Preliminary Analysis of Methamphetamine Detection in Lucilia sericata (Diptera: Calliphoridae) Reared in Methamphetamine-Treated Meat at Various Developmental Stages.

Background: It is possible to identify drugs and poisons present in cadavers by analyzing blowfly larvae and pupae collected during forensic autopsies. The main purpose of this study was to use Lucilia sericata's larvae and pupae to identify drugs and poisons present in human cadavers. Methods: In an investigation, immature L. sericata fed meat treated with methamphetamine (MA) at various concentrations (45, 90, and 180 ng/mg) were analyzed to detect MA. Acetylation derivatization and liquid-liquid extraction (LLE) were used as sample preparation methods prior to gas chromatography-mass spectrometry (GC-MS) analytical instrumentation to find MA. Results: According to this study, L. sericata can be used in toxicological testing to identify MA in a host body. All L. sericata larval stages, particularly the third stage larva, pupa and empty pupa tested were positive for MA. Larvae in their first instar produced weak peaks. The L. sericata post-feeding instar following the 45 ng/mg treatment showed the highest MA concentration. For the first time, derivatization using the acetylation approach was used to prepare samples, and successfully, excellent results were obtained. Conclusion: Low quantities of MA can be easily found in immature fly samples using GC-MS. It is important to analyze all samples including human tissues and insect samples, for postmortem drug testing. They can be utilized to find entire MA before they are excreted in excretory samples such as urine. Also, third instar larvae are a great and reliable sample for toxicological study.

First Report of Natural Infection of Phlebotomus mongolensis to Leishmania major and Leishmania turanica in the Endemic Foci of Zoonotic Cutaneous Leishmaniasis in Iran.

Background: The primary aim of this study is to determine infection to Leishmania parasites in the wild population of Phlebotomus caucasicus and Phlebotomus mongolensis using molecular methods in some important zoonotic cutaneous leishmaniasis foci in Iran. Methods: Sand flies were collected from active colonies of rodent burrows from 16 trapping sites using sticky trap paper. In order to detect and identify of Leishmania parasites in females Ph. caucasicus and Ph. mongolensis, the Nested-PCR amplification of ITS2-rDNA region was performed to generate amplicon with 245bp for Leishmania major, 206bp for L. gerbilli and 141bp for L. turanica. Results: In the current study we found DNA of different gerbil parasites such as L. major and L. turanica, and mixed infection of L. major/L. turanica in Ph. caucasicus and Ph. mongolensis. It should be noted that, in Iran, natural infection with Leishmania parasites is recorded for the first time in this study in Ph. mongolensis. Conclusion: Both species of Ph. caucasicus and Ph. mongolensis not only may participate in the ZCL transmission cycle between reservoir hosts, but also results of this study support the role of these species as secondary vectors in the transmission of leishmaniasis to humans.

Comparative Performance of Different Traps for Collection of Phlebotominae Sand Flies and Estimation of Biodiversity Indices in Three Endemic Leishmaniasis Foci in North Khorasan Province, Northeast of Iran.

BACKGROUND: Phlebotominae sand flies (Diptera: Psychodidae) are the vectors of leishmaniasis. There are different methods for sand fly collection with different performance. The purpose of the current study was to compare the effect of different traps for collection of Phlebotominae sand flies in three endemic leishmaniasis foci in North Khorasan Province, northeast of Iran. METHODS: Sand flies were collected using seven different traps from three villages, three times each twenty days during peak periods of seasonal activity in 2016. RESULTS: A total of 7253 sand flies were collected. The specimens belonged to19 species. Phlebotomus sergenti was the most predominant species in the study area. Light trap baited with Carbon dioxide (CLT) and sticky paper trap (SPT) caught 22.6% and 22.3% of sand flies respectively. Animal baited trap (ABT) and white Shannon trap (WST) caught significantly fewer sand flies than the other traps. The sex ratio was different by phlebotominae sand fly species and collection methods. The sex ratio was highest in SPT and lowest in black Shannon trap (BST). Species diversity and species richness in SPT were more than other traps. CONCLUSION: Our findings confirm that CLT and SPT are the most efficient sand fly collection methods. CLT is higher attractive for females and Phlebotomus genus and is an ideal method for monitoring the population of Phlebotomus genus during surveillance. SPT is an inexpensive, convenient and easy to be used to detect the presence of sand flies at low densities and provide a more realistic estimation of sand flies biodiversity.

