Origin and Dissemination of Plasmodium falciparum Drug-Resistance Mutations in South America
- 1Malaria Section, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore;
- 2Malariology Department, School of Public Health, Ministry of Health, and
- 3Immunology Institute, School of Medicine, Central University, Caracas, Venezuela
- Reprints or correspondence: Dr. Christopher V. Plowe, Malaria Section, Center for Vaccine Development, University of Maryland School of Medicine, 685 W. Baltimore St., HSF 480, Baltimore, MD 21201 (cplowe{at}medicine.umaryland.edu)
Abstract
Multidrug resistance is a major obstacle to the control of Plasmodium falciparum malaria, and its origins and modes of dissemination are imperfectly understood. In this study, haplotyping and microsatellite analysis of malaria from 5 regions of the South American Amazon support the conclusion that the parasite mutations conferring mid- and high-level resistance to the antifolate combination sulfadoxine-pyrimethamine have a common origin. Parasites harboring these mutations are also found to share drug-resistance alleles that confer a unique chloroquine resistance phenotype and to be similar at loci not linked to drug resistance, although not genetically identical. Since the 1980s, multidrug-resistant P. falciparum has spread in a north-northwest manner across the continent, from an origin likely in the lower Amazon. This study highlights the importance of continent-wide malaria-control policies and suggests that the containment of resistance to the next generation of therapies may be feasible
South America has experienced a resurgence of malaria in recent decades, coincident with both the reduction in household insecticide spraying since the 1960s [1] and the increase in parasite drug resistance [2]. Although Plasmodium vivax is the predominant pathogen causing human malaria in South and Central America and the Caribbean, the deadlier P. falciparum is also present in most regions in which malaria is endemic. Increasing rates of resistance to chloroquine and the antifolate combination sulfadoxine-pyrimethamine (SP) have largely removed these safe and affordable drugs from South America's antimalarial repertoire. Nonetheless, there is value in understanding the evolution and dissemination of resistance to these agents, if the lessons learned can be applied to forestall their failure in Africa or to extend the life of the next generation of therapies in South America
Both chloroquine and pyrimethamine were implemented in South America soon after their validation as antimalarial agents [3]. In the 1950s, during the global malaria-eradication campaign, they were exploited as malaria prophylactics, either by administration in weekly or monthly dosing regimens, as practiced by Gabaldon in Venezuela [4], or by incorporation in dietary salt, as conceived by Pinotti in Brazil [5]. Countries throughout the southern hemisphere adopted such regimens, but the campaigns were soon aborted. Mass dosing did not radically cure P. vivax infections [6], and, by the early 1960s, chloroquine-resistant cases of falciparum malaria began to be reported, beginning in areas where prophylaxis had been attempted in the previous decade
Chloroquine resistance in P. falciparum was first documented in 1960 in Venezuela [7] and soon afterwards in Colombia [8] and Brazil [9]. Later in the decade, resistance was reported in areas where the drug had not been mass-administered [10]. By the mid-1980s, chloroquine-treatment failure was widespread in the Amazon [11]. In the 1970s, pyrimethamine was formulated with sulfadoxine and reintroduced as SP, in part to counter chloroquine resistance [12, 13]. Pyrimethamine failure was not noted after the early mass-administration trials in Venezuela [6], but in vitro antifolate resistance was demonstrated in malaria strains from Brazil in the mid-1960s [14] and Venezuela in the 1970s [15]. Where reported, SP remained effective in treating chloroquine-resistant infections until the 1980s [16]. In 1981, low-level (RI [17]) in vivo SP resistance was identified in the Colombian Amazon [10]. By the mid-1980s, areas of Colombia and Brazil exhibited SP resistance failure rates >25% and >60%, respectively [10]. High-level (RIII) SP resistance was first reported in Brazil in 1982 [18]. When more South American countries replaced chloroquine with SP as the first-line antimalarial agent, including Peru as late as 1995 [19], SP resistance already had been documented in their locales, and it spread rapidly under the ensuing drug pressure. By the 1990s, SP was no more effective than chloroquine in much of South America, and mefloquine, tetracycline, and quinine were moved to the forefront, with the artemisinine derivatives reserved for complicated disease [2]
