Wilson et. al. Biotherapy Journal Review Spring 2016
Maggot Debridement Therapy Reviews
Wilson MR, Nigam Y, Jung W, Knight J, and Pritchard DI. The impacts of larval density and protease inhibition on feeding in medicinal larvae of the greenbottle fly Lucilia sericata. Med.Vet.Entomol. 2016; 30(1):1-7.
Larval therapy, the therapeutic use of blowfly larvae to treat chronic wounds, is primarily used in debridement. There are, however, gaps in current knowledge of the optimal clinical application of the therapy and mechanisms of action in the debridement process. Using an artificial assay, two studies were undertaken to investigate these aspects of larval debridement by Lucilia sericata Meigen (Diptera: Calliphoridae); the first studied the effects of the density of larvae on tissue digestion and larval mass, and the second considered the effects on the same parameters of incorporating protease inhibitors into the feeding substrate. The total mass of tissue digested increased with larval density until saturation was observed at 5.0-7.5 larvae/cm(2) . This range was considered optimal as lower doses resulted in the removal of less tissue and higher doses offered no additional tissue removal and appeared to exacerbate competition for feeding. In the second study, increased protease inhibitor concentration led to significant decreases in tissue digestion and larval mass, suggesting that serine proteases, particularly trypsin, may play major roles in larval digestion. Such information is important in elucidating the main constituents that make up larval digestive products and may be significant in the development of new therapies.
Introduction & Background to Study
The authors revisit the background to larval therapy which is increasingly used to remove necrotic and devitalized tissue from wounds, to prevent and fight infection and to stimulate wound healing. They briefly review recent biochemical and molecular approaches to the characterization of proteases and their corresponding gene sequences, and discuss the potential for these advances to translate into the development of new wound care therapies. However, Wilson et al. appear to be primarily motivated to shed light on the relationship between larval density and debridement activity, and on the role of proteases in larval debridement. They argue that determining optimal larval density and a better understanding of larval activity will lead to greater efficacy and cost-effectiveness in larval therapy
Materials & Methods
Larval activity assays were conducted using bagged medical maggots that were placed on pork substrate. Both the change in larval and substrate mass was recorded for the 48 hour feeding period at 32 oC, because larval mass gain is influenced by competition and the ability to digest/ingest substrate (i.e. debride the wound), and substrate mass loss is a direct measure of debridement activity.
In the density experiment, treatments included densities of between 0 and 15 larvae per cm2 applied via BioBag to 20g of pork. In the protease activity experiment, the authors added Kunitz soybean trypsin inhibitor and a high-activity soybean trypsin inhibitor to the pork substrate at concentrations of 1 to 20 mg per gram of pork. The authors used an optimal larval density of 5 larvae per cm2 for this experiment.
As for the larval density and debridement efficiency experiment, the authors clearly demonstrated that tissue loss increased for densities from 0 and 7.5 larvae per cm2 but that there was no further increase in tissue loss beyond 7.5 larvae per cm2. Larval mass was unaffected by increasing larval density until 5 larvae per cm2 beyond which larval mass decreased. Increasing larval densities did not lead to increased mortality of larvae.
In the protease inhibitor study, the addition of inhibitor had an inversely proportional effect on tissue loss and larval mass gain, meaning that increasing concentrations of inhibitor led to less tissue being consumed and less larval mass gained over the experimental period. There was no difference between the two types of soybean trypsin inhibitor nor did their concentration affect mortality rates. Interestingly, inhibitors also had an affect when there were no larvae present.
The authors conclude that the ideal application density for bagged L. sericata is between 5 and 7.5 larvae per cm2 of covered wound area, based on the maximal debridement activity at 7.5 larvae per cm2 and optimal weight gain for larvae up to a density of 5 larvae per cm2. The authors explain this with a steady increase in resource competition within the confined environment of the BioBag which eventually affects food availability. Over-application of larvae therefore does not convey additional debridement gain.
In the eyes of the authors, the inhibitor study confirmed the importance of trypsin for medical maggots to digest tissue and grow, which confirms the findings of other studies that investigated the relationship between trypsin and protein digestion. Moreover, results suggest that chymotrypsin has a lesser effect on larval digestion. The authors conclude “The present assays provide insight into effective dosing and how larval density affects debridement. In addition, the study confirms the significance of specific serine proteases to the mechanism of larval debridement.”
This is an important study because it provides an experimentally verified optimal larval density for application of bagged larvae, for which there was hitherto little experimental data available in the public arena. The authors freely acknowledge the limitations of such an assay in that it can only approximate the wound environment. Provided these limitations are kept in mind, the wound assay is an adequate approach and the results appear to be sound and conclusive.
What deserves discussion, however, is the influence of maggot enclosure on density-dependent debridement efficiency. Wilson and colleagues correctly pointed out that the BioBag restricts larval access to the wound and renders what is in nature a three-dimensional system into a flat two-dimensional system. In the wound, medical maggots can bury into the slough and necrotic tissue and therefore spread out in three dimensions and gain better access to their food source. Therefore, in BioBags one should expect density-dependent negative effects to occur at lower densities than under ‘free-range’ conditions, which has implications for larval dosage advice for free-range versus bagged application of medical maggots.
Furthermore, the authors suggest that in addition to being clinically inefficient, overapplication is also wasteful. Whether a few maggots more or less in a BioBag is a cost concern is a matter for producers to decide. However, reducing larval densities to levels recommended in this study without considering potential larval mortality in the wound may indeed decrease efficiency rather than improve it. Larval survival has been shown to be affected by wound aetiology (Čičková, et al. 2013, Evidence-based Comp Alt Med 2013), and other factors such as healthcare provider skill in applying and maintaining dressings, and patient behavior, also have an impact. A moderate surplus of maggots applied to wounds whether free-range or bagged should compensate for such mortality and still deliver adequate therapeutic benefit. Besides, the study suggests that over-application within BioBags has no detrimental effect on wound debridement efficiency. It merely reduces the growth rate of larvae which is of little consequence because they are disposed of after treatment.
To the reviewer’s mind, the paper would have been a more coherent body of work if the inhibitor study had been omitted and discussed in a separate publication with additional experimentation elucidating the relative roles of trypsin and chymotrypsin. More importantly, the density study has direct and practical implications for larval therapy while the inhibitor study aims to understand the role of larval proteases with a long-term view toward the harnessing of biochemical attributes of larval excretions and secretions for wound care pharmaceuticals.
In summary, the authors are to be congratulated for generating the experimental evidence for effective and efficient larval densities in the application of bagged maggots in maggot therapy.