Plasmodium falciparum Chloroquine-pfcrt Resistant Haplotypes in Brazilian Endemic Areas Four Decades after CQ Withdrawn

(1) Background: Malaria is a public health problem worldwide. Despite global efforts to control it, antimalarial drug resistance remains a great challenge. In 2009, our team identified, for the first time in Brazil, chloroquine (CQ)-susceptible Plasmodium falciparum parasites in isolates from the Brazilian Amazon. The present study extends those observations to include survey samples from 2010 to 2018 from the Amazonas and Acre states for the purpose of tracking pfcrt molecular changes in P. falciparum parasites. (2) Objective: to investigate SNPs in the P. falciparum gene associated with chemoresistance to CQ (pfcrt). (3) Methods: Sixty-six P. falciparum samples from the Amazonas and Acre states were collected from 2010 to 2018 in patients diagnosed at the Reference Research Center for Treatment and Diagnosis of Malaria (CPD-Mal/Fiocruz), FMT-HVD and Acre Health Units. These samples were subjected to PCR and DNA Sanger sequencing to identify mutations in pfcrt (C72S, M74I, N75E, and K76T). (4) Results: Of the 66 P. falciparum samples genotyped for pfcrt, 94% carried CQ-resistant genotypes and only 4 showed a CQ pfcrt sensitive-wild type genotype, i.e., 1 from Barcelos and 3 from Manaus. (5) Conclusion: CQ-resistant P. falciparum populations are fixed, and thus, CQ cannot be reintroduced in malaria falciparum therapy.


Introduction
Tens of thousands of years after the Plasmodia that infected hominids became established as parasites that cause disease in humans, malaria is still a major public health problem worldwide in the third millennium of the Christian era. According to the World Malaria Report, 247 million cases and 619 thousand malaria-related deaths were reported in 2021 [1]. Plasmodium falciparum is responsible for the most virulent and dangerous malaria in humans [1,2]. In 2021, in the Brazilian Amazon Basin, 138,988 cases-representing 99.92% of the Brazilian cases-were reported. Among them, 21,614 (15.55%) were caused by P. falciparum [2].
Despite numerous advances in the use and efficacy of vaccines, there is still heavy reliance on antimalarials for the prevention and treatment of malaria; these drugs are considered the most important malaria control measures [3]. However, with the continued use of antimalarials, P. falciparum gradually develops drug resistance that spreads rapidly [4]. Therefore, antimalarial drug resistance has become one of the major challenges in eliminating the disease [5]. The emergence of drug-resistant strains may be influenced by parasites and host factors, such as parasite mutation frequency, patient adherence to therapy, selection pressure, and host immunity, in addition to drug quality [6].
Before artemisinin-based combination therapies (ACTs) were approved worldwide as first-line therapy for uncomplicated falciparum malaria in 2007, chloroquine (CQ) was widely used in Brazil, especially up to the 1980s, to treat acute infections with P. falciparum as a safe, inexpensive, and effective antimalarial drug [7][8][9]. Mefloquine was then introduced as a therapeutic alternative for multidrug-resistant falciparum malaria; it was used until the introduction of ACTs in Brazil, with relative safety, alone or in association with artemisinin derivatives in cases of severe malaria and multidrug-resistant P. falciparum parasites [10]. Currently, after reports of cases of resistance to mefloquine [10], this drug is only used in combination with artesunate for the treatment of acute, uncomplicated malaria caused by P. falciparum. It is indicated for cases of P. falciparum mono-infection, as well as for mixed infections with P. vivax (with subsequent treatment of its hypnozoite forms).
Since the first reports of P. falciparum resistance to antimalarial drugs in the nineteenth century, molecular epidemiological surveillance has been essential for the early detection and prevention of the spread of resistant parasites [11,12] by identifying and monitoring genetic polymorphisms associated with parasite resistance, mainly single nucleotide polymorphisms (SNPs) [8,12].
Mutations in the P. falciparum chloroquine resistance transporter gene (pfcrt), a member of the drug metabolite transporter superfamily, have been associated with reduced susceptibility to CQ [11]. The K76T pfcrt polymorphism is considered the molecular marker of CQ resistance (CQR) [13] and is associated with CQ treatment failure [14,15]. However, studies have suggested that the K76T mutation does not act alone but in conjunction with other pfcrt mutations, such as those at positions 72, 73, 74, and 75 [16][17][18]. Thus, CQR strains of P. falciparum could carry triple CVIET (mostly in Southeast Asia and Africa) or double SVMNT mutants (South America) [19][20][21][22].
In 2009, our team identified, for the first time in Brazil, the presence of P. falciparum parasites sensitive to CQ in the Brazilian Amazon [23]. The present study extends these observations to include survey samples from 2010 to 2018 from the Amazonas and Acre. Due to the limitations of in vivo and in vitro studies to survey chemoresistant parasites in endemic areas where reinfections are common, molecular analysis of parasite mutations associated with chemoresistance is an important tool. These findings prompted us to conduct a study to track molecular changes in P. falciparum parasites through the investigation of SNPs in the pfcrt gene in parasites from the Amazonas and Acre Brazilian states.

