In vitro drug sensitivity of Plasmodium falciparum in Acre, Brazil.

In vitro drug sensitivity of Plasmodium falciparum in Acre, Brazil 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 chloroquine, amodiaquine, mefloquine, quinine and sulfadoxine/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 drugs has been reported in South America since 1961 (1, 2). Resistance to chloroquine was first observed in Colombia and Brazil (1, 2), and is now 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 on the resistance of P. falciparum to amodiaquine, quinine and mefloquine (4-6) on the South American continent. Multidrug resistance is a major public health concern, especially in the Amazon region of Brazil where immigration of non-immunes from the southern part of the country has increased over recent years. It was therefore decided to investigate ' Research Assistant, Institute of Tropical Medicine, Vienna, Austria. Requests for reprints should be sent to Dr P.G. Krems- ner, Institute of Tropical Medicine Berlin, Konigin-Elisabeth- Strasse 32, 1000 Berlin 19. 2 Associate Professor, Institute of Tropical Medicine, Berlin. 3 Associate Professor, Department of Chemotherapy, University of Vienna, Austria. 4 Medical Assistant, Institute of Tropical Medicine, Vienna, Austria. 5 Head, Institute of Tropical Medicine, Vienna, Austria. 6 Head, Superintendencia de Campanhas de Saude Publica, Acre, Brazil. 7 Formerly Chief, Research and Technical Intelligence, Malaria Action Programme, World Health Organization, Geneva, Switzerland. Reprint No. 4978 in vitro the response of P. falciparum to chloroquine, amodiaquine, quinine, mefloquine and sulfadoxine/ pyrimethamine in a "colonization" area in the state of Acre, Brazil. Patients and methods The study took place in Rio Branco, Acre, Brazil, which is situated at 680 longitude west and 10° lati- tude south in the Amazon rain forest. All patients included in the study had a monoinfection with P. falciparum, the parasitaemia being >500 and <90 000 asexual parasites per p1 of blood. The patients denied having taken any antimalarial medi- cation during the preceding four weeks. Before col- lection of the blood samples, informed consent was obtained from the patients or, in the case of children, from their parents. From January to June 1987, 161 patients were admitted to the study. The patients were 2 months to 62 years old and included 37 females and 124 males. For the in vitro tests, the Rieckmann microtech- nique (7) was applied following the standard pro- cedurea and using microtitration plates and culture media provided by WHO. From each patient, 0.2 ml venous blood was collected in heparinized tubes and a Instructions for the use of the microtest kit for the assessment of the response of Plasmodium falciparum to chloroquine and mefloquine in vitro. (Unpublished WHO document MAP/82.1, 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- tivity to chloroquine (83 Isolates) No. of isolates Drug showing complete % inhibition concentration inhibition of schizont of schizont (pmol/l blood) maturation maturation 0.2 0 (0) 8.42 0.4 2 (2.4) 30.02 0.8 9 (10.8) 53.18 1.14 13 (15.7) 64.95 1.6 31 (37.3) 76.27 3.2 54 (65.1) 94.93 6.4 72 (86.7) 99.76 a Figures in parentheses are percentages. mixed with 1.8 ml RPMI 1640 medium previously buffered with 25 mmol/l HEPES and 32 mmol/l NAHCO3. Aliquots (50 ,ul) of blood-mixture were pipetted into the wells of the microtitration plates, dosed with various drug quantities: namely, 1, 2, 4, 5.7, 8, 16 or 32 pmol chloroquine/well; 0.25, 0.5, 1, 2, 4, 8 or 16 pmol amodiaquine/well; 0.5, 1, 2, 4, 5.7, 8 or 16 pmol mefloquine/well; or 4, 8, 16, 32, 64, 128 and 256 pmol quinine/well. For assessment of resistance to sulfadoxine/ pyrimethamine, the blood was mixed with RPMI Table 2: Plasmodlum falciparum In Acre, Brazil: sensi- tivity to amodiaquine (89 isolates) No. of isolates Drug showing complete % inhibition concentration inhibition of schizont of schizont (pmol/l blood) maturation maturation 0.05 0 (0). 