Preliminary observations of the effect of methamphetamine in decomposing tissues on the development rate of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and implications of this effect on the estimations of postmortem intervals.

Larvae of Parasarcophaga ruficornis (Fabricius) (Diptera: Sarcophagidae) were reared on tissues from rabbits administered different dosages of methamphetamine to study the effects of this drug on development of this species. The rabbits were given 37.5, 71.4, and 142.9 mg of methamphetamine via ear vein infusion. From Hours 30 to 60, larvae feeding on tissues from rabbits receiving 71.4 and 142.9 mg of methamphetamine developed more rapidly than larvae from the control colony and those feeding on tissues from the rabbit receiving 37.5 mg of methamphetamine. The time required for pupariation was significantly greater for colonies fed on tissues from methamphetamine-dosed rabbits than for the control. These differences were sufficient to alter postmortem interval estimates based on larval development by up to 18 h and estimates based on puparial development by up to 48 h. The presence of methamphetamine or amphetamine could not be detected in Diptera larvae in this experiment using radioimmunoassay techniques, as there was a nonspecific reaction, resulting in a false positive.     

Determination of drug levels and the effect of diazepam on the growth of necrophagous flies of forensic importance in southeastern Brazil.

Larvae of Chrysomya albiceps (Wiedemann) and Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) were reared on tissues from rabbits administered twice the lethal dosage of diazepam in order to study the effects of this drug on the development of these two species. The rabbits were given 50mg of diazepam via ear vein infusion. From 18 to 54h, larvae feeding on tissues containing the drug developed more rapidly than larvae from the control colony for both fly species. The time required for pupariation and adult emergence was significantly greater for colony fed on tissues from diazepam dosed rabbits than for the control ones. These differences are significant for they are large enough to alter the estimate of postmortem interval based on fly development. The presence of diazepam could be detected through gas chromatography-mass spectrometer (GC-MS) in all rabbit samples and in almost all diptera samples in this experiment.     

Determination of Malathion levels and its effect on the development of Chrysomya megacephala (Fabricius) in South China

The present study determines the concentration of Malathion in rabbit tissues and Dipteral larvae feeding on those tissues. Malathion was found in all muscle and liver samples of the test rabbit, as well as larvae fed on it. Samples from the control rabbit and pupae from all colonies were negative for Malathion. Correlations were found between administered dosage, tissue concentrations and younger or prepuparial larvae. Effects of Malathion on the development rate of Chrysomya megacephala (Fabricius) were also reported. C. megacephala is the most common fly species found on corpses in South China during the early stages of decomposition. Significant differences in larval growth rate were both observed among the colonies fed on muscle and liver. The presence of Malathion in both muscle and liver appears to retard the normal growth rate of C. megacephala in larval stage. Larvae from all colonies fed on tissues from rabbits treated with Malathion were smaller and attained maximum length later than those from the control colony. Duration of the larval and pupal stages was both significantly prolonged for larvae on tissues from rabbit receiving Malathion than those from the control colony. The difference of the duration of the larval and pupal stages together from the muscle colonies would alter the postmortem interval estimation by up to 36 h. As for liver colonies, it would alter the postmortem interval estimation by up to 28 h. A significantly different duration of the larval and pupal stages from the muscle colonies would alter a postmortem estimate by up to 28 h relative to the liver colonies.     

Determination of drug levels in larvae of Protophormia terraenovae and Calliphora vicina (Diptera: Calliphoridae) reared on rabbit carcasses containing morphine

Two species of blow flies (Diptera: Calliphoridae) were reared on tissues from rabbits administered different dosages of morphine. These species, Protophormia terraenovae and Calliphora vicina are among the first wave of insects colonizing a dead body. Two series of 3 rabbits were given dosages of 10, 20, and 40 mg/h of morphine over a 3 h period via ear artery perfusion. A morphine blood level plateau was attained after 1 h of perfusion. Two other rabbits were used as controls. Samples of tissues collected from rabbits using a coelioscopic technique were determined to have morphine concentrations similar to those encountered in human overdoses and were correlated with dosages of morphine administered. All samples from control rabbits were negative for morphine. Larvae and puparia of both species were regularly collected from each rabbit for toxicological analysis. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were significantly lower than concentrations found in the tissues. There was a decrease in concentration in morphine observed in transition from feeding 3rd instar larva to puparium. A correlation between larval concentration and tissue concentration was found only in feeding 3rd instar larvae.     

