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Background: Anopheles stephensi is the main vector of malaria in Iran. This study aimed to determine the susceptibility of An. stephensi from the south of Iran to bendiocarb and to investigate biochemical and molecular resistance mechanisms in this species., Methods: Wild An. stephensi were collected from Hormozgan Province and reared to the adult stage. The susceptibility test was conducted according to the WHO protocols using bendiocarb impregnated papers supplied by WHO. Also, field An. Stephensi specimens were collected from south of Kerman and Sistan and Baluchistan Provinces. To determine the G119S mutation in the acetylcholinesterase (Ace1) gene, PCR-RFLP using AluI restriction enzyme and PCR direct-sequencing were performed for the three field populations and compared with the available GenBank data. Also, biochemical assays were performed to measure alpha and beta esterases, insensitive acetylcholinesterase, and oxidases in the strains., Results: The bioassay tests showed that the An. stephensi field strain was resistant to bendiocarb (mortality rate 89%). Ace1 gene analysis revealed no G119S in the three field populations. Blast search of sequences revealed 98-99% identity with the Ace1 gene from Pakistan and India respectively. Also, the results of biochemical tests revealed the high activity of non-sensitive acetylcholinesterase, alpha and beta-esterase in the resistant strain compared to the susceptible strain. No G119S was detected in this study additionally the enhanced enzyme activity of esterases and acetylcholinesterase suggesting that resistance was metabolic., Conclusion: The use of alternative malaria control methods and the implementation of resistance management strategies are suggested in the study area., (Copyright © 2023 The Authors. Published by Tehran University of Medical Sciences.)

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), β-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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Phlebotomine sand flies are vectors of Leishmania species, the causative agents of leishmaniasis in the world. Present study aimed to evaluate the bioecological aspects of sand flies in different ecotopes in Sari County, north of Iran., Methods: Sand flies were collected from four villages in mountainous, forest, plain and peri-urban areas monthly using sticky traps in May-October 2016. Mounted specimens were identified using valid identification keys under optical microscope. The Arc GIS 10.5 software was applied for showing the distribution of sand flies. Shannon-Weiner, Simpson and Evenness species diversity indices were calculated., Results: Generally, 334 specimens were captured and identified, namelly Phlebotomus kandelakii , Ph. papatasi , Ph. major , Ph. sergenti , Ph. longiductus , Ph. halepensis , Ph. tobbi , Sergentomyia dentata , Se. theodori , Se. sintoni , Se. antennata and Se. sumbarica . The most common species was Ph. kandelakii (n= 128, 38.32 %). The highest Simpson index (0.81) and abundance (N= 141) were recorded in the mountaineous area. Shannon diversity index was higher in the forest (H'= 1.53) and the highest evenness index was in the plain area (J'= 0.93). The highest richness (S= 9) and Shannon indices (H'= 1.57) were observed in June., Conclusions: Phlebotomus kandelakii , Ph. sergenti , Ph. tobbi , Ph. longiductus , Se. theodori , Se. antennata and Se. sumbarica were recorded for the first time in the study area. Since some species are incriminated for leishmaniasis transmission, further studies are required in the northern regions of Iran to timely control measures planning., Competing Interests: Conflicts of Interest Authors declare that there is no conflict of interest., (Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences.)

The present study was conducted to investigate the ecological aspects of sand flies in southwestern North Khorasan, in which cutaneous leishmaniasis caused by Leishmania major has been reported with the highest annual incidence in Iran. Sampling was carried out in four localities including: Khorasha (natural), Ghamiteh (natural), Jorbat (semi urban) and Brick kilns (urban), twice a month using 105 sticky paper traps from indoors and outdoors dwellings during May-December 2017. Specimens were removed from sticky papers, washed in acetone, preserved in 80% ethanol, mounted on microscopic slides by Puri's medium, and identified using valid morphological keys. Simpson (D), richness (S), Menhinick (D Mg ), Margalef (D Mn ), Shannon-Weiner (H'), evenness (J') were calculated for species diversity. The synanthropic index was determined for the first time in the area. Totally 517 specimens were collected, 47% in outdoors and 30.4% in human indoor dwellings and 22.6% in animal. Eight species of sand flies including 5 species of the genus Phlebotomus and 3 species of the genus Sergentomyia were identified. Phlebotomus papatasi and Sergentomyia sintoni were the most common and Eudominant species, active in all months, collected in the maximum number and percentage in September and August, respectively, and showed the highest abundance in outdoors. The synanthropic index ranged from 6.25 to 38.9 in the study area. The Shannon-Wiener index was estimated to be up to 1.4 and 1.37 in Khorasha and November, respectively, which showed the highest diversity due to maximal richness and evenness compared to other areas. High abundance of Ph. papatasi, as the main vector of cutaneous leishmaniasis, can enhance the potential risk of emerging CL in new areas, the data can be equally important when vector control measures are considered., (© 2022. The Author(s).)

Zoonotic cutaneous leishmaniasis (ZCL) is a form of disease, which is mostly caused by Leishmania major (L.major ) in the Old World with high incidence through early life. Recently, the high incidence of CL has been reported in Jajarm County, northeastern Iran. The aim of the present study was to investigate the epidemiological aspects of CL and to identify Leishmania spp. in the area. Between 2012 and 2018, patients with active lesions suspected of CL were examined. Diagnosis was performed by methods of clinical examination and direct smears. Conventional kDNA polymerase chain reaction (PCR) was used to identify species of Leishmania . During the seven-year study period, 629 subjects were diagnosed as CL cases by clinical and microscopic examination. Considering the population of the study area, the average incidence rate was 237.8 per 100,000 population. Most (29.4%) of the subjects with ZCL were aged <10 years. Most of the patients (58.7%) were male and the rest were female. Most ulcers were observed on the face (32.2%) and hands (23.7%), respectively. Most patients (90%) had two or more ulcers on their body. The highest incidence was in the autumn in October (32%). Our data revealed that an ongoing reemergence of ZCL focus caused by L.major has arisen in the study area. Moreover, direct PCR on the smears for identifying Leishmania showed 100% specificity, and the species was identified as L. major using species -specific kDNA PCR. The use of conventional PCR on skin smears seems to provide a valuable method for confirming the clinical diagnosis of ZCL, which is very specific and sensitive particularly for clinical correlative studies. Further investigations are needed to improve planning strategies of vectors and reservoirs populations., Competing Interests: None., (© 2021 Published by Elsevier Ltd on behalf of World Federation of Parasitologists.)

