(Diptera: Calliphoridae) in Maggot Debridement Therapy in Alexandria

DIRECT INJURY, MYIASIS, FORENSICS

An Accidental But Safe and Effective Use of Lucilia cuprina (Diptera: Calliphoridae) in Maggot Debridement Therapy in Alexandria, Egypt TAREK I. TANTAWI,1 KIRSTIN A. WILLIAMS,2

AND

MARTIN H. VILLET3

J. Med. Entomol. 47(3): 491Ð494 (2010); DOI: 10.1603/ME09183

ABSTRACT The calliphorid ßy, Lucilia cuprina (Wiedemann), is known to cause serious malign myiasis in animals, whereas its sibling species Lucilia sericata (Meigen) is commonly a carrion breeder and is used in maggot debridement therapy (MDT). The current study reports an accidental involvement of L. cuprina in MDT in Alexandria, Egypt, that has proved to be safe and effective. In November 2008, the laboratory colonies of L. sericata (the species regularly used in MDT) at the Faculty of Science, Alexandria University were renewed by Lucilia ßies collected as third instar larvae on exposed rabbit carcasses. Flies from the new colonies were successfully used to heal the diabetic foot wounds of two patients at Alexandria Main University Hospital. Analysis of DNA sequences and adult and larval morphology then revealed that these ßies were and still are L. cuprina. Breeding of this species in carrion in Alexandria is a new record. Despite the safety of this strain of L. cuprina in MDT, entomologists rearing blow ßies for the purpose of wound debridement should regularly maintain high quality assurance of their speciesÕ identity to avoid possible clinical complications that may result from the introduction of an unexpected and invasive species to their laboratory colonies. KEY WORDS Lucilia cuprina, Calliphoridae, maggot debridement therapy, myiasis, Egypt

Maggot debridement therapy (MDT) is an effective treatment for certain wounds that are not amenable to other forms of therapy (Sherman et al. 2000, Tantawi et al. 2007). It involves allowing maggots of certain species of blow ßies to consume the necrotic tissue in the wound, and to produce antibiotic substances that kill the pathogenic bacteria and promote tissue healing (Sherman et al. 2000; Nigam et al. 2006a,b). The species recommended for MDT is Lucilia sericata (Meigen) (Sherman et al. 2000, Nigam et al. 2006a). This species is a member of a large group of ßies that includes the genera Lucilia Robineau-Desvoidy and Hemipyrellia Townsend (Zumpt 1965). Molecular identiÞcation techniques were used to promote quality assurance in the identiÞcation of maggots used for MDT (Williams et al. 2008). This is important because the behavior of maggots varies between species. For instance, Lucilia cuprina (Wiedemann), the closest relative to L. sericata, is known to be involved in veterinary problems in sheep, and can lead to the death of the animals by rectal myiasis (Ullyett 1945, 1950; Zumpt 1965). MDT has been practiced at Alexandria Main University Hospital, Alexandria, Egypt, since 2000 using disinfected maggots of L. sericata from laboratory col1 Department of Zoology, Faculty of Science, Alexandria University, Moharrem Bey, Alexandria, Egypt (e-mail: t.tantawi@ yahoo.com). 2 Durban Natural Science Museum, Durban, 4000 South Africa. 3 Southern African Forensic Entomology Research Laboratory, Department of Zoology & Entomology, Rhodes University, Grahamstown, 6140 South Africa.