Toxicological Analysis of Insects on the Corpse: A Valuable Source of Information in Forensic Investigations.

BACKGROUND: Entomotoxicology as a subset of forensic entomology can be used by analysis of carcass feeding insects to detecting of drugs or toxins, as well as the cause and manner of death in cases of ante-mortem drugs intoxication. Morphine is one of the deacetylate metabolites of heroin. The aim of this study was to determine the presence and quantity of morphine in insects on the carcass and compare them with decomposing carcass. METHODS: Field of this study was in Chalabeh District and toxicological tests were carried out at the Department of Forensic Toxicology, Legal Medicine Center, Kermanshah, Iran in 2017. Morphine was inoculated into live rabbit as experimental model at concentrations of 12.5, 25, 50mg/ml, similar to those normally encountered in human overdoses, then quality and quantity of morphine were determined in insects such as Chrysomya albiceps (as the first wave of insect succession on human cadavers) fed on carcass. RESULTS: Quantitative assessment at larvae showed that morphine was detected in all larvae (feeding and post feeding stage) fed on tissues from carcasses administered morphine, except for post-feeding larvae from R1 which received 12.5mg/ml dosage of morphine. CONCLUSION: Necrophagous insects are an indicator on the scene of crime and a potential source of information about the antemortem situation. Detection of drug in insects which is actually a reflection of the cause of death is possible.

Richness and Diversity of Phlebotomine Sand Flies (Diptera: Psychodidae) in North Khorasan Province, Northeast of Iran.

BACKGROUND: We aimed to determine the species composition, richness and diversity indices of the Phlebotomine sand flies at different topographic condition in visceral (VL) and cutaneous leishmaniasis (CL) foci in the northeast of Iran. METHODS: This cross-sectional study was conducted during 2016 in North Khorasan Province. The sampling was focused on rural regions, where human cases of VL and/or CL were diagnosed and reported during last 5 years. Sand flies were collected three times each twenty days during peak periods of seasonal activity. Seven collection methods were used. Some Alpha and Beta diversity indices were calculated. RESULTS: Overall, 7253 sand flies were collected and identified. They were from 19 species of Phlebotominae sand-flies. Phlebotomus sergenti and Ph. papatasi were the most prevalent (84.9%) species in the study area. Species richness (S) was very different in three areas and were18, 8, and 4 respectively but Evenness (E) were 0.357, 0.345, and 0.380, so evenness was almost equal in the study areas. Shannon Index (H) and Margalef Richness Index were calculated 1.033, 0.718, 0.527 and 2.117, 0.8998, 0.4006 respectively. CONCLUSION: The sand fly fauna in North Khorasan Province was very rich and often included some of the most important proven or suspected vectors of leishmaniasis. Species diversity indices (Shannon index, and Simpson's index) were not high due to decreasing in evenness. The Margalef richness index could accurately reflect the biodiversity of sand flies between three subtidal locations.

Evaluation of Insect Succession Patterns and Carcass Weight Loss for the Estimation of Postmortem Interval.

This study assesses the succession of insects on rabbit carcass. The study was conducted in sunny and shaded sites in the west of Iran during four seasons in 2016 and 2017. Based on the results of this study, various factors such as body size, carcass location, and carcass injury could affect the pattern of insect succession on carcass. In this study, we estimated the elapsed time since death (R2 > 0.98, P = 0.00) based on carcass weight loss during the stages of decomposition and the cubic method. Jaccard analysis was performed to determine the similarity of insect taxa during decomposition of rabbit carrion in two different sites during a period of four seasons. Succession pattern analysis for necrophagous insects in both habitats showed a similarity between bloat and decay stages for each habitat. On the other hand, pairwise similarities in taxa were low at the fresh and dry stages, however increased at bloat and decay stages of decomposition. This study shows that succession has some limitations in determining the elapsed time of death. Therefore, the use of source of information such as a weight loss model seems to be essential.

Some epidemiological aspects of cutaneous leishmaniasis with emphasis on vectors and reservoirs of disease in the borderline of Iran and Iraq.

