TY - JOUR
AU1 - Dallavecchia, Daniele, L
AU2 - Ricardo,, Elisabete
AU3 - Aguiar, Valéria, M
AU4 - da Silva, Alexandre, S
AU5 - Rodrigues, Acácio, G
AB - Abstract Maggot debridement therapy (MDT) is a simple wound debridement technique. It is a natural treatment licensed by the Food and Drug Administration (FDA) and is increasingly used in the United States and in Europe. This treatment is safe when the larvae originate from laboratory stocks of eggs that have been sterilized. In this study, a simple, inexpensive microbe decontamination technique is described. It yields eggs that are free of chemical residues and are easy to handle, meeting the growing demand for medicinal larvae in hospitals or medical centers. Three treatments (T1, T2, T3) involving 3, 6, and 12 min of exposure to ultraviolet (UV-C) rays, respectively, were compared. Egg sterility was evaluated by culture in thioglycollate broth, incubated at 32°C ± 2.5°C under aerobic conditions for up to 14 d. The UV-C radiation sterilization process obtained satisfactory results after 12 min exposure (treatment 3). Larval viability was 57%, pupal viability was 54%, and 54% of the adults emerged. The sex ratio was 50%, within the expected values. There were no morphological abnormalities associated to the UV-C treatment in the flies. In conclusion sterilization by UV-C rays is indicated to obtain sterile larvae destined for MDT. decontamination, physical sterilization, maggot therapy, UV-C ray Resistance to antibiotics is a serious, growing global problem. Patients with wounds infected by resistant bacteria are at high risk of amputation or even death, particularly if these are wounds associated with co-existing diseases such as diabetes, large burns, and pressure ulcers. Such wound treatments are extremely costly, in particular, due to long hospitalization times (WHO 2017). Surgical wound debridement involves the mechanical removal of devitalized tissue from an infected injury. Sometimes it is the only option available since preparation of the wound bed requires removal of necrotic tissues infected with bacteria and/or biofilm, thereby exposing healthy tissue that will require further treatment and healing (Fowler and Van Rijswijk 1995). Medicinal larvae perform a quite meticulous debridement that is associated with their feeding process. Their buccal apparatus has hooks which, during feeding, tear the necrotic tissue and release digestive juices containing proteolytic enzymes, thereby liquefying the tissue which is then sucked by the larvae. This process is able to eliminate small fragments of tissue (including bone), reaching places that no other treatment does (Nitsche et al. 2010). The enzymes and small peptides released by the fly larvae, known as lucifensins, have strong antimicrobial action and destroy bacteria (Cerovsky and Bém 2014). Maggot debridement therapy (MDT) was approved by the Food and Drug Administration (FDA) in 2004 as an alternative therapy for wound debridement and has since been used by several professionals as a first-line treatment (Teick and Myers 1986, Mumcuoglu et al. 2001, Sherman 2014, Nassori and Hoomand 2017). In 2005, about 30,000 debridement treatments employing maggots were performed in Europe (Geary et al. 2009). Some species of Calliphoridae flies can cause the primary myiasis, that is, lay eggs on healthy skin by feeding on viable tissue, e.g., Cochliomyia genus. On the other hand, in the secondary myiasis the flies are attracted by the smell of the wound necrotic tissue. MDT is the intentional insertion of previously sterile larvae into wounds that are difficult to heal (Guimaraes and Pavavero 1999). Each larva is capable of removing 25 mg of necrotic material in 24 h (Mumcuoglu et al. 2001). The species most used in MDT is Lucilia sericata (Meigen, 1826) (Baer 1931, Sherman 2003, Contreras et al. 2005) but other species belonging to this family were also used: Lucilia caesar (Linnaeus, 1758) (Baer 1931), Lucilia cuprina (Wiedemann, 1830) (Aaron et al. 2009), Lucilia illustris (Meigen, 1826) (Leclercq 1990), Calliphora vicina (Robineau-Desvoidy, 1830) (Teick and Myers 1986) and Phormia regina (Robineau-Desvoidy, 1830) (Reames 1988). C. vicina (Robineau-Desvoidy 1830) is a species of fly from the Holartic region, being very common in Portugal and frequently found in urban areas (Smith 1986, Byrd and Castner 2010). It presents high reproductive capacity of egg mass, develops well under laboratory conditions, is a necrobiontophagous fly, thus possessing essential characteristics for use in MDT. When the physician-surgeon William Baer (1931) started to use MDT to cleanse wounds, some of his patients develop tetanus. He concluded that, to avoid this, the fly eggs needed to be sterilized before larvae hatch. From