Intercontinental Spread of Pyrimethamine-Resistant Malaria

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Science  20 Aug 2004:
Vol. 305, Issue 5687, pp. 1124
DOI: 10.1126/science.1098876


Here we present molecular evidence demonstrating that malaria parasites bearing high-level pyrimethamine resistance originally arrived in Africa from southeast Asia. The resistance alleles carried by these migrants are now spreading across Africa at an alarming rate, signaling the end of affordable malaria treatment and presenting sub-Saharan Africa with a public health crisis.

Here we present molecular evidence demonstrating that malaria parasites bearing high-level pyrimethamine resistance originally arrived in Africa from southeast Asia. Chloroquine (CQ) is being replaced by sulfadoxine pyrimethamine (SP) for treatment of Plasmodium falciparum malaria in Africa. Mutations in the dihydrofolate reductase (dhfr) gene of P. falciparum underlie resistance to pyrimethamine. Dhfr alleles with one (108N) or two (108N plus 51I or 108N plus 59R) mutations result in increased parasite clearance times. Infections bearing triple-mutant dhfr (108N, 51I, and 59R) have high treatment failure rates, and quadruple-mutant dhfr alleles (108N, 51I, 59R, and 164L) render parasites untreatable (1). Parasites with one to three mutations occur in Africa, whereas all four mutations are common in southeast Asia (2) and South America (3). Triple-mutant alleles are replacing other alleles in Africa (4).

It is generally assumed that pyrimethamine resistance has evolved multiple times (1), because it is selectable in the laboratory, has a simple genetic basis, and appears rapidly after SP introduction. However, analysis of microsatellites that flank dhfr in African parasites sampled from sites 4000 km apart demonstrate just three independent origins of double mutants and a single origin of the triple mutant (4). Similarly, dhfr alleles with two to four mutations have a single evolutionary origin across five southeast Asian countries (2).

Genotyping of eight microsatellite markers flanking dhfr in both southeast Asian and African parasites shows that the triple-mutant dhfr allele in Africa shares a common origin with dhfr alleles bearing two to four mutations in southeast Asia (Fig. 1). The predominant five-locus microsatellite haplotype (–10 kb to +0.5 kb) associated with triple-mutant dhfr in Africa is identical to that associated with dhfr alleles carrying two to four mutations in southeast Asia. In contrast, these five loci show high levels of variation (mean expected heterozygosity = 0.76) around sensitive dhfr alleles. Markers situated further from dhfr (–20 kb and >+6 kb) show higher polymorphism on chromosomes that carry resistant dhfr alleles, as expected, in a selective sweep (2, 5). However, the predominant alleles are the same on resistant chromosomes from both continents. In contrast, African double-mutant dhfr alleles have dissimilar flanking alleles, indicating independent origins (4). Because all southeast Asian dhfr alleles carrying >1 mutation have a single origin, the simplest explanation is that triple-mutant dhfr alleles spreading in Africa originated in southeast Asia.

Fig. 1.

dhfr alleles and flanking microsatellites of parasites from Africa and Thailand. The figure comprises data from 12 Thai parasites with two to four resistance mutations, 24 African parasites with triple-mutant alleles, and 18 African parasites with sensitive dhfr alleles. The four-letter codes describe amino acids present at positions 51, 59, 108, and 164 in the predicted dhfr protein (10). Amino acids conferring resistance are underlined, and dhfr alleles are shaded yellow, orange, red, and black in order of increasing resistance. Sensitive alleles are shaded turquoise. Allele lengths are shown for eight microsatellites positioned at –0.1, –4.4, –5.3, –10, and –20 kb upstream and +0.5, +6, and +10 kb downstream of dhfr. Dots and yellow shading indicate identical allele size to the predominant resistant haplotype (shown at right).

Alleles at the major CQ-resistance locus pfcrt also have a common origin in African and Asian parasites (5, 6). CQ-resistant pfcrt alleles and triple-mutant dhfr alleles may have arrived in Africa in the same parasite genome. Pyrimethamine resistance was widespread in Asia when CQ resistance was first recorded in Africa (7). Import of southeast Asian parasites has thus led to the demise of the two affordable drugs that have been the mainstay of malaria treatment in Africa.

Why did the triple-mutant allele not arise independently in Africa? Assuming a mutation rate of 10–9 per base per generation, we would expect 10 to 1000 independent origins of triple-mutant parasites in every infection (1010–12 parasites) containing double-mutant dhfr alleles. The implication is that complex compensatory mutations are required to restore parasite fitness.

Every year 30,000 malaria cases are imported into industrialized countries (8). The numbers of cases imported into Africa is unknown but likely to be substantial. Given that 67% of parasites sampled in Thailand, Cambodia, and Myanmar carry the 164L mutation in dhfr (2), as well as high levels of mefloquine and quinine resistance, it is only a matter of time before these invade and establish in Africa. We suggest that careful thought should be given to preventing further import of resistant parasites, perhaps by screening and treatment of passengers traveling from southeast Asia or South America to Africa. Widespread introduction of artemisinin-based combination therapy (9) could also help to minimize the foci from which resistant parasites can spread. Importantly, these data demonstrate that antimalarial drug resistance is an international problem requiring a coordinated international response.

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