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1.
Medicina (B.Aires) ; 79(6): 433-437, dic. 2019. graf, tab
Article in Spanish | LILACS | ID: biblio-1056749

ABSTRACT

Se analizó; de forma retrospectiva la presencia de anticuerpos só;©ricos IgG e IgM anti-Toxoplasma gondii en las embarazadas que concurrieron a siete hospitales del ó;rea Metropolitana de Buenos Aires durante 2006 y 2017. La prevalencia de infecció;n, medida como presencia de anticuerpos, en 2006 vs. 2017, fue: Hospital Alemán: 22 y 17% (p = 0.004), Hospital Fiorito: 44 y 33% (p < 0.001), Hospital Gandulfo: 30 y 34% (p 0.025), Hospital Grierson: 60 y 44% (p < 0.001), Hospital Rivadavia: 59 y 51% (p=0.003), Maternidad Sardá 47 y 39% (p < 0.001) y Hospital Thompson: 61 y 51% (p < 0.001). La comparació;n demostró; una disminució;n estadísticamente significativa de la seroprevalencia en seis hos pitales. Tambín disminuyeron significativamente la reactividad para IgM en 2017 respecto de 2006 y la seroprevalencia para T. gondii en el total de la població;n de embarazadas estudiadas, lo que significa un mayor nó;ºmero de mujeres susceptible de desarrollar infecció;n aguda durante el embarazo.


We analyzed the presence of IgG and IgM anti- Toxoplasma gondii, as a measure of infection, in pregnant women attending seven hospitals in the Metropolitan Area of Buenos Aires during 2006 and 2017. T. gondii seroprevalence in 2006 vs. 2017, was: Hospital Alemán: 22 and 17% (p = 0.004), Hospital Fiorito: 44 and 33% (p < 0.001), Hospital Gandulfo: 30 and 34% (p 0.025), Hospital Grierson 60 and 44% (p < 0.001), Hospital Rivadavia: 59 and 51% (p = 0.003), Hospital Sardá: 47 and 39% (p < 0. 001), and Hospital Thompson: 61 and 51% (p < 0.001). The comparison showed a significant decrease in seroprevalence in six hospitals. We also observed a significant decrease in the reactivity for IgM in 2017 compared to 2006 and in the seroprevalence for T. gondii in the overall population of pregnant women in the study. This means that a greater number of women are susceptible to develop acute infection during pregnancy.


Subject(s)
Humans , Female , Pregnancy , Adult , Middle Aged , Young Adult , Pregnancy Complications, Infectious/immunology , Toxoplasma/immunology , Antibodies, Protozoan/blood , Toxoplasmosis/immunology , Argentina/epidemiology , Pregnancy Complications, Infectious/epidemiology , Time Factors , Immunoglobulin G/blood , Immunoglobulin M/blood , Antibodies, Protozoan/immunology , Seroepidemiologic Studies , Toxoplasmosis/blood , Toxoplasmosis/epidemiology , Retrospective Studies , Risk Factors , Age Distribution , Hospitals/statistics & numerical data
2.
Mem. Inst. Oswaldo Cruz ; 114: e190158, 2019. graf
Article in English | LILACS | ID: biblio-1040629

ABSTRACT

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.


Subject(s)
Animals , Female , Mice , Phagocytosis/physiology , Plasmodium vivax/immunology , Antibodies, Protozoan/immunology , Protozoan Proteins/immunology , Merozoites/immunology , Plasmodium vivax/physiology , Merozoites/cytology , Flow Cytometry , Mice, Inbred BALB C
3.
Arq. bras. cardiol ; 110(4): 348-353, Apr. 2018. tab, graf
Article in English | LILACS | ID: biblio-888059

ABSTRACT

Abstract Background: Vimentin is a main structural protein of the cell, a component of intermediate cell filaments and immersed in cytoplasm. Vimentin is mimicked by some bacterial proteins and anti-vimentin antibodies occur in autoimmune cardiac disease, as rheumatic fever. In this work we studied vimentin distribution on LLC-MK2 cells infected with T. cruzi and anti-vimentin antibodies in sera from several clinical pictures of Chagas' disease or American Trypanosomiasis, in order to elucidate any vimentin involvement in the humoral response of this pathology. Objective: We standardized an indirect immunofluorescence assay (IFI) to determine sub cellular expression in either parasites and host cells, and ELISA to evaluate anti-vimentin antibodies in sera fron chagasic patients. Methods: We analyzed the distribution of vimentin in culture cells using indirect fluorescent assays, using as external controls anti-T. cruzi sera, derived from chronic infected patients for identification of the parasites in the same model. After infection and growth of T.cruzi amastigotes, those cells express larger amounts of vimentin, with heavy staining of cytoplasm outside the parasitophorous vacuole and some particle shadowing patterns, suggesting that vimentin are associated with cell cytoplasm. Anti-vimentin antibodies were present in most American trypanosomiasis samples, but notably, they are much more present in acute (76, 9%) or clinical defined syndromes, especially cardiac disease (87, 9%). Paradoxically, they were relatively infrequent in asymptomatic (25%) infected patients, which had a clearly positive serological reaction to parasite antigens, but had low frequency of anti-vimentin antibodies, similar to controls (2,5%). Conclusion: Our current data revealed that anti-vimentin antibodies induced during T. cruzi infection could be a marker of active disease in the host and its levels could also justify drug therapy in American Trypanosomiasis chronic infection, as a large group of asymptomatic patients would be submitted to treatment with frequent adverse reactions of the available drugs. Anti-vimentin antibodies could be a marker of cardiac muscle cell damage, appearing in American Trypanosomiasis patients during active muscle cell damage.


