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

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.

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. (1) 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), comdoi: 10 bined 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. (2) P. vivax resistance to CQ occurred later in 1989 in Papua New Guinea (3) and CQ monotherapy was ineffective. Following this seminal observation, numerous cases of CQ resistance were reported in Southeast Asia (4) and South America, (5,6) 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 pathway (7) . 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 documented 8) 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, (9) 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 solutionstained blood smears and by P. vivax cysteine-proteinase target gene polymerase chain reaction (PCR). (10) 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 (QIAGEN TM , 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.
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. 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.
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 vivo (13) 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. (14) 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. (14,15) The predominance of S117N followed by the double mutant 58R/117N (28%) was also analogous to those reported in India, (15) Afghanistan, (16) China, (17) Nepal, (18) Thailand, (19) Colombia, (20,21) French Guiana (19) and Brazil. (8) 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 Guiana (19,22) and Amazonas, Brazil. (8) 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, (19) reinforce the possible existence of geographic subdivision of different P. vivax parasites in samples from the old and new worlds.
Concerning the pvdhps gene, previous data indicated that mutations were mainly detected at codons A383G and A553G (14,21,23,24) and suggested that these mutations alone could be responsible for reduced sensitivity to sulfa and sulfones. (25,26) 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%), (7) Thai-Myanmar border (47%) (27) and Indonesia (50%). (9) Whereas, in a Colombian study investigating polymorphisms in pvdhps, the wild-type was the most frequently detected (71.6%); (21) the same was true in India (79%) (15) and also in Thai -Cambodian border (74%). (28) 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.
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.

AUTHORS' CONTRIBUTION
MFFC idealide the study, participate in the discussion and review the manuscript; LRG performed PCRs, analysis DNA sequencing and drafted the manuscript; CTDR, CLP and DM participated in the discussions and reviewed the final manuscript; AL performed DNA extraction and molecular diagnosis; PB recruited the patients. All authors read and approved the final manuscript.