Monitoring the density of Plasmodium spp. gametocytes in isolates from patient samples in the region of Porto Velho, Rondônia.

Gametocytes are the forms of the malaria parasite that are essential for the continuation of the transmission cycle to the vector Anopheles. This study aimed to evaluate the parasite density of Plasmodium spp gametocytes in samples from patients in the region of Porto Velho, Rondônia. Slides containing patient samples were selected from users who sought out care at the Center for Research in Tropical Medicine (CEPEM) during the period from January to December 2016. Samples of Plasmodium vivax and Plasmodium falciparum were selected for analysis of their respective gametocytes. In parallel, monitoring was performed in cultures of NF54 strain P. falciparum gametocytes. Of 248 thick smear slides (EG) evaluated in double blind, 142 (57.2%) were detected with P. vivax, of this total 47 (18.9%) had gametocytes, 1 (0.4%) with LVC negative diagnosis for gametocytes and 1 (0.4%) Pv + Pf (mixed malaria). Regarding P. falciparum, the total number of samples analyzed was 106 (42.7%), of which 20 (8.0%) had gametocytes detected, 6 (2.4%) LVC negative for gametocyte forms, and 3 (1.2%) Pv + Pf (mixed malaria), Plasmodium malariae species was not detected among the samples. The results showed that P. vivax gametocytes were present in the first days of symptoms, with a higher prevalence in patients with two crosses, a fact that was also observed in patients with P. falciparum regarding the prevalence of gametocytes. Faced with this problem, it is necessary to monitor the fluctuation of gametocytes, since these forms are responsible for continuing the malaria cycle within the mosquito vector.

Adenosine pathway regulates inflammation during Plasmodium vivax infection.

Background: Plasmodium spp. infection triggers the production of inflammatory cytokines that are essential for parasite control, and conversely responsible for symptoms of malaria. Monocytes play a role in host defense against Plasmodium vivax infection and represent the main source of inflammatory cytokines and reactive oxygen species. The anti-inflammatory cytokine IL-10 is a key regulator preventing exacerbated inflammatory responses. Studies suggested that different clinical presentations of malaria are strongly associated with an imbalance in the production of inflammatory and anti-inflammatory cytokines. Methods: A convenience sampling of peripheral blood mononuclear cells from Plasmodium vivax-infected patients and healthy donors were tested for the characterization of cytokine and adenosine production and the expression of ectonucleotidases and purinergic receptors. Results: Here we show that despite a strong inflammatory response, monocytes also bear a modulatory role during malaria. High levels of IL-10 are produced during P. vivax infection and its production can be triggered in monocytes by P. vivax-infected reticulocytes. Monocytes express high levels of ectonucleotidases, indicating their important role in extracellular ATP modulation and consequently in adenosine production. Plasmatic levels of adenosine are not altered in patients experiencing acute malaria; however, their monocyte subsets displayed an increased expression of P1 purinergic receptors. In addition, adenosine decreases Tumor Necrosis Factor production by monocytes, which was partially abolished with the blockage of the A2a receptor. Conclusion: Monocytes have a dual role, attempting to control both the P. vivax infection and the inflammatory response. Purinergic receptor modulators emerge as an untapped approach to ameliorate clinical malaria.

Acute Chagas Disease Caused by Trypanosoma cruzi TcIV and Transmitted by Panstrongylus geniculatus: Molecular Epidemiological Insights Provided by the First Documented Autochthonous Case in Rondônia, Southwestern Amazonia, Brazil.

Recurrent outbreaks of oral infection and isolated cases characterize the new epidemiological scenario of Chagas disease (CD) in the Brazilian Amazon. Acute Chagas disease (ACD) is common in Pará and Amazonas, Northeastern and Northwestern Brazilian Amazonia. In the present study, we describe the first molecularly characterized autochthonous case of ACD in Rondônia, Southwestern Amazonia. The patient, a 39-year-old male resident in the small city of Cujubim, presented typical ACD symptoms: fever, asthenia, myalgia, progressive dyspnea, swelling of the legs, and tiredness at minimal efforts, all compatible with ACD and indicative of cardiac involvement. A thick blood drop test revealed trypomastigote forms of Trypanosoma cruzi genotyped as TcIV. An epidemiological investigation ruled out oral infection, and support for vectorial transmission included the finding of Panstrongylus geniculatus positive for T. cruzi (TcIII and TcIV) inside the tent used by the patient when harvesting forest timber, and a circular cutaneous lesion resembling a chagoma of inoculation. Treatment with benznidazole led to blood parasite clearance as confirmed by molecular tests. Altogether, our findings fitted well into the ecological scenario where deforestation and colonization of forested areas represent an important risk factor to the adaptation of P. geniculatus to human habitats, favoring vectorial transmission of CD in the Amazonian region.

