In vitro susceptibility of Plasmodium falciparum collected from pyrimethamine—sulfadoxine sensitive and resistant areas in Thailand

Bulletin ofthe World Health Organization, 63 (3): 597-602 (1985) © World Health Organization 1985 In vitro susceptibility of Plasmodium falciparum collected from pyrimethamine-sulfadoxine sensitive and resistant areas in Thailand A. SABCHAREON, 1 T. CHONGSUPHAJAISIDDHI, 1 P. ATTANATH, 1 K. KANJANAPIPATKUL,1 E. B. DOBERSTYN,2 & L. SUEBSAENG2 Seventy Plasmodium falciparum isolates, collectedfrom two geographically separate areas of Thailand, were testedfor their in vitro responses to pyrimethamine, sulfadoxine, and a combination of these two drugs. The effects ofpyrimethamine andpyrimethamine- sulfadoxine combinations against P. falciparum isolates were found to be significantly greater in a northern area where the combined drug was an effective therapeutic agent than in a south-eastern area, near the Thai-Kampuchean border, where the combined drug was no longer effective. However, the actions of sulfadoxine against parasites obtainedfrom the two areas were not significantly different. There was no significant difference between the mean values ofplasma 4-aminobenzoic acid (PABA) infalciparum malariapatients and in healthy controls. The test for PABA determinations used in this study gave positive readings with both PABA and sulfadoxine. A countrywide in vitro study between 1977 and 1980 in Thailand showed that 96.8%o of 557 fresh Plasmodium falciparum isolates were resistant to chloroquine (1), and the clinical responses of malaria patients in the Hospital for Tropical Diseases, Bangkok, were most often RII and RIII responses. After the emergence of chloroquine resistance in 1962 (2), a combination of pyrimethamine and sulfadoxine (Fansidar)a became one of the main antimalarial drugs for the treatment of uncomplicated falciparum malaria in this country. Between 1967 and 1977 a single dose of Fansidar gave radical cure rates of 76.7-89.5% (3-6). However, studies in 1978-79 of various groups of falciparum malaria patients showed high failure rates of 50-90% after treatment with this drug combination (7-9). Between November 1980 and May 1981, an investigation in five separate areas of the country showed that, after a single full ' Department of Tropical Paediatrics, Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Requests for reprints should be addressed to Dr A. Sabchareon. 2 Malaria Division, Ministry of Public Health, Bangkok, Thailand. a Fansidar is a combination of pyrimethamine and sulfadoxine prepared by Hoffmann-La Roche, Basle, Switzerland. dose of 1.5 g sulfadoxine and 75 mg pyrimethamine (3 tablets of Fansidar), the radical cure rates in the north (Petchabun malaria clinic) (901o) and south (Sadoa malaria clinic) (82%) were significantly higher than those in the south-central (Chantaburi malaria clinic) (32%), north-eastern (Kuchinarai clinic) (39/o), and north-central (Mae Sot clinic) (42%) parts of the country (10). In the in vitro test, a culture medium containing 25-30 ,g/l of 4-aminobenzoic acid (PABA), which provides sufficient PABA for the growth and propa- gation of P.falciparum in continuous culture, has been used to evaluate the sensitivity to sulfadoxine, while Waymouth medium has been suggested for use in pyrimethamine and sulfadoxine testing (11). The in-vitro microtest of Rieckmann et al. (12) has been widely used in field evaluations of drug resistance in malaria, but the PABA concentrations in the test system were not measured. The object of the present study was to investigate the in-vitro sensitivity of fresh P.falciparum isolates (collected from patients living in Fansidar-sensitive and resistant areas) to pyrimethamine and sulfa- doxine, and to determine the plasma 4-aminobenzoic acid concentrations in malaria patients. 