Identificação, caracterização e determinação de padrões da resposta imune de epítopos imunogênicos de Plasmodium vivax / Identification, characterization and determination of immune response patterns of immunogenic epitopes of Plasmodium vivax

O Plasmodium vivax representa um grande desafio no controle da malária devido a sua vasta distribuição ao redor do globo, grande frequência de infecções sub microscópicas e habilidade de induzir recaídas em consequência das formas evolutivas que podem ficar latentes no fígado por longos períodos (hipnozoítos). O recente aumento de cepas de P. vivax resistentes aos fármacos disponíveis, a evolução de formas mais virulentas do parasito e a produção precoce de gametócitos, característica desta espécie, contribuem para classificar a malária vivax como um problema de saúde pública que merece atenção. Ainda que reconhecido por suas características biológicas peculiares e pelo agravamento recente de sua virulência, poucos investimentos têm sido feitos no desenvolvimento de ferramentas de controle para vivax. Portanto, o presente estudo teve como objetivo identificar e caracterizar novos alvos potenciais utilizando amostras de diferentes áreas endêmicas ao redor do mundo (Brasil, Mali, Camboja e Estados Unidos da América). Para tanto, investigamos e caracterizamos uma proteína recém descoberta na urina de pacientes naturalmente infectados (PvVir14); e descrevemos o potencial imunogênico de epítopos de células B de uma das proteínas mais bem caracterizadas de P. vivax, a PvAMA-1. Anti-IgG circulantes contra PvVir14 apareceram em 61% e 34.5% dos indivíduos do Brasil e Camboja, respectivamente, enquanto que indivíduos de Mali (infectados com falciparum e não expostos a vivax), tiveram 0% de reconhecimento. Ainda, os níveis de anti-PvVir14 correlacionaram-se com aqueles contra outros antígenos de vivax já bem caracterizados, como a PvCSP e a PvDBP, que foram reconhecidos por 7.6% e 42% dos indivíduos respectivamente. Com relação ao perfil celular, indivíduos sororreativos para PvVir14 apresentaram níveis significativamente maiores de células B atípicas circulantes (CD 21- CD 27-), sugerindo que tal tipo celular possa estar ligado à resposta anti-PvVir14. Entre as células T, os níveis de CD4+ e CD8+ diferiram entre indivíduos com e sem anticorpos contra PvVir14 (menor e maior expressão, respectivamente), enquanto que os níveis de células NKT foram mais expressivos em indivíduos sem anti-PvVir14. Se tratando da PvAMA-1, a antigenicidade dos peptídeos com epítopos para células B previamente selecionados foi avaliada através de múltiplos ensaios sorológicos utilizando amostras de indivíduos com infecção aguda por P.vivax do norte do Brasil. Os peptídeos sintéticos foram reconhecidos por 45.5%, 48.7% e 31.2% (PI, PII e PII, respectivamente) dos indivíduos selecionados para o estudo. Quando sintetizados em conjunto (tripeptídeo), a reatividade aumentou para 62%, porcentagem comparável àquela obtida pela proteína em sua forma e tamanho originais (57%). Além disso, a reatividade anti-IgG conta o tripeptídeo foi reduzida em 42% pós-depleção, indicando que tais epítopos podem ser responsáveis por parte considerável da imunogenicidade da proteína. Esses resultados representam uma excelente perspectiva na identificação de novos alvos com potencial imunogênico para compor uma vacina, ou auxiliar no desenvolvimento de outras medidas de controle, como testes diagnósticos, já que contemplar diversos alvos do ciclo de vida do parasito parece ser a chave para alcançar a resposta robusta e protetora que uma vacina contra a malária vivax precisa para ter sucesso.

Plasmodium vivax is a major challenge for malaria control due to its wide geographic distribution, high frequency of submicroscopic infections, and ability to induce relapses due to the latent forms present in the liver (hypnozoites). The recent increase in drug-resistant P. vivax strains, the evolution toward more virulent forms and the early production of gametocytes adds up to make P. vivax malaria a public health issue of increasing importance. Besides its tricky biological features and new awareness of its virulence, minimal investments have been made in vaccine discovery for P. vivax. Given that, this study aimed to discover and characterize potential new targets for future vaccine development using samples from different endemic areas around the world (Brazil, Mali, Cambodia and United States of America). For this purpose, we investigated and characterized a novel protein recently discovered in the urine of naturally infected subjects (PvVir14) and described the immunogenic potential of peptides from a well-known vivax protein (PvAMA-1), which has been proved to have important B cell epitopes that can induce specific immune response. Circulating antibodies against PvVir14 appeared in 61% and 34.5% of subjects from Brazil and Cambodia, respectively, versus none (0%) of the P. falciparum-infected subjects from Mali who have no exposure to P. vivax. PvVir14 antibody levels correlated with those against other well-characterized sporozoite/liver (PvCSP) and blood stage (PvDBP-RII) antigens, which were recognized by 7.6% and 42% of Brazilians, respectively. Concerning the cellular immune profiling of Brazilian subjects, PvVir14 seroreactive individuals displayed significantly higher levels of circulating atypical (CD21− CD27−) B cells, raising the possibility that atypical B cells may be contribute to the PvVir14 antibody response. Among T cells, CD4+ and CD8+ levels differed (lower and higher, respectively) between subjects with versus without antibodies to PvVir14, while NKT cell levels were higher in those without antibodies. As for PvAMA-1, the antigenicity of the selected B-cell peptides was assessed by multiple serological assays using sera from acute P.vivax infected subjects. The synthetic peptides were recognized by 45.5%, 48.7% and 32.2% of infected subjects for peptides I, II and III respectively. Moreover, when synthetized together (tripeptide), the reactivity increases up to 62%, which is comparable to the reactivity found against the whole protein PvAMA-1 (57%). Furthermore, IgG reactivity against the tripeptide after depletion was reduced by 42%, indicating that these epitopes may be responsible for a considerable part of the protein immunogenicity. These results represent an excellent perspective on discovering new targets with immunogenic potential to compose a vaccine, or even to assist the development of other control measures, such as diagnostic tools, since contemplating several targets seems to be the key to achieving a robust and protective response that a malaria vaccine needs to be successful.

