Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates

Plasmodium vivax control is now being hampered by drug resistance. Orthologous Plasmodium falciparum genes linked to chloroquine or sulfadoxine-pyrimethamine chemoresistance have been identified in P. vivax parasites, but few studies have been performed. The goal of the present work is to characterise pvmdr1 and pvdhfr genes in parasite isolates from a Brazilian endemic area where no molecular investigation had been previously conducted. The pvmdr1 analysis revealed the existence of single (85.7%) and double (14.3%) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2%) and triple (42.8%) mutant haplotypes. The implications of these findings are discussed

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

Submitted by Sandra Infurna ([email protected]) on 2019-08-22T17:36:24Z No. of bitstreams: 1 AlineLavigne_OtacilioMoreira_etal_IOC_2019.pdf: 458878 bytes, checksum: 4993b198c5212641c504f2cc9791d069 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-08-22T17:42:31Z (GMT) No. of bitstreams: 1 AlineLavigne_OtacilioMoreira_etal_IOC_2019.pdf: 458878 bytes, checksum: 4993b198c5212641c504f2cc9791d069 (MD5)Made available in DSpace on 2019-08-22T17:42:31Z (GMT). No. of bitstreams: 1 AlineLavigne_OtacilioMoreira_etal_IOC_2019.pdf: 458878 bytes, checksum: 4993b198c5212641c504f2cc9791d069 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Instituto de Medicina Social. Departamento de Epidemiologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.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

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

Submitted by Sandra Infurna ([email protected]) on 2016-12-27T12:33:32Z No. of bitstreams: 1 larissa2_gomes_etal_IOC_2016.pdf: 1019673 bytes, checksum: 37afb1dafa313ef83407b071d6b5f1c3 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-27T12:44:02Z (GMT) No. of bitstreams: 1 larissa2_gomes_etal_IOC_2016.pdf: 1019673 bytes, checksum: 37afb1dafa313ef83407b071d6b5f1c3 (MD5)Made available in DSpace on 2016-12-27T12:44:02Z (GMT). No. of bitstreams: 1 larissa2_gomes_etal_IOC_2016.pdf: 1019673 bytes, checksum: 37afb1dafa313ef83407b071d6b5f1c3 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa, Diagnóstico e Tratamento em Malária (CPD-Mal). Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa, Diagnóstico e Tratamento em Malária (CPD-Mal). Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa, Diagnóstico e Tratamento em Malária (CPD-Mal). Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa, Diagnóstico e Tratamento em Malária (CPD-Mal). Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisa, Diagnóstico e Tratamento em Malária (CPD-Mal). Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas (INI-IPEC). Laboratório de Doenças Febris Agudas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisa, Diagnóstico e Tratamento em Malária (CPD-Mal). Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas (INI-IPEC). Laboratório de Doenças Febris Agudas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa, Diagnóstico e Tratamento em Malária (CPD-Mal). Rio de Janeiro, RJ, Brasil.Institut Pasteur in Cambodia. Malaria Molecular Epidemiology Unit. Phnom Penh, Cambodia.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa, Diagnóstico e Tratamento em Malária (CPD-Mal). Rio de Janeiro, RJ, Brasil.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

Pfkelch13 Plasmodium falciparum Mutations in Huambo, Angola.

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

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

Submitted by Fábio Marques ([email protected]) on 2020-03-20T19:58:45Z No. of bitstreams: 1 Extensive Genetic_Patricia_Brasil_etal_INI_2020.pdf: 1629547 bytes, checksum: b147ad8b01b1a4dd498b8eb6e699ea99 (MD5)Approved for entry into archive by Regina Costa ([email protected]) on 2020-03-24T00:40:21Z (GMT) No. of bitstreams: 1 Extensive Genetic_Patricia_Brasil_etal_INI_2020.pdf: 1629547 bytes, checksum: b147ad8b01b1a4dd498b8eb6e699ea99 (MD5)Made available in DSpace on 2020-03-24T00:40:21Z (GMT). No. of bitstreams: 1 Extensive Genetic_Patricia_Brasil_etal_INI_2020.pdf: 1629547 bytes, checksum: b147ad8b01b1a4dd498b8eb6e699ea99 (MD5) Previous issue date: 2020Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz, Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Doenças Febris Agudas. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil / Centro Universitário Serra dos Órgãos. Teresópolis, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genética Molecular de Microrganismos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em Doenças Febris Agudas. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisas em Malária. Rio de Janeiro, RJ, Brasil / Centro de Pesquisa, Diagnóstico e Treinamento em Malária Reference Laboratory for Malaria in the Extra-Amazonian Region for the Brazilian Ministry of Health, SVS & Fiocruz. Rio de Janeiro, RJ, Brazil.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 efcacy. This study aimed to investigate the pvdbp-II genetic diversity in two Brazilian regions with diferent epidemiological patterns: the unstable transmission area in the Atlantic Forest (AF) of Rio de Janeiro and; the fxed 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 fanking pvdbp-II was amplifed 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 diferentiation 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 fndings could suggest the potential of these geographical clusters as population-specifc-signatures that may be useful to track the origin of infections. The sample size should be increased in order to confrm this possibility

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