Analysis of drug resistance marker genes of Plasmodium falciparum after implementation of artemisinin-based combination therapy in Pune district, India.

The global emergence and spread of malaria parasites resistant to antimalarial drugs is a major problem in malaria control and elimination. In this study, samples from Pune district were characterized to determine prevalence of molecular markers of resistance to chloroquine (pfcrt codons C72S, M74I, N75E, K76T and pfmdr-1 N86Y, Y184F), pyrimethamine (pfdhfr C50R, N51I, C59R, S108N), sulfadoxine (pfdhps, S436A, A437G, K540E, A581G), and artemisinin (pfkelch13, C580Y, R539T). The pfcrt K76T mutation was found in 78% samples as CVMNT, SVMNT and CVIET haplotype. The pfmdr-1 N86Y and Y184F mutations were found in 54% of samples. The pfdhfr double mutation C59R + S108N was present in 67% of samples, while the pfdhfr triple mutation (N51I + C59R + S108N) was not detected. The pfdhps mutations A437G and K540E were found in 67% of samples. Single mutants of pfdhps were rare, with K540E detected in only 6 patient samples. Similarly, pfdhps A581G was found in 13 of the isolates. The molecular markers associated with artemisinin resistance (mutations in pfkelch13 C580Y, R539T) were not detected in any of the isolates. These results suggest an emerging problem with multidrug-resistant P. falciparum. Though the genotype conventionally associated with artemisinin resistance was not observed, chloroquine-resistant genotype has reached complete fixation in the population. Moreover, the prevalence of mutations in both pfdhfr and pfdhps, with the presence of the quadruple mutant, indicates that continued monitoring is required to assess whether sulfadoxine-pyrimethamine can be used efficiently as a partner drug for artemisinin for the treatment of P. falciparum.

Hemozoin-induced activation of human monocytes toward M2-like phenotype is partially reversed by antimalarial drugs-chloroquine and artemisinin.

Plasmodium falciparum malaria is the most severe form of malaria with several complications. The malaria pigment-hemozoin (Hz) is associated with severe anemia, cytokine dysfunction, and immunosuppression, thus making it an interesting target for developing new strategies for antimalarial therapy. Monocytes (MO) in circulation actively ingest Hz released by Plasmodium parasites and secrete pro- and anti-inflammatory cytokines. M1 and M2 types represent the two major forms of MO/macrophages (MQ) with distinct phenotypes and opposing functions. Imbalance in the polarization of these types is reported in many infectious diseases. Though the association of Hz with immunosuppression is well documented, its role in activation of MO in context of M1/M2 phenotypes remains to be addressed. We report here that natural Hz drives human MO toward M2-like phenotype as evidenced by the expression of M2 signature markers. Hz-fed MO showed elevated transcript and secreted level of IL-10, CCL17, CCL1, expression of mannose-binding lectin receptor (CD206), and arginase activity. Hz attenuated HLA-DR expression, nitric oxide, and reactive oxygen species production, which are the features of M1 phenotype. Our data also implicate the involvement of p38 MAPK, PI3K/AKT, and NF-κB signaling pathways in skewing of Hz-fed MO toward M2-like type and suppression of mitogen-stimulated lymphocyte proliferation. Importantly, antimalarial drugs-chloroquine and artemisinin-partially reversed activation of Hz-induced MO toward M2-like phenotype. Considering the limitations in the current therapeutic options for malaria, we propose that these drugs may be re-examined for their potential as immunomodulators and candidates for adjunctive treatment in malaria.

IgG autoantibody to brain beta tubulin III associated with cytokine cluster-II discriminate cerebral malaria in central India.