Malaria en la región eco-epidemiológica del Caribe Colombiano, 1960-2019 / Malaria in the eco-epidemiological region of the Colombian Caribbean, 1960-2019

La malaria en Colombia tiene un comportamiento heterogéneo y variable, entre las regiones. Para establecer su comportamiento epidemiológico en la región del Caribe colombiano entre 1960 y 2019 se realizó un estudio observacional, descriptivo y retrospectivo a partir de registros del Ministerio de Salud y otras fuentes secundarias. Se definieron variables epidemiológicas y se elaboraron medidas de frecuencia y tendencia central. Se registraron 155 096 casos. Las décadas con mayor número de casos fueron 1990-1999 (20,5%) y 1980-1989 (18,9%). El promedio de casos por década fue de 25 849,3. Los índices parasitarios más elevados se registraron en 1970 (3,3 por 1000 habitantes) y 1981 (3,9 por 1000 habitantes). La especie Plasmodium vivax fue la más frecuente y la mayoría de la carga por grupos de edad se registró en menores de 29 años, entre 2010-2019. La malaria presentó un patrón endemo-epidémico de baja y muy baja intensidad de transmisión, con una tendencia al descenso.

Malaria has a heterogeneous and variable behavior among Colombian regions. In order to establish its epidemiological behavior in the Colombian Caribbean region between 1960 and 2019, we carried out an observational, descriptive and retrospective study based on records from the Ministry of Health and other secondary sources. We defined epidemiological variables and used measures of frequency and central tendency. A total of 155,096 cases were registered. The decades with the highest number of cases were 1990-1999 (20.5%) and 1980-1989 (18.9%). The average number of cases per decade was 25,849.3. The highest parasite rates were recorded in 1970 (3.3 per 1000 population) and 1981 (3.9 per 1000 population). Plasmodium vivax was the most frequent species and most of the burden by age group was found in people under 29 years of age, between 2010-2019. Malaria showed an endemic-epidemic pattern of low and very low transmission intensity, with a decreasing trend.

Malaria cerebral con pancitopenia por plasmodium vivax en la amazonía peruana: reporte de caso / Plasmodium vivax cerebral malaria with pancytopenia in the peruvian amazon: case report

RESUMEN Plasmodium vivax es la especie más común en la Amazonía peruana y ocasiona el 81% del total de casos de malaria. Presentamos el caso de un paciente adulto varón con malaria cerebral por Plasmodium vivax, que inicia con malestar general y fiebre, luego presenta convulsiones más de dos veces al día con pérdida de consciencia y limitación funcional motora. Se le realiza gota gruesa donde se observa trofozoítos de Plasmodium vivax y depresión de las tres series sanguíneas. Se inicia tratamiento con artesunato y clindamicina por cinco días, se le transfunde un paquete globular, y continua con primaquina por siete días. El paciente muestra mejoría clínica con secuela neurológica en extremidad inferior izquierda.

ABSTRACT Plasmodium vivax causes 81% of all malaria cases and is the most common species in the Peruvian Amazon. We present the case of a male patient with cerebral malaria caused by Plasmodium vivax, who had general malaise and fever, and then presented seizures more than twice a day with loss of consciousness and motor functional limitation. Plasmodium vivax trophozoites were detected by thick blood smear, besides, we also observed low counts of all three blood cell types. Treatment began with artesunate and clindamycin for five days, then one unit of packed red blood cells was transfused; treatment continued with primaquine for seven days. The patient showed clinical improvement with neurological sequelae in one lower limb.

Paludismo importado no falciparum: aspectos de interés para el tratamiento de los pacientes / Imported non-falciparum malaria: aspects of interest for patient therapeutic management

Introducción: El paludismo es una enfermedad febril aguda potencialmente mortal causada por parásitos transmitidos por el mosquito Anopheles. El paludismo no falciparum (PNF), producido por otras especies de Plasmodium, está menos documentado en la literatura internacional, a pesar de su prevalencia. Objetivos: Describir aspectos clínicos y epidemiológicos de interés para el tratamiento en pacientes ingresados con diagnóstico de PNF importado, y determinar la relación existente entre la respuesta al tratamiento y otras variables. Métodos: Se realizó un estudio transversal analítico de 89 pacientes adultos con PNF importado, ingresados en el Departamento de Medicina del Instituto de Medicina Tropical Pedro Kourí, entre enero de 1997 a diciembre de 2017. Se determinó la pauta de profilaxis y tratamiento según los criterios de las guías publicadas y los fármacos disponibles en Cuba, y la definición de paludismo complicado según la OMS en 2003. Hubo respuesta demorada al tratamiento, cuando el paciente demoraba más de 7 días en negativizar la gota gruesa. Resultados: Predominaron los pacientes del sexo masculino, y una media de edad de 37,2 años. El 55,1 por ciento de los pacientes provenía de la región de las Américas y en el 85,4 por ciento se aisló Plasmodium vivax. La respuesta al tratamiento fue excelente con los esquemas combinados utilizados a base de cloroquina. Fue significativa la relación existente entre la demorada respuesta al tratamiento con la gravedad del cuadro clínico y el estado no inmune de los pacientes. Conclusiones: El PNF es una importante causa de paludismo importado en pacientes provenientes de áreas endémicas, fundamentalmente de América. Se distingue por parasitemias bajas, un cuadro clínico caracterizado por fiebre, escalofríos, cefaleas y evolución hacia cuadros no complicados. La cloroquina fue el medicamento de elección, aunque la repuesta demorada al tratamiento no justifica su suspensión o variación(AU)

Introduction: Malaria is a potentially fatal acute febrile illness caused by parasites transmitted by the Anopheles mosquito. Non-falciparum malaria (NFM), caused by other Plasmodium species, is less documented in the international literature, despite its prevalence. Objectives: To describe clinical and epidemiological aspects of interest for the treatment of patients hospitalized with a diagnosis of imported NFM, and to determine the relationship between response to treatment and other variables. Methods: It was conducted an analytical cross-sectional study of 89 adult patients with imported NFM, admitted to the Department of Medicine of the Institute of Tropical Medicine Pedro Kourí, between January 1997 to December 2017. The prophylaxis and treatment guideline was determined according to the published guidelines and drugs available in Cuba, and the definition of severe malaria by WHO in 2003. There was delayed response to treatment when the patient took more than 7 days to become negative for thick blood smear. Results: Patients were predominantly male, with a mean age of 37.2 years. Plasmodium vivax was isolated in 85.4 percent of the patients and 55.1 percent were from the Americas region. The response to treatment was excellent with the chloroquine-based combination regimens used. The relationship between the delayed response to treatment and the severity of the clinical picture and the non-immune status of the patients was significant. Conclusions: NFM is an important cause of imported malaria in patients from endemic areas, mainly from the Americas. It is characterized by low parasitemia, clinical manifestations of fever, chills, headache and evolution towards uncomplicated symptoms. Chloroquine was the drug of choice, although the delayed response to treatment does not justify its suspension or variation(AU)

