Am. J. Trop. Med. Hyg., 66(5), 2002, pp. 487–491 Copyright © 2002 by The American Society of Tropical Medicine and Hygiene
POLYCLONAL PLASMODIUM FALCIPARUM MALARIA IN TRAVELERS AND SELECTION OF ANTIFOLATE MUTATIONS AFTER PROGUANIL PROPHYLAXIS ¨ RNERT, KAROLIN TENGSTAM, INGELA BERGGREN PALME, ULF BRONNER, MARIANNE LEBBAD, ANNA FA ¨ RKMAN ¨ TE SWEDBERG, AND ANDERS BJO GO Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden; Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Huddinge University Hospital, Huddinge, Sweden; Department of Pharmaceutical Biosciences, Division of Microbiology, Uppsala University, Uppsala, Sweden
Abstract. The polymorphism of malaria parasites will greatly influence the efficiency of antimalarial drugs and vaccines. This study determined the genetic diversity of Plasmodium falciparum infections in 107 travelers and estimated the importance of mutations in the parasite dihydrofolate reductase (dhfr) gene for clinical breakthrough during proguanil prophylaxis. Genotyping with regards to the three highly polymorphic antigen-coding regions (merozoite surface protein-1 [msp-1], msp-2, and the glutamate-rich protein [glurp]) revealed multiple genotypes (up to five) in 64% of the patients. Single genotype infections were mainly associated with prior intake of antimalarial drugs, but also with a shorter stay in a malaria-endemic area and low parasite density. Malaria breakthrough despite proguanil prophylaxis was always associated with mutations in the dhfr gene; always the Asn-108 mutation and often the Ile-51 and Arg-59 mutations. The Leu-164 mutation was found in four travelers from Africa. Travelers with limited time in an endemic area were often infected with polyclonal P. falciparum infections, which suggests that single mosquito inoculations are often composed of several genetically diverse parasites. Chemoprophylaxis reduces the number of infecting clones and selects for resistant parasites as shown for proguanil through mutations in the dhfr gene. prophylaxis. Point mutations in the dhfr gene in the parasite were analyzed in relation to previous intake of proguanil prophylaxis in the patients.
INTRODUCTION Malaria research during the last decades has focused on vaccine and drug development to reduce the high global morbidity and mortality caused by the Plasmodium falciparum parasite. Several antigens have been studied as potential components of a vaccine but molecular analyses have revealed extensive polymorphism in the parasite.1 Studies on mechanisms for drug resistance have also revealed genetic diversity with evidence that sequence variations in the gene coding for dihydrofolate reductase (DHFR) are correlated with resistance to antifolate drugs, mainly pyrimethamine (with sulfadoxine as Fansidar;威 F. Hoffmann LaRoche, Basel, Switzerland), and cycloguanil, the active metabolite of proguanil.2 Proguanil is now receiving increased interest because of its use both in prophylaxis and in new combination therapy, i.e., with atovaquone (Malarone;威 GlaxoSmithKline, UK). An understanding of the epidemiology of parasite diversity is thus important both in relation to the ability of parasites to escape antimalarial drugs as well as the immune system, including future vaccines. The development of polymerase chain reaction (PCR)-based methods to analyse genetic diversity has provided efficient tools for studies of the molecular epidemiology of malaria.3,4 Genetic regions coding for three vaccine candidate antigens (the two merozoite surface proteins [msp-1 block 2] and msp-2 and the glutamate-rich protein [glurp]) represent suitable markers for analyses of P. falciparum population diversity due to their extensive polymorphism. Plasmodium falciparum infections in individuals in endemic areas are generally composed of several concurrent diverse parasites, assumingly reflecting an accumulation of repeated infections. The aim of this study was to investigate the genetic diversity of P. falciparum infections in travelers with limited time in endemic areas and restricted exposure to malaria-infected mosquitoes. In Sweden, approximately 180 malaria patients are diagnosed yearly after traveling to malarious countries. We have genotyped the P. falciparum infections in approximately 100 such patients and related the findings to different host characteristics such as use of chemo-
PATIENTS, MATERIALS, AND METHODS The study included 107 patients (44 females and 63 males, including two children; age range ⳱ 1–67 years), with P. falciparum infections diagnosed or suspected by microscopy and confirmed by a PCR, who were admitted at the Infectious Diseases Departments at Danderyd and Huddinge University Hospitals in Stockholm, Sweden between 1995 and 1998. Venous blood was collected in Vacutainer威 tubes (Becton Dickinson, Rutherford, NJ) containing citrate before administration of antimalarial therapy and stored frozen. All samples were collected and analyzed with the informed consent of the patients. The study was approved by the Ethical Committee at the Karolinska Institutet (KI d NR 94:230). The patients’ clinical records were examined for several parameters: malaria-endemic country visited, reason and type of traveling, duration of the journey, number of days between travel and symptoms, number of days between onset of symptoms and admission, native origin, intake of antimalarial drug (prophylaxis and/or self treatment), and severity of disease (Table 1). Giemsa-stained blood smears were analyzed by light microscopy at a magnification of 1,000× at the routine diagnostic laboratory. Parasite densities were quantified in thin blood films as the percentage of infected erythrocytes. Low parasite densities were referred to as < 0.1%. Genotyping. Purification of DNA was performed by phenol-extraction. Plasmodium falciparum infections were first confirmed with a species-specific PCR method that targets the ssRNA genes.5 Positive samples were then analyzed with a nested PCR method that includes amplification of three genetic regions: msp-1 block 2, msp-2, and glurp.6 These regions are characterized by short sequences repeated a variable number of times in different parasite lines, thus representing length polymorphism. The three allelic types of the
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TABLE 1 Associations between multiclonal Plasmodium falciparum infections and clinical parameters Univariate analysis No.
