J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 72 http://jad.tums.ac.ir Published Online: March 31, 2023 Original Article Evaluation of the Time Course on the Effectiveness of WHO Standard Pyrethroid and Carbamate Impregnated Test Papers against Anopheles stephensi, the Main Malaria Vector in Iran Awat Dehghan1, Seyed Hassan Moosa-Kazemi1, Mohammad Mehdi Sedaghat1, *Hassan Vatandoost1,2, *Ahmad Ali Hanafi-Bojd1,3 1Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Environmental Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3Zoonoses Research Center, Tehran University of Medical Sciences, Tehran, Iran *Corresponding authors: Dr Ahmad Ali Hanafi-Bojd, E-mail: aahanafibojd@tums.ac.ir; Dr Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com (Received 27 Apr 2022; accepted 19 Mar 2023) Abstract Background: Anopheles stephensi is a major vector of malaria in some parts of the world. A standard method for de- termining resistance in adult mosquito populations is the bioassay test recommended by the world health organization (WHO). The papers used in this method have an expiry date. This study aimed to determine the effectiveness of outdat- ed susceptibility test papers for use in insecticide resistance monitoring programs. Methods: Beech and Bandar Abbas strains of An. stephensi were reared in the insectary. Permethrin 0.75%, Deltame- thrin 0.05%, and Bendiocarb 0.1% impregnated test papers prepared by Universiti Sains Malaysia were used. Probit analysis was used to analyze the results and prepare time-mortality regression lines of LT50 and LT90. Results: There was a difference in the mortality of both tested strains of An. stephensi was exposed to all tested insecti- cides. Both expired and not expired Permethrin and Deltamethrin papers induced 100% mortality at the diagnostic time (60min), but their insecticidal properties were reduced gradually in serial times. The highest efficacy of test papers was in the first trimester after the expiry date and decreased over time. Conclusion: At the diagnostic time of 60 minutes, the mortality rate of both dated and expired papers was 100% in the pyrethroid insecticides, even three years after expiry dates, if stored in the package provided by the producer, in a re- frigerator. This value was reduced to less than 100% in the expired papers of Bendiocarb comparing the dated papers that induced 100% mortality. Keywords: Susceptibility test; Anopheles stephensi; Insecticide impregnated papers; Expiration date Introduction Malaria is an infectious disease and one of the major health problems worldwide. In 2021, estimated 247 million cases of malaria occurred worldwide, resulting in 619,000 deaths (1). The disease is caused by Plasmodium parasites and is transmitted to humans by the bites of Anoph- eles mosquitoes (2). One of the methods to pre- vent and fight this disease is to control vec- tors in different ways. Due to the importance of transmitting this disease in Iran, especially in the southern and southeastern regions, vector control operations are carried out regularly in these areas. In the global malaria strategy, the use of insecticides has maintained its special place in control operations. Following the in- creasing use of various pesticides and the emer- gence of the phenomenon of physiological and behavioral resistance of vectors, major problems Copyright © 2023 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:aahanafibojd@tums.ac.ir mailto:hvatandoost1@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 73 http://jad.tums.ac.ir Published Online: March 31, 2023 have been created in malaria control programs. One of the most important vectors of malaria in Iran is Anopheles stephensi, whose distribu- tion is limited to the provinces of Sistan and Baluchestan, Hormozgan, Bushehr, Kerman, Fars, Khuzestan, Ilam, Lorestan, Kohgiluyeh and Boyer-Ahmad, and Kermanshah (3, 4). Exten- sive use of residual spraying to control malar- ia vectors has led to resistance of An. stephensi to DDT, Dieldrin, Malathion and other organ- ophosphates, Bendiocarb, and some pyrethroids in the Middle East (5). The first report of re- sistance in An. stephensi was to DDT in 1957 in the south of the country (6). So, Dieldrin replaced DDT in the indoor residual spraying (IRS) program, but unfortunately, three years after the application of dieldrin, in 