Prevalencia de malaria gestacional en Ecuador / Prevalence of gestational malaria in Ecuador

Introducción. La malaria gestacional, definida como la presencia de Plasmodium spp. en sangre periférica materna o el hallazgo del parásito en la placenta, es considerada un importante problema de salud pública en las regiones tropicales y subtropicales. Objetivo. Determinar la frecuencia de casos de malaria gestacional diagnosticados en Ecuador entre 2015 y 2018. Materiales y métodos. Se hizo un estudio descriptivo, retrospectivo y transversal. Resultados. Se determinaron 46 casos de malaria gestacional en el período evaluado, 25 por Plasmodium falciparum y 21 por Plasmodium vivax, siendo el 2018 el año con más casos. En cuanto a las variables de edad y trimestre de gestación, prevalecieron en el grupo de 20 a 29 años (46 %) y en el segundo trimestre (37 %). Solo se observó una diferencia significativa entre los casos por año y la especie parasitaria. Conclusión. La malaria gestacional en Ecuador ha aumentado en los últimos cinco años, por lo que es importante informar a las mujeres en estado de gravidez sobre las medidas preventivas para evitar el contagio con el parásito, dadas las graves consecuencias que conlleva para ellas y sus hijos.

Introduction: Gestational malaria, defined as the presence of Plasmodium spp. in maternal peripheral blood or in the placenta, is considered an important public health problem in tropical and subtropical regions. Objective: To determine the frequency of gestational malaria cases diagnosed in Ecuador between 2015 and 2018. Materials and methods: We conducted a descriptive, retrospective, and cross-sectional study. Results: There were 46 cases of gestational malaria between 2015 and 2018: 25 caused by Plasmodium falciparum and 21 by Plasmodium vivax. The year with the most cases in this period was 2018. The age group most affected was 20 to 29 years old with 21 cases (46%). Prevalence was found to be highest in the second trimester of pregnancy with 17 cases (37%). A significant difference was only observed between cases per year and parasitic species. Conclusion: The prevalence of gestational malaria in Ecuador increased in the last five years. Therefore, it is important to inform pregnant women about preventive measures to avoid infection given its serious consequences both for the mother and her unborn child.

Prevalencia de malaria en Aguarico, comunidad de la Amazonía ecuatoriana / Malaria prevalence in Aguarico, a community in the Ecuadorian Amazon

Objetivo. Determinar el estado actual de la prevalencia de Plasmodium en pacientes febriles que acuden al Hospital Básico Franklin Tello Nuevo Rocafuerte, cantón Aguarico, comparando con los datos de otros estudios epidemiológicos de la misma zona y la frontera con el vecino país de Perú. Métodos. Se realizó un estudio observacional descriptivo, retrospectivo de prevalencia. Desde 2011-2015 se recogieron 2.668 muestras de sangre capilar correspondientes al 55,04% de la población total del cantón Aguarico. Se empleó la técnica Gota Gruesa y Frotis coloreados con Giemsa para determinar positividad de Plasmodium. Resultados. El rango de variación de la prevalencia en los pobladores de las comunidades investigadas osciló entre 2,38% y 28,57%, detectándose mayor prevalencia en el sexo masculino (50,56 %). Estos hallazgos son similares a los estudios previos realizados entre 1992-1995, en la misma región del Aguarico. El Riesgo Relativo es (RR) es de 1,36 y el Odds Ratio (OR) fue de 1,71, siendo mayor el riesgo a desarrollar la enfermedad en los positivos. Conclusiones. Los datos de la investigación confirman la presencia de un foco autóctono de malaria producida por Plasmodium vivax en la selva amazónica ecuatoriana, excepto 2 casos de P. falciparum importados de Perú. Los casos diagnosticados clínicamente y mediante la técnica de la Gota Gruesa, fueron tratados con medicación antipalúdica con excelente adherencia al medicamento.

Objective. To determine the status of the prevalence of Plasmodium in febrile patients who attend the Franklin Tello Nuevo Rocafuerte Basic Hospital, Aguarico town, comparing the results obtained with data from other epidemiological studies in the same area, and places near the border with Peru. Methods. A descriptive, retrospective, observational study of prevalence was carried out. From 2011-2015 2,668 capillary blood samples were collected corresponding to 55.04% of the total population of the Aguarico town. The Thick Drop and Giemsa-stained smear technique was used to determine Plasmodium positivity. Results. The range of variation of the prevalence in the inhabitants of the investigated communities ranged between 2.38% and 28.57%, detecting a higher prevalence in males (50.56%). These findings are like previous studies carried out between 1992-1995, in the same Aguarico region. The Relative Risk (RR) is 1.36 and the Odds Ratio (OR) was 1.71, with the risk of developing the disease being greater in the positives. Conclusions. The research data confirm the presence of an autochthonous focus of malaria produced by Plasmodium vivax in the Ecuadorian Amazon rainforest, except for 2 cases of P. falciparum imported from Peru. The cases diagnosed clinically and using the thick gout technique were treated with antimalarial medication with excellent adherence to the medication.