Native origin Endemic Non-endemic Residency latest 5 years Endemic Non-endemic Gender Female Male Antimalarial drug No Yes Self treatment No Yes Travel time (days) ⱖ21 10,000 parasites/l) in which unspecific bands (due to high amount of DNA) were detected within one of the three markers, the infections were considered to be of single genotypes (e.g., 1 msp-1 K1 type, 2 msp-2-FC type, and 1 glurp ⳱ 1 genotype). This was a conservative approach that may have underestimated the number of genotypes. Univariate analyses testing the proportion with more than one genotype over different variables were done with chisquare tests. Multiple logistic regression was used to identify factors explaining the variation in the risk of having more than one genotype. Associations between different dhfr mutations were analyzed with McNemar’s test and correlations between mutations and proguanil intake were analyzed with Fisher’s exact test.
RESULTS The 107 patients had visited 38 malaria-endemic countries, mainly in sub-Saharan Africa (95), but also in Asia (9) and South America (3). The most frequently visited countries were the Gambia (17), Ghana (15), and Kenya (14). Although 49 patients originated from malaria-endemic areas (47 in Africa), a majority of them (39) had lived the last five years or more in a non-endemic area, mainly Sweden. Six patients were living in malarious areas but were in Sweden at the time of the malaria episode. Among the patients normally residing in Sweden, the time spent in the endemic area varied (7 days to 2 years, median ⳱ 32 days). Most patients (91) experienced mild episodes of malaria. Sixteen patients, however, were treated at the Intensive Care Unit (ICU) due to cerebral malaria, hyperparasitemia, or other severe manifestations. Multiple P. falciparum genotypes were detected in 68 patients (64%), i.e., 26 with two genotypes, 28 with three, 9 with four, and 5 with five. Single genotypes were detected in 39 patients. The number of genotypes was then correlated to several host characteristics. Multiple infection was associated with five characteristics in the univariate analysis (Table 1). After including these variables in a multiple logistic regression analysis, only three variables showed an important influence on multiplicity in this analysis, without interaction, namely, long duration of travel (> 21 days), absence of antimalarial drug intake, and high parasitemia (> 0.1%). The six patients living in an endemic area and only temporarily visiting Sweden were excluded from the univariate analysis of time in an endemic area, as well as from the consequent multiple regression analysis.
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TABLE 2 Patterns of mutations in the dihydrolate reductase (dhfr) gene detected in patients with and without proguanil prophylaxis Proguanil intake dhfr mutations*
No
1 9 8 2 1 0 4
2 6 1 1 4 9 7
3 15 9 3 5 9 11
25
30
55
108, 51, 59, 164 108, 51, 59 108, 51 108, 59 108 No mutation Mixed types Total
Total no. of patients
Yes
* Mutations 51, 59, and 164 were found only in combination with 108.