In vitro and ex vivo antiplasmodial activity of 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one) against circulating strains of Plasmodium spp. in the state of Rondônia, Brazil

Abstract Malaria is a disease caused by Plasmodium spp. protozoa. The ability of Plasmodium to develop resistance to current antimalarial drugs makes the study of chemotherapeutic alternatives extremely important. This study aimed to evaluate the antimalarial activity of compound 3286938 (1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one), which presents in its structure a 3,4,5-trimethoxyphenyl group, in vitro, using the W2 strain of P. falciparum and against circulating strains of P. vivax and P. falciparum from the state of Rondônia. The compound 3286938 obtained an IC50 of 24.4 µM against the W2 strain of P. falciparum, and against the circulating strains, it presented a median (MD)=38.7 µM for P. vivax and MD=6.7 µM for P. falciparum. As for toxicity, 3286938 showed CC50 > 500 µM for VERO and HepG2 strains with a selectivity index greater than 12.9, a ratio calculated for P. falciparum and P. vivax regarding Vero and HepG2 cells. The compound was not considered hemolytic in in vitro assays, thus indicating the specificity of its antiplasmodial action. Based on the results presented, and considering the unprecedented character of the compound, it can be concluded that 3286938 was shown to be promising for complementary in vitro and in vivo studies aiming to produce effective antiplasmodial action.

Asymptomatic Plasmodium vivax malaria in the Brazilian Amazon: Submicroscopic parasitemic blood infects Nyssorhynchus darlingi.

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria.

Plasmodium vivax Infection Alters Mitochondrial Metabolism in Human Monocytes.

Monocytes play an important role in the host defense against Plasmodium vivax as the main source of inflammatory cytokines and mitochondrial reactive oxygen species (mROS). Here, we show that monocyte metabolism is altered during human P. vivax malaria, with mitochondria playing a major function in this switch. The process involves a reprograming in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. P. vivax infection results in dysregulated mitochondrial gene expression and in altered membrane potential leading to mROS increase rather than ATP production. When monocytes were incubated with P. vivax-infected reticulocytes, mitochondria colocalized with phagolysosomes containing parasites representing an important source mROS. Importantly, the mitochondrial enzyme superoxide dismutase 2 (SOD2) is simultaneously induced in monocytes from malaria patients. Taken together, the monocyte metabolic reprograming with an increased mROS production may contribute to protective responses against P. vivax while triggering immunomodulatory mechanisms to circumvent tissue damage. IMPORTANCE Plasmodium vivax is the most widely distributed causative agent of human malaria. To achieve parasite control, the human immune system develops a substantial inflammatory response that is also responsible for the symptoms of the disease. Among the cells involved in this response, monocytes play an important role. Here, we show that monocyte metabolism is altered during malaria, with its mitochondria playing a major function in this switch. This change involves a reprograming process in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. The resulting altered mitochondrial membrane potential leads to an increase in mitochondrial reactive oxygen species rather than ATP. These data suggest that agents that change metabolism should be investigated and used with caution during malaria.

Single-Dose Tafenoquine to Prevent Relapse of Plasmodium vivax Malaria.