that time on many distinct methodologies have been used for sterilization of eggs, most of which involve the use of chemical products. The most commonly used procedure involves the use of sodium hypochlorite, often associated with boric acid (Sherman et al. 2014). Alternative sterilization methods have been used in different areas such as the food industry (Ingram and Roberts 1980, Faria 2001), i.e., using UV-C rays. For an efficient system of disinfection/ sterilization of a product by ultraviolet rays, attention should be paid to the intensity of the radiation, the time of contact with the microorganisms and the material to be used as packaging. The ultraviolet rays are divided in different wavelengths—UV-A: 315 and 400 nm, responsible for tanning in the human skin; UV-B: 280 and 315 nm, responsible for the synthesis of vitamin D in the body, burns in the body skin; UV-C: 200 and 280 nm, can cause genetic mutations; UV-V (empty): 100 and 200 nm, strongly absorbed by water and air, and can only be transmitted in a vacuum. The ozone layer filters out 100% of UV-C rays; therefore, artificial sources are used for disinfecting the air, water or surfaces and sterilization of certain materials. UV-C rays are only absorbed by some materials, such as polyethylene, polypropylene, polybutylene, ethylene-vinyl acetate, nylon, and ethylene-vinyl alcohol. The UV-C radiation efficiency is about 99.99% lethal against microorganisms. The process of eggs sterilization must have an efficient cost versus benefit, especially in relation to the time spent in the procedure. UV-C rays do not leave residues, compared to sterilization by chemical agents such as Formalin 5% and sodium hydroxide 1%, Sodium sulphite 2.5% and formaldehyde 2.5%, Orthophthaldehyde, Phenol 3% or sodium hypochlorite 0.5%, Glutaraldehyde 2% (Simmons 1934, Mumcuoglu et al. 1999, Contreras et al. 2005, Sherman et al. 2007, Dallavecchia et al. 2014). UV-C rays act fast, are effective and safe, It is known that a UV lamp with 254 nm of light wave has lethal germicidal activity on bacteria, viruses, fungi, and protozoa, requiring only a few minutes to sterilize materials (ICMSF 1980). The aim of this work was to test the efficiency of UV-C rays on egg surface sterilization of C. vicina, an abundant fly in Portugal, easy to create in the laboratory and with a potential for use in MDT. Materials and Methods Fly Colony A stock colony of C. vicina, maintained at the Laboratory of Diptera Studies, Microbiology Service, Department of Pathology, Faculty of Medicine of the University of Porto, was used. The colony was reared in three different transparent polyethylene cages (40 × 30 × 20) with top and front openings for aeration. The insects were kept, during winter, in a room with ventilation, at room temperature with natural light on a 10:14 (L:D) h cycle. Food and Substrate The larval diet based on carbohydrates (honey diluted in water in a ratio of 1:2) was offered daily to adult insects. For hydration, a container filled with 20 ml of water (water changed daily), was placed in each cage. For females’ ovary maturation, chicken gizzard was offered as a protein source during the first 5 d after the colony was born (Dallavecchia et al. 2015). Egg Collection To stimulate the oviposition of the stock colony (approximately 100 adults of the first generation), 12 g of chicken gizzard diet was inserted in each cage (n = 3) 12 h prior to the beginning of the experiment. One cage was selected, and the egg masses were transferred, with the aid of a sterile forceps to a Petri dish containing 1 ml of sterile distilled water to dissociate the eggs. From this mass, 90 eggs were collected using a sterile wet brush and distributed in four polypropylene boxes with three sections (triplicate), each containing 30 eggs. Three boxes were used for each of the different treatments of exposure to UV-C rays and one box as the control with no exposure to UV-C rays. Sterilization Treatments Three treatments were performed: T1, T2, T3, i.e., 3, 6, and 12 min of exposure, respectively, and a control without exposure to UV-C radiation (25W – 254 nm wavelength - Phillips), at a distance of 50 cm from the source. The distance from the source was calculated according to ICMSF (1980), which determines that a germicidal lamp of 50 W, positioned 1 m away from a target, has an intensity of 100 mW/cm2. According to the norms of the Brazilian Pharmacopoeia (Brasil 2010) for solid products, three eggs were separated from the lot of each treatment and incubated in tubes containing 5 ml of thioglycollate broth (Oxoid Ltd, United Kingdom) to confirm the efficacy of the sterilization. The tubes were incubated at 32°C ± 2.5°C in aerobiosis for up to 14 d. Daily visual check for signs of bacterial