Resumo Fundamento: A Vimentina é uma proteína estrutural importante da célula, um componente dos filamentos celulares intermediários e imersa no citoplasma. Algumas proteínas bacterianas imitam a Vimentina e anticorpos anti-vimentina ocorrem em doenças cardíacas auto-imunes, como a febre reumática. Neste trabalho, estudamos a distribuição de vimentina em células LLC-MK2 infectadas com T. Cruzi e anticorpos anti-vimentina em soros de várias imagens clínicas da doença de Chagas ou tripanossomíases americanas, a fim de elucidar qualquer implicação da vimentina na resposta humoral desta patologia. Objetivo: padronizamos um teste de imunofluorescência indireta (IFI) para determinar a expressão subcelular em parasitas e células hospedeiras, e ELISA para testar anticorpos anti-vimentina em soros de pacientes chagásicos. Métodos: analisamos a distribuição de vimentina em células de cultura usando ensaios fluorescentes indiretos, utilizando como controles externos soros anti-T. Cruzi, derivados de pacientes com infecção crônica para a identificação de parasitas no mesmo modelo. Após a infecção e o crescimento de amastigotas de T. Cruzi, essas células expressam grandes quantidades de vimentina, com forte coloração do citoplasma fora da vacuola parasitófora e alguns padrões de sombreamento das partículas, sugerindo que a vimentina está associada ao citoplasma da célula. Os anticorpos anti-vimentina estavam presentes na maioria das amostras americanas de tripanossomíases, mas estão notavelmente mais presentes em síndromes agudas ou clinicamente definidas (76,9%), especialmente em doenças cardíacas (87,9%). Paradoxalmente, eram relativamente infrequentes em pacientes infectados assintomáticos (25%), que apresentavam uma reação sorológica claramente positiva aos antígenos parasitas, mas apresentavam baixa frequência de anticorpos anti-vimentina, semelhante aos controles (2,5%). Conclusão: Nossos dados atuais revelaram que os anticorpos anti-vimentina induzidos durante a infecção por T. Cruzi poderiam ser um marcador de doença ativa no hospedeiro e seus níveis também poderiam justificar o tratamento farmacológico em infecção crônica com tripanossomíase americana, uma vez que um grande grupo de pacientes assintomáticos seria submetido a tratamento com reações adversas frequentes aos medicamentos disponíveis. Os anticorpos anti-vimentina poderiam ser um marcador de danos nas células do músculo cardíaco, que aparece em pacientes com tripanossomíase americana durante o dano das células musculares ativas.


Subject(s)
Humans , Animals , Trypanosoma cruzi/immunology , Vimentin/immunology , Antibodies, Protozoan/immunology , Chagas Disease/immunology , Antigens, Protozoan/immunology , Reference Values , Enzyme-Linked Immunosorbent Assay/methods , Antibodies, Protozoan/analysis , Cells, Cultured , Analysis of Variance , Statistics, Nonparametric , Fluorescent Antibody Technique, Indirect/methods , Macaca mulatta , Antigens, Protozoan/analysis
4.
Mem. Inst. Oswaldo Cruz ; 112(12): 850-856, Dec. 2017. tab, graf
Article in English | LILACS | ID: biblio-1040565

