Detection of pyrethroid resistance in Anopheles mosquitos.

Detection of pyrethroid resistance in Anopheles mosquitos S.M. Magesa,1 0. Aina,2 & C.F. Curtis3 Although pyrethroid insecticides are a promising means of controlling Anopheles malaria vectors, there is a need to monitor for resistance. It has been proposed that the results of the WHO-recommended testing method, involving exposure to impregnated paper for 1 hour, might be misleading because of knockdown during this period, and that exposure to a higher dose of pyrethroid for 2 minutes might be preferable. However, comparative tests with a susceptible and a permethrin-resistant strain of A. stephensi showed that exposure for 1 hour was at least as sensitive in detecting resistance as was the short exposure method. Introduction Pyrethroid insecticides impregnated into bednetsa or used for conventional residual house-spraying (1) are a promising weapon against malaria mosquitos, the rapid knockdown effect being an important advan- tage. However, there is concem that pyrethroid resistance may be selected in Anopheles mosquitos as a result of either antimalarial or agricultural ap- plications. For several years, WHO has made permethrin- and deltamethrin-impregnated papers available for use in standard susceptibility test kits. The diagnostic dosage recommended for permethrin has been 0.25% (w/v) with exposure lasting 1 hour (2). In 1992, the WHO Expert Committee on Vector Biology and Control recommended various diagnostic dosages of permethrin for different Anopheles species, but expo- sure for 1 hour continued to be recommended for all except one species (3). However, it has long been recognized that the quick knockdown caused by py- rethroids may give equivocal results (4). Susceptible mosquitos are not expected to spend the intended I hour absorbing insecticide through their tarsi: with 1 Research Scientist, National Institute for Medical Research, Amani Medical Research Centre, Muheza, Tanga, United Republic of Tanzania. 2 Medical Laboratory Scientific Officer, London School of Hygiene and Tropical Medicine, London, England. 3 Professor, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1 E 7HT, England. Requests for reprints should be sent to this author. a The use of impregnated bednets and other materials for vec- torborne disease control. A report of the WHO/VBC informal consultation held in Geneva, 14-18 February 1989. Unpublished document WHO/VBC 89.981, 1989. Reprint No. 5522 a vertical exposure tube they would be quickly knocked down and fall on to the plastic base of the kit, where they might recover before the end of the 24-hour holding period and be scored as survivors. Conversely, if the tube were kept horizontal and rela- tively tolerant mosquitos were knocked down they would lie on the impregnated paper and could be expected to absorb a large, possibly lethal, dose. Because of these problems, the Expert Committee recommended further studies on methods of testing for pyrethroid resistance (3). We report here the results we obtained in 1988 and 1994. Method Our work was based on the following considerations and expectations. * Tests are likely to be more meaningful if opera- tionally realistic dosages and substrates are employed. We therefore used nylon mosquito netting impregnated with permethrin according to the meth- od described by Hossain et al. (5) at a range of dos- ages similar to those used on bednets. The netting was used as a lining, backed by a paper stiffener, in WHO test kits. * Exposure should only be for 2 minutes so that no mosquitos are knocked down until after the exposure period, and all therefore have the same chance of absorbing insecticide through their tarsi. Such a short exposure also seems likely to give a more meaning- ful test since pyrethroids are irritants and mosquitos do not spend long periods resting on bednets that are impregnated with permethrin (6). * Possible diagnostic dosages and exposure times should be evaluated, not merely for their ability to kill susceptible insects reliably, but also to discrimi- Bulletin of the World Health Organization, 1994, 72 (5): 737-740 © World Health Organization 1994 737 S.M. Magesa et al. nate resistant from susceptible strains. An A. ste- phensi strain of Dubai origin, selected at the adult stage in the laboratory with permethrin by Laddoni & Townson (7), and designated Dub/Apr, provides one of the few clear-cut cases of pyrethroid-resistant Anopheles. Results and discussion In 1988 we compared the Dub/Apr resistant strain with the Beech susceptible strain of the same species using the testing system described above and also the conventional WHO system using