growth was repeated after an initial incubation period of 24 h. All experiments were carried out in triplicate and conducted in a Class II Biological Safety Cabin. To verify the viability of the larvae, the eggs sterilized by UV-C radiation (n = 27 per group) and the control were transferred to 30 g of chicken gizzard diet at a ratio of 1 g of diet per larvae to evaluate larval development. The experiments were performed at room temperature. The larvae, after abandoning the diet, were weighed individually on an analytical scale. The specimens were transferred to test tubes (20 × 200 mm) containing 1 g of sterile substrate (hay) for pupation and were labeled according to each treatment. Observations were conducted daily and were always performed at the same hour—12 h—until the emergence of adults. The following biological parameters were determined: body mass; duration of larval development; abnormality and sex ratio (SR). The SR was obtained as follows: SR = F / M + F, where F is the number of females and M the number of males. Statistical Analysis Chi-square and Fisher’s exact tests were used to prove the independence between the qualitative variables. For the analysis of larval weight, nonparametric tests (Kruskal Wallis) were chosen after the normality hypothesis was rejected by the Shapiro-Wilk test. In all tests, a significance level of 0.005 was considered. The tests were run in program R version 3.4.4. Results Microbiological sterility was confirmed by the growth test with thioglycollate broth. Treatments 1 and 2 (exposure to UV-C radiation for 3 and 6 min) were not efficient, as the microbiological sterility test (in triplicate) presented turbidity, indicating growth of microorganisms. Treatment 3 (12 min of exposure) showed to be efficient since there were no turbidity signals, i.e., microbial contamination in the tubes (in triplicate) with the sterilized egg samples (n = 3). Since the control was not treated by radiation it was expected to present microbial contamination in the tubes; therefore it was not tested in the thioglycollate broth. Considering the body mass of larvae both the control, T1 and T2 presented an average of 0.100 g for all larvae, with no variability in these treatments. T3 presented a mean weight of 0.104 g and a standard deviation of 0.0143, due to four larvae that presented higher weights, i.e., 0.125 g, 0.135 g, 0.150 g, and 0.175 g. However, according to the Kruskal-Wallis test (P = 0.01235) the difference of the larvae weight in the different treatments and control was not statistically significant. The larvae abandonment peak, pupariation and adult emergence peak obtained for C. vicina in the three different treatments and control are shown in Fig. 1. There were no differences in the peaks of larvae abandonment, pupariation and in the emergence of adults among the three treatments and control tested. The larval abandonment peak occurred on the sixth day after the transfer of the eggs to the diet. The pupariation occurred on the seventh day and the emergence of the adults on the 20th day. Fig. 1. View largeDownload slide Abandonment rate of Calliphora vicina maggots at pupation and emergence (%) from three treatments and control (27 maggots per replication). Control = without exposure to UV-C rays; T1 = 3 min exposure to UV-C rays; T2 = 6 min exposure to UV-C rays and T3 = 12 min exposure to UV-C rays. Fig. 1. View largeDownload slide Abandonment rate of Calliphora vicina maggots at pupation and emergence (%) from three treatments and control (27 maggots per replication). Control = without exposure to UV-C rays; T1 = 3 min exposure to UV-C rays; T2 = 6 min exposure to UV-C rays and T3 = 12 min exposure to UV-C rays. According to the Chi-square test comparing all treatments and control, there was no statistically significant difference (P = 0.3811) between the amount of eggs used in each treatment and the number of larvae that hatched, as well as the number of eggs larvae born until the development of the adult phase (P = 0.3915) (Table 1). Table 1. The viability of larval, pupal and neo-larvae to adult of Calliphora vicina after exposure to UV-C rays Treatment UV exposure time No. of individuals % Viabilities Eggsa Larvae Adults Larvae Pupae Adults Control w / exposure 81 42 41 52 51 51 T1 3 min 81 46 45 57 55 55 T2 6 min 81 35 35 43 43 43 T3 12 min 81 46 44 57 54 54 Treatment UV exposure time No. of individuals % Viabilities Eggsa Larvae Adults Larvae Pupae Adults Control w / exposure 81 42 41 52 51 51 T1 3 min 81 46 45 57 55 55 T2 6 min 81 35 35 43 43 43 T3 12 min 81 46 44 57 54 54 aEggs: Each replicate contained 27 eggs (n = 3 × 27) after the sterility test. Control = without exposure to UV-C rays; T1 = 3 min exposure to UV-C rays; T2 = 6 min exposure to