Background: Malaria is a major public health problem and life threatening parasitic vector-borne disease. For the first time, we established and report the molecular mechanism responsible for Anopheles gambiae s.l. resistance to pyrethroids and DDT from Yamaltu Deba, Southern Guinea Savanna, Northern-Nigeria., Methods: The susceptibility profile of An. gambiae s.l. to four insecticides (DDT 4%, bendiocarb 0.1%, malathion 5% and deltamethrin 0.05%) using 2-3 days old females from larvae collected from study area between August and November, 2018 was first established. Genomic DNA was then extracted from 318 mosquitoes using Livak DNA extraction protocol for specie identification and kdr genotyping. The mosquitoes were identified to species level and then 96 genotyped for L1014F and L1014S kdr target site mutations., Results: The mosquitoes were all resistant to DDT, bendiocarb and deltamethrin but fully susceptible to malathion. An. coluzzii was found to be the dominant sibling species (97.8%) followed by An. arabiensis (1.9%) and An. gambiae s.s (0.3%). The frequency of the L1014F kdr mutation was relatively higher (83.3%) than the L1014S (39%) in the three species studied. The L1014F showed a genotypic frequency of 75% resistance (RR), 17% heterozygous (RS) and 8% susceptible (SS) with an allelic frequency of 87% RR and 13% SS while the L1014S showed a genotypic frequency of RR (16%), RS (38%) and SS (46%) with an allelic frequency of 40% RR and 60% SS, respectively., Conclusion: This study reveals that both kdr mutations present simultaneously in Northern-Nigeria, however contribution of L1014F which is common in West Africa was more than twice of L1014S mutation found in East Africa., (Copyright © 2020 The Authors. Published by Tehran University of Medical Sciences.)

Background: Anaplasmosis and Ehrlichiosis are the most important tick-borne diseases. This study was conducted in three cities of Kerman Province in Iran to investigate the circulation of the bacteria in ticks collected from sheep., Methods: Ticks were collected from animals using Srkj forceps and transferred to the Entomology lab in cold chain. After specimen's identification, they kept at -70 °C. Tick DNA was extracted using Bioneers DNA extraction kits followed by Nested PCR technique to amplify ribosomal 16S rRNA gene to detect Anaplasma infection in ticks., Results: 472 sheep were examined from which 349 ticks were collected and identified in laboratory using valid keys. Tick specimens belonged to two genera and four species; Hyalomma marginatum (62.47%) was the most frequent and Hylomma asiaticum (5.73%) showed the least abundance. The infestation rate to different tick species was different in three regions of Kerman Province. Observation revealed that 24 specimens (58.3%) were positive for Anaplasma . There is a significant difference between male and female infection rate. However, there is no significant difference between these variables in each of these cities., Conclusion: This study shows high infection rates to Anaplasma in hard ticks. It is essential for health and veterinary authorities and farmers to use appropriate strategies to control ticks to reduce the infestation., (Copyright© Iranian Society of Medical Entomology & Tehran University of Medical Sciences.)

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%., (Copyright© Iranian Society of Medical Entomology & Tehran University of Medical Sciences.)

Background: Culex pipiens play an important role in transmission of infectious diseases. Vector control by chemical pesticides, leads inevitably to resistance development. Understanding the underlying resistance mechanisms can help improve the control programmes and insecticide resistance management., Methods: The total contents of cytochrome p450s and the activities of glutathione S-transferases, alpha- and beta-esterases and inhibition rates of acetylcholine esterase (by propoxur) were measured in the field population of Cx. pipiens collected from Sari County, North of Iran, in 2016 and the results were compared with those of the laboratory susceptible strain according to the biochemical assay methods of WHO for adult mosquitoes. Independent sample t -test was used to compare the mean values of enzyme activities/contents between filed and laboratory susceptible populations., Results: The enzyme ratio of cytochrome p450s, alpha- and beta-esterases in the field population was 2.07, 3.72 and 1.36 respectively when compared with the results of the laboratory population. Although not statistically significant, the mean GSTs activities in the field population was marginally less than the laboratory population (ER=0.92). Acetylcholinesterase was insensitive to propoxur in 62.82% of the individuals of the tested field population. There was a significant difference (P< 0.05) between all values of the activities/contents of the enzyme in the field population except for GSTs compared with the laboratory susceptible strain. The highest enzyme activity was related to alpha esterase., Conclusion: The present study showed a range of metabolic mechanisms, comprising p450s and esterases combined with target site insensitivity of AChE, contributing to organophosphate, carbamate and pyrethroid resistance in the field population of Cx. pipiens ., (Copyright© Iranian Society of Medical Entomology & Tehran University of Medical Sciences.)

A well-known strategy for managing pest resistance is application of mixture of pesticides. Conventionally formulated pesticides have several environmental incompatibilities. The use of biocompatible and biodegradable nanocarriers in formulating pesticides could improve environmental protection. In this study, a mixture of imidacloprid and lambda-cyhalothrin was co-encapsulated for the first time using liposomes as nanocarrier to simultaneously deliver these insecticides. Ethanol injection was used to produce self-assembled liposomes. The formed nanoliposomes were coated with different concentrations of chitosan. Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscope (SEM) and FT-IR spectroscopy. The encapsulation efficiencies of lambda-cyhalothrin and imidacloprid were about 93% and 51%, respectively. The insecticide carrying liposomes had a size and surface charge of 57 nm and +0.6 mV, respectively. The size and surface charge of the particles produced were increased to 69 nm and +31 mV after being coated with chitosan (0.1%, W/V). In this study, residual activity of technical grade imidacloprid, lambda-cyhalothrin and their mixture and the effect of adjuvants used in commercial and nano formulations of these insecticides on Myzus persicae Sulzer was investigated. The insecticidal effects and duration of residual activity of nano-formulations was correlated with concentration of chitosan in final formulation. In accordance with the life cycle of M. persicae, using the mixture of imidacloprid and lambda-cyhalothrin improves the residual effect over their use alone. The use of lipid nanocarriers makes the improvement even further and can be a better alternative to conventional combination of these insecticides due to their more environmental friendliness., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Head lice infestation is one of the main public health problems worldwide and a sanitary, cultural and economic indicator for communities' health. This study aims to estimate the prevalence of head lice infestation among female primary school children in selected districts of Mazandaran Province, Iran., Methods: In this cross-sectional study, 3033 female school children were recruited using the consensus method. Head lice infestation was defined as visible adult lice, nymph or egg with the naked eye. Data were described by percent frequency and analyzed using the chi-squared test and multivariate logistic regression models. A p-value less than 0.05 was considered significant., Results: Prevalence of head lice infestation among female school children was estimated at 7.9%. The adjusted odds ratios (OR) [95% confidence intervals (CI)] for rural residents, low level educated mothers, family size more than five and not combing their hair were 1.96 (1.41-2.78), 2.46 (1.35-4.49), 2.04 (1.12-3.70) and 3.94 (1.73-8.96), respectively. In addition, considering first grade students as a reference group, the ORs (95% CI) for second and third grades were 1.89 (1.18-3.05) and 1.69 (1.05-2.74), respectively., Conclusion: Our study showed that studying in urban schools, low educational level of mothers, grade, family size and no history of regular combing were predictors of head lice infestation., (©2019 Walter de Gruyter GmbH, Berlin/Boston.)