Cutaneous leishmaniasis (CL) is endemic and a major health problem in 17 provinces out of 31 in Iran. This study aimed to determine vectors and reservoirs of the disease using molecular techniques in the borderline of Iran and Iraq. Sand flies and rodents were sampled using sticky paper traps and metal wire live traps, respectively, in the selected villages. About 10% of archived confirmed human positive slides was randomly checked for Leishmania by PCR-RFLP assay. The female sand flies were dissected in alcohol 96% in a sterile condition, the head and two segments of the abdomen end permanently mounted for identification and the remaining of body used for DNA extraction. The direct parasitological tests were carried out on the stained slides of rodents for Leishmania as well as PCR-RFLP assay used for molecular detection of parasite. A total of 2050 sand flies were identified comprising of Phlebotomus papatasi, Sergentomyia sintoni, Se. clydei, Se. mervynae, Se. theodori, Se. dentate and Se. iranica. The Ph. papatasi was ranked as a prevailing sand fly species. Molecular tests on female sand flies revealed infection of Ph. papatasi to Leishmania major. Direct parasitology and molecular tests confirmed of 20% infection to L. major among the sole rodents species "Tatera indica". Due to wide dispersion of rodents colonies in the area and long favorite climate condition for sand flies, the CL foci will be provided the health risk for the religious tourists.

Susceptibility status of wild population of Phlebotomus sergenti (Diptera: Psychodidae) to different imagicides in a endemic focus of cutaneous leishmaniasis in northeast of Iran.

BACKGROUND & OBJECTIVES: Phlebotomine sandflies (Diptera: Psychodidae) transmit several important zoonotic diseases to humans and leishmaniasis is one of them. Two types of leishmaniasis, viz. visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are endemic in Iran. The main vector of anthroponotic cutaneous leishmaniasis (ACL) is Phlebotomus sergenti. The aim of the present study was to determine the susceptibility status of wild strain of P. sergenti to different imagicides of DDT, bendiocarb and permethrin at the median lethal time, LT50 level. METHODS: Sandflies were collected from selected village in North Khorasan Province, northeast of Iran from indoors using CDC light-traps. Susceptibility test was carried out against DDT (4%), bendiocarb (0.1%) and permethrin (0.75%) for all the females according to WHO method, and mortality was calculated. Species identification was carried out using the morphological keys. Data were analysed using probit regression analysis to determine the LT50 and LT90 values. RESULTS: In total, 851 female P. sergenti sandflies were tested. LT50 values to DDT (4%), Bendiocarb (0.1%) and permethrin (0.75%) were 15.4, 19.2 and 6.3 min respectively. The values for LT90 were 51.1, 47.4 and 18.6 min respectively. The mortality rates for 1 h exposure time to DDT, bendiocarb and permethrin were 89.8 ± 1.4; 93.6 ± 1.4; and 95.6 ± 1.7%, respectively. INTERPRETATION & CONCLUSION: The suscesptibility studies revealed development of resistance against DDT (4%) in the wild strain of P. sergenti population. Monitoring and mapping of insecticide resistance in the region is recommended for vector control.

Control of Cutaneous Leishmaniasis Using Deltamethrin Treated Nets in Comparison to Indoors Residual Spraying in a Rural Area of Iran.

BACKGROUND: Cutaneous leishmaniasis (CL) is an important vector-borne disease in Iran. The present study aimed to evaluate the efficacy of Deltamethrin treated nets in comparison to Indoors Residual Spraying (IRS) for the control of leishmaniasis in a new focal area of Iran. METHODS: The study was carried out in 5 villages in the west of Iran during 2012-2013. One village was selected to the application of Insecticide Treated Nets (ITNs, 25 mg/m2 deltamethrin) among residents. Four other villages were considered for IRS using deltamethrin emulsion, 2.5% EG and Aicon 10WP. Spraying was repeated after 3 month. Other control methods e.g. environmental managements, and treatment of positive cases were carried out in all villages. RESULTS: After 1 yr of trial, the cases of disease reduced from 50 to 2 and 128 to 5 for ITNs distributed nets and use of IRS in the villages, respectively. CONCLUSION: Reduction of CL cases in both methods were significant (P<0.001).

