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    Time-line scheme of genetic evidence for Plasmodium falciparum resistance to chloroquine and pyrimethamine in Indochina and the Western Pacific. Genotypes of A, the Plasmodium falciparum chloroqunie resistance transporter (pfcrt) gene and B, the dihydrofolate reductase (dhfr) gene obtained in this study and previous reports are combined. Genotypes of pfcrt and dhfr identified in this study are indicated by a star and those from cultured parasites are shown with the name of parasite strain in matching colors. Frequencies of pfcrt and dhfr genotypes are shown in pie charts with reference number in parenthesis. The blue arrow in B indicates the period when sulfadoxine-pyrimethamine (SP) was used as a first-line treatment in Thailand.

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Genetic Evidence for Plasmodium falciparum Resistance to Chloroquine and Pyrimethamine in Indochina and the Western Pacific between 1984 and 1998

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  • 1 Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan; Laboratory of Malariology, International Research Center of Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; Faculty of Human Care, Teikyo College, Tokyo, Japan; Department of Appropriate Technology Development and Transfer, Research Institute, International Medical Center of Japan, Tokyo, Japan; Research Unit for Advanced Preventive Medicine, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan

Plasmodium falciparum resistance to chloroquine and pyrimethamine is widely distributed in malaria-endemic areas. The origin and geographic spread of this drug resistance have been inferred mainly from records of clinical resistance (treatment failure). Identification of the Plasmodium falciparum chloroqunie resistance transporter (pfcrt) gene and the dihydrofolate reductase (dhfr) gene as target genes of chloroquine and pyrimethamine, respectively, has made it possible to trace the history of genetic resistance to these two drugs. However, evidence for genetic resistance has been limited because of scarcity of archival specimens. We examined genotypes of pfcrt and dhfr in Indochina (Thailand, Myanmar, and Laos) and the Western Pacific (the Philippines, Indonesia, and Papua New Guinea) between 1984 and 1998 by testing samples obtained from malaria cases imported to Japan. Results show that 96% (28 of 29) and 77% (20 of 26) of samples had resistant genotypes of pfcrt and dhfr, respectively, substantiating the inferred history of clinical resistance in these geographic areas during this period.

INTRODUCTION

Drug resistance imposes a serious burden in the treatment of Plasmodium falciparum malaria in tropical regions. Chloroquine (CQ) resistance in P. falciparum first appeared almost simultaneously in Southeast Asia and South America in the late 1950s.1 In Southeast Asia, failure of CQ treatment was reported in all countries in Indochina during the 1960s, with CQ resistance rapidly reaching the Western Pacific and Africa by the early 1970s. CQ resistance is currently prevalent in all malaria-endemic areas, except the Caribbean, China, and the Middle East.2

Sulfadoxine-pyrimethamine (SP) was used as an alternative drug for treatment of CQ-resistant malaria in Indochina in the late 1960s.3 However, resistance to SP rapidly emerged in malaria-endemic areas where CQ was replaced with SP.4 In Thailand, SP was used as a first-line treatment in the early 1970s,5,6 but it was abandoned in the early 1980s because of increased treatment failures.6,7 Laos and Myanmar also introduced SP in the early 1970s, but stopped its usage recently.6 SP was rarely used in Cambodia, Vietnam, and Malaysia for treatment of CQ-resistant malaria, but resistance to SP has also been prevalent in these countries.4,6 Resistance to SP was also observed in the Western Pacific countries of Indonesia, Papua New Guinea, and the Philippines in the 1980s.4 Thus, SP resistance is now highly prevalent in Indochina and the Western Pacific.2

The molecular basis of CQ and pyrimethamine (Pyr) resistance has been well characterized in P. falciparum.8 The target gene of CQ resistance is the P. falciparum CQ resistance transporter (pfcrt) gene, which encodes a putative transporter localized in the digestive vacuole membrane of the parasite. Among 10 amino acid substitutions within pfcrt identified among drug-resistant parasites, replacement of lysine with threonine at amino acid 76 is essential for conferring resistance to CQ.8 The target gene of Pyr is the dihydrofolate reductase (dhfr) gene, which encodes dihydrofolate reductase, a key enzyme in the folate biosynthetic pathway. Six amino acid substitutions within dhfr have been reported among Pyr-resistant parasites.9,10 An essential mutation for resistance is a substitution of serine with asparagine at amino acid 108.10,11 Additional point mutations (at 16, 51, 59, and 164) are associated with increased levels of Pyr resistance in vitro.10,11

