Perceived needs of disease vector control programs: A review and synthesis of (sub)national assessments from South Asia and the Middle East.

Systems for disease vector control should be effective, efficient, and flexible to be able to tackle contemporary challenges and threats in the control and elimination of vector-borne diseases. As a priority activity towards the strengthening of vector control systems, it has been advocated that countries conduct a vector-control needs assessment. A review was carried out of the perceived needs for disease vector control programs among eleven countries and subnational states in South Asia and the Middle East. In each country or state, independent teams conducted vector control needs assessment with engagement of stakeholders. Important weaknesses were described for malaria, dengue and leishmaniases regarding vector surveillance, insecticide susceptibility testing, monitoring and evaluation of operations, entomological capacity and laboratory infrastructure. In addition, community mobilization and intersectoral collaboration showed important gaps. Countries and states expressed concern about insecticide resistance that could reduce the continued effectiveness of interventions, which demands improved monitoring. Moreover, attainment of disease elimination necessitates enhanced vector surveillance. Vector control needs assessment provided a useful planning tool for systematic strengthening of vector control systems. A limitation in conducting the vector control needs assessment was that it is time- and resource-intensive. To increase the feasibility and utility of national assessments, an abridged version of the guidance should focus on operationally relevant topics of the assessment. Similar reviews are needed in other regions with different contextual conditions.

Kdr genotyping and the first report of V410L and V1016I kdr mutations in voltage-gated sodium channel gene in Aedes aegypti (Diptera: Culicidae) from Iran.

BACKGROUND: Aedes aegypti is the main vector of arboviral diseases worldwide. The species invaded and became established in southern Iran in 2020. Insecticide-based interventions are primarily used for its control. With insecticide resistance widespread, knowledge of resistance mechanisms is vital for informed deployment of insecticidal interventions, but information from Iranian Ae. aegypti is lacking. METHODS: Fifty-six Ae. aegypti specimens were collected from the port city of Bandar Lengeh in Hormozgan Province in the South of Iran in 2020 and screened for kdr mutations. The most common kdr mutations in Latin America and Asia (V410L, S989P, V1016G/I and F1534C), especially when present in combinations, are highly predictive of DDT and pyrethroid resistance were detected. Phylogenetic analyses based on the diversity of S989P and V1016G/I mutations were undertaken to assess the phylogeography of these kdr mutations. RESULTS: Genotyping all four kdr positions of V410L, S989P, V1016G/I and F1534C revealed that only 16 out of the 56 (28.57%) specimens were homozygous wild type for all kdr mutation sites. Six haplotypes including VSVF (0.537), VSVC (0.107), LSVF (0.016), LSIF (0.071), VPGC (0.257) and LPGC (0.011) were detected in this study. For the first time, 11 specimens harbouring the V410L mutation, and 8 samples with V1016I mutation were found. V410L and V1016I were coincided in 8 specimens. Also, six specimens contained 1016G/I double mutation which was not reported before. CONCLUSIONS: The relatively high frequency of these kdr mutations in Iranian Ae. aegypti indicates a population exhibiting substantial resistance to pyrethroid insecticides, which are used widely in control operations and household formulations. The detection of the 410L/1016I kdr mutant haplotype in Iranian Ae. aegypti suggests possible convergence of invasive populations from West Africa or Latin America. However, as Iran has very limited maritime/air connections with those African countries, a Latin American origin for the invasive Ae. aegypti in Iran is more plausible.

Assessment of Knowledge, Attitude and Practice of Inmates, Prison Staff and Soldiers Regarding Cimex lectularius (Hemiptera: Cimicidae) in Mazandaran Prisons.

Background: Bed bugs (Cimex lectularius) are blood-sucking insects that bite humans usually at night and cause nuisance, psychological and social problems. These insects are considered a health hazard in densely populated places such as prisons. The aim of this study was to evaluate the knowledge, attitude and practice of inmates, soldiers, and prison staff regarding bed bugs in Mazandaran prisons. Methods: This is a descriptive-analytical study in which 450 inmates and 210 staff (105 personnel and 105 soldiers) from prisons of Mazandaran Province were included in the study. Data on knowledge, attitude, and practice of the participants regarding bed bugs were collected using a structured questionnaire. To determine the relative content validity coefficient, the questionnaire was assessed by 10 specialists in terms of difficulty, inappropriateness, and ambiguity of the phrases. The reliability of the questionnaire was measured using Cronbach's Alpha and then the data were analyzed using SPSS software version 20. Results: The maximum mean scores of knowledges, attitude, and practice between the three target groups were 16.42%, 44.07% and 71.21% respectively. Prison staff had the highest knowledge and practice, and inmates showed the highest attitude regarding bed bugs. There was no significant difference between the knowledge and attitude of inmates, prison staff and soldiers (P> 0.05) but there was a significant difference in their practice (P< 0.05). Conclusion: Results of this study confirmed that all three groups of people in the prisons need appropriate education regarding all aspects of bed bug including its control.