BACKGROUND: Treatment of Plasmodium vivax malaria requires the clearing of asexual parasites, but relapse can be prevented only if dormant hypnozoites are cleared from the liver (a treatment termed "radical cure"). Tafenoquine is a single-dose 8-aminoquinoline that has recently been registered for the radical cure of P. vivax. METHODS: This multicenter, double-blind, double-dummy, parallel group, randomized, placebo-controlled trial was conducted in Ethiopia, Peru, Brazil, Cambodia, Thailand, and the Philippines. We enrolled 522 patients with microscopically confirmed P. vivax infection (>100 to <100,000 parasites per microliter) and normal glucose-6-phosphate dehydrogenase (G6PD) activity (with normal activity defined as ≥70% of the median value determined at each trial site among 36 healthy male volunteers who were otherwise not involved in the trial). All patients received a 3-day course of chloroquine (total dose of 1500 mg). In addition, patients were assigned to receive a single 300-mg dose of tafenoquine on day 1 or 2 (260 patients), placebo (133 patients), or a 15-mg dose of primaquine once daily for 14 days (129 patients). The primary outcome was the Kaplan-Meier estimated percentage of patients who were free from recurrence at 6 months, defined as P. vivax clearance without recurrent parasitemia. RESULTS: In the intention-to-treat population, the percentage of patients who were free from recurrence at 6 months was 62.4% in the tafenoquine group (95% confidence interval [CI], 54.9 to 69.0), 27.7% in the placebo group (95% CI, 19.6 to 36.6), and 69.6% in the primaquine group (95% CI, 60.2 to 77.1). The hazard ratio for the risk of recurrence was 0.30 (95% CI, 0.22 to 0.40) with tafenoquine as compared with placebo (P<0.001) and 0.26 (95% CI, 0.18 to 0.39) with primaquine as compared with placebo (P<0.001). Tafenoquine was associated with asymptomatic declines in hemoglobin levels, which resolved without intervention. CONCLUSIONS: Single-dose tafenoquine resulted in a significantly lower risk of P. vivax recurrence than placebo in patients with phenotypically normal G6PD activity. (Funded by GlaxoSmithKline and Medicines for Malaria Venture; DETECTIVE ClinicalTrials.gov number, NCT01376167 .).

Plasmodium vivax Infection Impairs Regulatory T-Cell Suppressive Function During Acute Malaria.

The balance between pro- and antiinflammatory mechanisms is essential to limit immune-mediated pathology, and CD4+ forkhead box P3 (Foxp3+) regulatory T cells (Treg) play an important role in this process. The expression of inhibitory receptors regulates cytokine production by Plasmodium vivax-specific T cells. Our goal was to assess the induction of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen (CTLA-4) on Treg during malaria and to evaluate their function. We found that P. vivax infection triggered an increase in circulating Treg and their expression of CTLA-4 and PD-1. Functional analysis demonstrated that Treg from malaria patients had impaired suppressive ability and PD-1+Treg displayed lower levels of Foxp3 and Helios, but had higher frequencies of T-box transcription factor+ and interferon-gamma+ cells than PD-1-Treg. Thus malaria infection alters the function of circulating Treg by triggering increased expression of PD-1 on Treg that is associated with decreased regulatory function and increased proinflammatory characteristics.

Efficacy and safety of artemisinin-based combination therapy and chloroquine with concomitant primaquine to treat Plasmodium vivax malaria in Brazil: an open label randomized clinical trial.

BACKGROUND: There is general international agreement that the importance of vivax malaria has been neglected, and there is a need for new treatment approaches in an effort to progress towards control and elimination in Latin America. This open label randomized clinical trial evaluated the efficacy and safety of three treatment regimens using either one of two fixed dose artemisinin-based combinations or chloroquine in combination with a short course of primaquine (7-9 days: total dose 3-4.2 mg/kg) in Brazil. The primary objective was establishing whether cure rates above 90% could be achieved in each arm. RESULTS: A total of 264 patients were followed up to day 63. The cure rate of all three treatment arms was greater than 90% at 28 and 42 days. Cure rates were below 90% in all three treatment groups at day 63, although the 95% confidence interval included 90% for all three treatments. Most of the adverse events were mild in all treatment arms. Only one of the three serious adverse events was related to the treatment and significant drops in haemoglobin were rare. CONCLUSION: This study demonstrated the efficacy and safety of all three regimens that were tested with 42-day cure rates that meet World Health Organization criteria. The efficacy and safety of artemisinin-based combination therapy regimens in this population offers the opportunity to treat all species of malaria with the same regimen, simplifying protocols for malaria control programmes and potentially contributing to elimination of both vivax and falciparum malaria. Trial registration RBR-79s56s.

T follicular helper cells regulate the activation of B lymphocytes and antibody production during Plasmodium vivax infection.

