Chloroquine-resistant falciparum malaria imported into the Netherlands*

Bulletin ofthe WorldHealth Organization, 63 (1): 101 - 108 (1985) © World Health Organization 1985 Chloroquine-resistant falciparum malaria imported into the Netherlands* J. C. F. M. WETSTEYN1 & A. DE GEUS2 A prospective study of imported chloroquine-resistant falciparum malaria in the Netherlands is described. From 1979 to 1983, 77non-immune patients were investigated; in 41 (53%) decreased sensitivity of Plasmodium falciparum to chloroquine could be con- firmed. Signs andsymptoms in thesepatients differfrom the classicalpicture. Resistance to sulfadoxine-pyrimethamine (Fansidar) was established in 6 patients. Parasitaemia was found twice during dapsone-pyrimethamine (Maloprim) prophylaxis. The implicationsfor advice on treatment and prophylaxis are discussed. INTRODUCTION Chloroquine has for many years been the drug of choice for the treatment of acute attacks of malaria and also for chemoprophylaxis. In 1960 chloroquine resistance of Plasmodiumfalciparum was reported in two patients from Colombia and was followed by further reports from south-east Asia (1). Since then, chloroquine resistance has spread to other regions, with increases in the number of resistant cases and in the severity of resistance. The first chloroquine- resistant case of falciparum malaria from East Africa was reported in 1979 (2). Non-immune patients, infected with a chloroquine- resistant strain of P.falciparum during prophylaxis with chloroquine, often present with symptoms and signs that are quite different from the classical features of an acute attack in a non-immune person. The appearance of chloroquine resistance has important implications for malaria control in endemic areas, for the treatment of patients, and for individual chemoprophylaxis. The present study, which describes observations on patients with imported chloroquine-resistant falciparum malaria in the Netherlands, is based on monitoring of cases of imported malaria by the malaria reference centresa which report clinical and parasitological data to WHO. * From the Institute of Tropical Hygiene, Royal Tropical Institute, Mauritskade 63, 1092-AD, Amsterdam, Netherlands. Specialist in Internal Medicine (tropical diseases). 2 Professor of Tropical Medicine. ' The three malaria reference centres in the Netherlands are as foliows: Institute of Tropical Hygiene, Amsterdam; Institute of Medical Parasitology, Nijmegen; and Institute of Tropical Medicine and Parasitology, Leiden. Drug resistance Resistance has been defined as "the ability of a parasite strain to survive and/or to multiply despite the administration and absorption of a drug given in doses equal to or higher than those usually recommended, but within the limits of tolerance of the subject" (1). Sensitivity (S) or resistance (R) of asexual parasites ofP.falciparum to aminoquinolines (see Fig. 1) is graded as follows (1, 3): S: clearance of asexual parasitaemia within 7 days of initiation of treatment, without subsequent recrudescence. RI: clearance of asexual parasitaemia as in sensitivity, followed by recrudescence. RII: marked reduction of asexual parasitaemia, but no clearance. RIII: no marked reduction of asexual parasit- aemia. Treatment with chloroquine in cases of RI or RII resistance results in initial clinical improvement (reduction of fever and declining parasitaemia), which is followed some days or weeks later by recrudescence. The interval is shorter in RII than in RI resistance; in RIII resistance no response of clinical disease or parasitaemia occurs. Appropriate treatment has been defined as a total dose of 25 mg chloroquine base per kg body weight, administered in 3-4 days (3). Methods to confirm chloroquine resistance of P. falciparum Clinical methods. A standard field test and an extended fi'eld test are available. In areas where reinfection is not possible, the extended field test (28 days) should be used. Sensitive and RI resistant 45 -101- J. C. F. M. WETSTEYN & A. DE GEUS | Sensitivity (S) AO%,u Patency level Ni RI Resistance, delayed recrudescence t, Patency level RI Resistance, early recrudescence - Patency level- Days after start of treatment on Day 0 |§ I~~~~~~~~~~~~~~~~~WHOS1554 STANDARD TEST 7-day observation EXTENDED TEST 28-day