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Human visceral leishmaniasis (HVL) is a neglected disease that occurs in 98 countries on five continents, and it is endemic in tropical and subtropical regions. In South America, the etiological agent of HVL is Leishmania infantum (syn. Leishmania chagasi), mainly transmitted through the bite of an infected sandfly female from the genus Lutzomyia. In American HVL endemic areas, the occurrence of asymptomatic infection is common, which contributes to the possibility of L. infantum transmission during a blood transfusion. To know the prevalence of L. infantum asymptomatic infection in blood donors from the microregion of Adamantina, we investigated 324 peripheral blood samples from donors through immunofluorescence (IFAT) and PCR-RFLP techniques. Seven blood samples (2.16%) tested positive for Leishmania by IFAT, and from those, six presented positive results by PCR (85.71%), which were later identified as L. infantum by RFLP. The presence of L. infantum in the peripheral blood of blood donors supported the hypothesis of transmission by blood transfusion and points to the need to include tests for visceral leishmaniasis in blood bank screening tests and pre-storage measures, especially in endemic areas to prevent the exponential increase of HVL by blood transfusion.
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Leishmania infantum , Leishmaniose Visceral , Psychodidae , Animais , Humanos , Feminino , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Doadores de Sangue , Infecções Assintomáticas , Reação em Cadeia da PolimeraseRESUMO
The species of the genus Leishmania are protozoa that are widely distributed from Asia to the Americas, affecting humans and wild and domestic animals. Little is known about infection by Leishmania in bats in the state of Maranhão, Brazil. The objective of this study was to investigate the presence of Leishmania in bats in Maranhão. Blood samples were collected from bat species for parasitological diagnosis. Samples of spleen and liver were collected for molecular analysis. All the blood cultures were negative. In two blood smears, organisms similar to amastigotes of Leishmania sp. were detected. Of the 116 samples, two spleen samples were positive and showed similarity to Leishmania infantum. Therefore, further studies are needed to elucidate whether bats take part in the epidemiological chain of leishmaniasis.
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Quirópteros , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Humanos , Animais , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/veterinária , Leishmaniose Visceral/parasitologia , Brasil/epidemiologia , Leishmaniose/epidemiologia , Leishmaniose/veterináriaRESUMO
Visceral leishmaniasis (VL) is an opportunistic disease in immunosuppressed individuals, who may present severe clinical conditions, such as the ones described in this patient. She lived in an endemic region for VL, and was possibly infected with L. (L.) infantum chagasi through the bite of a contaminated sand fly. This initial infection has triggered a pemphigus vulgaris condition by immunogenic proteins present in the mosquito's saliva. The immunosuppression caused by the use of high doses of corticosteroids to control the disease promoted a severe VL condition, with hepatosplenomegaly, thrombocytopenia and hemorrhages, requiring hospitalization and the onset of a subsequent SARS-CoV-2 infection. Due to the intensity of clinical manifestations related to VL, aggravated by COVID-19, she died two days after admission to the Clinical Hospital of Marilia Medical School (HC-Famema).
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COVID-19 , Coinfecção , Leishmaniose Visceral , Psychodidae , Animais , COVID-19/complicações , Feminino , Humanos , Leishmaniose Visceral/complicações , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/tratamento farmacológico , SARS-CoV-2RESUMO
The Santo André Regional Center from Adolfo Lutz Institute evaluated 91 537 samples by reverse transcription-polymerase chain reaction (RT-PCR) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from March 2020 to April 2021. The age, sex, and race of patients from three cities in southeastern Brazil, namely São Bernardo do Campo (SBC), Diadema, and Mauá were assessed in association to the rate of positive results using generalized linear models. Circulating lineages were obtained from GISAID and intralineage genetic variation was investigated employing Lasergene software. A declining number of reported cases around October to November 2020 separate two epidemic waves in the three cities. Mauá differed by the highest positive RT-PCR scores in January and February. GISAID classification of 38 SARS-CoV-2 complete genomic sequences showed the circulation of lineages P.1, B.1.1.28, P.2, B.1.1.332; P.1, P.2, B.1.1.28, B.1.1.33; and P.1, P.2 in SBC, Diadema and Mauá, respectively. Intralineage variation revealed a significant amino-acid substitution in the ORF3a encoding protein (A33S) present in four out of six (67%) P.1 Mauá isolates. As ORF3a encodes a nonselective Ca2+ permeable cation channel, supposed to interfere in airway homeostasis, specific mutations could increase its pathogenic effect resulting in a higher number of symptomatic individuals explaining why the second wave was more intense in Mauá city.