Blood Samples and Malaria Diagnosis
Samples were collected from P. falciparum-infected symptomatic patients who attended the Ambulatório de Síndromes Febris Agudas/Acute Febrile Syndrome Outpatient Clinic at the National Institute of Infectology (INI), Rio de Janeiro, a member of the Reference Center for Research, Diagnosis, and Training of Malaria-CPD-Mal/Fiocruz, RJ for the Extra-Amazonian region ( Figure 1). Independently of blood collection locality, P. falciparum diagnosis was made by light microscopy (Giemsa-stained thick blood droplets) in situ and by species-specific polymerase chain reaction (PCR) [24] at the Fiocruz Malaria Research Laboratory-the headquarters of the CPD-Mal-where the samples were stored. Table 1. Localities of P. falciparum parasite blood collection by year.

DNA Extraction, Amplification, and Sequencing
The DNA from 1 mL blood samples was extracted using a QIAamp™ DNA Blood Midi Kit (QIAGEN), according to the manufacturer's instructions. PCRs were performed to amplify the pfcrt fragment gene according to previously described protocols [25]. PCR products were analyzed by electrophoresis on 2% agarose gel and visualized under a UV transilluminator (DigiDoc-It, UVP, Upland, CA, USA). Each PCR product was purified using Wizard™ SV Gel and the PCR Clean-Up System (Promega, WI, USA), following the manufacturer's procedure. Purified DNA sequencing was carried out through Big Dye™ Terminator Cycle Sequencing Ready Reaction version 3.1 (Applied Biosystems, Carlsbad, CA, USA), with 3.2 μM of forward and reverse PCR primers. DNA sequences to investigate C72S, M74I, N75E/D, and K76T were determined using an ABI Prism DNA Ana-lyzer™ 3730 (Applied Biosystems, CA, USA), on the Fiocruz Genomic Platform PDTIS/Fi-

DNA Extraction, Amplification, and Sequencing
The DNA from 1 mL blood samples was extracted using a QIAamp™ DNA Blood Midi Kit (QIAGEN), according to the manufacturer's instructions. PCRs were performed to amplify the pfcrt fragment gene according to previously described protocols [25]. PCR products were analyzed by electrophoresis on 2% agarose gel and visualized under a UV transilluminator (DigiDoc-It, UVP, Upland, CA, USA). Each PCR product was purified using Wizard™ SV Gel and the PCR Clean-Up System (Promega, WI, USA), following the manufacturer's procedure. Purified DNA sequencing was carried out through Big Dye™ Terminator Cycle Sequencing Ready Reaction version 3.1 (Applied Biosystems, Carlsbad, CA, USA), with 3.2 µM of forward and reverse PCR primers. DNA sequences to investigate C72S, M74I, N75E/D, and K76T were determined using an ABI Prism DNA Analyzer™ 3730 (Applied Biosystems, CA, USA), on the Fiocruz Genomic Platform PDTIS/Fiocruz RPT01A. Nucleotide sequences were aligned using the ClustalW multiple sequence aligner in the BioEdit software [26]. The PF3D7_1343700 strain was used as a reference sequence (from PlasmoDB: http://www.plasmoDB.org, accessed on 20 March 2023). DNA sequences were deposited in GenBank (the NIH's genetic sequence database; www.ncbi/nlm/nih.gov/GenBank, accessed on 21 March 2023) with the accession numbers OQ672386-OQ672451.

