Serial studies on the evolution of chloroquine resistance in an area of East Africa receiving intermittent malaria chemosuppression

Bulletin ofthe WorldHealth Organization, 63 (1): 109 -118 (1985) c) World Health Organization 1985 Serial studies on the evolution of chloroquine resistance in an area of East Africa receiving intermittent malaria chemosuppression C. C. DRAPER,' G. BRUBAKER,2 A. GESER,3 V. A. E. B. KILIMALI,4 & W. H. WERNSDORFER5 Serial in vitro and in vivo testsfor chloroquine sensitivity of Plasmodium falciparum were carried outfrom 1979 to 1982 in an area of E. Africa where chemosuppression with chloroquine had been attempted since 1977. Within l iyears there weresigns ofa decreasing drug response. Chloroquine resistance was first detected in 1981 and this increased markedly in 1982. Other contributory causesfor the rise ofparasite rates in children were possibly a decline in the efficiency of the drug distribution system and also immunological factors. Evidence ofresistance to pyrimethamine was alsofound. Observations were made of the heterogeneity of the parasites' responses with emerging resistance. Implications for the future are discussed. The main objective of the Mara Scheme in the United Republic of Tanzania has been to look for any effects on the incidence of Burkitt's lymphoma as a result of chemosuppression of hyperendemic malaria in children. This project has generated new informa- tion, some of which has been published. The present paper, for example, deals with the drug sensitivity of the malaria parasites in the area concerned. An earlier brief communication (1) reviewed the situation up to 1980, at which time no overt resistance had occurred; the detection of resistance in 1981 and 1982 is described here. MATERIALS AND METHODS The area. The North and South Mara districts in the north-west of the United Republic of Tanzania lie to the east of Lake Victoria; the lowland study areas are at an altitude of about 1200 m above sea level. Surveys on many occasions before chemosuppression was started had shown that the point prevalence of malaria parasitaemia in children up to 10 years old varied from 30% to 40¾ in both North and South ' Ross Institute, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, England. Requests for reprints should be addressed to this author. 2 Shirati Hospital, Musoma, United Republic of Tanzania. 3International Agency for Research on Cancer, Lyon, France. 4 National Institute for Medical Research, Amani Centre, United Republic of Tanzania. 5 Malaria Action Programme, World Health Organization, 1211 Geneva 27, Switzerland. Mara; the infections were predominantly due to Plasmodium falciparum. Chemosuppression. Chemosuppression was carried out only in the North Mara lowlands, covering an area of 3000 km2 with a population of about 220 000, of whom 100 000 were children under the age of 10 years. Because of its cheapness and availability, chloroquine phosphate in the form of tablets was chosen for distribution from Shirati Hospital using the local community system; for example, balozis (leaders of a cluster of about ten households in villages) kept registers of children and acted as drug distributors. This has been described in detail elsewhere (2). From the second half of 1977 the intention was that all children up to 10 years old should receive chloroquine, given as tablets or fractions of tablets, in a dose of 5 mg/kg body weight every 2 weeks; the effectiveness of this form of mass drug administration in malaria suppression had earlier been shown in a small pilot trial. However, after an initial period of success the parasite rates seemed to be rising again after Ij years, and the fortnightly dose was therefore increased to 10 mg/kg at the end of 1979. In area A of North Mara weekly administration was attempted. All regular drug distribution was stopped at the beginning of 1982, but chloroquine distribution to the dispensaries was con- tinued in order to increase their supplies. No chemo- suppression was attempted in South Mara, the control area for comparison, which is separated from North Mara by the Mara River. Because this river can 4507 -109- C. C. DRAPER ET AL. 0 00 6 0 CD 0 6 LD 0) C. C-'Nf% CO 00_ 6 67 CO COD 6) COD . 