• 1.

    Delacollette C., Embonga B., Malengreau M. , 1983. Response to chloroquine of infections with Plasmodium falciparum in the Kivu region of Zaire. Preliminary observations. Ann. Soc. Belge Med. Trop., 63: 171173.

    • Search Google Scholar
    • Export Citation
  • 2.

    Nguyen-Dinh P., Schwartz I. K., Sexton J. D., Bomboto E., Botomwito B., Kalisa R., Ngimbi N. P., Wery M. , 1985. In vivo and in vitro susceptibility to chloroquine of Plasmodium falciparum in Kinshasa and Mbuji-Mayi, Zaire. Bull. W.H.O., 63: 325330.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ngimbi N. P., Wery M., Henry M. C., Mulumba M. P. , 1985. Response in vivo à la chloroquine au cours du traitement du paludisme a Plasmodium falciparum en region suburbaine de Kinshasa, Zaire. Ann. Soc. Belge Med. Trop., 65 (Suppl. 2): 123135.

    • Search Google Scholar
    • Export Citation
  • 4.

    Nguyen-Dinh P., Spencer H. C., Chemangey-Masaba S., Churchill F. C. , 1982. Susceptibility of Plasmodium falciparum to pyrimethamine and sulfadoxine-pyrimethamine in Kisumu, Kenya. Lancet, 1: 823825.

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    • Export Citation

 

 

 

Plasmodium Falciparumin Kinshasa, Zaire: In Vitro Drug Susceptibility Studies

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  • 1 * Malaria Branch, Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333
  • 2 † DepartMent of Pediatrics, Marna Yerno Hospital, Kinshasa, Zaire
  • 3 ‡ Institut National de Recherche Biomedicale, Kinshasa, Zaire
  • 4 § Ministry of Public Health and Social Affairs, Kinshasa, Zaire

In June 1986, Plasmodium falciparum parasites were collected from 33 children presenting at the Mama Yemo Hospital in Kinshasa (Zaire) and were successfully tested in vitro by a 48-hr reinvasion test for their susceptibility to various antimalarial drugs. In vitro resistance to chloroquine was found in 82% of the isolates, a marked increase over findings obtained by the same technique 3 years ago in Kinshasa. In vitro chloroquine resistance was not associated with a history of previous drug intake. The inhibitory endpoints for quinine varied from 0.03 to 1 µM, and correlated with the chloroquine endpoints in the corresponding isolates (r = 0.64). Pyrimethamine resistance in vitro was demonstrated in 52% of the isolates tested.

During the past 5 years chloroquine-resistant Plasmodium falciparum infections have been noted with increasing frequency in Zaire. The first cases were demonstrated in vivo in the eastem part of the country, in 1982.1 In 1983, combined in vivo and in vitro studies conducted in Kinshasa (western Zaire) and in Mbuji-Mayi (central Zaire) failed to detect resistance. 2 Since 1984 the occurrence of chloroquine resistance in Kinshasa has been demonstrated both in vivo (J. G. Breman, CDC, personal communication) and in vitro.3 To determine the extent of in vitro choroquine resistance in relation to in vitro response to other antimalarial drugs, we conducted drug resistance tests in Kinshasa in which we evaluated the response of P. falciparum to chloroquine, quinine, and pyrimethamine.

Materials and Methods

Patients

The patients were recruited among children presenting in June 1986 to the Pediatric Emergency Ward of Mama Yemo Hospital, the major health facility of Kinshasa. Criteria for admission to the study included symptoms suggestive of malaria, and infection with P. falciparum only, with at least 0.1% of the erythrocytes infected by asexual blood stage parasites, as demonstrated microscopically by Giemsa-stained thick and thin smears. While the accompanying adult was questioned about the child’s previous intake of antimalarial drugs in the last 2 weeks, such intake did not preclude admission to the study.

In vitro tests

Parasitized venous blood samples collected from all patients and stored in heparin for a maximum of 6 hr were used to perform in vitro drug susceptibility tests. A previously described 48- hr in vitro reinvasion test was used, with minor modifications.2, 4 In this test, the 48-hr period covers the entire cycle of asexual multiplication of P. falciparum, and allows the measurement of the effect of chloroquine and quinine (which prevent the formation of schizonts) as well as that of pyrimethamine (which acts mainly on the late trophozoite and schizont stages). Briefly, the parasitized blood was diluted 20-fold in culture medium consisting of RPMI 1640 supplemented with 30 mM HEPES buffer and 10% AB+ nonimmune human serum. In blood samples with high parasite densities (> 3%), the patients’ erythrocytes were diluted with uninfected washed human erythrocytes (group O+) to decrease the parasite density to more appropriate starting levels prior to dilution in culture medium. This culture material, where the erythrocytes had been resuspended at a hematocrit of approximately 2.5%, was distributed as 100 µl aliquots in microliter wells predosed with either no drugs (controls) or various concentrations of chloroquine, quinine, and pyrimethamine. The plates were incubated for 48 hr at 37°C in a candle jar, after which thin smears were made from the content of each well, to ascertain parasite multiplication over the 48-hr period. The drug concentration at which no parasite multiplication occurred (the inhibitory endpoint) was used to determine the in vitro susceptibility of the parasite to the drug tested.

