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Emergence of a dhfr Mutation Conferring High-Level Drug Resistance in Plasmodium falciparum Populations from Southwest Uganda

  1. Caroline Lynch1,
  2. Richard Pearce2,
  3. Hirva Pota2,
  4. Jonathan Cox1,
  5. Tarekegn A. Abeku1,
  6. John Rwakimari3,
  7. Inbarani Naidoo2,4,
  8. James Tibenderana1 and
  9. Cally Roper2
  1. 1 Disease Control and Vector Biology Unit, Department of Infectious Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom
  2. 2 Pathogen Molecular Biology Unit, Department of Infectious Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom
  3. 3 National Malaria Control Programme, Ministry of Health of Uganda, Kampala, Uganda
  4. 4 Malaria Lead Programme, Medical Research Council, Durban, South Africa
  1. Reprints or correspondence: Dr. Cally Roper, London School of Hygiene and Tropical Medicine, Keppel St., London, WC1E 7HT (cally.roper{at}lshtm.ac.uk).
 
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Abstract

The S108N, C59R, and N51I mutations in the Plasmodium falciparum gene that encodes dihydrofolate reductase, dhfr, confer resistance to pyrimethamine and are common in Africa. However, the I164L mutation, which confers high-level resistance, is rarely seen. We found a 14% prevalence of the I164L mutation among a sample of 51 patients with malaria in Kabale District in southwest Uganda in 2005 and a 4%prevalence among 72 patients with malaria in the neighboring district of Rukungiri during the same year. Surveillance at 6 sites across Uganda during 2002–2004 reported a single case of infection involving an I164L mutant, also in the southwest, suggesting that this is a regional hot spot. The spatial clustering and increasing prevalence of the I164L mutation is indicative of local transmission of the mutant. Targeted surveillance is needed to confirm the extent of the spread of the I164L mutation and to monitor the impact of I164L on the efficacy of antifolates for intermittent preventive treatment of pregnant women and/or infants with falciparum malaria.

The S108N, C59R, and N51I mutations in the Plasmodium falciparum gene dhfr, which encodes dihydrofolate reductase, confer resistance to pyrimethamine and are widespread in Africa. Parasites that carry an additional mutation, I164L, are highly resistant to sulfadoxine-pyrimethamine (SP) and, in addition, are resistant to chlorproguanil dapsone [1] and artesunate dapsone proguanil [2]. P. falciparum isolates with dhfr mutations that confer a high level of drug resistance became established in Southeast Asia 20 years ago, but the anticipated emergence of the same mutation among African parasites has not yet occurred. Isolated cases have demonstrated that the I164L mutation has the capacity to occur, but its failure to become established seems to indicate that some important precondition is not being met [3].

In a review of the published literature, we identified 94 surveys in 70 unique geographical localities in Africa where P. falciparum isolates have been tested for the I164L mutation. The 52 articles in which they are described are cited in the Appendix, which is available only in the electronic edition of the Journal, and a map with embedded links to these references is available online at http://www.drugresistancemaps.org. Of 6753 isolates tested, 99.8% were negative for I164L. Sites at which isolates without the I164L mutation were found are shown in figure 1.

Figure 1.
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Figure 1.

Recovery of Plasmodium falciparum isolates carrying the I164L mutation in the dhfr gene in Africa. A literature search identified 94 surveys in 70 unique localities where isolates were tested for I164L. Of the 6753 isolates tested, 99.8% were negative for I164L. Black circles indicate surveys where mutations were not found, and red circles indicate surveys where the mutation was detected, as well as its prevalence. Underlined data indicate prevalences from the current study.