ABSTRACT

BACKGROUND The surface of infected red blood cells (iRBCs) has been widely investigated because of the molecular complexity and pathogenesis mechanisms involved. Asymptomatic individuals are important in the field because they can perpetuate transmission as natural reservoirs and present a challenge for diagnosing malaria because of their low levels of circulating parasites. Recent studies of iRBC antibody recognition have shown that responses are quantitatively similar in symptomatic and asymptomatic infections, but no studies have characterised the plasmodial proteins targeted by this response. OBJECTIVES Our main objective was to identify Plasmodium falciparum proteins associated with iRBC ghosts recognised by antibodies in the sera of symptomatic and asymptomatic individuals in the Brazilian Amazon. METHODS We collected symptomatic and asymptomatic sera from patients residing in the Brazilian Amazon and P. falciparum iRBC ghosts to identify the proteins involved in natural antibody recognition by 2D-electrophoresis, western blotting, and high- resolution mass spectrometry. FINDINGS 2D gel-based immunoproteome analysis using symptomatic and asymptomatic sera identified 11 proteins with at least one unique peptide, such as chaperones HSP70-1 and HSP70-x, which likely are components of the secretion machinery/PTEX translocon. PfEMP1 is involved in antigenic variation in symptomatic infections and we found putative membrane proteins whose functions are unknown. MAIN FINDINGS Our results suggest a potential role of old and new proteins, such as antigenic variation proteins, iRBC remodelling, and membrane proteins, with no assigned functions related to the immune response against P. falciparum, providing insights into the pathogenesis, erythrocyte remodelling, and secretion machinery important for alternative diagnosis and/or malaria therapy.


Subject(s)
Humans , Plasmodium falciparum/immunology , Antibodies, Protozoan/genetics , Erythrocyte Membrane/parasitology , Antigens, Protozoan/genetics , Plasmodium falciparum/genetics , Mass Spectrometry , Antibodies, Protozoan/immunology , Electrophoresis, Gel, Two-Dimensional , Blotting, Western , Proteomics , Erythrocyte Membrane/immunology , Asymptomatic Infections , Antigens, Protozoan/immunology
5.
Rev. Soc. Bras. Med. Trop ; 49(4): 398-407, July-Aug. 2016. tab, graf
Article in English | LILACS | ID: lil-792794

ABSTRACT

Abstract: Visceral leishmaniasis (VL) is one of the most important tropical diseases worldwide. Although chemotherapy has been widely used to treat this disease, problems related to the development of parasite resistance and side effects associated with the compounds used have been noted. Hence, alternative approaches for VL control are desirable. Some methods, such as vector control and culling of infected dogs, are insufficiently effective, with the latter not ethically recommended. The development of vaccines to prevent VL is a feasible and desirable measure for disease control; for example, some vaccines designed to protect dogs against VL have recently been brought to market. These vaccines are based on the combination of parasite fractions or recombinant proteins with adjuvants that are able to induce cellular immune responses; however, their partial efficacy and the absence of a vaccine to protect against human leishmaniasis underline the need for characterization of new vaccine candidates. This review presents recent advances in control measures for VL based on vaccine development, describing extensively studied antigens, as well as new antigenic proteins recently identified using immuno-proteomic techniques.


Subject(s)
Humans , Animals , Dogs , Antibodies, Protozoan/immunology , Protozoan Vaccines/immunology , Leishmania/immunology , Leishmaniasis, Visceral/prevention & control , Antigens, Protozoan/immunology , Protozoan Proteins/immunology , Leishmania/classification
6.
Mem. Inst. Oswaldo Cruz ; 110(3): 445-451, 05/2015. graf
Article in English | LILACS | ID: lil-745966

ABSTRACT

Prevention of Trypanosoma cruzi infection in mammals likely depends on either prevention of the invading trypomastigotes from infecting host cells or the rapid recognition and killing of the newly infected cells by T. cruzi-specific T cells. We show here that multiple rounds of infection and cure (by drug therapy) fails to protect mice from reinfection, despite the generation of potent T cell responses. This disappointing result is similar to that obtained with many other vaccine protocols used in attempts to protect animals from T. cruzi infection. We have previously shown that immune recognition of T. cruzi infection is significantly delayed both at the systemic level and at the level of the infected host cell. The systemic delay appears to be the result of a stealth infection process that fails to trigger substantial innate recognition mechanisms while the delay at the cellular level is related to the immunodominance of highly variable gene family proteins, in particular those of the trans-sialidase family. Here we discuss how these previous studies and the new findings herein impact our thoughts on the potential of prophylactic vaccination to serve a productive role in the prevention of T. cruzi infection and Chagas disease.


Subject(s)
Animals , Female , Mice , Chagas Disease/immunology , Protozoan Vaccines/immunology , Trypanosoma cruzi/immunology , Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Chagas Disease/parasitology , Chagas Disease/prevention & control
7.
J. bras. nefrol ; 36(4): 430-436, Oct-Dec/2014. tab, graf
Article in Portuguese | LILACS | ID: lil-731139

