Treatment of Cutaneous Leishmaniasis lesion caused by Leishmania major Using Leech Therapy in BALB/c Mice.

BACKGROUND OBJECTIVES: Amphotericin B, Allopurinol, Glucantime and Beta-Glucan are the main drugs currently used for the treatment of cutaneous leishmaniasis. In present study, we evaluated the effect of medical leeches on BALB/c mice models infected with Leishmania parasites. METHODS: To create animal models of leishmaniasis, BALB/c mice were inoculated with the standard Leishmania major strain MRHO / IR / 75 / ER. Treatment was performed through blood sampling with leeches. We used medical leech (Hirudo medicinalis) species. This leech is a European medicinal leech, one of several species known used as medicinal leech. 22 mice infected with Leishmania major were used for the tests .The studied mice were divided into two groups which were named with the letters L and D. The duration of blood sampling was 25 min for initial treatment, 20 min for the 2nd blood sampling and 15 min for the third times and onwards. Meglumine antimoniate (Glucantime) is a pentavalent antimony (SbV) that recommended by the World Health Organization as a golden standard treatment for all kind of leishmaniasis, but many side effects are still reported. Criteria for anti-leshmania assessment of Leech were including: Leech saliva has an anti-leishmania effect by acting on the purin pathway. Leech saliva prevents the growth and multiplication of Leishmania by a mechanism of action similar to Glucantim (breaking ATP). RESULTS: The duration of leech treatment in BALB/c mice infected with L.major parasites was about 6-8 weeks. Out of the 22 leishmaniasis mice models that underwent treatment, 13 survived until the end of the treatment duration. The recovery rate was about 56%; lesions improved in five out of the 13 surviving mice models. Paired t-test showed a significant difference between the mean wound size at the beginning of treatment and the 8th week of treatment (PV <0.0001). INTERPRETATION CONCLUSION: Considering the fact that the standard drugs for the treatment of leishmaniasis have many side effects, alternative therapies such as leech therapy can be effectively used to treat these chronic wounds. There are about 20 active substances in leech saliva. These substances include antistasin, eglins, guamerin, hirudin, saratin, bdellins, complement, and carboxypeptidase inhibitors. Leech saliva is an anticoagulant, anti-inflammation and prevents the platelet. Leech therapy was effective in treating leishmaniasis wounds in BALB/c mice. In this study, there was complete wound healing in 56% of the mice models.

Rodents as vehicle for delivery of transgenic bacteria to make paratransgenic sand fly vectors of cutaneous leishmaniasis in field condition.

Vector-borne diseases, among them leishmaniasis, cause more than 700,000 deaths annually. The lack of an effective vaccination and the increasing resistance of sand flies to insecticides require the urgent development of innovative approaches to contain the disease. The use of engineered bacteria that express anti-parasite molecules (paratransgenesis) shows much promise. However, a challenge for implementation of this strategy is to devise means to introduce modified bacteria into sand flies in the field. In this study, we use rodent food bait as a delivery strategy to introduce two mCherry-fluorescent bacteria, Serratia AS1 and Enterobacter cloacae, into adult sand flies in field settings. Bacteria-infected food was provided to Rhombomys opimus rodents. These bacteria transiently pass through the rodent alimentary tract and are delivered to larval habitats with the rodent feces. The feces are ingested by sand fly larvae and, in the case of Serratia AS1, are trans-stadially transmitted to adults. This is the first report of targeting delivery of Serratia AS1 in a paratransgenic system to control transmission of leishmaniasis under field condition. This novel strategy shows promise for delivering transgenic bacteria to Leishmania vectors in the field.

New data on medically important scorpion species of Iran based on seven physiographic areas.

