J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 17 http://jad.tums.ac.ir Published Online: April 27, 2019 Original Article Comparison of CDC Bottle Bioassay with WHO Standard Method for Assessment Susceptibility Level of Malaria Vector, Anopheles stephensi to Three Imagicides *Hassan Vatandoost1,2; *Mohammad Reza Abai1,2; Morteza Akbari1; Ahmad Raeisi3; Hemn Yousefi1; Soraya Sheikhi1; Akbar Bagheri1 1Department of Medical Entomology and Vector Control, School of Public Health and National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran 2Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3Department of Malaria, Center for Disease Control (CDC), Ministry of Health and Medical Education, Tehran, Iran (Received 8 Jan 2018; accepted 16 Apr 2018) Abstract Background: The detection of insecticide resistance in natural populations of Anopheles vectors is absolutely necessary for malaria control. CDC bottle bioassay as a new tools has been employed for detecting the insecticide resistance. For a limit number of mosquito vectors, diagnostic doses and diagnostic times for some insecticides have already been deter- mined using this new assay. For the first time in the area, susceptibility levels of Anopheles stephensi was done with DDT, deltamethrin, and bendiocarb using CDC bottle bioassay and compared results with WHO standard test method. Methods: Anopheles stephensi were collected in larvae stage from the cisterns of drinking water in Chabahar port which considered as old malaria foci, Sistan and Baluchistan province. The field collected larvae were colonized at the insectary of School of Public Health (SPH), Tehran University of Medical Science. The susceptibility tests were carried out on sugar fed female mosquitoes aged 2–3 days, against DDT 4%, bendiocarb 1% and deltamethrin 0.05% using WHO and CDC susceptibility methods. The mortality and knockdown rates, as well as the parameters of regression analysis, includ- ing LT50 and LT90, was calculated separately for the WHO and CDC methods. Results: The 24h mortality rates of An. stephensi were 28.6% and 25.6% for DDT, 60.8% and 64.6% for bendiocarb and 100% for deltamethrin using both WHO and CDC assay at 30 and 60min respectively. The 50% lethal times (LT50) were estimated 44.9 and 66.2min, 38.9 and 81.8min and 0.7 and 15.0min respectively using both WHO and CDC susceptibility tests. Conclusion: The similar results of susceptibility levels were shown for DDT, bendiocarb and deltamethrin. The lethal times (LT50) showed significant difference using both WHO and CDC bioassay methods. Keywords: Susceptibility; Insecticide; WHO bioassay; CDC bioassay; Anopheles stephensi Introduction Malaria and other mosquito-borne diseases are the major problems worldwide. Currently, there are proven and effective tools to combat against vectors (1). More than 80% of malaria cases in the coun- try are reported from three provinces southern and southeastern areas of the country (2). The most routes of malaria cases are immigratio from eastern borders (3) which affected by ex- tensive population movement across the bor- der with Pakistan, There are several works on different aspects of malaria in the country which is useful for decision making (4). Among of Anopheles mosquitoes, Anopheles stephensi Lis- ton (Diptera: Culicidae) for both Plasmodium falciparum and P. vivax with the geographical range from the Middle East to India and China and southern slope of areas located in Zagros *Corresponding authors: Prof Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com, Mr Mohammad Reza Abai, E-mail: abaimr@tums.ac.ir J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 18 http://jad.tums.ac.ir Published Online: April 27, 2019 mountain chains (5). Recent studies in Iran have been revealed the presence of 31 Anopheles species including genotypes and sibling spe- cies, eight of them involved in malaria trans- mission in Iran (6). Vector control as the main measures used to reduce malaria transmission at the community level and considered as the most effective measure for eradicating malaria. It has been proven to be the only measure that can reduce malaria transmission from very high to a low level (7). Resistance to insecticides among the mosquito vectors is the most im- portant growing concerns in many countries and bioassays allow for the detection and charac- terization of insecticide resistance in a vector population. World Health Organization (WHO) susceptibility test is the main assay for assessing the susceptibility /resistance status among mos- quito vectors. CDC bottle bioassay is a new tool for assessing resistance to insecticides in the world (8). The goal of CDC bioassay is to meas- ure the mortality rate of members of a popula- tion at a given dose of the insecticide. In order to know the advantages, disadvantages and fea- tures, the WHO test compared with the CDC bottle bioassay using three insecticides against An. stephensi. The