0 a0) 0 C) - O 0 0 0) 0) cD b 0 r- 0 0) C _ 0) CY) CVI r- 00 0) C) 00 C') Ce C)0) C) 0) 0a 0) 00 r-. 0) CN 0) C- 0) v 6 C- 6 CD) It CD C-I 0D o .C CD c0 t t OD t OD ,:" oor- o oo o a0z 0)z0) 0 0) Z Z '- Z U)'v- 110 0)- C .'XE o x E - C ' w C co cm * E C 0 NC O C C u O C4p 0 0 -o OC 0._2 40. UJw U o w 0 n CD0 0 CD (D Cv7 (D 6 07) CD 0 CN CY) 6 0 C-I 0 6 0 0 C O0 00 -._ 0) CC ._ 0 0 CC CU 0 Cl) ._ -C u 0 Cl) Dl CHLOROQUINE RESISTANCE IN EAST AFRICA 1II be crossed at only two points, population movements were concentrated here. Malariometry. Groups of houses, from villages throughout the North Mara lowlands, were selected randomly each month so that totals of from 200 to 400 children were examined at each of the cross- sectional surveys. Samples were taken less frequently from the South Mara comparison zone. Giemsa- stained, thick blood films were used and parasites counted against 500 white blood cells. In the samples taken there were throughout approximately equal numbers of children in the 0-4-year and 5-10-year age group so that no bias was introduced with regard to age composition within the 0-10-year age group. Micro in vitro tests for drug sensitivity. The same groups of villages were studied from 1979 to 1982. As the blood was required for other purposes also, small (1-2 ml) samples of venous blood were taken into 15% phosphate-citrate-dextrose (as anticoagulant) from children aged a few months old and older children, who were found with moderate to high parasitaemia in a rapid survey done just beforehand. Blood samples were taken at ambient temperature to the field laboratory within a few hours. The tests were done by the standard microtechnique (3) using chloroquine-treated plates (supplied by WHO) in a concentration of 1 to 32 x 10- 12 mol/litre per well. These would give concentrations in the test of from 0.2 to 6.4x 10-6 mol/litre of undiluted blood. In 1979 and 1980, prior to the introduction of the current concentration levels, different chloroquine con- centrations were used (see Table 1), but this was of little importance since the concentration range was quite similar and the evaluation of results was based on a probit analysis of the log-dose response. The mefloquine plates contained 1 to 16 x 10 - 12 mol/litre per well, giving concentrations in undiluted blood of from 0.2 to 3.2 x 10 -6 mol/litre. No improvement in the maturation of parasites to schizonts was obtained by taking the blood for culture immediately into RPMI 1640 medium, by removing the donor's plasma, or by adding supplementary non-immune serum. A useful modification; however, was the inclusion of an extra control well in a separate untreated plate. After incubation of the tests for 24 hours at 38 °C in a plastic candle jar, housed in a custom-built incubator operated from car batteries, the extra control was harvested, rapidly dried and stained, and examined. This helped to indicate the best time for harvest of the test cultures, when maturation of parasites to schizonts would be maximal (yet without schizont disintegration), which varied from 24 to 40 hours of incubation, depending on the initial stage of development. Test results were included only if at least 10% of the parasites in the control wells had developed to schizonts, defined as parasites with three or more chromatin dots and based on the reading of 200 asexual parasites per well. All results were sent to WHO, Geneva, for probit analysis using the log-dose response test from 3-8 point assaya for the calculation, from grouped data, of regression lines and effective concentrations (EC). In vivo tests. A modification of the WHO alter- native test (3) was applied in all those children on whom in vitro tests were attempted. Their para- sitaemias ranged from 1000 to over 50 000 parasites per i1. Each was given, under supervision, a single dose of 10 mg chloroquine base per kg body weight, and after a week another blood film was taken from the child. In 1982 chloroquine syrup was used instead of tablets to give an equivalent dose to many of the younger children, and in this year also a number of children were dosed with 25 mg pyrimethamine instead of chloroquine. In 1979 and 1980 urine samples were obtained before and 24 hours after dosing to test for the presence of chloroquine by the Dill-Glazko eosin colour reaction. As this test was