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COVID-19 , SARS-CoV-2 , Brasil/epidemiologia , COVID-19/epidemiologia , Cidades/epidemiologia , Humanos , Fatores de Risco , SARS-CoV-2/genéticaRESUMO
Until mass vaccination befalls, control of the new betacoronavirus-associated severe acute respiratory syndrome pandemic (SARS-CoV-2) is based on decreasing virus circulation by social distancing and blocking transmission foci after diagnosis. Globally adopted SARS-CoV-2 diagnostic criteria embrace viral RNA detection by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) on nasopharynx secretions, which requires healthcare facilities and specialized personnel for sample collection. To develop an alternative protocol, hydrophilic cotton as the material and saliva as the source for biological sample collection in qRT-PCR/RT-endpoint-PCR SARS-CoV-2 diagnostic methods prepared with local consumables were evaluated using 99 archived nasopharynx samples previously diagnosed as positive for SARS-CoV-2 and 111 prospective saliva samples pared with nasopharynx samples from patients attending the local reference ABC Medical School diagnostic laboratory. The kappa agreement coefficient between the SARS-CoV-2 qRT-PCR and RT-endpoint-PCR was k = 0.97 (95 % CI 0.92-1.00) and k = 0.90 (95 % CI 0.81-0.99), respectively, on SARS-CoV-2-positive archived samples, with the initial qRT-PCR CT under 25. The agreement coefficient of the SARS-CoV-2 alternative saliva diagnostic protocol, when used to test the paired nasopharynx samples, was k = 0.79 (95 % CI 0.56-1,00). These data support that the SARS-CoV-2 diagnostic assay based on self-collected saliva on cotton represents an alternative protocol for mass diagnosis and epidemiological studies in low-income regions.
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COVID-19 , SARS-CoV-2 , Humanos , Nasofaringe , Pandemias , Estudos Prospectivos , RNA Viral/genética , Saliva , Manejo de EspécimesRESUMO
ABSTRACT Visceral leishmaniasis (VL) is an opportunistic disease in immunosuppressed individuals, who may present severe clinical conditions, such as the ones described in this patient. She lived in an endemic region for VL, and was possibly infected with L. (L.) infantum chagasi through the bite of a contaminated sand fly. This initial infection has triggered a pemphigus vulgaris condition by immunogenic proteins present in the mosquito's saliva. The immunosuppression caused by the use of high doses of corticosteroids to control the disease promoted a severe VL condition, with hepatosplenomegaly, thrombocytopenia and hemorrhages, requiring hospitalization and the onset of a subsequent SARS-CoV-2 infection. Due to the intensity of clinical manifestations related to VL, aggravated by COVID-19, she died two days after admission to the Clinical Hospital of Marilia Medical School (HC-Famema).
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Trypanosoma cruzi, the etiological agent for Chagas disease, is widely distributed in the Americas. Its hosts are humans and wild and domestic mammals, and its vectors are triatomine insects. Studies have indicated that domestic dogs are sentinel animals in the epidemiology of Chagas disease in endemic regions, including states in the Legal Amazon region of Brazil. In São Luís, the capital of Maranhão, a non-endemic state, the existence of a domestic cycle involving domestic rats has been proven, along with a wild cycle maintained by didelphids. However, no studies on T. cruzi infection in domestic animals in this locality have been conducted. The aim of this study was to investigate occurrence of T. cruzi in dogs living in the Itaqui Bacanga district of São Luís, Maranhão, by means of serological and molecular tests. Blood samples were obtained from 330 dogs and structured epidemiological questionnaires were applied to their keepers. These samples were used in the indirect immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Fisher's exact test was used for statistical calculations with the aim of identifying risk factors. Out of the 330 animals, 105 (31.8%) were reactive in IFAT, 46 (13.0%) in ELISA and 20 (6.0%) in both serological tests. The results were not significant (p > 0.05) when submitted to statistical analysis for the studied variables. From PCR, 58 samples (17.5%) were found to be positive and, of these, one (0.3%) showed similarity to T. cruzi after sequencing. These data demonstrate that dogs were exposed to and infected by T. cruzi. Thus, they can be considered sentinel animals for Chagas disease in the locality studied, which signals that there is a need for epidemiological surveillance actions.