There are 2612 and 80 scorpion species in the world and Iran, respectively. Iran is rich in climatic conditions and biodiversity. In this study, new data on scorpion fauna of different physiographic areas of Iran were obtained from various databases, including plains and deserts, eastern area of the Caspian Sea, western and central areas of the Caspian Sea, heights and foothills, the west coast of the Persian Gulf and the east coast of the Persian Gulf. Then, spatial distribution maps for scorpion species in different physiographic areas were prepared using ARC GIS software. There are three families (Buthidae, Hemiscorpiidae, and Scorpionidae), 19 genera, and 80 species and subspecies. In plains and deserts, there are three families, 15 genera, and 37 scorpion species; in the Caspian Sea area, there is only one family with four scorpions; in heights and foothills, there are three families, 14 genera, and 35 scorpion species; in the west coast of the Persian Gulf, there are three families, 13 genera, and 31 scorpion species, and finally, in the east coast of the Persian Gulf, there are three families, 14 genera, and 35 scorpion species. The Buthidae is the most prevalent family in Iran. Because of good climatological conditions on the east and west coast of the Persian Gulf, there are three families of scorpions, 18 genera, and 45 species. The majority of scorpion species inhabit this region, and the highest rate of scorpionism and death due to scorpion sting occurs in this part of Iran.

Molecular and Biochemical Detection of Insecticide Resistance in the Leishmania Vector, Phlebotomus papatasi (Diptera: Psychodidae) to Dichlorodiphenyltrichloroethane and Pyrethroids, in Central Iran.

The aim of the present study was to explore resistance markers and possible biochemical resistance mechanisms in the Phlebotomine sand fly Phlebotomus papatasi in Esfahan Province, central Iran. Homogenous resistant strains of sand flies were obtained by exposing P. papatasi collected from Esfahan to a single diagnostic dose of DDT. The adults from the colony were tested with papers impregnated with four pyrethroid insecticides: Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15%, and Lambdacyhalothrin 0.05% to determine levels of cross-resistance. To discover the presence of mutations, a 440 base pair fragment of the voltage gated sodium channel (VGSC) gene was amplified and sequenced in both directions for the susceptible and resistant colonies. We also assayed the amount of four enzymes that play a key role in insecticide detoxification in the resistant colonies. A resistance ratio (RR) of 2.52 folds was achieved during the selection of resistant strains. Sequence analysis revealed no knockdown resistance (kdr) mutations in the VGSC gene. Enzyme activity ratio of the resistant candidate and susceptible colonies were calculated for α-esterases (3.78), ß-esterases (3.72), mixed function oxidases (MFO) (3.21), and glutathione-S-transferases (GST) (1.59). No cross-resistance to the four pyrethroids insecticides was observed in the DDT resistant colony. The absence of kdr mutations in the VGSC gene suggests that alterations in esterase and MFO enzymes are responsible for the resistant of P. papatasi to DDT in central Iran. This information could have significant predictive utility in managing insecticide resistant in this Leishmania vector.

Baseline susceptibility of a wild strain of main vectors of leishmaniasis to WHO-recommended insecticides in southeastern Iran.

BACKGROUND: In Iran, both cutaneous leishmaniases (CL) and visceral leishmaniases (VL) are endemic, recording one of the 10 highest CL prevalence in the world. Parasites are transmitted by the bite of infected Phlebotomus sand fly females. Several sand fly species have been identified as vectors in the studied region of Kerman province. Residual spraying to control adult sand flies, is the only way to decrease the spreading of the diseases but, following control treatment against malaria vectors in endemic areas in Iran, resistance or tolerance to insecticides emerged in some sand fly species. The objective of this study was to survey insecticides susceptibility levels of 3 vector species in wild sand fly populations in different foci of the diseases in Kerman province. Ph. sergenti, and Ph. papatasi respectively vectors of anthroponotic and zoonotic cutaneous leishmaniases and for the first time Ph. alexandri one of the anthroponotic visceral leishmaniases vector were tested against: deltamethrin 0.05%, malathion 5%, dichloro-diphenyl-trichloroethane (DDT) 4%. MATERIALS AND METHODS: In leishmaniases endemic areas species specific sand fly sites were selected in Kerman province, and specimens were collected by manual aspirators at different time intervals during the spring and summer 2019. All the susceptibility tests were performed according to the WHO tube test recommended procedure. RESULTS: Twenty five blood-fed female sand flies from the region's prevalent species were used in each pooled test replicates. All wild specimens died within 60 min of exposure to DDT 4%, malathion 5%, and deltamethrin 0.05%, but the mortality rate for Ph. papatasi exposed to malathion and DDT was 91.6% and 66.6%, respectively. CONCLUSION: According to current study results, Ph. sergenti and Ph. alexandri are highly susceptible to all the evaluated insecticides in the study areas. However, Ph. papatasi was susceptible to deltamethrin (100% mortality), possibly resistant or tolerant to malathion (91.6% mortality), and confirmed to be resistant to DDT (66.6% mortality).

