Acessibilidade / Reportar erro

Lack of quadruple and quintuple mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax isolates from Brazilian endemic areas

Abstract

BACKGROUND AND OBJECTIVE

Brazil is responsible for a large number of Plasmodium vivax cases in America. Given the emergence of P. vivax parasites resistant to chloroquine and the effectiveness of antifolates in vivax malaria treatment together with a correlation between mutations in P. vivax dhfr and dhps genes and SP treatment failure, the point mutations in these genes were investigated.

METHODS

Blood samples from 54 patients experiencing vivax malaria symptomatic episodes in the Amazonian Region were investigated. Genomic DNA was extracted using a DNA extraction kit (QIAGENTM). Nested polymerase chain reaction (PCR) amplification was carried out followed by Sanger sequencing to detect single nucleotide polymorphisms (SNPs).

FINDINGS

All tested isolates showed non-synonymous mutations in pvdhfr gene: 117N (54/54, 100%) and 58R (25/54, 46%). Double mutant allele 58R/117N (FRTNI, 28%) was the most frequent followed by triple mutant alleles (58R/117N/173L, FRTNL, 11%; 58R/61M/117N, FRMNI, 5% 117N/173L, FSTNL, 4%) and quadruple mutant allele (58R/61M/117N/173L, FRMNL, 2%). A single mutation was observed at codon C383G in pvdhps gene (SGKAV, 48%).

CONCLUSION

No evidence of molecular signatures associated with P. vivax resistance to SP was observed in the Brazilian samples.

Key words:
P. vivax; malaria; pvdhfr; pvdhps; chemoresistance


Plasmodium vivax is the most geographically widespread human malaria parasite. It is prevalent mainly outside Africa including Asia, South and Central America, and the Middle East. In the Americas, the burden of vivax malaria mostly affects Venezuela and Brazil. In Brazil, malaria transmission occurs almost entirely (> 99% of the registered cases) within the northern Brazilian Amazon Region where both P. falciparum and P. vivax infections co-exist. In this area, P. vivax is the predominant species, responsible for 89% of 194,409 malaria cases reported in 2017.11. SVS - Secretaria de Vigilância em Saúde. 2018. Available from: http://portalms.saude.gov.br/svs.
http://portalms.saude.gov.br/svs...
Nowadays, falciparum malaria is treated with a 3-day fixed Artesunate+Mefloquine combination, according to Brazilian National Malaria Program guidelines, and a radical cure for P. vivax malaria is achieved with 25 mg/kg of CQ base for three days (maximum adult dose, 1.5 g for three days), combined with a short hypnozoitocidal regimen of 0.5 mg/kg/day of primaquine (PQ) base (maximum daily dose, 30 mg/day) for seven days in patients that weighed below 70 kg. As subtherapeutic PQ doses may lead to relapse in overweight patients, weight-adjusted PQ doses are now recommended in Brazil for patients over 70 kg.

P. falciparum resistance to chloroquine (CQ) observed in the 1980s greatly contributed to the emergence of falciparum malaria outbreaks across Amazon.22. Gama BE, Lacerda MVG, Daniel-Ribeiro CT, Ferreira-da-Cruz MF. Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control. Mem Inst Oswaldo Cruz. 2011; 106(Suppl. 1): 159-66.P. vivax resistance to CQ occurred later in 1989 in Papua New Guinea33. Rieckmann KH, Davis DR, Hulton DC. Plasmodium vivax resistance to chloroquine? Lancet. 1989; 18(2): 1183-4. and CQ monotherapy was ineffective. Following this seminal observation, numerous cases of CQ resistance were reported in Southeast Asia44. Thanh PV, Hong NV, Van NV, Louisa M, Baird K, Xa NX, et al. Confirmed Plasmodium vivax resistance to Chloroquine in Central Vietnam. Antimicrob Agents Chemother. 2015; 59(12): 7411-9. and South America,55. Soto J, Toledo J, Gutierrez P, Luzz M, Llinas N, Cedeno N, et al. Plasmodium vivax clinically resistant to chloroquine in Colombia. Am J Trop Med Hyg. 2001; 65(2): 90-3.,66. de Santana Filho FS, Arcanjo AR, Chehuan YM, Costa MR, Martinez-Espinosa FE, Vieira JL, et al. Chloroquine-resistant Plasmodium vivax, Brazilian Amazon. Emerg Infect Dis. 2007; 13(7): 1125-6. thus complicating the current international efforts for malaria control and elimination, and signalling the need for alternative drugs for vivax malaria treatment.

