Relapsing Plasmodium vivax malaria in a 12-year-old Brazilian girl: A case report.

Plasmodium vivax causes the vast majority of malaria cases in Brazil. The lifecycle of this parasite includes a latent stage in the liver, the hypnozoite. Reactivation of hypnozoites induces repeated relapses. We report a case of two relapses of vivax malaria in a teenage girl after conventional treatment with chloroquine and primaquine. Chloroquine prophylactic treatment for three months was prescribed with a favourable outcome of the case.

Clinical and immunological profiles of anaemia in children and adolescents with Plasmodium vivax malaria in the Pará state, Brazilian Amazon.

Children and adolescents are at great risk for developing iron deficiency anaemia worldwide. In the tropical areas, malaria and intestinal parasites may also play an important role in anaemia pathogenesis. This study aimed at evaluating clinical and immunological aspects of anaemia in children and adolescents with Plasmodium vivax malaria, in the Pará State, Brazil. A longitudinal study was performed in two Reference Centers for malaria diagnosis in the Brazilian Amazon in children and adolescents with malaria (n = 81), as compared to a control group (n = 40). Patients had blood drawn three times [before treatment (D0), after treatment (D7) and at the first cure control (D30)] and hemogram, autoantibody analysis (anticardiolipin, antibodies against normal RBC membrane components) and cytokine studies (TNF and IL-10) were performed. Stool samples were collected for a parasitological examination. Malaria patients had a 2.7-fold greater chance of anaemia than the control group. At D0, 66.1% of the patients had mild anaemia, 30.5% had moderate and 3.5% had severe anaemia. Positivity to intestinal helminths and/or protozoa at stool examinations had no influence on anaemia. Patients had significantly lower levels of plasmatic TNF than control individuals at D0. Low TNF levels were more prevalent among patients with moderate/severe anaemia than in those with mild anaemia and among anaemic patients than in anaemic controls. TNF levels were positively correlated with the haemoglobin rates and negatively correlated with the interval time elapsed between the onset of symptoms and diagnosis. Both plasma TNF levels and haemoglobin rates increased during the follow-up period. The IL-10 levels were lower in patients than in the controls at day 0 and decreased thereafter up to the end of treatment. Only the anti-anticardiolipin autoantibodies were associated with moderate/severe anaemia and, possibly by reacting with the parasite glycosylphosphatidylinositol (a powerful stimulator of TNF production), may have indirectly contributed to decrease the TNF levels, which could be involved in the malarial vivax anaemia of these children and adolescents. More studies addressing this issue are necessary to confirm these findings and to add more information on the multifactorial pathogenesis of the malarial anaemia.

Outbreak of human malaria caused by Plasmodium simium in the Atlantic Forest in Rio de Janeiro: a molecular epidemiological investigation.