Background: Anopheles stephensi is a key urban malaria vector in the Indian subcontinent and Middle East including south and southeast of Iran. Wide application of insecticides resulted in resistance of this species to various insecticides in these regions. This study was conducted to reveal the role of metabolic mechanisms in the development of resistance in An. stephensi to DDT and cyfluthrin., Methods: Field mosquito specimens were collected from Chabahar Seaport, southeast corner of Iran, in 2015. Insecticide susceptibility and enzyme assays were conducted as recommended by WHO., Results: Mean enzyme ratios were 3.95 and 3.04 for α- esterases and 2.40 and 1.97 for β- esterases in the DDT and cyfluthrin- resistant populations correspondingly compared with the susceptible strain. The GSTs enzyme mean activity ratios were 5.07 and 2.55 in the DDT and cyfluthrin- resistant populations compared with the susceptible beech strain. The cytochrome p450s enzyme ratios were 1.11 and 1.28 in the DDT and cyfluthrin- resistant populations respectively compared with the susceptible beech strain., Conclusion: Metabolic mechanisms play a crucial role in the development of DDT and cyfluthrin resistance in An. stephensi , therefore, further evaluation of the mechanisms involved as well as implementation of proper insecticide resistance management strategies are recommended.

The human flea, Pulex irritans, is the most important ectoparasite of humans. Intensive use of pyrethroids for its control has led to insecticide resistance. Monitoring pyrethroid resistance and its underlying mechanisms is essential for flea control. The aims of this study were to identify the susceptibility status of human flea to permethrin and to detect the presence of knockdown resistance (kdr) mutation and its frequency in populations of P. irritans. Adults of P. irritans were collected from Zanjan Province, northwest of Iran, during 2013-2017. Different populations of this flea were exposed to permethrin 0.75% for one and 8 h and then the mortality rate, as well as KD 50 and KD 95 times were calculated. Total RNA and gDNA of samples were extracted, and the fragments of cDNA encoding the partial voltage-gated sodium channel (VGSC) peptides were amplified using degenerated primers. Specific PCR and TaqMan real-time assays were conducted to characterize the vgsc gene and to detect the presence of mutation and genotyping of the populations. Mortality rates were in the range from 32% to 67% for one-hour and 73% to 90% for eight-hour exposure to permethrin 0.75%. KD 50 and KD 95 times varied in a range from 46 to 241 and 177 to 899 min, respectively. Sequencing of 70 amplified fragments of gDNA resulted in a 578-bp product. These fragments contained two introns (92 and 63 bp) and three exons (141, 189, and 92 bp) encoding 138 amino acids that encompassed IIS4-IIS6 and the partial linker between domains II and III of VGSC. All the studied populations showed L1014F mutation, substitution of CTT for TTT at the 1014 allele. The result of TaqMan assay for 624 samples showed 96.6% homogenous and 3.36% heterozygous mutant. The development of permethrin resistance and the presence of the L1014F mutation at high frequency in flea populations indicate that pyrethroids are likely ineffective in controlling human flea. Therefore, novel alternative control methods are needed to combat this human ectoparasite., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Noncommunicable diseases (NCDs) account for 76% of deaths in Iran, and this number is on the rise, in parallel with global rates. Many risk factors associated with NCDs are preventable; however, it is first necessary to conduct observational studies to identify relevant risk factors and the most appropriate approach to controlling them. Iran is a multiethnic country; therefore, in 2014 the Ministry of Health and Medical Education launched a nationwide cohort study-Prospective Epidemiological Research Studies in Iran (PERSIAN)-in order to identify the most prevalent NCDs among Iran's ethnic groups and to investigate effective methods of prevention. The PERSIAN study consists of 4 population-based cohorts; the adult component (the PERSIAN Cohort Study), described in this article, is a prospective cohort study including 180,000 persons aged 35-70 years from 18 distinct areas of Iran. Upon joining the cohort, participants respond to interviewer-administered questionnaires. Blood, urine, hair, and nail samples are collected and stored. To ensure consistency, centrally purchased equipment is sent to all sites, and the same team trains all personnel. Routine visits and quality assurance/control measures are taken to ensure protocol adherence. Participants are followed for 15 years postenrollment. The PERSIAN study is currently in the enrollment phase; cohort profiles will soon emerge.

Background: Culex pipiens is a mosquito species distributed in different parts of Iran. It is known as vector of some diseases as well as a nuisance insect. A successful control campaign needs to study the biology, ecology and susceptibility status of the target pest. The aim of this study was to investigate the susceptibility status of Culex pipiens to some insecticides recommended by WHO in north of Iran., Methods: Larvae were collected from different breeding places in Sari County by standard dipping method. They were reared to adult stage in an under standard condition. World Health Organization (WHO) standard test kits and the diagnostic dose of 12 imagicides were used for adults, while two larvicides were used against larvae., Results: The least and highest mortality rates after exposure to insecticides were 4.7% (Propoxur 0.1%) and 76.4% (Etofenprox 0.5%), respectively. Larvae showed high resistance to temephos (19.19%) and malathion (54.63%). Culex pipiens was found to be resistant to all used insecticides/larvicides at diagnostic dose., Conclusion: Intensive use of pesticides against agriculture and urban pests increased the resistance level of this species to different insecticides, although currently there is no control program against it. Study on the mechanisms of resistant in this species to different insecticides is recommended.