Antifolates, most notably sulfadoxine-pyrimethamine (SP), have been used as anti-malaria for P. falciparum treatment throughout the world because this combination is inexpensive, relatively safe, and requires only a single dose course treatment. SP had been available in Brazil since 1960s to treat CQ-resistant falciparum malaria but SP-resistant P. falciparum isolates appeared since 1990; SP is not used for malaria therapy in Brazil. Although resistant to antifolates, P. falciparum treatment has been well documented in many parts of the world, and P. vivax chemoresistance to SP is scarcely studied.

Sulfadoxine and pyrimethamine are competitive inhibitors of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), the two major proteins involved in folate biosynthesis pathway77. Tantiamornkul K, Pumpaibool T, Piriyapongsa J, Culleton R, Lek-Uthai U. The prevalence of molecular markers of drug resistance in Plasmodium vivax from the border regions of Thailand in 2008 and 2014. Int J Parasitol Drugs Drug Resist. 2018; 8(2): 229-37.. Polymorphisms in these two genes are the major factors associated with SP resistance.

Data on pvdhfr and pvdhps genotypes are available for many Southeast Asian countries. Such reports remain limited for some P. vivax endemic areas, notably South America. In Brazil, only one study characterising polymorphisms in pvdhfr gene was documented8) and there is no report on the frequency of single nucleotide polymorphism (SNP) in dhps gene in P. vivax clinical isolates from Brazilian endemic areas.

Given the emergence of P. vivax CQ resistant parasites and the effectiveness of antifolates in malaria vivax treatment together with a strong correlation between mutations in P. vivax dhfr and dhps genes and SP treatment failure,99. Asih PB, Marantina SS, Nababan R, Lobo NF, Rozi IE, Sumarto W, et al. Distribution of Plasmodium vivax pvdhfr and pvdhps alleles and their association with sulfadoxine-pyrimethamine treatment outcomes in Indonesia. Malar J. 2015; 14: 365-71. the present paper reports an investigation on the pattern of point mutations in pvdhfr and pvdhps genes in Brazilian isolates.

MATERIALS AND METHODS

Parasites isolates and DNA extraction - Blood samples from Amazon Region (Acre, Amapá, Amazonas, Rondônia and Pará) were collected from 54 patients presenting with vivax malaria from 2010 to 2016 at the Laboratório de Doenças Febris Agudas, INI-IPEC, Fiocruz, the Reference Clinical Laboratory for Malaria in the Extra-Amazon to the Brazilian Ministry of Health. All the clinical isolates were diagnosed as single P. vivax infections by light microscopic examination of Giemsa’s solution-stained blood smears and by P. vivax cysteine-proteinase target gene polymerase chain reaction (PCR).1010. Torres KL, Figueiredo DV, Zalis MG, Daniel-Ribeiro CT, Alecrim W, Ferreira-da-Cruz MF. Standardization of a very specific and sensitive single PCR for detection of Plasmodium vivax in low parasitized individuals and its usefulness for screening blood donors. Parasitol Res. 2006; 98(6): 519-24. The parasitaemia ranged from 960 to 19160 parasites/µL. All malaria patients presented with clinical signs and/or symptoms of uncomplicated malaria, such as fever, headache, and chills, and the baseline characteristics were similar. No significant difference in parasitaemia was observed among the studied Brazilian localities and all the Brazilian endemic states were hypoendemic malaria areas.

Genomic DNA was extracted using a commercially available DNA extraction kit (QIAGENTM, Frankfurt, Germany), following the manufacturer’s instructions. This study was performed according to the protocols previously approved by the Ethical Research Committees of Fiocruz (32839013.6.00005248). Patients were treated with CQ plus PQ, according to the Brazilian Ministry of Health recommendation for uncomplicated vivax malaria treatment and were followed up to 42 days. No treatment failure was detected during this period.

