M1 macrophage features in severe Plasmodium falciparum malaria patients with pulmonary oedema

Background Pulmonary oedema (PE) is a serious complication of Plasmodium falciparum malaria which can lead to acute lung injury in severe cases. Lung macrophages are activated during malaria infection due to a complex host-immune response. The molecular basis for macrophage polarization is still unclear but understanding the predominant subtypes could lead to new therapeutic strategies where the diseases present with lung involvement. The present study was designed to study the polarization of lung macrophages, as M1 or M2 macrophages, in the lungs of severe P. falciparum malaria patients, with and without evidence of PE. Methods Lung tissue samples, taken from patients who died from severe P. falciparum malaria, were categorized into severe malaria with PE and without PE (non-PE). Expression of surface markers (CD68+, all macrophages; CD40+, M1 macrophage; and CD163+, M2 macrophage) on activated lung macrophages was used to quantify M1/M2 macrophage subtypes. Results Lung injury was demonstrated in malaria patients with PE. The expression of CD40 (M1 macrophage) was prominent in the group of severe P. falciparum malaria patients with PE (63.44 ± 1.98%), compared to non-PE group (53.22 ± 3.85%, p < 0.05), whereas there was no difference observed for CD163 (M2 macrophage) between PE and non-PE groups. Conclusions The study demonstrates M1 polarization in lung tissues from severe P. falciparum malaria infections with PE. Understanding the nature of macrophage characterization in malaria infection may provide new insights into therapeutic approaches that could be deployed to reduce lung damage in severe P. falciparum malaria

Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules

INTRODUCTION: Oxidative stress has been demonstrated in malaria. The potential oxidative modification of lipoproteins derived from malaria patients was studied. These oxidized lipids may have role in pathogenesis of malaria. METHOD: The plasma lipid profile and existence of oxidized forms of very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) were investigated in malaria (17 mild and 24 severe patients) and 37 control subjects. Thiobarbituric acid reactive substances (TBARs), conjugated dienes, tryptophan fluorescence and fluidity of lipoproteins were determined as markers of oxidation. The biological effect of malarial lipoproteins was assessed by the expression of adhesion molecules on endothelial cells. RESULTS: Malarial lipoproteins had decreased cholesterol (except in VLDL) and phospholipid. The triglyceride levels were unchanged. The cholesterol/phospholipid ratio of LDL was decreased in malaria, but increased in VLDL and HDL. TBARs and conjugate dienes were increased in malarial lipoproteins, while the tryptophan fluorescence was decreased. The fluidity of lipoproteins was increased in malaria. These indicated the presence of oxidized lipoproteins in malaria by which the degree of oxidation was correlated with severity. Of three lipoproteins from malarial patients, LDL displayed the most pronounced oxidative modification. In addition, oxidized LDL from malaria patients increased endothelial expression of adhesion molecules. CONCLUSION: In malaria, the lipoproteins are oxidatively modified, and the degree of oxidation is related with severity. Oxidized LDL from malarial patients increases the endothelial expression of adhesion molecules. These suggest the role of oxidized lipoproteins, especially LDL, on the pathogenesis of disease

Suppression of Plasmodium falciparum by serum collected from a case of Plasmodium vivax infection.

BACKGROUND: It has frequently been reported that Plasmodium vivax suppressed Plasmodium falciparum and ameliorated disease severity in patients infected with these two species simultaneously. The authors investigate the hypothesis that immunological responses stimulated by P. vivax may play a role in suppressing co-infecting P. falciparum. METHODS: Sera, taken sequentially from one of the authors (YN) during experimental infection with P. vivax, were added to in vitro cultures of P. falciparum. Cross-reactive antibodies against P. falciparum antigens, and cytokines were measured in the sera. RESULTS: Significant growth inhibitory effects upon P. falciparum cultures (maximally 68% inhibition as compared to pre-illness average) were observed in the sera collected during an acute episode. Such inhibitory effects showed a strong positive temporal correlation with cross-reactive antibodies, especially IgM against P. falciparum schizont extract and, to a lesser degree, IgM against Merozoite Surface Protein (MSP)-119. Interleukin (IL)-12 showed the highest temporal correlation with P. vivax parasitaemia and with body temperatures in the volunteer. CONCLUSION: These results suggest the involvement by cross-reactive antibodies, especially IgM, in the interplay between plasmodial species. IL-12 may be one of direct mediators of fever induction by rupturing P. vivax schizonts, at least in some subjects. Future studies, preferably of epidemiological design, to reveal the association between cross-reactive IgM and cross-plasmodial interaction, are warranted