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Doença de Chagas , Doenças do Cão , Trypanosoma cruzi , Animais , Brasil/epidemiologia , Doença de Chagas/diagnóstico , Doença de Chagas/epidemiologia , Doença de Chagas/veterinária , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Cães , Mamíferos , Estudos Soroepidemiológicos , Trypanosoma cruzi/genéticaRESUMO
RESUMO A vigilância da qualidade dos esgotos sanitários pode representar uma ferramenta complementar para monitoramento de doenças infecciosas e prevenção de surtos epidêmicos, especialmente quando a capacidade para testes clínicos é limitada. Dessa maneira, o presente estudo descreve o detalhamento técnico de um método de baixo custo para a concentração e extração de ácidos nucleicos de amostras de esgoto sanitário como etapa prévia para a detecção de vírus e outros agentes patogênicos. Para validar a metodologia proposta, após as etapas de concentração e extração, analisaram-se a presença do ácido ribonucleico do SARS-CoV-2 (COVID-19) nas amostras, por meio de reação em cadeia da polimerase em tempo real. O ácido ribonucleico do vírus foi detectado em 80% das amostras de esgoto sanitário analisadas, comprovando o êxito do procedimento metodológico adotado. A detecção precoce de um patógeno associado ao trabalho de equipes multidisciplinares possibilita a prática da vigilância epidemiológica, que auxilia na tomada de decisões na Saúde Única — união indissociável entre a saúde animal, humana e ambiental.
ABSTRACT Sewage quality surveillance can represent a complementary tool for monitoring infectious diseases and preventing epidemic outbreaks, especially when the capacity for clinical testing is limited. Thus, the present study describes the technical details of a low-cost method for concentrating and extracting nucleic acids from sewage samples, as a preliminary step for the detection of viruses and other pathogens. To validate the proposed methodology, after the concentration and extraction steps, the presence of the SARS coronavirus-2 (COVID-19) in the samples was analyzed using real-time polymerase chain reaction. The virus' ribonucleic acid was detected in 80% of the sewage samples analyzed, proving the success of the methodological procedure adopted. The early detection of a pathogen associated with the work of multidisciplinary teams allows the practice of epidemiological surveillance, which assists in making decisions about One Health — an inseparable union between animal, human, and environmental health.