Situational Analysis of Visceral Leishmaniasis in the Most Important Endemic Area of the Disease in Iran.

BACKGROUND: Visceral leishmaniasis is one of the most important vector borne diseases in the world, transmitted by sand flies. Despite efforts to prevent the spread of the disease, cases continue worldwide. In Iran, the disease usually occurs in children under 10 years. In the absence of timely diagnosis and treatment, the mortality rate is 95-100%. The main objective of this study was to determine the spatial and temporal distribution of visceral leishmaniasis as well as its correlation with climatic factors for determining high-risk areas in an endemic focus in northwestern Iran. METHODS: In this cross-sectional study, data on VL cases were collected from local health centers in Ardabil Province, Iran during 2001-2015 to establish a geodatabase using ArcGIS10.3. Data analysis was conducted using SPSS23 and ArcMap Spatial Analyst. MaxEnt model was used to determine ecologically suitable nichesfor the disease. RESULTS: Two hotspots were found in Meshkinshahr and Germi counties with 59% and 23% of total cases, respectively. There was an increase in the incidence rate of VL in Ardabil County from 2.9 in 2009 to 9.2/100,000 population in 2015. There was no spatial autocorrelation between county and total number of cases (P> 0.05). Higher NDVI, lower altitude and southern aspects had positive effects on the presence probability of VL. CONCLUSION: The number of cases of this disease have been rising since 2013 and doubled in 2015. According to the derived distribution maps, the disease is spreading to new locations such as Ardabil and Namin counties.

Predicting the Distribution of Phlebotomus papatasi (Diptera: Psychodidae), the Primary Vector of Zoonotic Cutaneous Leishmaniasis, in Golestan Province of Iran Using Ecological Niche Modeling: Comparison of MaxEnt and GARP Models.

Zoonotic cutaneous leishmaniasis (ZCL) is a prevalent vector-borne disease in the Golestan province of Iran, with Phlebotomus papatasi (Scopoli, 1786) serving as the main vector. The aim of this study was to model the probability of presence of this species in the study area, and to determine the underlying factors affecting its distribution. Three villages were selected from each county of the province and visited monthly for investigating ZCL. Sticky paper traps were used for collecting the sand flies to determine the species present. The presence of Ph. papatasi was modeled using genetic algorithm for rule-set production (GARP) and maximum entropy (MaxEnt) techniques. Both models showed the central and northern parts of the province with lowland areas were more vulnerable to Ph. papatasi propagation, in comparison with the southern parts with mountainous and forest areas. The area under curve (AUC) of MaxEnt model for the training points was calculated as 0.90, indicating excellent performance of the model in predicting Ph. papatasi distribution. Jackknife test showed that the factors with the greatest influence in vector distribution were slope, vegetation cover, annual mean temperature, and altitude. By using ecological niche models, it is possible to identify areas with higher probability of presence of Ph. papatasi, which guides public health policy makers for planning better vector control interventions.

Spatial Analyses of the Relation between Rodent's Active Burrows and Incidence of Zoonotic Cutaneous Leishmaniasis in Golestan Province, Northeastern of Iran.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is one of the most important vector-borne diseases in Iran. Wild Rodents play as a reservoir. The main aim of this study was to determine spatial analyses of the relationship between rodent's active burrows and Incidence of ZCL in Golestan Province, north east of Iran. METHODS: The cross-sectional study was conducted in 59 rural districts in Golestan Province. Spatial distribution of rodent's active burrows, human cases of ZCL and Incidence of disease were collected, using Geographical Information Systems (GIS). The relationship of them were analyzed by Sperman test, SPSS software version No.13. RESULTS: The most number of rodents' active burrows, human positive cases (100 persons) and high Incidence of disease (35/1000) were observed in Korand rural district of Gonbad-e Kavoos County. There was significant correlation between the number of rodents active burrows with Incidence rate of disease (0.470, P< 0.001) as well as the number of cases in each districts (0.465, P< 0.001), There is high correlation between higher Incidence rate and human positive cases in districts with number of rodents' active burrows. CONCLUSION: Vicinity of wild rodents' burrows to villages plays an important role in transmission of ZCL to humans.

Molecular Detection of Leishmania major and L. turanica in Phlebotomus papatasi and First Natural Infection of P. salehi to L. major in North-East of Iran.