Effects of cocaine and heroin,and their combination,on the development rate of Calliphora vomitoria (Diptera: Calliphoridae)

Insects present on or near decomposing bodies are collected by forensic entomologists and used to estimate the post-mortem interval. Drugs metabolized by a person before death may affect the rate of development of insects feeding on the corpse. This study aimed to determine the effects of cocaine and heroin main metabolites on the development rate of the Calliphora vomitoria (Diptera: Calliphoridae) and their implications on minimum post-mortem interval determination. Groups of 250 eggs each were placed into four separate pots of 150 g of minced pork meat being either un-spiked, or spiked with benzoylecgonine, morphine, or a combination of both. Larval length (mm) and weight (mg) measurements were taken twice daily and the rate of development of the insects’ life cycle was monitored until eclosion. Results show that cocaine-fed larvae developed less in length and weight than the control group. Heroin-fed larvae showed a more fluctuating pattern, being smaller and lighter than the control group for most of their larval cycle, but overtaking them in both parameters towards pupation. Combination-fed larvae seemed to favour the effects of cocaine. The three conditions also had a significant impact on the length of the insects’ life cycle. Cocaine and drug combination treatments increased the length of the second and third instar stages, but led to the shortening of pupation and accelerated eclosion. Conversely, heroin treatment led to lengthier pupation. Interestingly, the effects of the drug combination seemed to mirror more precisely those of cocaine.These findings indicate that both cocaine and heroin, singularly and in combination, have sizable effects on blowflies’ development rates, potentially biasing post-mortem interval estimations.     

Effects of morphine in decomposing bodies on the development of Lucilia sericata (Diptera: Calliphoridae)

This study concerns the effects of morphine in tissues on the rate of development of Lucilia sericata (Diptera: Calliphoridae) using those tissues as a food source. Lucilia sericata is a species of fly commonly found on human corpses in Europe during the early stages of decomposition and thus of forensic interest. Three rabbits were administered 12.5, 25.0 and 50.0 mg/h of morphine chlorhydrate via ear perfusion over a period of 3 h. These dosages and duration of perfusion were calculated to give tissue concentrations of morphine similar to those encountered in fatal human overdoses. A fourth rabbit was used as a control. Following administration of the drug, rabbits were sacrificed and 400 eggs of Lucilia sericata, all of the same age, were placed in the eyes, nostrils and mouth of each rabbit. Developing larvae were sampled daily to determine growth rate and weight. Puparia and emerging adult flies were also sampled. Data were analyzed using analysis of variance (ANOVA) and Student's T-test. Results of this study show that an underestimation of the postmortem interval of 24 h is possible if the presence of morphine in tissues is not considered. This study demonstrates again the necessity of considering the possible effects of drugs in tissues on insect growth rates when estimating the postmortem interval using entomological techniques.     

Morphine perfused rabbits: a tool for experiments in forensic entomotoxicology

In order to establish an animal model for entomotoxicological studies, the kinetics of morphine elimination from blood after a single intravenous injection of morphine and the concentration of morphine in tissues following a continuous perfusion were studied. The aim of these experiments was to obtain controlled morphine tissue concentrations similar to those encountered in fatal human heroin overdoses. These tissues can be used as a food source for developing fly larvae in entomotoxicological studies. In the single injection experiment, seven rabbits were administered 1 or 2 mg/kg body weight of morphine chlorhydrate via the main ear artery. Blood samples of 200 microL were removed regularly via a catheter. Morphine concentration was determined using RIA techniques. Morphine was found to be first rapidly distributed and then slowly eliminated, following a two-exponential equation. Elimination of morphine from blood can be described as a two-compartment model. Constants of the equation were determined using the Kaleidagraph program. Using those constants, the main pharmacokinetics parameters were calculated. Results of these parameters showed the following: clearance from 13.3 to 16.2 L.h.1, half-life of the distribution phase from 0.6 to 0.9 min, and half-life of the elimination phase from 21 to 26 min. These results were used to calculate the rate of perfusion of morphine for rabbits to obtain desired, controlled, and constant concentrations of morphine in tissues. In the second experiment, three rabbits received a perfusion of morphine intravascularly at a rate of 2 mg/kg/h for a period of 3 h. These rabbits were sacrificed and analyses performed on several abdominal and thoracic organs. Results showed that the concentrations of morphine differed according to the organ analyzed, but were reproducible for organs between animals. These concentrations were similar to those normally encountered in cases of human death due to heroin overdoses.     