4558 -597 598 A. SABCHAREON ET AL. MATERIALS AND METHODS The study was begun on 12 July 1982 in the Petchabun malaria clinic (a Fansidar-sensitive area) and later in the Chantaburi malaria clinic (a Fansidar- resistant area) (10). The two clinics are approximately 500 kilometres apart. Patients attending the Petchabun clinic live in forested villages from which migration is minimal, whereas patients attending the Chantaburi clinic are generally migratory gem- mining workers. The study was completed on 26 October 1982. Only patients with a negative result in urine tests for evidence of prior ingestion of 4-aminoquinolines and sulfonamides, using the Dill-Glazko test (13) and the lignin test (14, respectively, were included in this study. After explanation of the trial, venous blood samples from each of the consenting uncomplicated falciparum malaria patients presenting at the Petchabum clinic and the Chantaburi clinic were collected into a heparinized syringe. Parasitized blood was thus taken from 83 patients; in vitro maturation to schizonts occurred in 70 isolates (84.3%) (40 from Petchabun and 30 from Chantaburi). All 70 isolates were tested for sensitivity to sulfadoxine and pyrimethamine-sulfadoxine, and 68 for pyrimethamine sensitivity. Initial parasitaemia in the Petchabun and Chantaburi patients was in the range 1510-44 107 and 907-53 520 ring forms per ul blood, respectively. Owing to a technical error, the erythrocyte volume fraction (ECF) of the Petchabun patients was not measured; the ECF of 36 Chantaburi patients was in the range 0.23-0.48 (mean, 0.36). The in vitro test system The blood samples were assayed immediately or within 1 hour after collection. They were tested for parasite sensitivity to pyrimethamine, sulfadoxine, and combined pyrimethamine and sulfadoxine (in a ratio of 1 to 200), using an adaptation of the microtest described by Rieckmann et al. (21) and Yisunsri & Rieckmann (15). The fixed drug ratio (1:200) of pyrimethamine and sulfadoxine was derived from plasma concentrations of the two drugs 24 hours after ingestion of Fansidar (16). The culture medium (Waymouth MB/752/1)b consisted of Waymouth formula powder 14.16 g/l, sodium bicarbonate 2.24 g/l, Hepes buffer 5.94 g/l, and gentamycin sulfate 8 mg/l, and the final pH was 7.4. The pure powder forms of pyrimethamine and sulfadoxine were kindly supplied by F. Hoffmann-La Roche & Co. Stock solutions of pyrimethamine and I Grand Island Biological Company, New York, USA. sulfadoxine were prepared separately. Pyrimetha- mine powder was dissolved with a small amount of 0.5% lactic acid, and the sulfadoxine powder was dissolved with a small amount of 50%0 sodium hydroxide. Double-distilled water was added itntil the concentrations of the stock solutions of pyrimetha- mine and sulfadoxine were 1Ox 10-3 mol/l and 2.5 x IO ' mol/l, respectively. Fifteen dilutions of each drug and of the drug combination were made in double-distilled water. The ranges of drug dilutions were: pyrimethamine 4 x 10- 8 to 3.6 x 10- mol/l, sulfadoxine I x 10- 5 to 7.2 x 10-3 mol/l, and pyrimethamine-sulfadoxine 4 x 10- 8 to 8 x 10-6 mol/l and 3.6 x 10- s to 7.2 x 10- 3 mol/l. The drug dilutions, the pre-dosed plates (flat-bottomed, 8 x 12 wells) with three control wells, and the medium were prepared just before entering each field. The plates and medium were kept in a refrigerator throughout the study period. Each well was inoculated with a mixture of 45 IA of Waymouth medium and 5 1sl of the patient's blood. The plates were gently rocked to dissolve the drug and then placed in a candle jar. After incubation in a covered water bathC at 38.5-40 IC for 48 hours, thick films were prepared from each well and stained with Giemsa solution. The number of apparently normal schizonts with three or more nuclei per 100 leukocytes was determined and expressed as a percentage of the mean of three control wells. Morphologically abnormal schizonts that did not have distinctly pink- stained chromatin and blue cytoplasm were also recorded. The minimum inhibitory concentration (MIC) of the drug was defined as the lowest concen- tration at which normal schizont formation was completely inhibited. The pH values of the blood-medium-drug mixtures ranged from 7.18 to 7.41, whereas the pH Qf the stock solutions of pyrimethamine and sulfadoxine were 2.1 and 9.9, respectively. Determination ofsulfadoxine and 4-aminobenzoic acid (PABA) Plasma collected from 79 patients in the Petchabun and Chantaburi clinics and plasma from 176 apparently healthy adults were tested for sulfadoxine and PABA concentrations. Determination of sulfa- doxine followed the methods previously described (17) and determination