web spaces and the surrounding areas were carefully dressed or covered with sterile roller gauge. The edges of the roller gauge were smeared with tincture iodine, which is one cm away from the wound edge. The web spaces were also smeared with the tincture iodine. Once, tincture iodine is dried, 15-20 maggots per cm square of wound were placed or inserted with Sterile five mmbrush. Finally, the wound was covered by air permeable cloth. The edges of the cloth were plastered, in such a way that, maggots should not escape from the wound area. The patients and family members were advised not to disturb the dressing and informed that they would see increased drainage and even bleeding.

[0055] After 48 hours of dressing, the wound cloth was removed and the maggots were then extracted from the wound with suction apparatus. If the wound was not completely debrided, the maggot eggs were reinserted or and the wound was covered again with permeable cloth. This cycle needed to be repeated two to four times, until full debridement was attained. All the patients were allowed to use their routine medication.

[0056] The results indicated that the patients exhibited complete debridement with the formation of granulation tissue within the wounds.

[0057] The maggot therapy also resulted in reduction of inflammatory reaction, which disappeared within 24-48 hours and developed erythema in the normal skin surrounding the wound.

[0058] The maggot therapy of the present invention is effective strategy for the treatment of complex, diabetic wounds and was also effective against dry and gangrenous wounds. The Blueggs thus prepared by the present invention is surgeon friendly in contrast to the existing live larvae maggot therapy .

[0059] The process of the preparation of maggot egg dressing according to the present invention facilitates debridement, disinfection and healing of the wound. The Blueggs of the present invention is safe, effective, and affordable option to the patients without risking for amputation of the limb.

Claims

Claims:We Claim:

1. A process for preparation of Maggot egg dressing for the treatment of diabetic foot ulcer, the process (100) comprising the steps of: a. collection of maggot eggs from a rearing cage of Green Bottle House Fly (Chrysomya) species (101) comprising the steps of; i. gently removing the maggot eggs from putrefied minced meat placed inside a rearing cage; ii. separating the Maggot Egg Clumps (MEC) using a plastic or wooden spatula coated with a soft impression material to avoid any damage to maggot eggs; and iii. transferring the separated egg mass into a sterile petri dish using a stainless-steel forceps; b. subjecting the separated MEC to pulverization (102) comprising the steps of; i. placing 450 mg to 600 mg of MEC in between two sponges of size in a range between 6” to 7”, that are plastered with a transparent film dressing and are placed in a stainless-steel bin containing two liters of soapnut solutionii. massaging MEC using a surgical glove and pulverizing in between the sponges r to separate the maggot egg clumps thus leaving the maggot eggs in the stainless- steel bin; iii. filtering the separated eggs using a Polyurethane Mesh Milk Filter (PMMF) of No. 100 and with a pore size of 150 microns;iv. collecting the filtered maggot eggs; wherein maggot eggs collected after pulverization is termed as Maggot Egg Mass (MEM); c. subjecting MEM to sterilization (103) comprising the steps of: i. transferring MEM into an 8” plastic blender bin consisting of 5% to 10% of povidone iodine solution using a stainless-steel spatula wherein a blade of the plastic blender is coated with the impression material; ii. subjecting the transferred MEM to blending at a speed of 100 RPM for a period of 3 to 5 minutes; iii. washing the blended MEM using 500 ml of sterile water and repeating the blending for at least two times; iv. filtering the washed MEM using PMMF filter of No. 100 and pore size of 150 microns under sterile tap water using a sterile brush; d. preparing Blueggs from sterilized MEM (104) comprising the steps of: i. transferring the MEM to 8” plastic blender using a sterile stainless-steel spatula wherein the blade of the plastic bender is coated with the impression material to form a soft coating; ii. blending the transferred MEM at a speed of 100 rpm using 100 ml of 0.1 % methylene blue solution; and iii. filtering the blended MEM using PMMF filter of No. 100 and pore size of 150 microns; wherein MEM appears blue in colour imparted by methylene blue and is termed as ‘Blueggs’ .

2. The process as claimed in Claim 1, wherein maggot eggs are selected from Chrysomya megacephala or Chrysomya rufifacies.

3. The process as claimed in Claim 1, wherein the pulverization separates MEC into mass of maggot eggs.

4. The process as claimed in Claim 1, wherein ‘Blueggs’ are sterile packed by filling 90-120 mg of ‘Blueggs’ in a sterile copper tube followed by packing in a sterile sachet and further packed in a sterile cuvette and termed as ‘Blucop’.

5. The process as claimed in Claim 1, wherein the sachet is made up of PMMF No. 100 of 150 microns.

6. The process as claimed in Claim 1, wherein ‘Blucop’ is packed as ‘Bluflask’ by using methylene blue ice cubes in a 500 ml thermal flask.

7. The process as claimed in Claim 1, wherein “Bluflask” exhibits a shelf life of 18 hours at a temperature of 5°C to 10°C and is ready to be used as dressing in the treatment of patient with diabetic foot ulcer.

8. The process as claimed in Claim 1, wherein, each maggot egg exhibits a width of 300-350 microns, length of 1400-1500 microns, weight of 0.10 mg to 0.13 mg.

9. The process as claimed in Claim 1, wherein maggot therapy exhibited debridement with the formation of granulation tissue within the wound.

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