Determination of drug levels in larvae of Lucilia sericata (Diptera: Calliphoridae) reared on rabbit carcasses containing morphine

This study concerns the determination of morphine concentrations in fly larvae reared on rabbits administered different concentrations of morphine and a correlation between concentrations of the drug in larvae and tissues. Three rabbits (R1, R2 and R3) were given dosages of 12.5, 25.0 and 50.0 mg/h of morphine over a 3 h period via continuous ear artery perfusion. These dosages and time of perfusion were calculated to create tissue concentrations of morphine similar to those encountered in human death due to overdose. Morphine blood level plateau was attained after 1 h of perfusion. A fourth rabbit was used as a control. To evaluate drug concentrations, tissues were sampled using a coelioscopic technique. Approximately 400 eggs of Lucilia sericata, all of the same age category, were placed in eyes, nostrils and mouth of each rabbit carcass. Larvae and puparia were regularly collected from each rabbit for toxicological analysis. The concentrations of the drug in the tissues sampled were determined to be similar to those normally encountered in human overdoses and were correlated with the dosage of morphine that had been administered. Morphine was detected in all larvae and pupae fed on tissues from carcasses administered morphine, except for puparia from the colony fed on the R1 animal which received 12.5 mg/h dosage of morphine. All samples from the control rabbit were negative for morphine. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were 30 to 100 times lower than the concentrations found in the tissues. A correlation between the tissue concentrations and larval concentrations was found in only 3rd instar larvae (80 to 140 h following hatching). No correlations were found between administered dosages, tissue concentrations and younger larvae, prepuparial larvae or puparia.     

Heroin, morphine, and hydromorphone determination in postmortem material by high performance liquid chromatography

A procedure has been developed for the simultaneous determination of heroin, morphine, and hydromorphone from postmortem tissues by reversed phase high performance liquid chromatography (HPLC) using electrochemical detection. This method permits the direct determination of unmetabolized heroin from antemortem or postmortem urine as evidence of illegal drug use. Presumptive confirmation of heroin was based on the ability to hydrolyze the HPLC heroin fraction to morphine. Heroin was also confirmed in urine by gas chromatographic/mass spectroscopic (GC/MS) analysis of the HPLC fraction. Analysis of postmortem blood, gastric contents, urine, and injection site tissues have revealed the presence of morphine and hydromorphone, while heroin has only been identified in urine.     

死后血液流动对氯胺酮在家兔尸体内再分布的影响

目的观察分析静脉注射氯胺酮家兔死后血液流动对体内药物浓度再分布影响。方法雄性新西兰大白兔随机分为实验组2组（各24只）,对照组（8只）;实验组家兔经耳缘静脉注入40mg/kg氯胺酮,1.5h后处死,其中一组立即结扎主动脉,另一组不结扎;家兔尸体仰卧位室温下保存,分别于死后0、3、6、12、24、48、72和96h解剖并采取组织和体液样本;对照组静脉注射等量生理盐水,同样方法解剖取相同样本。所有样本采用GC/MS和GC-NPD法检测样品中氯胺酮含量。结果两个实验组家兔尸体放置96h内氯胺酮含量,除尿液各时间点与0h以及相邻时间点之间比较均有显著性差异（P〈0.05）外,两组家兔其余各类样本与0h以及相邻时间点比较均无显著性差异（P〉0.05）。除尿液外,各类样本两个实验组之间均无显著性差异（P〉0.05）;结扎组和不结扎组心血与外周血中氯胺酮含量比值分别为0.90~1.03和0.90~1.02。结论静脉注射氯胺酮家兔死后的血液流动不是体内氯胺酮发生再分布的主要机制。     