Knowledge, attitude, and practice of healthcare workers regarding dengue fever in Mazandaran Province, northern Iran.

Background: Dengue fever is a rapidly emerging infection worldwide with a high public health burden. Adequate training of healthcare workers is essential to warrant the timely provision of health services to improve the outcome of dengue management. Methods: This is an analytical cross-sectional study, conducted to assess the knowledge, attitudes and practice (KAP) of healthcare workers regarding dengue from April 2021 to March 2022 in Mazandaran Province, northern Iran. Data was collected using a researcher-made structured questionnaire, prepared as Google Forms, and sent to target groups through social media and email. Data analysis was performed by SPSS 22 software using descriptive and inferential statistics (Chi-square) at a significant level of 5%. Results: Most of the respondents had heard about dengue (83.8%); media (32.7%) and academic education (25%) were the main sources of information. Respondents had less knowledge associated with dengue symptoms (52%) than prevention and control (69%), transmission (72.2%) and clinical management (81%). Based on the 70% cut-off point, the majority of the participants had a good attitude (81%) and practice (73%). However, only 49.6% of the respondents showed good practice regarding dengue local transmission. A significant difference was observed between participants knowledge on clinical management with occupation; attitude with gender and occupation; and practice with gender (p < 0.05). Conclusion: The results of this study revealed gaps in some dimensions of KAP in healthcare workers, therefore, a greater focus should be placed on future training programs to raise knowledge and attitude leading to sound practice and behavior for adequate management of dengue.

Entomological surveillance of invasive Aedes mosquitoes in Mazandaran Province, northern Iran from 2014 to 2020.

Mosquitoes are the most important vectors of serious infectious diseases in the world. Dengue, Zika, chikungunya and yellow fever are emerging and re-emerging infectious diseases, associated with the distribution of two key vectors i.e. Aedes aegypti and Aedes albopictus throughout the world including countries neighbouring Iran. Entomological surveillance was planned and performed monthly from May to December during 2014-2020 in selected counties of Mazandaran Province, North of Iran, by ovitrap, larval collection, hand catch and human baited trap. Overall, 4410 Aedes specimens including 2376 larvae (53.9%) and 2034 (46.1%) adults belonging to six species, namely Aedes vexans, Aedes geniculatus, Aedes caspius, Aedes echinus, Aedes pulcritarsis and Aedes flavescence were collected and morphologically identified. Over the seven years of surveillance, Ae. aegypti and Ae. albopictus were not found by any sampling method. Aedes vexans and Ae. geniculatus were the most abundant species, their populations peaked in October and November and was positively correlated with precipitation and relative humidity. Aedes flavescence was a new species record for the province. A flowchart for planning and implementation of invasive mosquito surveillance for provincial health authorities in the country is proposed. These surveillance efforts provide basic and timely information for the health system to act promptly on integrated and intensified surveillance and control programs should Ae. aegypti and Ae. albopictus detected in the province.

Entomological Surveillance of the Invasive Aedes Species at Higher-Priority Entry Points in Northern Iran: Exploratory Report on a Field Study.

