Chronic chagasic cardiomyopathy (CCC) progresses with parasite persistence, fibrosis, and electrical alterations associated with an unbalanced immune response such as high plasma levels of tumor necrosis factor (TNF) and nitric oxide (NO). Presently, the available treatments only mitigate the symptoms of CCC. To improve CCC prognosis, we interfered with the parasite load and unbalanced immune response using the trypanocidal drug benznidazole (Bz) and the immunoregulator pentoxifylline (PTX). C57BL/6 mice chronically infected with the Colombian strain of Trypanosoma cruzi and with signs of CCC were treated for 30 days with a suboptimal dose of Bz (25 mg/kg of body weight), PTX (20 mg/kg), or their combination (Bz plus PTX) and analyzed for electrocardiographic, histopathological, and immunological changes. Bz (76%) and Bz-plus-PTX (79%) therapies decreased parasite loads. Although the three therapies reduced myocarditis and fibrosis and ameliorated electrical alterations, only Bz plus PTX restored normal heart rate-corrected QT (QTc) intervals. Bz-plus-PTX-treated mice presented complementary effects of Bz and PTX, which reduced TNF expression (37%) in heart tissue and restored normal TNF receptor 1 expression on CD8+ T cells, respectively. Bz (85%) and PTX (70%) therapies reduced the expression of inducible nitric oxide synthase (iNOS/NOS2) in heart tissue, but only Bz (58%) reduced NO levels in serum. These effects were more pronounced after Bz-plus-PTX therapy. Moreover, 30 to 50 days after treatment cessation, reductions of the prolonged QTc and QRS intervals were sustained in Bz-plus-PTX-treated mice. Our findings support the importance of interfering with the etiological agent and immunological abnormalities to improve CCC prognosis, opening an opportunity for a better quality of life for Chagas' disease (CD) patients.

Laboratory tests for malaria are only performed if there is clinical suspicion of the disease, and a missed diagnosis contributes substantially to morbidity and mortality. Malaria parasites produce haemozoin, which is able to depolarize light and this allows the automated detection of malaria during routine complete blood count analysis (CBC) with some Abbott Cell-Dyn instruments. In this study, we evaluated the Cell-Dyn CD4000 with 831 blood samples submitted for malaria investigations. Samples were categorized as malaria negative (n = 417), convalescent malaria (n = 64) or malaria positive (n = 350) by reference to thin/thick film microscopy, 'rapid test' procedures, polymerase chain reaction analysis and clinical history. With regard to CD4000 depolarization analysis, a malaria positive CD4000 pattern was ascribed to samples that showed one or more abnormal depolarizing purple events, which corresponded to monocytes containing ingested malaria pigment (haemozoin). Positive CD4000 patterns were observed in 11 of 417, 50 of 64 and 281 of 350 of malaria negative, convalescent malaria and malaria positive samples respectively. The specificity and positive predictive values for malaria (active and convalescent) were very high (97.4 and 96.8%, respectively), while sensitivity and negative predictive values were 80.0 and 83.0% respectively. Depolarization analysis was particularly effective for Plasmodium falciparum malaria but there was lower detection sensitivity for White compared with Black African patients. CD4000 90 degrees depolarization vs 0 degrees analysis revealed a proportion of samples with small nonleucocyte-associated depolarizing particles. Appearance of such events in the form of a discrete cluster was associated with P. vivax rather than P. falciparum infection.

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8+ and CD4+ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110) or cardiopathic (mild, B1, n = 163; severe, C, n = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 −403 (rs2107538) CT heterozygotes (OR = 0.5, P-value = 0.04) and T carriers (OR = 0.5, P-value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5–CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1+ CD8+ T cells and CD14+ macrophages were decreased, while frequencies of CCR5+ cells were increased. Importantly, CCR1+CD14+ macrophages were mainly IL-10+, while CCR5+ cells were mostly TNF+. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5−/− mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1+CD8+ T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1+ cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation.

Chagas disease, caused by the parasite Trypanosoma cruzi, is endemic in Latin America. Its acute phase is associated with high parasitism, myocarditis and profound myocardial gene expression changes. A chronic phase ensues where 30% develop severe heart lesions. Mouse models of T. cruzi infection have been used to study heart damage in Chagas disease. The aim of this study was to provide an interactome between miRNAs and their targetome in Chagas heart disease by integrating gene and microRNA expression profiling data from hearts of T. cruzi infected mice. Gene expression profiling revealed enrichment in biological processes and pathways associated with immune response and metabolism. Pathways, functional and upstream regulator analysis of the intersections between predicted targets of differentially expressed microRNAs and differentially expressed mRNAs revealed enrichment in biological processes and pathways such as IFNγ, TNFα, NF-kB signaling signatures, CTL-mediated apoptosis, mitochondrial dysfunction, and Nrf2-modulated antioxidative responses. We also observed enrichment in other key heart disease-related processes like myocarditis, fibrosis, hypertrophy and arrhythmia. Our correlation study suggests that miRNAs may be implicated in the pathophysiological processes taking place the hearts of acutely T. cruzi-infected mice.

Platelet counts and automated detection of platelet clumps were evaluated by optical analysis with the Abbott CD4000 analyser (Abbott Diagnostics, Santa Clara, CA, USA) in this South African study of 828 samples referred for malaria investigations. Based on microscopy (Micro) and rapid tests (RT) for HRP2 protein and parasite-associated LDH, malaria negative samples (n = 417) were defined as Micro-, RT-. Convalescent cases (n = 64) were Micro-, RT+ and had a recent record of positive microscopy. Malaria positive cases were subdivided into Micro+ (n = 315) and Micro-, RT+, PCR+ (polymerase chain reaction) (n = 32) subgroups. The mean platelet count for Micro+ cases (89.7 x 10(9)/l) was significantly lower than both the malaria negative (mean 212.6 x 10(9)/l) and convalescent malaria (mean 152.8 x 10(9)/l) groups; 89% of microscopy positive cases were thrombocytopenic (< 150 x 10(9)/l) and 30% had severe thrombocytopenia (< 50 x 10(9)/l). For comparison, 32% of the 417 malaria negative samples were thrombocytopenic and 6% of these were severe. Two thirds of samples with parasitaemia above 10% had platelet counts of < 50 x 10(9)/l while the counts were largely independent of parasite numbers when the parasitaemia was below 10%. Thirty percent of samples with microscopically detectable parasites had a PltClmp flag compared to 13% of the malaria negative group but, when the actual platelet count was taken into account, it became apparent that appearance of the flag was primarily associated with thrombocytopenia per se rather than malaria status. In most samples with a PltClmp flag, the CD4000 optical platelet clump 'signature' was indicative of small platelet aggregates and giant platelets. Morphological examination confirmed the presence of varying numbers of small platelet aggregates (3-12 individual platelets), often together with increased giant platelets, in many samples with a PltClmp flag. The observations suggest that while patients with malaria may be predisposed to the development of thrombocytopenia, a reduced platelet count in some patients may also be due in part to pseudo-thrombocytopenia.