0 0.1 0 (0) 4.20 0.2 2 (2.2) 23.62 0.4 24 (27.0) 61.56 0.8 71 (79.8) 86.77 1.6 85 (95.5) 98.28 3.2 89 (100.0) 100.0 a Figures in parentheses are percentages. Table 3: Plasmodlum falciparum in Acre, Brazil: sensi- tivity to mefloquine (83 isolates) No. of isolates Drug showing complete % inhibition concentration inhibition of schizont of schizont (pmol/l blood) maturation maturation 0.1 0 (0). 3.59 0.2 2 (2.4) 30.66 0.4 18 (21.7) 74.15 0.8 66 (79.5) 99.21 1.14 80 (96.4) 99.96 1.6 82 (98.8) 99.99 3.2 83 (100.0) 100.0 a Figures in parentheses are percentages. 1640 medium containing lower concentrations of p- aminobenzoic acid (pABA) and folic acid.b Final concentrations of pyrimethamine were 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.63, 3.13, 6.25 and 12.5 imol/l blood with a constant ratio of pyrimethamine to sulfadoxine of 1: 80. Control wells without drug were included in all assays. The test plates were placed in a candle jar and incubated at 37.5°C for 24 to 32 hours. At the end of incubation, thick blood smears were prepared and stained with Giemsa (5%) for 10 minutes. For each drug concentration tested, the number of schizonts per 200 asexual parasites was determined. Isolates with a schizont maturation of less than 10% in the control wells were not used for evaluation. In the sulfadoxine/pyrimethamine tests, only schizonts with a normal morphology and at least 8 nuclei were counted. Drug resistance was assumed if schizont matu- ration was observed at 1.14 umol chloroquine/l, 0.4 umol amodiaquine/l, 3.2 iml mefloquine/l and 51.2 umol quinine/l, while resistance to sulfadoxine/ pyrimethamine was considered if there was less than 90% schizont inhibition at a concentration of 130 imol sulfadoxine and 1.63 imol pyrimethamine/l of blood. The results were statistically evaluated using log-dose response probit analysis.c Results In all, 83 isolates were evaluated for P. falciparum sensitivity to chloroquine, 89 for amodiaquine, 83 for mefloquine, 86 for quinine, and 25 for sulfadoxine/ pyrimethamine. The response pattern to chloroquine is shown in Table 1. Over 84% of the isolates are chloroquine-resistant (growth at 1.14 prmol/1). The data for various effective concentration (EC) levels are shown in Table 5. They are indicative of high resistance. A similar frequency of resistance (73%) was observed for amodiaquine (growth at 0.4 pmol/l) (Table 2); the EC data indicate substantial resistance (Table 5). No statistically significant difference could be detected between the response to chloroquine and that to amodiaquine (P > 0.05). As for mefloquine, all isolates were fully inhibited at 3.2 imol/l, indicat- ing high sensitivity to this drug (Table 3). This is also evident from the relatively low EC levels (Table 5). b In vitro microtest (Mark 11) for the assessment of the response of Plasmodium falciparum to chloroquine, mefloquine, quinine, sulfadoxine/pyrimethamine and amodiaquine. (Unpublished WHO document MAP/87.2, 1987). c Grab, B. & Wernsdorfer, W.H. Evaluation of in vitro tests for drug sensitivity in Plasmodium falciparum: probit analysis of log- dose response test from 3-8 point assay. (Unpublished WHO document MAL/83.990, 1983). WHO Bulletin Vol. 67 1989290 In vitro drug sensitivity of Plasmodlum talciparum In Acre, Brazil Table 4: Plasmodlum faiciperum In Acre, Brazil: sensi- tivity to quinine (86 Isolates) No. of