Data on chloroquine (CQ)-resistant Plasmodium vivax in LatinAmerica is limited, even with the current research efforts to sustain an efficient malaria control program in all these countries where P. vivax is endemic and where malaria still is a major public health issue. This study estimated in vivo CQ resistance in patients with uncomplicated P. vivax malaria, with use of CQ and primaquine simultaneously, in the Brazilian Amazon. Of a total of 135 enrolled subjects who accomplished the 28-day follow-up, parasitological failure was observed in 7 (5.2%) patients, in whom plasma CQ and desethylchloroquine (DCQ) concentrations were above 100 ng/dl. Univariate analysis showed that previous exposure to malaria and a higher initial mean parasitemia were associated with resistance but not with age or gender. In the multivariate analysis, only high initial parasitemia remained significant. Hemoglobin levels were similar at the beginning of the follow-up and were not associated with parasitemia. However, at day 3 and day 7, hemoglobin levels were significantly lower in patients presenting CQ resistance. The P. vivax dhfr (pvdhfr), pvmrp1, pvmdr1, and pvdhps gene mutations were not related to resistance in this small sample. P. vivax CQ resistance is already a problem in the Brazilian Amazon, which could be to some extent associated with the simultaneous report of anemia triggered by this parasite, a common complication of the disease in most of the areas of endemicity.

Peptidomimetic imidazolidin-4-one derivatives of primaquine (imidazoquines) recently displayed in vitro activity against blood schizonts of a chloroquine-resistant strain of Plasmodium falciparum. Preliminary studies with a subset of such imidazoquines showed them to both block transmission of P. berghei malaria from mouse to mosquito and be highly stable towards hydrolysis at physiological conditions. This prompted us to have deeper insight into the activity of imidazoquines against both Plasmodia and Pneumocystis carinii, on which primaquine is also active. Full assessment of the in vivo transmission-blocking activity of imidazoquines, in vitro tissue-schizontocidal activity on P. berghei-infected hepatocytes, and in vitro anti-P. carinii activity is now reported. All compounds were active in these biological assays, with generally lower activity than the parent drug. However, imidazoquines’ stability against both oxidative deamination and proteolytic degradation suggest that they will probably have higher oral bioavailability and lower hematotoxicity than primaquine, which might translate into higher therapeutic indexes.

BackgroundChloroquine (CQ) and primaquine (PQ) are still the drugs of choice to treat Plasmodium vivax malaria in many endemic areas, Brazil included. There is in vivo evidence for the P. vivax resistance to CQ in the Brazilian Amazon, where the increase in the proportion of P. vivax malaria parallels the increase of unusual clinical complications related to this species. In this study, in vitro CQ and mefloquine (MQ)-susceptibility of P. vivax isolates from the Western Brazilian Amazon was tested using the double-site enzyme-linked lactate dehydrogenase immunodetection (DELI) assay.MethodsA total of 112 P. vivax isolates were tested in vitro for CQ-susceptibility and out of these 47 were also tested for MQ-susceptibility. The DELI assay was used to detect P. vivax growth at 48-hour short-term culture in isolates with ring stages ranging from 50 to %. Each isolate was tested in triplicate and geometric means of IC50’s was obtained. Nineteen isolates were genetically characterized for pvdhfr, pvmrp1, pvmdr1 and pvdhps candidate genes likely related to CQ resistance (10 with IC50<40 nM and 9 with IC50 >100 nM).ResultsTwelve out of 112 isolates were considered resistant to CQ, resulting in 10.7% (IC95% 5.0-16.4), while 3 out of 47 (6.4%; IC95% 0.0-12.8) were resistant to MQ. A discrete correlation was observed between IC50’s of CQ and MQ (Spearman=0.294; p=0.045). For pvdhps gene, a non-synonymous mutation was found at codon 382 (S→C) in 5/8 CQ-sensitive samples and 1/9 CQ-resistant samples (p=0.027). The other molecular markers were not associated to CQ-susceptibility.ConclusionsIn vitro CQ-resistance estimated in this study, estimated by the DELI test, was very similar to that observed in clinical trials, suggesting that in vitro procedures developed by capable local laboratories are useful in the surveillance of CQ-resistance in the Amazon; concurrent Amazon P. vivax strains with both CQ and MQ resistance may be common; and a non-synonymous mutation at pvdhps codon 382 (S→C) was associated to in vitro susceptibility to CQ, needing further studies to be confirmed.