Table 1

In vitro response to chloroquine, pyrimethamine, and quinine of 33 Plasmodium falciparum isolates, Kinshasa, Zaire, 1986

Drug concentrations (µM )% of isolates tnhibued by:
ChloroquinePyrimethamineQuinine
0000
0.003NT*3NT
0.01048NT
0.0318483
0.0618NTNT
0.1304830
0.3914873
110057100
310097100
10NTNT100
30NTNT100

NT = not a test concentration.

Results

Patients

A total of 38 children participated in the study, whose ages ranged from 1 to 12 years (mean 5.0 years). Their parasitemias ranged from 0.3% to 39% of the erythrocytes infected (geometric mean 3.3%). Twelve patients (32%) presented with severe malaria: 3 children had cerebral malaria and 9 children had a parasite density > 10%. Twenty-one children (55%) were reported to have taken known antimalarial drugs during the 2 weeks before coming to the hospital, mostly chloroquine (18 children) and quinine (I child), or a combination of both (2 children).

In vitro tests

In 33 (87%) of the 38 tests, the parasites in the control wells multiplied at least two-fold, allowing interpretable results (Table 1). The initial parasitemias of the in vitro tests varied between 0.2% and 2.9%. (In 22 patients with high parasite densities, the patients' erythrocytes were diluted with uninfected erythrocytes.)

In the chloroquine tests, a concentration of 0.06 µM of the drug, previously shown to suppress multiplication of chloroquine-sensitive parasites,2 inhibited only 18% of the isolates successfully tested. The majority (82%) of the isolates were thus chloroquine-resistant in vitro. Of the 14 isolates from patients with no history of previous antimalarial drug intake, 10 (71%) were chloroquine-resistant in vitro.

In the quinine tests, the inhibitory endpoints varied between 0.03 and 1 µM, and correlated with the chloroquine endpoints observed among the corresponding isolates (r = 0.64, P < 0.01).

In the pyrimethamine tests, 48% of the isolates were sensitive to the drug in vitro, being inhibited by 0.03 µM,4 whereas the remainder were resistant, inhibition occurring only at concentrations up to 3 µM. No association was found between resistance in vitro to chloroquine and pyrimethamine.

Discussion

These studies in patients presenting to the Mama Yemo Hospital indicate a high prevalence of in vitro resistance to both chloroquine and pyrimethamine. The chloroquine results contrast sharply with those obtained with the same test in 1983, where uniform in vitro chloroquine sensitivity was found in all 22 P. falciparum isolates collected from asymptomatic schoolchildren in Kinshasa.2

While the Mama Yemo Hospital patients, frequently presenting after an initial failure of self-medication, might represent a population selected for a higher proportion of drug-resistant isolates, 71% of the isolates from patients with no history of previous antimalarial drug intake were in vitro chloroquine-resistant, and no association was found between history of previous drug intake and in vitro chloroquine resistance (Fisher’s exact test, P = 0.2).

The in vitro pyrimethamine results, indicating a high prevalence of resistance, might be attributed to the use of pyrimethamine in some malaria prophylaxis programs in Zaire. Whether the presence of such pyrimethamine-resistant parasites wiU result in an earlier appearance of resistance to sulfadoxine-pyrimethamine, following the widespread introduction of this drug combination, will have to be determined.

No conclusions on the in vivo efficacy of quinine can be drawn from the corresponding in vitro results, since correlations between in vivo and in vitro quinine responses are not yet well established. Studies to evaluate such correlations are urgently needed, since quinine will play an increasingly important role in the treatment of P. falciparum malaria in areas of chloroquine resistance.

ACKNOWLEDGMENTS

We thank Jonathan M. Mann, Bongo Lyamba, and Jean-Jacques Salaun for their support and advice, the laboratory staff of the Projet SIDA (Ministry of Health and Social Affairs, Kinshasa), and the nursing staff of the Mama Yemo Hospital for technical assistance.

  • 1.

    Delacollette C., Embonga B., Malengreau M. , 1983. Response to chloroquine of infections with Plasmodium falciparum in the Kivu region of Zaire. Preliminary observations. Ann. Soc. Belge Med. Trop., 63: 171173.

    • Search Google Scholar
    • Export Citation
  • 2.

    Nguyen-Dinh P., Schwartz I. K., Sexton J. D., Bomboto E., Botomwito B., Kalisa R., Ngimbi N. P., Wery M. , 1985. In vivo and in vitro susceptibility to chloroquine of Plasmodium falciparum in Kinshasa and Mbuji-Mayi, Zaire. Bull. W.H.O., 63: 325330.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ngimbi N. P., Wery M., Henry M. C., Mulumba M. P. , 1985. Response in vivo à la chloroquine au cours du traitement du paludisme a Plasmodium falciparum en region suburbaine de Kinshasa, Zaire. Ann. Soc. Belge Med. Trop., 65 (Suppl. 2): 123135.

    • Search Google Scholar
    • Export Citation
  • 4.

    Nguyen-Dinh P., Spencer H. C., Chemangey-Masaba S., Churchill F. C. , 1982. Susceptibility of Plasmodium falciparum to pyrimethamine and sulfadoxine-pyrimethamine in Kisumu, Kenya. Lancet, 1: 823825.

    • Search Google Scholar
    • Export Citation
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