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.

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.

Apoptose linfocitária na malária humana por P. Falciparum E. P. Vivax relação com a resposta imunitária ao parasito / Lymphocyte apoptosis in human malaria by P. Falciparum E. P. Vivax relation with the immune response to parasite

Para se avaliar o papel da apoptose linfocitária durante um episódio de malária não complicada por P. falciparum e P. vivax foram coletadas amostras de sangue de 35 pacientes com gota espessa positiva e de 17 indivíduos residentes na mesma área e sem história pregressa de malária. A apoptose das células CD4(positivo), CD8(positivo) e B foi analisada por citometria de fluxo ex vivo e após 96 horas de cultivo in vitro na presença ou não de antígenos plasmodiais. A expressão do antígeno Fas/APO 1 (CD95), as respostas imunes celular e humoral a antígenos do parasito, os percentuais/números das populações de células T e B circulantes, a ativação policlonal de células B e a reatividade de soros contra auto-antígenos também foram avaliadas. Observamos baixos percentuais de apoptose ex vivo nas células dos pacientes com malária. Entretanto, ainda ex vivo, verificamos um aumento dos percentuais de apoptose inicial nas células CD4 (positivo) e CD8 (positivo), assim como uma maior ativação das células CD4 (positivo) e níveis aumentados de IFN e de IL 10 e diminuídos de TNF, quando comparados com aqueles dos indivíduos sadios. Nenhuma diferença foi observada entre as concentrações plasmáticas de TNF e de IL 10 entre os pacientes com P. falciparum e com P. vivax, embora níveis mais elevados de IFN tivessem sido observados nos pacientes com P. vivax. Na análise ex vivo, não observamos correlação entre os níveis de citocinas e a ativação, viabilidade ou apoptose das CMSP. As respostas anticorpos anti-MSP-1 e anti PSS1 não estavam correlacionadas com a apoptose, a ativação celular ou com a parasitemia. Diferentemente, após 96h de cultura, os percentuais de apoptose estavam aumentados nas células CD4 (positivo), CD8 (positivo) e B e, mesmo na presença de estímulos, a resposta celular dos pacientes foi menor do que a dos indivíduos sadios. Anticorpos contra componentes da membrana dos eritrócitos, cardiolipina e ADN foram igualmente detectados tanto nos pacientes com malária como nos indivíduos clinicamente sadios. No entanto, anticorpos contra a actina foram mais frequentemente detectados nos pacientes com malária. Concluímos que os percentuais elevados de apoptose nessas células de pacientes infectados tanto pelo P. falciparum como pelo P. vivax poderia contribuir para a linfopenia associada à malária. Entretanto, a ausência de correlação entre apoptose, parasitemia, número de infecções prévias e de resposta proliferativa, especialmente à de células antígeno-específicas, sugere que a apoptose observada durante um episódio de malária não complicada poderia ser reflexo de uma resposta fisiológica do sistema imune que atuaria na tentativa de regular a ativação policlonal e manter o equilíbrio na densidade dessas populações celulares.

Genetic polymorphism of the serine rich antigen N-terminal region in Plasmodium falciparum field isolates from Brazil

In this work we investigated the frequency of polymorphism in exon II of the gene encoding most of the amino-terminal region of the serine rich antigen (SERA) in Plasmodium falciparum field samples. The blood samples were colleted from P. falciparum infected individuals in three areas of the Brazilian Amazon. Two fragments have been characterized by polymerase chain reaction: one of 175 bp corresponding to the repeat region with 5 octamer units and one other of 199 bp related to the 6 repeat octamer units of SERA protein. The 199 bp fragment was the predominant one in all the studied areas. The higher frequency of this fragment has not been described before and could be explained by an immunological selection of the plasmodial population in the infected individuals under study. Since repeat motifs in the amino-terminal region of SERA contain epitopes recognized by parasite-inhibitor antibodies, data reported here suggest that the analysis of the polymorphism of P. falciparum isolates in different geographical areas is a preliminary stage before the final drawing of an universal vaccine against malaria can be reached.