To obtain insight into the mechanisms that contribute to the pathogenesis of Plasmodium infections, we developed an improved rodent model that mimics human malaria closely by inducing cerebral malaria (CM) through sporozoite infection. We used this model to carry out a detailed study on isolated T cells recruited from the brains of mice during the development of CM. We compared several aspects of the immune response related to the experimental model of Plasmodium berghei ANKA infection induced by sporozoites in C57BL/6 mice and those related to a blood-stage infection. Our data show that in both models, oligoclonal TCRV␤4 ؉ , TCRV␤6 ؉ , TCRV␤8.1 ؉ , and TCRV␤11 ؉ major histocompatibility complex class I-restricted CD8 T cells were present in the brains of CM ؉ mice. These CD8 ؉ T cells display an activated phenotype, do not undergo apoptosis, secrete gamma interferon or tumor necrosis factor alpha, and are associated with the development of the neurological syndrome.Cerebral malaria (CM) continues to contribute to the deaths of more than two million people every year in areas of endemic infection (World Health Organization, 1998, http://www .who.int/inf-fs/en/fact094.html). Although the physiopathology of Plasmodium infection has been extensively investigated, we still know relatively little about the precise mechanisms that contribute to its pathogenesis, in particular during CM. Two main factors have been implicated: (i) the sequestration of Plasmodium falciparum-parasitized red blood cells (8,27,35,37) and leukocytes (33, 36, 38) within brain vessels and (ii) the involvement of T cells activated by Plasmodium antigens (29, 41). These two main mechanisms act together under the control of mediators of the inflammatory responses released during the infection such as tumor necrosis factor alpha (TNF-␣) and gamma interferon (IFN-␥) (13,14,15,21,22,24,25). The up regulation of adhesion molecules such as CD36, intercellular cell adhesion molecule 1 (ICAM-1), and thrombospondin, which lead to the adherence of infected erythrocytes and leukocytes to endothelial cells of the brain microvessels, is a common feature of the physiological events that occur during CM (4, 7, 15, 39).Host CD4 ϩ and CD8 ϩ T cells are involved in the development of fatal murine CM, as demonstrated by depletion of these cells with anti-CD4 or anti-CD8 monoclonal antibodies (MAb) and by using mice that are genetically deficient in the expression of either CD4 or CD8 (2,5,12,17,18,30,42). This suggests that the immunopathological process that occurs during CM involves both CD4 ϩ and CD8 ϩ T-cell subsets. However, the way in which CD4 ϩ and CD8 ϩ cells contribute to the development of pathogenicity during fatal CM remains to be elucidated.The purpose of this study, therefore, was to develop an alternative model for CM, using sporozoites of P. berghei ANKA strain clone 1. 49L to initiate the infection in order to compare the pathogenic T-cell responses that occur during sporozoite-and blood-stage-induced infection in mice with CM. Such responses wer...

BackgroundMozambique implemented artemisinin-based combinations therapy (ACT) using artemether-lumefantrine (AL) as the first-line treatment for uncomplicated malaria in 2009. AL remains highly efficacious, but widespread use may soon facilitate emergence of artemisinin tolerance/resistance. The prevalence of pfmdr1 different alleles in Maputo and Mozambique is not known, either after or before the introduction of ACT. Pfmdr1 molecular markers related to Plasmodium falciparum susceptibility were analysed before and after transition to ACT.MethodsA first group of samples was collected between June 2003 and June 2005 and a second group in the period between March 2010 and March 2012. Three alleles were analysed by PCR-RFLP: N86Y, Y184F and D1246Y, in the pfmdr1 gene.ResultsAlleles N86, 184F and D1246 increased from 19.5, 19.6 and 74.4% in 2003–2005 to 73.2, 22.7 and 96.7% in 2010–2012, respectively. After implementation of ACT (2010–2012), pfmdr1 haplotypes, either two- and three-codon basis, were generally less diverse than before the implementation of ACT (2003–2005). The prevalence of haplotypes N86-184Y, N86-D1246 and 184Y-D1246 increased from 12,2, 27.3 and 71.7% in 2003–2005 to 59.4, 84.3 and 78.6% in 2010–2012. The three-codon basis haplotypes NFD and NYD also increased significantly during the same period.ConclusionThe alleles N86 and 184 F and the triple haplotype N86-184 F-D1246 showed a significantly increased prevalence after introduction of ACT.