BACKGROUND: Arboviral diseases such as dengue, Zika, and chikungunya are transmitted by Aedes aegypti and Ae albopictus and are emerging global public health concerns. OBJECTIVE: This study aimed to provide up-to-date data on the occurrence of the invasive Aedes species in a given area as this is essential for planning and implementing timely control strategies. METHODS: Entomological surveillance was planned and carried out monthly from May 2018 to December 2019 at higher-priority entry points in Guilan Province, Northern Iran, using ovitraps, larval collection, and human-baited traps. Species richness (R), Simpson (D), evenness (E), and Shannon-Wiener indexes (H̕) were measured to better understand the diversity of the Aedes species. The Spearman correlation coefficient and regression models were used for data analysis. RESULTS: We collected a total of 3964 mosquito samples including 17.20% (682/3964) belonging to the Aedes species, from 3 genera and 13 species, and morphologically identified them from May 2018 to December 2019. Ae vexans and Ae geniculatus, which showed a peak in activity levels and population in October (226/564, 40.07% and 26/103, 25.2%), were the eudominant species (D=75.7%; D=21.2%) with constant (C=100) and frequent (C=66.7%) distributions, respectively. The population of Ae vexans had a significant positive correlation with precipitation (r=0.521; P=.009) and relative humidity (r=0.510; P=.01), whereas it was inversely associated with temperature (r=-0.432; P=.04). The Shannon-Wiener Index was up to 0.84 and 1.04 in the city of Rasht and in July, respectively. The rarefaction curve showed sufficiency in sampling efforts by reaching the asymptotic line at all spatial and temporal scales, except in Rasht and in October. CONCLUSIONS: Although no specimens of the Ae aegypti and Ae albopictus species were collected, this surveillance provides a better understanding of the native Aedes species in the northern regions of Iran. These data will assist the health system in future arbovirus research, and in the implementation of effective vector control and prevention strategies, should Ae aegypti and Ae albopictus be found in the province.

Determination of susceptibility levels of three different cockroach species including hospitals German cockroach, Blattella germanica L. (Blattodea: Blattellidae), to common insecticides, cypermethrin, propoxur and fenitrothion.

Objective: German cockroach is highly adapted to different environments like hospitals. This pest is an important carrier of pathogenic agents and allergic compounds. Hence, it is important to German cockroaches always be monitored and controlled. This study investigated the toxicity and resistance levels of technical-grade of cypermethrin, propoxur, and fenitrothion against adult males of four strains of the German cockroach. Methods: Four German cockroaches' strains including laboratory-reared susceptible strain (S) and three hospital-collected strains (B, F, and Z) were tested in this study. Adult male cockroaches were treated topically with three technical grade insecticides on the first abdominal segment of the insects using a hand micro-applicator. Results: Using topical application methods, fenitrothion showed the most toxicity to all four strains. The LD50 values of fenitrothion in the susceptible strain (S) and the hospital-collected strains B, F, and Z were 1.89, 21.48, 25.73, and 31.55 µg/grBW, respectively. All hospital-collected strains showed different resistant levels to all insecticides. The field-collected Z strain was the most resistant strain to cypermethrin, propoxur, and fenitrothion. The resistance ratios of strain Z to cypermethrin, propoxur, and fenitrothion were 10.9, 10.47 and 16.67, respectively. Conclusion: Based on the susceptibility and resistance ratios for each insecticide, we conclude that there are high rates of insecticide resistance in Blattella germanica from different hospitals of Sari County which can be caused by different insecticides treatment histories.

Global water quality changes posing threat of increasing infectious diseases, a case study on malaria vector Anopheles stephensi coping with the water pollutants using age-stage, two-sex life table method.

BACKGROUND: Water pollution due to uncontrolled release of chemical pollutants is an important global problem. Its effect on medically important insects, especially mosquitoes, is a critical issue in the epidemiology of mosquito-borne diseases. METHODS: In order to understand the effect of water pollutants on the demography of Anopheles stephensi, colonies were reared in clean, moderately and highly polluted water for three consecutive generations at 27 °C, 75% RH, and a photoperiod of 12:12 h (L:D). The demographic data of the 4th generation of An. stephensi were collected and analysed using the age-stage, two-sex life table. RESULTS: The intrinsic rate of increase (r), finite rate of increase (λ), mean fecundity (F) and net reproductive rate (R0) of An. stephensi in clean water were 0.2568 d-1, 1.2927 d-1, 251.72 eggs, and 109.08 offspring, respectively. These values were significantly higher than those obtained in moderately polluted water (r = 0.2302 d-1, λ = 1.2589 d-1, 196.04 eggs, and R0 = 65.35 offspring) and highly polluted water (r = 0.2282 d-1, λ = 1.2564 d-1, 182.45 eggs, and R0 = 62.03 offspring). Female adult longevity in moderately polluted (9.38 days) and highly polluted water (9.88 days) were significantly shorter than those reared in clean water (12.43 days), while no significant difference in the male adult longevity was observed among treatments. CONCLUSIONS: The results of this study showed that An. stephensi can partially adapt to water pollution and this may be sufficient to extend the range of mosquito-borne diseases.

Imported tires; a potential source for the entry of Aedes invasive mosquitoes to Iran.