isolates Drug showing complete % inhibition concentration inhibition of schizont of schizont (pmol/l blood) maturation maturation 0.8 0 (0)' 0 1.6 0 (0) 3.90 3.2 2 (2.3) 30.50 6.4 8 (11.6) 68.43 12.8 53 (61.6) 94.81 25.6 78 (90.7) 99.88 51.2 83 (96.5) 99.95 a Figures in parentheses are percentages. Resistance to quinine was encountered in three isolates which still showed schizont maturation at 51.2 imol/l (Table 4). EC data indicate a relatively low sensitivity to quinine (Table 5). Table 6 presents the results of sulfadoxine/ pyrimethamine. Only two out of the 25 isolates showed at least 90% inhibition of schizont matu- ration at 130 pmol sulfadoxine/l and 1.63 gmol pyrimethamine/l. At the highest concentration of 1000 pmol sulfadoxine/l and 12.5pmol pyrimeth- amine/l, only 14 out of the 25 isolates showed at least 90% inhibition of schizont maturation. Table 5: In vitro sensitivity of Plasmodlum falciperum In Acre, Brazil, 1987: effective concentrations (gmol/l) cal- culated by probit analysis for 10% (ECIO), 50% (EC5*), 90% (EC,O), 95% (EC,.) and 99% (EC,,) Inhibition Drug tested EC,0 EC50 EC9, EC95 EC99 Chloroquine 0.21 0.73 2.50 3.55 6.82 Amodiaquine 0.13 0.34 0.86 1.12 1.83 Mefloquine 0.14 0.27 0.52 0.63 0.91 Quinine 2.06 4.60 10.30 12.94 19.87 Table 6: Plasmodlum taiciperum In Acre, Brazil: sensi- tivity to sulfadoxine/pyrlmethamine (25 Isolates) Sulfadoxine/pyrimethamine No. of isolates showing concentrations 90% inhibition of (pmol/l blood) schizont maturation 1/0.0125 0 (0). 2/0.025 0 (0) 4/0.05 0 (0) 8/0.1 0 (0) 16/0.2 0 (0) 32/0.4 1 (4) 64/0.8 1 (4) 130/1.63 2 (8) 250/3.13 4 (16) 500/6.25 9 (36) 1000/12.5 14 (56) Figures in parentheses are percentages. Discussion The state of Acre in western Brazil is representative of the areas in the South American Amazon jungle which have recently been opened up. The great increase of migration of non-immune people towards the Amazon region and the mobility of populations within the region have led to a considerable rise in malaria transmission. In 1987, the incidence of malaria in Acre was 3750 cases per 100000 inhabitants (R.M. Rocha, unpublished observation). Appropriate medication is only available in Rio Branco, capital of the state of Acre. In the rural areas, malaria control is limited to visits by Ministry of Health guards, who apply residual insecticides and use amodiaquine for treat- ment. Self-medication, especially in the rural areas, is therefore frequent: in some instances, chloroquine is even mixed with the food or salt (personal observation). In the present study, a high degree of P. falciparum resistance to chloroquine was demon- strated, with EC50 and EC90 values of 0.73 umol/l and 6.82 umol/l, respectively, resulting in a relatively flat regression line. The EC levels observed in this study were much higher compared with the results from Gabon (8), but similar to data from Kenya (9). 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- tivity in Colombia and eastern Brazil, which was aggravated by uncontrolled population movements (4, 6, 9, 11). In vitro, 73% of the P. falciparum isolates were resistant to amodiaquine in this study. In vivo, an even higher percentage of amodiaquine resistance was demonstrated in a recent clinical trial in Acre (5). This equal inefficiency of both of these 4- 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 more effective drug. This difference may be explained by the fact that the earlier observations were made at the time of introducing amodiaquine as an alter- native drug, while, in Acre, amodiaquine had already been widely used. There was no evidence of in vitro resistance to mefloquine. All parasites were completely inhibited at 3.2 pmol/l; 80% of