Chloroquine susceptibility of Plasmodium falciparum in Haiti

Bulletin ofthe WorldHealth Organization, 61(6): 1017-1020(1983) © World Health Organization 1983 Chloroquine susceptibility of Plasmodium falciparum in Haiti Roc MAGLOIRE & PHUC NGUYEN-DINH 2 In January andFebruary 1982, in vivo and in vitro studies ofthe chloroquinesensitivity ofPlasmodium falciparum were conducted in Port-au-Prince, Haiti. Of19 infections tested in vivo using the WHO extended test, all but one were susceptible to the drug; the remaining case showed a recurrence ofparasitaemia on day 28. Of the 19 corresponding 48-hour in vitro tests, 16 provided interpretable results; 12 tests demonstrated sensitivity to the drug, while in the remaining 4, parasite multiplication was inhibited only at drug concentrations higher than that previously accepted as indicative of sensitivity. (These four included the isolatefrom thepatient who had a recurrence ofparasitaemia on day 28.) While these results provide no absolute demonstration of chloroquine resistance, they underline the needfor close monitoring of the susceptibility to chloroquine of P. falciparum in Haiti. Malaria due to Plasmodium falciparum represents a major public health problem in Haiti. While chloro- quine remains the antimalarial drug of choice in the country, several reports have appeared recently de- scribing apparent clinical unresponsiveness to chloro- quine of some P.falciparum infections. This has prompted the initiation of a countrywide programme of monitoring for drug resistance in P.falciparum. The findings of studies conducted in January and February 1982 in Port-au-Prince are summarized in this paper. MATERIALS AND METHODS Patients The subjects participating in the study were selected from patients attending the outpatient clinic of the Service national des Endemies majeures (SNEM) in Port-au-Prince with symptoms suggestive of malaria. Patients were included in the study if: (1) they had infection with P.falciparum only, diagnosed on Giemsa-stained thick and thin blood smears; (2) the parasite count was higher than 500 asexual parasites per 1u of blood (or more than 25 asexual parasites per 300 leukocytes, assuming an average leukocyte count of 6000 per ul of blood); (3) they had no critical illness; and (4) they had no history of drug intake during the previous two weeks, and no 4-aminoquino- ' Chief, Epidemiology Section, Service national des Endemies majeures, Ministry of Health, Port-au-Prince, Haiti. 2 Visiting Scientist, Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, Department of Health and Human Services, Atlanta, GA 30333, USA. Requests for reprints should be addressed to this author. lines in the urine (tested using the Dill-Glazko tech- nique (1)). The study procedures were explained in detail to each prospective subject and/or to the guard- ians of children less than 15 years old, and those who agreed to participate were entered in the study group. All infected patients, whether they were included in the study group or not, were treated with 25 mg of chloroquine per kg of body weight. In vivo tests The WHO extended field test (2) was carried out, as follows. Each patient was treated with chloroquine phosphate, given orally, in a total dose of 25 mg base per kg of body weight-10 mg/kg on the first day of treatment (day 0), 10 mg/kg on the following day (day 1), and 5 mg/kg on day 2. Absorption of the drug was confirmed by the presence of chloroquine in a urine sample collected on day 1 (1) and by excluding patients who reported vomiting within 12 hours of taking any dose of the drug. Blood smears were examined for asexual parasites daily for 7 days and then weekly for a further 21 days. In vitro tests A 48-hour in vitro test was carried out, as described previously (3, 4). Before administration of drug to the patient, a parasitized venous blood sample was collected. An aliquot of 0.2 ml of the sample was diluted with 4.8 ml of a complete culture medium, consisting of RPMI 1640 mediuma supplemented with 30 mmol/litre Hepes buffer and 100 ml/litre human nonimmune AB+ serum. The resulting 2% erythrocyte suspension was distributed into 16-mm ° From Gibco, Grand Island, New York, USA. 4369 -1017- 1018 R. MAGLOIRE & P. NGUYEN-DINH flat-bottomed wells containing different amounts of chloroquine to give final concentrations of either 0, 0.01, 0.03, 0.06, 0. 