onies descended from third instar larvae collected on exposed rabbit carcasses placed in the botanical garden of the Faculty of Science in the Moharrem Bey district of Alexandria. Since 1988, all of the Lucilia larvae collected by this regular manner were of L. sericata. The Þrst collections of this species were deÞnitively identiÞed by Prof. Bernard Greenberg (University of Illinois at Chicago, Chicago, IL) and the senior author, based on the morphology of both adults and larvae (Tantawi et al. 1996). Subsequent collected specimens were morphologically identiÞed by the latter only. Some of this work was part of a survey of carrion ßies in Alexandria that showed that adults of L. cuprina were extremely scarce and did not breed in carrion in that setting (Tantawi et al. 1996). The colonies were effective and safe in a variety of MDT cases (Tantawi et al. 2007). In November 2008, when the senior author returned from leave, the ßy colonies were renewed by collecting third instar larvae from exposed rabbit carcasses placed in the botanical garden as usual, based on many yearsÕ experience that the species would be L. sericata. As part of a quality assurance protocol, specimens from one laboratory cage of recently collected maggots were screened using molecular identiÞcation (Williams et al. 2008) and discovered to be L. cuprina. The colony had been used successfully for MDT before being screened. Subsequently, the carrion ßy community present in the botanical garden of Moharrem Bey was surveyed again, and it was found that L. cuprina and L. sericata were now both present in that setting.

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Table 1. Primers used to amplify and sequence the mitochondrial cytochrome oxidase I (CO1) gene (partial, 5ⴕ end), the nuclear 28S ribosomal RNA (28S) gene (D1–D2 fragment) and the nuclear Period (per) gene of specimens from the 2008 colony of Lucilia Gene 28S CO1 per

Primers

Source

Name

Sequence

D1.F D2.R C1-J-1709 C1-N-2353 PerF5 Per3primeR1

5⬘-CCCCCTGAATTTAAGCATAT-3⬘ 5⬘-GTTAGACTCCTTGGTCCGTG-3⬘ 5⬘-AATTGGGGGGTTYGGAAAYTG-3⬘ 5⬘-GCTCGTGTATCAACGTCTATTCC-3⬘ 5⬘-GCCTTCAGATACGGTCAAAC-3⬘ 5⬘-GATAGGCCTCCAGCATAGGG-3⬘

The presence of L. cuprina in carrion in Alexandria and its use in MDT are new records. This publication gives details of the molecular and morphological identiÞcation of the maggots, their occurrence in carrion in Alexandria, and preliminary evidence of the effectiveness of an Egyptian strain of L. cuprina in MDT. Materials and Methods Flies were sampled from a maggot therapy colony held at Alexandria, Egypt, that was established from ßies collected from rabbit carcasses placed in the botanical garden of the Faculty of Science, Moharrem Bey (31⬚11.4⬘N 29⬚54.5⬘E) in November 2008. Molecular Identification. One hind leg of each ßy was removed and DNA was extracted using a Qiagen DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA) according to the manufacturerÕs instructions. AmpliÞcations of the mitochondrial cytochrome oxidase I (CO1) gene, and the nuclear 28S ribosomal RNA (28S) and Period (per) genes using the polymerase chain reaction (PCR) were performed using the primers listed in Table 1. PCR products for the three genes were sequenced using the respective primers used for their ampliÞcation and an ABI 3730l Genetic Analyzer (Applied Biosystems, Foster City, CA). Additional sequences of L. sericata and L. cuprina were obtained from GenBank for comparison. The sequences were aligned using BioEdit v7.0.9 software (Hall 1999) and subsequently analyzed using maximum parsimony and bootstrap analysis in PAUP*4b10 (Swofford 2003). The new sequences were also compared against the GenBank database using BLAST (basic local alignment search tool) software (Altschul et al. 1997). Morphological Identification. Morphological identiÞcation of adults of L. cuprina was based on the number of occipital, humeral, and notopleural setae (Holloway 1991, Whitworth 2006). They were also compared with Egyptian and Turkish specimens of L. sericata deposited in the collection of the senior author. Morphological identiÞcation of larvae was based on the characters described by Smith (1986).

Stevens et al. 2002 Stevens et al. 2002 Simon et al. 2006 Simon et al. 2006 G.R. Warman, pers. comm. G.R. Warman, pers. comm.

