SHORT COMMUNICATION Human Wound Colonization by eximia and Chrysomya rufifacies (Diptera: ): Myiasis, Perimortem, or Postmortem Colonization?

1,2 3 1 MICHELLE R. SANFORD, TERRY L. WHITWORTH, AND DARSHAN R. PHATAK

J. Med. Entomol. 51(3): 716Ð719 (2014); DOI: http://dx.doi.org/10.1603/ME13229 ABSTRACT The infestation of human or tissues by ßy larvae has been given distinctive terminology depending on the timing and location of colonization. Wounds and oriÞces colonized by Diptera in a living human or animal are typically referred to as myiasis. When the colonization occurs after death, it is referred to as postmortem colonization and can be used to estimate the minimum postmortem interval. What happens when the human, as in the case presented here, has a necrotic limb while the human remains alive, at least for a short period of time? The case presented here documents perimortem wound colonization by Lucilia eximia (Wiedemann) and Chrysomya rufifacies (Macquart) and the considerations for approximating development temperatures and estimating the time of colonization (TOC). This represents the Þrst record of L. eximia in human myiasis in the United States and the Þrst record of the co-occurrence of L. eximia and C. rufifacies in human myiasis in the United States. The TOC was estimated using both ambient and body temperature. colonization before death complicates the estimation of TOC and minimum postmortem interval and illustrates the problem of temperature approximation in forensic entomology casework.

KEY WORDS forensic entomology, myiasis, time of colonization, casework

Myiasis, the infestation of living tissues by ßies, is well ring in Central and South American countries (Baum- documented in the tropics and neotropics (Fran- gartner and Greenberg 1985, Vianna et al. 1998, Ba- cesconi and Lupi 2012). Before the successful eradi- tista-da-Silva et al. 2011). It has been proposed for use cation of the primary screwworm ßy, Cochliomyia in wound debridement in Colombia (Echverri et al. hominivorax (americana) (Coquerel) (Diptera: Cal- 2010), suggesting that it is capable of feeding on ne- liphoridae), in the United States, myiasis of wildlife crotic wound tissue, but whether it prefers to lay eggs and domestic was common in Texas in wounds is unknown. L. eximia is a known carrion (Lindquist 1937, Knipling 1939) with reports in hu- breeding ßy species (Hall 1948). mans as well (Causey 1937, James 1947, Scott 1964, Four members of Chrysomya (Diptera: Calliphori- Macias et al. 1973). After the eradication program, dae) have been introduced to the Americas, including more reports of human myiasis caused by Lucilia (Pha- Chrysomya albiceps (Wiedemann), Chrysomya puto- enicia) sericata (Meigen) (Diptera: Calliphoridae) ria (Wiedemann), Chrysomya megacephala (F.), and (Rice and Nelson 1956, Horen 1967, Greenberg 1984, Chrysomya rufifacies (Macquart) (Baumgartner and Sherman 2000), Chrysomya spp. (Diptera: Calliphori- Greenberg 1984). Two of these species are currently dae) (Richard and Gerrish 1983), and Phormia regina present in North America. C. megacephala, introduced (Meigen) (Diptera: Calliphoridae) (Hall et al. 1986) from Africa (Baumgartner and Greenberg 1984), and were reported. C. rufifacies from the Australasian region (Wells et al. Lucilia (Phaenicia) eximia (Wiedemann) has been 1999). In parts of their native range, Thailand for reported as causing myiasis in animals (Diptera: Cal- example, they have been recorded simultaneously in liphoridae) (Madeira et al. 1989, Azeredo-Espin and human myiasis (Sukontason et al. 2005). In wet and Madeira 1996). This species has not been previously warm climatic zones of the Hawaiian Islands, these documented in association with human myiasis in the two species have also been collected in cases of cattle United States and is considered an uncommon species, myiasis (Zimmerman 1944, Shishido and Hardy 1969). observed most often in Texas and Florida (Whitworth However, to our knowledge L. eximia and C. rufifacies 2010). It is widespread throughout the tropics occur- have yet to be recorded together in human myiasis. Colonization by ßies after death is useful in esti- 1 Pathology Division, Harris County Institute of Forensic Sciences, mating the minimum postmortem interval (PMIMIN), 1885 Old Spanish Trail, Houston, TX 77054. a key task of a forensic entomologist. Species-speciÞc 2 Corresponding author, e-mail: [email protected]. 3 Department of Entomology, Washington State University, WA developmental data and local temperature data are 99164. used to work backwards to estimate the time of ßy egg

0022-2585/14/0716Ð0719$04.00/0 ᭧ 2014 Entomological Society of America May 2014 SANFORD ET AL.: PERIMORTEM COLONIZATION 717