Background: The aim of this study was to evaluate different concentrations of deltamethrin combined with formulated piperonyl butoxide (PBO) synergist on various surfaces against the wild strain of Anopheles stephensi , the main malaria vector in Southern Iran under semi-field condition., Methods: Four concentrations of deltamethrin WG 25% (Tagros) and PBO 800EC-UV (Endura) were prepared and sprayed on the pre-designed surfaces in accordance with WHO alliance line of the IRS Micronair®. The WHO's recommended bioassay kit and method was used during this study., Results: Comparing the mortality rate of mosquitoes, the results showed a significant difference between months after treatment of IRS (Indoor Residual Spraying) (P< 0.05) but didn't show any significant differences between days during the first and second months (P> 0.05).Statistical test revealed a significance difference between mortality rate of mosquitoes in exposing to concentrations of 1 and 4 (P< 0.05) which demonstrated effect of synergizing PBO on mortality rate., Conclusion: This research as the first semi-field trial on deltamethrin added to different concentrations of formulated PBO for IRS, indicates that deltamethrin+10X PBO is more effective than other concentrations. Therefore, using synergists can be suggested as a new tool for prevention of pyrethriod resistance, although more studies are recommended.

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 LT 50 and LT 90 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 LT 50 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., (Copyright © 2017. Published by Elsevier B.V.)

In the current study, the effects of the presence of symbiotic bacteria on the activity of the enzymes involved in An. stephensi resistance to temephos are evaluated for the first time. Four different strains (I. susceptible strain, II. resistant strain, III. resistant strain + antibiotic, and IV. resistant strain + bacteria) were considered in order to determine the possible effects of the symbiotic bacteria on their hosts' resistance to temephos. The median values of all enzymes of susceptible strain were compared with those of other resistant strains. The results of this study indicated a direct relationship between the presence of bacteria in the symbiotic organs of An. stephensi and resistance to temephos. The profile of enzymatic activities in the resistant strain changed to a susceptible status after adding antibiotic. The resistance of An. stephensi to temephos could be completely broken artificially by removing their bacterial symbionts in a resistant population.

Background: Anopheles mosquitoes are an important group of arthropods due to their role in transmission of malaria. The present study was conducted for determination of susceptibility status of Anopheles stephensi to different imagicides collected from malarious area in Chabahar city, Iran., Methods: In the present study seven insecticides including: DDT 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and etofenprox 0.5% were tested based on WHO method. Regression line was plotted for each insecticide using mortality of different exposure times. Bioassay data were analyzed using Probit software and the lethal time for 50% and 90% mortality (LT 50 and LT 90 ) values were calculated., Results: The susceptibility levels of field strain of An. stephensi to the discriminative dose of different imagicides were determined 100, 98, 96, 89, 82 and 62% for etofenprox, permethrin, deltamethrin, lambdacyhalothrin, cyfluthrin and DDT, respectively. Our finding indicated that An. stephensi is resistant to DDT, lambdacyhalothrin and cyfluthrin, and susceptible to etofenprox and permethrin and candidate of resistant to deltamethrin based on WHO criteria., Conclusion: Our findings indicated that An. stephensi is resistant to DDT and some pyrethroid insecticides which can be developed due to application of insecticides in health and agriculture. These results can provide a clue for future chemical control program in the study area.

Background: During recent years transmission of Dirofilaria immitis (dog heart worm) by Culex pipiens and West Nile virus have been reported from Iran. The present study was preformed for evaluating the susceptibility status of Cx. pipiens collected from capital city of Tehran, Iran., Methods: Four Insecticides including: DDT 4%, Lambdacyhalothrin 0.05%, Deltamethrin 0.05% and Cyfluthrin 0.15 % according to WHO standard methods were used for evaluating the susceptibility status of Cx. pipiens from Tehran moreover For comparison susceptibility status a Laboratory strain also was used. Bioassay data were analyzed using Probit program. The lethal time for 50% and 90% mortality (LT 50 and LT 90 ) values were calculated from regression line., Results: The susceptibility status of lab strain of Cx. pipiens revealed that it is susceptible to Lambdacyhalothrin, Deltamethrin, Cyfluthrin and resistant to DDT. Moreover cyfluthrin with LT 50 =36 seconds and DDT with LT 50 =3005 seconds had the least and most LT 50 s. Field population was resistance to all tested insecticides and DDT yielded no mortality., Conclusion: Highly resistance level against all WHO recommended imagicides were detected in field populations. We suggest more biochemical and molecular investigations to detect resistance mechanisms in the field population for further decision of vector control.

Objectives: Head lice infestation is one of the most important health problems, generally involving children aged 5-13 years. This study aims to estimate the prevalence of head lice infestation and its associated factors among primary school children using systematic review and meta-analysis methods., Methods: Different national and international databases were searched for selecting the relevant studies using appropriate keywords, Medical Subject Heading terms, and references. Relevant studies with acceptable quality for meta-analysis were selected having excluded duplicate and irrelevant articles, quality assessment, and application of inclusion/exclusion criteria. With calculating standard errors according to binomial distribution and also considering the Cochrane's Q test as well as I-squared index for heterogeneity, pediculosis prevalence rate was estimated using Stata SE V.11 software., Results: Forty studies met the inclusion criteria of this review and entered into the meta-analysis including 200,306 individuals. Using a random effect model, the prevalence (95% confidence interval) of head lice infestation among primary school children was estimated as 1.6% (1.2-2.05), 8.8% (7.6-9.9), and 7.4% (6.6-8.2) for boys, girls, and all the students, respectively. The infestation rate was found to be associated with low educational level of parents, long hair, family size, mother's job (housewife), father's job (worker/unemployed), using a common comb, lack of bathrooms in the house, and a low frequency of bathing., Conclusion: This meta-analysis revealed that the prevalence of head lice infestation among Iranian primary school children is relatively high with more prevalence among girls. We also found that economic, social, cultural, behavioral, and hygienic factors are associated with this infestation.

