Susceptibility testing and research on resistance

rf- r I WORLD HEALTH ORGANIZATION AFRTCAN REGION EXPERT ADVISORY COMMITTEE Third MeeEing Geneva r.3- 17 tenber 1982 The gene S. soubrense/ ORCANISATION MONDIALE DE LA SANTE REGION DE L'AFRIQUE ocP/EAC3.3 (ocP82,9) ORIGINAL: ENGLISH ONCHOCERCIASIS CONTROL PROCRAMME IN THE VOLTA RTVER BASIN AREA PROCRAMME DE LUTTE CONTRE L'ONCHOCERCOSE DANS LA RECION DU BASSIN DE LA VOLTA Tdt.: 333.12' 329.10'338.5? - T6l6x ONCHO 5241 UV SUSCEPTIBILITY TESTING AIID RESEARCH ON RESISTANCE The firsr half of 1982 has been a period of intense activity for the suscePti- biLity testing teams. Since 1 January, nearLy 14O tests have been carried out in various parts of the Progrnnrme area. The results may be discussed under several headings: 1. Tests for cross-resisEance A11 compounds proposed for trial have now been tested at leasc once on resis- EanE and non-resist.ant sErains. Ihe results are sunlnarized in Table 1' The threshhold for asserEing that cross-resistance is present is based on the relation- ship between suscePEibility tesEs results and treat6ent faiLures. Dr' D' Kurtak discusses the criteria in deEail in an annexed docr-ment entiEled "InterpreEation of Eests for cross-resisEancett. ral pattern, as previously rePorted, is that Ehe resistant strain of sanctipauli shows cross-resisEance tO oosE organophosPhates, but not f to carbamaEes, Pyrethroids, or chlorinate d hydrocarbons The organophosphates which show no cross-resistance or low leveLs of cross- resisEance are in the carboxyester group (azametiphos, dlchlorvos, detrachlorvinphos and trichLorphon). One of the characteristics of this grouP is its relatively high solubility in wat.er coopared with temephos. This could lead to their Partitioning in Che aqueous phase inside the insect's body, thus avoiding detoxifying enzymes in ehe lipid phase. At presenE azametiphos and tetrachlorivinphos are scheduled for testing in Eroughs against resistant larvae, although the latter has not performed $re11 as a larvicide. Chlorfenvinphos is another member of carboxyesEer group which should be tesced for cross-resistance. Although the teoephos derivatives do not show the same level of resistance as the parenE compound, cross-resistance is sCil1 evident. Temephos sulfone is scheduled for testing in troughs nonetheless. Temephos sulfoxide is not available in large quantiEies. In general, more comparaEive data betcreen suscePtibility tests and trough tesgs are needed. For specific compounds and formulations, iE would be very useful to know the relationship between increases in LC.,., in bqtrls and in troughs when testing the same popularions. This wouLd help id"the interPretation of cross- resistance Eests. 2. Variations in intensi ty and spectrum of is tance During the course of the cross-resistance tesEing, it has become evidenE chat aIl resistant populations are not equivalent, nor stable vrith tine. Chutes Cauthier,1- ocP/EAC3. 3 Page 2 on the lower Bandama, ltas maintaired the highest leve1 of resisEance. t'lortality at Ehediagnostic dose of temephos has been betr^reen 3 and 2OZ since November, 1981.Pirimiphos-methyl at O,25mg/1 produces a morrality of.2OZ. This stabilicy is nor due roinsecticide Pressure since flo temephos has been used since June, 198o nor chLorphoxim since November, 1981. Therefore it may be proposed thac the population is genetically stable and chere is little iumigration. 