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INTRODUCTION: With the arrival of the SARS-CoV-2 (Coronavirus 2 of severe acute respiratory syndrome) pandemic in Brazil, especially in the city of São Paulo, there was a need to apply social isolation policies associated with testing, covering all municipalities. The Clinical Analysis Laboratory of Centro Universitário FMABC was one of the first laboratories to receive certification and qualification to perform RT-PCR (reverse transcriptase reaction followed by polymerase chain reaction) tests in the metropolitan region of São Paulo OBJECTIVE: Aim to analyze the influence of adopting social isolation on the incidence of positivity in COVID-19 tests in the metropolitan region of São Paulo, Brazil METHODS: a descriptive study carried out from March to May 2020, epidemiological data were collected from each unit served and organized by the data controllership team of the Clinical Analysis Laboratory of FMABC. Epidemiological, demographic, and laboratory data were extracted from the Matrix® outpatient data management system. Clinically suspected cases and confirmed by laboratory tests (RT-PCR and serological tests) were entered. The tests were divided into serological tests using the RT-PCR molecular test, on samples of nasopharyngeal mucosal scrapings collected with sterile Swab RESULTS: It were evaluated PCR test and antibody presence (IgA, IgM and IgG) in blood samples of 16.297 patients. 22.718 tests were performed for the diagnosis of COVID-19, both RT-PCR (10.410 tests) and serological tests to detect anti-SARS-CoV-2 antibodies, IgA, IgM and IgG, a total of 16.297 patients were assessed, 63% women and 37% men. It was observed that the social isolation policies adopted during this period contained the massive expansion of contamination, at least while the social isolation rates were above 55% CONCLUSION: The data of this study demonstrated the effectiveness of social isolation in containing the positive contamination of SARS-CoV-2 in the metropolitan region of São Paulo, at least for the first three months
INTRODUÇÃO: com a chegada da pandemia de SARS-CoV-2 (Coronavirus 2 da síndrome respiratória aguda grave) ao Brasil, especialmente na cidade de São Paulo, houve a necessidade de aplicar medidas de distanciamento social associado a testagem, que abrangesse todos os municípios. A região metropolitana de São Paulo compreende 39 municípios e possui uma rede de laboratórios habilitados a realizar a testagem para a detecção do coronavírus, tanto testes sorológicos quanto moleculares. O Laboratório de Análises Clínicas do Centro Universitário ABC/FMABC foi um dos primeiros laboratórios a receber a certificação e habilitação para realizar os testes RT-PCR (reação da transcriptase reversa seguida pela reação em cadeia da polimerase) na região metropolitana de São Paulo OBJETIVO: analisar a influência da adoção do isolamento social na incidência de positividade nos testes de COVID-19 em região metropolitana de São Paulo, Brasil MÉTODO: estudo descritivo realizado no período de março a maio de 2020, os dados epidemiológicos foram coletados de cada unidade atendida e organizada pela equipe de controladoria de dados do Laboratório de Análises Clínicas da FMABC. Os dados epidemiológicos, demográficos e laboratoriais foram extraídos do sistema Matrix® de gerenciamento de dados ambulatoriais. Foram inseridos os casos clinicamente suspeitos e confirmados por testes de laboratório (RT-PCR e testes sorológicos). Os testes foram divididos em testes sorológicos no teste molecular RT-PCR, em amostras de raspado de mucosa nasofaríngea coletada com Swab estéril RESULTADOS: foram avaliados o teste de RT-PCR e a presença de anticorpos (IgA, IgM e IgG) em amostras de sangue de 16.297 pacientes. Foram realizados 22.718 testes para o diagnóstico de COVID-19, tanto RT-PCR (10.410 testes), quanto testes sorológicos para detecção de anticorpos anti-SARS-CoV-2, IgA, IgM e IgG, um total de 16.297 pacientes foram avaliados, 63% mulheres e 37% homens. Observou-se que as políticas de isolamento social adotadas nesse período continham a expansão massiva da contaminação, pelo menos enquanto as taxas de isolamento social eram superiores a 55% CONCLUSÃO: nossos dados demonstraram a efetividade do isolamento social na retenção da positividade da contaminação do SARS-CoV-2 nas cidades contempladas pelo serviço de testagem do Centro Universitário Saúde ABC, pelo menos nos três primeiros meses
Assuntos
Humanos , Masculino , Feminino , Recém-Nascido , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Quarentena , Distanciamento Físico , COVID-19/prevenção & controle , COVID-19/epidemiologia , Brasil/epidemiologia , Zonas Metropolitanas , Incidência , Teste de Ácido Nucleico para COVID-19 , COVID-19/diagnósticoRESUMO