Background & Objectives: This paper presents the results of a study on ecological parameters including diversity indices applied on populations of Culicidae mosquitoes (Diptera: Culicidae) from three rural areas (Darvishan, Chalmardi and Khairabad) of Neka township in Mazandaran province, northern Iran., Methods: Adult and larval stages of mosquitoes were systematically sampled to obtain data required for measuring diversity of mosquitoes in these areas. Alfa and beta indices were analyzed to compare mosquito diversity. A total of 5270 specimens belonging to four genera and 14 different species were collected and identified., Results: Evaluation of dominance structure of species showed that culicid mosquitoes of Neka rangelands include one eudominant species, two dominant species, two subdominant species, two rare species and eight subrare species when Heydemann classification was used., Interpretation & Conclusion: Data analysis revealed a higher diversity of mosquitoes in Khairabad with a low degree of interspecific dominance, while Darvishan and Chalmardi recorded the lowest observed diversity and a high degree of interspecific dominance. The cluster analysis based on Jaccard's index indicated the relationship between the categories and indicates that Darvishan and Khairabad are the closest categories in their specific composition.

Background: Anopheles stephensi is a sub-tropical species and has been considered as one of the most important vector of human malaria throughout the Middle East and South Asian region including the malarious areas of southern Iran. Current reports confirmed An. stephensi resistance to temephos in Oman and India. However, there is no comprehensive research on mechanisms of temephos resistance in An. stephensi in the literature. This study was designed in order to clarify the enzymatic and molecular mechanisms of temephos resistance in this species., Methods: Profile activities of α- and ß-esterases, mixed function oxidase (MFO), glutathione-S-transferase (GST), insensitive acetylcholinesterase, and para-nitrophenyl acetate (PNPA)-esterase enzymes were tested for An. stephensi strain with resistance ratio of 15.82 to temephos in comparison with susceptible strain., Results: Results showed that the mean activity of α-EST, GST and AChE enzymes were classified as altered indicating metabolic mechanisms have considerable role in resistance of An. stephensi to temephos. Molecular study using PCR-RFLP method to trace the G119S mutation in ACE-1 gene showed lack of the mutation responsible for organophosphate insecticide resistance in the temephos-selected strain of An. stephensi., Conclusion: This study showed that the altered enzymes but not targets site insensitivity of ACE-1 are responsible for temephos resistance in An. stephensi in south of Iran.

Background: Pyrethroid insecticide-treated bed nets (ITNs) help contribute to reducing malaria deaths in Africa, but their efficacy is threatened by insecticide resistance in some malaria mosquito vectors. We therefore assessed the evidence that resistance is attenuating the effect of ITNs on entomological outcomes., Methods and Findings: We included laboratory and field studies of African malaria vectors that measured resistance at the time of the study and used World Health Organization-recommended impregnation regimens. We reported mosquito mortality, blood feeding, induced exophily (premature exit of mosquitoes from the hut), deterrence, time to 50% or 95% knock-down, and percentage knock-down at 60 min. Publications were searched from 1 January 1980 to 31 December 2013 using MEDLINE, Cochrane Central Register of Controlled Trials, Science Citation Index Expanded, Social Sciences Citation Index, African Index Medicus, and CAB Abstracts. We stratified studies into three levels of insecticide resistance, and ITNs were compared with untreated bed nets (UTNs) using the risk difference (RD). Heterogeneity was explored visually and statistically. Included were 36 laboratory and 24 field studies, reported in 25 records. Studies tested and reported resistance inconsistently. Based on the meta-analytic results, the difference in mosquito mortality risk for ITNs compared to UTNs was lower in higher resistance categories. However, mortality risk was significantly higher for ITNs compared to UTNs regardless of resistance. For cone tests: low resistance, risk difference (RD) 0.86 (95% CI 0.72 to 1.01); moderate resistance, RD 0.71 (95% CI 0.53 to 0.88); high resistance, RD 0.56 (95% CI 0.17 to 0.95). For tunnel tests: low resistance, RD 0.74 (95% CI 0.61 to 0.87); moderate resistance, RD 0.50 (95% CI 0.40 to 0.60); high resistance, RD 0.39 (95% CI 0.24 to 0.54). For hut studies: low resistance, RD 0.56 (95% CI 0.43 to 0.68); moderate resistance, RD 0.39 (95% CI 0.16 to 0.61); high resistance, RD 0.35 (95% CI 0.27 to 0.43). However, with the exception of the moderate resistance category for tunnel tests, there was extremely high heterogeneity across studies in each resistance category (chi-squared test, p<0.00001, I² varied from 95% to 100%)., Conclusions: This meta-analysis found that ITNs are more effective than UTNs regardless of resistance. There appears to be a relationship between resistance and the RD for mosquito mortality in laboratory and field studies. However, the substantive heterogeneity in the studies' results and design may mask the true relationship between resistance and the RD, and the results need to be interpreted with caution. Our analysis suggests the potential for cumulative meta-analysis in entomological trials, but further field research in this area will require specialists in the field to work together to improve the quality of trials, and to standardise designs, assessment, and reporting of both resistance and entomological outcomes.

Background: Malaria still remains a public health problem in Iran. There are different vector control interventions such as insecticide spraying. The present study was carried out to determine the susceptibility status of Anopheles stephensi larvae to temephos as a national plan for monitoring and mapping of insecticide resistance., Methods: Eight different localities in two main malarious provinces were determined as field collecting sites. Mosquitoes were collected from the field and reared in an insectray. Susceptibility assays were carried out according to the WHO method. The laboratory reared susceptible Beech-Lab strain was used for comparison. Data were analyzed using Probit analysis to determine LC50 and LC90 values., Results: Susceptibility of An. stephensi to temephos indicated that the LC50 ranged from 0.0022 mg/l to 0.0141 mg/l. Although all field strains were susceptible to temephos, considerable variations in temephos resistance ratios of field strains were noticed from all the localities studied in comparison with the susceptible strain. A low level of resistance ratio was noticed in An. stephensi populations except for the Chabahar strain (RR= 4.27 fold). All field-collected An. stephensi populations exhibited homogeneity to the larvicide except for Bandar Abbas and Hormoodar village strains (P> 0.05%)., Conclusion: Due to intensive use of temephos in the neighboring countries and occurrence of resistant to this insecticide in the main malaria vector in the region, insecticide resistance gene may evolve in the populations of An. stephensi. If temephos be applied as a larvicide it should be used judiciously for resistance management, as rotation strategy.

