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In vitro drug sensitivity of in Acre, P.G. Kremsner,' G.M. Zotter,1 H. Feldmeier,2 W. Graninger,3 M. Kollaritsch,4 G. Wiedermann,5 R.M. Rocha,6 & W.H. Wernsdorfer7

In Acre, the westernmost state of Brazil in the Amazon region, the sensitivity of Plasmodium falciparum to , , , and /pyrimethamine was determined in vitro by the Rieckmann microtechnique. The study was performed between January and June 1987; the in vitro parasite responses to all antimalarial drugs were determined according to the recommendations of WHO. Of 83 isolates of P. falciparum, all were sensitive to mefloquine and of 87 isolates of P. falciparum, 84 (97%0) were sensitive to quinine. The EC50 for mefloquine was 0.27 pmol/l and for quinine 4.60 pmol/l. In contrast, 65 of 89 (73%0) and 70 of 83 (84%) isolates were resistant to amodiaquine and chloroquine, respectively; 11 isolates even grew at 6.4 pmol chloroquinell. The EC50 for amodiaquine was 0.34 pumol/l and for chloroquine 0.73 pmol/l. Sulfadoxine/pyrimethamine resistance was seen in 23 of 25 (92%) cases. These data clearly indicate that in the western part of the Amazon region the 4-aminoquinolines, as well as sulfadoxine/pyrimethamine, can no longer be recommended for the treatment of P. falciparum infections.

Resistance of Plasmodium falciparum to antimalarial in vitro the response of P. falciparum to chloroquine, drugs has been reported in South America since amodiaquine, quinine, mefloquine and sulfadoxine/ 1961 (1, 2). Resistance to chloroquine was first pyrimethamine in a "colonization" area in the state observed in Colombia and Brazil (1, 2), and is now of Acre, Brazil. common in many endemic areas of the Americas (3-6). In 1981, resistance to sulfadoxine/ pyrimethamine was confirmed in Colombia (3). So far, only insufficient information has been available Patients and methods on the resistance of P. falciparum to amodiaquine, The study took place in Rio Branco, Acre, Brazil, quinine and mefloquine (4-6) on the South American which is situated at 680 longitude west and 10° lati- continent. Multidrug resistance is a major public tude south in the Amazon rain forest. All patients health concern, especially in the Amazon region of included in the study had a monoinfection with P. Brazil where immigration of non-immunes from the falciparum, the parasitaemia being >500 and southern part of the country has increased over <90 000 asexual parasites per p1 of blood. The recent years. It was therefore decided to investigate patients denied having taken any antimalarial medi- cation during the preceding four weeks. Before col- was ' Research Assistant, Institute of Tropical Medicine, Vienna, lection of the blood samples, informed consent Austria. Requests for reprints should be sent to Dr P.G. Krems- obtained from the patients or, in the case of children, ner, Institute of Tropical Medicine Berlin, Konigin-Elisabeth- from their parents. Strasse 32, 1000 Berlin 19. From January to June 1987, 161 patients were 2 Associate Professor, Institute of Tropical Medicine, Berlin. admitted to the study. The patients were 2 months to 3 Associate Professor, Department of Chemotherapy, University 62 years old and included 37 females and 124 males. of Vienna, Austria. For the in vitro tests, the Rieckmann microtech- 4 Medical Assistant, Institute of Tropical Medicine, Vienna, nique (7) was applied following the standard pro- Austria. cedurea and using microtitration plates and culture 5 Head, Institute of Tropical Medicine, Vienna, Austria. media provided by WHO. From each patient, 0.2 ml 6 Head, Superintendencia de Campanhas de Saude Publica, venous blood was collected in heparinized tubes and Acre, Brazil. 7 Formerly Chief, Research and Technical Intelligence, a Instructions for the use of the microtest kit for the assessment Action Programme, World Health Organization, Geneva, of the response of Plasmodium falciparum to chloroquine and . mefloquine in vitro. (Unpublished WHO document MAP/82.1, Reprint No. 4978 1982).

Bulletin of the World Health Organization, 67 (3): 289-293 (1989) © World Health Organization 1989 289 P.G. Kremsner et al.