The limited information on I164L in Africa to date fall into 3 categories. The first category comprises data from investigations in which minority—and otherwise undetectable—mutant genotypes are subcloned from isolates recovered from infected persons [4, 5]. Such studies only identify minority strains that have probably arisen through de novo mutations in blood stage parasites during the course of an infection and are therefore not indicated in figure 1. A second category of analyses involves people with infection after unsuccessful treatment or after prophylaxis with antifolates. In Sweden, 4 travellers from Kenya and Ghana who were taking proguanil as prophylaxis were found to carry parasites with I164L [6], and I164L mutants were found in 1 individual after SP treatment in an in vivo study in Kampala during 1999 [7]. Similarly, during a 2002–2003 study in western Kenya of persons who received trimethoprim-sulfamethoxazole for complications associated with human immunodeficiency virus (HIV) infection, 4 people were found to be infected with I164L mutants [5], and during a 2001–2003 study in Malawi of a cohort of 85 patients who received SP prophylaxis, I164L was detected in parasites recovered from 4 pregnant women [8]. Figure 1 shows the prevalence of the I164L mutation at sites where it was found. As previously reported, these mutations are generally considered to have arisen de novo during mitotic division and to have become prevalent among parasites in an individual host during an extended period of drug exposure. To date, parasites with the I164L mutation do not appear to have been transmitted to other people to create a “second generation” of infections with I164L mutants. The explanation often given is that I164L is deleterious and that fitness costs have so far prevented the establishment of the I164L mutation in Africa [3]. The propagation of I164L mutants to second- and third-generation cases appears, so far, to have been a stumbling block to the spread of I164L, which perhaps awaits the evolution of compensating adaptations to overcome a fitness cost of the mutation.

A third category of observations, and perhaps the most worrying, involves I164L mutant infections in patients who had not previously received treatment. To date, reports of such cases have been rare: in 2004, the mutation was found in 1 of 167 isolates from symptomatic individuals in Bangui in the Central African Republic [9]. Francis et al. [10] screened 480 isolates from patients at 6 sites across Uganda (Kanungu, Tororo, Arua, Jinja, Mubende, and Apac) between 2002 and 2004 and found only 1 I164L mutant, which was from a patient in the southwestern site of Kanungu.

In this article, we report a cluster of I164L mutants recovered from patients presenting for malaria treatment at clinics in 2 districts, Kabale and Rukungiri, in southwestern Uganda. The presence of unprecedented numbers of geographically clustered single-genotype infections involving parasites with the I164L dhfr mutation indicates that local transmission of I164L mutants is occurring.

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Subjects, Materials, and Methods

Study area, subjects, and samples. The study was performed at reference health facilities in Bufundi (Kabale District) and Kebisoni (Rukungiri District), which serve a population of 4–7 parishes (Ugandan administrative boundaries from largest to smallest are the district, subcounty, parish, and village). The health facilities are part of the government public health system and act as referral facilities for other health centers in their respective subcounties. Since 2002, these facilities have acted as key sentinel sites in an early warning system for malaria epidemics, piloted by the Ugandan Ministry of Health and the Highland Malaria Project (HIMAL; available at: http://www.lshtm.ac.uk/dcvbu/himal). Blood spots were collected from patients at these facilities in 2005 as part of the HIMAL [11].

Bufundi health center is located at an altitude of 2200 m and has a catchment population of ∼ 18,000 persons residing at altitudes of 1700–2200 m. Kebisoni health center is located at an altitude of 1600 m and serves a population of ∼32,000 inhabitants living at altitudes of 1400–1600 m.

The travel history of patients was recorded by laboratory teams in the health facility. Patients with suspected malaria were asked about their next of kin, village of residence, travel during the past 3 months, area of travel (subcounty and district), date of departure, duration of travel, and reasons for travel. Data were verified in a separate study through household visits.

Malaria transmission in both locations is strongly seasonal, with peaks in transmission occurring in December (after the short rainy season) and April (after the long rainy season). Rates of malaria transmission are typically low in Bufundi: in 2004, the total number of suspected malaria cases seen at the health center was 4961, of which only 7% were subsequently confirmed parasitologically by rapid diagnostic testing (HIMAL, unpublished data). Kebisoni lies in a fringe area where malaria transmission is unstable and nearly hyperendemic. The total number of suspected malaria cases seen at Kebisoni in 2004 was 9115, of which 49% were confirmed by rapid diagnostic testing (HIMAL, unpublished data).