resistance parameters of this research allowed comparing data across other countries. To determine the diagnostic dose and the diag- nostic time for use in the CDC bottle bioassay, the assay was calibrated. Materials and Methods Mosquito tested The larvae of An. stephensi were collected from the artificial ponds in the urban area of Chabahar Port, Sistan and Baluchistan Prov- ince and colonized at the insectary of School of Public Health (SPH), Tehran University of Medical Science (TUMS). The rearing condi- tion was 29±1 and 60–65% relative humidity with 12L: 12D photoperiod cycle. Larvae were fed with fish flakes and adults were fed a sugar water solution consisting of 10% sucrose. For mosquito mass colonization, female mosqui- toes were fed on guinea pin hold in a restrainer. Females 3 to 4 days post emergence were used in all experiments. Insecticide materials The choice of deltamethrin and bendiocarb were based on conventional use in residual spray- ing in malaria foci and DDT selected as indica- tor insecticide for revealing of resistance. The insecticide impregnated papers were purchased with WHO representative in Penang, Malaysia. The technical active ingredient of DDT, bendi- ocarb and deltamethrin were provided from Eco- toxicology Laboratory, School of Public Health, Tehran University of Medical Sciences. WHO’s susceptibility test protocol In each replicate, 20 to 25 sugar-fed female mosquitoes aged 2–5 days gently released into the holding tubes with green dots (4–5 as treat- ed group and 1 as control one) lined inside each tube with untreated papers following the WHO’s protocol (9). After resting the mosquitoes 15 minutes at the vertical position, they gently blown into the exposure tubes with red dots lined inside the tubes with WHO’s insecticide impregnated papers including DDT 4%, bendi- ocarb 1% and deltamethrin 0.05%. The mortal- ity rate of An. stephensi was assessed at loga- rithmic trend around the diagnostic time. After each exposure time, the mosquitoes were trans- ferred back into the holding tubes marked with green dots and provided the cotton pad mois- tened with the 10% sucrose solution at top of the nets. The knockdown and mortality rates were recorded after 60 minutes and 24 hours respec- tively. The environmental condition of test room was 28±1 °C and 60–65% relative humidity. CDC bottle test protocol The CDC bottle test was conducted as a new surveillance tool for detecting the insecticide resistance among malaria vector at the given J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 19 http://jad.tums.ac.ir Published Online: April 27, 2019 diagnostic doses and the diagnostic exposure times. The stock solutions were prepared by add- ing 10 and 1.25µl of technical grade of DDT, deltamethrin and bendiocarb per 100ml pure ethanol. The inside surfaces of 250ml glass bottles were coated with 1ml of stock solution and control bottles were coated only with pure alcohol according to the CDC protocol. For each concentration five bottles were coated. The di- agnostic doses of DDT, deltamethrin and ben- diocarb for Anopheles species are respectively standardized as 100, 12.5 and 12.5µg/bottle (8). The coated bottles were placed in incubator equipped to ventilator and set temperature at 35 °C to allow to evaporate the solvent overnight. After the alcohol was fully evaporated, 15–25 mosquitoes aged 3–4 days fed with 10% su- crose solution were released to each bottle us- ing a hand aspirator. The exposure time was set to 30min and for possible estimating of median lethal time (LT50), different exposure times 0.5, 1.0, 1.9, 3.8, 7.5, 15, 30, 45, 60, 75, 90, 105 and 120min were used. The mosquitoes were re- moved from the bottles and sorted into “alive” and “knocked-down” groups. Mosquito groups were kept in separate paper cups with 10% su- crose solution under insectary conditions. After 24h, they were scored as alive or dead in order to determine delayed mortality. At least five rep- licates were performed for each exposure time. Statistical analysis and data interpretation The data interpretation for WHO suscepti- bility tests for susceptible/resistance status of Anopheles species were adopted according to the latest related criteria (1) as follows: 1. Susceptible status: When 98–100% mor- tality rate resulted at the recommended di- agnostic dose and time. 2. Tolerance status: When, 90–97% mortal- ity rate resulted at the recommended diag- nostic dose and time which suggests the pos- sibility of resistance and needs to be con- firmed. 