ABSTRACT

Introdução: Atualmente, é descrita elevada prevalência de hipovitaminose D no Lúpus Eritematoso Sistêmico (LES), a qual se associa a algumas manifestações clínicas e maior atividade inflamatória. Objetivo: Avaliar a associação entre insuficiência de vitamina D com LES e marcadores inflamatórios. Métodos: Estudo transversal, tendo sido avaliados 45 pacientes com LES e 24 controles sem a doença. Níveis de 25-hidroxivitamina D [25(OH)D] menores que 30 ng/mL foram considerados insuficientes. A atividade da doença foi avaliada pelo Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Foram avaliados, ainda, proteína C reativa ultrassensível (PCRus) e interleucina-6 (IL-6) para verificação do status inflamatório. Para avaliação do envolvimento renal, foram realizados análise de elementos anormais e sedimentoscopia urinárias (EAS), hematúria e piúria quantitativas, proteinúria e depuração de creatinina em urina de 24 horas e anti-DNA de dupla hélice sérico. Resultados: A prevalência de insuficiência de 25(OH)D foi de 55% nos pacientes lúpicos e 8% nos participantes controles (p = 0,001). A mediana da 25(OH)D foi menor nos pacientes do que no grupo controle. Os pacientes com insuficiência de 25(OH)D apresentaram níveis mais elevados de IL-6 e maior prevalência de hematúria ao EAS. Não houve correlação entre vitamina D, nefrite lúpica e SLEDAI. Conclusão: Em nosso estudo, a insuficiência de vitamina D foi mais prevalente em pacientes com LES e se associou com níveis mais elevados de IL-6 e presença de hematúria. .


Introduction: Nowadays it is described a high prevalence of hypovitaminosis D in Systemic Lupus Erythematosus (SLE), which is associated with some clinical manifestations and increased inflammatory activity. Objective: To evaluate the association between vitamin D insufficiency with SLE and inflammatory markers. Methods: Cross-sectional study, in which have been evaluated 45 SLE patients and 24 controls without the disease. Levels of 25-hydroxyvitamin D [25(OH) D] less than 30 ng/mL were considered inadequate. Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). High sensitivity C reactive protein (hsCRP) and interleukin-6 (IL-6) were evaluated for verification of the inflammatory status. For assessment of renal involvement, analysis of abnormal elements and urinay sediment (AES), quantitative hematuria and pyuria, proteinuria and creatinine clearance in 24-hour urine and serum anti-double stranded DNA were performed. Results: The prevalence of 25(OH)D insufficiency was 55% in SLE patients and 8% in the controls participants (p = 0.001). The median of 25(OH)D was lower in patients than in controls. Patients with insufficient 25(OH)D had higher levels of IL-6 and higher prevalence of hematuria in the AES. There was no correlation between vitamin D and SLEDAI or lupus nephritis. Conclusion: In our study, vitamin D deficiency was more prevalent in patients with SLE and was associated with higher levels of IL-6 and hematuria. .


Subject(s)
Animals , Rabbits , Antigens, Protozoan/immunology , Membrane Proteins/immunology , Protein Folding , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Sarcosine/analogs & derivatives , Antibodies, Protozoan/immunology , Antigens, Protozoan/biosynthesis , Antigens, Protozoan/genetics , Antigens, Protozoan/isolation & purification , Cysteine , Chromatography, Affinity/methods , Chromatography, Ion Exchange/methods , Edetic Acid , Endotoxins , Escherichia coli , Fermentation , Gene Expression , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Nickel , Protein Structure, Tertiary , Plasmodium falciparum/genetics , Protozoan Proteins/biosynthesis , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Sucrose
8.
Mem. Inst. Oswaldo Cruz ; 109(5): 598-601, 19/08/2014. tab, graf
Article in English | LILACS | ID: lil-720422

ABSTRACT

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms. .


Subject(s)
Animals , Humans , Antibodies, Protozoan/immunology , /immunology , Erythrocytes/parasitology , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Asymptomatic Infections , CHO Cells , Cricetulus , Cell Adhesion/genetics , Cell Adhesion/immunology , Erythrocytes/immunology , Flow Cytometry , Gene Expression Profiling , Malaria, Falciparum/parasitology , Protozoan Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction
10.
J. pediatr. (Rio J.) ; 90(4): 363-369, Jul-Aug/2014. tab, graf
Article in English | LILACS | ID: lil-720890

ABSTRACT

OBJECTIVES: to investigate the rate of positivity for immunoglobulin M anti-Toxoplasma gondii (Toxo-IgM) in newborns with congenital toxoplasmosis, and the age when these antibodies become negative. METHODS: patients with congenital toxoplasmosis who started monitoring in a congenital infection clinic between 1998 and 2009 were included. Inclusion criteria were routine maternal or neonatal serological screening; diagnostic confirmation by persistence of immunoglobulin G anti-Toxoplasma gondii at age > 12 months, and Toxo-IgM screening in the neonatal period. To calculate the frequency of positive Toxo-IgM, cases detected by neonatal screening were excluded. For the study of the age when Toxo-IgM results became negative, patients with negative Toxo-IgM since birth and those in whom it was not possible to identify the month when the negative result was achieved were excluded. RESULTS: among the 28 patients identified through maternal screening, 23 newborns had positive Toxo-IgM (82.1%, 95% CI: 64.7-93.1%). When adding the 37 patients identified by neonatal screening, Toxo-IgM was positive in the first month of life in 60 patients, and it was possible to identify when the result became negative in 51 of them. In 19.6% of patients, these antibodies were already negative at 30 days of life; and in 54.9%, at 90 days. Among the 65 patients included in the study, 40 (61.5%) had some clinical alteration. CONCLUSIONS: even with high sensitivity methods, newborns with congenital toxoplasmosis can have negative Toxo-IgM at birth. In those who have these antibodies, the positive period may be quite short. It is important not to interrupt the monitoring of infants with suspected congenital toxoplasmosis simply because they present a negative Toxo-IgM result. .