A series of 2(3H)-thiazole thiones 3-5 was synthesized and evaluated for tyrosinase inhibition and DPPH radical scavenging activities. Among them, 3-methyl-4-phenyl-2(3H)-thiazole thione (4a) showed good tyrosinase inhibitory activity, even better than that of the well-known tyrosinase inhibitor, namely, kojic acid. From the structure-activity point of view, although it was found that the phenolic hydroxyl group in prototype 3-5 might contribute to the scavenging activity against DPPH radicals, there was no correlation between the potency of tyrosinase inhibition and the presence of the phenolic moiety. The in silico ADME-Tox screening revealed that the drug-likeness and drug-score values of the most potent compound 4a were significantly higher than those of kojic acid., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To describe the midgut microbial diversity and to find the candidate bacteria for the genetic manipulation for the generation of paratransgenic Anopheline mosquitoes refractory to transmission of malaria, the microbiota of wild larvae and adult Anopheles stephensi mosquito midgut from southern Iran was studied using a conventional cell-free culture technique and analysis of a 16S ribosomal RNA (rRNA) gene sequence library. Forty species in 12 genera including seven Gram-negative Myroides, Chryseobacterium, Aeromonas, Pseudomonas, Klebsiella, Enterobacter and Shewanella and five Gram-positive Exiguobacterium, Enterococcus, Kocuria, Microbacterium and Rhodococcus bacteria were identified in the microbiota of the larvae midgut. Analysis of the adult midgut microbiota revealed presence of 25 Gram-negative species in five genera including Pseudomonas, Alcaligenes, Bordetella, Myroides and Aeromonas. Pseudomonas and Exiguobacterium with a frequency of 51% and 14% at the larval stage and Pseudomonas and Aeromonas with a frequency of 54% and 20% at the adult stage were the most common midgut symbionts. Pseudomonas, Aeromonas and Myroides genera have been isolated from both larvae and adult stages indicating possible trans-stadial transmission from larva to adult stage. Fast growth in cheap media, Gram negative, and being dominantly found in both larvae and adult stages, and presence in other malaria vectors makes Pseudomonas as a proper candidate for paratransgenesis of An. stephensi and other malaria vectors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This report presents the results of the first faunistic study of hard ticks in Qazvin province of Iran. The primary objective was to determine the species diversity and geographic distribution of hard ticks that parasitize domestic ruminants. Information about the abiotic preferences of these species has been provided. A total of 286 cattle, 1,053 goats, and 2,050 sheep were examined in 13 villages in 28 flocks distributed throughout the studied areas. Total direct body collections of ticks were made from each domestic ruminant. A total of 228 Ixodid specimens belonging to nine species in three different genera were recorded in the areas, including Boophilus annulatus (Say, 1821), Hyalomma anatolicum Koch, 1844, Hyalomma asiaticum (Schulze and Schlettke, 1929), Hyalomma detritum Schulze, 1919, Hyalomma dromedarii Koch, 1844, Hyalomma marginatum Koch 1844, Hyalomma schulzei Olenev, 1931, Rhipicephalus bursa Canestrini and Fanz, 1878 and Rhipicephalus sanguineus (Latreille, 1806). The most abundant species on sheep was R. sanguineus (46.92%), while B. annulatus (6.6%) found only on cattle. A finding of great significance was that R. sanguineus, the main vector of babesiosis, is firmly established throughout the counties. A further objective of the study was to compare the abundance of the major tick species on domestic ruminants. This was carried out at 19 sampling sites. The highest number of ticks was collected in July-August during the hot season.

The development of insecticide resistance in the German cockroach, Blattella germanica (L.) is a serious problem in controlling this medically important household pest. The insecticide resistance status in three hospital-collected strains of the German cockroach using four commonly used insecticides from different classes (permethrin, cypermethrin, bendiocarb and chlorpyrifos) was detected by topical bioassay method and preliminary information on possible involvement of monooxygenases in permethrin resistant strains employing synergist piperonyl butoxide (PBO) was obtained. For each insecticide, four to six concentrations resulting in >0% and <100% mortality were used. Three to six replicates of 10 cockroaches per concentration were conducted. For synergism studies, 100 μg PBO per gram body weight of cockroach as the maximum sublethal dose was administered to the first abdominal segment 1 h before insecticide treatment. The differences between LD50 (μg/g) values were considered statistically significant only when the 95% confidence intervals did not overlap. The resistance ratio and synergism ratio were calculated for each insecticide. All three hospital-collected strains of the German cockroach showed different levels of resistance to permethrin and cypermethrin based on resistance ratios compared with SUS strain. Permethrin and cypermethrin resistance ratios ranged from 11.61 to 17.64 and 11.45 to 26.45 at LD50 levels, respectively. Low to moderate levels of bendiocarb resistance and low level of chlorpyrifos resistance were also observed in the hospital-collected strains under study. The synergist piperonyl butoxide (PBO) significantly enhanced the toxicity of permethrin to all strains with different degrees of synergist ratio, 2.45-, 1.87-, 2.51- and 2.38-fold, suggesting monooxygenase involvement in permethrin resistance.

Background & Objectives: Phlebotomine sandflies are vectors of leishmaniases and other diseases. Long-lasting insecticidal nets (LLINs) as possible tools for control have not been widely tested against them. The objective of this study was to determine the efficacy of Olyset Net and PermaNet LLINs alongside a local brand, K-O Tab treated net (Supanet) against Phlebotomus duboscqi female sandflies., Methods: Four replicates of unwashed and 20x washed Olyset Nets and PermaNets, K-O Tab-treated and untreated Supanet and 'no net' treatments were evaluated against sandflies within the laboratory by tunnel tests and in semi-field conditions in the greenhouse model for their efficacy., Results: All bednets allowed entry of P. duboscqi sandflies and subsequent blood-feeding. Olyset net's blood feeding inhibition was significantly higher than that of Supanet in the laboratory but not in semi-field condition. Of the LLINs, only Olyset net had sandflies that could not feed significantly more than those of Supanet. Additionally, no significant efficacy difference was observed between LLINs washed 20x and unwashed ones. The only significant difference noted in number of sandflies that were found dead or paralyzed within bednets in the semi-field condition was between Olyset and K-O Tab treated Supanet. In the laboratory, unwashed Olyset had a significantly higher number of sandflies killed than all other bednet treatments., Conclusion: Olyset net use in areas where sandflies are nuisance biters and/or disease vectors could be more beneficial in preventing sandfly bites than other tested bednets. It is recommended that mesh sizes of LLINs should be smaller for control of sandflies than those used for control of mosquitoes.