Nested PCR and electrophoresis - Nested PCR amplification of pvdhfr and pvdhps were carried out as described previously.1111. Mint Lekweiry K, Boukhary AOMS, Gaillard T, Wurtz N, Bogreau H, Hafid JE, et al. Molecular surveillance of drug-resistant Plasmodium vivax using pvdhfr, pvdhps and pvmdr1 markers in Nouakchott, Mauritania. J Antimicrob Chemother. 2012; 67(2): 367-74. Ten point mutations were investigated: F57L/I, S58R, T61M, S117T/N and I173F/L for pvdhfr, and S382A, C383G, K512M/T/E, A553G and V585G for pvdhps. PCR products were analysed by ethidium bromide-stained agarose-gel (2%) electrophoresis.

DNA sequencing and SNPs detection - The 632 bp and 767 bp fragments generated by amplification of pvdhfr and pvdhps, respectively, were extracted and purified from gel using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) commercial kits. Briefly, the amplified fragments were sequenced using BigDye Terminator cycle sequencing ready reaction version 3.1 and ABI Prism DNA analyser 3730 (Applied Biosystems) at the Genomic Platform/PDTIS/Fiocruz.1212. Otto TD, Vasconcellos EA, Gomes LH, Moreira AS, Degrave WM, Mendonça-Lima L, et al. ChromaPipe: a pipeline for analysis, quality control and management for a DNA sequencing facility. Genet Mol Res. 2008; 7(3): 861-71. The direct DNA sequencing from PCR products were compared with the reference Sal I sequence of pvdhfr (GenBank X98123) and pvdhps (GenBank AY186730.1). Forward and reverse sequences were analysed using the free software, Bioedit Sequence Alignment Editor version 7.2.5. PCRs and DNA sequencing were randomly repeated to check possible sequence errors introduced during these stages.

RESULTS

All the 54 isolates sequenced for pvdhfr gene showed non-synonymous mutations: 117N (54/54; 100%) and 58R (25/54; 46%) mutant alleles were more frequent, while 173L (9/54; 17%) and 61M (4/54; 7%) were detected at lower frequencies. Mutation at position 57L was not found (Table I). The most common single mutant allele was 117N (27/54; 50%). This single mutant was more frequent in Acre (10/15; 66%), Amazonas (11/23; 52%) and Pará states (4/8; 50%), compared to Rondônia state (1/7; 14%), where double 58R+117N mutant was dominant (Table II). Independent of the year collection, Amazonas state showed the highest number of pvdhfr gene mutations (23/54; 42,5%), followed by Acre (15/54; 27,7%, Para (8/54; 15%) and Rondônia (7/54, 13%) (Tables III-VI). Apparently in 2011, Acre presented more pvdhfr gene mutations (7/15; 47%) than Amazonas (2/23; 8,6%) (Tables III-IV), but this difference could be related to the smaller number of Amazonas samples collected in 2011, because when percentages are compared instead of figures, 100% of Amazonas (2/2) and Acre samples (7/7) presented mutations in 2011.

The double 58R/117N allele (FRTNI, 28%) was the most common allele, contrasting with the frequencies of other dhfr double, triple, or quadruple mutant alleles, with lower frequencies: 58R/117N/173L (FRTNL, 11%), 58R/61M/117N (FRMNI, 5%), 117N/173L (FSTNL, 4%), and 58R/61M/117N/173L (FRMNL, 2%). In all localities, wild-type pvdhfr (FSTSI) was not observed (Table VII). The 58R/117N double mutant allele was detected in Acre (2/15; 13%), Rondônia (5/7; 71%), and Amazonas (8/23; 35%) while the 117N+173L only in Pará and Rondônia. The triple mutant allele 58R/117N/173L was found in all localities, except Rondônia, and the quadruple mutant 58R/61M/117N/173L was observed only in one isolate collected from Amazonas state (1/23; 4%) (Table VIII). The frequencies of double, triple, or quadruple mutants were not related to the year of collection (Tables III-VI).

