Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites

Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC₅₀ in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered.The World Health Organization (WHO) campaign to eradicate malaria in the 1950s and 1960s was initially largely successful in decreasing the burden of malaria. Drug failure eventually led to a resurgence in the number of malaria cases in the 1990s and caused vast numbers of deaths that could have been avoided through a better appreciation of the prevalence of drug-resistant malaria and more informed choices of first-line drugs (Greenwood et al. 2008). While malaria deaths are now likely to decline, primarily because of the introduction of artemisinin-based combination therapy (ACT) as well as increased insecticide spraying and the use of bed nets (Greenwood et al. 2008), this may only be temporary. Indeed, there has been a general increase in the parasite clearance times in ACT-treated Plasmodium falciparum malaria cases from near the Thai–Cambodian border, suggesting that case numbers may soon begin increasing (Dondorp et al. 2009).Remarkably, although artemisinin is used on tens of millions of individuals annually, we have little idea about how it acts or which genes contribute to resistance, confounding the community''s ability to monitor the spread of resistance using molecular markers and to deploy new therapies (Eastman and Fidock 2009). The fact that the genes involved in artemisinin resistance are not known is due to a variety of problems, including the fact that in vivo resistance has not been replicated in vitro (Dondorp et al. 2009). Additionally, the association of phenotypes with genotypes in P. falciparum is hampered by the difficulties in performing complementation studies due to extremely low transfection efficiencies and the fact that laboratory crosses of drug-sensitive and drug-resistant P. falciparum cannot be easily performed. Thus, it took more than 40 yr between the identification of chloroquine resistance in the field (Harinasuta et al. 1965) and confirmation that resistance is due to mutations in the chloroquine resistance transporter (pfcrt, MAL7P1.27) (Wellems et al. 1990; Fidock et al. 2000; Sidhu et al. 2002). While the recombinant progeny from one of the three extant crosses (Walliker et al. 1987; Wellems et al. 1990; Hayton et al. 2008) have most famously been used to map chloroquine resistance (Wellems et al. 1990), they have been used to map loci contributing to a wide variety of phenotypes that distinguish parental clones. For example, they have already been scored for a variety of different phenotypes that are related to drug sensitivity, including antifolate sensitivity (Wang et al. 2004b), quinine sensitivity (Ferdig et al. 2004), expression levels (Gonzales et al. 2008), and plasmodial surface ion channels (Alkhalil et al. 2009), but they could be scored for any phenotype that quantitatively distinguishes the parental strains Dd2 and HB3, such as propensity to mutate in the laboratory (Rathod et al. 1997). However, there are a limited number of phenotypes that distinguish these two laboratory strains that were derived from patients 40 yr ago. While more crosses would provide valuable data for many researchers interested in parasite genetics, there are ethical considerations associated with using primates in research. An alternative to creating new recombinant progeny is to find existing recombinant isolates that arose from recent meioses occurring in humans. Such parasites might be identified by performing full-genome analyses on parasites from a limited geographical area and could provide the malaria community with an unprecedented number of parasites differing in a variety of phenotypes for use in linkage or quantitative trait locus (QTL) studies.One attractive group of parasites for full genome investigation is from the Peruvian Amazon. Due to the low transmission rates it is expected that parasites isolated from an individual will be from a single clone infection. In addition, malaria was eradicated in the 1960s in this region but re-emerged with epidemics in the early 1990s (Aramburu Guarda et al. 1999; Branch et al. 2005), suggesting that the genomes might contain signatures of selective sweeps (Wootton et al. 2002; Roper et al. 2004). At first, malaria in this region was treated with chloroquine (first-line), sulfadoxine-pyrimethamine (second-line), and quinine with clindamycin or tetracycline (third-line) (Aramburu Guarda et al. 1999), but the emergence of resistance resulted in widespread chloroquine and antifolate treatment failure (Durand et al. 2007). Today, malaria remains hypoendemic with low levels of seasonal transmission of P. falciparum and P. vivax parasites in the region surrounding Iquitos, Peru (Roshanravan et al. 2003). Previous studies of parasites in the region describe only one or two independent haplotypes for important drug-resistance genes, suggesting a limited number of founders for this population (Bacon et al. 2009) and suggest that we might find recombinant progeny in this region.In this study, we performed genome scanning on 14 P. falciparum patient isolates from a limited geographical region. We show that the parasite population in the Peruvian Amazon is very closely related, with combinations of only two to four different genotypes for drug resistance genes, suggesting at most four parental haplotypes. Furthermore, some parasites taken from different patients who presented with symptoms were essentially clones of one another, while others were recent meiotic siblings that could be useful in linkage studies or eQTL analyses. Unexpectedly, genome scanning also revealed uncharacterized mutations in the apicoplast 23S rRNA that distinguished some Peruvian strains from the reference strain, 3D7. Because one of these mutations had been previously associated with lincosamide antibiotic resistance in the chloroplast and many bacterial species (Vester and Douthwaite 2001), sensitivity testing was performed revealing that the parasites harboring the mutation had indeed become resistant to clindamycin, a drug used in combination with quinine to treat pregnant women and infants for malaria in Peru. These are the first documented cases of resistance to this class of drugs in malaria and suggest that the use of lincosamide drugs in treating malaria should be reconsidered.