0n the Upper Bandama, however, the picture has changed. InJanuary 1982' che mortality at the diagnosric dose of chlorphoxim r^ras 73,42. Thi; figureincreased to 95,07" in April and looZ in June. At the ""." iir", the figures for temephos andpirimiphos-oethyl stayed constant, though at a slightly higher level than Chutes Gauthier.There are several practical conclusions to be taken from this observation: (a) Chlorphoxim resistance is genetically independent of temephos resistance and may be due to different factors. (b) Chlorphoxim resistance has regressed in part of its range in the absence of Pressure. This gives some hopes for alternation schemes, although it is still not knoqm whether the regression is sufficient to enable effectivefield treatments. (c) TesEs for cross-resistance should be done at Chutes Gauthier. Danangoro, on the Marahou6 has also been recently tested and gives 1OOZ mortalicy atthe diagnostic dose of chlorphoxio. 3 aphic distribution of resistance ,1 .1 The outlines of the area affected by resistance have not changed significant the beginning of the year. No S. soubrense/sanctipauli larvae have been found in ly since MaIi.Resistant larvae were present i n the Bui area of the Black Volta River in Ghana in January. They disappeared spontaneously in March and April, and reappeared in saall numbers in MayTheir presence is associated wi th very smal1 nunbers of dark wing-tufted biting flies A survey is being carried out on the susceptibility of S. soubrense/sanccipauli populat ionsin South-lrlesrern Ghana. dl (hough Ehe results are not yet complere, it seems that the susceptibility is somewhat lower than normal. In che absence of daca taken before the advent of resistance in Ivory Coast, these results are a bit difficult to interpret. 4. Effects of synergists ['Iith the aPPearance of resisEance Eo chlorphoxim, che synergistic effect of esreraseinhibicors such as DEF has become less pronounced. At chutes Gauthier, it is very difficultto demonstrate the synergism with temephos. This would seem to indicate that the phosphare esterases are not the sole mechanism of resistance. At Ehe same time trials with untreated 5. Genetic experiments In an attemPt to better understand Ehe genetics of the resistance, t$ro approaches havebeen tried. First, susceptibility tests have been done on the offspring of single females.Under good rearing eonditions, over 3oo larvae have been obtained, which allows gettingdata on a sample of reduced genetic variability. Although only three such tests have beendone thus far. the results show thac there is a wide variation in response to temephos even among the offspring of a single female. This implies a multifactoria-l resisrance. Secondly. attemPts are being made to measure the susceptibility of larvae from resistant females(Chutes Gauthier) mated to non-resistant males (Togo). At the present state of the techniques for colonization of S. damnosum, this is a difficult undertaking. A first populations of S. soubrense/sanctipauli, S.squamostun and S. damnosum s.str. have shor,m thatDEFproduc"""@"ig"mot;."g'."Esffi"m.wittrtemephos.This implies that these populations all possess phosphate esterase enzynes which aetoxity temephosto some extent. rt also implies that S. squamosrm and S. damnosum s.str. could developresistanceundercertainconditionsorffii-_pr"s".,EiT!@1ationdynamics. \ I \ ( rocP/EAC3.3 Page 3 aEEempE resulted in only two "hybrid" larvae. A second is under way. Capacity to give blood meals by the enema Eechnique would be a great help in these attempEs. If sufficient numbers of Fl larvae are obEained, an aEEemPt will be made to observe segregation in fhe F2 generation. 