OBJETIVOS: investigar a taxa de positividade para imunoglobulina M anti-Toxoplasma gondii (Toxo-IgM) em recém-nascidos com toxoplasmose congênita, e a idade de negativação desses anticorpos. MÉTODOS: foram incluídos pacientes com toxoplasmose congênita que iniciaram acompanhamento em uma clínica de infecções congênitas entre 1998 e 2009. Os critérios de inclusão foram toxoplasmose congênita detectada por triagem sorológica materna ou neonatal de rotina, confirmação diagnóstica por persistência de imunoglobulina G anti-Toxoplasma gondii com >12 meses e pesquisa de Toxo-IgM no período neonatal. Para o cálculo da frequência de positividade da Toxo-IgM foram excluídos os detectados por triagem neonatal. Para o estudo da época de negativação da Toxo-IgM foram excluídos os pacientes com Toxo-IgM negativa desde o nascimento e aqueles em que não foi possível identificar o mês da negativação. RESULTADOS: entre 28 pacientes detectados por triagem materna, 23 recém-nascidos tiveram Toxo-IgM positiva (82,1%, IC 95%: 64,7-93,1%). Somando-se os 37 pacientes detectados por triagem neonatal, a Toxo-IgM foi positiva no primeiro mês de vida em 60 pacientes e em 51 foi possível identificar a época de negativação. Em 19,6% dos pacientes esses anticorpos já eram negativos aos 30 dias e em 54,9% aos 90 dias. Entre os 65 pacientes incluídos no estudo, 40 (61,5%) apresentaram alguma alteração clínica. CONCLUSÕES: mesmo com métodos de alta sensibilidade, recém-nascidos com toxoplasmose congênita podem ter Toxo-IgM negativa ao nascer. Nos que apresentam esses anticorpos, o período de positividade pode ser bastante fugaz. É importante não interromper o monitoramento dos lactentes com suspeita de toxoplasmose ...


Subject(s)
Female , Humans , Infant , Infant, Newborn , Pregnancy , Antibodies, Protozoan/immunology , Immunoglobulin M/analysis , Toxoplasmosis, Congenital/immunology , Brazil , Cohort Studies , Fluorescent Antibody Technique/methods , Neonatal Screening/methods , Pregnancy Complications, Parasitic/immunology , Sensitivity and Specificity , Time Factors
11.
Rev. Soc. Bras. Med. Trop ; 47(1): 105-107, Jan-Feb/2014. tab, graf
Article in English | LILACS | ID: lil-703159

ABSTRACT

Introduction: The aim of this study was to evaluate the serological cross-reactivity between Leishmania sp. and other canine pathogens. Methods: Positive serum samples for Ehrlichia canis, Babesia canis, Toxoplasma gondii, Neospora caninum and Trypanosoma cruzi were tested using three serological methods enzyme linked immunosorbent assay (ELISA), indirect immunofluorescent antibody test (IFAT) and Kalazar Detect™, for canine visceral leishmaniasis. Results: Of the 57 dog samples tested, 24 (42.1%) tested positive using one of the three serological methods: 10/57 (17.5%) for ELISA, 11/57 (19.3%) for IFAT and 3/57 (5.3%) for Kalazar Detect™. Conclusions: Our results demonstrated that the presence of other infectious agents may lead to cross-reactivity on leishmaniasis serological tests. .


Subject(s)
Animals , Dogs , Antibodies, Bacterial/blood , Antibodies, Protozoan/blood , Dog Diseases/parasitology , Leishmaniasis, Visceral/parasitology , Antibodies, Bacterial/immunology , Antibodies, Protozoan/immunology , Babesia/immunology , Cross Reactions , Ehrlichia canis/immunology , Enzyme-Linked Immunosorbent Assay/veterinary , Fluorescent Antibody Technique, Indirect/veterinary , Leishmania infantum/immunology , Neospora/immunology , Toxoplasma/immunology , Trypanosoma cruzi/immunology
12.
Article in English | WPRIM | ID: wpr-70516

ABSTRACT

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.