Prevalence of antifolate drug resistance markers in Plasmodium vivax in China / 医学前沿

The dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes of Plasmodium vivax, as antifolate resistance-associated genes were used for drug resistance surveillance. A total of 375 P. vivax isolates collected from different geographical locations in China in 2009-2019 were used to sequence Pvdhfr and Pvdhps. The majority of the isolates harbored a mutant type allele for Pvdhfr (94.5%) and Pvdhps (68.2%). The most predominant point mutations were S117T/N (77.7%) in Pvdhfr and A383G (66.8%) in Pvdhps. Amino acid changes were identified at nine residues in Pvdhfr. A quadruple-mutant haplotype at 57, 58, 61, and 117 was the most frequent (57.4%) among 16 distinct Pvdhfr haplotypes. Mutations in Pvdhps were detected at six codons, and the double-mutant A383G/A553G was the most prevalent (39.3%). Pvdhfr exhibited a higher mutation prevalence and greater diversity than Pvdhps in China. Most isolates from Yunnan carried multiple mutant haplotypes, while the majority of samples from temperate regions and Hainan Island harbored the wild type or single mutant type. This study indicated that the antifolate resistance levels of P. vivax parasites were different across China and molecular markers could be used to rapidly monitor drug resistance. Results provided evidence for updating national drug policy and treatment guidelines.

PCR-RFLP Based genetic diversity of Plasmodium vivax genotypes in district Mardan, Pakistan / Diversidade genética baseada em PCR RFLP de genótipos de Plasmodium vivax no distrito de Mardan, Paquistão

Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3βgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3β genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp 3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3βgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3β genes of P. vivax isolates by using PCR/RFLP from District Mardan and [...].

O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3β, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3β, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp 3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3βgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3β de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados [...].

PCR-RFLP Based genetic diversity of Plasmodium vivax genotypes in district Mardan, Pakistan / Diversidade genética baseada em PCR-RFLP de genótipos de Plasmodium vivax no distrito de Mardan, Paquistão

Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3ßgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3ß genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3ßgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3ß genes of P. vivax isolates by using PCR/RFLP from District Mardan and showed a remarkable level of genetic diversity in the studied genes of circulating parasites in the study area. The results of this study will contribute in future studies about the genetic structure of parasite and vaccine development against the malaria.

O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3ß, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3ß, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3ßgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3ß de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados de parasitas circulantes na área de estudo. Os resultados desse estudo contribuirão em estudos futuros sobre a estrutura genética do parasita e o desenvolvimento de vacinas contra a malária.

Sistemas de expresión de proteínas recombinantes para el análisis funcional de antígenos de Plasmodium falciparum y Plasmodium vivax: una revisión / Recombinant Protein Expression Systems for Functional Analysis of Plasmodium falciparum and Plasmodium vivax Antigens: A Review / Sistemas de expressão de proteínas recombinantes para o analise funcional de antígenos de Plasmodium falciparum e Plasmodium vivax: uma revisão

Introducción: Para diseñar vacunas es necesario comprender la función de los antígenos de Plasmodium spp. in-volucrados en la invasión a células hospederas. Diferentes investigaciones han generado proteínas recombinantes utilizando sistemas de expresión heterólogos y así han obtenido moléculas semejantes a las nativas. Con estos avances se desarrollan estrategias que bloquean la infección de estos patógenos. Objetivo: Describir las características y los aspectos metodológicos más importantes de los sistemas de expresión de las proteínas recombinantes en estudios funcionales de Plasmodium spp. Metodología: Revisión descriptiva de estudios publicados en Pubmed, Science Direct, Embase y Medline, entre 2010 y 2020, que incluyeran sistemas recombinantes en células de Escherichia coli, de mamífero y sistemas libres de células, para estudios funcionales de antígenos de Plasmodium falciparum y Plasmodium vivax. Se revisaron 70 artículos originales y 58 cumplieron con los criterios establecidos. Resultados: Obtener proteínas recombinantes mediante un sistema procariota, de mayor rendimiento y bajo costo, ha permitido estudiar un número importante de antígenos. Los sistemas con células de mamífero y libres de células, que permiten modificaciones postraduccionales y plegamiento adecuado de moléculas, se usan para producir librerías de antígenos con estructura conformacional similar a la nativa. Conclusión: El estudio de los antígenos de Plasmodium spp. implicados en la infección y desarrollo de células diana requiere una adecuada selección del método de producción recombinante. El refinamiento de procesos de expresión en sistemas procariotas, eucariotas e in vitro, mediante ingeniería genética y cultivo celular, permitirá mejores rendimientos y menor costo.

Introduction: Understanding the function of Plasmodium spp. Antigens involved in invasion of host cells is necessary to design vaccines. Different studies have generated recombinant proteins using heterologous expression systems, obtaining molecules similar to native ones. These advances are es-sential to develop strategies that block the infection of these pathogens. Objective: Describe the most important characteristics and methodological aspects of recombinant protein expression systems in functional studies of Plasmodium spp. Methodology: Descriptive review of studies published in Pubmed, Science Direct, Embase and Medline, between 2010 and 2020, that included recombinant systems in Escherichia coli cells, mam-malian and cell-free, for functional studies of Plasmodium falciparum and Plasmodium vivax antigens. 70 original articles were reviewed, 58 met the established criteria. Results: Obtaining recombinant proteins by means of a prokaryotic system, with higher performance and low cost, has allowed functional studies of a significant number of antigens. Mammalian cell and cell free systems, which allow for post-translational modifications and adequate folding of molecules, are used to produce antigen libraries with native-like conformational structure. Conclusion:Plasmodium spp. antigen study involved in infection and development in target cells, re-quires adequate selection of the recombinant production method. The refinement of expression pro-cesses in prokaryotic, eukaryotic and in vitro systems, through genetic engineering and cell culture, will allow better yields and lower cost

Introdução: Para desenvolver vacinas, é necessário entender a função dos antígenos de Plasmodium spp. envolvidos na invasão das células hospedeiras. As pesquisas têm gerado proteínas recombinan-tes utilizando sistemas de expressão heterólogos para obter moléculas similares às nativas. Com estes avanços, estratégias que bloqueiam a infecção destes patógenos estão sendo desenvolvidas. Objetivo: Descrever as características mais importantes e aspectos metodológicos dos sistemas de expressão de proteínas recombinantes em estudos funcionais de Plasmodium spp. Metodologia: Revisão descritiva dos estudos publicados em Pubmed, Science Direct, Embase e Medline, entre 2010 e 2020, que incluíram sistemas recombinantes em células de Escherichia coli, de mamífero e sistemas livres de células, para estudos funcionais dos antígenos de Plasmodium fal-ciparum e Plasmodium vivax. Setenta artigos originais foram revisados e 58 preenchiam os critérios estabelecidos. Resultado: A obtenção de proteínas recombinantes usando um sistema procariótico, com maior rendimento e baixo custo, permitiu o estudo de um número significativo de antígenos. Sistemas de células mamíferas e sem células, que permitem modificações pós-tradução e dobramento adequado das moléculas, são usados para produzir bibliotecas de antígenos com uma estrutura semelhante à nativa. Conclusão: O estudo dos antígenos Plasmodium spp. envolvidos na infecção e no desenvolvimento das células-alvo requer uma seleção adequada do método de produção recombinante. O refinamento dos processos de expressão em sistemas procarióticos, eucarióticos e in vitro, através da engenharia genética e da cultura celular, permitirá melhores rendimentos e menores custos.