The molecular basis of P. falciparum antifolate resistance has been reviewed elsewhere [20]. An array of point mutations conferring amino acid alterations in the parasite's dihydrofolate reductase (DHFR), the target of pyrimethamine, and dihydropterate synthase (DHPS), where sulfonamides act, are found in antifolate-resistant P. falciparum. In the field, the mutations arise under antifolate pressure in a stepwise fashion, with successive mutations conferring higher levels of resistance [21]. The first dhfr mutation to arise is invariably S108N, which alone is insufficient to consistently confer in vivo resistance to pyrimethamine or SP. Two subsequent mutations in S108N dhfr N51I and C59R, arise singly and in combination as drug pressure is sustained and increase the level of pyrimethamine resistance to the threshold of in vivo failure. C50R is found in association with N51I and S108N in South America and confers midlevel resistance. A fifth mutation, I164L, in association with S108N and mutations at codon 51 and/or codon 59, confers levels of in vitro and in vivo resistance that mark the end of the useful life of SP. A 5-aa insertion after codon 30, termed the “Bolivia repeat” (BR), is an apparently silent polymorphism identified only in South America. A parallel array of mutations in dhps roughly follows the progression of dhfr polymorphisms under SP pressure, with a dhps A437G mutation appearing after the dhfr S108N mutation and K540E and A581G arising in conjunction with the subsequent dhfr mutations
The geographic prevalence of the mutations reflects both the duration of antifolate use and the level of therapeutic resistance. However, the modes by which antifolate resistance develops have not been elucidated. For example, it remains unclear whether the resistance mutations repeatedly arise de novo or spread by dissemination after rare or infrequent mutation events. This question would be difficult to address in sub-Saharan Africa, with its high vector-transmission intensity and parasite recombination rates. In this study, we have explored the evolution and dissemination of antifolate resistance in South America, where transmission intensity is low and parasite propagation frequently clonal [22]. Our findings reflect on the spread not only of antifolate but also of chloroquine resistance and have implications for new antimalarial regimens, as well as for malaria-control policies in South America
Materials and Methods
P. falciparum isolates and published lines.P. falciparum isolates and published parasite lines were from the following sources: for Colombia, isolates from pretreatment blood samples collected on filter papers during a 1999 SP efficacy study in Bueneventura [23]; for Venezuela, isolates from diagnostic smears on microscope slides collected in 1998–2000 from Bolivar State health clinics [24] and the VEN line established in 1987; for Brazil, isolates from blood samples collected on filter papers from the southern Amazon in 1996–1998 [25] and Brazilian lines PAD, DIV30, ICS, 7G8, and ECP; for Peru, isolates from pretreatment blood samples collected on filter papers from a 1997 SP efficacy study in Iquitos [26]; for Bolivia, isolates from blood samples collected on filter papers from northern Bolivia in 1994 [21]; for Haiti, a parasite line established from a 1997 isolate and 2 additional isolates; and for Honduras and Ecuador, the HB3 and Ecu1110 lines
Microsatellite and mutation analysisDNA was extracted from infected blood dried on filter papers or microscope slides, as described on our Web site (http://medschool.umaryland.edu/CVD/2002_pcr_asra.htm), and nested polymerase chain reactions (PCRs) were performed to amplify the dhfr 5′ untranslated region (UTR) and the first pppk-dhps intron. Amplicons were cloned at least twice into the pCR 2.1 vector (Invitrogen) and sequenced by standard methods. Cloning efficiency of the dhfr 5′ UTR was low, likely consequent to the repeat sequences. In 3 instances, PCR/cloning-induced alterations in the repeats were observed, and 2 assays on a single sample yielded repeats differing by 2–4 nt. In these cases, the majority result of 3 assays was used. Polymorphisms or artifacts other than the repeats under study are noted in tables 1, 2, and 3 but were not evaluated. Primary primers for the dhfr 5′ UTR were 5′-GAATAGATACATTTATTTATGAATC-3′ and 5′-TAGATACATTTATTTATGAATCTG-3′; secondary primers were 5′-TTTAAAAGGTTATATTAAGGGGAT-3′ and 5′-CATATGGCATAAATATCGAA-3′. Primary primers for the pppk-dhps intron were 5′-GGGAATGATAGAAGAAACGCTGT-3′ and 5′-GGGCAAGTAGGACGTATTAATAATTT-3′; secondary primers were 5′-GATTCTAGAAACTGCTCTGCACC-3′ and 5′-CAAGTAGGACGTATTAATAATTTTTCC-3′. The primary PCR program for the dhfr 5′ UTR was 1 cycle of 95°C for 5 min (primary denaturing); 45 cycles of 94°C for 30 s (denaturing), 42°C for 45 s (annealing), and 65°C for 45 s (extension); and then 1 cycle of 72°C for 5 min (final extension). The secondary PCR program was the same, except that only 20–35 cycles of the denaturing, annealing, and extension steps were done, and the final extension was 1 cycle of 72°C at 30 min. PCR programs for the pppk-dhps intron were the same, except that the temperature for the annealing step was 45°C
Allele-specific restriction analysis was performed for dhfr, dhps, pfmdr1 and pfcrt codons, as our Web site describes. Four microsatellite markers (ta99, ca1, ta87, and ta1) [27] were amplified by PCR as described on our Web site. Amplicons were run on 2.5% NuSieve GTG agarose gels (BMA) and sized using Gene Tools analysis software (Syngene), with a 100-bp ladder as reference. Products of each marker were examined in a single gel, and calculated sizes were rounded such that isolates of identical visual size were given identical values and isolates that were visibly different were given differing values. Thus, the size values presented in table 3 are approximate, and should only be interpreted as denoting similarity or variation among parasites
Results
dhfr anddhps in South AmericaTo track the origins and dissemination of the mutant dhfr and dhps we searched for polymorphisms in the noncoding sequences of these genes. dhfr is the upstream half of dhfr-ts which also encodes thymidylate synthase. The gene is intronless, but polymorphic repeats in the 5′ UTR have been identified in established strains [27]. We amplified and sequenced the 312 bp most proximal to the dhfr start codon and found 2 AT repeat regions, designated “AT-1” and “AT-2,” with size variation among South American parasites (figure 1A ). pppk-dhps which encodes a pyrophosphokinase upstream of dhps harbors 2 introns. We found 2 polymorphic domains, termed “AT” and “VR” (variable repeat), in the first intron (figure 1B ), but no substantial variation in the second intron. Therefore, the 5′ UTR of dhfr and the first intron of pppk-dhps were selected to characterize the antifolate-resistance mutations. We chose samples from 5 regions of the Amazon that represented the array of dhfr and dhps alleles found in South America and for which the polymorphic domains could be amplified, cloned, and sequenced by standard methods
Figure 1
dhfr-ts and pppk-dhps microsatellite sequences. A, Published Plasmodium falciparum (3D7 strain; GenBank accession no. U53326) sequence of the 5′ untranslated region of dhfr-ts with polymorphic repeat domains AT-1 and AT-2. Numbering denotes base location upstream from the dhfr-ts start codon. B Published P. falciparum (K1 strain; GenBank accession no. Z31584) sequence of the polymorphic region of the first intron of pppk-dhps with polymorphic repeat domains AT and VR (variable repeat). VR was imperfectly duplicated or triplicated in some pppk-dhps alleles. Numbering denotes base location from the pppk-dhps start codon
To date, the SP-resistance mutations detected in South America include the dhfr mutations S108N, N51E, C50R, I164L, and BR and the dhps mutations A437G, K540E, and A581G. Although we had previously reported the dhfr C59R in a subset of low-parasitemia Bolivian isolates [21], a more rigorous analysis of the samples failed to confirm this finding. As the dhfr mutation C59R has not since been detected in Brazil [25], Peru [26], Colombia [23], or Venezuela [24], it appears to be rare or absent in South America. We therefore identified the mutations dhfr S108N, dhfr N51I, and dhps A437G as the “primary antifolate-resistance mutations” and will refer to the mutations dhfr C50R, dhfr I164L, dhps K540E, and dhps A581G as the “secondary antifolate-resistance mutations” (SARMs). The dhfr mutation A16V/S108T, which confers cycloguanil, but not pyrimethamine, resistance was not addressed
