Chloroquine and mefloquine resistance profiles are not related to the circumsporozoite protein (CSP) VK210 subtypes in field isolates of Plasmodium vivax from Manaus, Brazilian Amazon

BACKGROUND The central repetitive region (CRR) of the Plasmodium vivax circumsporozoite surface protein (CSP) is composed of a repetitive sequence that is characterised by three variants: VK210, VK247 and P. vivax-like. The most important challenge in the treatment of P. vivax infection is the possibility of differential response based on the parasite genotype. OBJECTIVES To characterise the CSP variants in P. vivax isolates from individuals residing in a malaria-endemic region in Brazil and to profile these variants based on sensitivity to chloroquine and mefloquine. METHODS The CSP variants were determined by sequencing and the sensitivity of the P. vivax isolates to chloroquine and mefloquine was determined by Deli-test. FINDINGS Although five different allele sizes were amplified, the sequencing results showed that all of the isolates belonged to the VK210 variant. However, we observed substantial genetic diversity in the CRR, resulting in the identification of 10 different VK210 subtypes. The frequency of isolates that were resistant to chloroquine and mefloquine was 11.8 and 23.8%, respectively. However, we did not observe any difference in the frequency of the resistant isolates belonging to the VK210 subtypes. MAIN CONCLUSION The VK210 variant is the most frequently observed in the studied region and there is significant genetic variability in the CRR of the P. vivax CSP. Moreover, the antimalarial drug sensitivity profiles of the isolates does not seem to be related to the VK210 subtypes.

Alterations in cytokines and haematological parameters during the acute and convalescent phases of Plasmodium falciparum and Plasmodium vivax infections

Haematological and cytokine alterations in malaria are a broad and controversial subject in the literature. However, few studies have simultaneously evaluated various cytokines in a single patient group during the acute and convalescent phases of infection. The aim of this study was to sequentially characterise alterations in haematological patters and circulating plasma cytokine and chemokine levels in patients infected with Plasmodium vivax or Plasmodium falciparum from a Brazilian endemic area during the acute and convalescent phases of infection. During the acute phase, thrombocytopaenia, eosinopaenia, lymphopaenia and an increased number of band cells were observed in the majority of the patients. During the convalescent phase, the haematologic parameters returned to normal. During the acute phase, P. vivax and P. falciparum patients had significantly higher interleukin (IL)-6, IL-8, IL-17, interferon-γ, tumour necrosis factor (TNF)-α, macrophage inflammatory protein-1β and granulocyte-colony stimulating factor levels than controls and maintained high levels during the convalescent phase. IL-10 was detected at high concentrations during the acute phase, but returned to normal levels during the convalescent phase. Plasma IL-10 concentration was positively correlated with parasitaemia in P. vivax and P. falciparum-infected patients. The same was true for the TNF-α concentration in P. falciparum-infected patients. Finally, the haematological and cytokine profiles were similar between uncomplicated P. falciparum and P. vivax infections.

Genetic polymorphisms in the glutamate-rich protein of Plasmodium falciparum field isolates from a malaria-endemic area of Brazil

The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.

Cells and mediators of inflammation (C-reactive protein, nitric oxide, platelets and neutrophils) in the acute and convalescent phases of uncomplicated Plasmodium vivax and Plasmodium falciparum infection

The haematological changes and release of soluble mediators, particularly C-reactive protein (CRP) and nitric oxide (NO), during uncomplicated malaria have not been well studied, especially in Brazilian areas in which the disease is endemic. Therefore, the present study examined these factors in acute (day 0) and convalescent phase (day 15) patients infected with Plasmodium falciparum and Plasmodium vivax malaria in the Brazilian Amazon. Haematologic parameters were measured using automated cell counting, CRP levels were measured with ELISA and NO plasma levels were measured by the Griess reaction. Our data indicate that individuals with uncomplicated P. vivax and P. falciparum infection presented similar inflammatory profiles with respect to white blood cells, with high band cell production and a considerable degree of thrombocytopaenia during the acute phase of infection. Higher CRP levels were detected in acute P. vivax infection than in acute P. falciparum infection, while higher NO was detected in patients with acute and convalescent P. falciparum infections. Although changes in these mediators cannot predict malaria infection, the haematological aspects associated with malaria infection, especially the roles of platelets and band cells, need to be investigated further.