Subject(s)
Animals , Antibodies, Protozoan/immunology , Antibody Affinity , Antigens, Protozoan/chemistry , Gene Expression , Immunoglobulin G/blood , Mice, Inbred BALB C , Recombinant Proteins/chemistry , Serologic Tests/methods , Solubility , Toxoplasma/genetics , Toxoplasmosis/diagnosis
13.
Article in English | WPRIM | ID: wpr-7398

ABSTRACT

Toxoplasma gondii is an obligate intracellular protozoan that is distributed worldwide. Recently, several tests for avidity of Toxoplasma IgG antibodies have been introduced to help discriminate between recently acquired and distant infections. The study was conducted in Jawaharlal Nehru Medical College and Hospital, India from February 2011 to September 2012. Serum specimens were subjected to Toxoplasma IgM ELISA and IgG avidity ELISA test. Out of 48 patients with abortions, 17 (35.4%) were positive for IgM ELISA, and 8 (16.6%) had low IgG avidity antibodies. Out of 48 patients with other obstetric problems, 23 (47.9%) were positive for IgM ELISA, and 17 (35.4%) had low IgG avidity antibodies. Combining both groups on avidity test, only 25 of 40 (62.5%) IgM-positive women had low-avidity IgG antibodies suggesting a recent T. gondii infection in these women. More importantly, 15 (37.5%) of the IgM-positive women had high-avidity antibodies suggesting that the infection was acquired before gestation The relation of IgM seropositivity with the following risk factors was not found to be statistically significant; contact with cats (0.13), non-vegetarian food habits (0.05), and low socio-economic status (0.49). While, for IgG avidity ELISA, only contact with cats (0.01) was significantly associated with seropositivity. All other risk factors have P-values of >0.05 (not significant). IgG avidity test when used in combination with IgM test was a valuable assay for diagnosis of ongoing or recently acquired T. gondii infection in India.


Subject(s)
Abortion, Spontaneous/immunology , Adolescent , Adult , Animals , Antibodies, Protozoan/immunology , Antibody Affinity , Cats , Enzyme-Linked Immunosorbent Assay , Female , Food Contamination , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , India/epidemiology , Risk Factors , Seroepidemiologic Studies , Toxoplasma/immunology , Toxoplasmosis/epidemiology , Young Adult
14.
Mem. Inst. Oswaldo Cruz ; 108(5): 616-622, ago. 2013. tab, graf
Article in English | LILACS | ID: lil-680767

ABSTRACT

The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.


Subject(s)
Animals , Mice , Antibodies, Monoclonal/immunology , Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Giardia lamblia/immunology , Protozoan Proteins/immunology , Blotting, Western , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Giardiasis/immunology , Giardiasis/parasitology
15.
Mem. Inst. Oswaldo Cruz ; 108(1): 98-105, Feb. 2013. ilus, graf, tab
Article in English | LILACS | ID: lil-666051

ABSTRACT

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.


Subject(s)
Animals , Female , Humans , Mice , Antibodies, Protozoan/blood , Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Cell Adhesion Molecules/immunology , Malaria, Falciparum/immunology , Malaria, Vivax/immunology , Plasmodium falciparum/immunology , Plasmodium vivax/immunology , Protozoan Proteins/immunology , Brazil , Carrier State , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Erythrocytes/parasitology , Mice, Inbred BALB C , Malaria, Falciparum/parasitology , Malaria, Vivax/parasitology
16.
Mem. Inst. Oswaldo Cruz ; 107(3): 342-347, May 2012. graf, tab
Article in English | LILACS | ID: lil-624015

ABSTRACT

The aim of this work was to evaluate the utility of ELISA-based testing of total IgG (IgGt) antibodies and its subclasses (IgG1, IgG2, IgG3 and IgG4) against soluble (STAg) and recombinant (rSAG1 and rMIC3) antigens of Toxoplasma gondii for diagnosing congenital toxoplasmosis. Sera from 217 newborns initially testing positive for specific IgM in filter paper dried blood spots were tested for specific IgM and IgG by ELFA-VIDAS®. Congenital toxoplasmosis was confirmed in 175 and ruled out in 42 infants. The validity of the ELISA tests was determined using the persistence of IgG antibodies (ELFA-VIDAS® kit) at the end of 12 months, which is considered the reference test for the diagnosis of congenital toxoplasmosis. The frequency of positivity with IgGt against STAg, rSAG1 and rMIC3 was found in 97.2%, 96.3% and 80.2%, respectively, of the newborns with confirmed congenital toxoplasmosis. IgG1 reacted with all three antigens, while IgG3 and IgG4 reacted preferentially with rMIC3. Higher mean values of reactivity (sample optical density/cut-off) were found for all subclasses when using rMIC3. All of the antigens showed high sensitivity and low specificity in detecting anti-T. gondii IgGt and IgG1 and low sensitivity and high specificity in detecting IgG3 and IgG4. In conclusion, the combined detection of IgG antibody subclasses against recombinant toxoplasmic antigens may be useful for the early diagnosis of congenital toxoplasmosis.


