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Cad. Saúde Pública vol.22 n.12 Rio de Janeiro Dec. 2006
Mutações nos genes pfmdr1, cg2 e pfcrt em isolados de Plasmodium falciparum provenientes de localidades malarígenas dos Estados de Rondônia e Pará, Amazônia Legal Brasileira
Giselle Maria Rachid VianaI; Ricardo Luís Dantas MachadoII; Vanja Sueli Pachiano CalvosaI; Marinete Marins PóvoaI
ISeção de Parasitologia, Instituto Evandro Chagas, Belém, Brasil
IIDepartamento de Doenças Dermatológicas, Infecciosas e Parasitárias, Faculdade de Medicina de São José do Rio Preto, São Paulo, Brasil
The objectives of this study were to investigate the molecular basis for Plasmodium falciparum resistance to chloroquine in isolates from the Brazilian Amazon and to identify polymorphisms in the pfmdr1 gene, codons 184, 1042, and 1246, the kappa and gamma regions of the cg2 gene, and the K76T mutation of the pfcrt gene, in order to calculate the distribution of polymorphism within each target gene, comparing samples from distinct geographic areas, using allele-specific polymerase chain reaction (PCR) for the pfmdr gene and PCR plus restriction fragment length polymorphism (RFLP) for the cg2 and pfcrt genes. The sample consisted of 40 human blood isolates, already collected and morphologically diagnosed as carriers of P. falciparum parasites, from four localities: Porto Velho in Rondonia State and Maraba, Itaituba, and Tailandia in Pará State. Distribution of P. falciparum in vitro chloroquine resistance in the isolates was 100% for pfmdr1, cg2 gamma region, and pfcrt, except for the polymorphism in the cg2 kappa region, which was not found.
Plasmodium falciparum; Genetic Polymorphism; Chloroquine
O estudo foi desenvolvido para investigar a base molecular da resistência do Plasmodium falciparum à cloroquina em isolados da região Amazônica brasileira e identificar os polimorfismos nos códons TYR184PHE, ASN1042ASP e ASP1246TYR do gene pfmdr1, as regiões kappa e gamma do gene cg2 e a mutação K76T do gene pfcrt, a fim de determinar a distribuição percentual dos alelos de cada gene estudado, comparando amostras de áreas geográficas distintas, utilizando a reação em cadeia da polimerase (PCR) alelo-específica para o pfmdr1 e a PCR e o polimorfismo do comprimento do fragmento de restrição (RFLP) para os genes cg2 e pfcrt. A amostra foi constituída de quarenta isolados de sangue humano já coletados e microscopicamente diagnosticados com malária por P. falciparum das localidades de Porto Velho (Rondônia) e Marabá, Itaituba e Tailândia (Pará). A distribuição percentual da resistência in vitro do P. falciparum à cloroquina nas amostras estudadas foi de 100% de resistência para os genes pfmdr1, região gamma do cg2 e pfcrt. O polimorfismo na região kappa do gene cg2 não foi encontrado nas amostras estudadas.
Plasmodium falciparum; Polimorfismo Genético; Cloroquina
The first references to malaria cases from chloroquine-resistant Plasmodium falciparum date to 1960 in South America and Southeast Asia, and since then drug resistance has been viewed as a public health problem in various countries of the world 1.
In Africa, chloroquine-resistant P. falciparum malaria has been expanding since the 1970s. Currently, resistance to this drug is found in nearly all the endemic countries, especially in East Africa 2,3. In Brazil, the first reports of resistance to anti-malarials date to the early 20th century, when it was observed that quinine was losing its efficacy 4. In relation to chloroquine, clinical failure is already reaching nearly 100% in the majority of endemic areas in Brazil 1,5,6,7.