Characterization of an outbreak of malaria in a non-endemic zone on the coastal region of Ecuador / Caracterización de un brote de malaria en una zona no endémica de la región costera del Ecuador

Abstract | Introduction: Malaria is a vector-borne disease widely distributed in the Amazon region and the coastal area of northern Ecuador. Its epidemiology involves related factors such as human settlements, vector reproduction sites, mobility, productive activity, and the response capacity of health systems, among others. Objective: To describe malaria transmission by Plasmodium vivax in a non-endemic area of Ecuador by analyzing the epidemiological and entomological factors involved. Materials and methods: We conducted the epidemiological study of the cases reported in the Salinas canton and the characterization of vector breeding sites through captures of larvae and adult mosquitoes by human capture of resting mosquitoes. Results: We detected 21 cases of malaria with local transmission related to the presence of initial cases in Venezuelan migrant patients and identified Anopheles albimanus as the predominant vector in natural breeding sites such as estuaries, wells, and water channels. Conclusions: We detected an outbreak of malaria triggered by imported cases from Venezuela. Climatic, social, environmental, and ecological conditions have favored the development of the vector maintaining the transmission cycle. Strategies to control imported malaria should be multiple including early case detection and control of productive breeding sites to avoid local transmission.

Resumen | Introducción. La malaria o paludismo es una enfermedad transmitida por vectores, ampliamente distribuida en la región amazónica y en la zona costera del norte del Ecuador. Su epidemiología involucra factores relacionados, como asentamientos humanos, sitios de reproducción del vector, movilidad, actividad productiva y capacidad de respuesta de los sistemas de salud, entre otros.Objetivo. Describir la transmisión de malaria por Plasmodium vivax en un área no endémica de Ecuador, mediante el análisis de los factores epidemiológicos y entomológicos involucrados. Materiales y métodos. Se hizo el estudio epidemiológico de los casos reportados en el cantón de Salinas y la caracterización de criaderos del vector con capturas de larvas y adultos mediante la captura de mosquitos en reposo. Resultados. Se detectaron 21 casos de malaria con transmisión local relacionados con la presencia de casos iniciales importados de Venezuela. Se identificó Anopheles albimanuscomo el vector predominante en criaderos naturales como estuarios, pozos y canales de agua. Conclusiones. Se detectó un brote de malaria desencadenado por casos importados de Venezuela. Las condiciones climáticas, sociales, ambientales y ecológicas han favorecido el desarrollo del vector, manteniendo el ciclo de transmisión. Las estrategias para controlar la malaria importada deben ser multifacéticas, e incluir la detección temprana de casos y el control de criaderos productivos para evitar la transmisión local.

Nutritional determinants as potential contributors to anemia in children and adolescents with malaria by P. vivax / Determinantes nutricionais como potenciais contribuintes para anemia em crianças e adolescentes com malária por P. vivax / Determinantes nutricionales como posibles contribuyentes para la anemia en niños y adolescentes con malaria por P. Vivax

Background and Objectives: Nutrition, in addition to its physiological function, plays an important role in the recovery of individuals with malaria, a disease that still represents a serious public health problem in the world. The objective of this study was to assess nutritional determinants in the frequency of food intake and the occurrence of anemia in children and adolescents with P. vivax malaria. Methods: A cross-sectional analytical study was carried out between 2014 and 2015 in the Marajo Island. The hemoglobin level was measured by the colorimetric enzymatic reaction and a questionnaire of food intake frequency was used to assess the consumption of different types of food. Results: A total of 67 patients met the inclusion criteria, from which 62.7% were children and 37.3% were adolescents. There was a high consumption of ultra-processed foods in both age groups. Anemia occurred in 52.2% of patients, and in most of them it was moderate. There was no significant association between anemia and sex, age group or parasitemia at admission. However a significant association was found between anemia and the ingestion of ultra-processed foods. Conclusion: The ingestion of ultra-processed foods contributes to anemia in children and adolescent with malaria by P. vivax.(AU)

Justificativa e Objetivos: A nutrição, além de sua função fisiológica, desempenha um papel importante na recuperação de indivíduos com malária, uma doença que ainda representa um grave problema de saúde pública no mundo. O objetivo deste estudo é avaliar os determinantes nutricionais na frequência da ingestão alimentar e a ocorrência de anemia em crianças e adolescentes com malária por P. vivax. Métodos: Estudo transversal analítico, realizado entre 2014 e 2015 na ilha do Marajó. O nível de hemoglobina foi medido pela reação enzimática colorimétrica e um questionário de frequência de ingestão alimentar foi utilizado para avaliar o consumo de alimentos. Resultados: Um total de 67 pacientes atendeu aos critérios de inclusão do estudo, dos quais 62,7% eram crianças e 37,3% adolescentes. Houve alto consumo de alimentos ultraprocessados em ambas as faixas etárias. A anemia foi detectada em 52,2% dos pacientes e, na maioria deles, foi moderada. Não houve associação significativa entre anemia e sexo, faixa etária ou parasitemia na admissão. No entanto, encontramos uma associação significativa entre presença de anemia e ingestão de alimentos ultraprocessados. Conclusão: A ingestão de alimentos ultraprocessados contribui para a anemia em crianças e adolescentes com malária por P. vivax.(AU)