Subject(s)
Humans , Infant, Newborn , Antibodies, Protozoan/blood , Antigens, Protozoan/immunology , Immunoglobulin G/immunology , Toxoplasma/immunology , Toxoplasmosis, Congenital/diagnosis , Antibodies, Protozoan/immunology , Early Diagnosis , Enzyme-Linked Immunosorbent Assay , Immunoglobulin G/blood , Predictive Value of Tests , Prospective Studies , Reproducibility of Results , Recombinant Proteins/blood , Sensitivity and Specificity , Toxoplasmosis, Congenital/immunology
17.
Rev. Soc. Bras. Med. Trop ; 45(1): 35-44, Jan.-Feb. 2012. ilus
Article in English | LILACS | ID: lil-614906

ABSTRACT

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100 percent sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.


INTRODUÇÃO: O objetivo foi desenvolver um método sorológico in-house de alta especificidade e sensibilidade para diagnosticar e monitorar a morbidade da doença de Chagas. MÉTODOS: Para tal, a reatividade sorológica de IgG e subclasses foi testada em soros de pacientes chagásicos de Berilo, Vale do Jequitinhonha/MG/Brasil. A reatividade sorológica foi também avaliada em amostras de pacientes com outras doenças infecto-contagiosas relevantes, incluindo o HIV, vírus da hepatite C (VHC), sífilis (SYP), leishmaniose visceral (LV), leishmaniose tegumentar americana (LTA) e controles não infectados (NI) para verificar o desempenho do método. Outras análises foram feitas para avaliar a aplicabilidade desta metodologia no monitoramento da morbidade da doença de Chagas. Com este propósito os pacientes com doença de Chagas foram anteriormente classificados em três grupos: indeterminados (IND), cardíacos (CARD) e digestivos/mistos (DIG/Mis) conforme seu estado clínico. RESULTADOS: A análise da reatividade sorológica de IgG total na diluição 1:40 mostrou ser uma abordagem importante no diagnóstico da doença de Chagas (100 por cento de sensibilidade e especificidade e ausência de reação cruzada com as demais infecções). A análise das subclasses de IgG mostrou reação cruzada principalmente com NI, LV e LTA em todas as diluições. O grupo IND apresentou a maior reatividade para IgG3 e o grupo DIG/Mis apresentou nível mais elevado de IgG se comparados aos grupos IND e CARD. CONCLUSÕES: Estes achados demonstram que o método de ELISA in-house apresenta uma promissora aplicabilidade no diagnóstico diferencial e na avaliação da morbidade da doença de Chagas.


Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Antibodies, Protozoan/immunology , Chagas Disease/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/immunology , Trypanosoma cruzi/immunology , Antigen-Antibody Reactions , Antibodies, Protozoan/blood , Chagas Cardiomyopathy/diagnosis , Chagas Disease/complications , Diagnosis, Differential , Immunoglobulin G/blood , Sensitivity and Specificity
18.
Article in English | WPRIM | ID: wpr-223074

ABSTRACT

Congenital Neospora caninum infection was diagnosed in two Saanen goat kids from two distinct herds with a history of abortion and weak newborn goat kids in the Southern region of the State of Minas Gerais, Brazil. The first kid was weak at birth, had difficulty to rise and was unable to nurse. Gross lesions of porencephaly and hydrocephalus ex vacuo were seen. Multifocal necrosis, gliosis and non-supurative encephalitis were observed in the brain. Several parasitic cysts with a thick wall that reacted strongly only with polyclonal antiserum to Neospora caninum were seen in the cerebral cortex, brain stem and cerebellum. The second kid was born from a Neospora caninum seropositive mother that aborted in the last pregnancy. It was born without clinical signs. The diagnosis of neosporosis was based on antibody titer of 1:800 to N. caninum by indirect fluorescence antibody test obtained from blood collected before the goat kid ingested the colostrum and Neospora caninum DNA was detected by polymerase chain reaction and sequenced from placenta. This is the first report of neosporosis in goats in the southeast region of Brazil.


Subject(s)
Animals , Antibodies, Protozoan/immunology , Brazil , Coccidiosis/congenital , Female , Goat Diseases/congenital , Goats , Molecular Sequence Data , Neospora/genetics , Pregnancy
19.
Rev. Inst. Med. Trop. Säo Paulo ; 53(5): 283-289, Sept.-Oct. 2011. ilus, tab
Article in English | LILACS | ID: lil-602365

ABSTRACT

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.