Fig. 1. Autopsy photograph illustrating the extent of the larval ßy infestation of the lower left leg of the decedent. deposition (Catts and Goff 1992). The temperatures tures identiÞed Proteus mirabilis (Enterobacteriales: experienced by the developing larvae inßuence the Enterobacteriaceae) as a bacterial species in the left rate of development and the estimated age of the leg wound, left lung, spleen, and blood, and sepsis due larvae. When the larvae develop on dead tissue, am- to this bacterium was a contributing factor in the bient air temperature is used to approximate devel- decedentÕs cause of death. Additional autopsy Þndings opment temperature, as the body is expected to cool related to the decedentÕs cause of death included to ambient conditions. However, in cases of myiasis hypertensive and atherosclerotic cardiovascular dis- the temperature used to approximate development ease, chronic obstructive pulmonary disease, and cir- time is body temperature. Thus, the differentiation of rhosis. myiasis from postmortem colonization becomes crit- Specimens. A portion of the larval specimens col- ical to accurately estimate the PMIMIN or the time lected from the leg wound during autopsy were pro- when ßies may have Þrst deposited eggs, the time of cessed by hot water killing (Adams and Hall 2003), colonization (TOC; Byrd and Castner 2000). followed by placement in 70% ethanol, and a portion The case presented here describes the apparent were prepared for rearing on defrosted beef liver at infestation of a wound on a necrotic limb around the warm room temperature for conÞrmation of identiÞ- time of death (perimortem) of the individual and the cation. Because a large number of specimens were difÞculties present in determining how much time had collected, the remaining larvae were killed and pre- passed since the wound was colonized. This report served by direct placement into 70% ethanol. Larval also documents the Þrst record of human myiasis measurements and microscope photographs were caused by L. eximia and the Þrst published record of made using a Keyence VHX-600 digital microscope the co-occurrence of L. eximia and C. rufifacies in- (Keyence Corporation of America, Itasca, IL). volved in human myiasis in the United States. Estimate of TOC. Quality Controlled Local Clima- tological Data from the nearest weather station lo- cated at George Bush Intercontinental Airport, Hous- Materials and Methods ton, TX, (Station #12960) was obtained from The Case Details. A homeless, 60-yr-old male was found National Oceanic and Atmospheric AdministrationÕs nearly unresponsive outdoors behind a local business National Climatic Data Center (http://cdo.ncdc. in Houston, TX, pronounced dead shortly after arrival noaa.gov/qclcd/QCLCD?prior ϭ N) for May and to the emergency room and was transported to the June 2013. Degree hours were calculated in Microsoft Harris County Institute of Forensic Sciences on 4 June Excel 2007 (Microsoft Corporation, Portland, OR) us- 13. Larval ßy activity was noted around the decedentÕs ing a base 10ЊC developmental threshold by the ac- scrotum and penis, and extensive larval ßy activity was cumulated degree hour method (ADH; Higley and observed on the lower left leg near the ankle (Fig. 1); Haskell 2000). medical records noted that there was no pulse in this leg. The body was stored in the morgue cooler at Results and Discussion Ϸ7.2ЊC from the time of check-in until autopsy the following day, when specimens were collected. Bac- Two types of larvae were collected: a smooth larva terial samples were taken at the hospital for culturing and a spiny larva. The smooth type specimens were and identiÞcation by the hospital laboratory. The cul- third instar and tentatively identiÞed as L. eximia 718 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 51, no. 3

(Knipling 1939) or (Macquart) species (Gallagher et al. 2010). The only dataset lo- (Diptera: Calliphoridae) (Seago 1953). Additional cated for L. eximia was from a population derived from Þrst- and second-instar smooth larval specimens were Peru (Greenberg and Szyska 1984). Data for C. rufi- collected but these specimens were only identiÞed as facies were from a Florida population (Byrd and Butler Calliphoridae. The spiny specimens were identiÞed as 1997). However, using a more locally derived data set second instar C. rufifacies (Wells et al. 1999). Reared obtained at a different temperature of 28.3ЊC suggests adults were conÞrmed as L. eximia and C. rufifacies colonization of C. rufifacies (Flores 2013) may have (Whitworth 2010). To our knowledge, this represents occurred in the afternoon of 2 June 2013 closer to the the Þrst time that these two species have been impli- colonization time of L. eximia. The remaining source cated together in human myiasis. The decedent was of error then becomes the difference in the temper- colonized before death; however, the leg was essen- atures from which the development data were ob- tially dead and presumably cooler than the rest of the tained. body while the decedent remained alive before death The case presented here documents the Þrst report at the hospital. The lack of circulation to the leg would of human myiasis caused by L. eximia and the Þrst have cooled the temperature of the tissues that the ßy reported co-occurrence of L. eximia and C. rufifacies larvae fed upon, but it is not clear how long the limb in human myiasis. It is unusual to have such extensive was necrotic and hence how cool the temperature knowledge of a decedentÕs terminal events with re- may have been with respect to body temperature spect to insect colonization. In most instances where when the eggs were initially laid. the forensic entomologist is called upon to make an