of PABA concentrations fol- lowed the method described by Horwitz (18). Evaluation of the specificity of the methods used for PABA determination Plasma samples from four volunteers in the Department of Tropical Paediatrics, Faculty of ' From Lab-Line Instruments, Inc., Illinois, USA. SUSCEPTIBILITY OF PLASMODIUM FALCIPARUM IN THAILAND Tropical Medicine, Mahidol University, Bangkok, were collected daily for 6 days before and for 5 days after the administration of 3 tablets of Fansidar, and their sulfadoxine and PABA concentrations were determined. Data analysis The PABA concentration in each well was calcu- lated using the formula: PABA in the well = plasma PABA (I - ECF)/10. An assumed mean ECF of 0.36 was used for the Petchabun patients and individual ECF values were used for the Chantaburi patients in the calculation. Student's "t" test was used to compare the log- transformed values of the MICs of the drugs acting on the Petchabun and Chantaburi parasites. Owing to a non-normal distribution of the data, the geometric means and their S.E. were tabulated. RESULTS The 48-hour duration of the test meant that the parasites were exposed to the drugs for a full cycle of asexual multiplication. In the presence of effective concentrations of antifolate drug combinations, ring forms do not mature into schizonts with a normal appearance; normal merozoite formation and sub- sequent reinvasion of erythrocytes are mostly pre- vented. This was in marked contrast to the large numbers of small ring forms that were frequently observed in wells containing no drug. The susceptibilities of P.falciparum to the tested drugs are summarized in Table 1. There was no statis- tically significant difference between the MICs of sulfadoxine acting on the Petchabun and Chantaburi parasites. However, the MICs of pyrimethamine and the pyrimethamine-sulfadoxine combination acting on the Petchabun parasites were significantly lower than those on the Chantaburi parasites (P< 0.001 and < 0.001, respectively). The geometric mean values of the MICs of pyrimethamine and pyrimetha- mine-sulfadoxine on the Petchabun parasites were 3- fold and 2.6-fold lower than on the Chantaburi parasites, respectively. Synergism of the two drugs was consistently noted in all isolates studied. Thus, evaluation of the geometric means of the MICs of the drugs acting on the parasites from the two areas shows that the drug combination resulted in a 2.6-fold increase in the activity of pyrimethamine and a 8.5-fold increase in the activity of sulfadoxine. Plasma from 45 out of 79 patients (6007o) showed detectable levels of sulfadoxine ranging from 11 to 121 mg/l. However, plasma containing sulfadoxine from the four volunteers gave a false positive reading for PABA concentrations, as shown in Table 2. The unusually high plasma PABA concentrations in these 45 patients, ranging from 4320 to 47 440 ,g/l, were considered to be false positives and excluded. Plasma PABA concentrations in 176 controls and 34 patients whose plasma had no detectable sulfa- doxine and the calculated PABA concentration in Table 1. Minimal inhibitory concentrations of pyrimethamine, sulfadoxine, and combined pyrimethamine/sulfadoxine in in vitro tests with fresh P. falciparum isolates collected from the Petchabun and Chantaburi malaria clinics in Thailand, 1982 Minimal inhibitory concentrations (mol/Il Pyrimethamine Sulfadoxine Pyrimethamine/sulfadoxine Petchabun (n = 40) a Range 1.2x 10-7to 5.0x 10-6 1.6x 10-4to4.8x 10-3 8.0x 10 -8/1.6x 10-6to 2.0x 10 -6/4.0x 10-4 Geometric mean+SE 1.6x 10-6±0.02x 10-6 1.6x 10-3±0.02x 10-3 6.6x 10-7/1.5x 10-4+ 0.2x 10-7/0.2x 10-4 Median 2.0x 10-6 2.4x 10-3 8.0x 10-7/1.6x 10-4 Chantaburi (n = 30) a Range 1 x 10-6 to 2.Ox 10-5 4.0x 10-4 to 6.0x 10-3 1 x 10-6/2.0x 10-4 to 4.0x 10-6/8.0x 10-4 Geometric mean±SE 5.0x 10-60.03x 10-6 2.1 x 1003+0.03x 10-3 1.7x 106/3.5x 1 + 0.02 x 10-6/0.2 x 10-4 Median 5.0x 10-6 2.4x 10-3 2.0x 10-6/4.0x 10-4 a n = number of isolates studied; only 28 isolates from the Chantaburi clinic were available for the pyrimethamine assay. 