1, and 0.3 gmol/litre of medium or 0, 0.01, 0.03, 0.1, 0.3, and 1 Amol/litre of medium.b The plates were then incubated for 48 h at 37 °C in a candle jar. Thin smears were taken from the initial sample and from each well after 48 h, and parasite counts per 10 000 erythrocytes were obtained. The inhibition of parasite multiplication produced by the various concentrations of drug was assessed in order to determine the in vitro susceptibility of the isolate to chloroquine. RESULTS The results are summarized in Table 1. Of 22 patients entering the study group, 19 completed the b I smol of chloroquine per litre of medium is equivalent to 25 Amol of chloroquine per litre of blood. follow-up period. There were 8 males and 11 females, with ages ranging from 2 to 48 years (mean, 20.4 years, excluding one adult patient who did not know her age). All were residents of Port-au-Prince or its environs. The initial parasite counts ranged from 900 to 111 240 asexual parasites per ul of blood (geo- metric mean, 9345). In vivo tests In all 19 patients, peripheral blood smears were cleared of asexual parasites during the first week following treatment. The parasite clearance time varied from 1 to 6 days (mean, 3.3 days). In all but one patient, follow-up smears taken on days 7, 14, and 28 were negative. In one patient (patient 4), a 2-year-old girl, the parasites were cleared on day 3, and she had negative follow-up smears on days 3-7, 14, and 21; however, the smear taken on day 28 showed 5720 asexual parasites per ul of blood. Table 1. Results of in vivo and in vitro tests of the chloroquine susceptibility of Plasmodium falciparum in Haiti, January 1982 Chloroquine concen- tration producing Day of inhibition of parasite Patient Age Sex Parasite clearance of multiplication (years) count' parasitaemia (Amol/litre) 1 26 M 11 140 3 0.03 2 Adult F 15 400 3 0.03 3 20 F 10 320 6 0.03 4 2 F 5 080 3b 0.1 c 5 21 F 9 740 2 0.03 6 24 F 8 380 4 0.03 7 22 M 11 700 1 0.03 8 48 F 14 280 5 0.03 9 13 M 1 660 5 0.03 10 25 M 15680 1 0.06 11 18 F 900 4 NR d 13 7 M 24 980 2 0.03 14 24 M 1 200 4 NR 15 11 F 17 000 4 0.03 16 30 F 12 440 4 NR 17 13 F 1 900 3 0.03 19 28 M 44 800 4 0.06 21 19 M 16000 2 0.03 22 17 F 111 240 3 0.06 Asexual parasites per tl of blood. b This patient again had parasitaemia (5720 parasites per ul of blood) on day 28. ' There was no well with 0.06 jAmol/litre in this test. d NR: no interpretable result could be obtained. CHLOROQUINE SUSCEPTIBILITY OF P. FALCIPARUM IN HAITI In vitro tests In 16 of the 19 in vitro 48-hour tests, a satisfactory parasite multiplication in the control wells (i.e., at least 2-fold increase) provided interpretable results. In 12 of the 16 isolates, no parasite multiplication occurred in the wells containing 0.03 smol or more of chloroquine per litre of medium. For the remaining 4 isolates, parasite multiplication did occur in wells containing 0.03 lsmol of chloroquine per litre, but not in the wells containing the next higher concentration of the drug (0.06 gsmol/litre in 3 tests and 0.1 smol/ litre in one test). DISCUSSION The sensitivity of malaria parasites to chloroquine was documented both in vivo and in vitro in 12 of the 19 cases studied. The results of the in vivo tests satis- fied WHO criteria for sensitivity to chloroquine, i.e., clearance of the parasitaemia during the first week, with no recrudescence detected during the 28-day observation period. In the corresponding in vitro tests, the parasites failed to multiply in wells contain- ing 0.03 1tmol or more of chloroquine per litre of medium, a response pattern similar to that observed previously in chloroquine-sensitive isolates from West Africa (3) and Honduras (4). In 3 cases (patients 11, 14, and 16), the in vitro tests failed to give interpretable results. However, the para- site response in vivo met the WHO criteria for sensi- tivity to chloroquine. In 3 other cases (patients 10, 19, and 22), the in vivo tests showed the parasites to be sensitive to chloroquine, but the corresponding 48-hour in vitro tests indicated a decreased sensitivity of the isolates. Parasite multiplication still occurred in wells contain- ing 0.03 jmol of chloroquine per litre, thus failing to satisfy the accepted criteria for drug-sensitive isolates. However, complete inhibition was observed in wells containing 0.06 tmol/litre, an end-point markedly lower than that usually seen with chloro- quine-resistant parasites, such as those found in south-east Asia which were inhibited