Table 1: Plasmodlum falciparum in Acre, Brazil: sensi- 1640 medium containing lower concentrations of p- tivity to chloroquine (83 Isolates) aminobenzoic acid (pABA) and folic acid.b Final No. of isolates concentrations of pyrimethamine were 0.0125, 0.025, Drug showing complete % inhibition 0.05, 0.1, 0.2, 0.4, 0.8, 1.63, 3.13, 6.25 and 12.5 imol/l concentration inhibition of schizont of schizont blood with a constant ratio of pyrimethamine to (pmol/l blood) maturation maturation sulfadoxine of 1: 80. Control wells without drug were included in all assays. 0.2 0 (0) 8.42 0.4 2 (2.4) 30.02 The test plates were placed in a candle jar and 0.8 9 (10.8) 53.18 incubated at 37.5°C for 24 to 32 hours. At the end of 1.14 13 (15.7) 64.95 incubation, thick blood smears were prepared and 1.6 31 (37.3) 76.27 stained with Giemsa (5%) for 10 minutes. For each 3.2 54 (65.1) 94.93 6.4 72 (86.7) 99.76 drug concentration tested, the number of schizonts per 200 asexual parasites was determined. Isolates a Figures in parentheses are percentages. with a schizont maturation of less than 10% in the control wells were not used for evaluation. mixed with 1.8 ml RPMI 1640 medium previously In the sulfadoxine/pyrimethamine tests, only buffered with 25 mmol/l HEPES and 32 mmol/l schizonts with a normal morphology and at least 8 NAHCO3. Aliquots (50 ,ul) of blood-mixture were nuclei were counted. pipetted into the wells of the microtitration plates, Drug resistance was assumed if schizont matu- dosed with various drug quantities: namely, 1, 2, 4, ration was observed at 1.14 umol chloroquine/l, 0.4 5.7, 8, 16 or 32 pmol chloroquine/well; 0.25, 0.5, 1, 2, umol amodiaquine/l, 3.2 iml mefloquine/l and 51.2 4, 8 or 16 pmol amodiaquine/well; 0.5, 1, 2, 4, 5.7, 8 umol quinine/l, while resistance to sulfadoxine/ or 16 pmol mefloquine/well; or 4, 8, 16, 32, 64, 128 pyrimethamine was considered if there was less than and 256 pmol quinine/well. 90% schizont inhibition at a concentration of 130 For assessment of resistance to sulfadoxine/ imol sulfadoxine and 1.63 imol pyrimethamine/l of pyrimethamine, the blood was mixed with RPMI blood. The results were statistically evaluated using log-dose response probit analysis.c Table 2: Plasmodlum falciparum In Acre, Brazil: sensi- tivity to amodiaquine (89 isolates) Results No. of isolates Drug showing complete % inhibition In all, 83 isolates were evaluated for P. falciparum concentration inhibition of schizont of schizont sensitivity to chloroquine, 89 for amodiaquine, 83 for (pmol/l blood) maturation maturation mefloquine, 86 for quinine, and 25 for sulfadoxine/ pyrimethamine. The response pattern to chloroquine 0.05 0 (0). 0 is shown in Table 1. of are 0.1 0 (0) 4.20 Over 84% the isolates 0.2 2 (2.2) 23.62 chloroquine-resistant (growth at 1.14 prmol/1). The 0.4 24 (27.0) 61.56 data for various effective concentration (EC) levels 0.8 71 (79.8) 86.77 are shown in Table 5. They are indicative of high 1.6 85 (95.5) 98.28 resistance. 3.2 89 (100.0) 100.0 A similar frequency of resistance (73%) was a Figures in parentheses are percentages. observed for amodiaquine (growth at 0.4 pmol/l) (Table 2); the EC data indicate substantial resistance Table 3: Plasmodlum falciparum in Acre, Brazil: sensi- (Table 5). No statistically significant difference could tivity to mefloquine (83 isolates) be detected between the response to chloroquine and that to amodiaquine (P > 0.05). As for mefloquine, No. of isolates Drug showing complete % inhibition all isolates were fully inhibited at 3.2 imol/l, indicat- concentration inhibition of schizont of schizont ing high sensitivity to this drug (Table 3). This is also (pmol/l blood) maturation maturation evident from the relatively low EC levels (Table 5).