Sequence polymorphisms occur in the pfcrt and dhfr loci in field isolates of P. falciparum, and distributions of polymorphisms differ among geographic areas.1215 Confining the polymorphisms to Indochina and the Western Pacific, a pfcrt genotype of CVIET at positions 74, 75, and 76 (mutated residues are underlined) represents the most common CQ-resistant type in Indochina, and a pfcrt genotype carrying SVMNT at positions 72 and 76 is observed predominantly in the Western Pacific.12,13 In the Pyr-resistant dhfr polymorphism, triple (CIRNI at 51, 59, and 108) and quadruple (CIRNL at positions 51, 59, 108, and 164) mutants are prevalent in Indochina, and the double mutant (CNRNI) is the major dhfr genotype in the Western Pacific.14,15 Migration of genotypes conferring resistance to CQ and Pyr from Indochina to Africa have also been demonstrated.16,17

Previous evidence for drug resistance of P. falciparum has been derived from records of clinical resistance (treatment failure) and/or in vitro drug sensitivity tests. Obtaining genetic evidence for resistance to CQ and Pyr was not feasible until after the identification of target genes and mutation(s) involved in the drug resistance.4 It should be emphasized that genetic resistance and clinical resistance are not always consistent because selection of drug-resistant parasites results from the interplay of the parasite, drug, and human host, and is largely influenced by immune factors and the pharmacokinetics of the drugs.18,19

In areas highly endemic for malaria, such as tropical Africa, CQ and Pyr are still effective, although only partially, in persons infected with drug-resistant genotypes largely because of their immunity, which is acquired after repeated infections.20,21 In addition, discrepancies between in vitro test results and genotypes have been reported: isolates carrying resistant genotypes sometimes show susceptibility to drugs, and vice versa.22 Thus, the history of P. falciparum genetic resistance to drugs remains elusive. We therefore consider it is important to obtain genetic evidence of P. falciparum drug resistance to further understand the history of parasite resistance to these drugs.

In this study, we investigate polymorphisms in the pfcrt and dhfr loci in Indochina and the Western Pacific between 1984 and 1998 using archival samples. Results obtained show a high prevalence of resistance to CQ and Pyr during this period, extending back before genetic resistance was first reported in these areas, and substantiating the inferred history of clinical resistance in these geographic areas during this period.

MATERIALS AND METHODS

Parasite samples.

Blood samples used in this study were collected as part of a national surveillance system of imported malaria cases in 50 hospitals in Japan between 1984 and 1998, and stored as blood smears. All cases were diagnosed by examination of Giemsa-stained blood smears by two experienced microscopists. The total number of samples examined was 588, with 30–60 samples per year. Annual reported cases of imported malaria in Japan were approximately 100 during this period.23 Among the 588 cases, 229 samples were positive for P. falciparum, 347 for P. vivax, 7 for P. ovale, and 5 for P. malariae. Of the 229 P. falciparum samples, we examined 55 single P. falciparum infections that originated in Southeast Asia and the Western Pacific in this study. The remaining 174 cases from Africa, South Asia, and South America will be analyzed elsewhere.

Extraction of DNA.

Parasite DNA was extracted according to the method of Kimura and others.24 Briefly, Giemsa-stained slides were dipped in xylene and then in methanol to remove the immersion oil and dye. Each blood smear was scraped off a destained slide with an edge of a clean glass slide, and subjected to DNA purification using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). To avoid cross-contamination of parasite DNA sample by sample, each scraping off was done using a fresh glass slide on a plastic dish, which were disposed immediately after scraping off. A non-infected blood smear was also used as a negative control in the DNA extraction and polymerase chain reaction (PCR) amplification procedures. The purified DNA, eluted in 100 μL of elution buffer provided with the kit, was stored at 4°C.

Polymerase chain reaction.