TABLE I
Plasmodium vivax dhfr and dhps amino acid changes in 54 P. vivax isolates from Brazilian endemic areas
TABLE II
Number of alleles in dhfr and dhps genes observed among 54 Brazilian Plasmodium vivax isolates, according to sampling location

TABLE III
Number of alleles in dhfr and dhps genes observed among 23 Plasmodium vivax isolates from Amazônia, according to year of blood collection

Concerning pvdhps gene in 26 out of 54 (48%) isolates only a single mutation at codon C383G was detected. No other mutations, including 382A, 512M, 553G, and 585C, were found. The wild-type SCKAV (52%) and single haplotype SGKAV (48%) were observed at similar frequencies. The single mutant 383G was observed in isolates from Amazonas (13/23, 56%), Acre (8/15, 53%) and Pará (5/8, 62%) but not in isolates from Rondônia state (0/7) (Table II). Once again frequencies of pvdhps gene mutations were not related to the year of collection (Tables III-VI).

Combining pvdhfr and pvdhps alleles, only one haplotype (FRTNI for pvdhfr and SGKAV for pvdhps) was seen in three of the four study sites with a higher frequency in Amazonas state (where one pvdhfr quadruple mutant was detected) (Table IX). No pvdhfr or pvdhps quadruple or quintuple mutant haplotype, which might result in poor clinical response against antifolate drugs, was detected in any of the Brazilian localities investigated.

DISCUSSION

Mutations in pvdhfr and pvdhps genes have been found to be associated with antifolate drug resistance. Both in vivo1313. Marfurt J, de Monbrison F, Brega S, Barbollat L, Müller I, Sie A, et al. Molecular markers of in vivo Plasmodium vivax resistance to amodiaquine plus sulfadoxine-pyrimethamine: mutations in pvdhfr and pvmdr1. J Infect Dis. 2008; 198(3): 409-17. and in vitro assays suggested that these molecular markers may provide information about the trends of SP resistance in P. vivax. Here, we investigated SP resistance in vivax isolates by seeking specific point mutations in pvdhfr and pvdhps genes.

It has been postulated that pvdhfr 117N mutation might occur first, followed by S58R mutation.1414. Das S, Banik A, Hati AK, Roy S. Low prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) quadruple and quintuple mutant alleles associated with SP resistance in Plasmodium vivax isolates of West Bengal, India. Malar J. 2016; 15(1): 395-404. In this study, pvdhfr S117N was detected in all isolates followed by 58R (74%), 173L (17%), and 61M (7%) polymorphisms, supporting that S117N mutation is the first step in drug selection process. These data are similar to other observations done in areas where P. falciparum and P. vivax parasites co-exist.1414. Das S, Banik A, Hati AK, Roy S. Low prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) quadruple and quintuple mutant alleles associated with SP resistance in Plasmodium vivax isolates of West Bengal, India. Malar J. 2016; 15(1): 395-404.,1515. Ganguly S, Saha P, Chatterjee M, Maji AK. Prevalence of polymorphisms in antifolate drug resistance molecular marker genes pvdhfr and pvdhps in clinical isolates of Plasmodium vivax from Kolkata, India. Antimicrob Agents Chemother. 2014; 58(1): 196-200.