1960, cases of resistance to this insecticide were observed in the areas under the coverage of IRS (7). Malathion, Propoxur, Lambda-cyhalothrin, Del- tamethrin, and Bendiocarb, are other insecticides used for IRS in Iran, respectively. Unfortu- nately, recent studies in the last decade show An. stephensi is resistant to all groups of in- secticides in some parts of Iran (8–13). Increasing resistance to insecticides is a com- plex and dynamic process and depends on many factors. Increasing the dose of insecticide is not recommended to maintain effectiveness because it creates environmental problems and adversely affects human life. The resistance gene in the vector population may also increase dramati- cally. Often, when the number of resistant in- sects in a vector population increases, the ef- fectiveness of a particular insecticide decreas- es to the point that it must be replaced by an- other insecticide. Therefore, insecticide re- sistance management is a very important issue that should be considered in malaria control programs. Detection of insecticide resistance in natural vector populations is essential for ma- laria control. A standard method for determin- ing resistance in adult mosquito populations is the bioassay test recommended by the World Health Organization (14). In the WHO stand- ard method, mosquito specimens are exposed to a series of different insecticides using insecti- cide-impregnated papers at differential or di- agnostic doses. This method has been widely used in this field and gives acceptable results in detecting insecticide resistance for monitor- ing purposes. The papers used in this method have an expiration date, so due to the customs consideration in some countries, it is a time- consuming process from the date they are pro- duced until they reach the malaria vector con- trol authorities; and may expire by the appro- priate time for starting the susceptibility tests. In 1957, during studies on the expiration date of insecticide-impregnated papers in sus- ceptibility tests against Aedes aegypti using the WHO standard method, it was found no sig- nificant difference between the two groups of papers impregnated with DDT and Dieldrin with different ages after production (15). In review- ing previous research around the world, we found that no other study has been conducted in this field since that date. During these years, new insecticides have been introduced and used around the world to control malaria vectors. Insecticide-impregnated test papers have also changed a lot. This study aimed to answer the question of whether the expiration date listed on the sus- ceptibility test papers can be a deterrent to the use of these papers or influence their lethality. Materials and Methods Mosquito strains We used a field strain of this mosquito col- lected from Hormoodar Village (56.32 oE, 27.31 oN), Bandar Abbas County, southern Iran, as well as the Beech susceptible laboratory strain. Phenotypic resistance profile of the wild pop- ulation (Bandar Abbas) of An. stephensi has been identified in a previous study (9). The collected samples from the field were trans- ferred to the insectary and reared to establish the laboratory colony. The rearing conditions were at 30±2 °C, 65±5% relative humidity, and a light to dark period of 12 to 12 hours (16). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 74 http://jad.tums.ac.ir Published Online: March 31, 2023 An artificial blood-feeding device with human whole blood was used to feed the adult female mosquitoes and this operation was repeated every three days (17). The colony was fed on a 10% sucrose solution. Insecticides Three insecticides that are used by the Iran national malaria control program, including Per- methrin (in the long-lasting insecticide-treated nets (LLINs), Deltamethrin, and Bendiocarb (in IRS), were considered in this study. So, for this experiment, we used Deltamethrin 0.05%, Per- methrin 0.75%, and Bendiocarb 0.1% impreg- nated papers prepared by the WHO collabora- tion center, Universiti Sains Malaysia (USM) (Table 1). Two series of each insecticide were used in this study with different impregnation and expiry dates. These papers were provided to us by the Center for Disease Management of the Ministry of Health and kept in the refrig- erator under standard conditions. Susceptibility tests Tests