Molecular surveillance of the antifolate-resistant mutation I164L in imported african isolates of Plasmodium falciparum in Europe: sentinel data from TropNetEurop
- Ole Wichmann†1Email author,
- Tomas Jelinek1,
- Gabriele Peyerl-Hoffmann3,
- Nikolai Mühlberger1,
- Martin P Grobusch2, 4,
- Joaquim Gascon8,
- Alberto Matteelli10,
- Christoph Hatz11,
- Hermann Laferl12,
- Marco Schulze5,
- Gerd Burchard1, 6,
- Saraiva da Cunha14,
- Jiøi Beran15,
- Paul McWhinney16,
- Herwig Kollaritsch13,
- Peter Kern7,
- Juan Cuadros9,
- Michael Alifrangis17,
- Ida Gjørup17 and
- the European Network on Surveillance of Imported Infectious Diseases (TropNetEurop)
© Wichmann et al; licensee BioMed Central Ltd. 2003
Received: 06 May 2003
Accepted: 25 June 2003
Published: 25 June 2003
Malaria parasites that carry the DHFR-mutation I164L are not only highly resistant to sulfadoxine-pyrimethamine but also to the new antimalarial drug chlorproguanil-dapsone. The spread of this mutation in Africa would result in a public health disaster since there is a lack of effective alternatives that are both affordable and safe. Up to now, this mutation has only been described in Asian and Latin-American countries. The objective of this study was to assess the prevalence of this mutation in African isolates of Plasmodium falciparum that have been imported into Europe through travellers.
TropNetEurop is a network for the surveillance of travel-associated diseases and seems to cover approximately 12% of all malaria cases imported into Europe. Within this network we screened 277 imported African isolates of P. falciparum with the help of PCR- and enzyme-digestion-methods for the antifolate-resistant mutation I164L.
The I164L mutation was not detected in any of the isolates tested.
Continuous molecular surveillance of mutations in P. falciparum, as it is practised within TropNetEurop, is an essential tool for the understanding and early detection of the spread of antimalarial drug resistance in Africa.
Drug resistant malaria is a major problem in malaria control. At the present, there are few antimalarial drugs available in endemic areas which are both cheap and save. Since resistance to chloroquine has spread across sub-Saharan Africa, several countries have switched their first-line drug to the antifolate sulfadoxine-pyrimethamine .
Soon after sulfadoxine-pyrimethamine was introduced to malaria-control programs in the late 1960s, resistance to this drug was noted . The efficacy of sulfadoxine-pyrimethamine primarily depends on the sensitivity of the parasite to pyrimethamine . The dihydrofolate reductase domain of Plasmodium falciparum (pfDHFR) is the target of this drug. DHFR catalyzes the reduction of dihydrofolate to regenerate tetrahydrofolate which is required for one-carbon transfer reactions and deoxythymidylate synthesis of the parasites.
The discovery of changes in codons of the pfDHFR-gene strongly indicated that single amino acid changes lead to observed resistance. Mutations at amino acid position 51, 59, 108 and 164 have been shown to be linked with resistance of P. falciparum to antifolate antimalarials. A scheme for evolution of resistance could be derived as a result of stepwise mutations starting with the S108N mutation, which was shown to be the optimal mutation in leading to both the decreased binding affinity for inhibitors and the retention of enzyme activity [4, 5]. Absolute resistance is conferred by the addition of I164L mutation in the quadruple mutant form (N108/I51/R59/L164). In this fourth mutation the enzyme is about 1,000-fold less sensitive to pyrimethamine .
The I164L mutation also plays an important role in the development of resistance to the more potent antifolate combination chlorproguanil-dapsone: while the triple mutant allele (N108/I51/R59) has no great impact on the sensitivity of this drug, parasites that carry the quadruple mutant allele are resistant [7, 8]. This highly potent quadruple mutation was described first in South East Asia, later also in South America [9–11]. There is a concern that continued sulfadoxine-pyrimethamine pressure, as well as the widespread use of trimethoprim-sulfamethoxazole for prophylaxis against opportunistic infections in patients with AIDS in Africa, is going to select these highly resistant alleles . This would rapidly make sulfadoxine-pyrimethamine, as well as chlorproguanil-dapsone, ineffective in this region. For this reason continued surveillance of this mutation is needed to evaluate the prevalence, the distribution and the speed with which these populations might be selected.