BACKGROUND: Malaria was eliminated from southern and southeastern Brazil over 50 years ago. However, an increasing number of autochthonous episodes attributed to Plasmodium vivax have recently been reported from the Atlantic Forest region of Rio de Janeiro state. As the P vivax-like non-human primate malaria parasite species Plasmodium simium is locally enzootic, we performed a molecular epidemiological investigation to determine whether zoonotic malaria transmission is occurring. METHODS: We examined blood samples from patients presenting with signs or symptoms suggestive of malaria as well as from local howler monkeys by microscopy and PCR. Samples were included from individuals if they had a history of travel to or resided in areas within the Rio de Janeiro Atlantic Forest, but not if they had malaria prophylaxis, blood transfusion or tissue or organ transplantation, or had travelled to known malaria endemic areas in the preceding year. Additionally, we developed a molecular assay based on sequencing of the parasite mitochondrial genome to distinguish between P vivax and P simium, and applied this assay to 33 cases from outbreaks that occurred in 2015, and 2016. FINDINGS: A total of 49 autochthonous malaria cases were reported in 2015-16. Most patients were male, with a mean age of 44 years (SD 14·6), and 82% lived in urban areas of Rio de Janeiro state and had visited the Atlantic Forest for leisure or work-related activities. 33 cases were used for mitochondrial DNA sequencing. The assay was successfully performed for 28 samples, and all were shown to be P simium, indicative of zoonotic transmission of this species to human beings in this region. Sequencing of the whole mitochondrial genome of three of these cases showed that P simium is most closely related to P vivax parasites from South America. The malaria outbreaks in this region were caused by P simium, previously considered to be a monkey-specific malaria parasite, related to but distinct from P vivax, and which has never conclusively been shown to infect people before. INTERPRETATION: This unequivocal demonstration of zoonotic transmission, 50 years after the only previous report of P simium in people, leads to the possibility that this parasite has always infected people in this region, but that it has been consistently misdiagnosed as P vivax because of an absence of molecular typing techniques. Thorough screening of local non-human primates and mosquitoes (Anopheline) is required to evaluate the extent of this newly recognised zoonotic threat to public health and malaria elimination in Brazil. FUNDING: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado de Rio de Janeiro, The Brazilian National Council for Scientific and Technological Development (CNPq), JSPS Grant-in-Aid for scientific research, Secretary for Health Surveillance of the Brazilian Ministry of Health, Global Fund, Fundaçao de amparo à pesquisa do estado de Minas Gerais (Fapemig), and PRONEX Program of the CNPq.

New multilocus genotypes of Giardia lamblia human isolates.

Giardia lamblia is considered a species complex, whose members show little differences in their morphology, but have remarkable genetic variability. The aim of this study was to identify inter- and intra-assemblage genetic variation in G. lamblia among patients in Rio de Janeiro. The parasitological study was performed on faeces, and DNA was extracted from the samples which tested positive for G. lamblia. The genetic assemblages and subtypes were determined via multilocus sequence typing (MLST) using ß-giardin, triose phosphate isomerase and glutamate dehydrogenase gene loci. Fourteen assemblage A samples were successfully genotyped at the three MLST loci (bg/tpi/gdh). Two previously identified multilocus genotypes were found (AII-1 and AII-4), and two novel multilocus genotypes are proposed (AII-8, profile A2/A2/A4; AII-9, profile A3/A2/A2). Sequence analysis showed that assemblage B isolates have a higher nucleotide variation than those from assemblage A. Novel assemblage B sequences are described and most (66.7%) have heterogeneous nucleotides, which prevent the definition of multilocus genotypes. This is the first time that MLST has been used to characterise G. lamblia isolates in human clinical samples from Rio de Janeiro. In addition, MLST has enabled the detection of novel subtypes in both assemblages and the description of two novel multilocus genotypes in assemblage A. This study provides new insights into the genetic diversity of assemblage A and shows that MLST should be used to characterise G. lamblia both in Brazil and globally.

Associations of Giardia lamblia assemblages with HIV infections and symptomatology: HIV virus and assemblage B were they born to each other?

Giardia lamblia is an intestinal parasite that has an extensive genetic variation among isolates. This species is divided into eight different assemblages (A-H), but only assemblages A and B have been associated with human infections. Studies on the associations of G. lamblia assemblages and symptoms have been done but were inconclusive. The aim of this study was to correlate G. lamblia assemblages with symptoms in patients with and without HIV/AIDS and its association with the CD4T cell count. The cross-sectional survey was conducted among patients attending the Evandro Chagas National Institute of Infectious Diseases (INI/FIOCRUZ) in Rio de Janeiro from January 2011 to February 2015. Thirty-eight of 65 microscopically positive stool samples for G. lamblia were from HIV positive patients and 27 were from HIV negative patients. Of the HIV infected patients, 19 (55.9%) were genotyped as assemblage B of which 9 (47.4%) had a CD4Tcell count below 200cells/mm3. In addition, we found a greater number of samples belonging to assemblage B in symptomatic cases (11 of 19; 57.9%). Our data suggest that assemblage B is very likely to be found in HIV infected patients and probably the lower CD4T count gives advantages for assemblage B replication. Furthermore, assemblage B seems to be associated with symptomatology, particularly abdominal pain, asthenia, diarrhea, fever, headache and myalgia. This study provides information on G. lamblia assemblages and symptoms in patients with and without HIV/AIDS virus and their association with CD4Tcell counts.