started on 9 Dec 2020 and finished on 27 August 2021. Three to five-day-old nul- liparous female mosquitoes fed with sucrose 10% were used in insecticide susceptibility tests, according to the WHO standard method and using standard impregnated papers (15). Each experiment for each tested time consist- ed of four replications and two controls, and in each replication, 25 adult mosquitoes were tested. The tests were performed at serial times of 7.5, 15, 30, and 60 minutes with the diagnostic dose of each insecticide. Shorter times were used as needed. With this method, we tested the efficacy of the test papers in se- rial time under 60 minutes to be able to calcu- late the LT50 and to compare the LT50s be- tween the different groups of test papers. Ac- cording to the WHO instructions, each paper was used only 6 times (15). Then, the tested mosquitoes were kept in the insectary condi- tion for 24 hours. The number of dead speci- mens was recorded at the end of the above time. If the mosquitoes in the control group had no mortality or less than 5%, the test was considered correct. If the control mosquitoes had a mortality of 5–20%, the results were corrected with the Abbott formula. Also, if the control mortality was more than 20%, the test was unacceptable and repeated (15). Data analysis Probit software (18) was used to analyze the results and to prepare a lethal time regres- sion line. The results were displayed using de- scriptive tables and graphs. By comparing the mortality rate of the papers impregnated with a specific insecticide and its standard devia- tion with different expiration dates, the effect of time course on the lethality of these papers was investigated. Results Bendiocarb 0.1% Beech laboratory and Bandar Abbas strains of An. stephensi were tested with the papers produced in Aug. 2017, expired in Aug. 2020, and tested in Feb. 2021. These tests were per- formed in five serial times (225, 450, 900, 1800, and 3600 sec). The regression line equa- tions obtained from probit analysis were cal- culated as Y= 0.0007X+3.5474 and Y= 0.0007X+3.405 respectively (Fig. 1). Also, the two strains were tested with the papers produced in Aug. 2019, expired in Aug. 2022, and tested in May 2021. These tests were performed with seven serial times (56, 112, 225, 450, 900, 1800, 3600 sec). The regression line equations obtained from probit analysis were calculated as Y= 0.0023X+ 4.1386 and Y= 0.002X+3.645 (Fig. 1). The LT50 and LT90 values for the two Bendiocarb papers are presented in Fig. 2. Permethrin 0.75% Beech laboratory and Bandar Abbas strains of An. stephensi were tested with the papers produced in Aug. 2017, expired in Aug. 2018, and tested in March 2021. These tests were http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 75 http://jad.tums.ac.ir Published Online: March 31, 2023 performed in eight serial times (28, 56, 112, 225, 450, 900, 1800, and 3600 sec). The re- gression line equations obtained from probit analysis were calculated as Y= 0.0035X+ 4.5092 (Fig. 3) and Y= 0.0019X+4.6029 (Fig. 5), respectively. Also, the two strains were tested with the papers produced in Aug. 2019 and expired in Aug. 2020. These tests were performed with 8 serial times (28, 56, 112, 225, 450, 900, 1800, 3600 sec), three, six, and nine months after expiration. For the Beech strain, the regres- sion line equation obtained from probit analy- sis was calculated as Y= 0.0074X+4.4337, Y= 0.0036X+4.7234, and Y= 0.0032X+4.7962 for the 1st, 2nd, and 3rd trimesters, respectively (Fig. 3). The LT50 and LT90 values are presented in Fig. 4. The LT50 and LT90 values are pre- sented in Fig. 4. For Bandar Abbas strain, the regression line equations for the first, second, and third trimesters were Y= 0.0088X+ 4.1856, Y= 0.0051X+4.4706, and Y= 0.0039X+4.6843, respectively (Fig. 5). Deltamethrin 0.05% Beech laboratory and Bandar Abbas strains of An. stephensi were tested with the papers produced in Aug. 2017, expired in Aug. 2018, and tested in March 2021. These tests were performed in eight serial times (28, 56, 112, 225, 450, 900, 1800, and 3600 sec). The re- gression line equations obtained from probit analysis were calculated as Y= 0.0015X+4.7598 (Fig. 6) and Y= 0.0038X+4.0065 (Fig. 7), re- spectively. Also, the two strains were tested with the papers produced in Aug. 2019 and expired in Aug. 2020. These tests