Plasmodium falciparum: gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine.

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22-46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed.

Electrophoretic variants of enzymes in isolates of Plasmodium falciparum, P. malariae and P. vivax from Thailand.

A new electrophoretic variant of glucose phosphate isomerase (GPI), which we now denote GPI-3, has been found in isolates of Plasmodium falciparum from 6 patients, all of whom acquired the infection in the same region (in or near Prachinburi province) of Thailand. In other regions, from which 453 isolates have been tested, only GPI-1 and/or GPI-2 have been found. Two isolates of P. malariae from patients at Kanchanaburi showed a band of GPI activity on cellulose acetate gels at a cathodal position quite distinct from that of any previously known GPI variants in other human malaria parasites. Thirty-nine isolates of P. vivax from 3 regions of Thailand have been examined for variants of GPI and lactate dehydrogenase (LDH). Three forms of GPI were found, corresponding approximately in band positions to GPI-1, 2 and 3 of P. falciparum. The position of the band of LDH activity in P. vivax was the same in all the isolates examined, and different from that of LDH-1 in P. falciparum.

Variability in drug susceptibility amongst clones and isolates of Plasmodium falciparum.

Heterogeneity within isolates of Plasmodium falciparum in regard to drug susceptibility is described from studies with three Thai isolates and some clones derived from them. One isolate (T9), which before cloning was resistant in vitro to chloroquine and pyrimethamine, contained a diverse assortment of clones, some of which were sensitive to one or other, or both, of these drugs. Another isolate (CH150) was initially sensitive to mefloquine in vitro, but, on recrudescence of infection in the patient, developed a number of clones all of which had diminished susceptibility to mefloquine. Drug pressure in a laboratory culture of CH150 resulted in a similar change in susceptibility. Hence resistant clones are thought to have been present as a minority component of the original isolate CH150. On testing uncloned isolates at different times after growth in culture, drug susceptibility showed considerable variability, but clones remained stable. Reaction in vitro of these isolates to some other drugs (amodiaquine, Fansidar, quinine) is also described, and the results are discussed in relation to changes in drug resistance of malaria parasites which may occur in laboratory cultures and under field conditions.

Evidence of increased chloroquine sensitivity in Thai isolates of Plasmodium falciparum.

Sensitivity of Thai isolates of Plasmodium falciparum to chloroquine collected over the years 1978-1986 was measured by two methods: (i) by growth of previously cultured isolates for 72 h in presence of drug, and (ii) by the WHO standard in vitro microtest. During this period there were signs of a gradual increase in drug sensitivity, coinciding with the withdrawal of chloroquine for treatment of falciparum malaria in Thailand.

Polymorphism of proteins in malaria parasites following mefloquine treatment.

Proteins in malaria parasites (Plasmodium falciparum) isolated from a patient in Thailand before treatment, and after recrudescence of infection subsequent to mefloquine treatment, were compared by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis. Nine 'pre-treatment' and six 'recrudescent' clones were studied. Variants of the enzyme glucose phosphate isomerase were also noted and mefloquine susceptibility of each clone was measured by in vitro tests. The 'pre-treatment' isolate was found to contain at least four genetically distinct clones, all sensitive to mefloquine, while the 'recrudescent' isolate contained at least two other types of clone, both showing increased tolerance to mefloquine. These two more tolerant types of clone differed from all the sensitive ones studied in regard to several different protein variants as shown by 2D-PAGE analysis. It is concluded that at least two (and probably more) genetically distinct clones of parasites with increased tolerance to mefloquine were present in the parasite population before mefloquine treatment was given, and were selected under mefloquine pressure.

Susceptibility of Thai isolates of Plasmodium falciparum to artemisinine (qinghaosu) and artemether.

Eleven Thai isolates and one West African isolate of Plasmodium falciparum were tested for their susceptibility to the Chinese antimalarial drugs artemisinine (qinghaosu) and artemether. The isolates were cultivated by the Trager-Jensen candle-jar technique and exposed to the action of the drugs for 36-48 hours. Artemisinine inhibited growth of most isolates at 10(-7)-10(-8) mol/litre and artemether at 10(-8) mol/litre (with an initial parasitaemia of 0.5-1.0%). Slight variation in the sensitivity of different isolates was found, but there was no correlation between sensitivity to artemisinine or artemether and sensitivity to pyrimethamine, pyrimethamine/sulfadoxine, or chloroquine. The action of artemisinine and artemether was reduced when the initial parasitaemia of the treated cultures was raised.

Clonal diversity in a single isolate of the malaria parasite Plasmodium falciparum.