found to give anomalous results under local con- ditions, it was not used further. RESULTS Prevalence of malaria Fig. 1 shows the point prevalence of malaria para- sitaemia, by approximate 3-monthly intervals, in CHLORO CHLORO 55 5m per kg l0mg perk j 5wry 2 rwsk sry2ovm ks to B-E to A-E +omkly for A 10% N.MARAAtoE 40 I-\ 35 125 / 1 South M ,1S.MARAF,8G 15 -~ 10 fordrugsensitiviyN.MARAaoly o 01,,I II 1974 1975 1976 1977 1978 1979 1960 1961 1962 Years Fig. 1. Parasite rates in 0-10-year old children in areas A-E of North Mara and comparison areas F and G in South Mara, 1974-82. aGRAB, B. & WERNSDORFER, W. H. Evaluation of in vitro tests for drug sensitivity in P. falciparum: probit analysis of log/dose response tests from 3-8 point assay. (Unpublished document, WHO/MAL/83.990, 1983). C. C. DRAPER ET AL. North Mara and the comparison zone of South Mara. Although there was some variation in the parasite rates, mainly due to seasonal fluctuations, it is clear that following the start of the chemosuppression programme these fell in North Mara for 11 years but started to rise in 1979. Doubling the fortnightly dose of chloroquine, or trying to give the drug weekly in area A in North Mara, had little effect and from the end of 1980 onwards the North Mara rates were close to the range of those found in South Mara. In vitro tests The grouped results of the in vitro tests for chloro- quine sensitivity are shown in Table 1. These tests were done in November 1979, July 1980, March- April 1981, and March 1982. The results of the first (1979) and second (1980) series have been combined as they were similar. The increasing number of tests done in North Mara in 1981 and 1982 was largely due to a better success rate in the tests attempted. This rose from 20% in 1979 to over 50%o in 1982 and was largely attributable to the introduction of solar power generators for charging batteries for the incubator. Study of the inhibition of maturation at the different data points and of the ECs shows clearly that in North Mara there was a decrease in suscepti- bility of P.falciparum to chloroquine from 1979/80 to 1981 and a further decrease in 1982. Maturation of schizonts at a concentration of chloroquine of 1.14 x10-6 mol/litre or above is held to indicate resistance (3). In 1979/80, only 2 out of 80 isolates showed very scanty maturation near this concentra- tion, but in 1981 some 10 out of 60 isolates and in 1982 some 27 out of 139 isolates showed maturation, with a significant proportion at higher concentrations (Table 2). Fig. 2 compares the fitted regression lines for 1979/80 and for 1982 and shows clearly the change in susceptibility in the last year. Table 2 and Fig. 3 compare the data from separate analyses of susceptible and resistant parasite isolates *in 1982, and in Table 3 and Fig. 4 the resistant isolates 0'U D *Iu 120 A A liex, o4 Fig. 2. In vitro tests (see text) in N. Mara, 1979/80 and 1982. Log/probit regression lines for inhibition of schizont maturation at different chloroquine concentrations. *5N I go s o 40 S 30 5 X104 MIl cblroqkwlt Fig. 3. In vitro tests in N. Mara, 1982. Log/probit regression lines for inhibition of schizont maturation at different chloroquine concentrations for sensitive isolates (no maturation beyond concentration of 0.8 x 10-6 mol/l blood) and resistant isolates. Table 2. Analysis of in vitro tests for chloroquine sensitivity of P. falciparum in North Mara, 1982. Comparison of sensitive and resistant isolates showing schizont maturation at (or above) a drug concentration of 1.14 x 10-6 mol/litre blood Effective Percentage inhibition of schizont maturation concentrations No. of at following drug concentrations (x 10 -6 mol/l) (x 10-6 mol/l) Variance of isolates slope b 0.20 0.40 0.80 1.14 1.60 3.20 6.40 EC5o EC90 EC" Slope b (Sb2) Sensitive isolates 112 51.5 85.8 98.2 100 100 100 100 0.20 0.45 0.88 3.59 0.02 Resistant isolates 27 23.0 52.1 78.3 86.2 92.4 93.6 98.0 0.38 1.66 5.50 2.01 0.05 LI 112 CHLOROQUINE RESISTANCE IN EAST AFRICA .' 