dhfr 5′ UTR polymorphisms and mutationsAs table 1 shows, no specific-size polymorphisms in the AT-1 and AT-2 domains correlated with the identity of dhfr codons 108 and 51. However, there was an absolute association between the occurrence of the C50R or the I164L mutation and the presence of 9 TA repeats in the AT-2 domain in parasites from Bolivia, Brazil, Peru, and Venezuela. dhfr without the C50R and I164L mutations had AT-2 domains of varying sizes, although some harbored 9 repeats, including a wild-type dhfr gene from Ecuador. In addition to the 9-repeat AT-2 domain, all 7 C50R alleles contained 12 repeats in AT-1. This AT-1/AT-2 genotype was not unique to C50R dhfr; 2 infections from Colombia and the Brazilian 7G8 line established in 1984 [28] harbored the same sequences but did not have the C50R mutation. The AT-2 domain in I164L alleles exhibited slight variation; 3 of 5 isolates harbored 16 repeats, 1 had 14 repeats, and 1 had 15. However, all the I164L alleles carried the BR. Although the BR was detected in all I164L dhfr from infections in Bolivia [21] and Brazil [25], a subsequent study from Peru reported a small number of I164L dhfr lacking the BR (3 of 29 I164L isolates) [26]. We reexamined these 3 Peruvian isolates (results not shown) and found that only 1 actually harbored I164L in the absence of the BR. This unique allele lacked the codon-51 mutation, in addition to lacking the BR, which suggests that it was either derived from a recombination event or, less parsimoniously, represents an additional origin to the I164L that has not disseminated. This isolate also had wild-type dhps and pfcrt alleles (CVMNT; see below) not identified in other SARM isolates but present in the non-SARM parasites from Peru
The combined presence of the BR and 9 repeats in AT-1, in addition to modest variation in AT-2, define the 5 I164L dhfr examined from Peru, Brazil, and Bolivia. Taken together, these analyses suggest that the dhfr SARM has a single origin, from a common or similar parasite strain. However, our limited analysis could not track the S108N and N51I mutations, which, judging by reports of SP resistance, likely have been present in South and Central America and the Caribbean since the 1960s [14]. Their origins may have been multiple or may have occurred so long ago that the noncoding polymorphisms have since lost their integrity and/or link to the mutant dhfr as it disseminated
dhps intron polymorphisms and mutationsThe first intron of pppk-dhps is upstream of the dhps coding domain. As table 2 shows, polymorphisms in the intron were divergent in wild-type dhps as well as in dhps bearing only the inaugural mutation at codon 437. However, intron polymorphisms correlated with the combined presence of K540E and A581G in countries where these mutations have been detected (Venezuela, Peru, Brazil, and Bolivia). Thirteen of fourteen parasites with K540E/A581G alleles carried 16 repeats in the AT domain and no duplication of the VR sequence in other alleles. The single exception was from Brazil; that one had an intron repeat like that of the F48 isolate from Colombia. This discordant intron/mutation combination was likely consequent to a recombination event involving the allele identified in the other 13 samples. The Brazilian 7G8 line derived in 1984 bears a A437G dhps and 14-repeat intron sequence like that of the Brazilian ICS line established in the late 1990s, the dhps gene of which carries both A437G and A581G. These alleles may represent antecedents to dhps that acquired the K540E. In any case, our analysis supports the conclusion that the dhps SARMs prevalent in the Amazon have a common origin
pfcrt and SARMsAlmost all South American P. falciparum parasites carry the pfcrt K76T mutation critical to chloroquine resistance [29, 30]. We examined 11 contemporary isolates from Colombia [23], 16 from Bolivia [21], 10 from Brazil [25], 16 from Peru [26], and 168 from Venezuela [24] and found that all but 1 Venezuelan isolate carried the pfcrt K76T mutation. The HB3 Honduras line established in the 1980s, as well as 3 isolates obtained in 1997 from Haiti, retain the wild-type codon at codon 76. pfcrt codons 72, 74, and 75 are polymorphic in mutant pfcrt from the Americas, and their roles in chloroquine resistance are under investigation [30]
