Brundage et. al. Biotherapy Journal Review Summer 2016
Reviewer: Frank Stadler, BSc Hon, doctoral candidate
Brundage AL, Crippen TL, Tomberlin JK. Methods for external disinfection of blow fly (Diptera: Calliphoridae) eggs prior to use in wound debridement therapy. Wound Repair Regen. 2016. doi: 10.1111/wrr.12408.
Maggot debridement therapy (MDT) is the use of the larval stage of flies (i.e., Calliphoridae) to remove necrotic tissue and disinfect wounds. Effective MDT requires aseptic technique to prevent the unintentional introduction of pathogenic bacteria into a wound to be debrided; yet the external surface of Calliphoridae eggs is often heavily contaminated with bacteria. Studies of external disinfection of dipteran eggs have been reported, but neither their efficacy nor effect on egg viability has been adequately assessed. The present study evaluated the efficacy of ten disinfection techniques involving immersion, rinse, or a combination of both in formalin, Lysol, formaldehyde, bleach, ethanol, Sporgon, or benzalkonium chloride. All techniques resulted in significant decreases in culturable, aerobic bacterial load on Lucilia cuprina eggs. For L. cuprina, a 10 minute 3% Lysol immersion was the most efficacious, disinfecting 96.67% of egg samples, while resulting in 31.84% egg mortality. The 5% formalin immersion was least efficacious, disinfecting only 3.33% of L. cuprina egg samples, while resulting in 33.51% egg mortality. A formaldehyde immersion, one of the most commonly used disinfection techniques, was moderately effective, disinfecting 66.7% of egg samples, while resulting in 40.16% egg mortality. For Chrysomya rufifacies and Cochliomyia macellaria egg samples, the 10 minute 3% Lysol immersion disinfected 100% of the samples, and for Lucilia sericata, 80% of egg samples,
while resulting in 33.97%, 7.34%, and 36.96% egg mortality, respectively. H2CO disinfected 16.67% of Ch. rufifacies, 26.67% of C. macellaria, and 56.67% of L. sericata egg samples, while resulting in 21.98%, 10.18%, and 32.19% egg mortality, respectively. Due to its high disinfection efficacy and relatively low egg mortality, a 10 minutes 3% Lysol immersion is recommended for sterilizing Calliphoridae eggs prior to rearing of larvae for use in MDT.
The authors point out that while a number of papers outline various sterilization protocols and reagents for fly eggs in medical maggot production, there has been insufficient evaluation or validation of the efficacy of sterilization methods, not only regarding the sterility of the eggs but also regarding egg survival. In this study ten sterilization treatments were tested on eggs from Lucilia cuprina. Cochliomyia macellaria, Chrysomya rufifacies and Lucilia sericata were exposed to two treatments, H2CO and 10 minutes soak in Lysol.
Main experimental treatments to determine sterilization efficacy and eclosion rate included:
Immersion (for 5 minutes unless otherwise stated) in
- 5% formalin,
- 10% formalin,
- 3% Lysol,
- 3% Lysol for 10 minutes
- 5% H2CO
- 5% NaOCl followed by 5% H2CO.
- 10 cc 70% EtOH
- 30 cc 1% NaOCl
- 10 minutes immersion in ADBAC followed by rinse in 10 cc 70% EtOH
- Immersion in 95% EtOH then evaporation for 5 minutes followed by immersion in SporGon®.
Other experiments included
- Effect of agitation on sterilization efficiency and eclosion
- The testing of immersion tolerance for medical fly eggs,
- Identification of baseline contamination of unsterilized eggs,
- Chorion visualization to determine chorion damage
It is important to introduce here the polycarbonate filter system that was used to hold the eggs during sterilization treatments (Figure 1) because it is relevant to the discussion below.
Polycarbonate Filter Holder, 13 mm http://www.coleparmer.com/Product/Polycarbonate_Filter_Holder_13_mm_12_pk/EW-29550-40 (Accessed, 21 August 2016)
With regard to the sterilization efficacy experiments, please refer to the abstract for detailed results. Overall, the authors conclude from their findings that a 10 minute 3% Lysol immersion provides the best sterilization and eclosion results for Calliphorid eggs.