were performed with 8 serial times (28, 56, 112, 225, 450, 900, 1800, 3600 sec), three, six, and nine months after expiration. For the Beech strain, the regres- sion line equation obtained from probit analy- sis was calculated as Y= 0.0031X+4.6574, Y= 0.0015X+5.1529, and Y= 0.0016X+5.0414 for the 1st, 2nd, and 3rd trimesters, respectively (Fig. 6). The LT50 and LT90 values are presented in Fig. 7. For Bandar Abbas strain, the regres- sion line equations for the first, second, and third trimesters were Y= 0.0033X+4.3534, Y= 0.0017X+4.6184, and Y= 0.0008X+4.9212, re- spectively (Fig. 7). The LT50 and LT90 values are presented in Fig. 8. Fig. 1. Comparison of time-mortality regression lines due to exposure of Anopheles stephensi with Bendiocarb 0.1% with two different producing and expiration dates. Be= Beech strain, BA= Bandar Abbas strain http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 76 http://jad.tums.ac.ir Published Online: March 31, 2023 Table 1. Specifications of insecticides used in this study Insecticide Impregnation date Expiry date Batch number Control number Deltamethrin 0.05% August 2017 August 2018 DE 527 527 Permethrin 0.75% August 2017 August 2018 PE 428 428 Deltamethrin 0.05% August 2019 August 2020 DE 717 717 Permethrin 0.75% August 2019 August 2020 PE 594 594 Bendiocarb 0.1% August 2017 August 2020 BE 200 200 Bendiocarb 0.1% August 2019 August 2022 BE 272 272 Fig. 2. Comparison of LT50 and LT90 values of two strains of Anopheles stephensi after exposure with Bendiocarb 0.1% with two different expiration dates. Be= Beech strain, BA= Bandar Abbas strain. Error bars show standard deviation (SD) 2.00 4.00 6.00 8.00 10.00 1 10 100 1000 P r o b it m o r ta li ty Exposure time (second) probit2017 probit2019 three month after expired probit2019 six month after expired probit2019 nine month after expired Log. (probit2017) Log. (probit2019 three month after expired) Log. (probit2019 six month after expired) Log. (probit2019 nine month after expired) Fig. 3. Comparison of the regression lines of time-mortality due to exposure of Anopheles stephensi (Beech strain) to Permethrin 0.75% with two different production and expiration dates http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 77 http://jad.tums.ac.ir Published Online: March 31, 2023 i Fig. 4. Comparison of LT50 and LT90 values of two strains of Anopheles stephensi after exposure with Permethrin 0.75% with two different expiration dates. Be= Beech strain, BA= Bandar Abbas strain. Error bars show standard deviation (SD) 2.00 3.00 4.00 5.00 6.00 7.00 8.00 1 10 100 1000 P r o b it m o r ta li ty Exposure time (Second) probit2017 probit2019 three month after expired probit2019 six month after expired probit2019 Nine month after expired Log. (probit2017) Log. (probit2019 three month after expired) Log. (probit2019 six month after expired) Log. (probit2019 Nine month after expired) Fig. 5. Comparison of the regression lines of time-mortality due to exposure of Anopheles stephensi (Bandar Abbas strain) to Permethrin 0.75% with two different production and expiration dates http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 78 http://jad.tums.ac.ir Published Online: March 31, 2023 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 10 100 1000 P r o b it m o r ta li ty Exposure time (S econd) probit2017 probit2019 three month after expired probit2019 six month after expired probit2019 nine month after expired Log. (probit2017) Log. (probit2019 three month after expired) Log. (probit2019 six month after expired) Log. (probit2019 nine month after expired) Fig. 6. Comparison of the regression lines of time-mortality due to exposure of Anopheles stephensi (Beech strain) to Deltamethrin 0.05% with two different production and expiration dates 2.00 3.00 4.00 5.00 6.00 7.00 8.00 1 10 100 1000 10000 P r o b it m o r ta li ty Exposure time (S econd) probit2017 probit2019 three month after expired probit 2019 six month after expired probit 2019 nine month after expired Log. (probit2017) Log. (probit2019 three month after expired) Log. (probit 2019 six month after expired) Log. (probit 2019 nine month after expired) Fig. 7. Comparison of the regression lines of time-mortality due to exposure of Anopheles stephensi (Bandar Abbas strain) to Deltamethrin 0.05% with two different production and expiration dates