The predominance of S117N followed by the double mutant 58R/117N (28%) was also analogous to those reported in India,1515. Ganguly S, Saha P, Chatterjee M, Maji AK. Prevalence of polymorphisms in antifolate drug resistance molecular marker genes pvdhfr and pvdhps in clinical isolates of Plasmodium vivax from Kolkata, India. Antimicrob Agents Chemother. 2014; 58(1): 196-200. Afghanistan,1616. Zakeri S, Afsharpad M, Ghasemi F, Raeisi A, Safi N, Butt W, et al. Molecular surveillance of Plasmodium vivax dhfr and dhps mutations in isolates from Afghanistan. Malar J. 2010; 9: 75-82. China,1717. Huang B, Huang S, Su XZ, Tong X, Yan J, Li H, et al. Molecular surveillance of pvdhfr, pvdhps, and pvmdr-1 mutations in Plasmodium vivax isolates from Yunnan and Anhui provinces of China. Malar J. 2014; 13: 346-55. Nepal,1818. Ranjitkar S, Schousboe ML, Thomsen TT, Adhikari M, Kapel CM, Bygbjerg IC, et al. Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal. Malar J. 2011; 10: 75-82. Thailand,1919. Brega S, de Monbrison F, Severini C, Udomsangpetch R, Sutanto I, Ruckert P, et al. Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates. Antimicrob Agents Chemother. 2004; 48(7): 2581-7. Colombia,2020. Hawkins VN, Auliff A, Prajapati SK, Rungsihirunrat K, Hapuarachchi HC, Maestre A, et al. Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase. Malar J. 2008; 7: 72-83.,2121. Saralamba N, Nakeesathit S, Mayxay M, Newton PN, Osorio L, Kim JR, et al. Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia. Malar J. 2016; 15: 484-90. French Guiana1919. Brega S, de Monbrison F, Severini C, Udomsangpetch R, Sutanto I, Ruckert P, et al. Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates. Antimicrob Agents Chemother. 2004; 48(7): 2581-7. and Brazil.88. Gama BE, Oliveira NK, Souza JM, Daniel-Ribeiro CT, Ferreira-da-Cruz MF. Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates. Mem Inst Oswaldo Cruz. 2009; 104(7): 1009-11. The triple 58R/117N/173L pvdhfr mutant, not seen in P. vivax samples from Southeast Asian, where non-synonymous mutation in codon 173 comprises the change of I by F generating the 173F allele, was here detected in Amazonas, Acre, Amapá and Pará states and also in P. vivax parasites from French Guiana1919. Brega S, de Monbrison F, Severini C, Udomsangpetch R, Sutanto I, Ruckert P, et al. Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates. Antimicrob Agents Chemother. 2004; 48(7): 2581-7.,2222. Barnadas C, Musset L, Legrand E, Tichit M, Briolant S, Fusai T, et al. High prevalence and fixation of Plasmodium vivax dhfr/dhps mutations related to sulfadoxine/pyrimethamine resistance in French Guiana. Am J Trop Med Hyg. 2009; 81(1): 19-22. and Amazonas, Brazil.88. Gama BE, Oliveira NK, Souza JM, Daniel-Ribeiro CT, Ferreira-da-Cruz MF. Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates. Mem Inst Oswaldo Cruz. 2009; 104(7): 1009-11. Conversely, the non-synonymous mutation at position F57L not recorded in this study was exclusively reported in Southeast Asian samples; findings that could reflect different drug pressure history and selective processes in the old and new worlds. In fact, the genetic similarity of 173L SNP recorded for P. vivax parasites from two neighbouring South-American countries Brazil and French Guiana,1919. Brega S, de Monbrison F, Severini C, Udomsangpetch R, Sutanto I, Ruckert P, et al. Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates. Antimicrob Agents Chemother. 2004; 48(7): 2581-7. reinforce the possible existence of geographic subdivision of differentP. vivax parasites in samples from the old and new worlds.

TABLE IV
Number of alleles in dhfr and dhps genes observed among 15 Plasmodium vivax isolates from Acre, according to year of blood collection

TABLE V
Number of alleles in dhfr and dhps genes observed among eight Plasmodium vivax isolates from Pará, according to year of blood collection