The mean number of genotypes in the patients without chemoprophylaxis was 2.7. Thus, only six of the 38 patients without intake of antimalarials had single genotypes. However, three patients had four or five genotypes despite intake of chemoprophylaxis. The patients treated at the ICU did not differ in allelic types or multiplicity, even when corrected for parasite densities. Single genotype infection was mainly associated with intake of antimalarial drugs (P < 0.001) (Table 1). Intake of antimalarial drugs before hospital admittance was reported by 68 patients. Chemoprophylaxis was reported by 60 patients, i.e., chloroquine and proguanil combined (42), chloroquine alone (14), proguanil alone (2), or mefloquine alone (2). Previous treatment of suspected malaria episode was reported by 18 patients, mainly with chloroquine (8). Approximately half (30) of the patients who had taken prophylaxis reported poor compliance or intake of an antimalarial drug normally not recommended for the visited area, but type of prophylactic drug, level of compliance, or self-treatment did not per se affect the number of infecting genotypes. Analysis of dhfr mutations was performed in 55 randomly selected patients. Multiple infections, i.e., both wild and mutant types in any of the positions, were detected in 11 patients and were not included in the statistical analysis. Mutations in other positions than 108 were found only in combination with Asn-108 (Table 2). Thus, among the 35 patients with the Asn108 mutation, five had Leu-164, 27 had Ile-51, and 21 had Arg-59 mutations. Of the patients with quadruple mutations, two had traveled to Kenya, two to Ghana, and one to Thailand. Val-16 or Thr-108 mutations were not detected in any samples. The presence of Asn-108 and Ile-51 mutations was associated with the intake of proguanil (P ⳱ 0.002, respectively) (Table 3). A similar tendency was found for Arg-59 but this was not significant (P ⳱ 0.4). All patients who had taken proguanil had parasites with the Asn-108 mutation, except for
one patient who had mixed infection with both wild type Ser-108 and mutant Asn-108. However, this patient reported poor compliance. Among the samples with Asn-108, there was no significant correlations of proguanil intake and Ile-51 or Arg-59 mutations (P ⳱ 0.2 and P ⳱ 0.7, respectively). One patient in the group with proguanil prophylaxis had a single mutation, Asn-108. All others had at least one additional mutation (Table 2). However, multiple mutations were also frequent in patients without previous prophylaxis (Table 2). Thus, two of the three samples with quadruple mutations were found in this group. The allelic types of msp-1 block 2, msp-2, and dhfr mutations, as well as multiplicity, were equally distributed with regards to the different geographic regions: Africa (divided into East and West Africa at longitude 15°W), Asia, and South America. DISCUSSION Plasmodium falciparum infections in travelers returning to Sweden were mainly composed of multiple genotypes, especially in the absence of antimalarial chemoprophylaxis. The risk of travelers acquiring malaria infection in sub-Saharan African countries has been estimated to be 0.1–1%.8 It can therefore be assumed that malaria-infected short-term travelers have normally been inoculated by only few and most probably only one infective mosquito. Our findings therefore suggest that several genetically diverse parasites may be transmitted in single inoculations. In a high-transmission area, where individuals generally harbor polyclonal infections, mosquito blood meals are often composed of several different genotypes.9–11 During sexual reproduction of the parasite in the mosquito, a mixture of gametocytes of genetically distinct clones will generate new genetic variants through recombination events.9,12–14 However, the resulting genetic diversity of the subsequent sporozoite inoculation and eventually the blood stage infection is unknown, since in an endemic area, infections may include accumulation of parasites both in the mosquito and the human host.15 In our study, the genetic diversity of P. falciparum infections observed in short-term travelers implies that sporozoite inoculations may often be polyclonal. Similar conclusions were recently drawn in a detailed cloning study with analyses of a few P. falciparum isolates from travelers.16 The true diversity of sporozoite population may even be underestimated because not only chemoprophylaxis, but also immunity and natural loss, may reduce the number of diverse clones that eventually reach the blood and will be detected by our analyses. Moreover, the whole parasite population might not be represented in the peripheral blood at the time of sampling.17 Single genotype infections were found mostly in Swedish tourists of non-endemic origin. This group took more often
TABLE 3 Frequency of dihydrofolate reductase mutations in relation to proguanil prophylaxis* 108 Proguanil intake
Ser (wt)
Asn (mt)
Yes No
0 9
21 14
51 P
0.002
Asn (wt)
Ile (mt)
3 14
18 9
59 P
0.002
Cys (wt)
Arg (mt)
9 14
12 9
* By Fisher’s exact test. Double types were excluded. wt ⳱ wild type; mt ⳱ mutant type; NS ⳱ not significant.