UV-C rays and T3 = 12 min exposure to UV-C rays. View Large Table 1. The viability of larval, pupal and neo-larvae to adult of Calliphora vicina after exposure to UV-C rays Treatment UV exposure time No. of individuals % Viabilities Eggsa Larvae Adults Larvae Pupae Adults Control w / exposure 81 42 41 52 51 51 T1 3 min 81 46 45 57 55 55 T2 6 min 81 35 35 43 43 43 T3 12 min 81 46 44 57 54 54 Treatment UV exposure time No. of individuals % Viabilities Eggsa Larvae Adults Larvae Pupae Adults Control w / exposure 81 42 41 52 51 51 T1 3 min 81 46 45 57 55 55 T2 6 min 81 35 35 43 43 43 T3 12 min 81 46 44 57 54 54 aEggs: Each replicate contained 27 eggs (n = 3 × 27) after the sterility test. Control = without exposure to UV-C rays; T1 = 3 min exposure to UV-C rays; T2 = 6 min exposure to UV-C rays and T3 = 12 min exposure to UV-C rays. View Large As for the sex of the individuals, the results (Table 2) showed higher births of males in control and T2, whereas in the T1 and T3 similar births on both sexes occurred. However, excluding values non-born (NB) and using the Chi-square test according to the P value obtained (P = 0.5814), the sexual proportions found in the three treatments and control did not differ from the expected that is, the number of adults that emerged in each group was unaffected by UV-C radiation. Table 2. Detailed biological parameters of eggs hatched after UV-C sterilization Treatment No. of individuals per replicates % individuals Normality R1 R2 R3 Total NB M F % SRa Normality Anormality Control 18 12 12 42 1 63 37 37 40 1 T1 24 16 6 46 1 51 49 49 45 0 T2 11 4 20 35 0 57 43 43 34 1 T3 11 23 12 46 2 50 50 50 43 1 Treatment No. of individuals per replicates % individuals Normality R1 R2 R3 Total NB M F % SRa Normality Anormality Control 18 12 12 42 1 63 37 37 40 1 T1 24 16 6 46 1 51 49 49 45 0 T2 11 4 20 35 0 57 43 43 34 1 T3 11 23 12 46 2 50 50 50 43 1 NB = non-born, SR = sex ratio. aThe SR was obtained as follows: SR = F / M + F, where F is the number of females and M the number of males. View Large Table 2. Detailed biological parameters of eggs hatched after UV-C sterilization Treatment No. of individuals per replicates % individuals Normality R1 R2 R3 Total NB M F % SRa Normality Anormality Control 18 12 12 42 1 63 37 37 40 1 T1 24 16 6 46 1 51 49 49 45 0 T2 11 4 20 35 0 57 43 43 34 1 T3 11 23 12 46 2 50 50 50 43 1 Treatment No. of individuals per replicates % individuals Normality R1 R2 R3 Total NB M F % SRa Normality Anormality Control 18 12 12 42 1 63 37 37 40 1 T1 24 16 6 46 1 51 49 49 45 0 T2 11 4 20 35 0 57 43 43 34 1 T3 11 23 12 46 2 50 50 50 43 1 NB = non-born, SR = sex ratio. aThe SR was obtained as follows: SR = F / M + F, where F is the number of females and M the number of males. View Large All adults (100%) submitted to T1 were normal, i.e., they did not present morphological deformities; in T2, T3, and control, they presented a small number of morphological deformities in the individuals (absence of leg, broken wing). Using the Fisher exact test (P = 0.7075), it was observed that there was no statistically significant difference (Fig. 2). Fig. 2. View largeDownload slide Sterilization tests with thioglycollate broth were conducted for all treatments, in triplicate: (A) test tube, pertaining treatment 1, indicating contamination; (B) treatment 2, showing turbidity and indicating contamination; (C, D, and E) tubes in triplicate pertaining treatment 3, with culture medium and egg samples, where it is clear that the eggs were sterilized: there is no turbidity, indicating absence of contamination; (F) Tube containing single culture medium only, for comparison of transparency of the medium. Fig. 2. View largeDownload slide Sterilization tests with thioglycollate broth were conducted for all treatments, in triplicate: (A) test tube, pertaining treatment 1, indicating contamination; (B) treatment 2, showing turbidity and indicating contamination; (C, D, and E) tubes in triplicate pertaining treatment 3, with culture medium and egg samples, where it is clear that the eggs were sterilized: there is no turbidity, indicating absence of contamination; (F) Tube containing single culture medium only, for comparison of transparency of the medium. Discussion The surface of fly egg is extremely contaminated, since females oviposit in putrefying meats. Even the ovaries of laboratory-created fly are stimulated to develop in diets based on raw meat, liver or chicken gizzard (Sherman 2000, Ferraz et al. 2014, Dallavecchia et al. 2014). Germ-Free rooms are used in many countries to rear fly and uncontaminated eggs (Sherman 2000). This technology requires considerable seed capital to produce sterile medicinal larvae. Limsopatham et al. (2017) tested three chemical agents to sterilize eggs