Concerning the pvdhps gene, previous data indicated that mutations were mainly detected at codons A383G and A553G1414. Das S, Banik A, Hati AK, Roy S. Low prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) quadruple and quintuple mutant alleles associated with SP resistance in Plasmodium vivax isolates of West Bengal, India. Malar J. 2016; 15(1): 395-404.,2121. Saralamba N, Nakeesathit S, Mayxay M, Newton PN, Osorio L, Kim JR, et al. Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia. Malar J. 2016; 15: 484-90.,2323. Prajapati SK, Joshi H, Dev V, Dua VK. Molecular epidemiology of Plasmodium vivax anti-folate resistance in India. Malar J. 2011; 10: 102-8.,2424. Kuesap J, Rungsrihirunrat K, Thongdee P, Ruangweerayut R, Na-Bangchang K. Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand. Mem Inst Oswaldo Cruz. 2011; 106(Suppl. 1): 130-3. and suggested that these mutations alone could be responsible for reduced sensitivity to sulfa and sulfones.2525. Korsinczky M, Fischer K, Chen N, Baker J, Rickmann K, Cheng Q. Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site. Antimicrob Agents Chemother. 2004; 48(6): 2214-22.,2626. Imwong M, Pukrittayakamee S, Cheng Q, Moore C, Looareesuwan S, Snounou G, et al. Limited polymorphism in the dihydropteroate synthetase gene (dhps) of Plasmodium vivax isolates from Thailand. Antimicrob Agents Chemother. 2005; 49(10): 4393-5. In the present work, the wild-type (52%) and the mutated codon 383G (48%) were detected at similar frequencies among P. vivax isolates, similar to reports from Thai-Cambodian (53%),77. Tantiamornkul K, Pumpaibool T, Piriyapongsa J, Culleton R, Lek-Uthai U. The prevalence of molecular markers of drug resistance in Plasmodium vivax from the border regions of Thailand in 2008 and 2014. Int J Parasitol Drugs Drug Resist. 2018; 8(2): 229-37. Thai-Myanmar border (47%)2727. Thongdee P, Kuesap J, Rungsihirunrat K, Tippawangkosol P, Mungthin M, Na-Bangchang K. Distribution of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles in Plasmodium vivax isolates from Thailand. Acta Trop. 2013; 128(1): 137-43. and Indonesia (50%).99. Asih PB, Marantina SS, Nababan R, Lobo NF, Rozi IE, Sumarto W, et al. Distribution of Plasmodium vivax pvdhfr and pvdhps alleles and their association with sulfadoxine-pyrimethamine treatment outcomes in Indonesia. Malar J. 2015; 14: 365-71. Whereas, in a Colombian study investigating polymorphisms in pvdhps, the wild-type was the most frequently detected (71.6%);2121. Saralamba N, Nakeesathit S, Mayxay M, Newton PN, Osorio L, Kim JR, et al. Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia. Malar J. 2016; 15: 484-90. the same was true in India (79%)1515. Ganguly S, Saha P, Chatterjee M, Maji AK. Prevalence of polymorphisms in antifolate drug resistance molecular marker genes pvdhfr and pvdhps in clinical isolates of Plasmodium vivax from Kolkata, India. Antimicrob Agents Chemother. 2014; 58(1): 196-200. and also in Thai - Cambodian border (74%). Therefore, the pvdhps wild-type allele seems to be common in malaria endemic areas of the world, probably due to a low SP drug selection in the sympatric P. vivax populations of these countries. However, in Brazil, for example, SP or its analogues have been used for fever and antimicrobial therapy and, in this way, there continues to be a lengthy selection pressure for SP-resistant strains of P. vivax resulting to low frequencies of wild-type pvdhps parasites.

Amazonas state recorded the highest number of pvdhfr and pvdhps mutations. This finding could not be attributed to differences of antimalarial drug usage in Brazilian states because the malaria treatment in Brazil is the same all over the country. Besides that, SP has never been recommended for vivax malaria treatment and SP has been excluded from P. falciparum treatment since 1989. Thus, it is more reasonable suppose that more mutations were found in Amazonas due to the highest number of samples examined from this locality, as only one sample from Amazonas was from a border area of the Amazon with Acre - the second state that showed the greatest number of mutations. A study with a representative number of Amazonian state cases may help answer this question.

TABLE VI
Number of alleles in dhfr gene observed among seven Plasmodium vivax isolates from Rondônia, according to year of blood collection

TABLE VII
Deduced dhfr and dhps haplotype profiles in 54 Plasmodium vivax isolates from Brazilian endemic areas
TABLE VIII
Prevalence of Plasmodium vivax dhfr and dhps amino acid changes in 54 P. vivax isolates from Brazilian endemic areas
TABLE IX
Percentage of double mutant dhfr / single mutant dhps in 54 Plasmodium vivax samples according to Brazilian states

In conclusion, we found no molecular strong evidence of P. vivax SP resistance in recently collected Brazilian samples. As mutations in P. vivax dhps and dhfr genes provide a valuable tool for epidemiological surveillance of SP resistance, the prevalence of point mutations on these genetic markers of SP resistance should be assessed for providing information for future treatment policy with alternative antifolate drugs because of the appearance and dispersion of CQ resistance in malaria endemic areas.