observation Fig. 1. Five possible responses in the field test for sensi- tivity of falciparum malaria to chloroquine administration during the first three days. ---- Patent parasitaemia may reappear by day 5; in sensitive (S) strains, it is absent on and after day 6. ---- There may be an increase in parasitaemia in Rill resistance. (Note that it is not possible to distinguish between S and RI responses in the standard (7-day) test.) strains can be distinguished as the recrudescence usually occurs within 28 days. Parasitological methods. For in-vitro testing of drug sensitivity, the test commonly used is the maturation inhibition test, described by Rieckmann et al. (4). Maturation of a sensitive strain and of an RIII resistant strain is inhibited at a chloroquine con- centration of 0.5 ltmol/l and 5.0 imol/l, respectively. Chloroquine sensitivity can also be determined after in-vitro cultivation in a modified test, described by Nguyen-Dinh & Trager (5) and by Ponnudurai et al. (6). Pharmacological methods. A sensitive strain is eliminated by a chloroquine concentration in the serum of > 0.1 smol/l (30 ng/ml) which has to be maintained during 6 days after the start of the treat- ment (7). In chemoprophylaxis no parasitaemia should be present at a chloroquine concentration in serum of > 0.05 itmol/l (16 ng/ml) (8). The following are the prerequisites before a P.falciparum strain can be pronounced as chloroquine resistant (7): (1) demonstration of a recrudescence by clinical observation; (2) estimation of chloroquine in the urine to prove adequate absorption; (3) measurement of serum chloroquine levels to demonstrate an adequate drug concentration; (4) in-vitro sensitivity testing to confirm the clinical findings; (5) experimental infection of animals or volunteers with the isolated strain to reproduce in vivo the in- vitro results. The above prerequisites cannot always be fulfilled since measurement of drug levels or in-vitro testing is not always possible. Experimental infections with P.falciparum can only be performed in specialized institutions (9). An extended field test can only be performed reliably when reinfection is excluded. Because P.falciparum will be cleared with a lower dose of chloroquine in semi-immune patients than in non- immune persons and as in-vitro sensitivity testing can be modified by the presence of malaria antibodies, investigations in chloroquine-resistant falciparum malaria can be performed with success in non- malarious areas on non-immune patients. PATIENTS AND METHODS From January 1979 onwards we considered every case of falciparum malaria to be potentially chloro- quine resistant, even when the patient acquired the infection in areas not (yet) known to harbour chloroquine-resistant P.falciparum strains. The majority of these patients took the recommended antimalarial prophylactic drugs. Of the 89 patients with P.falciparum malaria observed, 12 were semi-immune inhabitants of endemic malarious areas and were excluded, leaving 77 patients for study. Of these, 35 were treated as out- patients, 20 were admitted to the hospital of the University of Amsterdam, and 7 were admitted to .I Z 0 a -a : 102 CHLOROQUINE-RESISTANT IMPORTED FALCIPARUM MALARIA other hospitals in Amsterdam. All were followed up after discharge in the outpatient clinic for tropical diseases in the Royal Tropical Institute. Fifteen patients were admitted to hospitals elsewhere in the Netherlands and advice on their treatment was given by telephone. Parasitological examinations of all blood slides were carried out in the laboratory for tropical diseases in the Royal Tropical Institute. In addition to physical examination (with careful attention to spleen size), several laboratory investigations were performed. In in-patients, these included daily thick and thin blood films for 1 week for malaria parasite microscopy, ESR and haemoglobin estimation, leukocyte count, platelet and reticulocyte counts, liver function tests, and serum creatinine. The serum chloroquine con- centration was measured on admission, as well as during and after treatment (Department of Clinical Pharmacology, University of Amsterdam, Dr C.J. van Boxtel). If possible, blood was taken for in-vitro sensitivity testing as described by Rieckmann et al. (4) and for in-vitro