INTRODUCTION: International trade of tires has been responsible for the introduction of invasive Aedes species into many countries. The present study aimed to determine the sources and volume of such trading in Iran and to establish and map points of entry for entomological surveillance. METHOD: A list of tire importers, type and number of tires and source of their shipment was compiled, and the map of the main location(s) of their depot in the country was produced from 2017 to 2018. Contamination of imported tires with mosquito eggs or larvae was also determined. The samples from all parts of the warehouse were taken randomly from 4 to 20 tires every 15 days. In tires with a trace of water inside, the existence of the egg and larva of Aedes mosquitoes was investigated by sticking the glue band to the hot spot of left water inside the unpacked tires. RESULTS: Approximately one-third of the annual tire requirements of Iran were imported from 15 countries, most of which were endemic to Aedes aegypti and or Ae. albopictus. It is obligatory to import only wrapped tires. About 10% of total country consumption is imported through informal markets and smuggled. However, the unofficially imported tires are usually not wrapped. The majority of tire imports were made through the southern ports of the country. The main sites for depots of imported tires were located in four Iranian provinces, namely Tehran, Isfahan, Fars, and Sistan and Baluchestan. The latter is extremely important given its border with Pakistan. Depot locations were mostly unstructured, and tires were kept in the open air. All these depot locations were at the edge of towns and residential areas. Priority areas for routine entomological surveillance were established. Such as the previous studies, surveillance of imported tires for the presence of Aedes eggs or larvae was negative in this study. DISCUSSION: Mapping the entry points of imported tires and their origin is crucial to determine and prioritize sites for entomological surveillance of invasive mosquito species. Strengthening collaboration with customs authorities and the association of tire importers is imperative in this effort. The development of national rules and regulations for tire import is necessary to minimize the danger of the introduction of invasive vector species into the country.

Biochemical Mechanism of Insecticide Resistance in Malaria Vector, Anopheles gambiae s.l in Nigeria.

BACKGROUND: Malaria is a parasitic vector-borne disease endemic in the tropical and subtropical countries of the world. The aim of this study was to investigate the current activities of the detoxification enzymes in resistant and susceptible Anopheles gambiae s.l. in northern Nigeria. METHODS: Anopheles larvae were collected from northeast and northwestern Nigeria between Aug and Nov 2018. Biochemical analyses was carried out on the mosquitoes exposed to various insecticides (deltamethrin, DDT, bendiocarb, malathion) to measure and compare the enzymatic activities of the major detoxification enzymes (P450, GSTs, Esterase). RESULTS: High levels of resistance was observed; DDT 37%-53% (95%, CI: 29-61), bendiocarb 44%-55% (CI: 39-60) and deltamethrin 74%-82% (CI: 70-86). However, these mosquitoes were found to be susceptible to malathion 99%-100% (CI: 98-100). The P450 and GSTs enzymes were found to be elevated in the resistant mosquitoes exposed to deltamethrin (1.0240±0.1902); (1.3088±1.2478), DDT (1.7703±1.4528); (1.7462±0.9418) and bendiocarb (1.1814±0.0918); (1.4479±1.0083) compared to the Kisumu strain (0.764±0.4226); (0.6508±0.6542), (0.3875±0.3482); (0.4072±0.4916) and (0.6672±0.3949); (0.7126±0.7259) at P<0.05. Similarly, the resistant mosquitoes expressed increased activity to esterase (0.7606±1.1477), (0.3269±1.1957) and (2.8203±0.6488) compared to their susceptible counterpart (0.6841±0.7597), (0.7032±0.5380) and (0.6398±0.4159) at P<0.05. The enzyme ratio was found to be: P450 (1.341, 4.568 and 1.77); GSTs (2.011, 4.288 and 2.031); Esterases (1.111, 0.469 and 4.408). One way Anova and single sample t-test were also conducted to determine the effect of the enzymes on the resistant and susceptible strains. CONCLUSION: High level of insecticide resistance was observed with significant elevation of detoxification enzymes activities in the resistant mosquitoes.

Preparation, characterisation and comparative toxicity of nanopermethrin against Anopheles stephensi and Culex pipiens.