The molecular basis for chloroquine resistance has been studied, but various aspects remain to be elucidated. In 1989, two homologues of the multiple drug resistant gene (mdr) were identified and denominated pfmdr1 and pfmdr2, located respectively in chromosomes 5 and 14 of P. falciparum. Polymorphisms in the pfmdr1 homologue have been associated with the chloroquine resistance phenotype 8. Although some studies have indicated a lack of association between the chloroquine resistance phenotype and point mutations in this gene 9,10, there is growing evidence of the role of the pfmdr1 gene in P. falciparum chloroquine resistance 5,6,11,12,13. Polymorphism in the Asn86Tyr codon has been related to chloroquine resistance in P. falciparum isolates in Nigeria 11, Guinea-Bissau 12, Gambia 14, and Malaysia 15. An association has also been demonstrated between polymorphism in the Asn1042Asp and Asp1246Tyr codons in the pfmdr1 gene and in vitro chloroquine resistance and absence of mutation in the Asn86Tyr codon in samples in Brazil 5.
After genetic crossing between a resistant strain (W2) and one sensitive to chloroquine (HB3), Su et al. 16 characterized the cg2 gene (region 36-kb of chromosome 7) as responsible for chloroquine resistance. The cg2 genotype is characterized by twelve point mutations and three length polymorphisms (kappa, gamma, and omega), which are associated with clones having chloroquine resistance phenotypes 16.
Currently, the study of molecular markers for resistance in malaria epidemiology focuses on polymorphism in the protein coded by the pfcrt gene (chloroquine-resistant P. falciparum related to the transporter), which contains 13 exons and is located in chromosome 7 of the parasite 17,18. Point mutations in this gene have been associated with in vitro chloroquine resistance in P. falciparum isolates from Africa, Southeast Asia, and South America, in which a mutation, substitution of lysine (K) with treonine (T) in position 76 (K76T), was present in all of the chloroquine-resistant samples and absent in all the isolates that were sensitive to this drug according to in vitro and in vivo analysis 2,19,20,21,22,23.
Despite the results of studies reporting flaws in the association between the K76T mutation of the pfcrt gene and chloroquine resistance 24,25, genetic transformation experiments with plasmids expressing mutant forms of pfcrt conferred resistance in the three different chloroquine-sensitive clones tested, thus demonstrating this gene's important role for in vitro chloroquine resistance 26.
The high rate of chloroquine-resistant P. falciparum cases in Brazil requires studies on the molecular basis of this resistance through the identification and characterization of genes with pfmdr1, cg2, and pfcrt, given that the studies conducted to date in the region (localities in the States of Amapá 5, Amazonas 7,22, Mato Grosso 6,7,22, and Rondônia 7,22) are scarce in light of the problem's geographic extension. It is equally important to produce knowledge allowing the development of new drugs and the application of anti-malarial prophylactic measures.
The objective of the current study was to investigate the molecular basis for P. falciparum chloroquine resistance in isolates from the Brazilian Amazon, by identifying polymorphisms in codons Asn1042Asp, Asp1246Tyr, and Tyr184Phe of the pfmdr1 gene, polymorphisms in (kappa) and (gamma) in the cg2 gene, and the K76T mutation in the pfcrt gene.
Material and methods
The selected study localities were Porto Velho (longitude 63° 55' 00''; latitude: 8° 40' 00''), Rondônia; Marabá (longitude 49° 07' 44.2''; latitude: 5° 23' 27.5''), Itaituba (longitude 55° 59' 27''; latitude: 4° 16' 33.3''), and Tailândia (longitude 48° 57' 07.2''; latitude: 2° 56' 06.6''), Pará, all located in the Brazilian Amazon.
In each study area, ten human blood samples were used that were already available in the blood samples bank at the Malaria Research Laboratory of the Evandro Chagas Institute (Instituto Evandro Chagas), diagnosed microscopically as P. falciparum malaria, totaling an overall sample of forty isolates (n = 40). The study was approved by the Research Ethics Committee of the Instituto Evandro Chagas.