164 P
NS
Ile (wt)
Leu (mt)
20 21
1 2
P
NS
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chemoprophylaxis and were most probably less exposed to malaria transmission than individuals of African origin, who reported longer duration of travel, often to more remote areas, and less protection against mosquitoes. Information about travel conditions was not sufficiently available for statistical analysis of these variables. However, the absence of antimalarial prophylaxis turned out to be the most important factor for multiple genotypes in the logistic regression analysis. Nevertheless, three patients were infected with four or five alleles despite chemoprophylaxis, again emphasising the potential diversity of single inoculations. The epidemiology of genetic diversity of antigens as well as chemotherapeutic targets such as dhfr is now receiving increased attention. The different allelic types of msp-1 and msp-2, as well as the different dhfr mutations, appear to be present in all the different geographic areas visited, but the sample size was too small for a more detailed analysis. However, in contrast to previous findings in endemic areas,18,19 no association was found between severity of disease and allelic types of msp-1 block2 or msp-2. The Asn-108 mutation appeared to be essential for resistance to proguanil prophylaxis. However, there was no significant evidence for an additional effect of Ile-51 and Arg-59 mutations. In laboratory-cultured parasite lines, a single Asn108 mutation has resulted in pyrimethamine resistance, whereas the accumulation of mutations (Asn-108, Ile-51, and Arg-59 and/or Leu-164 or Thr-108 and Val-16) was necessary for high levels of cycloguanil resistance.20,21 In endemic areas, the in vivo effect of chlorproguanil26 and the in vitro activity of cycloguanil22–26 have been associated with Asn-108 but generally in combination with Ile-51 and/or Arg-59. However, the importance of these additional mutations is not fully clear. They may appear only in combination with Asn-108,7,27 but without necessarily causing higher resistance than single Asn108 mutations.21,25 A Leu-164 mutation was rare and Val-16 and Thr-108 mutations were not detected in our patients. This confirms that these mutations are generally rare.27,28 A wide spectrum of dhfr mutations was also observed in the patients without previous proguanil prophylaxis. This most likely reflects a frequent natural occurrence of dhfr mutations in P. falciparum parasites and may be related to previous use of pyrimethamine-sulfadoxine in a specific area.7,27,28 Surprisingly, the Leu-164 mutation was found in four patients who had traveled to East and West Africa. This mutation has previously been reported only in two samples from Africa.29 The spread of the dhfr 164 mutation is believed to be a major threat to the use of antifolate drugs for treatment of malaria in Africa. To our knowledge, this is the first study in which dhfr mutations have been studied in relation to intake of proguanil prophylaxis. Previous in vivo studies on the association between antifolate resistance and dhfr mutations have only considered the blood stage effect of antifolate drugs and often in combination with synergistic sulfa components.22,26 However, the truly causal prophylactic effect of proguanil on the liver stage of the parasite may represent a highly separate effect than what is assessed by the blood stage. Furthermore, recent in vitro30 and in vivo31 observations suggest that proguanil may exhibit an antimalarial effect against the blood stage by a mode of action different and independent of the antifolate inhibition of its active metabolite cycloguanil. This in turn may not be relevant for the causal prophylactic effect, since
the antimalarial effect on the liver stage has been assigned to cycloguanil rather than proguanil.20 Mutations in the dhfr gene have been related to reduced activity of cycloguanil rather than proguanil.30 Therefore, our findings may indicate that cycloguanil accounts for the main prophylactic effect in the liver. If cycloguanil is critical for this effect, individuals with low concentrations of cycloguanil, i.e., poor metabolizers with mutations in the cytochrome-P450 CYP2C19 gene, may be less protected. However, we do not believe that this may have affected the results in our study group since these mutations are not common among Caucasians and Africans.32 In conclusion, our study shows that travelers with restricted time in an endemic area are often infected with several genetically diverse parasites. Chemoprophylaxis reduces the number of diverse parasites and selects for resistant parasites. The finding of point mutations in the dhfr gene in all patients with prior proguanil prophylaxis, as well as the reduced number of P. falciparum clones in these patients, provide convincing evidence of selection of drug-resistant parasites. Acknowledgments: We are most grateful to the patients and staff at the Department of Infectious Diseases and the Diagnostic Laboratories for Parasitology at Danderyd and Huddinge University Hospitals. Special thanks are given to Kerstin Engstro¨ m, Berit Emilsson, Gunilla Herrman, Lillemor Karlsson, Johanna Tegerstro¨ m, and Emilie Wahren for clinical and laboratory assistance. We also acknowledge Georges Snounou for the PCR method and the oligonucleotide primers and Jan Kowalski for statistical assistance. Financial support: The study was funded by the Swedish International Development Agency (Swe 95-076) and the Swedish Medical Research Council (K99-16X-000172-35A). Authors’ addresses: Anna Fa¨ rnert, Karolin Tengstam, Ulf Bronner, and Anders Bjo¨ rkman, Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Karolinska Hospital, SE-171 76 Stockholm, Sweden, Telephone: 46-8-517-7000, Fax: 46-8-517-71806. Ingela Berggren Palme, Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Huddinge, Sweden, Telephone: 46-8-585-8000. Marianne Lebbad, Department for Parasitology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden, Telephone: 46-8457-2526. Go¨ te Swedberg, Department of Pharmaceutical Biosciences, Division of Microbiology, Uppsala University, Box 581, SE751 23 Uppsala, Sweden, Telephone: 46-18-471-4619. Reprint requests: Anna Fa¨ rnert, Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden, Telephone: 46-8-517-7000, Fax: 46-8517-71806; E-mail:
[email protected].
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