INTRODUCTION: Trypanosoma cruzi infection triggers an inflammatory process with exacerbated production of cytokines that stimulate inflammatory and anti-inflammatory signals, including the efferent anti-inflammatory signal known as the anti-inflammatory cholinergic pathway. Thus, the use of anticholinesterase drugs, such as galantamine, could minimize the inflammatory process caused by this disease. METHODS: For the study at 30, 60, and 90 days, 120 Swiss mice were divided into three groups. Each group was subdivided into four subgroups: uninfected/untreated (CTRL), uninfected/treated (GAL), infected/untreated (INF), and infected/treated (GAL/INF). The infected groups were inoculated intraperitoneally with 0.1 ml of mouse blood containing 5 × 104 trypomastigote forms of the T. cruzi QM2 strain. The galantamine-treated groups received 5 mg/kg of galantamine orally, through pipetting. From each subgroup, the parameters of parasitemia, histopathological analysis, butyrylcholinesterase activity (BuChE), and functional study of the colon were evaluated. RESULTS: BuChE performance was observed when AChE was suppressed, with increased activity in the GAL/INF group similar to the INF group on the 30th day post infection, thus corroborating the absence of a significant difference in parasitic curves and histopathological analysis. CONCLUSIONS: The presence of an inflammatory process and nests of amastigotes, as well as evidence of reactivity to ACh and NOR, suggest that galantamine did not interfere with the colonic inflammatory response or even in colonic tissue parasitism at this stage of Chagas disease.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Butirilcolinesterase , Doença de Chagas/tratamento farmacológico , Galantamina , Camundongos , ParasitemiaRESUMO
SARS-CoV-2, the novel Coronavirus, was first detected in Wuhan, China, in December 2019, and has since spread rapidly, causing millions of deaths worldwide. As in most countries of the world, in Brazil, the consequences of the COVID-19 pandemic have been catastrophic. Several studies have reported the fecal shedding of SARS-CoV-2 RNA titers from infected symptomatic and asymptomatic individuals. Therefore, the quantification of SARS-CoV-2 in wastewater can be used to track the virus spread in a population. In this study, samples of untreated wastewater were collected for 44 weeks at five sampling sites in the ABC Region (São Paulo, Brazil), in order to evaluate the SARS-CoV-2 occurrence in the sewerage system. SARS-CoV-2 RNA titers were detected throughout the period, and the concentration ranged from 2.7 to 7.7 log10 genome copies.L-1, with peaks in the last weeks of monitoring. Furthermore, we observed a positive correlation between the viral load in wastewater and the epidemiological/clinical data, with the former preceding the latter by approximately two weeks. The COVID-19 prevalence for each sampling site was estimated via Monte-Carlo simulation using the wastewater viral load. The mean predicted prevalence ranged 0.05 to 0.38%, slightly higher than reported (0.016 ± 0.005%) in the ABC Region for the same period. These results highlight the viability of the wastewater surveillance for COVID-19 infection monitoring in the largest urban agglomeration in South America. This approach can be especially useful for health agencies and public decision-makers in predicting SARS-CoV-2 outbreaks, as well as in local tracing of infection clusters.
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COVID-19 , SARS-CoV-2 , Brasil , Humanos , Pandemias , RNA Viral/genética , Águas ResiduáriasRESUMO
Dengue virus, the etiological agent of dengue fever (DF) occurs in four genetically distinct serotypes (DENV1-4), being transmitted by female Aedes mosquitoes. DF incidence is increasing in Brazil, following vector dispersal, proliferation and DENV serotypes introduction, co-circulation and substitution. Medium- and small-sized cities in Sao Paulo State, such as Marilia (Midwest region), have been affected by huge epidemics. To understand the evolution of DENV epidemics in medium-sized cities, in this study a historical data on DENV incidence (2000-2015) in Marilia, was evaluated. Previous studies disclosed regional and specific DF outcomes associated with 2007 outbreak in that city, when co-circulating DENV1 and DENV3 presented different hematological profiles. In this study, characteristics of 2007 DENV epidemics were compared to the epidemiological, hematological and demographic outlines of the major outbreak of DENV1 in Marilia in 2015. DENV1 genetic diversity was assessed through capsid and pre-membrane junction encoding gene (CprM) sequencing. The results revealed circulation of DENV1 serotype from 2007 to 2015, with epidemics occurring every three-years until 2013 and then, increasing yearly. There were significant differences in hematological profiles of DENV1 patients between 2015 and 2007. CprM showed DENV1 genetic variability in 2015, contrasting with the unique sequence pattern in 2007. These results reinforce the regional and temporal characteristics of DENV epidemics that need local public health research to improve care for people and to limit the spread of new serotypes/genotypes to uninfected areas.
