BackgroundMalaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine–pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparumdhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively.MethodsBlood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing.ResultsIn the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr–pfdhps mutant was noted.Conclusionpfdhfr and pfdhps gene mutations in isolates from Lubango are suggestive of a low-grade SP resistance and IPT for pregnant women and infant based on SP treatment could be effective. Routine molecular studies targeting polymorphism in these two genes need to be routinely conducted at country level.

BackgroundPlasmodium 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.MethodsBlood 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.ResultsAmong 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.ConclusionsConsidering 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.

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 drugresistant strains, underscore the urgency of the development of vaccines against P. vivax. Polymorphisms on DBP-IIgene 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. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article' 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.

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.

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.

Artemisinin (ART) is recommended as the first-line drug for P. falciparum infections combined with a long-acting partner drug. The emergence of P. falciparum resistance to ART (ARTR) is a concern for malaria. The most feared threat remains the spread of ARTR from Southeast Asia to Africa or the independent emergence of ARTR in Africa, where malaria accounts for 93% of all malaria cases and 94% of deaths. To avoid this worst-case scenario, surveillance of Pfkelch13 mutations is essential. We investigated mutations of Pfkelch13 in 78 P. falciparum samples from Huambo, Angola. Most of the parasites had a wild-type Pfkelch13 allele. We identified one synonymous mutation (R471R) in 10 isolates and one non-synonymous mutation (A578S) in two samples. No Pfkelch13 validated or candidate ARTR mutants were identified. The finding suggests that there is little polymorphism in Pfkelch13 in Huambo. Since cases of late response to ART in Africa and the emergence of ARTR mutations in Rwanda and Uganda have been reported, efforts should be made toward continuous molecular surveillance of ARTR. Our study has some limitations. Since we analyzed P. falciparum parasites from a single health facility, the study may not be representative of all Angolan endemic areas.

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