Preparation and washing of samples
From each total blood sample, five 20µL drops were added to a fiberglass membrane measuring 2.5cm in diameter (Whatman International, Maidstone, UK), placed on Whatman filter paper measuring 5.5cm in diameter (Whatman International, Maidstone, UK). After drying the droplets at room temperature and properly identifying the samples, the membranes were stored in plastic recipients (BHL Limited, Poole, UK) at -20°C until submitting to washing according to Warhurst et al. 27.
Polymerase chain reaction (PCR)
One eighth of one drop of blood soaked into the membrane (the equivalent of 5µL of extracted DNA) and obtained with a sterile scalpel was used as the source of DNA. The following reagents were added to this part of the membrane for analysis of the pfmdr1, cg2, and pfcrt genes: 5µL of 10X buffer [tris-HCl 10mM, pH 8.3; gelatin 0.01%(p/v)]; KCl 10M; and MgCl2 15mM) (Bioline, Massachusetts, USA cat. M95801B); 2 to 2,5µL of each specific initiator for each region of the target gene; 0.75 to 1.0µL of each dNTP (2.5µmol Pharmacia Biotech) (final concentration 200µM); 0.25µL of Taq polymerase (Biotaq 5U/µL: 550 units Bioline M95801B); and sterile distilled water q.s.p. 50µL/tube. The initiator sequences and PCR conditions for codons 184, 1042, and 1246 of the pfmdr1 gene were conducted according to Grobusch et al. 13 and Adagu & Warhurst 28, for the kappa and gamma of the cg2 gene according to Adagu & Warhurst 28, and for the K76T mutation of the pfcrt gene according to the protocol by Christopher Plowe (University of Maryland, USA) 26.
In addition to the test samples, size 50 or 100bp markers were used, and as controls, types HB3 (standard sensitive clone, Honduras), 7G8 (standard resistant clone, Brazil), DD2 (standard resistant clone, Southeast Asia), and K1 (standard resistant clone, Thailand).
The tubes were centrifuged rapidly at 14,000rpm and placed in a thermal cycler (Hybaid OmniGene® Thermal Cycler) to be submitted to the specific amplification conditions for each region of the target gene.
The PCR products were fractionated electrophoretically in agarose gel at 2% for codons 184, 1042, and 1246 on the pfmdr1 gene, at 3% for the kappa and gamma regions of the cg2 gene, and at 1% for the K76T mutation of the pfcrt gene (Ultra pure agarose, BRL 155517-014), at 100 volts for one hour. Later, the agarose gel was stained with ethidium bromide (5µL/mL in TBE) for 30 minutes and the resulting bands were viewed under ultraviolet light (Fluo-Link, Flowgen) and photographed in a photodocumentation system (Kodak® Edas 290).
Restricted fragment length polymorphism (RFLP)
For the kappa region of the cg2 gene, the endonuclease Tse I was used. The PCR products with a pattern similar to DD2 or K1 produced three fragments with 558bp, 90bp, and 68bp, while the isolates with a pattern similar to HB3 produced two fragments with 632bp and 68bp. For the gamma region of this same gene, the restriction enzyme was Rca 1. The PCR products with a pattern similar to 7G8 produced two bands with 194bp and 97bp, while the isolates with a pattern similar to HB3 produced three bands with 119bp, 106bp, and 45bP. For the pfcrt gene, the APO 1 endonuclease was used, and the samples with a pattern similar to 7G8 produced a fragment with 134bp, and those with a pattern similar to the HB3 type produced two fragments, with 100bp and 34bp. Digestions were conducted at 65ºC in 10µL volume, using 0.2µL of restriction endonuclease (Tse I, Rca 1, or APO 1, according to the target gene), 1µL of buffer solution, 4.8µL of sterile distilled water, and 4µL of the PCR products in each Eppendorf tube. The restriction fragments had the sizes fractionated in agarose gel at 2% at 100 volts for one hour. Later, the agarose gel was stained with ethidium bromide (5µL/mL in TBE) for 30 minutes and the bands were viewed under ultraviolet light (Fluo-Link, Flowgen) and photographed in a photodocumentation system (Kodak® Edas 290).