http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 79 http://jad.tums.ac.ir Published Online: March 31, 2023 Fig. 8. Comparison of LT50 and LT90 values of two strains of Anopheles stephensi after exposure with Deltamethrin 0.05% with two different expiration dates. Be= Beech strain, BA= Bandar Abbas strain. Error bars show standard deviation (SD) Discussion Given that our study is the first in the world on susceptibility test papers produced by the WHO collaboration center in Malaysia (USM) and there are no similar studies for it, in this section, we discuss the insecticide used and the reasons for the decrease in the effectiveness of susceptibility test papers at different times after production. Although in a study conduct- ed in the 1960s using DDT test papers against Ae. aegypti, each set of papers killed very sim- ilar proportions of the tested mosquitos (15). In this study, we found in all tests performed using deltamethrin 0.05% and permethrin 0.75% according to the standard method of the WHO in the diagnostic time of 60 minutes, in both dated and outdated papers, the mortality rate was found to be 100% in Beech and Bandar Abbas strains of An. stephensi. Comparing the regression lines of time mortality obtained from probit analysis of Ben- diocarb insecticide against Beech strain of An. stephensi with different expiration dates, it is clear that the insecticidal properties decreased one year after the expiration date of Bendio carb 0.1% impregnated papers. Also, the val- ues of LT50 and LT90 in expired papers have increased 5.4 and 4.4 times, respectively (Fig. 2). Also, in the tests performed with 2017– 2020 Bendiocarb impregnated papers, the LT90 value is 3 times LT50, while for 2019–2022 Bendiocarb papers, the LT90 value is 3.7 times LT50. Comparing the regression lines of time mortality obtained from probit analysis of Ben- diocarb insecticide against Bandar Abbas strain of An. stephensi with different expiration dates, it is clear that the insecticidal properties de- creased one year after the expiration date of Bendiocarb 0.1% impregnated papers. Also, the values of LT50 and LT90 in expired papers have increased by 3.2 and 2.2 times, respectively (Fig. 2). Also, in the tests performed with 2017–2020 Bendiocarb impregnated papers, the LT90 val- ue is 2.5 times LT50, while for 2019–2022 Ben- diocarb papers, the LT90 value is 3.5 times LT50 (Fig. 2). Comparing the regression lines of time mor- tality obtained from probit analysis of Perme- thrin insecticide against Beech strain of An. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 80 http://jad.tums.ac.ir Published Online: March 31, 2023 stephensi with different expiry dates, the insec- ticidal properties decreased over time. Also, the LT50 and LT90 in the 2017–2018 papers have increased compared to the papers of 2019–2020 (1.5 times for LT50 and 2.3 times for LT90). The results showed that in the third trimester (9 months after the expiration of 2019–2020 papers), the values of LT50 and LT90 have in- creased by 1.04 times for LT50 and 1.7 times for LT90 compared to the first trimester (Fig. 4). Comparing the regression lines of time mor- tality obtained from probit analysis of Perme- thrin insecticide against Bandar Abbas strain of An. stephensi with different expiry dates, the in- secticidal properties decreased over time. Also, the LT50 and LT90 in the 2017–2018 papers have increased compared to the papers of 2019–2020 (2.1 times for LT50 and 6.3 times for LT90). The results showed that in the third trimester (9 months after the expiry date of 2019–2020 pa- pers), the values of LT50 and LT90 increased 1.7 times for LT50 and 3.8 times for LT90 com- pared to the first trimester (Fig. 4). Comparing the regression lines of time mor- tality obtained from probit analysis of Deltame- thrin insecticide against Beech strain of An. ste- phensi with different expiration dates, the in- secticidal properties decreased over time. Also, the LT50 and LT90 in the 2017–2018 papers have increased compared to the papers of 2019–2020 (1.7 times for LT50 and 2.3 times for LT90). The results showed that in the third trimester (9 months after the expiration of 2019–2020 pa- pers), the values of LT50 and LT90 increased by 1.4 times for LT50 and 1.4 times for LT90 com- pared to the first trimester (Fig. 8). Compared the regression lines of time mortality