We examined the pfcrt gene of the isolates mentioned above to estimate parasite heterogeneity at a locus unlinked to the SARMs and that had no known role in antifolate resistance. As is demonstrated in tables 2 and 3, 5 of the known permutations at codons 72–75 were detected [29]. Unexpectedly, the pfcrt alleles were unequally distributed among SARM and non-SARM P. falciparum (figure 2). All 11 of the Colombian non-SARM infections carried the CVMET allele originally identified in Colombian parasites [29]. The CVMNT allele first identified in an Ecuadorian line [29] was found in all (7 of 7) Peruvian isolates not harboring the SARMs. Venezuelan isolates lacking the SARMs carried 1 of 3 resistant alleles (CVMET, CVMNT, or CVIET) [24]. In contrast, the S(TCT)VMNT allele of the Brazilian line DIV was the exclusive pfcrt allele detected in the 16 Bolivian samples, as well as in most of the dhfr C50R and I164L isolates from Brazil (9 of 10), Peru (7 of 10), and Venezuela (31 of 33). The exceptions invariably harbored the S(AGT)VMNT of the Brazilian 7G8 line. Thus, 2 lines established from Brazilian isolates in the 1980s bear an SVMNT pfcrt allele that was subsequently carried by the South American SARM P. falciparum of the 1990s
Figure 2
Geographic distribution of Plasmodium falciparum drug-resistance alleles in South America. A, dhfr/dhps secondary antifolate-resistance mutations. B, pfcrt alleles. Black circles denote study sites from which samples were obtained from individuals with P. falciparum malaria. Insert shows the Amazon forest
pfmdr1 and SARMsIn light of the pfcrt allele distributions, we examined the pfmdr1 1246 codon of the parasites for which noncoding polymorphisms in dhfr and dhps were determined. Transfection studies have shown that the pfmdr1 D1246Y mutation enhances in vitro chloroquine resistance in mutant pfcrt parasites [32], but pfmdr1 has no known role in antifolate resistance. Although pfmdr1 is not linked to the other drug-resistance genes examined in this study, as tables 1 and 2 show, D1246Y was found in 92% (23 of 25) of parasites with SARMs but only 23% (3 of 13) of parasites without these mutations. Of the parasites that carried D1246Y, 89% (24 of 27) had either S(AGT)VMNT or S(TCT)VMNT pfcrt. Thus, pfmdr1 D1246Y is another drug-resistance mutation carried by SARM P. falciparum
Table 1
South American Plasmodium falciparum dhfr alleles and associated microsatellites and drug-resistance mutations
Table 2
South American Plasmodium falciparum dhps alleles and associated microsatellites and drug-resistance mutations
Additional microsatellite analysisThe limited quantities of DNA obtained from our samples prevented us from performing a phylogenetic analysis of the South American malaria parasites. We instead chose 4 microsatellite markers not linked to the drug-resistance mutations and assayed a different subset of samples of various origins and drug-resistance haplotypes for clonality. As table 3 shows, the SARM parasites had similar but not identical profiles. Sixteen of 17 and 19 of 19 SARM parasites yielded identically sized ta99 and ta1 microsatellites, respectively. In contrast, there were at least 5 different ca1 products among the SARMs and 2 of ta87 at roughly equal prevalence. With the exception of the 4 Venezuelan isolates carrying the dhfr C50R mutation, SARM parasites from a single origin also exhibited genetic variation. The non-SARM parasites that we examined exhibited both differences from and similarities to their more mutant counterparts. Thus, there is genetic heterogeneity among SARM parasites at loci unlinked to the drug-resistance mutations but an overall homogeneity consistent with a common origin
Table 3
Microsatellite analysis of South American Plasmodium falciparum at loci distinct from drug-resistance genes
Discussion
Our analysis of P. falciparum from 5 locations in the Amazon suggests that the SARMs conferring mid- and high-level SP resistance have a common origin. First, sequences in coding and noncoding regions within or flanking these genes show identical or nearly identical polymorphic patterns among South American parasites harboring the SARM alleles. The existence of the few mutants that have exceptional microsatellites can be attributed to mitotic drift or infrequent recombination events between the SARM alleles and their less mutant antecedents. Intragenic recombination has been demonstrated in P. falciparum [33], and regions of the Amazon, such as Venezuela and Peru, continue to maintain cohabiting populations of antifolate-resistant and -sensitive malaria parasites [24, 26]. Second, alleles of 2 genes involved in chloroquine resistance that are not linked to dhfr and dhps tracked with the SARMs. Either of 2 pfcrt alleles first identified in Brazilian parasite lines from the 1980s and subsequently detected in all or most Brazilian and Bolivian isolates from the 1990s were found in all of the SARM P. falciparum examined in this study but only in a minority without the mutations. pfmdr1 D1246Y also was predominant among SARM malaria parasites. The association of the SARMs with the mutant pfmdr1 allele, as well as with 2 resistant pfcrt alleles carrying a unique C72S consequent to mutually exclusive nucleotide substitutions, suggests that the SARMs may have spread by the selective pressures of both the antifolates and the 4-aminoquinolines. A recent study has shown that P. falciparum harboring the SVMNT pfcrt allele exhibited diminished verapamil reversibility of in vitro chloroquine resistance relative to parasites harboring other resistant pfcrt alleles [31]