Background & Objectives: Temperature plays a significant role in insect's development where a rise in temperature, accelerates the insect's metabolic rates, increases egg production and makes blood feeding more frequent. It also shortens the time period required for the development of pathogens within insects. Visceral leishmaniasis (VL) is one of the most important vector-borne diseases transmitted by different sandfly species. In this study, a phenological model was used to estimate the number of generations, peak activity and temporal variability of sandflies in the main VL foci in northwest Iran., Methods: Development requirements of different life stages of a Phlebotomus papatasi laboratory colony were measured and were subjected to the formula for calculation of accumulated degree day (ADD) for field sandflies using the online soft (UC IPM), using horizontal cut-off method and single triangle model. Sandflies population dynamics was monitored in the field during the seasonal activity in the region and its association with the ADD was tested using SAS software., Results: Populations of sandflies accommodated well with the amount of accumulated degree days (ADD) in the region. During the seasonal activity, a total of 639 ADD were produced which was enough to support one complete life cycle and growth of the next generation up to late larval instar. Larvae of the second generation hibernate through winter and the first adult population appears in the mid to late June of the next year when they receive at least 182 ADD from the beginning of the spring. The highest population density of sandflies was observed in early August, followed by a rapid decrease in early September, with the adult population disappearing completely in late September. This is the first degree day model related to sandflies in the most important VL foci of Iran., Interpretation & Conclusion: Further studies in various regions with variable climate are recommended in order to better estimate and understand the development time, population dynamics and activities of the vectors which in turn could be used in proper implementation of effective vector control programmes.

Hard tick infestation is an important problem in both traditional and industrial animal husbandry as well as in veterinary and public health. Several acaricides have been used to control tick infestation in Iran. Poor control has been attributed to acaricide resistance in tick populations although this has not been demonstrated experimentally. In this study, susceptibility status to propetamphos (Blotic) of 2-3-week-old larvae of Rhipicephalus bursa, one of the most common tick species in Iran, was evaluated using the FAO recommended larval packet test (LPT) method with some modifications. Log dosage probit analysis of propetamphos dose-response regression lines produced a resistance ratio based on LC(50) in the most resistant strain of approximately 103-fold. Biochemical assays suggested that the tick strains contained multiple insecticide resistance mechanisms involving elevation of esterases, GSTs, MFOs and alteration of acetylcholinesterase. In light of this data, practical implications for pest and pesticide resistance management strategies are discussed.

Background: The German cockroach is an important household insect pest mechanically involved in transmission of a variety of diseases to humans. Different classes of insecticides have extensively been used for its control leading to insecticide resistance development. Hence, for an optimal control of this pest, the status and underlying mechanisms of insecticide resistance should be studied in this group of insects., Methods: Adult German cockroaches were collected from Imam and Bouali Cina Hospitals (Sari, Iran) and subjected to bioassay using jar test method. The results were compared to those of a susceptible laboratory strain. Biochemical assays of esterases, monooxigenases and glutathione S-transferase (GST) levels were undertaken on German cockroaches from Imam and Bouali Cina Hospitals and the results were compared to a susceptible laboratory strain., Results: The LT50 values of the three strains were 20.24+/-2.2, 19.87+/-2.3 and 8.89+/-0.26 for permethrin; 19.3+/-3.05, 17.6+/-0.68 and 8.8+/-0.99 for deltamethrin; 19.64+/-2.9, 18.66+/-3.45 and 8.64+/-0.62 min for cypermethrin, respectively. The mean -esterase activity of Imam and Bouali Cina Hospitals and susceptible strains were 6.941x10(-4), 6.940x10(-4) and 8.01x10(-5) nmol/min/mg protein; the mean -esterase activity in those strains were 5.8x10(-4), 4.25x10(-4) and 7.28x10(-5) nmol/min/mg protein; the mean content of p450 in the above-mentioned strains were 5.64x10(-6), 1.89x10(-6) and 1.2x10(-6) nmol/mg protein; the mean GST activity were 6.66x10(-2), 0.102 and 5.72x10(-2) mol/min/mg protein, respectively., Conclusion: The LT50 values and also the mean activity of all enzyme groups in field strains were significantly different from those of the susceptible strain, indicating a vigour tolerance to insecticides and pyrethroids in particular. Hence, insecticide resistance monitoring techniques should be put in place and also resistance management strategies and measures should be considered implementing in the area.

Background: Electronic mosquito repellents (EMRs) are marketed to prevent mosquitoes biting and to prevent malaria., Objectives: To assess whether EMRs prevent mosquito bites, and to assess any evidence of an effect on malaria infection., Search Strategy: In August 2006, we searched the Cochrane Infectious Diseases Group Specialized Register, CENTRAL, MEDLINE, EMBASE, LILACS, Cambridge Scientific Abstracts, and the Science Citation Index. We also checked conference proceedings, contacted international specialist centres and EMR manufacturers, and checked reference lists., Selection Criteria: Field entomological studies, which controlled for geographic site, time, and attractiveness of human participants, of EMRs for preventing mosquito bites; and randomized and quasi-randomized controlled trials of EMRs to prevent malaria infection., Data Collection and Analysis: Two authors assessed trial quality, and extracted and analysed the data., Main Results: Ten field entomological studies met the inclusion criteria. All 10 studies found that there was no difference in the number of mosquitoes caught from the bare body parts of the human participants with or without an EMR. No randomized or quasi-randomized controlled trials on the efficacy of EMR on malaria infection were found., Authors' Conclusions: Field entomological studies confirm that EMRs have no effect on preventing mosquito bites. Therefore there is no justification for marketing them to prevent malaria infection.