Although the importance of humoral immunity to malaria has been established, factors that control antibody production are poorly understood. Follicular helper T cells (Tfh cells) are pivotal for generating high-affinity, long-lived antibody responses. While it has been proposed that expansion of antigen-specific Tfh cells, interleukin (IL) 21 production and robust germinal center formation are associated with protection against malaria in mice, whether Tfh cells are found during Plasmodium vivax (P. vivax) infection and if they play a role during disease remains unknown. Our goal was to define the role of Tfh cells during P. vivax malaria. We demonstrate that P. vivax infection triggers IL-21 production and an increase in Tfh cells (PD-1+ICOS+CXCR5+CD45RO+CD4+CD3+). As expected, FACS-sorted Tfh cells, the primary source of IL-21, induced immunoglobulin production by purified naïve B cells. Furthermore, we found that P. vivax infection alters the B cell compartment and these alterations were dependent on the number of previous infections. First exposure leads to increased proportions of activated and atypical memory B cells and decreased frequencies of classical memory B cells, whereas patients that experienced multiple episodes displayed lower proportions of atypical B cells and higher frequencies of classical memory B cells. Despite the limited sample size, but consistent with the latter finding, the data suggest that patients who had more than five infections harbored more Tfh cells and produce more specific antibodies. P. vivax infection triggers IL-21 production by Tfh that impact B cell responses in humans.

On the cytokine/chemokine network during Plasmodium vivax malaria: new insights to understand the disease.

BACKGROUND: The clinical outcome of malaria depends on the delicate balance between pro-inflammatory and immunomodulatory cytokine responses triggered during infection. Despite the numerous reports on characterization of plasma levels of cytokines/chemokines, there is no consensus on the profile of these mediators during blood stage malaria. The identification of acute phase biomarkers might contribute to a better understanding of the disease, allowing the use of more effective therapeutic approaches to prevent the progression towards severe disease. In the present study, the plasma levels of cytokines and chemokines and their association with parasitaemia and number of previous malaria episodes were evaluated in Plasmodium vivax-infected patients during acute and convalescence phase, as well as in healthy donors. METHODS: Samples of plasma were obtained from peripheral blood samples from four different groups: P. vivax-infected, P. vivax-treated, endemic control and malaria-naïve control. The cytokine (IL-6, IL-10, IL-17, IL-27, TGF-ß, IFN-γ and TNF) and chemokine (MCP-1/CCL2, IP-10/CXCL10 and RANTES/CCL5) plasma levels were measured by CBA or ELISA. The network analysis was performed using Spearman correlation coefficient. RESULTS: Plasmodium vivax infection induced a pro-inflammatory response driven by IL-6 and IL-17 associated with an immunomodulatory profile mediated by IL-10 and TGF-ß. In addition, a reduction was observed of IFN-γ plasma levels in P. vivax group. A lower level of IL-27 was observed in endemic control group in comparison to malaria-naïve control group. No significant results were found for IL-12p40 and TNF. It was also observed that P. vivax infection promoted higher levels of MCP-1/CCL2 and IP-10/CXCL10 and lower levels of RANTES/CCL5. The plasma level of IL-10 was elevated in patients with high parasitaemia and with more than five previous malaria episodes. Furthermore, association profile between cytokine and chemokine levels were observed by correlation network analysis indicating signature patterns associated with different parasitaemia levels. CONCLUSIONS: The P. vivax infection triggers a balanced immune response mediated by IL-6 and MCP-1/CCL2, which is modulated by IL-10. In addition, the results indicated that IL-10 plasma levels are influenced by parasitaemia and number of previous malaria episodes.

Induction of Inhibitory Receptors on T Cells During Plasmodium vivax Malaria Impairs Cytokine Production.

The function and regulation of the immune response triggered during malaria is complex and poorly understood, and there is a particular paucity of studies conducted in humans infected with Plasmodium vivax. While it has been proposed that T-cell-effector responses are crucial for protection against blood-stage malaria in mice, the mechanisms behind this in humans remain poorly understood. Experimental models of malaria have shown that the regulatory molecules, cytotoxic T-lymphocyte attenuator-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and programmed death-1 (PD-1) are involved in the functional impairment of T cells during infection. Our goal was to define the role of these molecules during P. vivax malaria. We demonstrate that infection triggers the expression of regulatory molecules on T cells. The pattern of expression differs in CD4(+) and CD8(+) T cells. Higher frequencies of CD4(+) express more than 1 regulatory molecule compared to CD8(+) T cells. Moreover, lower proportions of CD4(+) T cells coexpress regulatory molecules, but are still able to proliferate. Importantly, simultaneously blockade of the CLTA-4, PD-1, and T-cell immunoglobulin and mucin-3 signaling restores the cytokine production by antigen-specific cells. These data support the hypothesis that upregulation of inhibitory receptors on T cells during P. vivax malaria impairs parasite-specific T-cell effector function.

Phenotypic profiling of CD8(+) T cells during Plasmodium vivax blood-stage infection.