Monitoring of Laboratory Reared of Phlebotomus papatasi (Diptera: Psychodidae), Main Vector of Zoonotic Cutaneous Leishmaniasis to Different Imagicides in Hyper endemic Areas, Esfahan Province, Iran.

BACKGROUND: In domestic and per domestic area, insecticides such as DDT, malathion, fenitrothion, propoxur and, more recently, synthetic pyrethroids such as deltamethrin and lambda-cyhalothrin, have been successfully used to control sand flies in many countries. The present study reports the results of time-mortality bioassay to DDT 4%, lambda-cyhalothrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and deltamethrin 0.05% in recently colonized Phlebotomus papatasi populations in Iran. METHODS: The insecticide susceptibility status of P. papatasi laboratory population was assessed during 2016-2017, following the standard WHO technique for mosquito (WHO, 2013) based on diagnostic dose. Sand flies collected from rural area of Badrood (Matin Abad), Natanz County, Esfahan Province, using aspirator. RESULTS: Susceptibility test to DDT and pyrethroids was assessed on 3534 laboratory-reared P. papatasi (1746 females and 1788 males). The LT 50 and LT 90 values were measured using probit analysis and regression lines. The test results against males of P. papatasi revealed that LT 50 values to DDT 4%, Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% were 439.28, 108.90, 97.75, 5.00 and 57.84 seconds. The figures for females were 641.62, 136.15, 146.44, 8.71 and 72.69 seconds, respectively. CONCLUSION: According to presented results, the reared population of sand flies collected from a hyper-endemic region of Esfahan Province is still susceptible to prethroids and Resistance candidate to DDT 4%.

Study on Periplaneta americana (Blattodea: Blattidae) Fungal Infections in Hospital Sewer System, Esfahan City, Iran, 2017.

BACKGROUND: American cockroaches contaminated with pathogens inside hospital manholes can be one of the major problems that health care systems face. OBJECTIVES: The aim of this study was to investigate the fungal infections of American cockroaches in the Esfahan hospital sewage network. The principle goal of the study was about the roaches as a vector of fungi and other pathogens. METHOD: The type of study was descriptive-analytical. A total of 55 American cockroach specimens from the manhole walls of the sewerage system of 7 large hospitals were captured. Samples were taken from the surface of the body, digestive tract, and haemocoel of cockroaches. The specimens were then cultured on Sabouraud dextrose agar separately, and fungi were identified according to the macroscopic and microscopic characteristics. RESULTS: All cockroaches collected from hospitals were infected with fungi. Among the 24 (13 infected and 11 noninfected) (44%) female cockroaches and 31 (18 infected and 13 noninfected) (56%) male cockroaches, it was identified that 40.00% was infected with Aspergillus niger, 3.64% with Rhizopus, 7.27% with Penicillium, and 5.45% with Mucor. 6 cockroaches had no yeast contamination. 17 (30.91%) cockroaches were contaminated with Candida glabrata, 23 (41.82%) cockroaches were contaminated with Candida krusei, and 22 (40%) cockroaches were contaminated with other yeast species. The results of this study showed that Candida krusei had the highest prevalence among the isolated fungi with 35.37% of the digestive system and Aspergillus niger with 70.97% of the surface of the cockroach body. CONCLUSION: The results emphasized the role played by cockroaches as potential pathogenic vectors in hospital environments. Therefore, suitable management is needed for controlling this insect to prevent disease transmission in hospitals.