In relation to codons 184 and 1042 on the pfmdr1 gene, all 40 isolates showed a profile similar to type 7G8 (the standard resistant clone for Brazil) according to the allele-specific PCR technique (Figures 1 and 2). For codon 1246 on the same gene, the study samples showed a similar profile to 7G8, except for samples 808/98, 809/98, 810/98, 813/98, 815/98, 816/98, and 817/98 from the municipality of Porto Velho, which showed a profile similar to K1 (standard resistant clone for Thailand). For the kappa region of the cg2 gene, all 40 isolates showed a profile similar to HB3 (standard sensitive clone for Honduras) in both tests (PCR and RFLP) (Figure 3), while for the gamma region of this same gene, all 40 samples showed a profile similar to type 7G8 (standard resistant clone for Brazil) in both tests (Figure 4).
The mutant allele K76T of the pfcrt gene was found in all 40 samples. Amplification of a 134bp region containing codon 76 was obtained in these 40 isolates by nested PCR and confirmed by RFLP using the APO 1 endonuclease, presenting a profile similar to 7G8 (standard resistant clone, Brazil) (Table 1) (Figure 5).
The molecular mechanisms for the development of chloroquine resistance by the parasite are still not completely clear, but many candidate molecular markers have been identified 20,21,29 and there is evidence that P. falciparum chloroquine resistance is a multigenic event 2,10,16.
In relation to the pfmdr1 gene, the 40 isolates were tested for the TYR184PHE, ASN1042ASP, and ASP1246TYR mutations associated with the chloroquine resistance phenotype from samples in Africa, Asia, and South America 5,11,12,13,30,31. Although all the isolates tested for the TYR184PHE mutation presented a resistance pattern similar to 7G8 (standard Brazilian resistant clone), changes in this codon appear not to be correlated with chloroquine resistance 29. Meanwhile the presence of a profile similar to clone 7G8 in the ASN1042ASP and ASP1246TYR mutations in the samples corroborates previous studies reporting the association between polymorphisms in these codons and in vitro P. falciparum chloroquine resistance in South American samples 2,5,6. The seven samples from the municipality of Porto Velho (808/98, 809/98, 810/98, 813/98, 815/98, 816/98, and 817/98) with profile similar to K1 (standard resistant clone, Thailand), indicate that previously rare mutations in South America are now emerging on this continent 23,29.
In transfection studies, Reed et al. 32 reported that the switch from aspargine to tyrosine on codon 1246 of pfmdr1 increased the chloroquine resistance in a clone known to be resistant and carrying the pfcrt mutant. The association between pfmdr1 and in vitro chloroquine resistance has not been confirmed by all the studies 9,10, indicating that there are diverse chloroquine resistance mechanisms 10 and that the pfmdr1 gene does not play a primary role in conferring chloroquine resistance in P. falciparum 2,26.
In the k (kappa) region of cg2, the second candidate gene for the resistance phenotype to this 4-aminoquinoline in P. falciparum, a 100% sensitivity pattern was found in the isolates, although studies report an association between repetitions in the kappa region and failures in sensitivity to chloroquine in samples from Africa 28,29. These findings suggest that kappa repetitions in the cg2 gene are not important genetic markers in isolates from the Brazilian Amazon, unlike the g (gamma) region of this same gene, which presented a pattern similar to the 7G8 clone in the 40 study samples. Thus, this result together with that of Calvosa et al. 33, who found 100% in vitro chloroquine resistance in the same samples in the municipality of Marabá, suggest a significant correlation between gamma repetitions in cg2 and in vitro chloroquine resistance in samples from the Brazilian Amazon.
The presence of mutations in the pfmdr1 and cg2 genes in the same samples suggests a selective pressure for its installation and maintenance in a given geographic area, in addition to other factors favoring this drug pressure, like the malaria transmission patterns, the degree of population immunity, population migration, pharmacokinetic factors, and indiscriminate use and/or sub-therapeutic dosage of antimalarials 20,21.