Immersion tolerance results are somewhat intriguing and will be discussed below in more detail. In summary, Ch. Rufifacies and C. macellaria exhibited high eclosion rates of above 88%. L. cuprina was the most sensitive to length of immersion with a statistically significant drop in eclosion rate for longer immersion times. However, even at 10 minutes eclosion was still high (76.3% +- 7.1). The most surprising result was that for L. sericata. Eclosion was below 50% and not significantly different between the treatments. Even eggs that were not immersed had very low eclosion of (47.3%).
First up, the authors are to be congratulated for contributing experimental data to the discussion of sterilization efficacy in maggot debridement therapy. Confidence in the quality and sterility of medical maggots is key to making this treatment more popular and more widely available.
This brief review will mainly focus on 1) validity and comparability of test outcomes of this study with the wider literature and medical maggot production practice, 2) the unusually low eclosion rate of L. sericata in the immersion experiment compared with the additional sterilization experiment conducted with L. sericata, and 3) general relevance of findings for medical maggot production and maggot debridement therapy.
Validity and comparability of sterilization efficacy testing.
The authors conducted sterility testing using reagents that have been used by researchers and medial maggot producers as published in the literature as far back as the early 20th century. It is important to note that sterility testing was not conducted using the same methodology as described in the respective literature or practiced in commercial laboratories. To the reviewer’s knowledge, there has been no report of the use of a polycarbonate filter holder (Fig 1) for egg sterilization in MDT.
Independent of the chemical reagents used there is a clear difference in the protocols, procedures for sterilization described in this paper compared to the literature and practice. First, in many labs sterilization is conducted in open vessels such as beakers or larger test tubes and greater volumes of sterilization reagent are used to treat and rinse the eggs. The polycarbonate filter holder appears to only hold a volume of 2.5 mL for immersion. This does not allow for sufficiently generous washing of eggs. Moreover, when the sterilization reagent and rinse are pushed through the filter, the eggs may be pressed against the filter paper preventing proper circumferential removal of bacteria from the chorion. Second, deagglutination in practice involves usually both mechanical separation of eggs and surfactant treatment with NaOCl or sodium sulfite. Only treatments f) and h) used this approach with NaOCl and with higher sterilization rates. Third, the most used species for MDT is L. sericata and to a lesser degree L. cuprina. It should therefore be a priority to thoroughly test sterilization methods on these two species. The authors argue for preferential use of L. cuprina on the basis of its sensitivity to immersion. It appears to the reviewer that for the study to have practical use, both L. sericata and L. cuprina should have been tested in equal measure or preference should have been given to L. sericata.
In short, while this study compares the respective sterilization options on a level playing field with the same albeit new methodology, it does not replicate and evaluate sterilization procedures reported in the literature. This is somewhat problematic because the poor sterilization outcomes reported in this study for many of the commonly used reagents implies inefficient and perhaps even inadequate sterilization of medial maggots used for MDT. However, this is unlikely because any commercial producer faced with such poor sterilization outcomes would rapidly improve the methodology in order to avoid costly waste of resources due to rejection of production batches. Moreover, it must be stressed and made explicit to the uninitiated reader that best practice medical maggot production does include sterility testing of maggots before application in MDT. This ensures that contaminated batches if they should occur are discarded and not used in treatment.
To conclude, this study is invaluable as it establishes the superiority of Lysol as a disinfection reagent under given experimental conditions. The problem, however, is the widely differing experimental methods employed here and elsewhere and the limitations this imposes on generalizability of findings. In fact, it appears to the reviewer that the objectives of this research have been achieved only in part.
Low eclosion rates for L. sericata
The authors report very poor eclosion rates (<50%) for L. sericata in the immersion experiment compared to the other species tested, even for those maggots not immersed in water. This appears highly unusual particularly when considering that in the sterilization experiment, eclosion rates of around 70% were noted for L. sericata.
Relevance and implication of this study
The reviewer would like to emphasize that quality control in best practice medical production ensures that no contaminated medical maggots reach the patient. The outcomes of this study are likely the consequence of methodological differences and the different equipment used compared to sterilization protocols described in the literature. Nevertheless, the authors have clearly established that under their experimental conditions, Lysol is the best sterilization reagent.
With regard to egg mortality due to sterilization, it should be noted that there is probably an overproduction and surplus of eggs in commercial production meaning that higher mortality rates can probably be tolerated in favor of high sterility. After all, only successfully eclosed larvae are packaged and used for MDT. Higher egg mortality would only be a problem if eggs instead of larvae were packaged and dispatched for MDT treatment.