Avaliação do potencial das proteínas de superfície de merozoítas de Plasmodium vivax (PvMSP-9 e PvMSP-3) como candidatas a compor uma vacina contra a malária / Assessing the potential of the surface proteins of merozoites of Plasmodium vivax (PvMSP-9 and PvMSP-3) as candidates to compose a malaria vaccine

As proteínas 9 e 3 de Superfície de Merozoítas de P. vivax (PvMSP9 e PvMSP3) vem sendo consideradas como importantes candidatas potenciais a uma vacina antimalárica principalmente pela: 1) localização na superfície do merozoíta; 2) homologia com plasmódios simianos e P. falciparum; 3) habilidade de IgG específicas em inibir a invasão dos merozoítas; 4) alta imunogenicidade em ensaios realizados em modelos animais. Entretanto, estudo da resposta imune em humanos para determinar o potencial de ambas como candidatas a vacina, embora fundamental, não havia sido realizado. Deste modo, descrevemos, em 3 artigos, o potencial das proteínas PvMSP9 e PvMSP3 como candidatas a compor uma vacina antimalárica através da caracterização da resposta imune naturalmente adquirida de indivíduos residentes em áreas endêmica. No primeiro trabalho, demonstramos que a PvMSP9 apresenta epitopos de células T fortemente reconhecidos e que são capazes de estimular a produção de IFN-gama e IL-4, além de uma potente resposta imune humoral com alta frequência e índices de reatividade de anticorpos IgG e subclasses (principalmente IgG1 e IgG2) para as regiões repetitivas espécie-específicas. No segundo trabalho, observamos que os epitopos de célula T descritos, apresentam propriedades promíscuas, sendo reconhecidos por indivíduos naturalmente expostos que apresentam diferentes grupos alélicos de HLA-DR e HLA-DQ, demonstrando que caso estes peptídeos fossem utilizados como imunógenos não haveria influência do genótipo de HLA na resposta imune celular. Por fim, observamos que a PvMSP3 é altamente reconhecida por anticorpos naturalmente adquiridos por indivíduos de área endêmica de malária, com altas frequências e concentrações de anticorpos citofílicos IgG1 e IgG3. Além disso, pudemos demonstrar, por spot-synthesis, que existem 25 epitopos de células B distribuídos por toda a extensão da proteína, passando por regiões conservadas e polimórficas. Deste modo, os dados gerados nesses trabalhos demonstram que as proteínas PvMSP9 e PvMSP3 são candidatas potenciais a compor uma vacina antimalárica, apresentando epitopos de células T e B capazes de gerar uma marcante resposta imune, com altos títulos de anticorpos citofílicos e uma potente resposta celular.

Estudo das respostas imunes celular e humoral frente à proteína 9 de superfície de merozoítas de plasmodium vivax (PVMSP-9), em indivíduos naturalmente expostos à infecção / Study of cellular and humoral immune responses against the protein surface 9 of merozoites of Plasmodium vivax (PvMSP-9) in individuals naturally exposed to infection

A proteína 9 de superfície de merozoíta de Plasmodium vivax (PvMSP9) apresenta homologia com diversas espécies incluindo a proteína ABRA de P. falciparum. PvMSP9 é composta por 979 aminoácidos, possui uma sequência sinal hidrofóbica e um grupo de 4 cisteínas na região N-terminal (PvMSP9-NT), e dois blocos de sequências repetitivas espécie específicas na região C-terminal (PvMSP9-RI e PvMSP9-RII). Estudos anteriores demonstraram que anticorpos monoclonais dirigidos para o P. vivax e anticorpos policlonais contra a MSP9 de P. cynomolgi são capazes de inibir a invasão de eritrócitos in vitro e que a PvMSP9 e altamente imunogênica em camundongos. No presente estudo, avaliamos a reatividade celular e de anticorpos contra a PvMSP9 em indivíduos naturalmente expostos à infecção. Em um estudo transversal realizado em Porto Velho (RO), células e soro de indivíduos de Ribeirinha (comunidade nativa da região que reside nas margens do rio Madeira, n=188), e Colina (comunidade de migrantes que vivem em áreas rurais perto da capital Porto Velho, n=122), foram testados por ELISPOT quanto à produção de IFN-gama e IL-4 sob estímulo de 11 peptídeos sintéticos, e por ELISA quanto a reatividade dos anticorpos IgG utilizando proteínas recombinantes representando a PvMSP-9. Nossos resultados mostram que o perfil de citocinas na resposta celular, foi diferente nas duas comunidades. Em Ribeirinha, a população nativa, a frequência de indivíduos positivos para a secreção das citocinas IFN-gama e IL-4 após estímulo com os peptídeos sintéticos foi similar. Os peptídeos mais imunogênicos induzindo a produção de IFN-gama foram pK (29,5por cento) e pA (23,8por cento) e de IL-4, pL (22,7por cento) e pA (17,3por cento). Em Colina, a população migrante, a frequência de positivos é principalmente para a secreção de IFN-gama, onde pE (29.5por cento) e pL (30.3por cento) foram os mais imunogênicos, e para a secreção de IL-4 as maiores frequências observadas...