599 A. SABCHAREON ET AL. Table 2. Plasma PABA and total sulfadoxine concentrations in four volunteer subjects collected 6 days before and 5 days after the administration of 3 tablets of Fansidar (pyrimethamine-sulfadoxine) Before administration of After administration of pyrimethamine-sulfadoxine pyrimethamine-sulfadoxine PABA (,tg/l) Sulfadoxine (mg/I) PABA (Agll) Sulfadoxine (mg/I) 1.' Range 30-111 - 50 530-61 613 146-177 Mean 68 0 55 789 161 2. Range 37-74 - 65 798-75 563 205-271 Mean 61 0 72 621 232 3. Range 30-111 - 50 375-73 780 145-242 Mean 56 0 62 744 194 4. Range 74-185 - 52 080-62 620 148-215 Mean 111 0 57 433 171 The numbers 1-4 refer to the four volunteer subjects tested. Table 3. Range, mean and median values of PABA concentrations in plasma (from 176 control subjects and 34 patients), in which sulfadoxine was not detectable, and the calculated PABA concentrations in wells containing a blood-medium-drug mixture from the 34 patients Plasma PABA concentration (Ag/l) PABA concentration in wells containing blood-medium-drug mixture (yg/l) Controls Patients Range 20-271.4 80-160 4.6-36.9 Mean 98.7 273.8 17.4 Median 75.8 240.0 16.2 wells containing blood-medium-drug mixtures are summarized in Table 3. There was no significant difference between the plasma PABA concentrations in the patients and the controls. DISCUSSION The results of this study show that the degree of sulfadoxine susceptibilities of the parasites collected from the two areas were similar and that the higher susceptibility of the Petchabun parasites to pyrime- thamine and the pyrimethamine-sulfadoxine combin- ation was therefore due to the pyrimethamine suscep- tibility of the parasites. The in vitro pyrimethamine and sulfadoxine assays indicate a wide range of sensitivity of the P.falci- parum isolates in Thailand to these drugs. However, there is a positive correlation between the in vitro sensitivity to pyrimethamine-sulfadoxine in the present study and the in vivo response to a single dose of 3 tablets of Fansidar recently reported by Pinichpongse et al. (10), the time interval between these two studies being approximately one year. Our results fully support the view that the in vitro test could be used to detect the presence and prevalence of P.falciparum resistance to the drug combination. However, after the completion of this study, Desjardins (personal communication, 1983) found that the growth rates of P.falciparum were affected by both the PABA and folic acid concentrations in the culture medium, and that the 1:80 ratio of pyrime- thamine to sulfadoxine in the drug combination test approaches the optimum ratio of the independently determined activities of each drug in vitro. Chulay and coworkers determined the sensitivities of two isolates of parasites to the drugs by 3H-hypoxanthine incorporation techniques and reported that the growing parasites in modified RPMI medium (with no PABA and no folic acid) were inhibited by 1o 6 mol/l of sulfadoxine. A 1000-fold reduction of 600 SUSCEPTIBILITY OF PLASMODIUM FALCIPARUM IN THAILAND 601 sulfadoxine activity was observed in the medium con- taining 0.01 mg folic acid/I. They also noted that in normal RPMI medium 1640 (containing I mg folic acid and I mg PABA per litre) there was about 10- fold reduction in pyrimethamine activity and that sulfadoxine did not cause 50%7o parasite inhibition, even at concentrations exceeding the achievable blood concentrations. They found that medium containing PABA at concentrations above 0.5 mg/l was a growth inhibitor (19). Waymouth MB/752/ 1 medium used in our study contains 0.4 mg folic acid/l and no PABA. It is therefore apparent that there is a need for a standardized field test for these drugs, and this subject was discussed at a recent meeting. d The report of the meeting proposed that the WHO standardized test for detecting the susceptibility of P.falciparum to pyrimethamine and sulfadoxine in the field should utilize a modified RPMI 1640 medium with low con- centrations of PABA (0.5 Ag/l) and folic acid (10 itg/l), because this has been found to be the most d WERNSDORFER, W. H. & DESJARDINS, R. E. Report on informal discussions of investigators engaged in research on anti- folate antimalarials, Bangkok, 27-29 April 1983. suitable medium considering the control growth rates of the parasites and the effects on the test of both PABA and folic acid. In addition, alkalization with NaOH for dissolving sulfadoxine in the culture medium