3. Resistance status: When less than 90% mor- tality rate resulted at the recommended diag- nostic dose and time. The data interpretation of the CDC bottle test was adopted according to the related pro- tocol. The susceptibility thresholds was con- sidered at the diagnostic time of 30 minutes for all insecticide tested at the recommended diag- nostic doses (8): 1. Susceptible status: The tested mosquitoes with insecticide-coated bottles are considered susceptible when died before the diagnostic time. 2. Resistance status: If tested mosquitoes sur- vive beyond diagnostic time, these survivors represent a proportion of the population with some degree of resistance. The susceptibility tests can be carried out beyond the diagnostic time to evaluate the intensity of resistance. The data from the CDC bottle bioassay us- ing test mosquitoes need to be compared with data from susceptible mosquitoes or from a pop- ulation that will serve as baseline. Resistance thresholds for each insecticide can be deter- mined by calibrating the CDC bottle bioassay. The bioassay data were analyzed using the probit program and the values for the lethal times 50% and 90% mortality (LT50, LT90) and 95% confidence interval were estimated. The Abbott’s formula was not applied to the mortality rate in treated and control group were less than 5% in all experiments (10). The re- gression line of the tested insecticides were plotted using Microsoft Excel (ver. 2013). Results Susceptibility to DDT The 24h mortality rate of An. stephensi was 28.6% and 25.6 % exposed respectively 30min and 60min to DDT using WHO and CDC as- says (Table 1). The logarithmic exposure times of DDT were ranged from 15 to 120min for CDC bottle test and from 30 to 240 minutes for J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 20 http://jad.tums.ac.ir Published Online: April 27, 2019 WHO test. The 50% lethal times (LT50) were estimated 44.9min and 66.2min respectively. The DDT regression equations of the mortality An. stephensi were set as Y= -6.4192+3.5250X and Y= -4.9652+3.0045X with WHO and CDC assays (Table 2). Susceptibility to bendiocarb The 24h mortality rates of An. stephensi were 60.8% and 64.6% exposed 30min and 60 min to bendiocarb using WHO and CDC assays (Table 1). The exposure times of bendiocarb were ranged from 7.5 to 60min for WHO tests and from 15 to 120 minutes for CDC bottle tests. The 50% lethal times (LT50) were 38.9min and 81.8min respectively. The regression equations of the mortality An. stephensi were set as Y= - 4.6479+2.9241X and Y= -4.4782+2.3413X with WHO and CDC assays (Fig. 2). Susceptibility to deltamethrin The 24h mortality rates of An. stephensi were 100% exposed 30min and 60min to deltame- thrin using WHO and CDC assays (Table 1). The exposure times of deltamethrin were ranged from 0.5 to 120min for WHO tests and from 15 to 120 minutes for CDC bottle tests. The 50% lethal times (LT50) were 0.8min and 15.4min respectively. The regression equations of the mortality An. stephensi were set as Y= 0.3371+2.7535X and Y= -3.4038+2.8646X with WHO and CDC assays (Fig. 3). Table 1. Susceptibility data recorded according to both WHO and CDC methods against An. stephensi Insecticides Susceptibility method Range of exposure time (min) Total tested Range of mortality rate (%) at loga- rithmic exposure time Mortality rate (%) after 24h maintenance Resistance status DDT WHO 30–240 371 18.6–100 28.6 (60min) R* CDC 15–120 1107 13.9–100 25.6 (30min) R Bendiocarb WHO 7.5–60 369 13.3–97.8 60.8 (60min) R CDC 15–120 389 57.3–100 64.6 (30min) R Deltamethrin WHO 0.47–120 380 28.3–100 100 (60min) S** CDC 15–120 1080 100–100 100 (30min) S * Resistance ** Susceptible Table 2. Probit regression line parameters of Anopheles stephensi exposed to different insecticides with WHO method Insecticide Suscepti- bility assay A B±SE LT50, 95% C.I. (Min) LT90, 95% C.I. (Min) X2 (df) P value DDT 4% WHO -6.4192 3.5250±0.298 59.3593 66.2271 73.6338 131.8797 152.9699 185.3884 15.265 (2) 0.01 DDT 1% CDC -4.9652 3.0045±0.151 42.0031 44.9348 48.0928 107.3886 119.9894 136.7425 81.874 (2) 0.01 Bendiocarb 1% WHO -4.6479 2.9241±0.262 34.4114 38.8617 43.7645 88.9256 106.6132 135.9259 9.045 (2) 0.05 J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 21 http://jad.tums.ac.ir Published Online: April 27, 2019 Bendiocarb 1% CDC -4.4782 2.3413±0.254 70.1951 81.7899 98.9898 209.6077 288.4610 464.5222 21.696 (2) 0.05 Deltamethrin 0.05% WHO 0.3371 2.7535±0.249 0.651 0.7543 0.8571 1.8701 2.2029 2.7275 4.545 (2) 0.05 Deltamethrin 1% CDC - 3.4038 2.8646±0.346 12.3959 15.4251 18.0780 36.7971 43.2137 54.0015 46.788 (2) 0.05 Fig. 1. Mortality rate and regression analysis of bioassays of Anopheles stephensi (Chabahar strain) exposed to DDT using WHO and CDC methods Fig. 2. Mortality rate and regression analysis of bioassays of Anopheles stephensi (Chabahar strain) exposed to bendiocarb using WHO and CDC methods, 2015–2016 Table 2. Continued … J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 22 http://jad.tums.ac.ir Published Online: April 27, 2019 Fig. 3. Mortality rate and regression analysis of bioassays of Anopheles stephensi (Chabahar strain) exposed to del- tamethrin using WHO and CDC methods, 2015–2016 Discussion The WHO standard method is a major and widely used method in the country, but the CDC bottle bioassay method was used for the first time in