MDT. Two patients received two cycles of maggots, each of 4 d, administered following the method described by Tantawi et al. (2007). The interval between the two cycles was 1 wk. Results Molecular Identification. The DNA extracts ampliÞed without complication, yielding sequences that totaled to 1978 bp (Table 2). Maximum Parsimony analysis placed the specimens in a clade with L. cuprina with bootstrap support of 99%. BLAST comparisons of the Egyptian specimens with the GenBank database showed a 99 Ð100% match with specimens of L. cuprina from Australia (COI and 28S genes) and New Zealand (per gene) (Table 2). Morphology. The adults were morphologically typical of L. cuprina. In almost all of the specimens examined, the central occipital area had no more than one seta below the inner ventral setae, the humeral callus had no more than three small setulae along its posterior margin, and the notopleuron had no more than three small setulae on its posterior border. This is the Þrst time since 1988 that these characters had been seen in the Alexandria University laboratory except for a few specimens. The adults of the new population also had a dull coppery color (characteristic of L. cuprina) in contrast with the older ones which were bright green (typical of L. sericata). Similarly, in the larvae in the new colony, the distance between the inner dorsal tubercles of the last abdominal segment was greater than the distance between each inner tubercle and its adjacent medial tubercle, identifying them as L. cuprina. All of the larvae in older laboratory colonies, Þeld carrion studies, and forensic cases since 1988 showed a distance between their inner tubercles that was equal to the distance between each inner tubercle and its medial neighbor, indicating that they were L. sericata. MDT. The Þrst patient was a 45-yr-old diabetic male with two ulcers on his left foot. One ulcer had a surface area of 140 cm2 and was located on the lateral aspect

Table 2. Sequence lengths and match statistics for BLAST searches of the GenBank database for the three genes used to confirm the identification of the 2008 colony of Lucilia flies Gene

Length (bp)

Match

Sequence no.

Species

Geographical origin

28S CO1 per

656 576 746

100% 99% 99%

AJ417709.1 AJ417706.1 Y19108.1

L. cuprina L. cuprina L. cuprina

Townsville, Australia Townsville, Australia Wallaceville, New Zealand

May 2010

TANTAWI ET AL.: Lucilia cuprina IN MAGGOT DEBRIDEMENT THERAPY

of the foot. The other ulcer had a surface area of 20 cm2 and was located on the plantar surface. Necrotic tissue covered ⬎90% of the area of both ulcers. The lateral wound was fully closed after 4 wk of the Þrst cycle of maggots, whereas the plantar wound closed after 13 wk of the Þrst cycle of maggots. The lateral wound was completely debrided by the Þrst cycle of maggots and the second cycle of maggots was intentionally applied to the clean wound to enhance healing. The second patient was a 62-yr-old male suffering from a diabetic heel ulcer on his left foot. The ulcer had a surface area of 25 cm2 of which ⬎90% was covered with necrotic tissue. This ulcer was fully healed after 11 wk of the Þrst cycle of maggots. Discussion Occurrence of L. cuprina. The primary lesson learned from this situation is that any new population of a particular ßy species must be taxonomically identiÞed before establishing a colony of it in the laboratory, especially where sibling species coexist. This afÞrms the lesson of a previous case reported by Williams et al. (2008), where a South African colony thought to be L. cuprina was identiÞed as L. sericata using DNA sequences. The authors of the current study are aware that if the introduced species had been applied to the wounds of their patients and found to be invasive, the patients may have suffered. Trapping adults of Lucilia ßies from the botanical garden in May 2009 showed that both L. sericata and L. cuprina coexist there at present, and suggests that the character of the ßy community there has changed recently. The presence of L. cuprina in carrion in Alexandria is a new record (see Tantawi et al. 1996, Tantawi and El-Kady 1997). This species has not been found in carrion in Alexandria since monitoring began in 1988. Spradberry (1991) stated that L. cuprina, under most conditions, survives poorly in carrion and its persistence is largely linked to its ability to infest sheep. However, this species is now readily breeding in rabbit carcasses in Alexandria. This calls attention to the need to consider the presence of L. cuprina in future cases of forensic entomology in Alexandria. One possible explanation of this new observation is human agency (Williams and Villet 2006). For example, exotic plants are commonly introduced in the botanical garden from different regions of Egypt and elsewhere in the world for scientiÞc study. Pupae of L. cuprina might have been associated with these plants. It is also possible that an upsurge in the L. cuprina population in Alexandria is due to the effects of climate change (Williams and Villet 2006), which may promote migration or population growth. L. cuprina is abundant in Cairo, 220 km across the Nile delta from Alexandria (T.I. Tantawi, unpublished data). Effectiveness of L. cuprina in MDT. MDT is essentially a controlled therapeutic myiasis in which the primary role of the medical entomologist is to select a safe and effective species and strain (Sherman et al. 2000, Sherman 2009). It is common wisdom that L. cuprina is a rural ßy that causes sheep traumatic and