We amplified a 190-basepair region in the second exon of pfcrt, which contained the polymorphic regions of amino acid residues 72–76, by using the nested PCR method described by Djimde and others.25 Primers were the same as those previously described.25 For amplification of dhfr, we targeted three regions of approximately 190 basepairs that covered four polymorphic residues involved in Pyr resistance. The PCR amplification was performed using Phusion™ high-fidelity DNA polymerase (New England Biolabs, Beverly, MA) in a 50-μL reaction mixture containing 1 μL of extracted DNA, 1 μL (10 mM) of each dNTP mixture (0.2 mM each), 10 μL of 5× Phusion HF buffer, 0.5 μL of DNA polymerase, and 0.5 μM of primers described below.

The regions and primers used were 1) a fragment flanking amino acids 51 and 59 with outer primers 21F (5′-GCC ATA TGT GCA TGT TGT AAG GTT GAA AGC-3′) and 22R (5′-CTT ATA TTT CAA TTT TTC ATA TTT TGA TTC-3′), and inner primers 23F (5′-TGT TGT AAG GTT GAA AGC AAA AAT GAG GGG-3′) and 24R (5′-TTT TTC ATA TTT TGA TTC ATT CAC ATA TGT-3′); 2) a fragment flanking amino acid 108 with outer primers 25F (5′-TGT AAA TAT TTA AAC AAA GAA ACT GTG GAT-3′) and 26R (5′-TTC ATC AAA ATC TTC TTT TTT TAA GGT TCT-3′), and inner primers 27F (5′-GAA ACT GTG GAT AAT GTA AAT GAT ATG CCT-3′) and 28R (5′-TTC TTT TTT TAA GGT TCT AGA CAA TAT AAC-3′); and 3) a fragment flanking amino acid 164 with outer primers 29F (5′-GAA GAT TTT GAT GAA GAT GTT TAT ATC ATT-3′) and 33R (5′-AAA TAC ATC ACA TTC ATA TGT ACT ATT TAT-3′), and inner primers 31F (5′-GTT TAT ATC ATT AAC AAA GTT GAA GAT CTA-3′) and 34R (5′-ACA TTC ATA TGT ACT ATT TAT TCT AGT AAA-3′). The PCR conditions to amplify the pfcrt and dhfr fragments were 98°C for 30 seconds, then 98°C for 10 seconds, 45°C for 20 seconds, and 72°C for 20 seconds for 40 cycles for the first amplification, and 98°C for 30 seconds, then 98°C for 10 seconds, 50°C for 20 seconds, and 72°C for 20 seconds for 30 cycles for the second amplification. Amplified fragments were subjected to direct sequencing with an ABI3730xl DNA sequencer (Bio Matrix Research, Inc., Tokyo, Japan). One sample isolated from the Philippines in 1998 had a mixed infection, showing both a wild-type and CQ-resistant pfcrt genotype (CVIET), as detected by double peaks in an electropherogram. Sequences were confirmed by sequencing two independent amplicons obtained from individual original templates.

RESULTS

Polymorphism in pfcrt.

Of the 55 samples subjected to PCR, amplification of the pfcrt fragment was successful for 29 samples. The low rate of successful amplification was probably caused by low parasite numbers in the blood smears, many of which had parasitemias < 0.03%. Thus, the success rate was 64% for samples with parasitemias > 0.03%, and 33% for those samples with parasitemias < 0.03%, which is consistent with a low rate of successful amplification using blood smear samples with low parasitemias.24 The polymorphisms observed are shown in Table 1. Records of previously reported pfcrt genotypes are combined with the present results and shown in a time-line scheme (Figure 1A). All of our samples showed a CQ-resistant pfcrt genotype, with the exception of a sample isolated from the Philippines in 1985. The CVIET CQ-resistant pfcrt genotype was present in samples from Myanmar and Laos collected in 1994. This date is five years earlier than the first reported record of this CQ-resistant pfcrt polymorphism in those two countries.6

In samples from Thailand, the CVIET genotype was detected in 1984, 1991, and 1992. An earlier presence of this resistant genotype has been noted in some culture-adapted parasite strains isolated from Thailand: the K1 strain isolated in 1979,26 the Indochina III strain in 1984,27 and the TM284 strain in 1990.28 The prevalence of this CQ-resistant genotype was 100% in 1995 in Thailand.6,29 Thus, our results suggest the persistence of this resistant genotype in Thailand from at least 1979 to the present time. The CVIDT polymorphism, a variant of CVIET, was reported in Cambodia in 2001 and 2004,30,31 and was also detected in two of our 1998 samples from Laos and/or Thailand. These findings suggest an earlier presence of this resistant genotype in central Indochina (Laos/Thailand/Cambodia) than previously reported. The Papua New Guinea form of CQ-resistant pfcrt genotype (SVMNT), was not detected in our limited samples from Indochina.