the isolates were already fully inhibited at 0.8 umol/l and the EC50 and EC90 were 0.27 umol/l and 0.91 imol/l, respectively, indicative of high sensitivity. Furthermore, the steep regression is a sign of relatively high sensitivity to mefloquine. These data are similar to those recently obtained in Thailand (13). The EC50 and EC99 levels for quinine were 4.60 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- tively high incidence of quinine resistance has been reported (11). In Acre, however, 97% of the isolates were inhibited by quinine concentrations below the generally accepted threshold of 51.2 pmol/l for in 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. In the test system for measuring sensitivity to sulfadoxine/pyrimethamine, schizonts were con- sidered to be mature only if they had at least eight nuclei of normal appearance. Moreover, optimized conditions were used for sulfadoxine/pyrimethamine testing as described by Sabchareon et al. (14). In a different test system, there was evidence that in vitro growth of P. falciparum at the concentration of 3.22 imol sulfadoxine/1 and 0.1 ,mol pyrimethamine/l correlated in vivo with recurrent parasitaemia (15). In the present study, data obtained with the WHO standard test system permitted a prediction of resist- ance to sulfadoxine/pyrimethamine in all P. falci- parum infections tested. After a single-dose treatment with sulfadoxine/pyrimethamine, maximum plasma concentrations between 0.8 and 1.63 umol pyrimeth- amine per litre of blood are reached (16). If the latter concentration is taken as the threshold level for in vitro resistance, then 92% of the cases investigated were resistant. These findings correspond closely with clinical observations from neighbouring areas in Colombia (4). Conclusions The observations made in the present study indicate that in the western part of the Amazon area of Brazil the 4-aminoquinolines, as well as the combination of sulfadoxine/pyrimethamine have practically lost their efficacy in the treatment of P. falciparum infec- tions. In this area, quinine remains an effective drug when used correctly. However, compliance problems often arise due to the side-effects of quinine. Prospects of overcoming these constraints by com- bining a short course of quinine with other drugs are 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 on an unequivocal microscopic diagnosis of falci- parum malaria followed by the administration of an adequate mefloquine dose so as to prevent undue drug pressure and thus delay the occurrence of resistance. Acknowledgements This study was supported by grant P 6233M from the Fonds zur Forderung der Wissenschaftlichen Forschung (FFWF), Vienna, Austria; P.G. Kremsner was on a fellow- ship at SUCAM, Acre, Brazil, in 1987 after an official invi- tation from the Ministry of Health, Brazil. We thank Dr A. Cruz Marques and Dr H. Stemberger for their assistance with these studies and Leitz, Austria, for making a Laborlux K-microscope available. Resume Pharmacosensibilite In vitro de Plasmodlum falciparum dans l'etat d'Acre (Bresil) Dans l'Etat d'Acre, I'Etat le plus occidental du Bresil, situe dans la region de l'Amazonie, la sensi- bilite de Plasmodium falciparum a la chloroquine, I'amodiaquine, la mefloquine, la quinine et I'association sulfadoxine/pyrimethamine a ete mesuree in vitro a I'aide de la microtechnique de- veloppee par Rieckmann. Dans le cadre de cette enquete, les epreuves ont ete faites entre les mois de janvier et juin 1987. Pour tous les anti- paludiques soumis a l'preuve in vitro, la reponse du parasite a ete mesuree suivant les recomman- dations de l'Organisation mondiale de la Sante. Sur 83 isolements de P. falciparum, tous