Correction: Balancing selection and high genetic diversity of Plasmodium vivax circumsporozoite central region in parasites from Brazilian Amazon and Rio de Janeiro Atlantic Forest.

[This corrects the article DOI: 10.1371/journal.pone.0241426.].

Balancing selection and high genetic diversity of Plasmodium vivax circumsporozoite central region in parasites from Brazilian Amazon and Rio de Janeiro Atlantic Forest.

Circumsporozoite protein (CSP) is the primary pre-erythrocytic vaccine target in Plasmodium species. Knowledge about their genetic diversity can help predict vaccine efficacy and the spread of novel parasite variants. Thus, we investigated pvcsp gene polymorphisms in 219 isolates (136 from Brazilian Amazon [BA], 71 from Rio de Janeiro Atlantic Forest [AF], and 12 from non-Brazilian countries [NB]). Forty-eight polymorphic sites were detected, 46 in the central repeat region (CR), and two in the C-terminal region. Also, the CR presents InDels and a variable number of repeats. All samples correspond to the VK210 variant, and 24 VK210 subtypes based on CR. Nucleotide diversity (π = 0.0135) generated a significant number of haplotypes (168) with low genetic differentiation between the Brazilian regions (Fst = 0.208). The haplotype network revealed similar distances among the BA and AF regions. The linkage disequilibrium indicates that recombination does not seem to be acting in diversity, reinforcing natural selection's role in accelerating adaptive evolution. The high diversity (low Fst) and polymorphism frequencies could be indicators of balancing selection. Although malaria in BA and AF have distinct vector species and different host immune pressures, consistent genetic signature was found in two regions. The immunodominant B-cell epitope mapped in the CR varies from seven to 19 repeats. The CR T-cell epitope is conserved only in 39 samples. Concerning to C-terminal region, the Th2R epitope presented nonsynonymous SNP only in 6% of Brazilian samples, and the Th3R epitope remained conserved in all studied regions. We conclude that, although the uneven distribution of alleles may jeopardize the deployment of vaccines directed to a specific variable locus, a unique vaccine formulation could protect populations in all Brazilian regions.

Exploration of Plasmodium vivax merozoite surface proteins 1 and 7 genetic diversity in Brazilian Amazon and Rio de Janeiro Atlantic Forest.

Plasmodium vivax merozoite surface proteins (PvMSP) 1 and 7 are considered vaccine targets. Genetic diversity knowledge is crucial to assess their potential as immunogens and to provide insights about population structure in different epidemiological contexts. Here, we investigate the variability of pvmsp-142, pvmsp-7E, and pvmsp-7F genes in 227 samples from the Brazilian Amazon (BA) and Rio de Janeiro Atlantic Forest (AF). pvmsp-142 has 63 polymorphisms - 57 nonsynonymous - generating a nucleotide diversity of π = 0.009 in AF, and π = 0.018 in BA. In pvmsp-7E, 134 polymorphisms - 103 nonsynonymous - generate the nucleotide diversity of π = 0.027 in AF, and π = 0.042 in BA. The pvmsp-7F has only two SNPs - A610G and A1054T -, with nucleotide diversity of π = 0.0004 in AF, and π = 0.0007 in BA. The haplotype diversity of pvmsp-142, pvmsp-7E, and pvmsp-7F genes is 0.997, 1.00, and 0.649, respectively. None of the pvmsp-142 or pvmsp-7E sequences are identical to the Salvador 1 strain's sequence. Conversely, most of pvmsp-7F sequences (94/48%) are identical to Sal-1. We evaluated eight B-cell epitopes in pvmsp-7E, four of them showed higher nucleotide diversity compared to pvmsp-7E's epitopes. Positive selection was detected in pvmsp-142, pvmsp-7E central region, and pvmsp-7F with Tajima's D. In pvmsp-7E, the significant nucleotide and haplotype diversities with low genetic differentiation, could be indicative of balancing selection. The genetic differentiation of pvmsp-142 (0.315) and pvmsp-7F (0.354) genes between AF and BA regions is significant, which is not the case for pvmsp-7E (0.193). We conclude that pvmsp-142 and pvmsp-7E have great genetic diversity even in AF region, an enclosure area with deficient transmission levels of P. vivax zoonotic malaria. In both Brazilian regions, pvmsp-119, pvmsp-7E, and pvmsp-7F are conserved, most likely due to their roles in parasite survival, and could be considered potential targets for a "blood-stage vaccine".

Extensive genetic diversity of Plasmodium vivax dbp-II in Rio de Janeiro Atlantic Forest and Brazilian Amazon Basin: evidence of positive selection.

BACKGROUND: Plasmodium vivax is the most widespread human malaria parasite outside Africa and is the predominant parasite in the Americas. Increasing reports of P. vivax disease severity, together with the emergence of drug-resistant strains, underscore the urgency of the development of vaccines against P. vivax. Polymorphisms on DBP-II-gene could act as an immune evasion mechanism and, consequently, limited the vaccine efficacy. This study aimed to investigate the pvdbp-II genetic diversity in two Brazilian regions with different epidemiological patterns: the unstable transmission area in the Atlantic Forest (AF) of Rio de Janeiro and; the fixed malaria-endemic area in Brazilian Amazon (BA). METHODS: 216 Brazilian P. vivax infected blood samples, diagnosed by microscopic examination and PCR, were investigated. The region flanking pvdbp-II was amplified by PCR and sequenced. Genetic polymorphisms of pvdbp-II were estimated based on the number of segregating sites and nucleotide and haplotype diversities; the degree of differentiation between-regions was evaluated applying Wright's statistics. Natural selection was calculated using the rate of nonsynonymous per synonymous substitutions with the Z-test, and the evolutionary distance was estimated based on the reconstructed tree. RESULTS: 79 samples from AF and 137 from BA were successfully sequenced. The analyses showed 28 polymorphic sites distributed in 21 codons, with only 5% of the samples Salvador 1 type. The highest rates of polymorphic sites were found in B- and T cell epitopes. Unexpectedly, the nucleotide diversity in pvdbp-II was higher in AF (0.01) than in BA (0.008). Among the 28 SNPs detected, 18 are shared between P. vivax isolates from AF and BA regions, but 8 SNPs were exclusively detected in AF-I322S, K371N, E385Q, E385T, K386T, K411N, I419L and I419R-and 2 (N375D and I419M) arose exclusively in BA. These findings could suggest the potential of these geographical clusters as population-specific-signatures that may be useful to track the origin of infections. The sample size should be increased in order to confirm this possibility. CONCLUSIONS: The results highlight that the pvdbp-II polymorphisms are positively selected by host's immune pressure. The characterization of pvdbp-II polymorphisms might be useful for designing effective DBP-II-based vaccines.

Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria.

BACKGROUND: The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE: Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS: The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS: The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/µL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/µL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/µL in SYBR™ Green and 1 p/µL in TaqMan™ and conventional PCR. CONCLUSION: Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.

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.

Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria

BACKGROUND The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.

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.

Absence of K13 Polymorphism in Plasmodium falciparum from Brazilian Areas Where the Parasite Is Endemic.

Plasmodium falciparum artemisinin-resistant parasites can be evaluated by examining polymorphisms in the kelch (PfK13) domain. A total of 69 samples from patients with falciparum malaria were analyzed. All samples were from areas in states in Brazil where the parasite was endemic: Acre (n = 14), Amapá (n = 15), Amazonas (n = 30), and Pará (n = 10). After DNA alignment with the 3D7 reference sequence, all samples were found to be wild type. These data provide a baseline for PfK13 and reinforce the pertinence of artemisinin combination therapy in Brazilian areas.

Plasmodium vivax mdr1 genotypes in isolates from successfully cured patients living in endemic and non-endemic Brazilian areas.

BACKGROUND: Plasmodium vivax is the most widely distributed species causing the highest number of malaria cases in the world. In Brazil, P. vivax is responsible for approximately 84 % of reported cases. In the absence of a vaccine, control strategies are based on the management of cases through rapid diagnosis and adequate treatment, in addition to vector control measures. The approaches used to investigate P. vivax resistance to chloroquine (CQ) were exclusively in vivo studies because of the difficulty in keeping parasites in continuous in vitro culture. In view of the limitations related to follow-up of patients and to assessing the plasma dosage of CQ and its metabolites, an alternative approach to monitor chemo-resistance (QR) is to use molecular markers. Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene pvmdr1 are putative determinants of CQ resistance (CQR), but such SNPs in P. vivax isolates from patients with good response to treatment should be further explored. The aim of this study is to investigate the mutations in the gene, supposedly associated to QR, in P. vivax isolates from successfully cured patients, living in Brazilian endemic and non-endemic areas. METHODS: Blood samples were collected from 49 vivax malaria patients from endemic (Amazon Basin: 45) and non-endemic (Atlantic Forest: four) Brazilian regions and analysed for SNPs in the CQR-related P. vivax gene (pvmdr1), using PCR-based methods. RESULTS: Among the 49 isolates genetically characterized for the gene pvmdr1, 34 (70 %) presented at least one mutation. T958M mutant alleles were the most frequent (73 %) followed Y976F (15 %) and F1076L (12 %). Single mutation was detected in 24 (70.5 %) isolates and double mutations in ten (29.5 %). The most common single mutant genotype was the 958M/Y976/F1076 (79 %), followed by 976F/F1076 (21 %) whereas 958M/Y976/1076L (60 %) and 976F/1076L (40 %) double mutant genotypes were detected. Single mutant profile was observed only in isolates from Amazon Basin, although double mutants were found both in the Amazon and Atlantic Forest regions. Interestingly, the genotype 958M/Y976/1076L was present in all isolates from the Atlantic Forest in the Rio de Janeiro State. CONCLUSIONS: Considering that primaquine (PQ) efficacy is highly dependent on concurrent administration of a blood schizontocidal agent and that PQ could not circumvent CQR, together with the fact that no pvmdr1 mutation should be expected in successfully cured patients, these findings seem to indicate that the pvmdr1 gene is not a reliable marker of CQR. Further investigations are needed to define a reliable molecular marker for monitoring P. vivax CQR in P. vivax populations.

Dengue infection as a potential trigger of an imported Plasmodium ovale malaria relapse or a long incubation period in a non-endemic malaria region.

OBJECTIVES: To report that dengue fever (DF) could have triggered Plasmodium ovale wallikeri malaria. METHODS: A retrospective case report of P. ovale malaria and DF in a single patient in Rio de Janeiro, Brazil, who had lived in Angola, is presented. RESULTS: On the second week of illness, the patient was referred to our research service. As symptoms had persisted up to day 14, malaria was also considered, based on the patient's long-standing epidemiological history. On day 16 of illness, a thick blood smear was positive for P. ovale (3480 parasites/mm(3)), PCR for malaria was positive for P. ovale wallikeri, and the kinetics of dengue virus (DENV) antibodies suggested a recent primary dengue infection. CONCLUSIONS: Concurrent infections of DENV and malaria have rarely been reported; the actual impact of these sequential or simultaneous infections remains unknown. Therefore, DF must be considered as a potential co-morbidity for malaria, because of its influence on fluid electrolyte management. The case presented showed consistent temporal, clinical, and laboratory evidence that the relapse or the long incubation period of P. ovale malaria may have been triggered by a recent DF episode. To the authors' knowledge, this is the first report of DENV and P. ovale co-infection.