cultivation and sensitivity testing of isolates adapted to culture, as described by Ponnudurai et al. (6) (Institute of Medical Para- sitology, University of Nijmegen, Professor J.H.E.Th. Meuwissen). In our outpatient clinic, investigations were limited to preparation of thick and thin blood films for parasite microscopy twice weekly, and to determination of haematological values and chloroquine concentration of the serum once weekly. A total of 56 patients could be followed up for a period of 4-12 weeks; attention was given to spleen size, parasitaemia, haemoglobin and reticulocyte levels, and serum concentrations of chloroquine. We used the following criteria to establish chloro- quine resistance: (1) Chloroquine resistance was proved when (a) a recrudescence was observed after appropriate treat- ment with chloroquine, and/or (b) an in-vitro sensi- tivity test showed inhibition of maturation at a chloroquine concentration of > 0.5 tmol/l. (2) Chloroquine resistance was presumed when (a) asexual parasites were present in the blood at a chloroquine concentration of > 0.05 gmol/l (16 ng/ml) (8), and/or (b) total inhibition of the isolated P.falciparum strain, adapted to in-vitro cultivation, occurred at chloroquine levels of > 0.1 smol/l (30 ng/ml) (6). From 1979 to 1981 we treated adult patients with a standard dose of 1500 mg chloroquine base in 3 days; after 1 January 1982 a total dose of 25 mg/kg body weight was prescribed, to be taken in 3-4 days. When a recrudescence occurred, sulfadoxine 1500 mg with pyrimethamine 75 mg (3 tablets of Fansidar) were given, sometimes in combination with 600 mg of quinine (three times a day for 3-4 days). RESULTS From 1 January 1979 until 1 January 1983 a total of 461 malaria patients were registered in the Netherlands; 136 were diagnosed in Amsterdam, 89 being infected with P.falciparum. Twelve of these were excluded because they were autochthonous inhabitants of endemic malarious areas, leaving 77 patients (Table 1), of whom 20 were female and 57 .male; 69 were adults and 8 were children under 15 years old; 54 (70%) were infected in East Africa. Of the 77 patients, 41 (53%) had chloroquine-resistant falciparum malaria (26 proved and 15 presumed); 11 were females and 30 were males; there were 36 adults and 5 children, the mean age being 30.8 years (range, 2.5-59 years). Table 1. Total number of patients with malaria,in the Netherlands and with falciparum malaria in the whole country and in Amsterdam, together with the number of chloroquine-resistant cases, 1979-82 No. of falciparum malaria cases No. of chloroquine-resistant cases Total number of Year malaria patients Netherlands Amsterdam Proved Presumed Total 1979 113 49 15 0 3 3 1980 101 33 15 4 2 6 1981 128 51 23 12° 1 13 1982 119 60 36 1 ob 9 19 Total 461 193 89 (77)c 26 15 41 a Includes 1 patient with resistance to sulfadoxine-pyrimethamine. b Includes 5 patients with resistance to sulfadoxine-pyrimethamine. c In brackets is given the number of non-immune cases. 103 J. C. F. M. WETSTEYN & A. DE GEUS Table 2. Places of origin of chloroquine-resistant falciparum malaria imported into the Netherlands No. of patients with Area Falciparum malaria Chloroquine resistance United Republic of Tanzania 31 24 Kenya 19 9 East Africa 2a 2° Suriname 4 3 Indonesia 3 2 South America 1 b 1 b a These patients visited several countries in East Africa. b This patient visited several countries in South America. The countries or areas where the infection was contracted are shown in Table 2. Depending on the patients' main symptoms on presentation, three groups could be distinguished: (1) Acute high fever, often with chills. This was the major sign in 31 patients (75%). (2) "Flu-like" symptoms: slight to moderate fever, headache and general malaise for some time. These were found in 3 patients (7%). (3) Anaemia. Seven patients (18%) complained of tiredness due to anaemia; no significant fever occurred. The case histories below illustrate these three groups. Case histories Patient A, a 56-year-old male, visited Kenya from 14 June to 14 July 1982. For malaria prophylaxis he took chloroquine sulfate (Nivaquine) tablets, 100 mg per day, which he continued after he developed fever (40.4°C) and rigors. On physical examination in the outpatient clinic for tropical diseases