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often fatal myiasis (termed sheep strike) in Africa and Australia, while L. sericata occupies the carrion niche and feeds on dead tissue (Ullyett 1950, Zumpt 1956, Meskin 1986). This would make L. sericata a good candidate for MDT (Thomas et al. 1996, www.zoobiotic. co.uk), but it is not as clear that L. cuprina is a suitable candidate because it eats live tissue, and for this reason it has been avoided in MDT. However, L. sericata commonly causes sheep strike in England (Hall and Wall 1995, Stevens and Wall 1997), so there is reason to assess L. cuprina for MDT. Between 2000 and August 2008, L. sericata was the species used in MDT at Alexandria Main University Hospital (Tantawi et al. 2007), after which maggots from the colony of L. cuprina established in November 2008 were successfully used to completely debride and close neuroischemic foot ulcers in two patients. That the Þrst patientÕs lateral wound, which was completely debrided by the Þrst cycle of maggots, did not incur trauma during the second cycle of maggots clearly provides evidence that this strain of L. cuprina is safe, and encourages full clinical trials. There is also evidence that L. cuprina is therapeutic in at least some wounds in Malaysia (Paul et al. 2009), where L. sericata is absent, and the recent accidental but successful use of this species in MDT in Egypt indicates that it may be an effective and safe species just like L. sericata. It is not known why Lucilia species attack the healthy tissue of sheep and do not do the same to human tissue (Nigam et al. 2006a). This implies that further research in this direction is needed, for both medical and veterinary science. The current study also highlights the question of whether different strains of the same species can be more or less invasive, safe, or effective. There are certain strains of L. sericata that have been known to invade healthy tissue (Cavusoglu et al. 2009). Because the number of ßy species used therapeutically is limited at present (Sherman et al. 2000, Paul et al. 2009), the potential use of more species should be encouraged. Different species may also be better suited to different wound types (Church and Courtenay 2002). Chrysomya albiceps (Wiedemann) has been reported as an occasional myiasis-causing ßy (Ullyett 1950, Zumpt 1965), but it has been used successfully in MDT in Alexandria since 2000 (Church and Courtenay 2002, Kotb et al. 2002, Thomas 2006). However, the maggots of L. sericata were more effective in debridement than those of C. albiceps (Kotb et al. 2002), perhaps due to differences in the potency of the proteolytic enzymes of the two species. Second and third instar larvae of C. albiceps are facultative predators on larvae of the same and other species, whereas those of L. sericata are purely scavengers throughout their entire feeding period (Ullyett 1950, Zumpt 1965). If C. albiceps, which differs somewhat from L. sericata in its biology, can be used safely for MDT, then giving attention to L. cuprina may also be fruitful for MDT.

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JOURNAL OF MEDICAL ENTOMOLOGY Acknowledgments

We thank Guy R. Warman (University of Auckland, New Zealand) for generously providing information about unpublished per gene primer sequences; our anonymous reviewers for their comments; and Rhodes University for funding.

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