OBJECTIVES: To assess the effectiveness of nanopermethrin as a potential new formulation for pest and vector control. METHODS: Permethrin nanoparticles were prepared by the ionic gelation method and its structure and the formulations were designed using Box-Behnken statistical technique. The effect of independent variables (Chitosan/Permethrin ratio, tripolyphosphate quantity, sonication time) on the properties of nanoparticles was investigated to determine the optimal formulation. RESULTS: The size of the nanoparticles ranged from 135.27 ± 5.88 to 539.5 ± 24.01 nm and the insecticide entrapment efficiency per cent (EE%) ranged from 7.72 ± 1.36 to 63.59 ± 3.17%. Anopheles stephensi larvae were then bioassayed with the nanopermethrin and compared with the results of the bioassay with the mother molecule of permethrin using a standard WHO-recommended mosquito larval bioassay kit. LC50 with permethrin and nanopermethrin on larvae of An. stephensi were 0.125 and 0.026 ppm showing a 4.8 times difference. The LC50 for permethrin and nanopermethrin on Culex pipiens were 0.003 and 0.00032 ppm, respectively, showing a 9.4-fold difference. CONCLUSION: Nanopermethrin is much more potent than its mother molecule against larvae of An. stephensi and Cx. pipiens.

Current Status of Insecticide Susceptibility in the Principal Malaria Vector, Anopheles gambiae in Three Northern States of Nigeria.

BACKGROUND: Malaria is a major public health problem in Nigeria with 97% of its population with high morbidity and mortality. Mosquitoes play an important role in the transmission of malaria parasites. This study was conducted to evaluate the current resistance status of Anopheles gambiae to insecticides. METHODS: Larvae of An. gambiae was collected from three zones; A, B and C differentiated on the basis of variation in agricultural ecosystems between August and November, 2018 in the northeast and northwestern parts of Nigeria. They were carefully reared to adult stage and insecticidal susceptibility tests were conducted. RESULTS: The mosquitoes tested showed high levels of resistance to all the insecticides used with the exception of malathion. Study zone A, recorded 74% mortality after 24h to deltamethrin compared to 81% from zone B and 82% from zone C, respectively. Mosquitoes from zone B exposed to DDT had the highest level of resistance at 37% compared to 40% and 53% from zones A and C, respectively. Resistant to bendiocarb was also observed, with zone A having the lowest mortality of 44% compared to 48% from zone C and 55% from Zone B, respectively. According to the results of knockdown tests, mosquitoes from Zone A exposed to deltamethrin recorded the lowest knockdown across the study locations while zone B recorded the lowest knockdown for DDT. CONCLUSION: The results of the study provide an insight into the current status of An. gambiae to four major insecticides in northern Nigeria as guideline for mosquitocontrol.

Population Fluctuations and Abundance Indices of Mosquitoes (Diptera: Culicid), as the Potential Bridge Vectors of Pathogens to Humans and Animals in Mazandaran Province, Northern Iran.

BACKGROUND: Seasonal activity patterns of mosquitoes are essential as baseline knowledge to understand the transmission dynamics of vector-borne diseases. This study was conducted to evaluate the monthly dynamics of the mosquito populations and their relation to meteorological factors in Mazandaran Province, north of Iran. METHODS: Mosquito adults and larvae were collected from 16 counties of Mazandaran Province using different sampling techniques, once a month from May to December 2014. "Index of Species Abundance" (ISA) along with "Standardized ISA" (SISA) was used for assessing the most abundant species of mosquitoes based on the explanations of Robert and Hsi. Pearson's correlation coefficient (R) was used to assess the relationships between the monthly population fluctuations and meteorological variables. RESULTS: Overall, 23750 mosquitoes belonging to four genera and nineteen species were collected and identified. The highest population density of mosquitoes was in July and the lowest in May. The ISA/SISA indices for Culex pipiens were both 1 for larvae and 1.25/0.973 for adults in total catch performed in human dwellings. For Cx. tritaeniorhynchus, the ISA/SISA were 1.68/0.938 in pit shelter method. A significant positive correlation was observed between population fluctuations of Cx. tritaeniorhynchus and mean temperature (R: 0.766, P< 0.027). CONCLUSION: The results indicated that the mosquitoes are more active in July, and Cx. pipiens and Cx. tritaeniorhynchus were the most abundant species. Considering the potential of these species as vectors of numerous pathogens, control programs can be planed based on their monthly activity pattern in the area.

Zika; a continuous global threat to public health.