BACKGROUND: Leishmaniasis is an important public health disease in many developing countries as well in Iran. The main objective of this study was to investigate on leishmania infection of wild caught sand flies in an endemic focus of disease in Esfarayen district, north east of Iran. METHODS: Sand flies were collected by sticky papers and mounted in a drop of Puri's medium for species identification. Polymerase chain reaction techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism were used for identification of DNA of Leishmania parasites within infected sand flies. RESULTS: Among the collected female sand flies, two species of Phlebotomus papatasi and Phlebotomus salehi were found naturally infected with Leishmania major. Furthermore, mixed infection of Leishmania turanica and L. major was observed in one specimen of P. papatasi. Sequence analysis revealed two parasite ITS1 haplotypes including three L. major with accession numbers: KJ425408, KJ425407, KM056403 and one L. turanica. (KJ425406). The haplotype of L. major was identical (100%) to several L. major sequences deposited in GenBank, including isolates from Iran, (Gen Bank accession nos.AY573187, KC505421, KJ194178) and Uzbekistan (Accession no.FN677357). CONCLUSION: To our knowledge, this is the first detection of L. major within wild caught P. salehi in northeast of Iran.

Diversity of Sand Flies (Diptera: Psychodidae) in Endemic Focus of Visceral Leishmaniasis in Azar Shahr District, East Azarbaijan Province, North West of Iran.

BACKGROUND: There are nearly 1000 species of Phlebotomine sand flies in 6 genera, of which only two, Phlebotomus in the old world and Lutzomyia in the new world are medically important. Globally, leishmaniasis prevalent in 98 countries and affects estimated 12 million people with almost two million new cases per year. Some rural areas of Azarshahr District in East Azarbaijan Province have been reported to be endemic for visceral leishmaniasis. This study is the first attempt to determine the species diversity and density in a new focus of visceral leishmaniasis in Azarshahr District, East Azarbaijan Province, Iran. METHODS: Sand flies were collected form indoor and outdoor biweekly using sticky traps. Diversity index of the collected sand flies within different villages were estimated by the Shannon-Weaver. RESULTS: The activity of the sand flies extended from April to October with one peak in August. Diversity of sand flies within study area were estimated as 0.917, 1.867, 1.339, 1.673, and 1.562 in Almalodash, Jaragil, Segaiesh, Amirdizaj, and Germezgol Vvillages, respectively. CONCLUSION: Identifying the diversity and seasonal abundance of the collected species is of importance for prediction of the period of maximum risk for leishmaniasis transmission and for the successful implementation of a control program. Species diversity is one of the most important factors in ecological studies.

Application of Flumethrin Pour-On on Reservoir Dogs and Its Efficacy against Sand Flies in Endemic Focus of Visceral Leishmaniasis, Meshkinshahr, Iran.

BACKGROUND: Visceral leishmaniasis (VL) is one of the most important parasitic zoonotic diseases in the world. Domestic dogs are the main domestic reservoirs of VL in endemic foci of Iran. Various methods, including vaccination, treatment of dogs, detection and removal of infected dogs have different results around the world. General policy on control of canine visceral leishmaniasis is protection of them from sand fly bites. The aim of this study was evaluation of pour-on application of flumethrin on dogs against blood-feeding and mortality of field-caught sand flies. METHODS: Once every 20 days from May untill September 2013, the treated and control dogs were exposed with field caught sandflies for 2 hours under bed net traps. After the exposure time, both alive and dead sand flies were transferred in netted cups to the laboratory. The mortality rate of them was assessed after 24 hours. The blood-fed or unfed conditions were determined 2 hours after exposure to the dogs under stereomicroscope. RESULTS: The blood feeding index was varied from 12.0 to 25.0 % and 53.0 to 58.0 % for treated and control dogs respectively (P< 0.0001). The blood feeding inhibition was 75.0-87.0 % and 41.0-46.0 % for the control and treated dogs (P< 0.0001), respectively.The total mortality rate was 94.0-100 % and 19.0-58.0 % respectively for the treated and control groups (P< 0.001). CONCLUSTION: Application of pour-on flumethrin on dogs caused 90-100 % mortality until 2.5 month and inhibited the blood-feeding of sand flies.