Geospatial distribution of intestinal parasitic infections in Rio de Janeiro (Brazil) and its association with social determinants.

BACKGROUND: Intestinal parasitic infections remain among the most common infectious diseases worldwide. This study aimed to estimate their prevalence and provide a detailed analysis of geographical distribution of intestinal parasites in the metropolitan region of Rio de Janeiro, considering demographic, socio-economic, and epidemiological contextual factors. METHODS/PRINCIPAL FINDINGS: The cross-section survey was conducted among individuals attending the Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ, RJ) during the period from April 2012 to February 2015. Stool samples were collected and processed by sedimentation, flotation, Kato-Katz, Baermann-Moraes and Graham methods, iron haematoxylin staining and safranin staining. Of the 3245 individuals analysed, 569 (17.5%) were infected with at least one parasite. The most common protozoa were Endolimax nana (28.8%), Entamoeba coli (14.8%), Complex Entamoeba histolytica/Entamoeba dispar (13.5%), Blastocystis hominis (12.7%), and Giardia lamblia (8.1%). Strongyloides stercoralis (4.3%), Schistosoma mansoni (3.3%), Ascaris lumbricoides (1.6%), and hookworms (1.5%) were the most frequent helminths. There was a high frequency of contamination by protozoa (87%), and multiple infections were observed in 141 participants (24.8%). A positive association between age (young children) and gender (male) with intestinal parasites was observed. Geospatial distribution of the detected intestinal parasitic infections was not random or homogeneous, but was influenced by socioeconomic conditions (through the material deprivation index (MDI)). Participants classified in the highest levels of deprivation had higher risk of having intestinal parasites. CONCLUSIONS/SIGNIFICANCE: This study provides the first epidemiological information on the prevalence and distribution of intestinal parasitic infections in the Rio de Janeiro metropolitan area. Intestinal parasites, especially protozoa, are highly prevalent, indicating that parasitic infections are still a serious public health problem. MDI showed that intestinal parasites were strongly associated with the socioeconomic status of the population, thus making it possible to identify social vulnerable areas.

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.

Safety, efficacy and pharmacokinetic evaluations of a new coated chloroquine tablet in a single-arm open-label non-comparative trial in Brazil: a step towards a user-friendly malaria vivax treatment.

BACKGROUND: Malaria remains a major public health problem, with half the world population at risk of contracting malaria. The effects of Plasmodium vivax on prosperity and longevity have been highlighted in several recent clinical case reports. The first line of vivax treatment drugs has seen no radical innovation for more than 60 years. This study introduces a subtle incremental innovation to vivax treatment: a chloroquine and primaquine co-blister. The co-blister includes a new chloroquine formulation incorporating coated tablets to mask the drug's bitter taste and user-friendly packaging containing tablets of each drug, which may improve patient adherence and facilitate the appropriate use of the drugs. This new formulation will replace the non-coated chloroquine distributed in Brazil. METHODS: Patients were orally treated with 150 mg coated chloroquine tablets for 3 days: an initial 450 mg dose, followed by two 300 mg doses. The patients were treated concomitantly with two 15 mg primaquine tablets for 7-9 days, according to their weight. The primary objective of this study was to prove parasitological and clinical cure rates above 90 % by day 28. RESULTS: This single-arm open-label non-comparative trial was conducted according to the WHO recommended methodology for the surveillance of anti-malarial drug efficacy in the Brazilian Amazon. On day 28, the parasitological and clinical response was adequate in 98.8 % of patients (CI 95 % 93.4-100 %). The success rate on day 3 was 100 %, and the cumulative success rate by day 28 was 98.8 % (CI 95 % 91.7-99.8 %). There were no serious adverse events, with most adverse events classified as mild. The pharmacokinetic parameters of chloroquine analysed in whole blood dry spot samples showed mean (coefficient of variation) Cmax and AUC0-t values of 374.44 (0.35) and 3700.43 (0.36) ng/mL, respectively. DISCUSSION: This study reports an appropriate safety and efficacy profile of a new formulation of coated chloroquine tablets for vivax malaria treatment in the Brazilian Amazon. The cure rates meet the WHO efficacy criteria, supporting current Brazilian guidelines and the use of the formulation for vivax malaria treatment. Nevertheless, further studies should be conducted to address adherence and the effectiveness of the formulation. Trial registration RBR-77q7t3-UTN: U1111-1121-2982. Registered 10th May 2011.

