Plasmodium falciparum Chloroquine-pfcrt Resistant Haplotypes in Brazilian Endemic Areas Four Decades after CQ Withdrawn.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

Spatial Dispersal of Epstein-Barr Virus in South America Reveals an African American Variant in Brazilian Lymphomas.

Epstein−Barr virus (EBV) is a saliva-borne É£-herpesvirus associated with benign and malignant lymphoproliferation. EBV-mediated tumorigenic mechanisms are not fully understood and may be related to viral genetic variations. In this work, we characterize the genetic diversity of EBV from Brazil, assessing 82 samples derived from saliva from asymptomatic carriers (n = 45), biopsies of benign reactive hyperplasia (n = 4), and lymphomas (n = 33). Phylogenetic and phylogeographic analysis of the entire coding region of the LMP-1 was performed. Additionally, type 1/type 2 distinction by the EBNA3C gene and Zp variants were evaluated. Our results revealed a high diversity of EBV in Brazil, with the co-circulation of four main clades, described here as: Mediterranean (40.2%, n = 33), Raji/Argentine (39%, n = 32), B95-8 (6.1%, n = 5), and Asian II (1.2%, n = 1). The Raji/Argentine and Mediterranean clades were the most prevalent in South America (45% and 28%, respectively). The Raji/Argentine clade was associated with polymorphisms I124V/I152L, del30 bp, and ins15 bp (p < 0.0001, to all clades) and with a high haplotype diversity related to EBV type and Zp variants. We found that a Raji/Argentine subclade spread primarily from Brazil and later to other South American countries. Although no LMP1 variant has been directly associated with disease, the Raji/Argentine clade was predominantly clustered with lymphomas (61%) and the Mediterranean clade with non-malignant cases (59%) (p = 0.1). These data highlight the high genetic diversity of EBV circulating in Brazil, calling attention to a Raji-related variant with great recombination potential in Brazilian lymphomas.

Molecular aspects of Chikungunya virus infections in cancer patients.

BACKGROUND: Chikungunya virus (CHIKV) is an arbovirus that can cause chronic and debilitating manifestations. The first autochthonous case in Rio de Janeiro state was diagnosed in 2015, and an outbreak was declared in 2016. OBJECTIVE: The aim of this work was to evaluate CHIKV viral load in serum, plasma and urine in cancer patients to determine the best sample for diagnosis, as well as perform molecular characterisation and phylogenetic analysis of circulating strains. METHODS: Paired serum, plasma and urine collected from 31 cancer patients were tested by real-time quantitative polymerase chain reaction (qPCR) and a segment of the CHIKV E1 gene was sequenced. FINDINGS: We detected 11 CHIKV+ oncological patients. Paired samples analyses of nine patients showed a different pattern of detection. Also, a higher viral load in plasma (6.84 log10) and serum (6.07 log10) vs urine (3.76 log10) was found. Phylogenetic analysis and molecular characterisation revealed East/Central/Southern Africa (ECSA) genotype circulation and three amino acids substitutions (E1-K211T, E1-M269V, E1-T288I) in positive patients. MAIN CONCLUSION: The results indicate the bioequivalence of serum and plasma for CHIKV diagnosis, with urine being an important complement. ECSA genotype was circulating among patients in the period of the 2016 outbreak with K211T, M269V and T288I substitution.

Molecular aspects of Chikungunya virus infections in cancer patients

BACKGROUND Chikungunya virus (CHIKV) is an arbovirus that can cause chronic and debilitating manifestations. The first autochthonous case in Rio de Janeiro state was diagnosed in 2015, and an outbreak was declared in 2016. OBJECTIVE The aim of this work was to evaluate CHIKV viral load in serum, plasma and urine in cancer patients to determine the best sample for diagnosis, as well as perform molecular characterisation and phylogenetic analysis of circulating strains. METHODS Paired serum, plasma and urine collected from 31 cancer patients were tested by real-time quantitative polymerase chain reaction (qPCR) and a segment of the CHIKV E1 gene was sequenced. FINDINGS We detected 11 CHIKV+ oncological patients. Paired samples analyses of nine patients showed a different pattern of detection. Also, a higher viral load in plasma (6.84 log10) and serum (6.07 log10) vs urine (3.76 log10) was found. Phylogenetic analysis and molecular characterisation revealed East/Central/Southern Africa (ECSA) genotype circulation and three amino acids substitutions (E1-K211T, E1-M269V, E1-T288I) in positive patients. MAIN CONCLUSION The results indicate the bioequivalence of serum and plasma for CHIKV diagnosis, with urine being an important complement. ECSA genotype was circulating among patients in the period of the 2016 outbreak with K211T, M269V and T288I substitution.

Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control.

In Brazil, malaria still remains a clinically important febrile syndrome for local populations and travelers, occurring mostly in the Amazon Basin. This review aims to report the main efforts employed to control this disease since the 1940s and the emergence of Plasmodium falciparum and Plasmodium vivax chemoresistance to chloroquine and sulphadoxine-pyrimethamine among other drugs. Additionally, in vivo, in vitro and molecular studies as well as malaria chemoresistance consequences on disease morbidity and policy treatment guidelines were commented.

Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control

In Brazil, malaria still remains a clinically important febrile syndrome for local populations and travelers, occurring mostly in the Amazon Basin. This review aims to report the main efforts employed to control this disease since the 1940s and the emergence of Plasmodium falciparum and Plasmodium vivax chemoresistance to chloroquine and sulphadoxine-pyrimethamine among other drugs. Additionally, in vivo, in vitro and molecular studies as well as malaria chemoresistance consequences on disease morbidity and policy treatment guidelines were commented.

Tipagem de marcadores moleculares e de genes potencialmente associados à resistência a antimaláricos em isolados de Plasmodium falciparum e P. vivaz do Brasil / Typing of molecular markers and genes potentially associated with resistance to antimalarial drugs in isolates of Plasmodium falciparum and P. Brazil's lively

A malária, doença infecciosa causada por parasitas do gênero Plasmodium, é um conhecido flagelo das populações humanas desde a antiguidade. Na falta de uma vacina, a política de controle baseia-se principalmente no diagnostico rápido e no tratamento dos casos. Devido ao impacto da quimiorresistência parasitária no controle, faz-se necessário o constante monitoramento da eficácia de drogas. Portanto, para contextualizar o Brasil no panorama mundial, nosso objetivo se constituiu em realizar um estudo descritivo sobre a ocorrência de mutações (SNPs) nos genes considerados marcadores moleculares associados à resistência como pfcrt, pfmdr1, pfdhfr, pfdhps e pvdhfr, assim como em genes potencialmente associados à quimiorresistência (pfatpase6 e pvmdr1) em isolados de P. falciparum e P. vivax coletados nas cidades Amazônicas de Porto Velho, Paragominas e Manaus. Para tal, utilizamos PCRs seguidas do sequenciamento de DNA. Ao investigarmos a ocorrência de mutações nos genes pfcrt, pfdhfr e pfdhps em amostras de P. falciparum provenientes de Porto Velho e Paragominas foi possível detectar 1 isolado apresentando um haplótipo ou perfil de sensibilidade CVMNK no gene pfcrt e ACNSVI no gene pfdhfr e, além disso, pudemos observar uma redução no numero de mutações no gene pfdhps, ao confrontarmos os nossos resultados com os de um estudo retrospectivo. Em relação ao P. vivax, a análise do gene pvdmr1 num conjunto de amostras provenientes de Paragominas revelou o predomínio de mutações no códon 976 do gene pvmdr1 (preliminarmente associado com a resistência à cloroquina) e a presença de haplótipos duplo-mutante FRTNI e triplo-mutante FRTNL para o gene pvdhfr. dando continuidade à caracterização de P. falciparum no Brasil, foi estabelecido um registro de base da ocorrência de mutações nos genes pfmdr1 e pfatpase6, em amostras procedentes de Porto Velho, Paragominas e Manaus. Tal análise permitiu a identificação de um haplótipo prevalente NEF/CDVY no pfmdr1, assim como identificação de mutantes em um único códon ou duplo-mutantes (630 e/ou 402) no caso do gene pfatpase6. também não foi encontrada a mutação 769N, associada com a diminuição da susceptibilidade ao arteméter. Concluímos que: (a) existem no Brasil popluações parasitárias de P. falcipoarum portando haplótipos sensíveis para o gene pfcrt e o pfdhfr; (b) a mutação no códon 976 no gene pfmdr1 pode não ser válida para o monitoramento da resistência à cloroquina em isolados brasileiros de P. vivax; (c) as populações de P. vivax avaliadas apresentaram mutações no gene pvdhfr, apesar de nunca ter sido instituído o tratamento com sulfadoxina-pirimetamina para malária vivax e; (d) as amostras de P. falciparum avaliadas não portavam todos os alelos do gene pfmdr1 associados a um potencial desenvolvimento de resistência à combinação arteméter-lumefantrina.

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.

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 percent) and double (14.3 percent) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2 percent) and triple (42.8 percent) mutant haplotypes. The implications of these findings are discussed.

Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparum parasites in a Brazilian endemic area.