on 23 July he did not look ill, or anaemic or jaundiced, and the liver and spleen were not palpable. Laboratory data: haemoglobin was 11.1 mmol/l; a thick blood film showed approximately 480 P.falciparum rings/mm3; and the serum chloroquine level was 0.27 itmol/l (88.6 ng/ml). In-vitro culture was unsuccessful and the Rieckmann test could not be performed because of the low parasitaemia and high serum chloroquine concentration. He was treated with 2000 mg chloro- quine base (25 mg/kg) over 4 days. The temperature became normal in 3 days, at which time a thick film showed an occasional ring. Thick films were examined twice weekly and remained negative. The patient complained of headache (without fever) on 10 August. The thick film then contained approximately 300 P.falciparum rings/mm3. He was immediately treated with sulfadoxine-pyrimethamine (Fansidar), 3 tablets, but had fever associated with a rigor the same evening. He felt completely well on 9 September but in the thick film a sporadic P.falciparum ring could still be found. He was then treated with tetracycline, 250 mg q.i.d. for 10 days, and quinine, 600mg t.i.d. for 7 days. Further follow- up showed no recrudescence. Comment: RI resistance (recrudescence, high pro- phylactic serum chloroquine level), combined with Fansidar resistance. Fever was the leading symptom. Patient B, a 46-year-old male, visited the outpatient clinic for tropical diseases on 12 September 1979 for his yearly medical examination. Since 1956 he had worked in the tropics, and more recently in the United Republic of Tanzania. He arrived in the Netherlands in August and continued taking 300 mg chloroquine base weekly as a prophylactic. He said he suffered from flu from 7 to 11 September. Physical examin- ation: not ill, no anaemia or jaundice, and the spleen tip could just be felt. Laboratory data: haemoglobin was 7.7 mmol/l, and a thick blood film showed approximately 600 P.falciparum rings/mm3. He was admitted to hospital and treated with 2100 mg chloro- quine base (27.5 mg/kg) in 4 days. Trophozoites disappeared by 14 September; the patient was followed up until 31 October but trophozoites were not found. Inhibition of cultured P.falciparum occurred only at a chloroquine level of 0.1 tmol/l (30 ng/ml). Comment: clinically sensitive case (disappearance of parasites in 2 days), not supported by in-vitro results. Flu-like symptoms earlier but no fever when examined; mild anaemia. Patient C, a 23-year-old male, visited the United Republic of Tanzania from the end ofMay until early 104 CHLOROQUINE-RESISTANT IMPORTED FALCIPARUM MALARIA July 1980. He took chloroquine sulfate, 100 mg per day, while abroad and continued this chemo- suppression for 4 weeks after returning to Europe. During this period he was on holiday in France and started complaining of headache and tiredness with- out fever. He was treated with iron tablets and was seen by a specialist physician on 18 August because of persistent anaemia. On admission on 28 August, 7 weeks after leaving Africa, he had no fever and the liver and spleen were not enlarged. Laboratory data: haemoglobin was 4.6 mmol/l, reticulocytes, 10.5%. Trophozoites of P.falciparum were present in the thin blood film but they were not counted. He was treated with 1800 mg chloroquine base (25 mg/kg) in 4 days. The thick film was negative on 4 September and the haemoglobin was 6.0 mmol/l. During follow- up he developed high fever and headache on 25 September. On readmission, he was fairly ill, temperature 39.4 IC, liver and spleen not enlarged. Laboratory data: trophozoites easily found in a thin blood film (0.1% parasitaemia). On our advice he was then treated with sulfadoxine-pyrimethamine (Fansidar), 3 tablets, and quinine, 600 mg t.i.d. for 3 days. He was followed up at the outpatient clinic for tropical diseases and no recrudescence occurred. Comment: RI resistance (recrudescence); severe anaemia was the leading symptom. DISCUSSION Chloroquine-resistant falciparum malaria should be suspected in all patients suffering from malaria during, or shortly after termination of, chemo- suppression with chloroquine and in those having a recrudescence after treatment with chloroquine. Criteria Chloroquine resistance was confirmed in 41 (53%) of the 77 patients with falciparum