Clones of an isolate of Plasmodium falciparum from Mae Sod (Thailand) were prepared by a dilution procedure. Some of the parasite cultures thus obtained have been typed for the following characters: (i) electrophoretic variants of three enzymes; (ii) susceptibility to chloroquine and pyrimethamine; (iii) antigen diversities recognized by ten strain-specific monoclonal antibodies; (iv) presence or absence of knobs on infected erythrocytes and (v) two-dimensional PAGE variants of seven proteins. Amongst the clones there was variation involving each of these five characters. At least seven different types of clones were found in ten cultures produced by dilution. The amount of phenotypic variation within a single isolate has thus been shown to be surprisingly great. Variations in drug susceptibility and antigens are considered to be particularly important in view of their relevance to anti-malarial treatments.

Susceptibility of Plasmodium falciparum to five drugs: an in vitro study of isolates mainly from Thailand.

This paper describes the results of testing the susceptibility of 60 isolates of the human malaria parasite Plasmodium falciparum from Thailand, and single isolates from five other countries, to five drugs: chloroquine, pyrimethamine, quinine, mefloquine and amodiaquine. The Thai isolates were obtained from patients in three different regions of the country (Chantaburi, Songkhla and Mae Sod), and were first grown in culture by the Trager-Jensen candle-jar technique. Samples were then exposed to a range of concentrations of the five drugs, in Falcon microtest culture wells, for 72 hours, with daily changes of medium (with or without added drug solutions). Presence or absence of parasites was then determined by microscope observations on thin-film Giemsa-stained preparations. Most Thai isolates showed a minimum inhibitory concentration (MIC) for chloroquine of 10(-6) M or higher, and were classified as highly resistant, though one cloned isolate was as sensitive to this drug as a chloroquine-sensitive isolate from West Africa. Similarly most Thai isolates showed a very high resistance to pyrimethamine (MIC 10(-4) M to 10(-6) M), but a few clones were sensitive (MIC 10(-9)) to it. Susceptibility to quinine showed some variation (MIC varied between 10(-6) M and 10(-8) M), and some isolates were thought to be incapable of responding to a therapeutically permissible dose of this drug. Little variation was found in the reaction of any of the isolates to mefloquine or amodiaquine, and by the in vitro technique used in this study, it was found that chloroquine-resistant and chloroquine-sensitive isolates were equally susceptible to amodiaquine. In general the survey showed the existence of a marked correlation between development of drug resistance of Plasmodium falciparum and the extent to which a given drug had been used in Thailand.

Enzyme typing of some isolates of Plasmodium falciparum from Thailand.

One hundred and eighty nine isolates of Plasmodium falciparum collected in Thailand, and eleven originating from Cambodia, have been typed by starch-gel electrophoresis of six enzymes (GPI, LDH, GDH, PGD, ADA, PEPE). Substantial polymorphism was found only with GPI. Occasional variants occurred with ADA, while the other four enzymes appeared to be invariant by the tests used. The results are compared with those of similar studies on African isolates, and lead to the provisional conclusion that P. falciparum isolates from different different endemic areas constitute a single, world wide species, containing potentially interbreeding individual organisms.

Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests.

In vitro drug resistance tests of ten isolates of Plasmodium falciparum from three different collection points in Central Thailand have been carried out, and the results compared with those of similar tests with a drug-sensitive West African isolate. Judged by concentration of drug tolerated, the Thai isolates appeared to be about 10 times as resistant to chloroquine, and usually about 10(5) times as resistant to pyrimethamine, as the African isolate. A little variation amongst the Thai isolates was detected.

The genetics of drug resistance in malaria parasites.

The available experimental data on the genetics of drug resistance in malaria parasites are reviewed. Seven possible mechanisms for the origin of drug resistance are considered, and it is pointed out that spontaneous gene mutation is probably the most important. Experiments on the production of pyrimethamine-resistant and chloroquine-resistant strains of rodent Plasmodium species, and on the inheritance of such drug resistance, are reviewed. Relevant biochemical data are also considered in relation to the genetics of drug resistance. Studies on competition between drug-sensitive and drug-resistant parasites in mixed populations of rodent plasmodia are described. The implications of these findings for drug resistance in P. falciparum are discussed.

Alterations in membrane proteins of mouse erythrocytes infected with different species and strains of malaria parasites.

1. The membrane fraction, prepared by hypotonic lysis, of mouse red cells infected with Plasmodium berghei, P. yoelii YM, P. yoelii 17 X, P. yoelii 33 X, P. vinckei or P. chabaudi shows significant alterations from normal in protein composition as observed by dodecylsulphate-polyacrylamide gel electrophoresis. 2. There is a reduction in intensity of various protein bands, notably bands I and II (spectrin), of membranes prepared from infected red cells. 3. New bands are observed as a result of infection, the intensity and location of which depend on the parasite species and strain. A new band of apparent molecular weight 150,000 appears with a strong intensity in P. yoelii YM infection, with a moderate intensity in P. berghei infection, and with a weak intensity in P. vinckei and P. chabaudi infection. In P. yoelii 17X and 33X infection, multiple weak bands are seen in the molecular weight range 120,000-210,000.