Financial support:

POM (Fiocruz), PNCM, Secretaria de Vigilância em Saúde, Ministério da Saúde. CTDR and MFFC are recipients of a Research Productivity Fellowship from the CNPQ and FAPERJ as Cientistas do Nosso Estado. LRG received a doctoral fellowship from FAPERJ.

REFERENCES

  • 1
    SVS - Secretaria de Vigilância em Saúde. 2018. Available from: http://portalms.saude.gov.br/svs
    » http://portalms.saude.gov.br/svs
  • 2
    Gama BE, Lacerda MVG, Daniel-Ribeiro CT, Ferreira-da-Cruz MF. Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control. Mem Inst Oswaldo Cruz. 2011; 106(Suppl. 1): 159-66.
  • 3
    Rieckmann KH, Davis DR, Hulton DC. Plasmodium vivax resistance to chloroquine? Lancet. 1989; 18(2): 1183-4.
  • 4
    Thanh PV, Hong NV, Van NV, Louisa M, Baird K, Xa NX, et al. Confirmed Plasmodium vivax resistance to Chloroquine in Central Vietnam. Antimicrob Agents Chemother. 2015; 59(12): 7411-9.
  • 5
    Soto J, Toledo J, Gutierrez P, Luzz M, Llinas N, Cedeno N, et al. Plasmodium vivax clinically resistant to chloroquine in Colombia. Am J Trop Med Hyg. 2001; 65(2): 90-3.
  • 6
    de Santana Filho FS, Arcanjo AR, Chehuan YM, Costa MR, Martinez-Espinosa FE, Vieira JL, et al. Chloroquine-resistant Plasmodium vivax, Brazilian Amazon. Emerg Infect Dis. 2007; 13(7): 1125-6.
  • 7
    Tantiamornkul K, Pumpaibool T, Piriyapongsa J, Culleton R, Lek-Uthai U. The prevalence of molecular markers of drug resistance in Plasmodium vivax from the border regions of Thailand in 2008 and 2014. Int J Parasitol Drugs Drug Resist. 2018; 8(2): 229-37.
  • 8
    Gama BE, Oliveira NK, Souza JM, Daniel-Ribeiro CT, Ferreira-da-Cruz MF. Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates. Mem Inst Oswaldo Cruz. 2009; 104(7): 1009-11.
  • 9
    Asih PB, Marantina SS, Nababan R, Lobo NF, Rozi IE, Sumarto W, et al. Distribution of Plasmodium vivax pvdhfr and pvdhps alleles and their association with sulfadoxine-pyrimethamine treatment outcomes in Indonesia. Malar J. 2015; 14: 365-71.
  • 10
    Torres KL, Figueiredo DV, Zalis MG, Daniel-Ribeiro CT, Alecrim W, Ferreira-da-Cruz MF. Standardization of a very specific and sensitive single PCR for detection of Plasmodium vivax in low parasitized individuals and its usefulness for screening blood donors. Parasitol Res. 2006; 98(6): 519-24.
  • 11
    Mint Lekweiry K, Boukhary AOMS, Gaillard T, Wurtz N, Bogreau H, Hafid JE, et al. Molecular surveillance of drug-resistant Plasmodium vivax using pvdhfr, pvdhps and pvmdr1 markers in Nouakchott, Mauritania. J Antimicrob Chemother. 2012; 67(2): 367-74.
  • 12
    Otto TD, Vasconcellos EA, Gomes LH, Moreira AS, Degrave WM, Mendonça-Lima L, et al. ChromaPipe: a pipeline for analysis, quality control and management for a DNA sequencing facility. Genet Mol Res. 2008; 7(3): 861-71.
  • 13
    Marfurt J, de Monbrison F, Brega S, Barbollat L, Müller I, Sie A, et al. Molecular markers of in vivo Plasmodium vivax resistance to amodiaquine plus sulfadoxine-pyrimethamine: mutations in pvdhfr and pvmdr1. J Infect Dis. 2008; 198(3): 409-17.
  • 14