TropNetEurop is a European surveillance system that covers approximately 12% of all imported malaria cases in Europe . Travellers can be used as a highly sensitive surveillance tool to detect development of drug-resistance in endemic areas, as it is suggested that most of them carry a monoclonal Plasmodium strain. Initial data on molecular surveillance, not including the I164L mutation, have already been published elsewhere .
Since there are only limited data available on the prevalence of the I164L mutation throughout Africa, the purpose of this survey was to screen a sample of P. falciparum imported from Africa for this particular antifolate mutation.
Material and methods
The European Network on Imported Infectious Disease Surveillance (TropNetEurop) has been established in 1999 and, in 2001, covered approximately 12% of all imported malaria cases in Western and Central Europe . At the present 45 clinical sites in Europe are members of the network with more than 57,000 patients seen post-travel annually.
Within the infrastructure of TropNetEurop several member sites started in 2001 to collect blood samples from patients diagnosed with a P. falciparum infection. 10 μl of whole blood from each patient were dotted on Whatman 3 MM® chromatography paper and air-dried at room temperature before initiation of treatment. The filter paper disks were sent to Munich for further preparation.
For this survey we screened all blood samples that were obtained between January 2001 and July 2002 from travellers returning from Africa with a microscopically confirmed P. falciparum infection.
Parasite DNA and Polymerase chain reaction
Parasite DNA was prepared from the dried blood spots on the filter paper by the Chelex method as described by Kain and Lanar . Nested mutation-specific PCR was done as previously described for analysis of the DHFR 164 mutation site [16, 17].
Restriction fragment length polymorphisms
A volume of 4 μl of PCR product was incubated for three hours at a temperature of 37°C with the mutation specific restriction enzyme DraI according to the manufacturer's instructions (New England Biolabs, Beverly, MA, USA). To discriminate between the two variants of codon 164, the 522 bp PCR product was digested with DraI to detect the leucine mutation (143 bp). The digestion products were separated by electrophoresis in a 1% SeaKem™ plus 1% NuSieve™ gel (FMC BioProducts, Rockland, ME, USA) containing ethidium bromide. DNA from established laboratory strains of P. falciparum served as controls of PCR and enzyme digests. Gels were recorded by photography.
Over the 18-month time period we collected 277 blood samples from patients with a microscopically confirmed P. falciparum infection acquired in Africa. The DNA from all blood samples was amplified by nested PCR systems as described above. 271 samples showed the expected bands at 522 bp for the DHFR gene, while six samples turned out to be negative for P. falciparum DNA.
Frequencies of geographical regions in which the observed Plasmodium falciparum infection has been acquired
Wild type (%)
After digestion none of the 271 isolates revealed the leucine-164 mutation. All 271 isolates of P. falciparum presented as the wild type with the amino acid isoleucine in position 164.
The burden of falciparum malaria is carried mainly by tropical Africa. Since resistance to Chloroquine is now widespread in Africa, the antifolate combination sulfadoxine-pyrimethamine (SP) is often the only affordable alternative.
Unfortunately, SP is particularly prone to the rapid emergence of resistance . There is evidence, that in vitro pyrimethamine chemosensitivity will be predictive of in vivo SP efficacy, and that parasitological resistance in Africa is primarily due to the triple mutant in DHFR: S108N, N51I and C59R [19–21].
At the moment, the only drug available in Africa to deal with SP failure is often further treatment with SP. Researchers are, therefore, seeking new, safe and inexpensive, second line drugs or combination regimens. Among these regimes chlorproguanil-dapsone is considered a potential replacement for SP in Africa because of its effectiveness against infections associated with triple mutants in DHFR, its short half-life and its low price [21–23]. However, the efficacy of combinations involving old drugs may be short-lived, since resistance-conferring mutations already exist. In the case of chlorproguanil-dapsone, the change in DHFR enzyme structure resulting from the I164L mutation is known to be associated with resistance to this drug [19, 20].