A recombinant glutathione S-transferase (GST)-based pyrethroid quantification assay was field-tested in Ifakara, Tanzania. Initial laboratory tests suggested that all reagents used in the assay should be sufficiently stable for field use, provided that domestic refrigeration facilities were available. Insecticide-impregnated bednets were collected from a region where a social marketing programme was in progress. A total of 100 bednets were collected and the assay plus standard HPLC analysis was performed on the residues extracted from four replicate areas of each net. Insecticide residue estimations for assays performed on white and pale green bednet samples were accurate when compared with residue analysis by HPLC. However, for dark green or blue bednets, there was no correlation between the GST-based assay and HPLC pyrethroid quantification results. The assay failure with the dark coloured nets was caused by the extraction of the dyes along with the insecticide, which subsequently interfered with the GST assay. When the same samples were analysed by HPLC, the dyes were separated from the insecticide by reverse phase column chromatography and hence did not affect the results.

Glutathione transferases (GSTs) are a diverse family of enzymes found ubiquitously in aerobic organisms. They play a central role in the detoxification of both endogenous and xenobiotic compounds and are also involved in intracellular transport, biosynthesis of hormones and protection against oxidative stress. Interest in insect GSTs has primarily focused on their role in insecticide resistance. GSTs can metabolize insecticides by facilitating their reductive dehydrochlorination or by conjugation reactions with reduced glutathione, to produce water-soluble metabolites that are more readily excreted. In addition, they contribute to the removal of toxic oxygen free radical species produced through the action of pesticides. Annotation of the Anopheles gambiae and Drosophila melanogaster genomes has revealed the full extent of this enzyme family in insects. This mini review describes the insect GST enzyme family, focusing specifically on their role in conferring insecticide resistance.

Insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides have become one of the most promising interventions to prevent malaria in highly endemic areas. Despite the large body of experience documenting their health impact and the best way to distribute them, some key practical issues remain unresolved. For example, the duration of effective life of a net under field conditions is unknown. The most important factor affecting net effectiveness is the issue of regular re-treatment with insecticide. Washing is also an important determinant of insecticide longevity in the field. Trials were undertaken to provide some essential field information on ITNs within the site of an extended ITN programme in the Morogoro region of Tanzania. It was found that 45% of all nets were in bad condition (defined as more than seven large holes). It is concluded that an effective 'life' for polyester nets is 2-3 years. Further, two-thirds of the 20% of nets that were reported as having been re-treated within the last 12 months had less than 5 mg/m(2) of insecticide. According to the World Health Organization this is insufficient to be effective. People reported that they washed their nets four to seven times per year, usually with soap. Observations showed that such washing does not harm the nets and that the wash-water was unlikely to have an impact on the environment. Finally, bioassays were carried out with Anopheles gambiae on polyester netting with 0.5, 2, 5, 10 and 30 mg/m(2) of deltamethrin, alphacypermethrin and lambdacyhalothrin to assess the effectiveness of pyrethroids. The results confirmed that even with low insecticide concentrations, nets can still provide partial protection.

The mosquito Anopheles stephensi Liston (Diptera: Culicidae) is the urban vector of malaria in several countries of the Middle East and Indian subcontinent. Extensive use of residual insecticide spraying for malaria vector control has selected An. stephensi resistance to DDT, dieldrin, malathion and other organophosphates throughout much of its range and to pyrethroids in the Middle East. Metabolic resistance mechanisms and insensitivity to pyrethroids, so-called knockdown resistance (kdr), have previously been reported in An. stephensi. Here we provide molecular data supporting the hypothesis that a kdr-like pyrethroid-resistance mechanism is present in An. stephensi. We found that larvae of a pyrethroid-selected strain from Dubai (DUB-R) were 182-fold resistant to permethin, compared with a standard susceptible strain of An. stephensi. Activities of some enzymes likely to confer pyrethroid-resistance (i.e. esterases, monooxygenases and glutathione S-transferases) were significantly higher in the permethrin-resistant than in the susceptible strain, but the use of synergists--piperonyl butoxide (PBO) to inhibit monooxygenases and/or tribufos (DEF) to inhibit esterases--did not fully prevent resistance in larvae (permethrin LC50 reduced by only 51-68%), indicating the involvement of another mechanism. From both strains of An. stephensi, we obtained a 237-bp fragment of genomic DNA encoding segment 6 of domain II of the para type voltage-gated sodium channel, i.e. the putative kdr locus. By sequencing this 237 bp fragment, we identified one point mutation difference involving a single A-T base change encoding a leucine to phenylalanine amino acid substitution in the pyrethroid-resistant strain. This mutation appears to be homologous with those detected in An. gambiae and other insects with kdr-like resistance. A diagnostic polymerase chain reaction assay using nested primers was therefore designed to detect this mechanism in An. stephensi.

Recombinant glutathione S-transferase (agGST1-6) from the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) was expressed in Escherichia coli using a pET3a vector system. The expressed enzyme was biochemically active with reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Activity of agGST1-6 with GSH and CDNB was inhibited to different degrees by both alpha-cyano and non-alpha-cyano pyrethroid insecticides. This inhibition was used to develop an assay for quantification of pyrethroids. Standard curves of insecticide concentration against percentage of enzyme inhibition or volume of iodine solution were established by spectrophotometry and iodine volumetric titration, respectively, for permethrin and deltamethrin. These assays allowed estimation of pyrethroid concentrations both spectrophotometrically and visually. For the residue assay of each insecticide, a cut-off point of 50% of the initial pyrethroid impregnation concentration was used, which should differentiate between biologically active and inactive treated bednets. The cross-reactivity of the primary permethrin photodegradants (3-phenoxyalcohol and 3-phenoxybenzoic acid) with the recombinant agGST1-6 was assayed in the same system. No agGST1-6 inhibition by the insecticide metabolites was observed, suggesting that the system is unaffected by primary permethrin metabolites and will accurately measure insecticide parent compound concentrations. The estimated pyrethroid insecticide concentrations, given spectrophotometrically and by iodine titration assay, were comparable to those obtained by direct HPLC quantification of residues extracted from bednets. Hence, it should be relatively easy to adapt this method to produce a test kit for residue quantification in the field.