obtained from probit analysis of Deltamethrin insecticide against Bandar Abbas strain of An. stephensi with different expiry dates, the insecticidal prop- erties decreased over time. Also, the LT50 and LT90 in the 2017–2018 papers have increased compared to the papers of 2019–2020 (1.4 times for LT50 and 2 times for LT90). The results showed that in the third trimester (9 months af- ter the expiry date of 2019–2020 papers), the values of LT50 and LT90 increased 1.1 times for LT50 and 1.7 times for LT90 compared to the first trimester (Fig. 8). The decrease in mortality and the increase in LT50 and LT90 are due to the decrease in the insecticidal properties of the insecticides used, which can be due to several reasons, including breaking and decomposing part of the insecti- cide or evaporating it later. This issue should be further investigated and the volume of in- secticide residues on susceptibility test papers should be measured with special facilities and methods (19, 20). Also, another reason for the decrease in the quality of these papers over time can be related to the storage conditions and how these papers are stored. In the interval between tests, the papers should be stored in their orig- inal plastic box, to be sealed with adhesive tapes, and stored in the refrigerator at 4 °C or, if this is not possible, in a dark cupboard at room temperature. Papers stored at 4 °C should be exposed to room temperature before use. In- secticide-impregnated papers should never be exposed to direct sunlight (14). In general, by comparing the mortality rates of two strains of Bandar Abbas and Beech at different serial times in the presence of Ben- diocarb 0.1%, Permethrin 0.75%, and Deltame- thrin 0.05%, we conclude that the susceptible laboratory strain in exposure to insecticide-im- pregnated papers is more sensitive because it had a higher mortality rate. The results of the most recent study in Bandar Abbas showed this species was resistant to Bendiocarb 0.1%, Permethrin 0.75%, and Deltamethrin 0.05% (9). The lower mortality rate in the Bandar Abbas strain could be due to the presence of re- sistance genes or metabolic mechanisms in the natural population of this strain because this strain was collected from the field and used after breeding in the insectary. Synergist bio- assays on the DDT and Permethrin-resistant la- boratory strains of An. stephensi from Iran in- dicated the metabolic resistance in this species (21). Another study in Iran confirmed that met- abolic mechanisms play a critical role in the http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 72–82 A Dehghan et al.: Evaluation of the … 81 http://jad.tums.ac.ir Published Online: March 31, 2023 resistance of An. stephensi to Cyfluthrin, a py- rethroid insecticide (12). It is recommended that additional studies be performed on insecticide residues and their metabolites on the WHO susceptibility test pa- pers at different times after production. Due to the special conditions of the Covid-19 pandem- ic, access to papers with different production dates was not possible for this study. This is the limitation of this research. Conclusions At the diagnostic time of 60 minutes, the mortality rate of dated and expired papers was equal in the pyrethroid insecticides, even three years after expiry dates, if stored in their stand- ard package in the refrigerator. About the Ben- diocarb, the mortality rate of the tested mos- quitoes was less than 100% in the expired test papers. Due to the high cost of test papers for the countries which are involved in malaria diseases, especially in the African region, the results of this experiment are interesting. By the way, to chemically analyze the residual amount of insecticides or their metabolites on the pa- pers using HPLC or GC is suggested to have an idea about the active ingredient remaining on the papers on different dates. Acknowledgments We thank the staff of the mosquito insec- tary, Department of Vector Biology and Con- trol of Diseases, School of Public Health, Teh- ran University of Medical Sciences, for their cooperation in breeding mosquitoes. Special thanks to Mr A Badzohre for his help in doing tests and Dr F Nikpour and the Center for Dis- ease Management of the Ministry of Health for providing the susceptibility test papers. 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