Experience with SP in South East Asia and South America has shown that continued use of SP will select for the quadruple mutant DHFR, including I164L. So far, mutations of this potency have not been observed in Africa in any study using standard PCR analyses [10, 17, 21, 23–27]. The I164L mutation was also not found in any of the isolates we analyzed within this survey.
Recently, Hastings et al.  developed a yeast-based system that is sensitive enough to identify point mutations in the DHFR gene even if they are rare. With the help of this technique alleles that encoded the I164L mutations were isolated from 3 of 6 patient samples with SP treatment failure from Tanzania. However, mutations of all kinds occur during DNA replication, but not all establish themselves and are functional.
Overall, efficient surveillance systems to detect and monitor resistance to SP are required, since malaria infections will become fully resistant to SP and chlorproguanil-dapsone with the selection of the DHFR mutation I164L in Africa. For this reason, several national and regional networks of sentinel sites have been set up in recent years and collaborate in order to monitor antimalarial drug resistance. The East African Network for Monitoring Antimalarial Treatment (EANMAT), for example, started in 1998 to monitor drug resistance with standard in vivo tests . TropNetEurop, as the largest network for infectious diseases acquired by travellers worldwide, can function as an additional sensitive tool in the surveillance of antimalarial drug resistance.
We wish to thank all TropNetEurop site staff, who has been invaluable in collecting material and data. Financial support from Dr. Democh Maurmeier Stiftung and the Förderprogramm für Forschung und Lehre der Medizinischen Fakultät, both at Ludwig-Maximilians-University, Munich, Germany, is gratefully acknowledged.
- White NJ, Nosten F, Looareesuwan S, Watkins WM, Marsh K, Snow RW, Kokwaro G, Ouma J, Hien TT, Molyneux ME, Taylor TE, Newbold CI, Ruebush II TK, Danis M, Greenwood BM, Anderson RM, Olliaro P: Averting a malaria disaster. Lancet. 1999, 353: 1965-1967. 10.1016/S0140-6736(98)07367-X.View ArticlePubMedGoogle Scholar
- Bjorkman A, Phillips-Howard PA: The epidemiology of drug-resistant malaria. Trans R Soc Trop Med Hyg. 1990, 84: 177-180.View ArticlePubMedGoogle Scholar
- Sibley CH, Hyde JE, Sims PFG, Plowe CV, Kublin JG, Mberu EK, Cowman AF, Winstanley PA, Watkins WM, Nzila AM: Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what is next?. Trends Parasitol. 2001, 17: 582-588. 10.1016/S1471-4922(01)02085-2.View ArticlePubMedGoogle Scholar
- Sirawaraporn W, Yongkiettrakul S, Sirawaraporn R, Yuthavong Y, Santi DV: Plasmodium falciparum: Asparagine mutant as residue 108 of dihydrofolate reductase is an optimal antifolate-resistant single mutation. Exp Parasitol. 1997, 87: 245-252. 10.1006/expr.1997.4221.View ArticlePubMedGoogle Scholar
- Yuthavong Y: Basis for antifolate action and resistance in malaria. Microbes Infect. 2002, 4: 175-182. 10.1016/S1286-4579(01)01525-8.View ArticlePubMedGoogle Scholar
- Plowe CV, Kublin JG, Doumbo OK: P. falciparum dihydrofolate reductase and dihydropteroate synthase mutations: epidemiology and the role in clinical resistance to antifolates. Drug Resist Updat. 1998, 1: 389-396.View ArticlePubMedGoogle Scholar