Activation of nuclear factor kappa B in peripheral blood mononuclear cells from malaria patients

BACKGROUND: Malaria parasites and their products can activate a specific immune response by stimulating cytokine production in the host’s immune cells. Transcription nuclear factor kappa B (NF-κB) is an important regulator for the control of many pro-inflammatory genes, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). The activation and expression of NF-κB p65 in peripheral blood mononuclear cells (PBMCs) of malaria patients were investigated and correlated with the levels of IL-10 and TNF to study the nature of NF-κB p65 and its linkage to inflammatory cytokines. METHODS: The sample group comprised 33 patients admitted with malaria caused by Plasmodium vivax (n = 11), uncomplicated Plasmodium falciparum (n = 11), and complicated Plasmodium falciparum (n = 11). Peripheral blood was collected at admission and on day 7 for PBMC isolation. Healthy subjects were used as a control group. The expressions of NF-κB p65 in the PBMCs from malaria patients and the plasma levels of IL-10 and TNF were measured by using enzyme-linked immunosorbent assay (ELISA). The immunofluorescence technique was used to determine NF-κB nuclear translocation. RESULTS: At admission, patients with P. vivax and uncomplicated P. falciparum had significantly elevated phospho-NF-κB p65 levels in the PBMCs compared with those of healthy controls. However, patients with complicated P. falciparum malaria had decreased levels of phospho-NF-κB p65. On day 7 post-treatment, significantly increased phospho-NF-κB p65 was found in the PBMCs of patients with complicated P. falciparum, compared with healthy controls. The plasma level of IL-10 was elevated in day 0 in patients with complicated P. falciparum malaria and was found to be negatively correlated with phospho-NF-κB p65 level (r(s) = −0.630, p = 0.038). However, there was no correlation between phospho-NF-κB p65 expression and TNF level in patients with complicated P. falciparum malaria. CONCLUSIONS: This is the first report demonstrating alterations in NF-κB p65 activity in the PBMCs of malaria patients. The altered lower features of NF-κB p65 in the PBMCs of patients with complicated P. falciparum at admission could be due to a suppressive effect of high IL-10 associated with complicated P. falciparum malaria

Whole blood angiopoietin-1 and -2 levels discriminate cerebral and severe (non-cerebral) malaria from uncomplicated malaria

<p>Abstract</p> <p>Background</p> <p>Severe and cerebral malaria are associated with endothelial activation. Angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) are major regulators of endothelial activation and integrity. The aim of this study was to investigate the clinical utility of whole blood angiopoietin (ANG) levels as biomarkers of disease severity in <it>Plasmodium falciparum </it>malaria.</p> <p>Methods</p> <p>The utility of whole blood ANG levels was examined in Thai patients to distinguish cerebral (CM; n = 87) and severe (non-cerebral) malaria (SM; n = 36) from uncomplicated malaria (UM; n = 70). Comparative statistics are reported using a non-parametric univariate analysis (Kruskal-Wallis test or Chi-squared test, as appropriate). Multivariate binary logistic regression was used to examine differences in whole blood protein levels between groups (UM, SM, CM), adjusting for differences due to ethnicity, age, parasitaemia and sex. Receiver operating characteristic curve analysis was used to assess the diagnostic accuracy of the ANGs in their ability to distinguish between UM, SM and CM. Cumulative organ injury scores were obtained for patients with severe disease based on the presence of acute renal failure, jaundice, severe anaemia, circulatory collapse or coma.</p> <p>Results</p> <p>ANG-1 and ANG-2 were readily detectable in whole blood. Compared to UM there were significant decreases in ANG-1 (p < 0.001) and significant increases in ANG-2 (p < 0.001) levels and the ratio of ANG-2: ANG-1 (p < 0.001) observed in patients with SM and CM. This effect was independent of covariates (ethnicity, age, parasitaemia, sex). Further, there was a significant decrease in ANG-1 levels in patients with SM (non-cerebral) versus CM (p < 0.001). In participants with severe disease, ANG-2, but not ANG-1, levels correlated with cumulative organ injury scores; however, ANG-1 correlated with the presence of renal dysfunction and coma. Receiver operating characteristic curve analysis demonstrated that the level of ANG-1, the level of ANG-2 or the ratio of ANG-2: ANG-1 discriminated between individuals with UM and SM (area under the curve, p-value: ANG-2, 0.763, p < 0.001; ANG-1, 0.884, p < 0.001; Ratio, 0.857, p < 0.001) or UM and CM (area under the curve, p-value: ANG-2, 0.772, p < 0.001; ANG-1, 0.778, p < 0.001; Ratio, 0.820, p < 0.001).</p> <p>Conclusions</p> <p>These results suggest that whole blood ANG-1/2 levels are promising clinically informative biomarkers of disease severity in malarial syndromes.</p