6. Enzyoe sEudies preliminary studies indicated that resistant flies aPPear to have greater esterase activity and there are differences in the frequencies of certain banding Patternsbetween susceptible and resistant populations in some areas. However, the handling of material before preservation and the physiological state of the samples can affect the interpretaEion of the enz)me activities, and it is very important to standardize techniques' Investigations carried out in Liverpool, UK early on in the year showed that a substan- Eial amount of variation exists in enzyrne acEiviEy and banding Pattern in both susceptible and resistant populations. Hor"rever, it was not knowrl whether this variation reflected real polp.orphism or was due to the state of the material. Using standardized methods of holding the flies pior to conservaEion, flies from Chutes Gauthier, Lower Bandama, Badikha and Souroukoudougou (UPper Bandana) were examined' Variation in the activity does exist but at this stage it is difficult to say whether this is relaced Eo the variation in resistance or is due to a minor difference in techniqueopreliminary examination of larvae collected at different times of the day indicates that acgivity is trigtrer in Ehe early morning, but this is from a very small sample size and results are only preliminarY. Suscepcible larvae of the Soubrense / s anc Eipauli differenc frequency of a slow esterase than resistanE for Ehe esEerase found in resistant populations, the value in distinguishing resistant individuals ' Susceptible and resisEant flies and larvae have been collected Eo continue investiga- Eions on the possibility of a clinical esEerase activity and Co examine further the geographically separated resistant populations. A carboxyesterase assay using the spectrophoEomeE.er is to be investigated as a more sensiEive method of detecting differences in esterase activiEy. group from T6t6tou, R. Mono, have a larvae, buE as Ehey are polymorphic banding Pattern is unlikely to be of 7 a'! v- L ocP/EAC3. 3 Page 4 Table 1 Co;npountl aza":ietiphos bronofos carbaryl ch1 orpyr if o s-ne'i.try I de l- ta;;re tirr rn dichlorvos etri;afoe fenchlorvos f entir.ion iodenpnos methorychlor piriniphos-ne tnyl propetanphos propoxur temephos sul.fone(Upp':r kncia:ra) tenephos sulfoxii.e(Ctrutes Gautiiier) l-- sL1.as o! Ti s,ls Lr(r,-i. c?L] Ss-llli s T5,l.r'Lt,'c[ .1 Cross - Re s is tance 0 x 0 x 0 0 X 1\ x x o temephos sulfoxid.e ,--(Urp?r Bzno.ana) x 0 x 2 ? ? te tr:ci:lorvinphos trichlorphon rt^ttrr. Itrn. = 1oq6 nunber of tests uith resistant }arvae. nunber of tests vlithnorresistant larvae. not reached vith resistant strain. ! \ Group .Lj50 resist.. LCcn nor:.iaL - (;m/ru) * LC 95 resist#)'U .)) nofrE,l_ Lirit LC100 norrnal stant organophosphate organophosphate carbairiiate or;ailopnosphate pyrethroid. organopl:osphate or6ai;.ophosphate or6e.ncpirosphate cr3ancthosphate orSanophosphate chlorinated. hytuo. carbon o.86(2/2) 2.5 G/1) r.1 Q/r) t1., Q/5) o.7z()/2) t.o (i/z) 5.7 0/4) g.z (t/t) B-z (2/t) i.s (2/2) o.46(e/e) orpnoplicsphai:e 6.1 O/3) organophosphate t.5 3/r) t).2 (z/4) v.6 (q/z) o.7,G/'6) ).2 (1/t) organoplrosphate or;anophosohate ).2 (2/r) tt.'t Gh) or5anophosphate Or,.:anOPhoSP:rate carbanate orp.nophosoirate L.2 6.r 1., 15.1 o.15 1.1 10.1 4.6 2=,7 10. / 0^88 o'l)a t ct 8.2 20.7 0.86 12 a2 0.Bi 1 ? 1 12 0., 1 U 2.5 2.0 8.0 1.0 I ? 2.5 1.2 o 20 1.0 4 ocP/EAC3. 3 Page 5 Annex INTERPRETATION OF RESTILTS OF TESTS FOR CROSS-RESISTANCE WITH LARVAE OF SIMI LII]M DAI'INOSIM s.1' bY Dr. D. Kurcakr EntomologisE' VecEor Control Unit, OCP (May 1982) The procedure in testing for cross-resistance is to run suscePcibility tesEs with both normal and resistan! strains and look for differences in the LCSO:LCSS' uPPer limit of Lc-...ratioLc^.l,jc.^,andsunrivalatthediagnosticconcentrdEionl-Increasesinthese ;;lgg..";;-"r.i9li"-i8"i"i"". strain may indicare cross-resistance' meaning Ehat the insecticide involved would be useless as a replacelDenc insecticide in the area where resistance occurs. First, Ehe data For LC'O and The slopes of two 1og dose-probic mortality lines may be considered co differ signifi- canEIY when: ZQ,.r, I TheproblemistojudgeEhesignificanceofchangesintheParametels. shoul-d be tesred to see if the differences are sEatistically significant' LC95, the confidence intervaLs should not overlap' vrhere v i) r.so or =bL-bc s..2(b1)*".". 