In the Western Pacific countries (Indonesia, Papua New Guinea, and the Philippines), most samples (n = 16) showed the CQ-resistant pfcrt polymorphism SVMNT. This genotype was detected in a sample from Indonesia from 1991. This date is much earlier than previous records from Indonesia collected in 1999 and 2002.32,33 We identified another pfcrt genotype (CVMNN) from Indonesia in 1986 and it is reported that this CVMNN mutant exhibits resistance to CQ in vitro.34 In Papua New Guinea, all samples (n = 8) isolated between 1986 and 1998 showed the SVMNT pfcrt genotype, which is consistent with the report by Mehlotra and others.35 In contrast, in samples collected from Papua New Guinea between 1956 and 1965, all carried the wild-type pfcrt genotype.36 Our results, together with these records, suggest that the SVMNT genotype, which appeared before 1982, persisted until 1998 (and probably until now). In samples from the Philippines, the SVMNT genotype was detected in samples from 1985, six years earlier than the first record of this type in 1991.37 We also detected the wild-type (CVMNK) pfcrt genotype in samples from the Philippines in 1985 and 1998. A substantial parasite population showed the wild-type pfcrt genotype present in 1997,29,38 which suggested the persistence and co-prevalence of both CQ-resistant and CQ-sensitive parasite populations in the Philippines.

Polymorphism in dhfr.

Of the 29 samples sequenced for pfcrt, 26 samples were successfully sequenced for three dhfr fragments, encompassing four polymorphic sites. Results are shown in Table 1 and Figure 1B. The triple mutant Pyr-resistant dhfr genotype CIRNI was present in samples from Thailand collected in 1984 (n = 2). This triple mutant dhfr is identical to the genotype of the Indochina III strain in 1984 (Figure 1).27 Thus, the CIRNI genotype was already prevalent as early as 1984 in Thailand. In Laos, the double mutant Pyr-resistant dhfr genotype CNRNI was first reported in 1999.14 In our study, this double mutant was detected in isolates from Laos from 1994 and 1998 (n = 4), which suggested the presence of this double mutant in Laos at least five years earlier than previously recorded. In Myanmar, one sample isolated in 1997 was wild-type (CNCSI). However in 1999, dhfr polymorphism reportedly consisted of 90% Pyr-resistant genotypes (CNRNI, CNRNL, and CIRNL).14 It is not known whether a Pyr-resistant dhfr genotype was present in Myanmar before 1999.

In the Western Pacific, the triple mutant dhfr genotype was not found, but the wild-type, single, and double mutant dhfr genotypes were detected. Wild-type dhfr was present between 1985 and 1990 in Papua New Guinea (n = 3) and the Philippines (n = 2). The Pyr-resistant dhfr (single mutant) genotype CNCNI was detected in samples from Indonesia, Papua New Guinea, and the Philippines between 1985 and 1986, and also in one isolate from Papua New Guinea in 1998. The double mutant dhfr (CNRNI) was obtained from samples from Indonesia in 1991, Papua New Guinea in 1987, and the Philippines in 1985. These dates are much earlier than the first record of this double mutant type in 1996 from Indonesia and Papua New Guinea.39,40 In addition, the presence of these Pyr-resistant dhfr mutant genotypes have not been previously reported in the Philippines.

DISCUSSION

The aim of this study was to obtain genetic evidence of P. falciparum drug resistance to CQ and Pyr in Indochina and the Western Pacific between 1984 and 1998, during which time reports of pfcrt and dhfr genotypes have been limited.35,36 Our results obtained with archival samples present genetic evidence of resistance of this parasite to CQ and Pyr during this period. Most of our samples (96%, 28 of 29) had a CQ-resistant pfcrt genotype, and there was a clear geographic separation of two resistant genotypes: CVIET in Indochina and SVMNT in the Western Pacific (Figure 1). Genetic resistance to Pyr was somewhat lower in frequency (77%, 20 of 26) than CQ resistance. The fact that Pyr was introduced in Indochina approximately 20 years later than CQ for treatment failure of P. falciparum malaria is consistent with the late spread of Pyr resistance in these areas.1,4 Also, single, double, and triple mutants of dhfr were detected. This situation reflects the present distribution of dhfr polymorphism in these areas (Figure 1).