- Nzila-Mounda A, Mberu EK, Sibley CH, Plowe CV, Winstanley PA, Watkins WM: Kenyan Plasmodium falciparum field isolates: correlation between pyrimethamine and chlorcycloguanil activity in vitro and point mutations in the dihydrofolate reductase gene. Antimicrob Agents Chemother. 1998, 42: 164-169.PubMed CentralPubMedGoogle Scholar
- Wilairatana P, Kyle DE, Looareesuwan S, Chinwongprom K, Amradee S, White NJ, Watkins WM: Poor efficacy of antimalarial biguanide-dapsone combinations in the treatment of acute, uncomplicated falciparum malaria in Thailand. Ann Trop Med Parasitol. 1997, 91: 125-132. 10.1080/00034989760662.View ArticlePubMedGoogle Scholar
- Basco LK, Eldin de Pecoulas P, Wilson CM, Le Bras J, Mazabraud A: Point mutations in the dihydrofolate reductase-thymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparum. Mol Biochem Parasitol. 1995, 69: 135-138. 10.1016/0166-6851(94)00207-4.View ArticlePubMedGoogle Scholar
- Plowe CV, Cortese JF, Djimde A, Nwanyanwu OC, Watkins WM, Winstanley PA, Estrada-Franco JG, Mollinedo RE, Avita JC, Cespedes JL, Carter D, Doumbo OK: Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance. J Infect Dis. 1997, 176: 1590-1596.View ArticlePubMedGoogle Scholar
- Masimirembwa CM, Phuong-dung N, Phuc BQ, Duc-Dao L, Sy ND, Sköld O, Swedberg G: Molecular epidemiology of Plasmodium falciparum antifolate resistance in Vietnam: genotyping for resistance variants of dihydropteroate synthase and dihydrofolate reductase. Int J Antimicrob Agents. 1999, 12: 203-211. 10.1016/S0924-8579(99)00061-8.View ArticlePubMedGoogle Scholar
- Wongsrichanalai C, Pickard AL, Wernsdorfer WH, Meshnick SR: Epidemiology of drug-resistant malaria. Lancet Infect Dis. 2002, 2: 209-218. 10.1016/S1473-3099(02)00239-6.View ArticlePubMedGoogle Scholar
- Jelinek T, Schulte C, Behrens R, Grobusch MP, Coulaud JP, Bisoffi Z, Martteello A, Clerinx J, Corachan M, Puente S, Gjorup I, Harms G, Kollaritsch H, Kotlowski A, Bjorkmann A, Delmont JP, Knobloch J, Nielson LN, Cuadros J, Hatz C, Beran J, Schmid ML, Schulze M, Lopez-Velez R, Fleischer K, Kapaun A, McWhinney P, Kern P, Atougia J, Fry G, da Cunha S, Boecken G: Imported Falciparum malaria in Europe: sentinel surveillance data from the European network on surveillance of imported infectious diseases. Clin Infect Dis. 2002, 34: 572-576. 10.1086/338235.View ArticlePubMedGoogle Scholar
- Jelinek T, Peyerl-Hoffmann G, Mühlberger N, Wichmann O, Schmider N, Grobusch MP, Corachán M, Hatz C, Harms G, Laferl H, McWhinney P, Schulze M, Kollaritsch H, da Cunha S, Beran J, Kern P, Gjørup I, Cuadros J: Molecular surveillance of drug resistance through imported isolates of Plasmodium falciparum in Europe: sentinel data from TropNetEurop. Malar J. 2002, 1: 11-10.1186/1475-2875-1-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Kain KC, Lanar DE: Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood. J Clin Microbiol. 1991, 29: 1171-1174.PubMed CentralPubMedGoogle Scholar
- Duraisingh MT, Curtis J, Warhurst DC: Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Exp Parasitol. 1998, 89: 1-8. 10.1006/expr.1998.4274.View ArticlePubMedGoogle Scholar
- Jelinek T, Rønn AM, Lemnge MM, Curtis J, Mhina J, Duraisingh MT, Bygbjerg IC, Warhurst DC: Polymorphisms in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes of Plasmodium falciparum and in vivo resistance to sulphadoxine/pyrimethamine in isolates from Tanzania. Trop Med Int Health. 1998, 3: 605-609. 10.1046/j.1365-3156.1998.00280.x.View ArticlePubMedGoogle Scholar
