Chloroquine-resistant isolates of Plasmodium falciparum with alternative CG2 omega repeat length polymorphisms.

A particular polymorphism in the cg2 gene has previously been linked to chloroquine resistance in reference isolates of Plasmodium falciparum. To assess the association of this polymorphism with chloroquine resistance in field specimens of P. falciparum, we analyzed the omega repeat region of the cg2 gene in 47 isolates of P. falciparum collected in the Ingwavuma District of northern KwaZulu-Natal, South Africa. Polymerase chain reaction (PCR) primers, which were designed to amplify the region of DNA surrounding the omega repeat, were used to obtain omega repeat PCR products from the field isolates. The PCR product for each isolate varied in length, depending on the number of cg2 omega repeats for that isolate. We found that several in vivo and in vitro chloroquine-resistant isolates of P. falciparum did not have the expected 16 omega repeats. These results suggest that the link between the cg2 polymorphism and chloroquine resistance identified previously may not apply in all malarious areas.

Drug-resistant Plasmodium falciparum malaria in the eastern Transvaal.

In March 1993, a study was undertaken in the Komatipoort/Malelane area to monitor the in vitro sensitivity of Plasmodium falciparum to antimalarial drugs currently in use in South Africa. Of the 12 isolates collected, 7 were successfully tested for sensitivity to chloroquine and quinine, 6 for mefloquine susceptibility, and 5 for sensitivity to Fansidar. Four of the isolates were resistant to chloroquine at RIII level, 1 at RII level, and 2 were sensitive. All isolates were found to be sensitive to both quinine and mefloquine. Results suggested possible resistance to Fansidar. These findings have implications for tourists travelling to this area.

In vitro sensitivity of southern African isolates of Plasmodium falciparum to halofantrine.

Twenty southern African isolates of Plasmodium falciparum and a 'control' Gambian strain were tested in vitro for their sensitivity to halofantrine. The concentration required to inhibit 50% of parasite growth, the IC50, ranged from 0.039 to 15.000 nmol/litre, with a mean of 4.619 nmol/litre. These IC50 values were comparable with those obtained in studies carried out in West Africa but were higher than the IC50 of South-East Asian isolates. All 21 isolates examined in the present study had minimum inhibitory concentrations (MIC) of 32 nmol/litre or less, with a median MIC value of 8 nmol/litre. Halofantrine was equally active against chloroquine-sensitive and chloroquine-resistant isolates and was also active against pyrimethamine-resistant strains. Indications are that this drug would be suitable for the treatment of P. falciparum malaria in the southern African region.

Xanthine oxidase inhibits growth of Plasmodium falciparum in human erythrocytes in vitro.

Malaria parasites, unable to synthesize purine de novo, use host-derived hypoxanthine preferentially as purine source. In a previous study (1990. J. Biol. Chem. 265:6562-6568), we noted that xanthine oxidase rapidly and completely depleted hypoxanthine in human erythrocytes, not by crossing the erythrocyte membrane, but rather by creating a concentration gradient which facilitated hypoxanthine efflux. We therefore investigated the ability of xanthine oxidase to inhibit growth of FCR-3, a chloroquine-resistant strain of Plasmodium falciparum in human erythrocytes in vitro. Parasites were cultured in human group O+ erythrocytes in medium supplemented, as required, with xanthine oxidase or chloroquine. Parasite viability was assessed by uptake of radiolabeled glycine and adenosine triphosphate-derived purine into protein and nucleic acid, respectively, by nucleic acid accumulation, by L-lactate production, and by microscopic appearance. On average, a 90% inhibition of growth was observed after 72 h of incubation in 20 mU/ml xanthine oxidase. Inhibition was notably greater than that exerted by 10(-7) M chloroquine (less than 10%) over a comparable period. The IC50 for xanthine oxidase was estimated at 0.2 mU/ml, compared to 1.5 x 10(-7) M for chloroquine. Inhibition was completely reversed by excess hypoxanthine, but was unaffected by oxygen radical scavengers, including superoxide dismutase and catalase. The data confirms that a supply of host-derived hypoxanthine is critical for nucleic acid synthesis in P. falciparum, and that depletion of erythrocyte hypoxanthine pools of chloroquine-resistant malaria infection in humans. of chloroquine-resistant malaria infection in humans.

In vitro sensitivity of southern African reference isolates of Plasmodium falciparum to chloroquine and pyrimethamine.

The in vitro sensitivity to chloroquine and pyrimethamine of 19 culture-adapted southern African reference isolates of Plasmodium falciparum was determined using a 48-hour assay. Four isolates collected in KwaZulu, Natal, were sensitive to chloroquine, and one of these was sensitive to the drug in vivo. Eight isolates from KwaZulu or Mozambique were resistant to chloroquine in vitro. Six of these isolates were chloroquine-resistant in varying degrees in vivo. Four of five isolates from north-eastern Transvaal and two clinically chloroquine-resistant Malawian isolates were resistant to chloroquine in vitro. A wide range of pyrimethamine susceptibilities was detected (0.01 mumol/l to greater than 3.0 mumol/l), although most isolates were inhibited at 0.1 mumol/l, indicating a low level of resistance. These results confirm the presence of both chloroquine and pyrimethamine resistance in the endemic areas of South Africa. This has serious implications for the prophylaxis and treatment of P. falciparum malaria in South Africa.