Indeed, as the geographic distribution of the drug-resistance mutations suggests, there has been a north-northwestward invasion of multidrug-resistant P. falciparum across South America, from an origin likely in the lower Amazon. Our previous identification of a Bolivian dhfr harboring both the C50R and I164L [21] attests to the sustained establishment of these polymorphisms in Bolivia. Notably, it was near the Brazil-Bolivia border that the first American cases of RIII-level antifolate resistance were documented in the 1980s [18]
Our typing at the drug-resistance loci demonstrates genetic similarities among the multidrug-resistant P. falciparum of South America. Microsatellite analysis using 4 additional markers revealed that parasites with identical drug-resistance haplotypes are also similar at loci not linked to the known resistance mutations but are not genetically identical. The existence of infrequent parasites with disassociated SARM/pfcrt /pfmdr1 allelic patterns, most often detected in Peru and Venezuela, further supports the conclusion that there is a limited degree of recombination between resident and invading pathogens
There are implications for public health in the present survey. Despite the episodic and unstable nature of South American malaria, a focally originated mutant that emerges under selective pressure can nonetheless become widespread in a matter of years. Many factors likely promote the dissemination of resistance in South America. Itinerant deforesting labor practices in the Amazon, along with unregulated establishment of pioneer settlements, maintain a mobile, susceptible host population. A reduced emphasis on vector control, compounded by the presence of insecticide-refractory Anopheles mosquitoes, promotes the spread of disease in less remote areas. Bacterial antifolate resistance has been rampant in South America, in part as a result of the unregulated distribution of antibiotics [34]; the unprescribed use of antimalarial agents is also likely of consequence. The Amazon's low vector-transmission intensity and limited acquired immunity to malaria among inhabitants may help sustain fitness-reducing mutations such as dhfr I164L [35] by limiting the frequency of recombination events that may exclude these mutations [36] and reduce direct competition among parasites. There is also an intensification of drug pressure in the absence of asymptomatic disease. Nonetheless, our evidence for the focal origin of P. falciparum drug-resistance speaks to the value of cogent continent-wide malaria-control and -treatment policies, as well for the implementation of molecular assays for the surveillance of drug resistance. Our findings support the use of combination therapies for malaria that target multiple gene products and metabolic nodes, to restrict the development and spread of resistance to individual agents. Lessons from the history of the antimalarial antifolates and the modes by which resistance arose and disseminated may help prolong the life span of the next generation of chemotherapies for this recalcitrant disease
Acknowledgments
This paper is dedicated to David F. Clyde, for his pioneering work in the epidemiology of malarial drug resistance. We thank Mariano Zalis, James Kublin, Fabian Mendez, and Jose Estrada-Franco, for generously providing samples, and Thomas Wellems and Xin-zhuan Su, for providing DNA from parasite strains
Footnotes
-
↵Informed consent was obtained from subjects or their parents or guardians, following the human experimentation guidelines of the US Department of Health and Human Services and/or those of the authors' institutions
Financial support: Inmunología Asociación Civil, Venezuela; National Institute of Allergy and Infectious Diseases (grant R01-AI-44824)
-
↵Deceased
- Received April 15, 2002.
- Revision received May 30, 2002.
- © 2002 by the Infectious Diseases Society of America
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