The difÞculty in approximating the temperatures estimate of TOC and PMIMIN knowledge about my- experienced by the developing larvae directly affects iasis and medical history will be unknown, a consid- the estimated TOC. The highest possible temperature eration of premortem colonization may be a signiÞ- the larvae may have experienced would have been the cant consideration especially when medical history decedentÕs body temperature and the lowest possible suggests conditions that may predispose an individual temperature was likely ambient air temperature. to skin ulcers and difÞculties in peripheral circulation. Therefore, a range encompassing these two extremes Without the unusual knowledge about this speciÞc was generated and the actual time of ßy colonization case, the estimated TOC would have overestimated likely falls within this range. A conservative estimate the PMI by as much as 2 d. based on completion of the second instar of L. eximia at 21.7Ð26.0ЊC (cyclic temperature; Greenberg and Acknowledgments Szyska 1984) places colonization of the leg in the late evening of 1 June 2013 or the early morning of 2 June We thank Jeffery Tomberlin, Ph.D., for peer review of the 2013 using the ambient air temperatures obtained original case report. We also wish to acknowledge the sup- from the nearest weather station. Using an estimated port of the Harris County Institute of Forensic Sciences. We body temperature of 37ЊC, the ADH for completion of also thank an anonymous reviewer for comments that greatly second instar L. eximia (1121.1 ADH) may have been improved the manuscript. reached if the eggs had been laid late on 2 June 2013 or early on 3 June 2013. The ADH for the completion References Cited of the Þrst-instar C. rufifacies larvae at 26.7ЊC (cyclic Adams, Z., and M.J.R. Hall. 2003. Methods used for the temperature; Byrd and Butler 1997) would have been killing and preservation of blowßy larvae, and their effect met if eggs had been laid in the afternoon of 3 June on post-mortem larval length. Forensic Sci. Int. 138: 50Ð 2013 using ambient air temperatures from the nearest 61. weather station. Based on body temperature and de- Azeredo-Espin, A.M.L., and N. G. Madeira. 1996. Primary velopment at 32.2ЊC (Byrd and Butler 1997), the ADH myiasis in dog caused by Phaenicia eximia (Diptera:Cal- would have been met if the eggs had been laid late in liphoridae) and preliminary mitochondrial DNA analysis the day of 3 June 2013. These conservative estimates of the species in Brazil. J. Med. Entomol. 33: 839Ð843. suggest that L. eximia likely colonized between the 1 Batista-da-Silva, J. A., G. E. Moya-Borja, R. P. De Mello, and and 3 June 2013 and C. rufifacies colonized later on 3 M.M.D.C. Queiroz. 2011. Abundance and richness of Calliphoridae (Diptera) of public health importance in June 2013. We would propose that the colonization the Tingua´ Biological Reserve , Nova Iguac¸u (RJ), Brazil. was perimortem, occurring around the time of the Entomotropica 26: 137Ð142. terminal events leading to the decedentÕs death. If the Baumgartner, D. L., and B. Greenberg. 1984. The genus decedent had been found without all the details re- Chrysomya (Diptera: Calliphoridae) in the New World. garding his terminal events, the estimated PMIMIN J. Med. Entomol. 21: 105Ð113. would have been inaccurate and potentially mislead- Baumgartner, D. L., and B. Greenberg. 1985. Distribution ing because of the unknown amount of time between and medical ecology of the blow ßies (Diptera: Calli- the TOC and the decedentÕs death. phoridae) of Peru. Ann. Entomol. Soc. Am. 78: 565Ð587. Another potential source of error in the estimate of Byrd, J. H., and J. F. Butler. 1997. Effects of temperature on Chrysomya rufifacies (Diptera: Calliphoridae) develop- TOC is that temperature-based development data sets ment. J. Med. Entomol. 34: 353Ð358. available for L. eximia and C. rufifacies were not local. Byrd, J. H., and J. L. Castner. 2000. of forensic im- Using data from a different locally adapted population portance, pp. 43Ð78. In J. H. Bryd and J. L. Castner (eds.), may not accurately reßect the development and tem- Forensic entomology: the utility of in legal perature response of a local population of the same ßy investigations. CRC, Boca Raton, FL. May 2014 SANFORD ET AL.: PERIMORTEM FLY COLONIZATION 719

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