The first-line treatment for malaria at the time of the study was SP, although national policy has since changed first-line treatment to artemether/lumefantrine, with SP reserved for intermittent preventive treatment during pregnancy. Community-based distribution of Homapak (SP and chloroquine prepackaged for children aged 2 months to 5 years [Kampala Pharmaceutical Industries]) is provided free of charge to children who present with fever to community-based medicine distributors working on behalf of the national home-based management of fever strategy, which began in Rukungiri during 2002 and in Kabale during 2005. Artemether/lumefantrine will replace SP and chloroquine in the home-based management of fever strategy throughout the country in 2008. Health care in public facilities is free of charge in Uganda. The Ministry of Health [12] reported per capita use of health facilities to be 1.01 and 0.6 visits per annum for Kabale and Rukungiri, respectively.

Fingerprick blood spot samples were obtained for symptomatic patients of all ages, after confirmatory diagnosis of P. falciparum infection by means of a Paracheck rapid test, from May through December 2005. Blood spots were air dried on Whatman no. 3 filter paper, sealed in plastic bags with a desiccant, and stored at room temperature for molecular genotyping.

Scientific and ethical permissions were given by the Uganda National Council for Science and Technology (UNSCT HS 35) and the ethics committee of the London School of Hygiene and Tropical Medicine (London, United Kingdom). Consent was obtained from all individuals or their guardians before collection of samples.

Molecular analysis, genotyping, and sequencing. DNA was prepared from blood spots by means of a chelex extraction method, and polymerase chain reaction (PCR) amplification of dhfr and dhps (the gene that encodes dihydropteroate synthase) was performed as described elsewhere [13]. The amplified PCR products were screened for dhfr and dhps sequence variants at the following 10 loci where single nucleotide polymorphisms (SNPs) are known to exist: codons 50, 51, 59, 108, and 164 in dhfr and codons 436, 437, 540, 581, and 613 in dhps.

PCR products were spotted in a 12-by-8 grid, cross-linked onto nylon membranes, and probed for sequence polymorphisms by hybridization to specific oligonucleotide probes, as described elsewhere [13]. The probed blots were visualized with ECF substrate and evaluated using a phosphoimager (GE Healthcare). Output was recorded by digitally captured images of the chemifluorescent signal.

The stringency and specificity of the hybridization process was confirmed by inclusion of a series of 4 controls with a known single genotype variant sequence. All blots with nonspecifically bound probes were stripped and reprobed. A SNP was considered to be present in the PCR product when the intensity of signal was higher than the background signal. The presence, absence, and relative intensity of hybridization signal was recorded for every probe at each locus. A sample was considered to have a single haplotype when only 1 sequence variant was found at each locus. Blood samples were categorized as having a single, a majority, or a mixed form of sequences for every SNP locus. Majority- and mixed-sequence infections were differentiated according to the relative intensity of the signal.

To examine the evolutionary origins of dhfr haplotypes that confer drug resistance, we studied polymorphic microsatellite repeats in the flanking region of dhfr on chromosome 4. We analyzed microsatellite markers located 0.3 kb, 4.4 kb, and 5.3 kb upstream from codon 108 by amplifying each locus by PCR and measuring the fragment size on an ABI 3730 sequencer with Genemapper software (Applied Biosystems). A seminested PCR design was used; the primer sequences and PCR reaction conditions are described elsewhere [14].

PCR products were purified using a kit from Qiagen. Cycle sequencing of samples loaded on the ABI 3730 capillary system was performed using the BigDye V 3.1 kit (Applied Biosystems). Sequence reads were checked by eye and edited using Seqman (DNAstar). The presence of SNPs was confirmed by reads through both forward and reverse strands.

Statistical analysis. Statistical comparison of allele frequencies in dhfr and dhps at the various loci was performed with the Fisher exact test, using Stata (StataCorp). The calculation of binomial exact 95% confidence intervals was also performed using Stata. For individuals infected with P. falciparum, the 2-tailed Fisher exact test was used to compare the travel histories of mutant-positive patients with those of mutant-negative patients. A Mantel-Haenszel test was used to compare travel histories among parasite-positive and parasite-negative patients.

