Homologues of the mammalian multidrug resistance gene have been identified in isolates and clones of Plasmodium falciparum and designated pfmdr1 and pfmdr2. Mutations in pfmdr1 have been associated with chloroquine resistance but confirmation could not be obtained in a genetic cross. We have examined the copy number and expression of pfmdr1 and pfmdr2 in chloroquine-sensitive and -resistant P. falciparum and have found no relationship between the copy number of either gene and chloroquine resistance. However, a marked correlation was seen between levels of mRNA transcribed for each gene and chloroquine resistance. Two transcripts of pfmdr1 were detected, and in the asexual blood cycle an 8 kb transcript appeared first, followed by the appearance of a 7 kb species.

In order to visualize low-pH compartments of Plasmodium berghei strains we have used a basic congener of dinitrophenol, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) which concentrates in acidic compartments, and can be detected by immunocytochemistry with anti-dinitrophenol antibodies. We have demonstrated that in a P. berghei chloroquine-sensitive strain (N strain), DAMP accumulates in the endocytic vacuoles where haemoglobin degradation is occurring. These compartments which have recently been shown to concentrate 4-aminoquinoline drugs (Moreau, Prensier, Maalla & Fortier, 1986) have an acidic pH. Conversely DAMP was found scattered all over the cytoplasm in a P. berghei chloroquine-resistant strain; the same phenomenon was previously observed (Moreau et al. 1986) in the localization of a 4-aminoquinoline on this same strain. Monensin-induced swelling of acidic compartments (Boss & Morre, 1984) was used as a complementary method for the determination of low-pH compartments on P. berghei strains. All the data reported here suggest that chloroquine resistance in P. berghei RC may be related to an impairment in the acidification of endocytic vesicles.

Subtherapeutic doses of chloroquine have been reported to enhance infectivity of drug-resistant Plasmodium species to their vectors. In this investigation, Plasmodium yoelii nigeriensis N67 strain showed enhanced infectivity to mosquitoes when stimulated by chloroquine. Both sensitive and resistant clones were derived from the N67 strain by dilution, showing that this strain is polymorphic for the resistant trait. A resistant subline was derived by selection under drug pressure from a chloroquine-sensitive clone, but neither the sensitive nor the resistant clones or sublines showed enhancement of infectivity in the presence of chloroquine. This suggests that the enhancement of infectivity shown by the N67 strain is a response to chloroquine stimulation shown only by certain of the genotypes within it, and that this response to chloroquine is not a trait causally connected with the genes coding for chloroquine resistance.

Mutations in pfcrt K76T are associated with chloroquine resistance in Plasmodium falciparum. Previous studies of K76T mutations in Senegal reported the association of T76 with in vitro-resistant isolates, but this mutation was also prevalent in chloroquine-sensitive isolates. This suggests involvement of additional genetic loci in modulating chloroquine resistance. Additional pfcrt polymorphisms at codons A220S, Q271E, N326S and R371I have been found in chloroquine-resistant isolates. We wanted to test if sequential acquisition of mutations at these codons leads to in vitro chloroquine resistance. Stepwise accumulation of mutations was not detected, rather there was almost complete linkage between the pfcrt K76T mutation and polymorphisms in these codons. Therefore these additional polymorphisms do not enhance the correlation between pfcrt T76 and chloroquine resistance in Senegal. These data suggest that in vitro chloroquine resistance requires the genetic background of the pfcrt K76T mutation and additional mutations in genetic loci outside the pfcrt gene.

In view of the recent discovery (Molecular Cell6, 861–871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology119, 343–348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine.

The in vitro activity of artemether against 63 African isolates of Plasmodium falciparum from Libreville, Gabon was evaluated using an isotopic drug susceptibility semi-microtest. The 50% inhibitory concentration (IC50) values for artemether were in a narrow range from 0·8 to 34·8 nm (mean IC50 5·0 nm) and the 95% confidence interval (CI95%) was 3·6–6·3 nm. In vitro decreased susceptibility or resistance were observed with artemether (14%), to chloroquine (90%), to quinine (32%). Isolate susceptibility to amodiaquine and halofantrine was high i.e. 100% and 98%, respectively. There was a significant positive correlation between responses to artemether and amodiaquine (r2=0·45, P<0·001), artemether and chloroquine (r2=0·36, P<0·001), artemether and quinine (r2=0·31, P<0·001), and artemether and halofantrine r2=0·19, P<0·01). Positive correlation between these drugs suggests in vitro cross-resistance or at least common features in drug uptake and/or mode of action or resistance.

Plasmodium falciparum resistance to chloroquine has been described in many parts of the world particularly in Africa where malaria is endemic. High levels of chloroquine resistance in our study area, Lambarene–Gabon, has led to the use of an alternative regimen for treatment and prevention of P. falciparum infection. In this study, we examined the in vitro chloroquine sensitivity of 15 isolates from this area and assessed the prevalence of a putative chloroquine resistance associated Pfmdr1 polymorphism (Asn86Tyr) using a novel allele-specific polymerase chain reaction (PCR). Only 4 of the isolates examined were chloroquine sensitive. The allele-specific PCR shows that all 15 isolates carried the variant (86Tyr) codon. Eleven of these were resistant to chloroquine suggesting a 73% agreement between chloroquine resistance phenotype and the point mutation. This molecular marker was examined in a further 73 Gabonese isolates, where 58 (79·5%) showed 86Tyr and 15 (20·5%) showed 86Asn. In all, 4 (4·5%) of the 88 isolates assessed carry both mutant and wild-type codons, suggesting mixed parasite populations. The incomplete agreement found between chloroquine resistance phenotype and Pfmdr1 (86Tyr) polymorphism would support the view that other genetic factors as well as Pfmdr1 may be involved in chloroquine resistance. While our results suggest a high prevalence of 86Tyr polymorphism in Lambarene, the Asp1246Tyr polymorphism (a point mutation which to date has only been associated with South American P. falciparum) seems to be absent in our study area.