2 (uz) and bl, b2 = slopes and s'e' = sEandard error of the slope' However, even if the differences are statisticalty significant. it is stitl not clear how this vould relate ro Ehe performance of the product in the field' To indicate how susceptibility data may be related to field efficacity' the Ewo attached tables present Ehe changes which had occurred in the susceptibility parameEers when Ereatment failures with Eemephos and chlorphoxim first occurred. The fact that Ehe parameters represent an oPera- rionally significant level of resistance was confirmed at ChuEes Gauthier by perforning "minigulte.i t""t" of efficacity ac Ehe same time as the suscepcibility tescs (Guillet et at, lggo). In the miniguEters, Ereatment aE the nomral dose of Eemephos (o.1 mg/l for 10 minutes) caused very little detachment of larvae. Even a dose of O'4 mg/l for 1O minutes (4 times nomral) gave only abouE 5OZ detachment. Direcc observation of the larvae in rhe breeding site also showed that the overdose was not effective' Ir is rhus reasonable co believe that if similar differences in susceptibility parameters are obtained with other insecticides' their use would result in treatment failures urith resistanE larvae at any Practical dosing rate. IC is not, however, advisable to depend on a single Parameter. A11 Che ParaEeters should be compared in search for evidence of cross-resistance. In the case of resistance to Cemephos, for example, the LC5O.changed very lirCle, while the LC95 increased considerably. In the case of chlorphoii'ni resistance, the changes in the tsro parameEers were similar. An increase in LC95 of 4 or more times is certainly significant. An increase in the LCg5lLC5g ratio is very characteristic of temephos resistance, trhile there is little change trith chlorphoxim resistance' a 7 OCP/EAC3. 3 Page 6 Annex An increase of 4 times in the upper limit of the LC166 occurred in both cases: Survival at the diagnostic concentration is a very sensitive indicator. The presence of even a few surviving larvae is significant. Certainly i-f, 2 or more of t,he parameters show increases characteristic of resistance, one can conclude that cross-resistance exisEs. In doubtful cases, minigutters or other methods which test efficacity under rnore or less realistic treatment condiEions may be needed. References cited GuilIec, P., Escaffre, M., Ou6draogo, M., et Qui116v616, D. (I98O). Mise en 6vidence dtune r6sistance au temephos dans le complexe Sinulium damnostun S. sanc E uli et S. soubrense) en C6te dtlvoire. (Zone du Progranrme de LutEe conEre I Onchocercose dans la r6gion du bassin de 1a Volta), Cah.o.R s. T.0 M., s6rie Ent. n6d. et Parasitol. 18 (3),29L-299. Kurtak, D., Ou6draogo, M.r Ocran, M., T616, B., and Guillet. P. (1982). Preliminary note on Ehe appearance in Ivory Coast of Resistance to Chlorphoxim in Simulium soubrense/sanctipauli larvae alread'y resistant to Temephos (Abate (R)). WHO/VBC mimeo doc. in press. i I ; ocP/EAC3.3 Page 7 Annex I Table I Increases in susceptibilicy parameters when Eemephos resisrance was first confirmed (May, 198O) at ChuEes Gauthier, Bandama River, (daca frorn Guillet et al' 1980). (young larvae) Table 2 Increases in susceptibility parameters in the early stages of chlorphoxim resistance (Sarakro, Comoe River' Ivory Coast, December, 1981). (Data fron Kurtak et al, 1982) (mature Larvae) *Diagnostic concentration is usually established as twice Ehe concentration which kills 1OOZ of normal larvae. l. ( LCso LC95 Limit LCTOO Lc95/Lc59 7 survival atdiagnos tic concentra tion* 1. 3X 5.8X 4X 5.2X normal 0 resistant 82 LC5o LCgs Linit LCtoo LC95lLC19o Z survival atdiagnostic concentration* 2.8X 3. 7X 4X 1 .3X normal O resistant 5.32 7