- Winstanley PA: Chemotherapy for falciparum malaria: the armoury, the problems and the prospects. sitol Today. 2000, 16: 146-153. 10.1016/S0169-4758(99)01622-1.Google Scholar
- Peterson DS, Milhous WK, Wellems TE: Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria. Proc Natl Acad Sci USA. 1990, 87: 3018-22.PubMed CentralView ArticlePubMedGoogle Scholar
- Watkins WM, Mberu PA, Winstanley PA, Plowe CV: The efficacy of antifolate antimalarial combinations in Africa: a predictive model based on pharmacodynamic and pharmacokinetic analyses. Parasitol Today. 1997, 13: 459-464. 10.1016/S0169-4758(97)01124-1.View ArticlePubMedGoogle Scholar
- Mutabingwa T, Nzila A, Mberu E, Nduati E, Winstanley P, Hills E, Watkins W: Chlorproguanil-dapsone for treatment of drug-resistant falciparum malaria in Tanzania. Lancet. 2001, 358: 1218-1223. 10.1016/S0140-6736(01)06344-9.View ArticlePubMedGoogle Scholar
- Winstanley PA, Watkins W, Muhia D, Szwandt S, Amukoye E, Marsh K: Chlorproguanil/dapsone for uncomplicated Plasmodium falciparum malaria in young children: pharmacokinetics and therapeutic range. Trans R Soc Trop Med Hyg. 1997, 91: 322-327.View ArticlePubMedGoogle Scholar
- Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, Mukadam RAG, Rogerson SJ, Lescano AG, Molyneux ME, Winstanley PA, Chimpeni P, Taylor TE, Plowe CV: Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis. 2002, 185: 380-8. 10.1086/338566.View ArticlePubMedGoogle Scholar
- Nzila AM, Mberu EK, Sulo J, Dayo H, Winstanley PA, Sibley CH, Watkins WM: Towards an understanding of the mechanism of pyrimethamnine-sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenian parasites. Antimicrob Agents Chemother. 2000, 44: 991-6. 10.1128/AAC.44.4.991-996.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Eberl KJ, Jelinek T, Aida AO, Peyerl-Hoffmann G, Heuschkel C, el Valy AO, Christophel EM: Prevalence of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum isolates from southern Mauritania. Trop Med Int Health. 2001, 6: 756-760. 10.1046/j.1365-3156.2001.00791.x.View ArticlePubMedGoogle Scholar
- Khalil I, Alifrangis M, Ronn AM, Gabar HA, Jelinek T, Satti GM, Bygbjerg IC: Pyrimethamine/sulfadoxine combination in the treatment of uncomplicated falciparum malaria: relation between dihydropteroate synthase/dihydrofolate reductase genotypes, sulfadoxine plasma levels, and treatment outcome. Am J Trop Med Hyg. 2002, 67: 225-229.PubMedGoogle Scholar
- Curits J, Maxwell CA, Msuya FHM, Mkongewa S, Alloueche A, Warhurst DC: Mutations in dhfr in Plasmodium falciparum infections selected by chlorproguanil-dapsone treatment. J Infect Dis. 2002, 186: 1861-1864. 10.1086/345765.View ArticleGoogle Scholar
- Hastings MD, Bates SJ, Blackstone EA, Monks SM, Mutabingwa TK, Sibley CH: Highly pyrimethamine-resistant alleles of dihydrofolate reductase in isolates of Plasmodium falciparum from Tanzania. Trans R Soc Trop Med Hyg. 2002, 96: 674-676.View ArticlePubMedGoogle Scholar
- Mutabingwa TK, Watkins WM, d'Alessandro U: Monitoring of drug-resistant malaria in Africa. Lancet. 2002, 360: 875-10.1016/S0140-6736(02)09978-6.View ArticlePubMedGoogle Scholar
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