The evidential value of intra-alveolar haemosiderin-macrophages in cases of sudden infant death syndrome (SIDS)

This study aimed to determine the effect of cocaine on the development and growth of immature and adult blowflies, in an attempt to better understand the impacts of such effects on postmortem interval (PMI) estimation. Twice the lethal dose of cocaine was injected into rabbits. The control animals were injected only with saline solution. Experimental and control rabbits were autopsied, and portions of their livers were exposed to newly eclosed larvae of Chrysomya putoria and Chrysomya albiceps. Larvae were weighed individually every 6 h, up 54 h of exposure. The larvae were then placed on an artificial diet to continue their development. Pupariation time, adult emergence and adult longevity were also analysed. The larvae of both species that fed on the cocaine-containing livers developed faster than those that fed on the livers of the control animals, leading to the conclusion that cocaine influences and stimulates larval growth. The difference in growth between the control and treated flies was best observed from 12 h of exposure onward. This finding has important implications for forensic investigations.     

The effects of larval crowding and food type on the size and development of the blowfly, Calliphora vomitoria

The use of entomological evidence in the estimation of the post mortem interval (PMI) often depends on the size and developmental stage of blowfly larvae collected from a corpse. Therefore, factors which can have an effect on the larval size and growth rate can have implications for reliable PMI determinations. This study explores the competitive effects of larval overcrowding on Calliphora vomitoria reared on three different pig tissues--liver, brain and muscle. The competitive feeding environment within the more crowded larval cultures resulted in increased development rates and the production of undersized larvae and adults. Variation in the extent of these effects was observed on each of the three body tissues, highlighting the importance of documenting the positions from which entomological evidence is recovered from a corpse.     

Quantifying the Temperature of Maggot Masses and its Relationship to Decomposition

Numerous Calliphoridae species have been observed to form larval aggregations during the feeding stage of development, resulting in localized increases in temperature. This study investigates the relationship between maggot numbers in a mass and heat generation. Single‐species aggregations (Lucilia sericata) of various sizes (50–2500 individuals) were reared in the laboratory at a constant ambient temperature of 22°C. Internal mass temperatures were recorded every 5 min throughout the feeding stage of development. Results showed that mass temperatures increased with mass numbers (p‐value < 0.001), ranging from 2.5 to 14°C above ambient. A minimum mass size of 1200 produced overall temperatures that were significantly warmer than ambient, diverging away from 22°C after c. 26 h. These results indicate that the microclimate of a mass has the potential to differ significantly from ambient, which may be influencing larval development rates and should therefore be factored into mPMI estimates to increase accuracy.     

Preliminary observations on the effects of hydrocortisone and sodium methohexital on development of Sarcophaga (Curranea) tibialis Macquart (Diptera: Sarcophagidae), and implications for estimating post mortem interval.

Larvae of Sarcophaga (Curranea) tibialis (S. tibialis) were reared at constant temperature on chicken liver treated with a steroid or a barbiturate at concentrations that would be lethal, half-lethal and twice-lethal doses for humans. Trends to greater mortality at higher drug concentrations were not statistically significant. Larvae exposed to either drug took significantly longer to reach pupation compared to those in the control, while larvae exposed to sodium methohexital passed through pupation significantly faster than those in the control. No systematic relationship was found between drug concentration and development time of larvae or pupae. The total developmental period from hatching to eclosion did not differ between treatments, implying that estimates of post mortem intervals- (PMI) based on the emergence of adult flies will not be affected by the involvement of these drugs in a case. On the other hand, anomalous pupation spans may indicate the presence of barbiturates. These findings are compared with patterns found in another fly fed other contaminants.     