is generally not recommended because of its variable effect on the parasite growth rate. Currently, investigators who collaborated in the studies on the WHO standard technique are evaluating the drugs' effect by comparing the number of parasites present at the end of the incubation period with the number at the beginning, i.e., the quantitative effect on parasite invasion of new red cells. It may be noted that the methodology used in our study may not necessarily meet all the required criteria. However, the synergistic effects of pyrime- thamine and sulfadoxine in combination confirm previous reports (20, 21). The samples for PABA determination must be checked to see that they are sulfonamide-free, because any cross-reaction is most likely to be due to the structural similarity of PABA and the sulfonamides. Sulfadoxine was detected in 60%1o of lignin-negative plasma from patients, which shows that the urine lignin test for sulfonamides is not a sensitive test. ACKNOWLEDGEMENTS We should like to express our gratitude to Dr Surin Pinichpongse, Director of the Malaria Division, Ministry of Public Health, Bangkok, for permission to conduct the study at the Petchabun and Chantaburi malaria clinics, and thank the staff of these clinics for their help and cooperation. We are grateful to Dr W. Wernsdorfer and Dr G. Childs for their suggestions and correction of the manuscript, and thank Dr Chariya Brockelman and Miss Perapan Tan-Ariya for their help with the PABA determinations. We are also indebted to all the patients who participated in this study which was supported by grants from the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. RESUME SENSIBILITE IN VITRO DE PLASMODIUM FALCIPARUM RECUEILLIS DANS DES ZONES SENSIBLES ET DES ZONES RESISTANTES A LA PYRIMETHAMINE-SULFADOXINE Une etude a e effectuee pour observer et comparer la sensibilite in vitro d'isolements frais de P.falciparum recueillis dans des zones recemment signalees comme sensibles a la pyrimethamine-sulfadoxine (Fansidar) (dispensaire antipaludique de Petchabun) et des zones resistantes a ce medicament (dispensaire antipaludique de Chantaburi), en Thailande. Les malades frequentant le dispensaire de Petchabun, situe dans le nord, habitaient dans des villages forestiers d'ou les migrations etaient insignifiantes. Ceux qui frequentaient le dispensaire de Chantaburi, situe dans le sud-est, pres de la frontiere avec le Kampuchea, etaient des travailleurs de mines de pierres precieuses. A partir de 83 malades atteints de paludisme a P.falci- parum qui ont frequente les deux dispensaires de juillet a octobre 1982, on a preleve des echantillons de sang et on les a examines dans l'heure suivante pour determiner la sensibilite a la pyrimethamine, la sulfadoxine et la pyrime- thamine-sulfadoxine, au moyen d'une adaptation du micro- test decrit par Rieckmann et al. en 1978 et Yisunsri & Rieckmann en 1980. La plus faible concentration de medica- ment inhibant completement la formation normale de schizontes a e prise comme concentration minimale inhibitrice. On a observe une maturation in vitro jusqu'au stade de schizontes dans 70 isolements (84,3 %) a savoir 40 provenant du dispensaire antipaludique de Petchabun et 30 de celui de Chantaburi. L'action de la pyrimethamine et des asso- 602 A. SABCHAREON ET AL. ciations pyrimethamine-sulfadoxine s'est revelee nettement plus forte a l'egard des isolements de P.falciparum prove- nant de Petchabun qu'a 1'egard de ceux de Chantaburi (P < 0,001), mais l'effet de la sulfadoxine sur les parasites provenant des deux zones n'etait pas significativement dif- ferent. Les effets synergiques des deux medicaments ont e constamment observes dans tous les isolements examines. Le pH du melange sang-milieu-medicament dans les cupules (allant de 7,18 a 7,41) ainsi que les concentrations de PABA dans les cupules (de 4,6 a 36,9 ,g/1) ne doivent pas avoir perturbe la croissance des parasites ni influe sur les valeurs de la concentration minimale inhibitrice observee dans la presente etude. Les differences entre les concentrations plas- matiques de PABA chez 34 malades et 176 temoins n'etaient pas significatives, ces concentrations etant comprises entre 20 et 271 pg/l. 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