Molecular Characterization of Giardia lamblia: First Report of Assemblage B in Human Isolates from Rio de Janeiro (Brazil).

BACKGROUND: Despite the high prevalence of giardiasis, the genetic characterization of Giardia lamblia has been poorly documented in Brazil and molecular epidemiology research has only been conducted in the last few years. The aim of this study was to determine the prevalence of different G. lamblia assemblages and detect mixed infections among patients with giardiasis. METHODS AND PRINCIPAL FINDINGS: The cross-section survey was conducted among patients attending the FIOCRUZ in Rio de Janeiro. In order to discriminate the genetic assemblages/sub-assemblages, G. lamblia isolates were characterized by PCR-RFLP and qPCR using four loci genes (bg, gdh, tpi and orfC4). Of the 65 positive samples, 41 (63.1%) were successfully amplified by nested-PCR of bg and gdh genes. Among them, 16 were typed as sub-assemblage AII, 7 as BIII, 4 as BIV and 8 as a mixture of BIII and BIV. After the analysis by qPCR assay, a total of 55 (84.6%) samples were amplified using at least one locus: bg gene was amplified in 38 (58.5%) samples, gdh in 41 (63.1%), tpi in 39 (60%), and orfC4 in 39 (60%). Multilocus genotyping results showed that 29 (52.7%) samples belonged to Assemblage A and 26 (47.3%) samples belonged to Assemblage B. In 2011 and 2012, 20 (74.1%) samples belonged to Assemblage A and 7 (25.9%) belonged to Assemblage B. In subsequent years (2013-2015) there was a predominance of Assemblage B, 19 (67.9%) versus 9 (32.1%) Assemblage A. CONCLUSIONS: This is the first time that Assemblage B of G. lamblia was reported in human clinical samples from Rio de Janeiro (Brazil) and is the first report about genetic characterization using four genes. The qPCR assemblage-specific showed no mixed infections by Assemblages A and B. A switch in genetic profile over the years was observed, firstly predominance of Assemblage A and lastly of Assemblage B.

Dengue infection as a potential trigger of an imported Plasmodium ovale malaria relapse or a long incubation period in a non-endemic malaria region.

OBJECTIVES: To report that dengue fever (DF) could have triggered Plasmodium ovale wallikeri malaria. METHODS: A retrospective case report of P. ovale malaria and DF in a single patient in Rio de Janeiro, Brazil, who had lived in Angola, is presented. RESULTS: On the second week of illness, the patient was referred to our research service. As symptoms had persisted up to day 14, malaria was also considered, based on the patient's long-standing epidemiological history. On day 16 of illness, a thick blood smear was positive for P. ovale (3480 parasites/mm(3)), PCR for malaria was positive for P. ovale wallikeri, and the kinetics of dengue virus (DENV) antibodies suggested a recent primary dengue infection. CONCLUSIONS: Concurrent infections of DENV and malaria have rarely been reported; the actual impact of these sequential or simultaneous infections remains unknown. Therefore, DF must be considered as a potential co-morbidity for malaria, because of its influence on fluid electrolyte management. The case presented showed consistent temporal, clinical, and laboratory evidence that the relapse or the long incubation period of P. ovale malaria may have been triggered by a recent DF episode. To the authors' knowledge, this is the first report of DENV and P. ovale co-infection.

Intestinal helminth coinfection is associated with mucosal lesions and poor response to therapy in American tegumentary leishmaniasis.