order to compare the resistance levels. Infor- mation about the susceptibility of the vectors to insecticide is essential for chemical interventions, so routine determining the susceptibility levels of malaria vector is an integral part of vector control program. In this study, two important methods were used to perform susceptibility levels on ma- laria vector, An. stephensi that has been colo- nized at the insectary of SPH, TUMS which the first generation transported from Chabahar dis- trict have been showed resistance or tolerance to several types of insecticides including orga- nochlorine, pyrethroid and carbamate (11). Ap- proximately in all previously studies on suscep- tibility status of An. stephensi in Iran, resistance to DDT have been reported, although tolerance to DDT only reported from southeastern part of the country (12). This is the first study for using CDC bottle test for detecting insecticide resistance in the mosquito vectors in Iran. There are some published studies on using CDC bottle test for detecting DDT resistance on disease vectors with 30min exposure time including An. gambiae in Madagascar with 99 % mortality rate (13), same species in Nigeria with 25% mortality (14, 15), An. gambiae in Zambia with 90% (16) and on An. nuneztovari in Columbia with 85% mortality (17). Our find- ing also showed highly resistance of An. ste- phensi to DDT with similar values (25.6% and 28.6%) for mortality rate using both test meth- ods. This is the second evidence of similarity of the results using both CDC and WHO bioassay method for revealing of susceptibility status com- pared the first one (22). The CDC bioassay con- ducted in the Macha, Zambia, the DDT at 300 μg/bottle, with delayed mortality 13–69% (18). The CDC bioassay method was also applied J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 23 http://jad.tums.ac.ir Published Online: April 27, 2019 to reveal the resistance status of malaria vec- tors to bendiocarb, with 30min exposure time including An. gambiae in the Benin with 78.9- 100% mortality depending to different tested localities (19, 20), An. gambiae in northern Be- nin with 100% mortality both in the field (16) and laboratory condition (18). The CDC bottle assay test showed that An. gambiae s.l. was ful- ly susceptible to bendiocarb in Nigeria (15) and in almost all of the 18 sampled districts in Ethi- opia except in Omonada, which 25% mortality was recorded (14). Regarding An. gambiae from Tanguieta site, a mortality of 78.9% was rec- orded after exposure to CDC bottles treated with bendiocarb showing an indication of resistance of this population to this insecticide (20). In this study, An. stephensi showed 57.3– 100% mortality (Table 1). Regarding Anophe- les gambiae s.l. population from the Tanguieta, Benin, resistant to bendiocarb was reported ac- cording to both WHO and CDC methods with mortality rates of 56.14% and 78.94% respec- tively (16). With WHO tube test, the exposure of An. stephensi to the bendiocarb 0.1% showed 13.3–97.8% in this study, and 71.4% on An. stephensi in other study carried out in the Nikshahr, southeastern Iran (23). Due to the extensive use of pyrethroids both for indoor residual spraying and impreg- nating bed nets and some reports for resistance occurrence among the Anopheles vectors (23- 24), the comparative study using WHO and CDC were carried out for revealing the re- sistance level in the world. This study revealed the susceptibility of An. stephensi to the del- tamethrin (Table 1, Fig. 3). Gorouhi et al. in 2015 indicated that field strain An. stephensi is the resistant candidate to deltamethrin (25). A bioassays conducted in north and south Benin, 100% mortality of An. gambiae was also shown to deltamethrin with CDC assay at 12.5μg/bottle and 30min exposure time (21) as well as with deltamethrin 0.05% impregnated paper (22). The CDC insecticide susceptibility test was used on An. funestus and An. Masca- rensis in three districts including Farafangana, Fenerive Est and Vavatenina in the Zambia and revealed the susceptibility of these species to the deltamethrin (13) and the high resistance of An. gambiae was revealed 0–90% mortality in Nigeria (15). In other study in Dangbo district, West Africa, the resistance of An. gambiae to deltamethrin was indicated both with CDC and WHO bioassay methods respectively with 73.8% and 50.8% mortality (27). The increas- ing of kdr allelic frequency correlated with the CDC bioassay data on Malanville and Sure- Lere population An. gambiae (27, 16) as well as the biochemical assays were implicated the mono-oxygenase enzymes as mechanisms of pyrethroid resistance in An. gambiae from Misserete, West Africa (28, 29). A diagnostic dose of 10μg a.i./bottle was identified as the most sensitive discriminating dose for charac- terizing resistance in An. darlingi and Ae. ae- gypti and both the bottle assay and the WHO assay were equally able to differentiate del- tamethrin-resistant