of Chrysomya megacephala and L. cuprina with success in the sterilization process. However, during the hatching of the eggs, the sterility test showed contamination probably due to the transfer of the eggs to the culture medium or, due to the fact that the eggs of L. cuprina, (seen under a scanning microscope) present grooves where the detergents cannot reach. In the case of UV-C sterilization, this phenomenon does not occur, as the UV-C rays cross the wall of the eggs uniformly, like a scanner, eliminating all bacteria. Proven fact in the sterility test using thioglycollate broth, incubated for 14 d without presenting bacterial growth. Masri et al. (2005) dissociated the eggs of L. cuprina with chlorhexidine and then sterilized with UV-C rays (wavelength 190 nm) every 1-min intervals, up to 10 min. According to the author, sterilization was effective between 7 and 10 min of exposure. In our work, the dissociation of the eggs was done with ultrapure water and the effective time period for sterilization was longer—12 min of exposure to UV-C rays (wavelength of 254 nm). This difference may be related to the fact that chlorhexidine is a disinfectant, whereas water does not interfere with the microbial load of the eggs. The body mass of the larvae did not vary significantly in the treatments performed (except in T3). This homogeneity in the weight of the larvae is related to the chicken gizzard diet. In a previous study (Dallavecchia et al. 2015), when comparing the two diets (beef and chicken gizzard) the same pattern of homogeneity was observed. The abandonment rate did not vary, larvae, pupae and adult emergence peaked on the same day in all treatments (6, 7 and 20, respectively). The same was observed in the work of Dallavecchia et al. (2014), who sterilized Chrysomia putoria eggs with 2% glutaraldehyde. This constancy in insect development may also be related to the chicken gizzard diet, being advantageous for large-scale laboratory production. In our test, the larval hatching rate was greater than 40%, unlike Masri et al. (2005), which presented a viability of 14–16%. The authors claim that UV-C rays may have destroyed the embryos inside the eggs; however, chlorhexidine used may have been responsible for drying the eggs’ surface, preventing larvae from hatching. According to quadratic analysis and the Fisher (1930) principles, a population only shows stability when the SR is 1:1. In this study, the sexual rates of individuals tested in the three treatments with UV-C radiation and control indicated population stability. According to Fisher, a deviation in this SR is not evolutionarily stable, because gradual increases will occur in future generations, in relation to the proportion of the sex observed in smaller numbers. Most clinicians who use larval therapy acquire the medicinal larvae from large laboratories (van Der Plas et al. 2009); those who make their own productions do not usually keep track of biological parameters of their flies, such as mass, SR or viability. Only T1 produced 100% of normal adult individuals (T2, T3, and control = 99%); however, the difference was not statistically significant. Ferraz et al. (2014), tested different concentrations of ciprofloxacin antibiotic in the development of C. putoria (Diptera: Calliphoridae) and showed a 100% normality for the control, T1 = 96%, T2 = 98% and T3 = 99%, similar to the values found in our study. The emergence of the adult happens through an alternation between dilation and contraction of the liquid of the ptilinum (Smith 1986, Gennard 2007), located in the head, that breaks the puparium allowing the displacement out of the pupa. During this phase, some flies cannot complete this course without loss of material. This fact may explain the mentioned abnormalities (two flies with one wing broken and one without a leg) and not the mutagenic action of the UV-C rays. Sterilization by UV-C rays is widely used in the food industry (Ingram and Roberts 1980, Faria 2001) to disinfect various types of products and their containers. It has also been used to inactivate eggs of parasitic insects (Brownell et al. 2006). It is an efficient method of sterilization; however, the use of specific material that absorbs the UVC rays is required, as well as the control of the ideal time period for not disabling the eggs of the flies. 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TI - Efficacy of UV-C Ray Sterilization of Calliphora vicina (Diptera: Calliphoridae) Eggs for Use in Maggot Debridement Therapy
JF - Journal of Medical Entomology
DO - 10.1093/jme/tjy140
DA - 2019-01-08
UR - https://www.deepdyve.com/lp/oxford-university-press/efficacy-of-uv-c-ray-sterilization-of-calliphora-vicina-diptera-ttVU56ZSSf
SP - 40
VL - 56
IS - 1
DP - DeepDyve
ER -