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Results

The prevalences of point mutations at 4 codons in dhfr are shown in table 1. The I164L mutant was detected in 3 cases in Rukungiri and in 7 in Kabale, and dhfr in 5 infections had a mixture of mutant and wild-type codons. Sequencing analysis confirmed the presence of the I164L mutant in all 10 P. falciparum infections, giving an estimated I164L prevalence of 14% in Kabale and 4.2% in Rukungiri.

Figure 2.
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Figure 2.

Point mutations at codons 51, 59, 108, and 164 of dhfr (white, wild-type; black, mutant; and grey, mixed) and associated microsatellite polymorphisms at loci 0.1 kb, 4.4 kb, and 5.3 kb upstream of dhfr. The I164L mutation was associated with the following 2 lineages: A, the N51I+C59R+S108N triple-mutant lineage with ancestral microsatellite haplotype (108 bp, 177 bp, and 203 bp), and B, the N51I+S108N double-mutant lineage with ancestral microsatellite haplotype (88 bp, 179 bp, and 193 bp). The association between point mutations and microsatellites in unmixed infections should be given greater weight than those in mixed infections in which multiple single-nucleotide polymorphisms and/or microsatellite alleles are found.

Table 1.
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Table 1.

Prevalences of wild-type and mutant codons in the dhfr and dhps genes of Plasmodium falciparum among patients with malaria who visited health facilities in Bufundi (Kabale District) and Kebisoni (Rukungiri District), Uganda.

In both districts, the prevalence of mutations was high; indeed, wild-type sequences at codon 108 and codon 51 were absent, whereas the wild-type sequence at codon 59 was rare in both Rukungiri (2 unmixed and 1 mixed cases) and Kabale (3 unmixed and 3 mixed cases). As expected from previous studies in Africa, the N108T and C50R mutations were absent.

The prevalences of point mutations at 5 codon positions in dhps are shown in table 1. The smaller denominator for dhps is explained by use of a PCR amplification reaction that was less sensitive for dhps than for dhfr. Almost all infections were found to contain the A437G and K540E mutations. Only 2 infections, 1 in Kabale (mixed) and 1 in Rukungiri (unmixed), lacked point mutations at these codons in dhps. No mutations at codon 436 were present. The A581G mutation was common in isolates from Kabale (45% of infections) and Rukungiri (46% of infections), and no mutations were found at codon 613.

Point mutation haplotype frequencies are shown in table 2. To determine the relative abundance of different allelic haplotypes in the parasite population, 1 haplotype per infection was counted, and mixed infections in which haplotypes could not be resolved were omitted. Therefore, reported frequency data include only isolates that were unmixed or that carried a predominating majority and in which the allelic haplotype could be confidently identified. There was a clear predominance of the dhfr triple-mutant haplotype (S108N+C59R+N51I) in both districts, with a small number of double-mutant alleles (N51I+S108N). Interestingly, although the majority of I164L mutations were found in association with the S108N+C59R+N51I triple-mutant allele, in one case the I164L mutation was found with the S108N+N51I allele, which indicates that I164L has occurred independently on both the triple-mutant and double-mutant haplotypes.

Table 2.
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Table 2.

Point mutation haplotype frequencies in the dhfr and dhps genes of Plasmodium falciparum among patients with malaria who visited health facilities in Bufundi (Kabale District) and Kebisoni (Rukungiri District), Uganda.