The most severe clinical form of American tegumentary leishmaniasis (ATL) due to Leishmania braziliensis is mucosal leishmaniasis (ML), characterized by destructive lesions in the facial mucosa. We performed a retrospective cohort study of 109 ATL patients from Rio de Janeiro State, Brazil, where ATL is caused by L. braziliensis, to evaluate the influence of intestinal parasite coinfections in the clinical course of ATL. Parasitological stool examination (PSE) was performed with samples from all patients by the sedimentation, Kato-Katz and Baermann-Moraes methods. The diagnosis of ATL was made from lesion biopsies by direct observation of amastigotes in Giemsa-stained imprints, isolation of Leishmania promastigotes or histopathological examination. All patients were treated with meglumine antimoniate. Patients with positive PSE had a frequency of mucosal lesions significantly higher than those with negative PSE (p<0.005). The same was observed for infections with helminths in general (p<0.05), with nematodes (p<0.05) and with Ascaris lumbricoides (p<0.05), but not for protozoan infections. Patients with intestinal parasites had poor response to therapy (therapeutic failure or relapse) significantly more frequently than the patients with negative stool examination (p<0.005). A similar difference (p<0.005) was observed between patients with positive and negative results for intestinal helminths, but not for intestinal protozoa. Patients with positive PSE took significantly longer to heal than those with negative PSE (p<0.005). A similar difference was observed for intestinal helminth infections (p<0.005), but not for protozoan infections. Our results indicate a deleterious influence of intestinal helminth infections in the clinical course of ATL and evidence for the first time an association between ML and these coinfections, particularly with nematodes and A. lumbricoides.

NO-Donor Dihydroartemisinin Derivatives as Multitarget Agents for the Treatment of Cerebral Malaria.

Hybrid products in which the dihydroartemisinin scaffold is combined with NO-donor furoxan and NONOate moieties have been synthesized and studied as potential tools for the treatment of cerebral malaria (CM). The designed products were able to dilate rat aorta strips precontracted with phenylephrine with a NO-dependent mechanism. All hybrid compounds showed preserved antiplasmodial activity in vitro and in vivo against Plasmodium berghei ANKA, comparable to artesunate and artemether. Hybrid 10, selected for additional studies, was capable of increasing survival of mice with late-stage CM from 27.5% to 51.6% compared with artemether. Artemisinin-NO-donor hybrid compounds show promise as potential new drugs for treating cerebral malaria.

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.

Cerebral tissue oxygenation impairment during experimental cerebral malaria.

Ischemia and hypoxia have been implicated in cerebral malaria (CM) pathogenesis, although direct measurements of hypoxia have not been conducted. C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) develop a neurological syndrome known as experimental cerebral malaria (ECM), whereas BALB/c mice are resistant to ECM. In this study, intravital microscopy methods were used to quantify hemodynamic changes, vascular/tissue oxygen (O2) tension (PO2), and perivascular pH in vivo in ECM and non-ECM models, employing a closed cranial window model. ECM mice on day 6 of infection showed marked decreases in pial blood flow, vascular (arteriolar, venular), and perivascular PO2, perivascular pH, and systemic hemoglobin levels. Changes were more dramatic in mice with late-stage ECM compared with mice with early-stage ECM. These changes led to drastic decreases in O2 delivery to the brain tissue. In addition, ECM animals required a greater PO2 gradient to extract the same amount of O2 compared with non-infected animals, as the pial tissues extract O2 from the steepest portion of the blood O2 equilibrium curve. ECM animals also showed increased leukocyte adherence in postcapillary venules, and the intensity of adhesion was inversely correlated with blood flow and O2 extraction. PbA-infected BALB/c mice displayed no neurological signs on day 6 and while they did show changes similar to those observed in C57BL/6 mice (decreased pial blood flow, vascular/tissue PO2, perivascular pH, hemoglobin levels), non-ECM animals preserved superior perfusion and oxygenation compared with ECM animals at similar anemia and parasitemia levels, resulting in better O2 delivery and O2 extraction by the brain tissue. In conclusion, direct quantitative assessment of pial hemodynamics and oxygenation in vivo revealed that ECM is associated with severe progressive brain tissue hypoxia and acidosis.