PCR-DHPLC法检测硅藻SSU基因在溺死鉴定中的应用

目的采用PCR-DHPLC法检测硅藻SSU基因,评估其在溺死鉴定中的应用价值。方法 60只实验兔随机分为生前溺死（水中溺毙）、死后入水（空气栓塞致死后入水）、对照组（空气栓塞致死后不做处理）;溺死人体脏器组织;取各组织检材提取硅藻DNA,PCR扩增硅藻特异的核糖体小亚基（SSU）片段,用琼脂糖凝胶电泳检测、DHPLC检测分析。结果 6份硝酸消化法检测阴性的溺死人体器官组织检材经PCR及琼脂糖凝胶电泳检出5例阳性。生前溺死组肺、肝、肾硅藻检出率分别为100%、90%、85%,死后入水组仅肺检出硅藻（15%）,对照组各组织均为阴性;生前溺死组检出率明显高于死后入水组（P〈0.05）。10份溺死人体器官组织检材采用DHPLC法检出硅藻种类明显多于微波消解-扫描电镜法（P〈0.05）。脏器检出硅藻种类与溺死点水样一致。结论采用PCR-DHPLC法检测硅藻SSU基因,有助于溺死鉴定和溺死地点的推断,具有法医学应用价值。     

Maggot development during morgue storage and its effect on estimating the post-mortem interval

When insect evidence is obtained during autopsy, forensic entomologists make decisions regarding the effects of low-temperature (-1 degrees C to 4 degrees C) storage of the body and associated insects when estimating the post-mortem interval (PMI). To determine the effects of storage in a morgue cooler on the temperature of maggot masses, temperatures inside and outside of body bags containing a human cadaver and porcine cadavers (seven replicates) were measured during storage. Temperatures remained significantly higher (p<0.05) inside of the body bags relative to the cooler, and remained at levels sufficient for maggot feeding and development. If the assumption that no insect development takes place during preautopsy refrigeration is made, potential error rates in PMI estimation of 8.6-12.8% occur. The potential for blow fly larvae to undergo significant development while being stored in the morgue is a possibility that forensic entomologists should consider during an investigation involving samples collected from autopsy. Case and experimental evidence also demonstrate that substantial tissue loss can occur from maggot feeding during morgue storage.     

Fatal intoxication as a consequence of intranasal administration (snorting) or pulmonary inhalation (smoking) of heroin

In recent years we have noticed an increasing proportion of mortalities resulting from an overdose of heroin that involve routes of administration other than injection. Of 239 cases of fatal heroin intoxication examined at our department during the period 1997-2000, 18 deaths were associated with non-parental administration. Seven of these fatalities were experienced heroin users who had begun to use more sporadically, seven were recreational "party-users", while the remaining four persons had relapsed into heroin use following long periods of abstinence. The median blood morphine concentration of these non-injectors was 0.095 microg/g (range: 0.02-0.67 microg/g), significantly lower than that of the injectors. Concurrent use of alcohol, other illicit drugs and/or pharmaceutical preparations was observed in 17 of the 18 cases. However, there were no statistically significant differences between the victims of heroin intoxication by injection or by other routes with respect to the proportion who had simultaneously consumed alcohol or benzodiazepines. Pathological alterations like lung fibrosis, liver cirrhosis, endocarditis, etc. were not found to play a significant role in any of the 18 mortalities. We conclude that snorting or smoking heroin probably involves a reduced risk of obtaining high blood concentrations of morphine but still constitutes a considerable risk of lethal outcome due to high variability in blood concentrations. Furthermore, decreased tolerance resulting from periods of reduced or sporadic use appears to be an important risk factor in connection with heroin overdosing by snorting or smoking, which indicate that some heroin addicts may inaccurately assume that these routes of administration are safe when resuming their use of heroin after a period of abstinence.     

The introduction of CCTV and associated changes in heroin purchase and injection settings in Footscray,Victoria, Australia

Objectives

In June 2011, closed-circuit television (CCTV) was introduced in Footscray (a suburb of Melbourne, Australia) to help deter street-based drug trading. We investigate whether there were subsequent shifts in the settings (e.g., street, house) in which heroin was purchased or injected by people who inject drugs (PWID).