BACKGROUND: The goal of the present study was the characterization of Plasmodium falciparum genes associated to malaria drug resistance (pfcrt, pfdhfr and pfdhps), in samples from two Brazilian localities. METHODS: Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct DNA sequencing. RESULTS: Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from Paragominas, that showed a SP-sensitive genotype. CONCLUSION: This study is the first to document the sensitivity of P. falciparum parasites to CQ and SP in Brazilian field samples. The importance of these findings is discussed.

Padronização de reações de polimerização em cadeia(PCRs) convencional e em tempo real gênero-específicas para a detecção dos parasitas da malária em indivíduos pauciparasitados / Standardization of reactions of PCRs conventional and real time genus specific for the detection of the parasites of the malaria in low-grade individuals

A malária até hoje permanece como uma doença de alta prevalência nas áreas tropicais e subtropicais.Em regiões de transmissão perene, muitos indivíduos após várias infecções podem permanecer assintomáticos com parasitemias submicroscópicas necessitando de técnicas mais sensíveis do que o exame microscópico para a detecção dos parasitas.Essa condição dificulta o diagnóstico individual...Dentro dessa ótica nos propusemos a desenvolver PCRs gênero específicas convencional e em tempo real para a detecção dos parasitas da malária em indivíduos pauciparasitados.Para a padronização e validação das PCRs foram utilizadas 84 amostras de sangue no caso da PCR convencional e 33 dessas amostras no caso da PCR em tempo real,as quais foram coletadas nas cidades de Paragominas(PA)e Porto Velho(RO)...Ambas PCRs apresentaram a mesma especificidade frente a amostras clínicas de HBV e HIV,tendo sido assegurada frente a controles negativos de áreas endêmicas,no caso da PCR convencional.O limite de detecção para ambas PCRs foi de 0,5 parasita/µL.Contrariamente...Considerando as amostras extraídas pela técnica de fenol-clorofórmio,as PCRs de gênero mostraram 100 por cento de concordância de resultados quando comparadas com as PCRs espécie-específicas e com o exame microscópico realizado em nosso laboratório.Entretanto,quando consideramos o exame microscópico realizado em Paragominas,os resultados desse exame concordaram 98,8 por cento(n=84)com a PCR convencional e 96,9 por cento(n=33)com a PCR em tempo real.Assim,embora o tempo de realização do fenol-clorofórmio seja um fator limitante para sua utilização em larga escala,além da sua toxicidade,ele se mostrou como o método mais eficiente para a extração de DNA, seguido pelas técnicas das colunas e de Kawasaki.A escolha entre a técnica de PCR em seu formato convencional e a em tempo real... certamente deverá ser norteada pela disponibilidade de recursos financeiros. Devido ao seu menor custo,a PCR convencional poderá ser utilizada em...

Glycosylation influencing on fusion activity of HA and HEF structures of influenza viruses

Desde os tempos mais remotos, os vírus influenza têm sido o agente causal de doenças respiratórias letais. O seu processo de replicação ocorre nas células epiteliais do trato respiratório, causando uma síndrome respiratória aguda, na qual as glicoproteínas hemaglutinina (HA) e hemaglutinina-esterase-fusão (HEF), ancoradas na superfície dos vírus influenza A e C, respectivamente, são responsáveis pelos processos de fusão durante o ciclo de replicação viral. Ao contrário dos processos de adsorção, sialidase e esterase, atividades inatas desempenhadas por estas mesmas estruturas, a fusão depende de uma glicosilação e clivagem protéica prévias, para que o vírus se torne infeccioso. A glicosilação das proteínas de superfície está associada principalmente com a antigenicidade e com a estabilidade das proteínas de fusão. O presente trabalho analisa a influência da de-glicosilação sobre a atividade de fusão, usando vírus influenza A e C como modelos de estudo. A de-glicosilação parcial provocou significativa redução da atividade fusogênica, induzindo uma redução de 51,0 por cento, 87,5 por cento, 95,5 por cento e 79,3 por cento em relação às amostras de vírus influenza A/Memphis/102/ 72, A/FM/1/47, C/Taylor/1233/47 e C/Paris/1/67, respectivamente, acarretando também eventuais aumentos desta atividade biológica para certas amostras: 10,15 por cento (pH 5,8), 59,47 por cento (pH 5,8), 32,55 por cento (pH 5,8) e 80,7 por cento (pH 5,4) com relação às amostras de vírus influenza A/Memphis/102/72, A/FM/1/ 47, C/Taylor/1233/47 e A/Paris/1/67, respectivamente. Os resultados permitem concluir que o nível de glicosilação mostra-se estreitamente relacionado com a estabilidade das proteínas, causando o processo deglicosilação uma influência significativa sobre a atividade fusogênica viral.