Point-of-Care Strategies Applied to Malaria Diagnosis

Rapid and specific diagnosis of malaria remains one of the main strategies to fight the disease. The diagnosis is made primarily by the simple and low-cost thick drop technique, considered the gold standard test. However, the requirement for good quality microscopes and well-trained personnel often lead to inaccurate diagnosis, especially in cases of mixed infections or low parasitemia. Although PCR-based tests can help in these situations, this technique requires large and sensitive equipments, being unsuitable for point of care (POC) settings. A myriad of POC diagnostic tests have being developed in the last years, relying on molecular methods but also on novel strategies. New platforms, miniaturization techniques, and multiplexing possibilities promise great potential to improve disease diagnostics through fast and accurate detection of cases, even at remote places. Here, we will address the main POC strategies developed for the diagnosis of malaria, highlighting their strengths and weakness as POC applications

Type I Interferon Transcriptional Signature in Neutrophils and Low-Density Granulocytes Are Associated with Tissue Damage in Malaria

Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. However, the role of these polymorphonuclear cells in mediating either the resistance or the pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, enhanced release of reactive oxygen species and myeloperoxidase, and a high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed intense recruitment of neutrophils to liver sinusoids. Neutrophil migration and IL-1beta and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggest that type I interferon signaling has a central role in neutrophil activation and malaria pathogenesis

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

Submitted by Repositório Arca ([email protected]) on 2019-04-24T17:38:50Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-08-13T14:21:37Z (GMT) No. of bitstreams: 2 ve_ Hojo-Souza_Natália_etal_INI_2015.pdf: 1172050 bytes, checksum: b1948378bfee8669ad90694b3aa2cb60 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-13T14:21:37Z (GMT). No. of bitstreams: 2 ve_ Hojo-Souza_Natália_etal_INI_2015.pdf: 1172050 bytes, checksum: b1948378bfee8669ad90694b3aa2cb60 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro. RJ, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.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

Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil

Several arboviruses have emerged and/or re-emerged in North, Central and SouthAmerican countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list

Distinctive genetic structure and selection patterns in Plasmodium vivax from South Asia and East Africa.

Despite the high burden of Plasmodium vivax malaria in South Asian countries, the genetic diversity of circulating parasite populations is not well described. Determinants of antimalarial drug susceptibility for P. vivax in the region have not been characterised. Our genomic analysis of global P. vivax (n = 558) establishes South Asian isolates (n = 92) as a distinct subpopulation, which shares ancestry with some East African and South East Asian parasites. Signals of positive selection are linked to drug resistance-associated loci including pvkelch10, pvmrp1, pvdhfr and pvdhps, and two loci linked to P. vivax invasion of reticulocytes, pvrbp1a and pvrbp1b. Significant identity-by-descent was found in extended chromosome regions common to P. vivax from India and Ethiopia, including the pvdbp gene associated with Duffy blood group binding. Our investigation provides new understanding of global P. vivax population structure and genomic diversity, and genetic evidence of recent directional selection in this important human pathogen

Neutrophil Paralysis in Plasmodium vivax Malaria

Plasmodium vivax is responsible for approximately 60–80% of the malaria cases in the world, and contributes to significant social and economic instability in the developing countries of Latin America and Asia. The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. The innate immune receptors recognize Plasmodium sp. and initiate a broad spectrum of host defense mechanisms that mediate resistance to infection. However, the innate immune response is the classic “two-edged sword”, and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α during acute malaria. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Unexpectedly, we noticed an impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. We proposed that neutrophil paralysis is in part responsible for the enhanced susceptibility to bacterial infection observed in malaria patients

Type I Interferon Transcriptional Signature in Neutrophils and Low-Density Granulocytes Are Associated with Tissue Damage in Malaria

Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. However, the role of these polymorphonuclear cells in mediating either the resistance or the pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, enhanced release of reactive oxygen species and myeloperoxidase, and a high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed intense recruitment of neutrophils to liver sinusoids. Neutrophil migration and IL-1β and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggest that type I interferon signaling has a central role in neutrophil activation and malaria pathogenesis.status: publishe

DNA-Containing Immunocomplexes Promote Inflammasome Assembly and Release of Pyrogenic Cytokines by CD14+ CD16+ CD64high CD32low Inflammatory Monocytes from Malaria Patients

Submitted by Nuzia Santos ([email protected]) on 2016-04-05T17:25:39Z No. of bitstreams: 1 DNA-Containing Immunocomplex (...) Monocytes from Malaria Patients.pdf: 4339793 bytes, checksum: 1b4586b816c6971792523290aa455335 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-04-05T17:31:16Z (GMT) No. of bitstreams: 1 DNA-Containing Immunocomplex (...) Monocytes from Malaria Patients.pdf: 4339793 bytes, checksum: 1b4586b816c6971792523290aa455335 (MD5)Made available in DSpace on 2016-04-05T17:31:16Z (GMT). No. of bitstreams: 1 DNA-Containing Immunocomplex (...) Monocytes from Malaria Patients.pdf: 4339793 bytes, checksum: 1b4586b816c6971792523290aa455335 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil.University of Massachusetts Medical School. Division of Infectious Diseases and Immunology. Worcester, MA, USA / Centro Internacional de Entrenamiento e Investigaciones Medicas. Cali, Colombia.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / University of Massachusetts Medical School. Division of Infectious Diseases and Immunology. Worcester, MA, USA.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brasil.University of Massachusetts Medical School. Division of Infectious Diseases and Immunology. Worcester, MA, USA.Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.University of California San Diego. School of Medicine. San Diego, CA, USA.The Feinstein Institute for Medical Research. Center for Autoimmune and Musculoskeletal Diseases. Manhasset, NY, USA.University of Massachusetts Medical School. Division of Infectious Diseases and Immunology. Worcester, MA, USA.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / University of Massachusetts Medical School. Division of Infectious Diseases and Immunology. Worcester, MA, USA.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / University of Massachusetts Medical School. Division of Infectious Diseases and Immunology. Worcester, MA, USA / Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brasil.High levels of circulating immunocomplexes (ICs) are found in patients with either infectious or sterile inflammation. We report that patients with either Plasmodium falciparum or Plasmodium vivax malaria have increased levels of circulating anti-DNA antibodies and ICs containing parasite DNA. Upon stimulation with malaria-induced ICs, monocytes express an NF-κB transcriptional signature. The main source of IC-induced proinflammatory cytokines (i.e., tumor necrosis factor alpha [TNF-α] and interleukin-1β [IL-1β])in peripheral blood mononuclear cells from acute malaria patients was found to be a CD14+ CD16 (FcγRIIIA)+ CD64 (FcγRI)high CD32 (FcγRIIB)low monocyte subset. Monocytes from convalescent patients were predominantly of the classical phenotype (CD14+ CD16−) that produces high levels of IL-10 and lower levels of TNF-α and IL-1β in response to ICs. Finally, we report a novel role for the proinflammatory activity of ICs by demonstrating their ability to induce inflammasome assembly and caspase-1 activation in human monocytes. These findings illuminate our understanding of the pathogenic role of ICs and monocyte subsets and may be relevant for future development of immunity-based interventions with broad applications to systemic inflammatory diseases

Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil

ABSTRACT Several arboviruses have emerged and/or re-emerged in North, Central and South-American countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list