BACKGROUND: The main processes in the pathogenesis of cerebral malaria caused by Plasmodium falciparum involved sequestration of parasitized red blood cells and immunopathological responses. Among immune factors, IgG autoantibodies to brain antigens are increased in P. falciparum infected patients and correlate with disease severity in African children. Nevertheless, their role in the pathophysiology of cerebral malaria (CM) is not fully defined. We extended our analysis to an Indian population with genetic backgrounds and endemic and environmental status different from Africa to determine if these autoantibodies could be either a biomarker or a risk factor of developing CM. METHODS/PRINCIPAL FINDINGS: We investigated the significance of these self-reactive antibodies in clinically well-defined groups of P. falciparum infected patients manifesting mild malaria (MM), severe non-cerebral malaria (SM), or cerebral malaria (CM) and in control subjects from Gondia, a malaria epidemic site in central India using quantitative immunoprinting and multivariate statistical analyses. A two-fold complete-linkage hierarchical clustering allows classifying the different patient groups and to distinguish the CM from the others on the basis of their profile of IgG reactivity to brain proteins defined by PANAMA Blot. We identified beta tubulin III (TBB3) as a novel discriminant brain antigen in the prevalence of CM. In addition, circulating IgG from CM patients highly react with recombinant TBB3. Overall, correspondence analyses based on singular value decomposition show a strong correlation between IgG anti-TBB3 and elevated concentration of cluster-II cytokine (IFNgamma, IL1beta, TNFalpha, TGFbeta) previously demonstrated to be a predictor of CM in the same population. CONCLUSIONS/SIGNIFICANCE: Collectively, these findings validate the relationship between antibody response to brain induced by P. falciparum infection and plasma cytokine patterns with clinical outcome of malaria. They also provide significant insight into the immune mechanisms associated to CM by the identification of TBB3 as a new disease-specific marker and potential therapeutic target.

Total and functional parasite specific IgE responses in Plasmodium falciparum-infected patients exhibiting different clinical status.

BACKGROUND: There is an increase of serum levels of IgE during Plasmodium falciparum infections in individuals living in endemic areas. These IgEs either protect against malaria or increase malaria pathogenesis. To get an insight into the exact role played by IgE in the outcome of P. falciparum infection, total IgE levels and functional anti-parasite IgE response were studied in children and adults, from two different endemic areas Gabon and India, exhibiting either uncomplicated malaria, severe non cerebral malaria or cerebral malaria, in comparison with control individuals. METHODOLOGY AND RESULTS: Blood samples were collected from controls and P. falciparum-infected patients before treatment on the day of hospitalization (day 0) in India and, in addition, on days 7 and 30 after treatment in Gabon. Total IgE levels were determined by ELISA and functional P. falciparum-specific IgE were estimated using a mast cell line RBL-2H3 transfected with a human Fcepsilon RI alpha-chain that triggers degranulation upon human IgE cross-linking. Mann Whitney and Kruskall Wallis tests were used to compare groups and the Spearman test was used for correlations. Total IgE levels were confirmed to increase upon infection and differ with level of transmission and age but were not directly related to the disease phenotype. All studied groups exhibited functional parasite-specific IgEs able to induce mast cell degranulation in vitro in the presence of P. falciparum antigens. Plasma IgE levels correlated with those of IL-10 in uncomplicated malaria patients from Gabon. In Indian patients, plasma IFN-gamma , TNF and IL-10 levels were significantly correlated with IgE concentrations in all groups. CONCLUSION: Circulating levels of total IgE do not appear to correlate with protection or pathology, or with anti-inflammatory cytokine pattern bias during malaria. On the contrary, the P. falciparum-specific IgE response seems to contribute to the control of parasites, since functional activity was higher in asymptomatic and uncomplicated malaria patients than in severe or cerebral malaria groups.

Modulation of cytokine profiles by malaria pigment--hemozoin: role of IL-10 in suppression of proliferative responses of mitogen stimulated human PBMC.

The malaria parasite pigment hemozoin (Hz) is internalized by circulating and resident phagocytes and modulates their functions. We report here that Hz from Plasmodium falciparum inhibits proliferative responses of PHA stimulated human peripheral blood mononuclear cells (PBMC) in a dose dependent manner. Hz phagocytosed monocyte/macrophages (MO/MQ) secreted high levels of IL-10, IL-1beta and TNF-alpha, but inhibition of proliferation was mediated by IL-10 alone which was reversed by neutralization of the cytokine. Drastic decrease in the levels of IL-2, IL-12 and IFN-gamma were observed in supernatants from PBMC stimulated in the presence of Hz loaded MO/MQ cells. Exogenous addition of these cytokines did not abrogate immunosuppression indicating the inability of these cytokines to enhance proliferation in the presence of IL-10. We provide additional data that the IL-10 levels correlated positively with the load of Hz in the MO/MQ. Kinetics of IL-10 secretion analyzed up to day 6 in MO/MQ cultures fed with Hz revealed that high levels of IL-10 were secreted during the first 48 h after ingestion and decreased drastically at later time points.