Justificación y objetivos: La nutrición, además de su función fisiológica, juega un papel importante en la recuperación de las personas con malaria, una enfermedad que todavía representa un importante problema de salud pública en el mundo. El objetivo de este estudio es evaluar los determinantes nutricionales en la frecuencia del consumo de alimentos y la ocurrencia de anemia en niños y adolescentes con malaria por P. vivax. Métodos: se realizó un estudio analítico transversal entre 2014 y 2015, en la Isla de Marajó. El nivel de hemoglobina fue evaluado por ensayos enzimáticos colorimétricos y se utilizó un cuestionario de frecuencia de consumo de alimentos para evaluar el consumo. Resultados: Un total de 67 pacientes cumplió los criterios de inclusión en el estudio, de los cuales el 62,7% eran niños y el 37,3% adolescentes. Se registró un alto consumo de alimentos ultraprocesados en niños y adolescentes. La anemia se detectó en el 52,2% de los pacientes, de carácter moderada principalmente. No se encontró una asociación significativa entre anemia y sexo, grupo de edad o parasitemia al ingreso. Sin embargo, se encontró una asociación significativa entre la anemia y la ingestión de alimentos ultraprocesados. Conclusión: La ingesta de alimentos ultraprocesados se asocia con la presencia de anemia en niños y adolescentes con malaria por P. vivax.(AU)

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening

BACKGROUND Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Avaliação da resposta de anticorpos IgG contra diferentes variantes alélicas do Antígeno 1 de Membrana Apical (AMA-1) de Plasmodium vivax em indivíduos de áreas endêmicas de malária / Evaluation of IgG antibody response against different variants of Plasmodium vivax apical membrane antigen 1 (AMA-1) in individuals from malaria endemic areas

O Plasmodium vivax é a espécie com maior distribuição geográfica no mundo e a que predomina nas Américas, incluindo o Brasil. Comparado ao Plasmodium falciparum, poucas vacinas contra o P. vivax encontram-se em fase de testes clínicos. Um dos antígenos de formas sanguíneas de P. vivax candidato a vacina é o Antígeno 1 de Membrana Apical (PvAMA-1). Entretanto, a diversidade antigênica do mesmo na natureza representa um grande desafio para seu uso no desenvolvimento de uma vacina de ampla cobertura. No presente estudo, avaliamos se os polimorfismos de sequências já descritos são capazes de influenciar na eficácia de uma vacina baseada em PvAMA-1. Para isso, geramos 9 proteínas recombinantes a partir da levedura Pichia pastoris, as quais são representativas de diferentes variantes alélicas do antígeno PvAMA-1, a saber: Belem, Chesson I, Sal-1, Indonesia XIX, SK0814, TC103, PNG_05_ESP, PNG_62_MU e PNG_68_MAS. Após expressão e purificação das proteínas selecionadas, avaliamos comparativamente por ELISA a resposta de anticorpos IgG naturalmente adquiridos em indivíduos expostos a malária, procedentes da Região Amazônica. Todas as proteínas foram obtidas com rendimento e pureza apropriados para os estudos propostos. A prevalência total de indivíduos expostos a malária com anticorpos contra PvAMA-1 Belem foi de 53,68%, em 611 amostras de soro testadas. Entre 100 das amostras sorologicamente positivas para PvAMA-1 Belem, os maiores valores de DO492 foram obtidos para as variantes Chesson I, SK0814 e Sal-1, sugerindo que epítopos comuns ou de reatividade cruzada estão sendo reconhecidos nessas variantes. Por outro lado, níveis mais baixos de DO492 foram obtidos para as variantes Indonesia XIX, TC103, PNG_05_ESP, PNG_62_MU e PNG_68_MAS, o que pode significar que essas variantes são menos prevalentes ou não circulam no Brasil. Soros policlonais de camundongos C57BL/6 previamente imunizados com PvAMA-1 Belem foram testados quanto ao reconhecimento das diferentes variantes por ELISA. Nossos resultados demonstraram que as variantes Chesson I, Indonesia XIX, SK0814, Sal-1 e a proteína homóloga foram predominantemente reconhecidas. Por fim, ensaios de competição baseados em ELISA revelaram que as proteínas Chesson I, Indonesia XIX, SK0814 e Sal-1, na fase solúvel, foram capazes de inibir a ligação de anticorpos à variante Belem aderida a placa, sugerindo a presença de epítopos comuns ou de reatividade cruzada entre as mesmas. Nossos dados sugerem que uma vacina baseada na variante PvAMA-1 Belem gera anticorpos variante-transcendentes. Entretanto, para gerar uma vacina universal baseada em PvAMA-1, uma formulação multi-alélica, incluindo variantes da Tailândia e Papua Nova Guiné, deverão ser testadas

Plasmodium vivax has the largest geographical distribution Plasmodium species in the world, and is predominant in the Americas, including Brazil. Fewer P. vivax vaccines than P. falciparum vaccines have successfully reached clinical trials. One of the candidate antigens for a blood-stage P. vivax vaccine is the apical membrane antigen 1 (PvAMA-1). However, the high natural variability found in this antigen presents a major challenge for its development into a wide-range vaccine. In the present study, we evaluated whether sequence polymorphisms would influence a vaccine based on PvAMA-1. To achieve this, we generated 9 recombinant proteins from the yeast Pichia pastoris, representative of different allelic variants of the PvAMA-1 antigen: Belem, Chesson I, Sal-1, Indonesia XIX, SK0814, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS. After expression and purification of these proteins, we compared, by ELISA and IgG blocking, the natural acquired response from malaria-exposed individuals in the Amazon Region. All proteins selected had the appropriate yield and purity for the proposed studies. The total prevalence of malaria-exposed individuals with reactivity to PvAMA-1 Belem was 53,68%, from 611 serum samples tested. One hundred of these serologically positive samples were further tested against recombinant proteins representing the other allelic variants. The highest OD values resulted from Sal-1, Chesson I and SK0814 variants, suggesting that common epitopes or cross-reactivity exist across the variants. On the other hand, the lowest OD values resulted from the variants Indonesia XIX, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS, which may mean these variants are less prevalent or do not circulate in Brazil. Polyclonal sera from C57BL/6 mice immunized with PvAMA-1 Belem were tested for recognition of different variants by ELISA. Our results showed that the variants Chesson I, Sal-1, Indonesia XIX, SK0814 and the homologous protein were predominantly recognized. Lastly, ELISA-based competition assays revealed that Chesson I, Sal-1, Indonesia XIX and SK0814 proteins were able to inhibit antibody binding to the Belem variant, suggesting the presence of common epitopes or cross-reactivity between these variants. Our data suggest that a vaccine based on the PvAMA-1 Belem variant displays strain-transcendent antibodies. However, to generate a universal vaccine based on PvAMA-1, a multiallelic formulation including variants from Thailand and Papua New Guinea must be tested