In vitro sensitivity of southern African reference isolates of Plasmodium falciparum to chloroquine and pyrimethamine

The in vitro sensitivity to chloroquine and pyrimethamine of 19 culture-adapted southern African reference isolates of Plasmodium falciparum was determined using a 48-hour assay. Four isolates collected in KwaZulu, Natal, were sensitive to chloroquine, and one of these was sensitive to the drug in vivo. Eight isolates from KwaZulu or Mozambique were resistant to chloroquine in vitro. Six of these isolates were chloroquine-resistant in varying degrees in vivo. Four of five isolates from north-eastern Transvaal and two clinically chloroquine-resistant Malawian isolates were resistant to chloroquine in vitro. A wide range of pyrimethamine susceptibilities was detected (0.01 mumol/l to greater than 3.0 mumol/l), although most isolates were inhibited at 0.1 mumol/l, indicating a low level of resistance. These results confirm the presence of both chloroquine and pyrimethamine resistance in the endemic areas of South Africa. This has serious implications for the prophylaxis and treatment of P. falciparum malaria in South Africa

Chloroquine-resistant Plasmodium falciparum malaria in the Kavango region of Namibia.

The sensitivity to chloroquine of Plasmodium falciparum from the Kavango region of Namibia was determined by a 24-hour test in vitro. Twenty-six isolates were successfully tested, of which 11 were resistant to a low degree, schizogony being inhibited at 8 pmol/well. The results of the Dill-Glazko test for the presence of 4-aminoquinolines in urine indicate that chloroquine is not widely used in the area.

Enzyme typing of southern African isolates of Plasmodium falciparum by polyacrylamide gel electrophoresis.

Nineteen southern African isolates of Plasmodium falciparum were typed by polyacrylamide gel electrophoresis, using 5 enzymes (glucose phosphate isomerase, adenosine deaminase, lactate dehydrogenase, NADP-dependent glutamate dehydrogenase and 6-phosphogluconate dehydrogenase). Limited variation was found amongst the isolates and the frequencies of variants were similar to those of isolates from other parts of the world. Eight of the isolates contained 2 forms of glucose phosphate isomerase, indicating clonal heterogeneity. One of these 8 isolates also contained 2 forms of adenosine deaminase and another showed 2 forms of lactate dehydrogenase.

Typing of southern African isolates of Plasmodium falciparum using monoclonal antibodies.

Antigenic diversity among 19 southern African isolates of Plasmodium falciparum was demonstrated using a panel of 9 monoclonal antibodies. Parasites obtained from single patients were heterogeneous. The antigen composition of 9 isolates was not stable with time in culture, particularly not with respect to 4 of the monoclonal antibodies. By the end of the investigation, 70% of isolates displayed an identical antigen pattern which was markedly different to any obtained in other parts of the world. Differences may be due to geographic origin of parasites or to variation in culture conditions.

In vitro confirmation of chloroquine-resistant Plasmodium falciparum malaria in KwaZulu.

In May 1987 and January 1988 the chloroquine sensitivity of Plasmodium falciparum in the Ubombo and Ingwavuma districts of KwaZulu was determined by a modified in vitro microtest in which the patients' plasma was replaced with non-immune human AB serum and the test plates were incubated in an atmosphere of 3% oxygen, 4% carbon dioxide and 93% nitrogen. A success rate of 74% was achieved using this technique. All of 23 successfully tested isolates from malaria patients reporting to clinics and a hospital in these areas were found to be resistant to chloroquine, schizogony being inhibited at 32 pmol per well in the majority of tests. Seventy-five per cent of the isolates obtained through active surveillance in the Ubombo district were found to be resistant in varying degrees. Malarial parasites collected from clinics and a hospital in the endemic area did not change markedly in their in vitro response to chloroquine during the 8-month period May 1987-January 1988.

In vitro cultivation of southern African strains of Plasmodium falciparum and gametocytogenesis.

Sixteen southern African isolates of Plasmodium falciparum were successfully established and maintained in gassed tissue culture flasks which were either held in a static position or shaken. Shaken cultures yielded 48-hour growth rates ranging from 3.9-fold to 9.5-fold and peak parasitaemias ranging from 9% to 27%. Growth rates in static flasks were lower, ranging from 1.8-fold to 4.2-fold every 48 hours with peak parasitaemias ranging from 4% to 12%. Four of the established isolates were treated so as to promote gametocytogenesis and the rate of gametocyte production was found to be high. The mean percentage gametocytes by day 20-25 was 4.2% of erythrocytes, which represented approximately 50% of total parasites.