Assessing the insecticide susceptibility status of field population of Phlebotomus papatasi (Diptera: Psychodidae) in a hyperendemic area of zoonotic cutaneous leishmaniasis in Esfahan Province, Central Iran.

Leishmaniasis is a Neglected Tropical Disease (NTD) and emerging parasitic infection that affect mainly poor regions around the world. This study aimed to determine the baseline susceptibility of Phlebotomus papatasi to commonly used insecticides in a hyper endemic area using WHO standard procedure in central Iran. A total of 4-5 replicates containing 120-200 sand flies were used for each insecticide. Baseline susceptibility to DDT and pyrethroids was assessed on 5326 specimens collected from the study area. The LT50 and LT90 values were measured according to the World Health Organisation test using probit analysis and regression lines. The test results against males P. papatasi revealed that LT50 values to DDT 4%,Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% were 564.07, 38.08, 1.95, 0.60 and 9.78s and the figures for females were 584.44, 110.10, 11.64, 1.53 and 16.91s, respectively. Our results indicated that P. papatasi as the main cutaneous leishmaniasis vector was susceptible to Cyfluthrin 0.15%, Lambdacyhalothrin 0.05%, Permethrin 0.75% and Deltamethrin 0.05% and tolerant to DDT 4%. This study was carried out in one out of many Leishmaniasis foci in Iran. We recommend that future studies incorporate other regions and use the same procedure for monitoring and evaluating sand fly resistance. Also, WHO can provide a specific guideline and create a test kit for sand fly resistance monitoring and for applying susceptibility test because the tubes prepared for mosquitoes are not actually fit for sand flies.

NRAMP1 gene polymorphisms and cutaneous leishmaniasis: An evaluation on host susceptibility and treatment outcome.

BACKGROUND & OBJECTIVES: Association between polymorphisms in the natural resistance associated macrophage protein 1 (NRAMP1) gene and susceptibility to cutaneous leishmaniasis (CL) has been demonstrated worldwide; however, the reported results were inconsistent. This study aimed to determine the association of NRAMP1 variants with susceptibility to CL infection and patients' response to treatment in Isfahan province of Iran. METHODS: Peripheral blood samples were collected from 150 patients with CL and 136 healthy controls. The CL patients were treated with intralesional injection of meglumine antimoniate. The polymorphic variants at NRAMP1 (A318V and D543N) were analyzed using PCR-RFLP. The chi-square test and Fisher's exact test were used to compare frequencies of alleles and genotypes of polymorphisms between patient and healthy control populations. RESULTS: There was a statistically significant difference in the D543N (rs17235409) polymorphism between the CL patients and healthy controls (p=0.008). However, no significant association was detected for A318V (rs201565523) polymorphism between groups (p=0.26). In addition, there was a lack of association between D543N and A318V genotypes with response to treatment (p=0.54 and p=0.31, respectively). INTERPRETATION & CONCLUSION: The results indicated that genetic variations of D543N (rs17235409) might be associated with susceptibility to CL infection. These data may be used for detection of sensitive individuals and prevention of CL in endemic areas.

Assessing the Fauna of Aquatic Insects for Possible Use for Malaria Vector Control in Large River, Central Iran.