BACKGROUND: For a long time, the role of CD8(+) T cells in blood-stage malaria was not considered important because erythrocytes do not express major histocompatibility complex (MHC) class I proteins. While recent evidences suggest that CD8(+) T cells may play an important role during the erythrocytic phase of infection by eliminating parasites, CD8(+) T cells might also contribute to modulate the host response through production of regulatory cytokines. Thus, the role of CD8(+) T cells during blood-stage malaria is unclear. Here, we report the phenotypic profiling of CD8(+) T cells subsets from patients with uncomplicated symptomatic P. vivax malaria. METHODS: Blood samples were collected from 20 Plasmodium vivax-infected individuals and 12 healthy individuals. Immunophenotyping was conducted by flow cytometry. Plasma levels of IFN-γ, TNF-α and IL-10 were determined by ELISA/CBA. Unpaired t-test or Mann-Whitney test was used depending on the data distribution. RESULTS: P. vivax-infected subjects had lower percentages and absolute numbers of CD8(+)CD45RA(+) and CD8(+)CD45RO(+) T cells when compared to uninfected individuals (p ≤ 0.0002). A significantly lower absolute number of circulating CD8(+)CD45(+)CCR7(+) cells (p = 0.002) was observed in P. vivax-infected individuals indicating that infection reduces the number of central memory T cells. Cytokine expression was significantly reduced in the naïve T cells from infected individuals compared with negative controls, as shown by lower numbers of IFN-γ(+) (p = 0.001), TNF-α(+) (p < 0.0001) and IL-10(+) (p < 0.0001) CD8(+) T cells. Despite the reduction in the number of CD8(+) memory T cells producing IFN-γ (p < 0.0001), P. vivax-infected individuals demonstrated a significant increase in memory CD8(+)TNF-α(+) (p = 0.016) and CD8(+)IL-10(+) (p = 0.004) cells. Positive correlations were observed between absolute numbers of CD8(+)IL-10(+) and numbers of CD8(+)IFN-γ(+) (p < 0.001) and CD8(+)TNF-α(+) T cells (p ≤ 0.0001). Finally, an increase in the plasma levels of TNF-α (p = 0.017) and IL-10 (p = 0.006) and a decrease in the IFN-γ plasma level (p <0.0001) were observed in the P. vivax-infected individuals. CONCLUSIONS: P. vivax infection reduces the numbers of different subsets of CD8(+) T cells, particularly the memory cells, during blood-stage of infection and enhances the number of CD8(+) memory T cells expressing IL-10, which positively correlates with the number of cells expressing TNF-α and IFN-γ.

CD4+ T cells apoptosis in Plasmodium vivax infection is mediated by activation of both intrinsic and extrinsic pathways.

BACKGROUND: Reduction in the number of circulating blood lymphocytes (lymphocytopaenia) has been reported during clinical episodes of malaria and is normalized after treatment with anti-malaria drugs. While this phenomenon is well established in malaria infection, the underlying mechanisms are still not fully elucidated. In the present study, the occurrence of apoptosis and its pathways in CD4+ T cells was investigated in naturally Plasmodium vivax-infected individuals from a Brazilian endemic area (Porto Velho - RO). METHODS: Blood samples were collected from P. vivax-infected individuals and healthy donors. The apoptosis was characterized by cell staining with Annexin V/FITC and propidium iodide and the apoptosis-associated gene expression profile was carried out using RT2 Profiler PCR Array-Human Apoptosis. The plasma TNF level was determined by ELISA. The unpaired t-test or Mann-Whitney test was applied according to the data distribution. RESULTS: Plasmodium vivax-infected individuals present low number of leukocytes and lymphocytes with a higher percentage of CD4+ T cells in early and/or late apoptosis. Increased gene expression was observed for TNFRSF1B and Bid, associated with a reduction of Bcl-2, in individuals with P. vivax malaria. Furthermore, these individuals showed increased plasma levels of TNF compared to malaria-naive donors. CONCLUSIONS: The results of the present study suggest that P. vivax infection induces apoptosis of CD4+ T cells mediated by two types of signaling: by activation of the TNFR1 death receptor (extrinsic pathway), which is amplified by Bid, and by decreased expression of the anti-apoptotic protein Bcl-2 (intrinsic pathway). The T lymphocytes apoptosis could reflect a strategy of immune evasion triggered by the parasite, enabling their persistence but also limiting the occurrence of immunopathology.

The CD14+CD16+ inflammatory monocyte subset displays increased mitochondrial activity and effector function during acute Plasmodium vivax malaria.