0 o o o 0 co .A * >e 0 Q 4) a N N c C (g cQ> o X L m r 4'5 NL) O cO LO -Dco L)x u° LU ( OA CCuZ c* (4) 00- 0) 4oP c o SCu eC o .fE °E t ,_E °s 0~~~~~( .0 Qc ~ ~ 0r O~~~~~C( Cu3 _- C w 3 ~0(A~~~~~~( 113 C. C. DRAPER ET AL. ti 'aI£1 ________-=' n (n -58) jN 70 I. 50 0030 10 - - - - - 5 -l I. -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 501 Fig. 4. In vitro tests in N. Mara, 1982. Comparison of resistant isolates only, with log/probit regression lines for inhibition of schizont maturation at different chloroquine concentrations according to limiting concentrations at which maturation occurs. have been analysed further according to their levels of resistance. Although the numbers are small, in itself a cause of increasing heterogeneity of the grouped data, it can be seen that the goodness of fit of the regression lines to the data points decreases with increasing resistance. This suggests that the resistant isolates are composed of heterogeneous populations of parasites, and conforms with observations in Thailand (16). A similar picture was seen with the 1981 results from North Mara when the 9 resistant isolates were analysed separately from the sensitive isolates. In the smaller series of tests in South Mara in 1981 (Table 1) there was one isolate which showed maturation at a chloroquine concentration of 1.14 x 10-6 mol/litre and the grouped results show a sensitivity between that of North Mara of 1979/80 and that of South Mara in 1981. They are perhaps indicative of incipient resistance there. The results for the mefloquine tests are given in Table 4 and Fig. 5. One out of 59 isolates (case number 107) showed schizont maturation up to a drug Fig. 5; In vitro tests in N.Mara, 1982. Log/probit regression lines for inhibition of schizont maturation at different mefloquine concentrations. Sensitive isolates. concentration of 3.2 x 10- 6 mol/litre. This isolate was also chloroquine resistant. Excluding this isolate the other test results are all in the range of full sensitivity to mefloquine. These findings suggest a sporadic occurrence of isolates with reduced mefloquine sensitivity, which should be considered in the context of the report on one case of primary mefloquine-resistant falciparum malaria which had originated in the United Republic of Tanzania (12). In vivo tests The results of the in vivo tests are summarized in Fig. 6. As mentioned in the Methods section above, no reliable check could be made to see that the administered chloroquine was absorbed and was not vomited later. Transmission was also known to be continuous at the time of the tests and obviously increasing between 1979 and 1982, and it is more than likely that some of the failures in the clearance of parasites might have been due to infections that were in the prepatent stage at the time of dosing. These Table 4. Analysis of in vitro tests for mefloquine sensitivity of P. falciparum in North Mara, 1982, with and without one case of markedly reduced sensitivity (see text) Effective Percentage inhibition of schizont maturation concentrations No. of at following drug concentrations ( x 10 -6mol/l) (x 10-6 mol/l) tests 0.10 0.20 0.40 0.80 1.14 1.60 3.20 ECso ECso EC,9 All tests 59 27.5 51.5 84.1 92.8 98.0 99.0 99.3 0.18 0.61 1.68 AN tests excluding case No. 107 58 26.5 53.0 86.6 94.4 99.4 99.9 100 0.17 0.51 1.22 U U 730 20 10 S 0.05 i Z 0.4 me 1.14 1.0 1&Zx1 Ml _afloqshMn Mood e 114 CHLOROQUINE RESISTANCE IN EAST AFRICA CHILDREN G-4 YEARS of m I it 90 80 70 0 50 40 30 20 10 0 90 80 70 60 50 40 30 20 10 0 I 1979/ -19 1979/9 9.8 1981 1982 Years CHI LDREN 5-11 YEARS 1981 1982 -S .So:^-^:@:.:w: >w< *:-:w:-:w:-:w: :-:-:>:-:v:-:v: >:-:>:-:v:-:v: s:z:z:z:.:+:.: :w:-:w:-:w:-:^: :nK3: .. . -. .-. ::::::::::..... .. - :::-::-::- :-::-::::: *:::-::-:: w: :,:,:,:.:: ...-...-..... ^, -.-.-. ,:. ',:.:.:.: ..''.'.. _ 1982w - % - F w 6-6 6D-wh . b*s....8z- 3. .:-,:.:,:,:-- :,:.:':':.:,: ,:',:-:,: :,:. l ,.......:,:.:.,:,:.: ..,:, ,:.:,. *,:,:,:,:..,: l::-::::: l::>::: ,:.:,:::: :,:,:,:.:,:.: *: ::: *.- . . -. 