malaria. In 26 (34%) this was proved, based on recrudescence or the in-vitro sensitivity test. In 15 (19%) the resistance was presumed, based on the presence of asexual parasites at the required chloroquine serum level or using the in-vitro sensitivity test of the cultured P.falciparum strain. In the first years of our study (1979 and 1980) the expected recrudescence sometimes failed to appear; we observed few patients with falciparum malaria despite the recommended chemoprophylaxis, and after treatment no recrudescences occurred (see case history of patient B). During the last year of our study the criterion of recrudescence could no longer be used. Because of the increasing severity of chloro- quine resistance, treatment with chloroquine could not be justified and alternative antimalarial drugs were prescribed. In-vitro testing (1.b and 2.b) (4, 6) was rarely possible owing to the low parasitaemia and/or the presence of chloroquine in the patients' serum. Instead, the prophylactic serum chloroquine con- centration had to be used as the criterion (2.a.) (8) because waiting for a recrudescence could not be justified. Source of the infection The majority of the patients acquired their infection in East Africa; chloroquine-resistant strains were only acquired in the United Republic of Tanzania and Kenya. The prevalence of chloroquine- resistance is, however, much higher and more serious in south-east Asia. Our findings only reflect that greater numbers of our Dutch patients were exposed to chloroquine-resistant P.falciparum strains in East Africa than elsewhere. An increasing magnitude of chloroquine resistance in these patients became evident during the study. Symptoms and signs The symptoms and signs observed in these patients with P.falciparum malaria were different from the classical clinical presentation. The non-immune host who is not protected by a suppressive drug, and is infected with P.falciparum becomes seriously and progressively ill with fever, anaemia, and spleno- megaly. The anaemia is due to haemolysis and increases rapidly. When no treatment is given, severe complications such as renal failure or cerebral malaria can occur. In thick and thin blood films asexual parasites are easily found. In the beginning all our patients were admitted to hospital for treatment and observation until the fever abated and there was no parasitaemia. Sometimes the patients had to be persuaded to be admitted, because they did not feel very ill. On observation, their clinical condition was good, although high fever with chills occurred. There were no serious complications, which is in contrast to the patients who contract falciparum malaria when not covered by antimalarial drugs for chemosuppression. After several such admissions to hospital without any complications we decided to treat these cases as outpatients. Investigations of the thick blood films showed that trophozoites were few. When only a thin film is examined, the parasites may not be detected, thereby delaying diagnosis and treatment. Both the patient and the doctor may not have con- sidered the possibility of malaria because chemo- prophylaxis was presumed to be adequate. It is therefore advisable to make thick blood films in all 105 J. C. F. M. WETSTEYN & A. DE GEUS Table 3. Comparison of clinical features, parasitaemia, and complications in 10 patients with classical malaria (no prophylactic chemosuppression) and in 41 patients with chloroquine-resistant P. falciparum malaria with chemo- suppression Proportion positive among cases of: Parameters Classical malaria Chloroquine-resistant falciparum malaria Fever 10/10 (100)" 31/41 (75)° Seriously ill 5/10 (50) 4/37 (11) Haemoglobin: < 8 mmol/l 4/10 (40) 18/39 (46) Splenomegaly 7/10 (70) 23/39 (59) Parasitaemia: > 1 % of erythrocytes infected 5/7 (72) 3/33 (9) Complications (renal failure, cerebral malaria) 4/10 (40) 0/41 (0) a Figures in parentheses are percentages. patients who have recently returned from tropical areas and who present with vague symptoms such as flu or fatigue. Repeated examination of thick films is necessary. In 75% of our patients fever was the leading symptom (Table 3), but no correlation with parasite density was found. It was remarkable that during follow-up after treatment, reticulocytosis often did