Transdermal glyceryl trinitrate as an effective adjunctive treatment with artemether for late-stage experimental cerebral malaria.

Cerebral malaria (CM) is associated with low nitric oxide (NO) bioavailability, cerebrovascular constriction, occlusion, and hypoperfusion. Administration of exogenous NO partially prevents the neurological syndrome and associated vascular pathology in an experimental CM (ECM) mouse model. In this study, we evaluated the effects of transdermal glyceryl trinitrate in preventing ECM and, in combination with artemether, rescuing late-stage ECM mice from mortality. The glyceryl trinitrate and/or artemether effect on survival and clinical recovery was evaluated in C57BL/6 mice infected with P. berghei ANKA. NO synthase (NOS) expression in mouse brain was determined by Western blots. Mean arterial pressure (MAP) and pial arteriolar diameter were monitored using a tail-cuff blood pressure system and a cranial window preparation, respectively. Preventative administration of glyceryl trinitrate at 0.025 mg/h decreased ECM mortality from 67 to 11% and downregulated inducible NOS expression in the brain. When administered as adjunctive rescue therapy with artemether, glyceryl trinitrate increased survival from 47 to 79%. The adjunctive therapy caused a sustained reversal of pial arteriolar vasoconstriction in ECM mice, an effect not observed with artemether alone. Glyceryl trinitrate induced a 13% decrease in MAP in uninfected mice but did not further affect MAP in hypotensive ECM mice. Glyceryl trinitrate, when combined with artemether, was an effective adjunctive rescue treatment for ECM. This treatment ameliorated pial arteriolar vasospasm and did not significantly affect MAP. These results indicate that transdermal glyceryl trinitrate has potential to be considered as a candidate for adjunctive therapy for CM.

Nitric oxide synthase dysfunction contributes to impaired cerebroarteriolar reactivity in experimental cerebral malaria.

Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment.

Slow and continuous delivery of a low dose of nimodipine improves survival and electrocardiogram parameters in rescue therapy of mice with experimental cerebral malaria.

BACKGROUND: Human cerebral malaria (HCM) is a life-threatening complication caused by Plasmodium falciparum infection that continues to be a major global health problem despite optimal anti-malarial treatment. In the experimental model of cerebral malaria (ECM) by Plasmodium berghei ANKA, bolus administration of nimodipine at high doses together with artemether, increases survival of mice with ECM. However, the dose and administration route used is associated with cardiovascular side effects such as hypotension and bradycardia in humans and mice, which could preclude its potential use as adjunctive treatment in HCM. METHODS: In the present study, alternative delivery systems for nimodipine during late-stage ECM in association with artesunate were searched to define optimal protocols to achieve maximum efficacy in increasing survival in rescue therapy while causing the least cardiac side effects. The baseline electrocardiogram (ECG) and arterial pressure characteristics of uninfected control animals and of mice with ECM and its response upon rescue treatment with artesunate associated or not with nimodipine is also analysed. RESULTS: Nimodipine, given at 0.5 mg/kg/day via a slow and continuous delivery system by osmotic pumps, increases survival of mice with ECM when used as adjunctive treatment to artesunate. Mice with ECM showed hypotension and ECG changes, including bradycardia and increases in PR, QRS, QTc and ST interval duration. ECM mice also show increased QTc dispersion, heart rate variability (HRV), RMSSD, low frequency (LF) and high frequency (HF) bands of the power spectrum. Both sympathetic and parasympathetic inputs to the heart were increased, but there was a predominance of sympathetic tone as demonstrated by an increased LF/HF ratio. Nimodipine potentiated bradycardia when given by bolus injection, but not when via osmotic pumps. In addition, nimodipine shortened PR duration and improved HRV, RMSSD, LF and HF powers in mice with ECM. In addition, nimodipine did not increased hypotension or decreased the speed of arterial pressure recovery when used in rescue therapy with artesunate. CONCLUSIONS: These data show that slow and continuous delivery of lower doses of nimodipine improves survival of mice with ECM in rescue therapy with artesunate while showing a safer profile in terms of cardiovascular effects.