    Das S, Banik A, Hati AK, Roy S. Low prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) quadruple and quintuple mutant alleles associated with SP resistance in Plasmodium vivax isolates of West Bengal, India. Malar J. 2016; 15(1): 395-404.
  • 15
    Ganguly S, Saha P, Chatterjee M, Maji AK. Prevalence of polymorphisms in antifolate drug resistance molecular marker genes pvdhfr and pvdhps in clinical isolates of Plasmodium vivax from Kolkata, India. Antimicrob Agents Chemother. 2014; 58(1): 196-200.
  • 16
    Zakeri S, Afsharpad M, Ghasemi F, Raeisi A, Safi N, Butt W, et al. Molecular surveillance of Plasmodium vivax dhfr and dhps mutations in isolates from Afghanistan. Malar J. 2010; 9: 75-82.
  • 17
    Huang B, Huang S, Su XZ, Tong X, Yan J, Li H, et al. Molecular surveillance of pvdhfr, pvdhps, and pvmdr-1 mutations in Plasmodium vivax isolates from Yunnan and Anhui provinces of China. Malar J. 2014; 13: 346-55.
  • 18
    Ranjitkar S, Schousboe ML, Thomsen TT, Adhikari M, Kapel CM, Bygbjerg IC, et al. Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal. Malar J. 2011; 10: 75-82.
  • 19
    Brega S, de Monbrison F, Severini C, Udomsangpetch R, Sutanto I, Ruckert P, et al. Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates. Antimicrob Agents Chemother. 2004; 48(7): 2581-7.
  • 20
    Hawkins VN, Auliff A, Prajapati SK, Rungsihirunrat K, Hapuarachchi HC, Maestre A, et al. Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase. Malar J. 2008; 7: 72-83.
  • 21
    Saralamba N, Nakeesathit S, Mayxay M, Newton PN, Osorio L, Kim JR, et al. Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia. Malar J. 2016; 15: 484-90.
  • 22
    Barnadas C, Musset L, Legrand E, Tichit M, Briolant S, Fusai T, et al. High prevalence and fixation of Plasmodium vivax dhfr/dhps mutations related to sulfadoxine/pyrimethamine resistance in French Guiana. Am J Trop Med Hyg. 2009; 81(1): 19-22.
  • 23
    Prajapati SK, Joshi H, Dev V, Dua VK. Molecular epidemiology of Plasmodium vivax anti-folate resistance in India. Malar J. 2011; 10: 102-8.
  • 24
    Kuesap J, Rungsrihirunrat K, Thongdee P, Ruangweerayut R, Na-Bangchang K. Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand. Mem Inst Oswaldo Cruz. 2011; 106(Suppl. 1): 130-3.
  • 25
    Korsinczky M, Fischer K, Chen N, Baker J, Rickmann K, Cheng Q. Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site. Antimicrob Agents Chemother. 2004; 48(6): 2214-22.
  • 26
    Imwong M, Pukrittayakamee S, Cheng Q, Moore C, Looareesuwan S, Snounou G, et al. Limited polymorphism in the dihydropteroate synthetase gene (dhps) of Plasmodium vivax isolates from Thailand. Antimicrob Agents Chemother. 2005; 49(10): 4393-5.
  • 27
    Thongdee P, Kuesap J, Rungsihirunrat K, Tippawangkosol P, Mungthin M, Na-Bangchang K. Distribution of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles in Plasmodium vivax isolates from Thailand. Acta Trop. 2013; 128(1): 137-43.

Publication Dates

  • Publication in this collection
    04 Feb 2019
  • Date of issue
    2019

History

  • Received
    04 Sept 2018
  • Accepted
    26 Dec 2018
Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
E-mail: memorias@fiocruz.br