Efficacy of DB289 in Thai Patients with Plasmodium vivax or Acute, Uncomplicated Plasmodium falciparum Infections

BackgroundDB289 is the orally active prodrug of the diamidine DB75, which was developed for the treatment of human African trypanosomiasis MethodsWe tested the safety and efficacy of DB289 for the treatment of Plasmodium vivax and acute, uncomplicated P. falciparum infections in an open-label pilot study at the Hospital for Tropical Diseases in Bangkok. Nine patients with P. vivax infections and 23 patients with P. falciparum infections were admitted and treated with 100 mg of DB289 given orally twice a day for 5 days and were followed for 28 days. Patients with P. vivax infections were also treated with primaquine on days 10-23 ResultsAll patients cleared parasites by day 7, with a mean±SD clearance time of 43±41 h. One patient with a P. vivax infection had a recurrence of parasitemia on day 9. Of the 23 patients with P. falciparum infections, 3 had recurrences of parasitemia caused by P. vivax and 2 had recurrences of parasitemia caused by P. falciparum. In only 1 of 2 recurrences of parasitemia caused by P. falciparum were the parasites genotypically distinct from the infecting parasites the patient had at enrollment, which means there was a 96% cure rate ConclusionsDB289 is a promising new antimalarial compound that could become an important component of new antimalarial combination

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure

Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein

Misclassification of Drug Failures in Plasmodium falciparum Clinical Trials in Southeast Asia

Most trials of antimalarials occur in areas where reinfections are possible. For Plasmodium falciparum, reinfections are distinguished from recrudescences by PCR analysis of 3 polymorphic genes. However, the validity of this approach has never been rigorously tested. We tested for misclassification in 6 patients from clinical trials in Thailand and Cambodia who were classified as reinfected by the standard PCR protocol. Using heteroduplex tracking assays and direct DNA sequencing, we found that 5 of 6 (83%) patients were misclassified. Misclassification in this manner overestimates the efficacy of antimalarials and delays recognition of decreasing therapeutic efficacy, thus delaying potential policy changes

Deployment of Early Diagnosis and Mefloquine- Artesunate Treatment of Falciparum Malaria in Thailand: The Tak Malaria Initiative

BACKGROUND: Early diagnosis and treatment with artesunate-mefloquine combination therapy (MAS) have reduced the transmission of falciparum malaria dramatically and halted the progression of mefloquine resistance in camps for displaced persons along the Thai-Burmese border, an area of low and seasonal transmission of multidrug-resistant Plasmodium falciparum. We extended the same combination drug strategy to all other communities (estimated population 450,000) living in five border districts of Tak province in northwestern Thailand. METHODS AND FINDINGS: Existing health structures were reinforced. Village volunteers were trained to use rapid diagnostic tests and to treat positive cases with MAS. Cases of malaria, hospitalizations, and malaria-related deaths were recorded in the 6 y before, during, and after the Tak Malaria Initiative (TMI) intervention. Cross-sectional surveys were conducted before and during the TMI period. P. falciparum malaria cases fell by 34% (95% confidence interval [CI], 33.5–34.4) and hospitalisations for falciparum malaria fell by 39% (95% CI, 37.0–39.9) during the TMI period, while hospitalisations for P. vivax malaria remained constant. There were 32 deaths attributed to malaria during, and 22 after the TMI, a 51.5% (95% CI, 39.0–63.9) reduction compared to the average of the previous 3 y. Cross-sectional surveys indicated that P. vivax had become the predominant species in Thai villages, but not in populations living on the Myanmar side of the border. In the displaced persons population, where the original deployment took place 7 y before the TMI, the transmission of P. falciparum continued to be suppressed, the incidence of falciparum malaria remained low, and the in vivo efficacy of the 3-d MAS remained high. CONCLUSIONS: In the remote malarious north western border area of Thailand, the early detection of malaria by trained village volunteers, using rapid diagnostic tests and treatment with mefloquine-artesunate was feasible and reduced the morbidity and mortality of multidrug-resistant P. falciparum