Zika virus is transmitted by Aedes mosquitoes, especially, Ae. aegypti and Ae. albopictus. About 80% of the cases do not manifest any symptoms, and it is a self-limiting, mild viral infection. In 20% of the cases and only in fraction of those who do show the symptoms, important complications including Guillaine Barre'syndrome and microcephaly may occur. The emergence of Zika in 2016 in Brazil spreading to about 70 other countries prompted the WHO officials to declare the disease a Public Health Emergency with International Concern (PHEIC). This has led to increased concerns in health authorities of almost all countries making them embark on the strengthened human and vector surveillance, vector control and clinical management of the disease. Although the main vectors of the disease have not yet been able to establish in Iran, because of their occurrence in neighboring countries as well as increased global travel and trade, the country established a national advisory committee for capacity building, vector and human surveillance and case management of Aedes-borne diseases. This study aims at performing a literature review about global situation of Zika and Aedes mosquitoes, their distribution, biology and ecology from the past to present and the threat posed to Iran. Aedes aegypti was historically present in the checklist of Iranian mosquitoes and Ae. albopictus has recently been collected from Southern Iran, however, the species has apparently failed to establish in the country as comprehensive follow up entomological surveillance could not reproduce the findings. Although Zika was not detected in Iran, considering the expansion in tourism, travel and trade to and from Zika infected and Aedes infested countries, suitable climate and favorable prediction for establishment of Aedes vectors, Iran may well be at risk of invasion of Aedes vector species and the diseases they carry. Therefore, this review is of value particularly to health authorities in Iran and other WHO Eastern Mediterranean countries for sustained vigilance and preparedness for early detection and response, including vector control.

Evolution of insecticide resistance and its mechanisms in Anopheles stephensi in the WHO Eastern Mediterranean Region.

BACKGROUND: While Iran is on the path to eliminating malaria, the disease with 4.9 million estimated cases and 9300 estimated deaths in 2018 remains a serious health problem in the World Health Organization (WHO) Eastern Mediterranean Region. Anopheles stephensi is the main malaria vector in Iran and its range extends from Iraq to western China. Recently, the vector invaded new territories in Sri Lanka and countries in the Horn of Africa. Insecticide resistance in An. stephensi is a potential issue in controlling the spread of this vector. METHODS: Data were collated from national and international databases, including PubMed, Google Scholar, Scopus, ScienceDirect, SID, and IranMedex using appropriate search terms. RESULTS: Indoor residual spaying (IRS) with DDT was piloted in Iran in 1945 and subsequently used in the malaria eradication programme. Resistance to DDT in An. stephensi was detected in Iran, Iraq, Pakistan, and Saudi Arabia in the late 1960s. Malathion was used for malaria control in Iran in 1967, then propoxur in 1978, followed by pirimiphos-methyl from 1992 to 1994. The pyrethroid insecticide lambda-cyhalothrin was used from 1994 to 2003 followed by deltamethrin IRS and long-lasting insecticidal nets (LLINs). Some of these insecticides with the same sequence were used in other malaria-endemic countries of the region. Pyrethroid resistance was detected in An. stephensi in Afghanistan in 2010, in 2011 in India and in 2012 in Iran. The newly invaded population of An. stephensi in Ethiopia was resistant to insecticides of all four major insecticide classes. Different mechanisms of insecticide resistance, including metabolic and insecticide target site insensitivity, have been developed in An. stephensi. Resistance to DDT was initially glutathione S-transferase based. Target site knockdown resistance was later selected by pyrethroids. Esterases and altered acetylcholinesterase are the underlying cause of organophosphate resistance and cytochrome p450s were involved in pyrethroid metabolic resistance. CONCLUSIONS: Anopheles stephensi is a major malaria vector in Iran and many countries in the region and beyond. The species is leading in terms of development of insecticide resistance as well as developing a variety of resistance mechanisms. Knowledge of the evolution of insecticide resistance and their underlying mechanisms, in particular, are important to Iran, considering the final steps the country is taking towards malaria elimination, but also to other countries in the region for their battle against malaria. This systematic review may also be of value to countries and territories newly invaded by this species, especially in the Horn of Africa, where the malaria situation is already dire.

Status of insecticide resistance and its biochemical and molecular mechanisms in Anopheles stephensi (Diptera: Culicidae) from Afghanistan.