These results of genetic evidence for drug resistance are generally consistent with the history of clinical resistance in Indochina and the Western Pacific.32,4042 Thus, the present study has substantiated a widely distributed idea that treatment failures were ascribed to genetic resistance to these drugs in Indochina and the Western Pacific. We were unable to find an association of genetic resistance with clinical resistance in our samples because records of drug treatment were accessible to only two malaria patients: one who traveled to Thailand in 1992, and the other who traveled to Papua New Guinea in 1998 (Table 1). The first patient cured after receiving quinine, and the second patient, who had parasites of the CVIET type CQ-resistant pfcrt genotype and the CIRNI type Pyr-resistant dhfr genotype, died after being treated with quinine and SP.

Additionally, when combined with results of previous reports, our study has three interesting findings. First, a CQ-resistant pfcrt genotype (SVMNT) was co-prevalent with a CQ-sensitive genotype in the Philippines in 1998, which is consistent with a relatively high prevalence (30%) of the CQ-sensitive pfcrt genotype in 1997.29 Treatment with CQ is still effective in more than half of P. falciparum-infected patients in this country.43 Notably, the persistence of this CQ-sensitive pfcrt genotype is in sharp contrast to countries, such as Thailand, the Solomon Islands and Vanuatu,6,44,45 where there is 100% prevalence of the CQ-resistant pfcrt genotype. Second, the CVIET pfcrt genotype was present in 1998 in the Philippines. Together with reports showing the same type in 1991 and 2002,37,38 this finding suggests the persistence of this resistant type throughout the 1990s in the Philippines. This CQ-resistant form of pfcrt was also reported in Indonesia in 1999 and 2002.32,33 It remains to be clarified whether the CVIET genotype originated independently in the Western Pacific or was imported from Indochina.38 Third, the CVMNN pfcrt genotype was present in Indonesia. One field isolate of this type was reported from Indonesia in 2002.33 Importantly, the CVMNN mutant, which was obtained from in vitro culture under CQ pressure, showed resistance to CQ.34 The pfcrt mutant carrying N at residue 76, an amino acid change other than T that results in CQ resistance, can occur in field isolates.

The efficacy of Pyr in treatment of persons with P. falciparum infections and the prevalence of both wild-type and Pyr-resistant genotypes of dhfr currently vary in the countries of Indochina and the Western Pacific.14,15,4042 Together with these reports, our present finding of the three resistant dhfr genotypes (single, double, and triple mutants), as well as the wild-type genotype, between 1984 and 1998 may reflect different histories of the use of Pyr in these areas. Thailand is the only country that introduced SP as a first-line treatment in the mid 1970s in Asia.5,6 However, in vivo/in vitro resistance to SP reached 100% in the 1980s.7 As a result, the drug policy of Thailand was then switched to mefloquine as a first-line treatment in the mid 1980s.6 We identified dhfr triple mutants in two Thai samples collected in 1984, which is consistent with the change of drug policy in the mid 1980s. We did not detect the dhfr quadruple mutant, which currently accounts for the highest population of dhfr mutants in Thailand,15 in our archival samples. The quadruple mutant was first identified in samples collected from Thailand in 1995.14 We report of dhfr polymorphism in the Philippines, which shows the wild-type and resistant-type dhfr genotypes (single and double mutants) in the 1980s and 1990s.

In conclusion, our analysis of archival samples shows genetic evidence for a wide distribution of P. falciparum resistance to CQ and Pyr in Indochina and the Western Pacific during the 1980s and 1990s. It also sheds light on the history of drug resistance in these areas, supporting previous records of clinical resistance during this period.