We analyzed microsatellite sequences located 0.3 kb, 4.4 kb, and 5.3 kb upstream from codon 108 of the dhfr gene by measuring the size of PCR-amplified products on a capillary sequencer. Microsatellite alleles flanking the major point mutation haplotypes are illustrated in figure 2. The common microsatellite haplotype flanking the dhfr triple mutant (S108N+C59R+N51I) involves alleles 108 bp, 177 bp, and 203 bp at the 0.1 kb, 4.4 kb, and 5.3 kb loci, respectively (figure 2A). This haplotype is common to triple-mutant alleles throughout Africa and originally derived from Southeast Asia. Microsatellites flanking the S108N+C59R+N51I+I164L quadruplemutant allele show that this allele has evolved through the acquisition of I164L on the triple-mutant background. Two samples were mixed at codon 59 and codon 164, perhaps indicating that, in these patients, the S108N+C59R+N51I+I164L quadruplemutant allele was mixed with a S108N+N51I double-mutant allele or even that the C59R mutation had reverted. Interpretation of haplotypic associations on the basis of samples from mixed infections needs caution however. Microsatellite and SNP PCR reactions can have different thresholds for the detection of minority genotypes, and consequently haplotype associations can only be established with absolute certainty in unmixed infections. The flanking microsatellites around the double-mutant S108N+N51I alleles (figure 2B) had a major haplotype, which consisted of alleles 88 bp, 179 bp, and 193 bp (with evidence of recombination among the more distantly linked microsatellite loci in 2 isolates). This indicates that the lineage arose independently from the triple-mutant lineage. I164L occurred with S108N+N51I on the same microsatellite background, showing that the I164L mutation has arisen independently on the S108N+N51I double-mutant lineage as well as on the more common triple-mutant lineage.

In Bufundi subcounty of Kabale District, of the 51 blood spots analyzed, 7 were found to have the I164L mutation. Travel data in the weeks before presentation at the health facility were available for 6 of the patients, all of whom had travelled outside of Kabale District in the previous 4 – 6 weeks (table 3). A comparison of the travel histories of patients with the I164L mutant with those of patients without it revealed no significant difference in the tendency to travel outside the district during the 6 weeks before presentation at the health facility. Five of the patients had travelled to work in tea plantations in the Toro region or in Kibaale, which also contains tea plantations. These districts are ∼180 km and ∼300 km, respectively, from Kabale by air. One subject had travelled to the Wakisso District near Kampala.

Table 3.
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Table 3.

Demographic and travel history for 10 cases of malaria for which an I164L Plasmodium falciparum mutant was recovered.

Table A1.
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Table A1.

Parameters and findings of PubMed and Medline literature searches.

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Discussion

Detection of the I164L mutation at prevalences of 4% and 14% among newly presenting cases of P. falciparum malaria in Rukungiri and Kabale indicates that parasites carrying this mutation are relatively common in this area. There have been 2 previous reports of I164L mutants in Uganda. The first, in 1999, represented a single observation in a case of posttreatment relapse in Kampala [7]. The second was also a single observation from a 2002–2004 survey of 480 isolates from 6 sites across Uganda [10]. The study found a single I164L mutant among 80 pretreatment samples (prevalence, 1.25%) from Kanungu in southwest Uganda. This might suggest that southwest Uganda is a focus of I164L, yet the travel histories of the I164L mutant—positive patients in the current study, together with recent observations in western Kenya [5], Malawi [8], and the Central African Republic [9], seem to confirm that the I164L mutation is emerging regionally.

The majority of the samples with the I164L mutant originated from Bufundi health center, Kabale, which is located only a few kilometers from the Rwandan border. This is an area of very low malaria transmission, where <10% of specimens typically test positive for parasites by means of a rapid diagnostic test. During the time of the study, patients with confirmed malaria who presented to Kabale health facility were significantly more likely to have travelled in the previous 6 weeks (P < .01).Of the 6 people with I164L mutant for whom a travel history was recorded, all had travelled during the 6 weeks before presentation, which suggests that the health center catchment is not the epicenter of the I164L-associated outbreak. Most patients had travelled to the western region of Uganda, a high-altitude area with a low transmission rate in which employment on tea plantations can be obtained.

More-intense analysis at a fine spatial resolution will be required to adequately describe the geographical distribution of the I164L mutation in Uganda. There may be one distinct geographical focus from which highly resistant parasites are being dispersed; alternatively, the mutation may have begun to emerge de novo across the region in a large number of infected individuals, perhaps as a result of strong drug pressure and/or the fixation of point mutations at the other resistance-determining codons in the dhfr and dhps genes of parasites in these areas.