etaient sensibles a la mefloquine et sur 87 isolements, 84 (97%) 1'etaient a la quinine. La concentration effi- cace a 50% (CE50) 'tait de 0,27 pmol/I de sang pour la mefloquine et de 4,60 4umol/I pour la quinine. En revanche, 65 isolements sur 89 (73%) etaient resistants a l'amodiaquine et 70 sur 83 (84%) a la chloroquine. II a ete observe dans ,1 isolements une maturation du parasite a une con- centration de 6,4 imol de chloroquine par litre de sang. Une resistance a la sulfadoxine/ pyrimethamine a ete demontree dans 23 cas sur 25 (92%). Les resultats de cette enquete indiquent que, dans la partie occidentale de l'Amazonie, l'utilisation des amino-4 quinoleines ou de I'association sulfadoxine/pyrimethamine ne peut plus etre recommandee pour le traitement des infections a P. falciparum. References 1. Moore, D.V. & Lanier, S.R. Observations on two Plas- modium infections with an abnormal response to chloroquine. American journal of tropical medicine and hygiene, 10: 5-9 (1961). 2. Rodrigues, D.P. [Cases of Plasmodium falciparum malaria resistant to treatment with chloroquine.] Arquivos de higiene e saude publica, 26: 231-235 (1961) (in Portuguese). 3. Espinal, C. et al. [Resistance of Plasmodium falci- 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.] Biomedica, 1: 213-217 (1981) (in Spanish). 4. Espinal, C. et al. Sensitivity of Plasmodium falci- parum to antimalarial drugs in Colombia. American journal of tropical medicine and hygiene, 34: 675-680 (1985). 5. Kremsner, P.G. et al. Amodiaquine-resistant malaria in Brazil. Lancet, 2: 684 (1987). 6. Vasconcelos, M.A. & Rosarlo, V.E. [Sensitivity tests on samples of Plasmodium falciparum from the lower Amazon (Brazil).] Revista brasileira de malar- iologia e doencas tropicais, 35: 21-28 (1983) (in Portuguese). 7. Rleckmann, K.H. et al. Drug sensitivity of Plasmo- dium falciparum. An in vitro microtechnique. Lancet, 1: 22-23 (1978). 8. Burchard, G.D. et al. Plasmodium falciparum malaria: resistance to chloroquine but sensitivity to mefloquine in the Gabon. A prospective in vitro study. Tropenmedizin und Parasitologie, 35: 1-4 (1984). 9. Spencer, H.C. Drug-resistant malaria-changing pat- terns mean difficult decisions. Transactions of the Royal Society of Tropical Medicine and Hygiene, 79: 748-758 (1985). 10. Cruz Marques, A. Migrations and the dissemination of malaria in Brazil. Memorias do Instituto de Oswaldo Cruz, 81 (suppl. 2): 17-30 (1986). 11. Reyes, S. [Malaria infections with Plasmodium falci- parum resistant to treatment with chloroquine. Situ- ation in Brazil (1960-1981).] Revista brasileira de malariologia e doencas tropicais, 33: 109-130 (1981) (in Portuguese). 12. Looaresuwan, S. et al. Intravenous amodiaquine and oral amodiaquine/erythromycin in the treatment of chloroquine-resistant falciparum malaria. Lancet, 2: 805-808 (1985). 13. Suebsang, L. et al. Sensitivity to quinine and meflo- quine of Plasmodium falciparum in Thailand. Bulletin of the World Health Organization, 64: 759-765 (1986). 14. Sabehareon, A. et al. Evaluation of an in vitro test method for the assessment of sensitivity of Plasmo- dium falciparum to pyrimethamine and sulfadoxine. Bulletin of the World Health Organization, 65: 345-352 (1987). 15. Spencer, H.C. et al. A new in vitro test for pyrimethamine/sulfadoxine susceptibility of Plasmo- dium falciparum and its correlation with in vivo resistance in Kenya. Bulletin of the World Health Organization, 62: 615-621 (1984). 16. Weldekamm, E. et al. Plasma concentrations of pyri- methamine and sulfadoxine and evaluation of phar- macokinetic data by computerized curve fitting. Bulletin of the World Health Organization, 60: 115-122 (1982). WHO Bulletin Vol. 67 1989 293