1"2 Years NM SM PYR. NM Fig. 6. In vivo tests with single doses of chloroquine, 1 Omg/kg (cross-hatched), or pyrimethamine, 25mg (stippled), in North Mara (NM) and South Mara (SM), 1979-82. factors could contribute to the fairly low clearance rates achieved in the younger children. It should be noted that the clearance rates in the 0-4-year age group (70-85%) were consistently lower than those in the 5-11 -year age group (90-95%) in 1979/80 and in 1981, when resistance was substantiated in vitro. In 1982, when resistance had increased, the clearance rate in the 0-4-year age group was significantly reduced to 50% but remained within its former range for the older age group. Similarly, the clearance rate with pyrimethamine was lower in the 0-4-year than in the 5-11-year age group despite the fact that the former received a relatively larger drug dose. These are indications of resistance to pyrimethamine. Among the children who had simultaneous in vivo and in vitro tests in 1982, 5 in the 0-4-year group had chloroquine-resistant isolates and only one of these had cleared parasites by day 7; on the other hand, 6 out of the 7 children in the 5-1 1-year group who had resistant isolates had cleared the parasitaemia by day 7. The results of the in vivo tests in South Mara of 1981 are similar to those of North Mara in 1979/80. DISCUSSION Since 1978, chloroquine-resistant P.falciparum has been detected on many occasions in East Africa in non-immune visitors and temporary residents (4). Only recently have a few surveys shown such resistance to be present in the indigenous population (5, 6). However, it is not known whether chloroquine- resistant P.falciparum was imported from south-east Asia by infected people or mosquitos, or whether it developed locally, and there is little information on how widespread this resistance is, or its degree, or its correlation with chemosuppression or treatment of the local people. The surveys reported here strongly suggest that intermittent chemosuppression with evidently subcurative doses of chloroquine was able, after several years, to select for a considerable degree of resistance. In 1982 about 20% of the isolates studied showed resistance, the majority at a low level, but a significant number also at relatively high levels. This occurred in a generally semi-immune population subjected to intensive malaria transmission. A limited survey done in 1981 at Mto-wa-Mbu, some 200 miles to the east ofMara, showed evidence of a lesser degree of resistance in an area where for many years chloro- quinized salt had been used, although not for the immediately preceding few years (7). This may imply that the biological advantages of chloroquine- resistant P.falciparum may be different in East Africa compared with south-east Asia, but this question cannot be answered in the absence of longitudinal, long-term studies. Moreover, our observations suggest some biological advantage of the chloroquine-resistant parasites, evident from the significantly higher control growth of chloroquine- resistant versus chloroquine-sensitive P.falciparum in the 1982 series. The sequence of events, and the impact of chloro- quine resistance in Mara, cannot yet be clearly dis- entangled from other variables. The parasite rates in children in North Mara started to rise in 1979, two S;w - - - | Sr 115 C. C. DRAPER ET AL. years before any manifest chloroquine resistance was detected. It is probable that this was partly due to some decline in the efficiency of the drug delivery system after the initial enthusiasm. This was sug- gested by a survey done in 1982 (2), which found some popular resistance to taking chloroquine regularly because of its bitter taste and side-effects, such as vomiting and itching (the latter affecting up to 50% of those interviewed). However, the EC" for 1979/80 was already close to the critical level which would reflect in vivo failure with 25 mg chloroquine/kg body weight. The suppressive doses applied were much lower of course. A third factor is obviously immunological since an age-related difference is only found in the in vivo tests, but not in the in vitro tests. Possibly, repeated small doses of chloroquine may be sufficient to reduce the antigenic stimulus of malaria infection without markedly altering the prevalence of parasitaemia. Hence the well recognized interaction between immunity and drug effects may be disturbed, and larger doses of the medicament may be required for clearing the infection. In the 1960s, in north- eastern Tanzania it was noted that increasing doses of chloroquine were needed to suppress parasitaemia as compared with 6 to 10 years previously (8, 9); the cause of this phenomenon was thought to be immuno- logical rather than due to the emergence of resistance. Furthermore, a "rebound" effect was noted after stopping chemosuppression