Mutations in the pfcrt gene are associated with in vivo chloroquine resistance in Africa and in vitro resistance in South America 2,17,21,22,23,34 and allele transfection studies with this gene, like the K76T mutation, have shown that the latter confers in vitro resistance to chloroquine 26,35.
In this study, 100% of the study isolates in the four municipalities in the Brazilian Amazon presented the K76T genotype, similar to the 7G8 type (standard resistant clone for Brazil). This finding agrees with the work of Vieira et al. 22, who found a high prevalence of this mutation in samples from the Brazilian Amazon, although this mutation is not absolute, since it can be found both in chloroquine-resistant samples and those sensitive to this drug, suggesting that other factors control the expression of the resistance phenotype 24,25,36. Possible reasons for this fact may be associated with the involvement of the host response, like the immune status, which can cause negative conversion of parasitemia, regardless of whether the sample is resistant to chloroquine. In addition, the drug's absorption and individual metabolic rates can also influence the type of response to chloroquine treatment. Another potential factor is the possibility that other pfcrt mutations, like those in codons 72, 74, 75, 97, 144, 148, 160, 194, 220, 271, 326, 333, 356, and 371, are also associated with the resistance phenotype 34,35,37, in addition to the fact that P. falciparum has more transport proteins than those already known to influence the pathogen's physiology 30.
There is evidence that chloroquine resistance is a multigenic phenomenon and that the K76T mutation in the pfcrt gene is necessary, but not sufficient, to confer resistance 20. Therefore, other studies are needed to evaluate the role of pfcrt as a reliable genetic marker for in vivo response to chloroquine 21.
The presence of polymorphisms in the pfmdr1, cg2, and pfcrt genes in the same samples indicates that these loci are important genetic markers for in vitro chloroquine resistance 38 and that this connection is maintained by the drug's selective pressure 19.
The presence of the ASN1042ASP and ASP1246TYR mutations in the pfmdr1 gene in the samples from the Brazilian Amazon suggests that these codons are important markers for in vitro chloroquine resistance in P. falciparum isolates from South America; however, the k (kappa) region of the cg2 gene is not a relevant marker for P. falciparum resistance to 4-aminoquinoline in samples from the Brazilian Amazon. Meanwhile, the presence of polymorphism in the g (gamma) region of the cg2 gene in the study isolates suggests an association between in vitro P. falciparum chloroquine resistance and this mutation. The presence of the K76T mutation in the pfcrt gene in all the study samples suggests that this mutation is an important genetic marker for in vitro P. falciparum resistance to chloroquine. The identification of the mutations in the pfmdr1, cg2, and pfcrt loci in samples from the Brazilian Amazon suggests that they are important genetic markers for in vitro P. falciparum chloroquine resistance and that the chloroquine resistance phenotype in P. falciparum is a multigenic process, indicating a possible local clonal expansion of P. falciparum chloroquine resistance, selected by the indiscriminate or inadequate use of this drug in the Brazilian Amazon.
G. M. R. Viana, R. L. D. Machado, and V. S. P. Calvosa collected the samples. M. M. Póvoa, G. M. R. Viana, and R. L. D. Machado participated in the tests, data analysis, discussion of the results, and drafting of the article.
The authors wish to thank technicians José Maria de Souza Nascimento and José Mario Veloso Peres from the Laboratório de Pesquisas em Malária, Seção de Parasitologia, Instituto Evandro Chagas, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for providing a Master's grant to Giselle Maria Rachid Viana during her graduate studies in the Biology of Infectious and Parasitic Agents at the Universidade Federal do Pará.
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M. M. Póvoa
Laboratório de Pesquisas em Malária
Seção de Parasitologia
Instituto Evandro Chagas
Rod. BR-316, km 7, s/n
Ananindeua, PA 67030-000, Brasil
Submitted on 04/Jul/2005
Final version resubmitted on 25/Oct/2005
Approved on 08/Nov/2005