not develop in spite of severe haemolytic anaemia. This is unlike the experience in the classical presentation. In many patients this lack of reticulo- cytosis appeared to be associated with later recrudescence. It is to be expected that this symptomatology will become less common when the grade of resistance increases. If chloroquine resistance spreads to other countries where chloroquine-resistant falciparum malaria has not yet been found, then the unusual clinical features described here will be seen again. The most probable explanation for this mild course of P.falciparum infection is the coexistence of a degree of immunity with protection by the suppressive drug, because chloroquine, with its long half-life (10), only suppresses parasitaemia while the immunological mechanisms are stimulated. Treatment of resistant cases Owing to the rapidly changing malaria situation in the world, firm recommendations on treatment are not possible. If the patient used the recommended suppressive dose of chloroquine, resistance can of course be suspected. If there are no complications and parasitaemia is low (< lo), a course of chloroquine can be given, adjusted to body weight. Thick blood films should be made daily for 7 days to confirm or disprove the parasitaemia. If the blood is cleared of parasites on day 7, once or twice weekly blood films (taken for 4 weeks) will show a re-appearance of parasites before the fever supervenes. If, however, complications are present and/or the parasitaemia is high, blood should be taken for in-vitro sensitivity testing and/or estimation of chloroquine concen- tration of the serum. Sulfadoxine-pyrimethamine should then be prescribed (unless contra-indications are present) together with quinine, 600 mg t.i.d. for 3-4 days. In 1981 and 1982 we observed a recrudescence after sulfadoxine-pyrimethamine in 6 patients who had acquired the infection in the United Republic of Tanzania (4 patients), Kenya, and Suriname (11, 12). Failure of treatment with sulfadoxine- pyrimethamine may not be due to resistance, as host factors in the metabolism of this drug combination may be involved (13, 14). We have treated these patients with quinine, 600 mg t.i.d. for 7 days and tetracycline, 250 mg q.i.d. for 10 days (15). No recru- descences occurred. Chemoprophylaxis In 1979-80, malaria attacks appeared 1-2 weeks after a course of chemosuppression with chloroquine base in a dose of 300 mg weekly or 100 mg daily. In 1981-82 the attacks occurred during the chemo- suppression, even with 100 mg chloroquine base daily! Twice a failure of dapsone-pyrimethamine (Maloprim) prophylaxis was observed. P.falciparum has also become increasingly resistant to sulfadoxine- pyrimethamine, and sometimes serious reactions have been reported during prophylaxis with these drugs (16, 17). Full protection can therefore no longer be guaranteed. In the Netherlands, we therefore resorted to a combination of proguanil, 100 mg per day, and chloroquine, 300 mg base per week (18). This com- bination has been suggested for use in areas with chloroquine-resistant P.falciparum strains, but has 106 CHLOROQUINE-RESISTANT IMPORTED FALCIPARUM MALARIA 107 not yet been tested extensively (19, 20). Cases of failure should be treated with sulfadoxine- pyrimethamine in a single dose of 3 tablets, if possible combined with quinine, 600 mg t.i.d. for 3-4 days. Conclusion Close observation of patients with chloroquine- resistant P.falciparum malaria in non-endemic areas is of value in relation to the situation in endemic malarious areas. A low grade of resistance will emerge first in the non-immune traveller (2, 21 ) or expatriate, while the endemic population will still be protected by acquired partial immunity. An increase in the grade of resistance will also first be noticed in non-immune persons. ACKNOWLEDGEMENT We thank Dr B. J. Brabin for kindly reviewing the manuscript. RtSUMt PALUDISME A FALCIPARUM CHLOROQUINO-RESISTANT IMPORTt AUX PAYS-BAS L'article decrit une etude prospective du paludisme a falciparum chloroquino-r6sistant aux Pays-Bas. De 1979 a 1983, 89 malades atteints de paludisme a falciparum ont ete 6tudies. Sur les 77 cas non immuns, 54 (70%) avaient contracte l'infection en Afrique orientale. Chez 41 d'entre eux (53%), une sensibilite r6duite de