in children: new infections temporarily caused parasitaemias higher than those found before suppression and were associated with an increase in clinical symptoms (10). The differences found in the in vivo tests throughout 1979-82 in North and South Mara between younger and older children support these observations. An extensive series of measurements of immuno- fluorescent antibodies was made over the years in North and South Mara, which will be the subject of a further communication; it suggests a fall of antibody titres following several years of chemosuppression. Similarly, in the survey at Mto-wa-Mbu, referred to above, there was a suggestion of a fall in antibodies when it was compared with other places (11). It can be assumed that immunofluorescent antibodies must be related to protective immunity in some way, since a drop in the seropositivity rate and geometric mean titre (GMT) reflects reduced host/parasite contact. Evidence of a relatively fast drop in seropositivity rates, GMTs, and IgM levels as a result of inter- vention with mass drug administration and/or vector control measures was provided in the Garki research project, Nigeria, which also demonstrated the rebound effect during the post-intervention phase (15). A temporary decline of protective immunity would also favour the selection, propagation, and spread of resistant parasite populations. The different response to chloroquine of younger and older children in areas with intensive malaria transmission has just been referred to. The impact of established chloroquine resistance is likely to be most obvious at first in persons with no (or little) immunity, such as the very young and those coming from non-malarious areas. When clinical symptoms of infection necessitate treatment, larger doses of chloroquine than previously used will have to be given. As happened in south-east Asia, it may ultimately be necessary to change to alternative drugs. The increasing heterogeneity of response in vitro which we observed in isolates of increasing resistance is of interest. It confirms evidence that natural populations of malaria parasites are of mixed sensitivity (16) and strengthens the suggestion that drug pressure will reduce the sensitive part of the population and select the resistant individuals (3). A higher reproduction of chloroquine-resistant as com- pared to chloroquine-sensitive parasites (16) would explain the marked population heterogeneity during the transition phase. The significant difference in schizont maturation observed between chloroquine- resistant and chloroquine-sensitive isolates in the 1982 series may be accepted as corroborative evidence. There has been one previous report of possible primary mefloquine resistance in the United Republic of Tanzania (12). The in vitro findings in North Mara merit a wider investigation of mefloquine sensitivity both by in vitro and in vivo testing since the response of 1 out of 59 isolates showed a relatively low sensitivity. The presence of some resistance to pyrimethamine in North Mara is not surprising as this phenomenon has been known for many years to exist in East Africa (13) although its distribution has not been accurately mapped in the absence of suitable assay methods. Pyrimethamine resistance was recently found in the Kisumu area of Kenya, which is adjacent to North Mara (14). Further investigations are indicated for which appropriate in vitro techniques are urgently required. Finally, there are signs that chloroquine-resistant P.falciparum selected in North Mara may be spread- ing into South Mara, but this may be difficult to sub- stantiate since South Mara is exposed to migration also from other areas and as chloroquine-resistant P.falciparum shows by now a multifocal distribution in the United Republic of Tanzania. 