by 0.3-1 imol/ litre (P. Nguyen-Dinh et al., unpublished data, 1980). This apparent discrepancy may signify either that the end-point previously accepted as indicating chloro- quine sensitivity in vitro is not absolute, or that the three isolates contained parasites whose resistance was demonstrable in vitro but not in vivo. It is possible that recrudescence would have occurred after the standard 28-day observation period, as reported elsewhere (5, 6). Alternatively, the parasites might have been prevented from expressing their chloro- quine resistance in vivo because of the combined inhibitory effects of the drug and the patient's immune defence. In one case (patient 4), after an initial clearance of parasitaemia by day 3, parasites were detected again on day 28. This may have resulted either from a rein- fection of the patient (who remained in an area with apparent active transmission of malaria) or from a delayed parasite recrudescence, compatible with an RI level of chloroquine resistance (2). The latter hypothesis would appear to be supported by the corresponding in vitro test, where complete inhibition did not occur at 0.03 umol/litre, but at the next higher concentration of chloroquine (in this case, 0.1 mol/ litre). CONCLUSION The majority of the P.falciparum infections tested in this study were found to be sensitive to chloro- quine. A decreased in vitro susceptibility to the drug was apparent in 4 isolates, one of which was as- sociated with an infection pattern compatible with an RI level of resistance. These latter findings do not constitute an absolute demonstration of chloroquine resistance. They do indicate, however, the need for further field studies to monitor the response to chloroquine of P.falciparum in Haiti. ACKNOWLEDGEMENTS We thank Dr L. Lafontant, Director of the Service national des End6mies majeures (SNEM), for his support, and the personnel of the SNEM/Port-au-Prince, especially Mrs G. Francois, Mr F. Eugene, and Mr F. Severe, for their technical assistance. This work received financial assistance from the Japanese Government, the US Agency for International Development, and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 1019 1020 R. MAGLOIRE & P. NGUYEN-DINH RESUME SENSIBILITE DE PLASMODIUM FALCIPARUM A LA CHLOROQUINE EN HAITI En janvier et fevrier 1982, des etudes in vivo et in vitro sur la sensibilite de Plasmodium falciparum a la chloroquine ont e faites a Port-au-Prince, Haiti. Sur les 19 infections soumises in vivo a 1'epreuve prolong&e OMS, toutes sauf une se sont averees sensibles au medicament; celle qui ne l'etait pas se differenciait par une reapparition de la parasi- temie au jour 28. Sur les 19 tests correspondants, 16 ont donne des resultats interpretables; 12 tests ont montr6 une sensibilite in vitro, tandis que dans les 4 autres, l'inhibition de la multiplication des parasites n'a e observ&e qu'a des concentrations plus elevees que celles qui 6taient jusque-la acceptees comme revelant in vitro une sensibilite a la chloroquine. L'un de ces tests a et fait sur un isolement obtenu a partir du patient chez lequel il y avait eu r6appa- rition de la parasitemie au jour 28. Ces resultats ne consti- tuent pas une preuve absolue de la resistance A la chloro- quine, mais ils mettent en lumiere la necessite pour l'avenir d'une surveillance etroite de la sensibilite de P.falciparum A la chloroquine en Haiti. REFERENCES 1. LELIJVELD, J. & KORTMANN, H. The eosin colour test of Dill and Glazko: a simple field test to detect chloro- quine in urine. Bulletin of the World Health Organiz- ation, 42: 477-479 (1970). 2. WHO Technical Report Series, No. 529, 1973. Chemo- therapy of malaria and resistance to antimalarials: report of a WHO Scientific Group), pp. 30-39. 3. NGUYEN-DINH, P. & TRAGER, W. Plasmodium fakci- parum in vitro: Determination of chloroquine sensi- tivity of three new strains by a modified 48-hour test. American journal oftropical medicine and hygiene, 29: 339-342 (1980). 4. NGUYEN-DINH, P. ET AL. Assessment of chloroquine sensitivity of Plasmodium falciparum in Choluteca, Honduras. Bulletin of the World Health Organization, 59: 641-646 (1981). 5. CAMPBELL, C. C. ET AL. Chloroquine-resistant Plas- modium falciparum from East Africa: Cultivation and drug sensitivity of the Tanzanian I/CDC strain from an American tourist. Lancet, 2: 1151-1154 (1979). 6. FOGH, S. ET AL. 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