and susceptible An. albi- manus populations (30). There are some published studies on using CDC bottle assay for detecting pyrethroids re- sistance of malaria vectors with 3min exposure time including An. gambiae in Madagascar with 99% mortality rate (13), An. gambiae in Nigeria with 25% mortality (14, 15), An. gam- biae in Zambia with 90% (16) and on An. nun- eztovari in Columbia with 85% mortality (17). The current study emphasizes that the re- sults of both WHO and CDC bioassays were similar. Another recent study was also empha- sizes that both WHO and CDC bioassays give similar results with regard to mosquito suscep- tibility to deltamethrin and bendiocarb insecti- cides (21). The efficacy Wheaton coated bottle with deltamethrin could be used at least three times during four consecutive days in labora- tory conditions (22). WHO method requires more mosquitoes J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 24 http://jad.tums.ac.ir Published Online: April 27, 2019 than CDC method, the comparison between the results of both methods is clear. When the WHO susceptibility kit is not readily availa- ble, bottle bioassays can be used to determine insecticide resistance status of mosquito pop- ulations. WHO bioassays utilize cylinder plastic tubes whereas CDC bottles bioassays use 250 ml Wheaton bottles which are made of glass. World Health organization (WHO) papers do not need to be treated by oneself before their utilization because they are ordered in the im- pregnated form. Conversely, CDC bottles need to be coated with insecticide by oneself before each bioassay. In fact, the shelf life and reuse of prepared bottles are still not well docu- mented or studied in laboratory conditions (21). However, in field conditions, the studies of Pe- rea et al. showed that bottles treated with 10μg ai deltamethrin per bottle could be stored for at least 14 days and reused on three occasions (27). The major advantages of the bottle as- says are that any concentration of a custom in- secticide (pure or formulated) may be evalu- ated and the technique is simple, rapid and economical. One of the disadvantages of who method is the transfer of mosquitoes to the tubes, which can damage the mosquitoes and cause disturbances in the test results so requires care during WHO susceptibility tests. This prob- lem is partly resolved in the CDC method be- cause CDC bottles bioassays do not need mos- quitoes to be transferred from the exposure bot- tle. In WHO susceptibility tests mosquitoes must remain in recovery period (stable conditions of temperature and relative humidity) during the 24 hours after exposure to insecticide im- pregnated paper. The environmental conditions that mosquitoes have in recovery period can affect the test results, which is one of the dis- advantages of this method. In CDC bottle bi- oassays method, this problem has been solved. The CDC bottles need to be clean, dry and coat- ed with insecticide by oneself before each bi- oassay that takes a long time. If the bottles are contaminated before the coating, there is an error in the test results and this is one of the disadvantages of CDC bottle bioassays meth- od. The assessment of the diagnostic dosage and time for each insecticide used against ma- laria vectors, in each region and for each of the main vector species is absolutely necessary. According to the CDC method, any concen- tration of any insecticide (pure or formulated) may be evaluated. WHO insecticide susceptibility test is the most common method for assessing resistance status in Iran. In this study, for the first time in the country, CDC bottle bioassay method has been used to evaluate the level of mosquito sus- ceptibility to the conventional insecticides, and additional supplementary studies are required. Conclusion The current study emphasizes that the results of both WHO and CDC bioassays were similar with the studied insecticides with no significant difference in the related values. The CDC bot- tle bioassay may be applied as part of a broader insecticide resistance monitoring program es- pecially combined with results of bioassays us- ing synergists and those of biochemical and molecular assays. Also the CDC bioassay have been used for determining of biochemical mech- anisms that involve the detoxifying enzymes. Acknowledgements The authors would like to appreciate the ef- forts of Mrs Rafi, staff of the Culicidae Insec- tary, SPH, TUMS for their technical assistance in mass colonization of field collected An. ste- phensi. This study was funded and supported by Tehran University of Medical Sciences (Grant No. 9211263018). References 1. World Health Organization (2016) World J Arthropod-Borne Dis, March 2019, 13(1): 17–26 H Vatandoost et al.: Comparison of CDC … 25 http://jad.tums.ac.ir Published Online: April 27, 2019 Malaria Report 2016. World Health Or- ganization, Geneva, Switzerland, p. 136. 2. 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