Caracterização imunológica da formulação vacinal baseada em VLP (Virus Like Particle) da Proteína Circumsporozoíta de Plasmodium vivax / Immunological characterization of the VLP (Virus Like Particle) vaccine formulation fused to Circumsporozoite Protein of Plasmodium vivax

O Plasmodium vivax é a espécie mais comum de parasita causador da malária humana encontrada fora da África, com maior endemicidade na Ásia, América Central e do Sul e Oceania. Embora o Plasmodium falciparum cause a maioria do número de mortes, o P. vivax pode levar à malária grave e resultar em morbimortalidade significativa. O desenvolvimento de uma vacina protetora será um passo importante para a eliminação da malária. Recentemente, uma formulação contendo as três variantes alélicas da proteína circumsporozoíta de P. vivax (PvCSP - All epitopes) induziu proteção parcial em camundongos após desafio com esporozoíto híbrido Plasmodium berghei (Pb), no qual as repetições centrais do PbCSP foram substituídas por repetições PvCSP-VK210 (esporozoítos Pb/Pv). No presente estudo, a proteína quimérica PvCSP contendo as variantes alélicas (VK210, VK247 e P. vivax-like) fusionadas com a proteína de nucleocapsídeo do vírus da caxumba (formando partículas semelhantes a nucleocapsídeos ou do inglês, NLP - Núcleo Like Particles) na ausência (NLP-CSPR) ou na presença do domínio C-terminal (CT) conservado da PvCSP (NLP-CSPCT). Para a realização do estudo selecionamos os adjuvantes Poly (I:C), um RNA sintético de dupla fita, agonista do receptor Toll do tipo 3 (TLR3) ou o adjuvante Montanide ISA 720, uma emulação óleo em agua. Para obter uma forte resposta imune, a levedura Pichia pastoris foi usada para expressar as proteínas recombinantes na forma de NLPs. Camundongos foram imunizados com cada uma das proteínas recombinantes em combinação com os adjuvantes citados. Embora ambas as NLPs tenham sido capazes de gerar uma forte resposta imune, com altos níveis de títulos e longevidade, apenas a formulação contendo a proteína NLP-CSPCT na presença do adjuvante Poly (I:C) foi selecionada para ser explorada em experimentos futuros. Esta proteína em combinação com o adjuvante Poly (I:C) induziu alta frequência de células secretoras de anticorpos específicas para o antígeno homólogo nos dias 5 e 30, no baço e na medula óssea, respectivamente. Altos títulos de IgG contra as 3 variantes de PvCSP foram detectados nos soros. Posteriormente camundongos imunizados com NLP-CSPCT foram desafiados com esporozoítos Pb/Pv e a parasitemia no 5º dia demonstrou proteção estéril em 30% dos camundongos desafiados. Portanto, a formulação vacinal gerada neste estudo tem potencial para ser explorada no desenvolvimento de uma vacina universal contra a malária causada por P. vivax

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP--All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein (assembling into nucleo like particles - NLP) in the absence (NLP-CSPR) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSPCT). To carry out the study, we selected the adjuvants Poly (I:C), a synthetic double-stranded RNA, Toll-like receptor 3 (TLR3) agonist or Montanide ISA 720 adjuvant, an oil-water emulation. To elicit stronger immune response, Pichia pastoris yeast was used to produce the NLPs. Mice were immunized with each recombinant protein in combination with above. Although both NLPs were able to generate stronger immune response, with high antibodies titer levels and longevity, formulation containing NLP-CSPCT in the presence of Poly (I:C) was selected to be explored in future experiments. NLP-CSPCT with Poly (I:C) adjuvant presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, immunized mice with NLP-CSPCT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.

Caracterização da resposta imune celular e humoral frente ao antígeno protetor rico em cisteína de p. Vivax (pvcyrpa) em indivíduos da Amazônia brasileira / Characterization of the cellular and humoral immune response to the protective cysteine-rich antigen of p. Vivax (pvcyrpa) in individuals from the Brazilian Amazon

As infecções causadas por Plasmodium vivax representam um grave problema de saúde pública, sendo esta espécie plasmodial considerada uma das mais difíceis de se eliminar. Por esta razão a pesquisa de antígenos candidatos vacinais específicos para P. vivax necessita de impulso, uma vez que atualmente existe uma única candidata vacinal em ensaios clínicos na iniciativa global de desenvolvimento de vacinas anti-maláricas. Neste cenário, a proteína PvCyRPA, presente em merozoítas de P. vivax surgiu como uma promissora candidata vacinal. Ensaios em P. falciparum demonstraram que anticorpos contra esta proteína são capazes de impedir a invasão de eritrócitos tanto in vitro como in vivo, além disso, essa proteína foi indicada em uma ampla biblioteca de antígenos de P. vivax como uma das proteínas com maior potencial protetor, mesmo com baixos níveis de anticorpos. Nesse aspecto, o objetivo deste trabalho foi caracterizar as respostas imune humoral e celular frente à proteína PvCyRPA em uma população da Amazônia brasileira naturalmente exposta à malária e correlacionar as respostas encontradas com parâmetros de exposição/proteção. Nos resultados obtidos verificou-se que a PvCyRPA é naturalmente imunogênica na população de estudo, e que a presença de anticorpos IgM contra esta proteína parece ser um indicativo de infecções recentes. Os anticorpos citofílicos IgG3 produzidos nessa população estão correlacionados com o número de infecções anteriores. A proteína apresenta uma sobreposição importante de epítopos de células B e T e as sequências correspondentes aos epítopos de células T foram capazes de induzir a produção de INF-γ em ensaios de ELISPOT. Estes dados representam a primeira caracterização da resposta imune frente à PvCyRPA em populações brasileiras e reforçam o potencial deste antígeno como um candidato vacinal para P. vivax. (AU)