0.1 0 (0). 3.59 b 0.2 2 (2.4) 30.66 In vitro microtest (Mark 11) for the assessment of the response 0.4 18 (21.7) 74.15 of Plasmodium falciparum to chloroquine, mefloquine, quinine, 0.8 66 (79.5) 99.21 sulfadoxine/pyrimethamine and amodiaquine. (Unpublished WHO 1.14 80 (96.4) 99.96 document MAP/87.2, 1987). 1.6 82 (98.8) 99.99 c Grab, B. & Wernsdorfer, W.H. Evaluation of in vitro tests for 3.2 83 (100.0) 100.0 drug sensitivity in Plasmodium falciparum: probit analysis of log- dose response test from 3-8 point assay. (Unpublished WHO a Figures in parentheses are percentages. document MAL/83.990, 1983).

290 WHO Bulletin Vol. 67 1989 In vitro drug sensitivity of Plasmodlum talciparum In Acre, Brazil

Table 4: Plasmodlum faiciperum In Acre, Brazil: sensi- tivity to quinine (86 Isolates) Discussion The state of Acre in western Brazil is representative No. of isolates of the areas in the South American Amazon jungle Drug showing complete % inhibition concentration inhibition of schizont of schizont which have recently been opened up. The great (pmol/l blood) maturation maturation increase of migration of non-immune people towards the Amazon region and the mobility of populations 0.8 0 (0)' 0 within the region have led to a considerable rise in 1.6 0 (0) 3.90 3.2 2 (2.3) 30.50 malaria transmission. 6.4 8 (11.6) 68.43 In 1987, the incidence of malaria in Acre was 12.8 53 (61.6) 94.81 3750 cases per 100000 inhabitants (R.M. Rocha, 25.6 78 (90.7) 99.88 unpublished observation). Appropriate medication is 51.2 83 (96.5) 99.95 only available in Rio Branco, capital of the state of a Figures in parentheses are percentages. Acre. In the rural areas, malaria control is limited to visits by Ministry of Health guards, who apply Resistance to quinine was encountered in three residual insecticides and use amodiaquine for treat- isolates which still showed schizont maturation at ment. Self-medication, especially in the rural areas, is 51.2 imol/l (Table 4). EC data indicate a relatively therefore frequent: in some instances, chloroquine is low sensitivity to quinine (Table 5). even mixed with the food or salt (personal Table 6 presents the results of sulfadoxine/ observation). In the present study, a high degree of pyrimethamine. Only two out of the 25 isolates P. falciparum resistance to chloroquine was demon- showed at least 90% inhibition of schizont matu- strated, with EC50 and EC90 values of 0.73 umol/l ration at 130 pmol sulfadoxine/l and 1.63 gmol and 6.82 umol/l, respectively, resulting in a relatively pyrimethamine/l. At the highest concentration of flat regression line. The EC levels observed in this 1000 pmol sulfadoxine/l and 12.5pmol pyrimeth- study were much higher compared with the results amine/l, only 14 out of the 25 isolates showed at from Gabon (8), but similar to data from Kenya (9). least 90% inhibition of schizont maturation. The high frequency of chloroquine resistance in the state of Acre is hardly surprising since earlier studies had shown a serious decrease in chloroquine sensi- Table 5: In vitro sensitivity of Plasmodlum falciperum In in eastern was Acre, Brazil, 1987: effective concentrations (gmol/l) cal- tivity Colombia and Brazil, which culated by probit analysis for 10% (ECIO), 50% (EC5*), aggravated by uncontrolled population movements 90% (EC,O), 95% (EC,.) and 99% (EC,,) Inhibition (4, 6, 9, 11). In vitro, 73% of the P. falciparum isolates were Drug tested EC,0 EC50 EC9, EC95 EC99 resistant to amodiaquine in this study. In vivo, an even higher percentage of amodiaquine resistance Chloroquine 0.21 0.73 2.50 3.55 6.82 Amodiaquine 0.13 0.34 0.86 1.12 1.83 was demonstrated in a recent in Acre Mefloquine 0.14 0.27 0.52 0.63 0.91 (5). This equal inefficiency of both of these 4- Quinine 2.06 4.60 10.30 12.94 19.87 aminoquinolines is at variance with other reports from South America, Africa and Asia (4, 9, 12), where amodiaquine has been found to be essentially the Table 6: Plasmodlum taiciperum In Acre, Brazil: sensi- more effective drug. This difference may be explained tivity to sulfadoxine/pyrlmethamine (25 Isolates) by the fact that the earlier observations were made Sulfadoxine/pyrimethamine No. of isolates showing at the time of introducing amodiaquine as an alter- concentrations 90% inhibition of native drug, while, in Acre, amodiaquine had already (pmol/l blood) schizont maturation been widely used. There was no evidence of in vitro resistance to 1/0.0125 0 (0). 2/0.025 0 (0) mefloquine. All parasites were completely inhibited 4/0.05 0 (0) at 3.2 pmol/l; 80% of the isolates were already fully 8/0.1 0 (0) inhibited at 0.8 umol/l and the EC50 and EC90 were 16/0.2 0 (0) 0.27 umol/l and 0.91 imol/l, respectively, indicative 32/0.4 1 (4) 64/0.8 1 (4) of high sensitivity. Furthermore, the steep regression 130/1.63 2 (8) is a sign of relatively high sensitivity to mefloquine. 250/3.13 4 (16) These data are similar to those recently obtained in 500/6.25 9 (36) Thailand (13). 1000/12.5 14 (56) The EC50 and EC99 levels for quinine were 4.60 Figures in parentheses are percentages. and 19.87 umol/l of blood, respectively. These values