Table 1

Polymorphisms of Plasmodium falciparum pfcrt and dhfr genes in archival samples collected from Indochina and the Western Pacific between 1984 and 1998*

pfcrt
IndochinaWestern Pacific
DateMyanmarThailandThailand/Laos †LaosIndonesiaPapua New GuineaThe Philippines
1984CVIET (2)
1985CVMNK (1)
SVMNT (2)
1986CVMNN (1)SVMNT (3)SVMNT (1)
1987SVMNT (1)
1988
1989
1990SVMNT (1)
1991CVIET (1)SVMNT (1)SVMNT (1)
1992CVIET (1)SVMNT (1)
1993
1994CVIET (1)CVIET (1)SVMNT (1)
1995SVMNT (1)
1996
1997CVIET (1)SVMNT (1)SVMNT (1)
1998CVIET (1)CVIDT (1)SVMNT (1)CVIET (1)‡
CVIDT (1)CVMNK (1)‡
Total (n = 29)2422388
dhfr
IndochinaWestern Pacific
DateMyanmarThailandThailand/Laos †LaosIndonesiaPapua New GuineaThe Philippines
* Values in parentheses indicate number of samples. Mutated residues are in bold and underlined. pfcrt = P. falciparum chloroquine resistance transporter; dhfr = dihydrofolate reductase; ND = not done.
†Persons visited both countries.
‡Mixed infection of two distinct pfcrt genotypes.
1984CIRNI (2)
1985CNCSI (1)
CNCNI (1)
CNRNI (1)
1986CNCNI (1)CNCSI (2)CNCSI (1)
CNCNI (1)
1987CNRNI (1)
1988
1989
1990CNCSI (1)
1991CIRNI (1)CNRNI (1)CNRNI (1)
1992ND (1)CNRNI (1)
1993
1994ND (1)CNRNI (1)CNRNI (1)
1995CNRNI (1)
1996
1997CNCSI (1)CNRNI (1)CNRNI (1)
1998CNRNI (2)CNRNI (1)CNCNI (1)ND (1)
Total (n = 26)1322387
Figure 1.
Figure 1.

Time-line scheme of genetic evidence for Plasmodium falciparum resistance to chloroquine and pyrimethamine in Indochina and the Western Pacific. Genotypes of A, the Plasmodium falciparum chloroqunie resistance transporter (pfcrt) gene and B, the dihydrofolate reductase (dhfr) gene obtained in this study and previous reports are combined. Genotypes of pfcrt and dhfr identified in this study are indicated by a star and those from cultured parasites are shown with the name of parasite strain in matching colors. Frequencies of pfcrt and dhfr genotypes are shown in pie charts with reference number in parenthesis. The blue arrow in B indicates the period when sulfadoxine-pyrimethamine (SP) was used as a first-line treatment in Thailand.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 4; 10.4269/ajtmh.2008.79.613

*

Address correspondence to Yumiko Saito-Nakano, Department of Parasitology, National Institute of Infectious Diseases, Fault, Toyama 1-23-1 True, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail: yumiko@nih.go.jp

Authors’ addresses: Yumiko Saito-Nakano, Hiroshi Ohmae, and Takuro Endo, Department of Parasitology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan, Tel: 81-3-5285-1111, Fax: 81-3-5285-1173. Kazuyuki Tanabe, Laboratory of Malariology, International Research Center of Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan, Tel: 81-6-6879-4260, Fax: 81-6-6879-4262. Kiseko Kamei, Faculty of Human Care, Teikyo College, Honmachi 6-31-1, Shibuya-ku, Tokyo 151-0071, Japan, Tel: 81-3-3376-4321, Fax: 81-3-3379-0492. Moritoshi Iwagami, Kanako Komaki-Yasuda, and Shigeyuki Kano, Department of Appropriate Technology Development and Transfer, Research Institute, International Medical Center of Japan, Toyama 1-21-1, Shinjiku-ku, Tokyo 162-8655, Japan, Tel: +81-3-3202-7181, Fax: +81-3-3202-7364. Shinichiro Kawazu, Research Unit for Advanced Preventive Medicine, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho 2-13, Obihiro, Hokkaido 080-8555, Japan, Tel: 81-155-495846, Fax: 81-155-495643.

Financial support: This work was supported by a Grant-in-Aid from the Ministry of Health, Labor and Welfare (grant H17-Shinkouippan-019) of Japan.

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Author Notes

Reprint requests: Yumiko Saito-Nakano, Department of Parasitology, National Institute of Infectious Diseases, Fault, Toyama 1-23-1 True, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan, Tel: +81-3-5285-1111, Fax: +81-3-5285-1173, E-mail: yumiko@nih.go.jp.
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