Insects with over 30,000 aquatic species are known as very successful arthropods in freshwater habitats. Some of them are applied as biological indicators for water quality control, as well as the main food supply for fishes and amphibians. The faunistic studies are the basic step in entomological researches; the current study was carried out emphasizing on the fauna of aquatic insects in Karaj River, northern Iran. A field study was carried out in six various sampling site of Karaj River during spring 2013. The aquatic insects were collected using several methods such as D-frame nets, dipping and direct search on river floor stones. Specimens were collected and preserved in Ethanol and identified by standard identification keys. Totally, 211 samples were collected belonging to three orders; Plecoptera, Trichoptera and Ephemeroptera. Seven genuses (Perla, Isoperla, Hydropsyche, Cheumatopsyche, Baetis, Heptagenia and Maccafferium) from five families (Perlidae, Perlodidae, Hydropsychidae, Batidae, Heptagenidae) were identified. The most predominant order was Plecoptera followed by Trichoptera. Karaj River is a main and important river, which provides almost all of water of Karaj dam. So, identification of aquatic species which exist in this river is vital and further studies about systematic and ecological investigations should be performed. Also, monitoring of aquatic biota by trained health personnel can be a critical step to describe water quality in this river. Understanding the fauna of aquatic insects will provide a clue for possible biological control of medically important aquatic insects such as Anopheles as the malaria vectors.

Effect of integrated pest management on controlling zoonotic cutaneous leishmaniasis in Emamzadeh Agha Ali Abbas (AS) District, Isfahan province, 2006-2009.

BACKGROUND: Cutaneous leishmaniasis (CL) is still considered as a health problem in the world. Several methods of control in different regions, together with obtaining integrated information on its natural foci, are needed to decrease its prevalence. This study was designed to evaluate the effects of simultaneous interventions on CL control. MATERIALS AND METHODS: A standard questionnaire was used to identify patients among pilgrims to Emamzadeh Agha Ali Abbas (Isfahan Province, Iran). Subsequently, three methods of controlling the disease, including, spraying residential buildings with Baygon, baiting with zinc phosphide poisons, changing the vegetative cover of the region, improving the environment, and mounting a mesh on all doors and windows of buildings in residential areas were used. The control measures were then evaluated by comparing the number of pilgrims affected by CL after and before the interventions. RESULTS: While 23 pilgrims (1.4%) were affected with CL before the intervention (pretest), five (0.3%) persons were found to have CL after taking control measures. The Chi-square test did not indicate any significant difference in the relative frequency of CL (P = 0.731). CONCLUSION: The only scientific method for preventing and controlling zoonotic CL (ZCL) is a combination of the control methods (improving the environment and fighting off the disease districts and vectors) together with changing the vegetative cover of the region. Any measure for controlling this disease must be taken and programmed in accordance with the relevant experts' views, in coordination with the participation of other organizations and the society.

Topical effectiveness of different concentrations of nanosilver solution on Leishmania major lesions in Balb/c mice.

BACKGROUND & OBJECTIVES: Cutaneous leishmaniasis is an infection caused by protozoan genus Leishmania. Although glucantime is commonly used for the treatment of leishmaniasis, it has some side effects including increased liver enzymes and electrocardiogram changes. In addition, the drug is expensive, the injection is painful, and research shows that resistance of parasite to glucantime is growing in different parts of the world. Therefore, scientists are paying more attention to develop new drugs such as nanosilver solution. The present study is an attempt to evaluate the in vivo topical effects of different concentrations of nanosilver solution in the treatment of leishmaniasis lesions. METHODS: In all, 90 female Balb/c mice aged 6-8 wk were infected with 2×10 power 6 viable stationary-phase promastigotes in the base of tail. Different concentrations (60, 80, 120, 130 and 2000 ppm) nanosilver solution were used in the present study to test the efficacy in the treatment of lesions. Clinical control of the infection trends was conducted weekly for 5 wk by measuring lesion diameter with standard Kulis-Vernieh. Data were analyzed by paired t-test, analysis of variance (ANOVA), and Tukey test. RESULTS: Mean lesion diameter pre- and post-treatment did not significantly differ between different treatment groups (p >0.05). Likewise, a significant difference in splenic parasite load was also not observed between different treatment groups. INTERPRETATION & CONCLUSION: Based on our results, different concentrations of nanosilver are ineffective in reducing mean sizes of lesions.