WHO Bulletin Vol. 67 1989 2911 P.G. Kremsner et al. are also similar to data from Thailand where a rela- (FFWF), Vienna, Austria; P.G. Kremsner was on a fellow- tively high incidence of quinine resistance has been ship at SUCAM, Acre, Brazil, in 1987 after an official invi- reported (11). In Acre, however, 97% of the isolates tation from the Ministry of Health, Brazil. below the We thank Dr A. Cruz Marques and Dr H. Stemberger were inhibited by quinine concentrations for their assistance with these studies and Leitz, Austria, generally accepted threshold of 51.2 pmol/l for in for making a Laborlux K-microscope available. vitro resistance. This agrees with the results of quinine treatment in the study area. Virtually all cases of falciparum malaria correctly treated with a 10-day quinine regimen were cured. Resume In the test system for measuring sensitivity to sulfadoxine/pyrimethamine, schizonts were con- Pharmacosensibilite In vitro de Plasmodlum sidered to be mature only if they had at least eight falciparum dans l'etat d'Acre (Bresil) nuclei of normal appearance. Moreover, optimized Dans l'Etat d'Acre, I'Etat le plus occidental du conditions were used for sulfadoxine/pyrimethamine Bresil, situe dans la region de l'Amazonie, la sensi- et al. (14). In a testing as described by Sabchareon bilite de Plasmodium falciparum a la chloroquine, different test system, there was evidence that in vitro I'amodiaquine, la mefloquine, la quinine et growth of P. falciparum at the concentration of 3.22 I'association sulfadoxine/pyrimethamine a ete imol sulfadoxine/1 and 0.1 ,mol pyrimethamine/l mesuree in vitro a I'aide de la microtechnique de- correlated in vivo with recurrent parasitaemia (15). In veloppee par Rieckmann. Dans le cadre de cette the present study, data obtained with the WHO les ont ete faites entre les mois standard test system permitted a prediction of resist- enquete, epreuves in all P. falci- de janvier et juin 1987. Pour tous les anti- ance to sulfadoxine/pyrimethamine paludiques soumis a l'preuve in vitro, la reponse parum infections tested. After a single-dose treatment du parasite a ete mesuree suivant les recomman- with sulfadoxine/pyrimethamine, maximum plasma dations de l'Organisation mondiale de la Sante. concentrations between 0.8 and 1.63 umol pyrimeth- Sur 83 isolements de P. falciparum, tous etaient amine per litre of blood are reached (16). If the latter sensibles a la mefloquine et sur 87 isolements, 84 concentration is taken as the threshold level for in (97%) 1'etaient a la quinine. La concentration effi- vitro resistance, then 92% of the cases investigated cace a 50% (CE50) 'tait de 0,27 pmol/I de sang were resistant. These findings correspond closely areas pour la mefloquine et de 4,60 4umol/I pour la with clinical observations from neighbouring quinine. En revanche, 65 isolements sur 89 (73%) in Colombia (4). etaient resistants a l'amodiaquine et 70 sur 83 (84%) a la chloroquine. II a ete observe dans ,1 Conclusions isolements une maturation du parasite a une con- The observations made in the present study indicate centration de 6,4 imol de chloroquine par litre de that in the western part of the Amazon area of Brazil sang. Une resistance a la sulfadoxine/ the 4-aminoquinolines, as well as the combination of pyrimethamine a ete demontree dans 23 cas sur sulfadoxine/pyrimethamine have practically lost 25 (92%). their efficacy in the treatment of P. falciparum infec- Les resultats de cette enquete indiquent que, tions. In this area, quinine remains an effective drug dans la partie occidentale de l'Amazonie, when used correctly. However, compliance problems l'utilisation des amino-4 quinoleines ou de often arise due to the side-effects of quinine. I'association sulfadoxine/pyrimethamine ne peut Prospects of overcoming these constraints by com- plus etre recommandee pour le traitement des bining a short course of quinine with other drugs are infections a P. falciparum. limited because of the lack of suitable partner com- pounds. Single-dose treatment with mefloquine is a feasible alternative, but this should be strictly based References on an unequivocal microscopic diagnosis of falci- followed administration of an 1. Moore, D.V. & Lanier, S.R. Observations on two Plas- parum malaria by the modium infections with an abnormal response to adequate mefloquine dose so as to prevent undue chloroquine. American journal of tropical medicine drug pressure and thus delay the occurrence of and hygiene, 10: 5-9 (1961). resistance. 2. Rodrigues, D.P. [Cases of Plasmodium falciparum malaria resistant to treatment with chloroquine.] Arquivos de higiene e saude publica, 26: 231-235 Acknowledgements (1961) (in Portuguese). This study was supported by grant P 6233M from the 3. Espinal, C. et al. [Resistance of Plasmodium falci- Fonds zur Forderung der Wissenschaftlichen Forschung parum to the combination sulfadoxine-pyrimeth-