Analysis of microsatellite sequence flanking resistance loci can give important clues about the geographical origins of mutant lineages. One study of microsatellite loci around dhfr with resistance-conferring mutations in Thailand, Myanmar, Laos, Cambodia, and Vietnam compared all allelic combinations of point mutations at dhfr with or without the I164L mutation and found that all derived from 1 common ancestor [15]. Microsatellite analysis of dhfr with resistance-conferring mutations from African parasites has shown that the triple mutant (N51I+C59R+S108N) shares ancestry with resistance-conferring dhfr in parasites from Southeast Asia [16]. The triplemutant allele derived from Asia has been recorded at sites across the African continent [5, 14, 17, 18].

Unlike those in Southeast Asia, all dhfr alleles with resistance-conferring mutations in Africa do not share a common ancestor. The double-mutant dhfr alleles found in southeast Africa belong to a restricted number of independently derived lineages [14], which are probably of African origin. Although the triplemutant allele was largely fixed in Kabale and Rukungiri, there was a small number of double-mutant N51I+S108N alleles, which were associated with a microsatellite haplotype lineage (88 bp, 176 bp, and 193 bp) that has been seen previously in Tanzania, South Africa [14], and Kenya [5]. We found the I164L mutation on both the Asian-derived triple-mutant background (in 9 of 10 cases) and on the African N51I+S108N double-mutant background (in 1 of 10 cases). Although we can say with some certainty that the mutation on the African background has occurred de novo in Africa, it is not possible to distinguish whether the I164L mutation on an Asian background has occurred de novo or was imported. If there was multiple recurrence of I164L within a lineage, it would be masked by the deeper shared ancestry of the progenitor triple- or double-mutant dhfr alleles. The observation of multiple origins is surprising because of the rare observation of I164L in Africa. This seems to suggest that constraints on the emergence of I164L mutations have been overcome by circumstances specific to this region.

Selection due to antifolate use has been strong in this part of Africa in recent years. Antifolates such as SP, which are used as the first-line treatment for uncomplicated malaria and malaria prophylaxis in pregnancy, are also used for management of the complications of HIV infection. Homapaks are distributed for home management of fever in children, although the extent to which they contribute to drug-associated selection pressure is uncertain. In areas of low transmission, such as Kabale, the majority of malaria cases are in adults (presumably because they are at greater risk when travelling outside the district to areas of higher transmission), so with fewer malaria cases among children, the use of Homapak is expected to be limited. It is, however, difficult to be certain that children are the sole users of Homapak.

As a consequence of drug pressure, the dhfr triple mutant (N51I+C59R+S108N) and the dhps double mutant (A437G+K540E) are approaching fixation in many parts of east Africa. Such a genetic background may be permissive for the emergence of the I164L mutation and, perhaps, also the A581G mutation in dhps. Adaptation to SP may include additional resistance-conferring mutations at genes other than dhfr and dhps, which either compensate for deficiencies in folate metabolism caused by the mutations in dhfr and dhps or up-regulate folate transport [19–21].

It will be important to monitor the emergence and spread of highly resistant I164L mutants in Africa in the coming years. Gene flow has historically played a central role in the establishment of resistance to chloroquine and SP at sites across Africa, so the spread of these mutants has the potential to quickly become a wider regional problem. The work of regional drug resistance networks will be important in monitoring the developing situation.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Gates Malaria Partnership.

  • The sponsors of this study had no role in designing the study; collecting, analyzing, and interpreting the data; or writing the report.

  • Received August 28, 2007.
  • Accepted December 5, 2007.
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Appendix

Literature searches were performed in June 2007 and again in October 2007, using PubMed and Medline (table A1). Inclusion criteria were “Africa” and studies that mentioned codon 164 of dhfr. We identified 52 articles in which codon 164 was analyzed in P. falciparum isolates recovered from patients in Africa who received a diagnosis of malaria [5, 7–9, 11, 14, 15, 19, 22–65]. Georeferenced data points are shown in the map in figure 1 and are available at http://www.drugresistancemaps.org.

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  1. J Infect Dis. (2008) 197 (11): 1598-1604. doi: 10.1086/587845
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