Infections caused by Plasmodium vivax represent a serious public health problem, and this plasmodial species is considered one of the most difficult to eliminate. For this reason, the search for specific vaccine candidate antigens for P. vivax needs to be boosted, since there is currently a single vaccine candidate in clinical trials in the global anti-malarial vaccine development initiative. In this scenario, the protein PvCyRPA, present in P. vivax merozoites has emerged as a promising vaccine candidate. Tests on P. falciparum have shown that antibodies against this protein are able to prevent erythrocyte invasion both in vitro and in vivo. In addition, this protein has been indicated in a wide library of P. vivax antigens as one of the proteins with the greatest protective potential, even with low levels of antibodies. In this regard, the objective of this work was to characterize the humoral and cellular immune responses against PvCyRPA protein in a population of Brazilian Amazon naturally exposed to malaria and to correlate the responses found with exposure / protection parameters. The results obtained showed that PvCyRPA is naturally immunogenic in the study population, and that the presence of IgM antibodies against this protein seems to be an indicative of recent infections. The cytophilic IgG3 antibodies produced in this population seems to be correlated with the number of previous infections. The protein has an important overlap of B and T cell epitopes and the sequences corresponding to the T cell epitopes were able to induce the production of INF-γ in ELISPOT assays. These data represent the first characterization of the immune response to PvCyRPA in Brazilian populations and reinforce the potential of this antigen as a vaccine candidate for P. vivax. (AU)

Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain is conserved in field isolates from Brazilian Amazon

In the present study, we investigated the genetic diversity of Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain in two municipalities of the main malaria hotspot in Brazil, i.e., the Juruá Valley, and observed complete sequence identity among all P. vivax field isolates and the Sal-1 reference strain. Analysis of PvMCA1 catalytic domain in different P. vivax genomic sequences publicly available also revealed a high degree of conservation worldwide, with very few amino acid substitutions that were not related to putative histidine and cysteine catalytic residues, whose involvement with the active site of protease was herein predicted by molecular modeling. The genetic conservation presented by PvMCA1 may contribute to its eligibility as a druggable target candidate in vivax malaria.

Molecular variation of Plasmodium vivax dehydrofolate reductase in Mexico and Nicaragua contrasts with that occurring in South America / Variación molecular de la dehidrofolato reductasa de Plasmodium vivax en México y Nicaragua contrasta con la que ocurre en Sudamérica

Abstract: Objective: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. Materials and methods: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. Results: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. Conclusions: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.

Resumen: Objetivo: Determinar mutaciones en la dihydrofolato reductasa deP. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. Material y métodos: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. Resultados: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. Conclusiones: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.

Assessment of the efficacy and safety of chloroquine monotherapy for the treatment of acute uncomplicated gestational malaria caused by P. vivax, Córdoba, Colombia, 2015-2017 / Evaluación de la eficacia y seguridad de la monoterapia con cloroquina para tratar malaria gestacional aguda no complicada debida p. vivax, córdoba, colombia, 2015-2017

RESUMEN Objetivo: determinar la eficacia y seguridad de la monoterapia con cloroquina en gestantes colombianas con ataque agudo no complicado de malaria vivax (MGV). Materiales y métodos: estudio de cohorte prospectiva en pacientes gestantes que consultaron de manera espontánea entre 1 febrero de 2015 y 31 diciembre de 2017 a los puestos de malaria o de control prenatal en dos poblaciones de Colombia, en quienes se confirmó el diagnóstico de Plasmodium vivax mediante gota gruesa y qPCR (quantitative polymerase chain reaction). Se midieron variables sociodemográficas, falla terapéutica (FT) y eventos adversos serios a los 28 días y la frecuencia de recurrencia-recaída (RR) con seguimiento de 120 días. Se aplicó el protocolo de la OMS para evaluar la eficacia de monoterapia con cloroquina (m-CQ). Resultados: se captaron 47 gestantes; en el seguimiento de 28 días no hubo pérdidas y hubo 4,2 % (2/47) de FT. En el seguimiento de 45 mujeres entre los días 29 y 120 hubo 11 pérdidas (24,4 % = 11/45) y 13 RR con frecuencia que varió entre 29 y 53 % según el tipo de análisis. Conclusiones: la cloroquina conserva muy alta eficacia para curar el ataque agudo de malaria vivax en malaria gestacional (MG) en Colombia, y continúa siendo una buena opción para el tratamiento de la fase aguda. La frecuencia de RR es alta. Se requieren estudios que evalúen alternativas terapéuticas en la MG. Hay urgente necesidad de disponer de medicamentos o procedimientos que reduzcan ese altísimo riesgo.

ABSTRACT Objective: To determine the efficacy of chloroquine monotherapy in Colombian pregnant women with acute uncomplicated malaria vivax Materials and methods: Prospective cohort study in pregnant women who presented of their own accord between February 1, 2015 and December 31, 2017 to malaria or prenatal care centers in two Colombian towns and in whom the diagnosis of Plasmodium vivax was confirmed by means of blood spot test and and quantitative polymerase chain reaction (qPCR). Measured variables included sociodemographics, therapeutic failure (TF) and serious adverse events at 28 days and frequency of recurrence-relap (RR) over a follow-up period of 120 days. The WHO protocol was applied for the assessment of monotherapy with cloroquine (m-CQ) efficacy. Results: Overall, 47 pregnant women were identified. During the 28-day follow-up period there were no losses, and there were two cases of TP (4.2%=2/47). Of the 45 women followed between 29 and 120 days, 11 were lost (24.4%=11/45) and there were 13 cases of RR, with an RR frequency ranging between 29 and 53 % depending on the type of analysis. Conclusions: Chloroquine is still highly effective as a cure of acute malaria vivax attack in GM in Colombia, and continues to be a good option for the treatment of acute phase GM. The RR frequency is high. Studies are required that evaluate therapeutic alternatives in MG. There is a pressing need for medications and/or procedures that can help reduce this very high risk.

Increased platelet distribution width and reduced IL-2 and IL-12 are associated with thrombocytopenia in Plasmodium vivax malaria

BACKGROUND Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated. OBJECTIVE To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in Plasmodium vivax infected (Pv-MAL) patients. METHODS Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). FINDINGS Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1β and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values. MAIN CONCLUSIONS Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated P. vivax malaria.