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax-infected patients display significant increase in circulating monocytes, which were defined as CD14(+)CD16- (classical), CD14(+)CD16(+) (inflammatory), and CD14loCD16(+) (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16(+) cells, in particular the CD14(+)CD16(+) monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14(+) were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14(+)CD16(+) monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14(+)CD16(+) cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.

Selective intermittent preventive treatment of vivax malaria: reduction of malaria incidence in an open cohort study in brazilian Amazon.

In children, the Intermittent Preventive Treatment (IPTc), currently called Seasonal Malaria Chemoprevention (SMC), was considered effective on malaria control due to the reduction of its incidence in Papua New Guinea and in some areas with seasonal malaria in Africa. However, the IPT has not been indicated because of its association with drug resistance and for hindering natural immunity development. Thus, we evaluated the alternative IPT impact on malaria incidence in three riverside communities on Madeira River, in the municipality of Porto Velho, RO. We denominate this scheme Selective Intermittent Preventive Treatment (SIPT). The SIPT consists in a weekly dose of two 150 mg chloroquine tablets for 12 weeks, for adults, and an equivalent dose for children, after complete supervised treatment for P. vivax infection. This scheme is recommend by Brazilian Health Ministry to avoid frequent relapses. The clinic parasitological and epidemiological surveillance showed a significant reduction on vivax malaria incidence. The results showed a reduction on relapses and recurrence of malaria after SIPT implementation. The SIPT can be effective on vivax malaria control in localities with high transmission risk in the Brazilian Amazon.

Low sensitivity of malaria rapid diagnostic tests stored at room temperature in the Brazilian Amazon Region.

INTRODUCTION: In remote areas of the Amazon Region, diagnosis of malaria by microscopy is practically impossible. This study aimed to evaluate the performance of two rapid diagnostic tests (RDTs) targeting different malaria antigens stored at room temperature in the Brazilian Amazon Region. METHODOLOGY: Performance of the OptiMal Pf/Pan test and ICT-Now Pf/Pan test was analyzed retrospectively in 1,627 and 1,602 blood samples, respectively. Tests were performed over a 15-month period. Kits were stored at room temperature in five community health centres located in the Brazilian Amazon Region. RDT results were compared with thick blood smear (TBS) results to determine sensitivity, specificity, and accuracy of the RDT. RESULTS: The sensitivities of the OptiMal Pf/Pan test were 79.7% for Plasmodium falciparum malaria diagnosis and 85.7% for non-P. falciparum infections. The results showed a crude agreement of 88.5% for P. falciparum, and 88.3% for non-P. falciparum infections (Kappa index = 0.74 and 0.75, respectively). For the ICT-Now Pf/Pan test (CI 95%), the sensitivities were 87.9% for P. falciparum malaria diagnosis and 72.5% for non-P. falciparum infection. Crude agreement between the ICT-Now Pf/Pan test and TBS was 91.4% for P. falciparum and 79.7% for non-P. falciparum infection. The Kappa index was 0.81 and 0.59 for the final diagnosis of P. falciparum and non-P. falciparum, respectively. Higher levels of parasitaemia were associated with higher crude agreement between RDT and TBS. CONCLUSIONS: The sensitivities of RDTs stored at room temperature over a 15-month period and performed in field conditions were lower than those previously reported.

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax.

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.

Asymptomatic infection with Plasmodium falciparum and Plasmodium vivax in the Brazilian Amazon Basin: to treat or not to treat?

In this study, we determined whether the treatment of asymptomatic parasites carriers (APCs), which are frequently found in the riverside localities of the Brazilian Amazon that are highly endemic for malaria, would decrease the local malaria incidence by decreasing the overall pool of parasites available to infect mosquitoes. In one village, the treatment of the 19 Plasmodium falciparum-infected APCs identified among the 270 residents led to a clear reduction (Z = -2.39, p = 0.017) in the incidence of clinical cases, suggesting that treatment of APCs is useful for controlling falciparum malaria. For vivax malaria, 120 APCs were identified among the 716 residents living in five villages. Comparing the monthly incidence of vivax malaria in two villages where the APCs were treated with the incidence in two villages where APCs were not treated yielded contradictory results and no clear differences in the incidence were observed (Z = -0.09, p = 0.933). Interestingly, a follow-up study showed that the frequency of clinical relapse in both the treated and untreated APCs was similar to the frequency seen in patients treated for primary clinical infections, thus indicating that vivax clinical immunity in the population is not species specific but only strain specific.