paper impregnated with 0.25% permethrin (equivalent to 97 mg/mi2) and a 1-hour exposure. In each case the tests were con- ducted at 25 °C and the mosquitos were held for 24 hours after exposure before mortality was recorded. Exposure for 2 minutes to 200 mg permethrin/ m2 of netting almost completely discriminated between the two strains, but a few of the Beech strain sur- vived and a few of the Dub/Apr strain were killed by the next higher dose tested (Table 1). The LD50 val- ues calculated from the results for the range of doses tested indicated a resistance ratio of 370:1. Contrary to expectation, the standard WHO test was better at discriminating between the two strains: at exposures of 1-2 hours there was a 100% kill of the Beech strain, whether the exposure tubes were held vertically or horizontally, while none of the Dub/Apr strain was killed (Table 2). Only when the exposure time was increased to 4 hours did a few specimens of the latter strain die. We repeated the tests in 1994, by which time the Dub/Apr strain was considerably less resistant than it had been in 1988. On netting, none of the doses came near to discriminating the strains. The tests with 0.25% permethrin papers and with 0.1% lambda-cyhalothrin papers gave the results shown in Table 2. Exposure for 1-2 hours permitted consid- erable, but nevertheless incomplete, discrimination to be made between the two stocks for each of the pyrethroids. There was little difference in the results with the tubes in the vertical or horizontal positions. Unexpected results in resistance tests are com- monly ascribed to the use of old test papers. In an effort to quantify the effect of repeated use, we exposed successive batches of the Beech stock to the same paper, keeping it refrigerated between tests. With a 1-hour exposure in the horizontal position, mortality was 100% until the seventeenth use of the same paper, by which stage a total of 255 mosquitos had been exposed. The mortality dropped to 50-90% for the next 50 mosquitos exposed, even though the WHO-recommended expiry date of the paper had not been reached. We therefore suggest that a test paper Table 1: Percentages of Beech (susceptible) and Dub/Apr (resistant) strains of Anopheles stephens! knocked down 1 hour after and dead 24 hours after exposure for 2 minutes to various doses of permethrin on nylon netting Beech strain Dub/Apr strain Dose % knocked % % knocked % (mg/M2) down dead No.a down dead No. 25 20.0 21.2 85 0 0 106 50 80.4 52.2 92 0 0 102 100 96.1 83.3 78 0 0 100 200 91.6 97.6 83 0 0 104 400 100.0 100.0 77 3.4 1.7 118 800 - - 0 17.3 3.8 104 1 600 - - 0 79.0 10.0 100 2 500 - - 0 88.4 12.5 104 3 200 - - 0 67.0 17.5 97 LD50(mg/m2) 47 17 400 a No. of mosquitos tested. should not be used to expose more than a total of 150 mosquitos. The results shown in Table 2 have increased our confidence that the WHO-recommended test system is likely to identify cases of pyrethroid resistance. It is being used with 0.025% (w/v) deltamethrin papers by the Antiparasitic Diseases Institute, Sichuan Prov- ince, China, to test for resistance in an area where 2.25 million bednets have been impregnated with deltamethrin (8, 9). However, we also advocate test- ing under conditions that are as realistic as possible, including those where the mosquitos are free-flying but are stimulated to land on an impregnated net (10). Pyrethroid-resistance genes of various levels of protective efficacy can be expected to arise; it is important to detect them at an early stage and to assess their likely impact on control operations. Note added in proof. It has recently been found that better discrimination can be obtained by obser- ving the time for knockdown of mosquitos left con- fined in a WHO bioassay core in contact with an impregnated net-all of the Beech stock are knocked down several minutes before the first of the Dub/Apr stock. Acknowledgements Most of the above work was carried out during the tenure of a WHO training fellowship by S.M.M. O.A. and C.F.C. are funded by the British Medical Research Council. We are grateful to Dr H. Townson for providing the Dub/Apr strain. This paper is published with the permission of the Tanzanian National Institute for Medical Research. WHO Bulletin OMS. Vol 72 1994738 Detection of pyrethroid resistance in Anopheles mosquitos Table 2: Proportions of the Beech and Dub/Apr strains of Anopheles stephensi knocked down at the end of various exposure periods to papers impregnated with 0.25% permethrin or 0.1% lambda-cyhalothrin and the proportions dead after 24 hours Beech strain Dub/Apr strain Tubes vertical Tubes horizontal Tubes vertical Tubes horizontalExposure time % knocked % % knocked % % knocked % % knocked % (hours) down dead No.a down dead No.8 down dead No.8 down dead No.8 Permethrin: 1988 results 0.25 6.5 71.4 105 6.2 50.0 128 0 0 72 - - 0 0.5 59.2 79.9 189 68.0 75.0 172 0 0 69 - - 0 1 98.1 100.0 109 100.0 100.0 124 0 0 74 - - 0 2 100.0 100.0 100 100.0 100.0 119 0 0 76 - - 0 4 - - 0 - - 0 8.6 12.3 105 - - 0 6 - - 0 - - 0 35.4 45.5 79 - - 0 8 - - 0 - - 0 47.4 61.8 76 - - 0 1994 results 0.25 19.0 48.0 100 26.6 52.0 75 10.0 10.0 100 0 15.3 130 0.5 44.4 60.0 90 41.2 66.2 80 10.0 10.0 100 0 2.0 100 1 85.8 77.5 120 81.9 80.1 111 14.4 9.6 125 0 5.1 135 2 98.1 99.0 110 97.1 98.5 140 36.9 50.7 130 32.8 47.2 125 4 100.0 100.0 90 100.0 100.0 90 36.3 41.8 110 38.0 40.9 105 6 - - 0 - - 0 37.0 35.1 108 76.1 72.3 105 8 - - 0 - - 0 97.5 100.0 40 95.0 95.0 45 Lambda-cyhalothrin: 1994 results 0.25 52.5 90.8 120 50.8 94.0 118 12.5 29.1 120 7.2 12.0 125 0.5 97.7 98.8 87 76.0 96.0 75 26.3 21.0 95 35.7 24.2 70 1 93.6 92.7 110 98.0 96.1 105 40.0 25.6 125 43.7 46.8 160 2 97.6 98.8 85 100.0 100.0 80 86.0 43.3 150 85.1 40.7 135 4 98.5 100.0 70 100.0 100.0 72 98.1 94.5 110 98.3 81.6 120 6 - - 0 - - 0 94.0 90.0 50 90.0 86.0 50 8 - - 0 - - 0 100.0 100.0 40 100.0 100.0 40 a No. of mosquitos tested. Resume D6tection de la r6sistance aux pyr6thrinoides chez les anophbles Les insecticides a base de pyr6thrino7des consti- tuent un moyen de lutte prometteur contre les anopheles vecteurs du paludisme. On peut en impr6gner les moustiquaires ou les vaporiser de faqon conventionnelle. Comme ces substances sont de plus en plus employ6es, il est necessaire de surveiller la r6sistance aux pyr6thrino7des. Les pyr6thrino7des ((assommentb) rapidement les insectes et l'on a pens6 que l'epreuve de sensibi- lit6 recommandee par l'OMS, qui expose les moustiques pendant une heure a du papier impre- gn6 avec une solution de permethrine a 0,25% (ce qui equivaut a 97 mg/M2), pourrait etre une source d'erreur a cause de cet effet de knock- down pendant la periode d'exposition. A I'aide d'une souche sensible d'A. stephensi, et d'une souche de la meme espece originaire de DoubaT, ayant 6te choisie pour sa resistance a la perme- thrine, nous avons compar6 la m6thode classique, avec des durees d'exposition allant de 15 min a 8 heures, a une exposition pendant 2 min a une moustiquaire impr6gnee de permethrine (25 a 3200 mg/M2). Contrairement aux r6sultats atten- dus, il s'est aver6 que la m6thode classique 6tait au moins aussi sensible pour d6celer la r6sistan- ce que 1'exposition pendant 2 min. On applique donc la premiere en Chine, ou les moustiquaires impr6gnees sont utilisees a grande 6chelle, pour surveiller la resistance. Si l'on decele un cas de resistance, il est recommande d'effectuer des epreuves sur des moustiques volant librement, WHO Bulletin OMS. Vol 72 1994 739 S.M. Magesa et al. afin d'6valuer la probabilite que cette resistance ait un impact sur les operations de lutte. References 1. Matola YG et al. Epidemiologic assessment of the impact of lambdacyhalothrin (OMS-3021) on the transmission of malaria in a rural area in Tanzania. Bulletin de la Soci6t6 frangaise de Parasitologie, 1990, 8(suppl. 2): 1202. 2. Resistance of vectors and reservoirs of disease to pesticides. Tenth Report of the WHO Expert Com- mittee on Vector Biology and Control. Geneva, World Health Organization, 1986 (WHO Technical Report Series, No. 737). 3. Vector resistance to pesticides. Fifteenth report of the WHO Expert Committee on Vector Biology and Control. Geneva, World Health Organization, 1992 (WHO Technical Report Series, No. 818). 4. Hemingway J. Modification of the adult resistance test kit to measure knockdown rates. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1980, 74: 677. 5. Hossain Ml, Curtis CF, Heekin JP. Assays of per- methrin-impregnated fabrics and bioassays with mosquitos. Bulletin of entomological research, 1989, 79: 299-308. 6. Hossain Ml, Curtis CF. Permethrin-impregnated bednets: behavioural and killing effects on mosqui- tos. Medical and veterinary entomology, 1989, 3: 367-376. 7. Malcolm C. Current status of pyrethroid resistance in anophelines. Parasitology today, 1988, 4: Si 3-Si 5. 8. Curtis CF. Annotated abstract of "Controlling mos- quito vectors and malaria with deltamethrin in Sichu- an, China". Tropical diseases bulletin, 1992, 89: R1-R6. 9. Cheng Hailu et al. Large-scale spraying of bednets to control mosquito vectors and malaria in Sichuan, China. Bulletin of the World Health Organization, in press. 10. Curtis CF, Hill N, Kassim SH. Are there effective resistance management systems for vectors of human diseases? Biological journal of the Linnean Society, 1992, 48: 3-18. 740 WHO Bulletin OMS. Vol 72 1994