BACKGROUND: Insecticide resistance of Anopheles stephensi, the main malaria vector in eastern Afghanistan, has been reported previously. This study describes the biochemical and molecular mechanisms of resistance to facilitate effective vector control and insecticide resistance management. METHODS: Mosquito larvae were collected from the provinces of Kunar, Laghman and Nangarhar from 2014 to 2017. The susceptibility of the reared 3-4 days old adults was tested with deltamethrin 0.05%, bendiocarb 0.1%, malathion 5%, permethrin 0.75% and DDT 4%. Cytochrome P450 content and general esterase, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities were measured in the three field populations and the results were compared with those of the laboratory susceptible An. stephensi Beech strain. Two separate allele-specific PCR assays were used to identify L1014, L1014F and L1014S mutations in the voltage gated sodium channel gene of An. stephensi. Probit analysis, ANOVA and Hardy-Weinberg equilibrium were used to analyse bioassay, biochemical assay and gene frequency data respectively. RESULTS: The population of An. stephensi from Kunar was susceptible to bendiocarb, apart from this, all populations were resistant to all the other insecticides tested. The differences between all values for cytochrome P450s, general esterases, GSTs and AChE inhibition rates in the Kunar, Laghman and Nangarhar populations were statistically significant when compared to the Beech strain, excluding GST activities between Kunar and Beech due to the high standard deviation in Kunar. The three different sodium channel alleles [L1014 (wild type), L1014F (kdr west) and L1014S (kdr east)] were all segregated in the Afghan populations. The frequencies of kdr east mutation were 22.9%, 32.7% and 35% in Kunar, Laghman and Nangarhar populations respectively. Kdr west was at the lowest frequency of 4.44%. CONCLUSIONS: Resistance to different groups of insecticides in the field populations of An. stephensi from Kunar, Laghman and Nangarhar Provinces of Afghanistan is caused by a range of metabolic and site insensitivity mechanisms, including esterases, cytochrome P450s and GSTs combined with AChE and sodium channel target site insensitivity. The intensity and frequency of these mechanisms are increasing in these populations, calling for urgent reorientation of vector control programmes and implementation of insecticide resistance management strategies.

Fauna, Ecological Characteristics, and Checklist of the Mosquitoes in Mazandaran Province, Northern Iran.

Mosquitoes are important vectors of human and animal diseases. This study updates current knowledge on fauna, dominance, and distribution of mosquitoes in Mazandaran Province, Northern Iran, to inform disease control effort. Larval collections, using standard dippers or droppers, and adult collections, using total catches, shelter pits, CDC light traps, and human landing catches, were performed monthly in 30 villages across 16 counties, from May to December 2014. Ovitraps, baited with hay infusion as oviposition attractants or stimulants for Aedes (Stegomyia) mosquitoes, were installed in each village and inspected weekly for eggs. Lactophenol and Berlese media were used for preserving and mounting specimens. Overall, 36,024 mosquito specimens (19,840 larvae and 16,184 adults) belonging to 4 genera and 20 species were morphologically identified. The dominance and distribution indices showed that Culex pipiens s.s. was the eudominant species with a constant distribution of larvae (D = 69.07%, C = 100%) and adults (D = 31.86%, C = 100%), followed by Cx tritaeniorhynchus (D = 38.14%, C = 100%) and Anopheles maculipennis s.l. (D = 11.05%, C = 100%) as adults. Aedes vexans was the dominant (7.85%) species, but it had a sporadic (20%) distribution. Culex torrentium and Culiseta morsitans were added as the new species to the checklist of mosquitoes in Mazandaran Province. Due to the potential role, Cx. pipiens s.s. as a vector of various pathogens, further ecological studies are recommended.

Correlation between mosquito larval density and their habitat physicochemical characteristics in Mazandaran Province, northern Iran.

Characteristics of mosquito larval habitats are important in determining whether they can survive and successfully complete their developmental stages. Therefore, data on the ecological factors affecting mosquito density and abundance especially the physicochemical properties of water of their breeding sites, can possibly be helpful in implementing larval management programs. Mosquito larvae were collected using a standard 350 ml dipper from fixed habitats including: artificial pool, river edge, creek and etc, in 30 villages of 16 counties from May-December 2014. Water samples were collected during larval collection and temperature (°C), acidity (pH), turbidity (NTU), electrical conductivity (µS/cm), alkalinity (mg/l CaCO3), total hardness (mg/l), nitrate (mg/l), chloride (mg/l), phosphate (mg/l) and sulphate (mg/l) were measured using standard methods. Spearman correlation coefficient, Kruskal-Wallis test of nonparametric analysis, Chi-square (χ2) analysis, regression analysis and C8 interspecific correlation coefficient were used for data analysis. A total of 7,566 mosquito larvae belonging to 15 species representing three genera were collected from fixed larval breeding places. Culex pipiens was the dominant species except in four villages where An. maculipennis s.l. and Cx. torrentium were predominant. There was a significant positive correlation between the density of Cx. pipiens and electrical conductivity, alkalinity, total hardness and chloride, whereas no significant negative correlation was observed between physicochemical factors and larval density. The highest interspecific association of up to 0.596 was observed between An. maculipennis s.l/An. pseudopictus followed by up to 0.435 between An. maculipennis s.l/An. hyrcanus and An. hyrcanus/An. pseudopictus. The correlations observed between physicochemical factors and larval density, can possibly confirm the effect of these parameters on the breeding activities of mosquitoes, and may be indicative of the presence of certain mosquito fauna in a given region.