Methods

Using heroin purchase data from the Melbourne Injecting Drug User Cohort Study, multinomial logistic models with fixed effects for CCTV introduction were used to estimate the percentage of: (1) heroin purchased on the street, from mobile dealers and in house settings; and (2) heroin injections occurring in street, car, public toilet, and house settings. Displacement effects were investigated with a logistic model capturing the likelihood of traveling to Footscray to purchase heroin.

Results

Following CCTV introduction, the percentage of heroin injections occurring in public toilet settings decreased by 13 % (95 % CI ?27 %, ?0 %). This was accompanied by a non-significant increase in the percentage of heroin injections in street settings of 23 % (95 % CI ?1 %, +41 %). Changes in other settings were small and non-significant. No suburb displacement effects were found.

Conclusions

The introduction of CCTV in Footscray may have displaced PWID who previously injected heroin in public toilets to street settings. Apart from this, Footscray’s street-based heroin market operates much as it did before CCTV.

     

重伤家兔体内Cu及代谢酶变化与MOF及死亡关系

目的探讨兔重伤后机体内铜（Cu）含量及其代谢酶铜锌超氧化物歧化酶（Cu/Zn-SOD）、细胞色素c氧化酶（CCO）活性变化与继发性多器官衰竭（MOF）及死亡的关系。方法参照国际通用的创伤程度量化评分标准（ISS）,建立致MOF的家兔重伤模型,检测伤后不同时段血清及组织Cu含量与Cu/Zn-SOD和CCO的活性,分析其变化规律与继发MOF及死亡的关系。结果①重伤组家兔（包括死亡组家兔）血清Cu含量在伤后12h显著下降,36h后逐步回复;脑、心、肺组织中Cu含量伤后下降,肝脏组织Cu含量于伤后12h显著升高,36h后回落。伤后死亡组家兔脑、心、肺、肝Cu含量均较对照组降低,其中脑、肝组织Cu含量下降显著。②重伤组家兔血清Cu/Zn-SOD活性伤后12h下降3,6h之后呈升高的趋势,肝脏组织中的Cu/Zn-SOD活性伤后持续显著升高。③重伤后家兔（包括死亡组家兔）大脑皮质、心、肝CCO活性变化不明显,但脑干组织CCO活性显著升高。伤后死亡组家兔脑、心、肺、肝等器官的大体及组织学呈MOF改变。结论严重创伤可引发兔血清和组织Cu水平及Cu/Zn-SOD、CCO活性变化,这些变化可能与继发多器官衰竭和死亡相关。     

Third molar development according to chronological age in populations from Spanish and Magrebian origin

Spain is frequently the entrance country into the European Union for undocumented immigrants, especially those from the Magreb (Northern Africa). Forensic age estimates for these persons are difficult because systematic studies of dental maturity are lacking. Three different populations were analyzed to determine the pattern of development of third molars as a tool for age estimation in people of different ethnic and geographic origin. Orthopantomograms from two different populations of Spanish origin (Galicia in northwestern continental Spain, and Ceuta, a Spanish province in Northern Africa) were compared to radiographs of molars from a Magrebian population (Northern Africa) resident in Ceuta. Orthopantomograms were obtained from a private dental clinic (n=344) in Galicia and from the Public Oral Health Services (n=228) in Ceuta. We looked for differences in third molar mineralization (determined with the Demirjian scale) that might serve as age indicators (older versus younger than 18 years). Differences in maturation patterns were found between sexes and populations of origin. Mineralization of tooth 38 was more advanced in males than females among subjects 18 years of age and older in all three populations. Moreover, mineralization of tooth 38 in subjects aged 18 years and older was significantly slower in the Spanish-Galicia population than in the Magrebian-Ceuta population. We found no significant differences between Magrebian and Spanish individuals from Ceuta. We conclude that differences in tooth 38 mineralization may be related more with socio-geographical than ethnic origin (ancestry), and thus constitute evidence of the relevance of socio-geographic rather than genetic factors in third molar development. We used ROC analysis to determine the accuracy of the examiner's ability to correctly estimate age as younger or older than 18 years. The results suggest that Demirjian stage for tooth 38 can be considered a good indicator of age in all three populations.