292 WHO Bulletin Vol. 67 1989 In vitro drug sensitivity of Plasmodlum faiciparum in Acre, Brazil

amine. Description of 3 primary cases in Colombia.] 11. Reyes, S. [Malaria infections with Plasmodium falci- Biomedica, 1: 213-217 (1981) (in Spanish). parum resistant to treatment with chloroquine. Situ- 4. Espinal, C. et al. Sensitivity of Plasmodium falci- ation in Brazil (1960-1981).] Revista brasileira de parum to antimalarial drugs in Colombia. American malariologia e doencas tropicais, 33: 109-130 (1981) journal of tropical medicine and hygiene, 34: 675-680 (in Portuguese). (1985). 12. Looaresuwan, S. et al. Intravenous amodiaquine and 5. Kremsner, P.G. et al. Amodiaquine-resistant malaria oral amodiaquine/erythromycin in the treatment of in Brazil. Lancet, 2: 684 (1987). chloroquine-resistant falciparum malaria. Lancet, 2: 6. Vasconcelos, M.A. & Rosarlo, V.E. [Sensitivity tests 805-808 (1985). on samples of Plasmodium falciparum from the 13. Suebsang, L. et al. Sensitivity to quinine and meflo- lower Amazon (Brazil).] Revista brasileira de malar- quine of Plasmodium falciparum in Thailand. Bulletin iologia e doencas tropicais, 35: 21-28 (1983) (in of the World Health Organization, 64: 759-765 (1986). Portuguese). 14. Sabehareon, A. et al. Evaluation of an in vitro test 7. Rleckmann, K.H. et al. Drug sensitivity of Plasmo- method for the assessment of sensitivity of Plasmo- dium falciparum. An in vitro microtechnique. Lancet, dium falciparum to pyrimethamine and sulfadoxine. 1: 22-23 (1978). Bulletin of the World Health Organization, 65: 8. Burchard, G.D. et al. Plasmodium falciparum 345-352 (1987). malaria: resistance to chloroquine but sensitivity to 15. Spencer, H.C. et al. A new in vitro test for mefloquine in the Gabon. A prospective in vitro pyrimethamine/sulfadoxine susceptibility of Plasmo- study. Tropenmedizin und Parasitologie, 35: 1-4 dium falciparum and its correlation with in vivo (1984). resistance in Kenya. Bulletin of the World Health 9. Spencer, H.C. Drug-resistant malaria-changing pat- Organization, 62: 615-621 (1984). terns mean difficult decisions. Transactions of the 16. Weldekamm, E. et al. Plasma concentrations of pyri- Royal Society of Tropical Medicine and Hygiene, 79: methamine and sulfadoxine and evaluation of phar- 748-758 (1985). macokinetic data by computerized curve fitting. 10. Cruz Marques, A. Migrations and the dissemination Bulletin of the World Health Organization, 60: of malaria in Brazil. Memorias do Instituto de 115-122 (1982). Oswaldo Cruz, 81 (suppl. 2): 17-30 (1986).

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