116 CHLOROQUINE RESISTANCE IN EAST AFRICA 117 ACKNOWLEDGEMENTS This work was supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. Of the many people who assisted, we would particularly like to thank Mr Z. Siso and Mr D. Juma of Shirati Hospital and Dr E. S. Mwasha, formerly Regional Medical Officer for Mara. We also thank Dr L. Molineaux ofWHO for helpful comments and suggestions. RESUMt ETUDES EN SERIE SUR L'EVOLUTION DE LA CHLOROQUINO-RESISTANCE DANS UNE REGION D'AFRIQUE DE L'EST O0 L'ON PRATIQUE UNE CHIMIOSUPPRESSION ANTIPALUDIQUE INTERMITTENTE L'objet de cet article est de d6crire les effets qu'une chimiosuppression antipaludique conduite dans une region d'hyperendemie d'Afrique de l'Est a eus sur la sensibilite medicamenteuse des plasmodies. L'etude s'est deroul& dans deux districts d'une zone de plateaux du nord-ouest de la R6publique-Unie de la Tanzanie, North Mara et South Mara. A North Mara, la chimiosuppression par la chloro- quine avait ete tent6e des 1977 mais, apres un succes initial, l'indice plasmodique avait recommence a augmenter au bout de II an. La chimiosuppression n'a jamais et6 essay6e A South Mara, qui sert de zone t6moin. Chaque mois, des groupes de maisons ont e choisis au hasard dans des villages r6partis sur l'ensemble de plateaux de North Mara de facon qu'on dispose de 200-400 enfants pour chaque enquete transversale, 1'etude se faisant par examen d'un frottis epais et numeration des plasmodies pour 500 globules blancs. Le meme groupe de villages a e utilis6 de 1979 a 1982 en vue d'epreuves seriees in vitro et in vivo destinees a tester la sensibilit6 a la chloroquine de Plasmodium falciparum. Les epreuves in vitro ont ete effectu6es en novembre 1979, juillet 1980, mars-avril 1981 et mars 1982. Elles ont montre que, a North Mara, la sensibilite de P.falciparum A la chloroquine avait diminue de 1979/80 (resultats groupes) A 1981 puis, de nouveau, en 1982. L'analyse des parasites sensibles et r6sistants isoles en 1982 a montre que les premiers regroupaient sans doute des populations parasitaires h6terogenes. Les reponses de plus en plus het6rogenes observees sur les isolements de resistance croissante corroborent l'id&e selon laquelle les populations naturelles d'h6matozoaires r6unissent des individus de sensibilit6 in6gale, de sorte que la pression m6dicamenteuse devrait r6duire la partie sensible de la population en assurant la selection des individus r6sistants. Tous les enfants pour lesquels on a pratique une epreuve in vitro ont egalement fait l'object d'une 6preuve in vivo consistant dans une variante de l'epreuve OMS A dose unique. Les resultats ont montre que le taux d'elimination dans le groupe d'Age 0-4 ans (70-85%) etait r6gulierement plus faible que chez les 5-11 ans (90-92%) en 1979/80 ainsi qu'en 1981, date a laquelle la resistance a pu etre 6tablie in vitro. En 1982, alors que la resistance avait augmente, le taux d'elimination avait notablement baisse, tombant a 50% dans le groupe d'age 0-4 ans tandis qu'il restait dans le meme intervalle pour les sujets plus ages. En outre, on a observ6 des signes de r6sistance a la pyrimethamine. Les r6sultats ainsi obtenus dans cette enquete donnent tout lieu de penser qu'une chimiosuppression par la chloroquine, pratiqu&e de faqon intermittente a des doses evidemment insuffisantes pour entrainer la guerison, a pu conduire en quelques ann6es, par s6lection, a une pharmacor6sistance tres forte. En 1982, environ 20% des isolements 6tudi6s manifestaient une telle r6sistance, faible le plus souvent mais relativement elev6e dans un nombre non n6gligeable de cas. II n'est pas facile de distinguer clairement des autres variables l'enchafnement des evenements et les repercussions de la chloroquino-r6sistance a Mara. Le taux .d'infestation chez les enfants de North Mara s'est mis a augmenter en 1978, c'est-a-dire deux ans avant qu'apparaisse un signe manifeste de resistance a la chloroquine. 11 est probable que cela tient en partie a une certaine perte d'efficacite du systeme de distribution du medicament ainsi qu'a des facteurs immunologiques, vu qu'une difference en rapport avec l'age n'a e observee que dans les epreuves in vivo. Mais il ne faut pas perdre de vue qu'on s'est servi de doses insuffisantes pour determiner la guerison et qui n'auraient pas permis, chez des sujets non immunises, d'eliminer l'infestation au niveau de reponse constate in vitro en 1979/80. Enfin, il est peut-etre difficile de confirmer les signes d'une propagation a South Mara des souches de P.falciparum devenues chloroquino-resistantes a North Mara par selection, car le premier district est le siege de mouvements migratoires sans compter que P.falciparum a une distribution multifocale en Tanzanie. REFERENCES 1. 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