Evidence of metabolic mechanisms playing a role in multiple insecticides resistance in Anopheles stephensi populations from Afghanistan.

BACKGROUND: Malaria is endemic in most parts of Afghanistan and insecticide-based vector control measures are central in controlling the disease. Insecticide resistance in the main malaria vector Anopheles stephensi from Afghanistan is increasing and attempts should be made to determine the underlying resistance mechanisms for its adequate management. METHODS: The contents of cytochrome P450s, esterases, glutathione S-transferases (GSTs) and acetylcholine esterase (AChE) activities were measured in the Kunar and Nangarhar populations of An. stephensi from Afghanistan and the results were compared with those of the susceptible Beech strain using the World Health Organization approved biochemical assay methods for adult mosquitoes. RESULTS: The cytochrome P450s enzyme ratios were 2.23- and 2.54-fold in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The enzyme ratios for esterases with alpha-naphthyl acetate were 1.45 and 2.11 and with beta-naphthyl acetate were 1.62 and 1.85 in the Kunar and Nangarhar populations respectively compared with the susceptible Beech strain. Esterase ratios with para-nitrophenyl acetate (pNPA) were 1.61 and 1.75 in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The GSTs enzyme ratios were 1.33 and 1.8 in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The inhibition of AChE was 70.9 in the susceptible Beech strain, and 56.7 and 51.5 in the Kunar and Nangarhar populations. The differences between all values of the enzymes activities/contents and AChE inhibition rates in the Kunar and Nangarhar populations were statistically significant when compared with those of the susceptible Beech strain. CONCLUSIONS: Based on the results, the reported resistance to pyrethroid and organophosphate insecticides, and tolerance to bendiocarb in the Kunar and Nangarhar populations of An. stephensi from Afghanistan are likely to be caused by a range of metabolic mechanisms, including esterases, P450s and GSTs combined with target site insensitivity in AChE.

Pyrethroid resistance in Iranian field populations of Rhipicephalus (Boophilus) annulatus.

Resistance to acaricides in ticks is becoming increasingly widespread throughout the world; therefore, tick control requires resistance monitoring for each tick species. The aims of this study were to monitor the susceptibility status of the cattle tick, Rhipicephalus (Boophilus) annulatus (Acari: Ixodidae), against pyrethroid acaricides from Mazandaran Province, northern Iran, and where resistance was evident, and establish the possible underlying mechanisms. Fully engorged adult R. (B.) annulatus females collected on cattle from Mazandaran Province. Twenty-nine tick populations produced 10-18days old larvae and bioassayed with cypermethrin and λ-cyhalothrin by larval packet test and the levels of detoxification enzymes were measured. Population AM-29 had a maximum resistance ratio (RR99) of 20.21 to cypermethrin and 53.57% of the tick populations were resistant at LC99 level. With λ-cyhalothrin, 17.86% of the tick populations were resistant and AM-29 was the most resistant population with RR99=4.54. AM-29 also showed significant elevation of cytochrome P450 monooxygenase (P450) and glutathione S-transferase (GST) (2.76- and 2.39-fold, respectively) (P<0.001). Rhipicephalus (Boophilus) annulatus showed resistance to pyrethroid insecticides with elevated levels of P450, GST and para-nitrophenyl acetate (p-NPA) in resistant populations. Operational failure was noted in controlling R. (B.) annulatus by pyrethroid insecticides, therefore alternative pest management measures should be adopted in Iran. For the first time, a new estimate of insecticide resistance based on effective dose recommended by the pesticide manufacturer termed Operational Dose Ratio (ODR) is defined and discussed.