S-nitrosoglutathione prevents experimental cerebral malaria.

Administration of the exogenous nitric oxide (NO) donor dipropylenetriamine-NONOate (DPTA-NO) to mice during Plasmodium berghei ANKA (PbA) infection largely prevents development of experimental cerebral malaria (ECM). However, a high dose (1 mg/mouse twice a day) is necessary and causes potent side effects such as marked hypotension. In the present study we evaluated whether an alternative, physiologically relevant NO donor, S-nitrosoglutathione (GSNO), was able to prevent ECM at lower doses with minimal side effects. Prophylactic treatment with high (3.5 mg), intermediate (0.35 mg) or low (0.035 mg) doses of GSNO decreased incidence of ECM in PbA-infected mice, decreasing also edema, leukocyte accumulation and hemorrhage incidence in the brain. The high dose inhibited parasite growth and also induced transient hypotension. Low and intermediate doses had no or only mild effects on parasitemia, blood pressure, and heart rate compared to saline-treated mice. PbA infection decreased brain total and reduced (GSH) glutathione levels. Brain levels of oxidized (GSSG) glutathione and the GSH/GSSG ratio were positively correlated with temperature and motor behavior. Low and intermediate doses of GSNO failed to restore the depleted brain total glutathione and GSH levels, suggesting that ECM prevention by GSNO was probably related to other effects such as inhibition of inflammation and vascular protection. These results indicate that ECM is associated with depletion of the brain glutathione pool and that GSNO is able to prevent ECM development in a wide range of doses, decreasing brain inflammation and inducing milder cardiovascular side effects.

Efficacy of different nitric oxide-based strategies in preventing experimental cerebral malaria by Plasmodium berghei ANKA.

BACKGROUND: Low nitric oxide (NO) bioavailability plays a role in the pathogenesis of human as well as of experimental cerebral malaria (ECM) caused by Plasmodium berghei ANKA (PbA). ECM is partially prevented by administration of the NO-donor dipropylenetriamine NONOate (DPTA-NO) at high concentration (1 mg/mouse), which also induces major side effects such as a sharp drop in blood pressure. We asked whether alternative strategies to improve NO bioavailability with minor side effects would also be effective in preventing ECM. METHODOLOGY/PRINCIPAL FINDINGS: Mice were infected with PbA and prophylactically treated twice a day with bolus injections of L-arginine, Nω-hydroxy-nor-Arginine (nor-NOHA), tetrahydrobiopterin (BH4), separately or combined, sodium nitrite, sildenafil or sildenafil plus DPTA-NO starting on day 0 of infection. L-arginine and BH4 supplementation, with or without arginase inhibition by nor-NOHA, increased plasma nitrite levels but failed to protect against ECM development. Accordingly, prophylactic treatment with continuous delivery of L-arginine using osmotic pumps also did not improve survival. Similar outcomes were observed with sodium nitrite sildenafil (aimed at inhibiting phosphodiesterase-5) or with DPTA-NO. However, sildenafil (0.1 mg/mouse) in combination with a lower dose (0.1 mg/mouse) of DPTA-NO decreased ECM incidence (82 ± 7.4% mortality in the saline group and 38 ± 10.6% in the treated group; p<0.05). The combined prophylactic therapy did not aggravate anemia, had delayed effects in systolic, diastolic and mean arterial blood pressure and induced lower effects in pulse pressure when compared to DPTA-NO 1 mg/mouse. CONCLUSIONS/SIGNIFICANCE: These data show that sildenafil lowers the amount of NO-donor needed to prevent ECM, resulting also in lesser side effects. Prophylactic L-arginine when given in bolus or continuous delivery and bolus BH4 supplementation, with or without arginase inhibition, were able to increase NO bioavailability in PbA-infected mice but failed to decrease ECM incidence in the doses and protocol used.