P.falciparum a la chloroquine a pu etre confirm6e. Au cours de 1'6tude, le nombre de malades et le degre de resistance ont augment6; 73% des malades ayant requ une chimioprophylaxie convenable ont ete infectes par une souche de P.falciparum chloroquino-resistante; lorsque la prophylaxie etait insuffisante, 25% des malades etaient infectes par des souches r6sistantes. Chez ces malades, les signes et sympt6mes s'ecartaient du tableau clinique classique du paludisme, avec trois symptomes pre- dominants: fievre, malaise general et anemie. La resistance a la sulfadoxine-pyrimethamine (Fansidar) a &e demontree chez six malades. Dans deux cas, on a trouve une parasitemie au cours d'une prophylaxie a la dapsone-pyrim6thamine (Maloprim). Les cons6quences de ces observations en ce qui concerne le traitement et la prophylaxie sont envisagees. REFERENCES 1. BRUCE-CHWATT, L. J. Drug resistance in malaria. In: Chemotherapy of malaria, 2nd ed., Geneva, World Health Organization, 1981, pp. 102-118. 2. FOGH, S. ET AL. Chloroquine-resistant Plasmodium falciparum malaria in Kenya. Transactions ofthe Royal Society of Tropical Medicine and Hygiene, 73: 228-229 (1979). 3. WHO Technical Report Series, No. 529, 1973 (Chemotherapy of malaria and resistance to anti- malarials: report of a WHO Scientific Group). 4. RIECKMANN, K. H. ET AL. Drug sensitivity of Plasmodium falciparum: an in-vitro microtechnique. Lancet, 1: 22-23 (1979). 5. NGUYEN-DINH, P. & TRAGER, W. Plasmodium falciparum in vitro: determination of chloroquine sensitivity of three new strains by a modified 48-hour test. American journal of tropical medicine and hygiene, 29: 339-342 (1980). 6. PONNUDURAI, T. ET AL. Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in vitro culture. Tropical and geographical medicine, 33: 50-54 (1981). 7. EBISAWA, I. & FUKUYAMA, T. Chloroquine resistance of Plasmodium falciparum in West Irian and East Kalimantan. Annals of tropical medicine and parasitology, 69: 275-282 (1975). 8. BROHULT, J. ET AL. The concentration of chloroquine in serum during short and long-term malaria prophylaxis with standard and "double" dosage in non-immunes: clinical implications. Annals of tropical medicine and parasitology, 73: 401-404 (1979). 9. CAMPBELL, C. C. ET AL. Chloroquine-resistant Plasmodium fakciparum from East Africa: cultivation and drug sensitivity of the Tanzanian I/CDC strain from an American tourist. Lancet, 2: 1151-1154 (1979). 108 -J. C. F. M. WETSTEYN & A. DE GEUS 10. OOSTERHUIS, B. ET AL. HPLC-analysis and preliminary pharmacokinetic parameter estimations of chloro- quine. Pharmaceutisch Weekblad (Scientific edition), 3: 263-267 (1981). 11. GEUS, A. DE, ET AL. A case of Fansidar-resistant Plasmodium falciparum infection from Tanzania. Tropical and geographical medicine, 34: 261-263 (1982). 12. VLEUGELS, M. P. H. ET AL. Fansidar-resistant Plas- modiumfalciparum infection from Tanzania. Tropical and geographical medicine, 34: 263-266 (1982). 13. TRENHOLME, G. M. ET AL. Host failure in treatment of malaria with sulfalene and pyrimethamine. Annals of internal medicine, 82: 219-223 (1975). 14. WILLIAMS, R. L. ET AL. Acetylator phenotype and response of individuals infected with a chloroquine- resistant strain of Plasmodium falciparum to sulfalene and pyrimethamine. American journal of tropical medicine and hygiene, 24: 734-739 (1975). 15. REACHER, M. ET AL. Drug therapy for Plasmodium falciparum malaria resistant to pyrimethamine- sulfadoxine (Fansidar). Lancet, 2: 1066-1068 (1981). 16. VESTERGAARD OLSEN, V. ET AL. Serious reactions during malaria prophylaxis with pyrimethamine- sulfadoxine. Lancet, 2: 994 (1982). 17. HORNSTEIN, O. P. & RUPRECHT, K. W. Fansidar- induced Stevens-Johnson syndrome. New England journal of medicine, 307: 1529-1530 (1982). 18. GEUS, A. DE & KAAY, H. J. VAN DER. Malariaprofylaxe. Nederlands Tijdschrift voor Geneeskunde, 127: 1252-1255 (1983) (in Dutch). 19. BRUCE-CHWATT, L. J. Chemoprophylaxis of malaria in Africa: the spent "magic bullet". British medical journal, 285: 674-676 (1982). 20. COLBOURNE, M. Malaria prophylaxis for travellers to East Africa. Communicable diseases report, 83 (21): 3-4 (1983). 21. STAHEL, E. ET AL. Pyrimethamine/sulfadoxine- resistantfalciparum malaria acquired at Dar es Salaam, Tanzania. Lancet, 1: 1118-1119 (1982).