A imunofluorescência indireta é o método recomendado para o diagnóstico de leishmaniose visceral em cães, entretanto, a acurácia dessa técnica é baixa e seu uso em grande escala é limitado. Uma vez que o ELISA não apresenta essas limitações, essa técnica poderia ser uma opção para a detecção de IgG ou subclasses IgG1 e IgG2 específicas. A ehrlichiose canina é um importante diagnóstico diferencial de Leishmaniose Visceral Americana (LVA). O presente estudo comparou o ELISA usando antígenos de Leishmania chagasi e Leishmania braziliensis para a detecção de IgG e subclasses anti-Leishmania em amostras de soro de 37 cães naturalmente infectados com L. chagasi (LVA) e em amostras de quatro cães co-infectados (CI). A ocorrência de reatividade cruzada foi investigada em amostras de soro controle de 17 animais saudáveis (HC) e 35 de infectados por Ehrlichia canis (EC). A média de densidade óptica obtida para a detecção de IgG foi significantemente maior quando o antígeno de L. chagasi foi usado e também mais elevada no subgrupo LVs (sintomático) quando comparado ao subgrupo LVa (assintomático). A correlação entre IgG e IgG1 foi baixa. O presente resultado sugere que ELISA IgG empregando antígeno homólogo, produz os melhores resultados, permitindo o diagnóstico de infecção assintomática por L. chagasi e a discriminação entre casos de LVA e ehrlichiose em cães.


Subject(s)
Animals , Dogs , Antibodies, Protozoan/blood , Antigens, Protozoan/immunology , Dog Diseases/diagnosis , Immunoglobulin G/immunology , Leishmaniasis, Visceral/veterinary , Antibodies, Protozoan/immunology , Case-Control Studies , Dog Diseases/immunology , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Immunoglobulin G/blood , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/immunology , Predictive Value of Tests , Sensitivity and Specificity
20.
Invest. clín ; 52(3): 216-229, sep. 2011. ilus, tab
Article in Spanish | LILACS | ID: lil-659212

ABSTRACT

En la leishmaniosis experimental, la función de los anticuerpos no está completamente clara, ya que algunos autores consideran que dichas proteínas no participan en la protección contra la infección; sin embargo, estudios histopatológicos en lesiones con leishmaniosis humana y experimental, muestran infiltrados de células plasmáticas positivas para IgA y secreción de IgM, IgG e IgA que podrían mediar la formación de complejos inmunológicos con antígenos parasitarios o propios, favoreciendo la necrosis lo que conlleva a la eliminación del parásito. En este trabajo se determinó si la IgA sérica en el modelo murino posee reactividad específica contra antígenos de Leishmania (Leishmania) mexicana de utilidad diagnóstica. Para ello, se utilizaron ratones susceptibles y resistentes a leishmaniosis cutánea, demostrándose mediante ELISA indirecta que la IgA sérica de ratones susceptibles es elevada en comparación con la producida por ratones resistentes. Aunque otros estudios en modelos murinos demuestran que la IgG sérica de ratones infectados con L. (L) mexicana presenta reactividad cruzada con antígenos parasitarios no relacionados obtenidos de Trypanosoma cruzi, al analizar la especificidad de IgA por antígenos de L. (L) mexicana y T. cruzi, mediante Western Blot, se demostró que la IgA sérica de ratones infectados con T. cruzi también reaccionan con antígenos de L. (L) mexicana, estos hallazgos sugieren que la IgA puede ser útil para el manejo clínico y pronóstico de la enfermedad.


In experimental leishmaniasis, the role of antibodies is not entirely clear, as some authors consider that these proteins are not involved in protection against infection. However, histopathological studies in human and experimental leishmaniasis lesions, show plasma cell infiltrates positive for IgA and secretion of IgM, IgG and IgA could mediate the formation of immune complexes with parasite antigens or self components, favoring necrosis leading to the elimination of the parasite. In this study, we determined if the serum IgA in the murine model has specific reactivity against antigens of Leishmania (Leishmania) mexicana of diagnostic utility. To do this, we used mice either susceptible or resistant to cutaneous leishmaniasis, and demonstrated by indirect ELISA that serum IgA is elevated in susceptible mice compared with that produced by resistant mice. Although other studies in murine models show that the serum IgG from mice infected with L. (L) mexicana present cross reactivity with unrelated parasite antigens derived from Trypanosoma cruzi, the analysis of the specificity of IgA by antigens of L. (L) mexicana and T. cruzi, by Western Blot, showed that the IgA serum of mice infected with T. cruzi reacts too with antigens of L. (L) mexicana. These findings suggest that IgA may be useful for the clinical management and prognosis of the disease.


Subject(s)
Animals , Female , Mice , Antibodies, Protozoan/blood , Antigens, Protozoan/immunology , Immunoglobulin A/blood , Leishmania mexicana/immunology , Leishmaniasis, Cutaneous/immunology , Antibody Specificity , Antibodies, Protozoan/immunology , Antigens, Protozoan/isolation & purification , Cross Reactions , Disease Resistance , Disease Susceptibility , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Immunoglobulin A/immunology , Leishmania mexicana/growth & development , Leishmaniasis, Cutaneous/blood , Mice, Inbred BALB C , Molecular Weight , Trypanosoma cruzi/immunology
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