Discussion
The P. falciparum has demonstrated its ability to develop resistance to all drugs that have been used against it on a large scale, continuously threatening global efforts to control malaria, a leading infectious cause of human morbidity and mortality. Although Africa bears by far the heaviest burden of malaria, CQ-resistant parasites first emerged in Southeast Asia and America [27]. This fact underscores the importance of studying and understanding the genotype of circulating parasites in malaria-endemic areas, since the strong pressure of drugs can lead to the establishment of drug resistance alleles, even if they generate a fitness cost for parasites in the absence of drug pressure [28]. Therefore, understanding the evolution of drug target genes under changing drug policy is crucial for drug efficacy monitoring using molecular markers.
Polymorphisms in the amino acid positions 72-76 of the pfcrt gene are reliable markers for CQR of P. falciparum parasites, of which K76T mutation is predominant [16,29]. In our study, the high prevalence of the 76T allele in isolates from Acre and Amazonas agrees with other studies in Brazil [30,31], and 76T mutation was found in two CQR haplotypes, CVIET and SVMNT, which was consistent with our initial hypothesis.
The CVIET haplotype is predominant in many African [21,32] and Southeast Asian countries in which CQ has been withdrawn for at least ten years after [33][34][35]; however, it has also been observed in P. falciparum parasites from South America [36], while SVMNT is dominant in South America and Oceania [37]. In Brazil, the CVIET haplotype was rarely encountered (it was found in only one sample from the municipality of Manaus), as previously seen in isolates from Amazonas and Rondônia [38]. This low CVIET haplotype frequency in Brazilian isolates suggests that this allele might have been recently introduced through human migration between Africa and South America.
On the other hand, the SVMNT haplotype is mainly detected in South America and is rarely found in Africa [21,39] and Southeast Asia [33][34][35]. A study released in 2022 claimed that the SVMNT haplotype originated independently in South America [22], and it was suggested that this haplotype might be responsible for the initial CQR sweeps across the Amazon in the early 1960s [38]. SVMNT was more prevalent than other mutant haplotypes found in this survey, corroborating previous findings in Brazil [24] and showing its persistence, despite a decline in CQ use.
Our team reported, for the first time, the presence of wild-type haplotypes circulating in Brazilian isolates [23]. Now, almost ten years later, we found this haplotype in only four isolates from the Amazonas state (three from Manaus and one from Barcelos). Considering that in Brazil, CQ has not been used to treat P. falciparum since the 1980s, a higher percentage of parasites sensitive to CQ would theoretically be expected. In fact, up to 90% of the samples showing a reversal of pfcrt from the CQ-resistant to the CQ-sensitive genotype were taken 19 years after the withdrawal of CQ in Kenya, in contrast to the results observed in the present study [40]. Thus, the high level of K76T pfcrt mutations observed in Brazilian endemic areas is suggestive of a sustained CQ pressure on P. falciparum parasites. In fact, CQ is used in the treatment of vivax malaria, leading to continuous exposure to this drug. Alternatively, the presence of a K76T mutation might have a positive effect on the fitness of the parasite, settling down in the parasitic population of the region, or lesser opportunities for competition because of a lower rate of polyclonal infections and a relative lack of competing wild-type parasites [41] Additionally, C350R substitution on the pfcrt gene could also participate in the restoration of CQ susceptibility, as suggested elsewhere [28]. Since the C350R mutation is in exon 10, and the primers we used flank the exon 2 region, comprising amino acids located at codons 43-91, studies are in progress to answer this question.

Conclusions
We conclude that the P. falciparum SVMNT haplotype is fixed in Brazilian endemic areas. This notwithstanding, molecular surveillance of the P. falciparum pfcrt gene to monitor trends in the emergence and spread of CQ-sensitive P. falciparum haplotypes in parasites in Brazilian endemic areas can help to understand the evolutionary dynamics of antimalarial drug resistance in the Amazon Basin, where more than 99% of Brazilian malaria cases occur and where P. falciparum resistance to CQ keeps being the rule.   Data Availability Statement: Data supporting the conclusions of this article are included within the article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.