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Vírus da Dengue/genética , Dengue/epidemiologia , Surtos de Doenças , Adolescente , Adulto , Idoso , Animais , Brasil/epidemiologia , Criança , Dengue/transmissão , Vírus da Dengue/classificação , Vírus da Dengue/isolamento & purificação , Feminino , Humanos , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Mosquitos Vetores , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorogrupo , Sorotipagem , Adulto JovemRESUMO
Studies on infectious and emerging diseases caused by bats have been increasing worldwide due to their well-recognised status as a reservoir species for various infectious agents as well as their close relationship to humans and animals. This study reports the molecular frequency and diversity of the parasites belonging to the Sarcocystidae family in bats in São Paulo state, Brazil. A total of 2892 tissue samples (brain and pectoral muscle/heart homogenates) from 1921 bats belonging to 36 species were collected, and the Sarcocystidae protozoan 18S ribosomal RNA encoding genes (18S rDNA) were detected by nested PCR and Sanger sequencing. The relative prevalence of Sarcocystidae species was 4.7% (91/1921) among 16 bat species, including insectivorous (n = 65), frugivorous (n = 13) and nectarivorous (n = 11) bats. From 66 sequenced positive samples, 50 were found to be suitable for analysis. Ten samples from insectivorous and nectarivorous bats showed 100% similarity with Neospora caninum (n = 1), Hammondia hammondi (n = 1), Cystoisospora canis (n = 1), Nephroisospora eptesici (n = 1), Sarcocystis (Frenkelia) glareoli (n = 1), and Toxoplasma gondii (n = 5). The 45 non-T. gondii samples revealed 15 different 18S rDNA alleles with identities varying from 96.1 to 100% with several Sarcocystidae species, which might suggest that bats can harbour a large variety of Sarcocystidae organisms. From the five T. gondii-positive tissue samples, three samples from two different bat specimens of the insectivorous Eumops glacinus were characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers, revealing the non-archetypal ToxoDB genotypes #6 (type BrI), which is one of the most prevalent in different hosts and regions from Brazil, and #69. We recommend the inclusion of T. gondii as a differential diagnosis for rabies and other neurological syndromes in bats.
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Abstract INTRODUCTION: Trypanosoma cruzi infection triggers an inflammatory process with exacerbated production of cytokines that stimulate inflammatory and anti-inflammatory signals, including the efferent anti-inflammatory signal known as the anti-inflammatory cholinergic pathway. Thus, the use of anticholinesterase drugs, such as galantamine, could minimize the inflammatory process caused by this disease. METHODS For the study at 30, 60, and 90 days, 120 Swiss mice were divided into three groups. Each group was subdivided into four subgroups: uninfected/untreated (CTRL), uninfected/treated (GAL), infected/untreated (INF), and infected/treated (GAL/INF). The infected groups were inoculated intraperitoneally with 0.1 ml of mouse blood containing 5 × 104 trypomastigote forms of the T. cruzi QM2 strain. The galantamine-treated groups received 5 mg/kg of galantamine orally, through pipetting. From each subgroup, the parameters of parasitemia, histopathological analysis, butyrylcholinesterase activity (BuChE), and functional study of the colon were evaluated. RESULTS: BuChE performance was observed when AChE was suppressed, with increased activity in the GAL/INF group similar to the INF group on the 30th day post infection, thus corroborating the absence of a significant difference in parasitic curves and histopathological analysis. CONCLUSIONS: The presence of an inflammatory process and nests of amastigotes, as well as evidence of reactivity to ACh and NOR, suggest that galantamine did not interfere with the colonic inflammatory response or even in colonic tissue parasitism at this stage of Chagas disease.
Assuntos
Animais , Camundongos , Trypanosoma cruzi , Doença de Chagas/tratamento farmacológico , Butirilcolinesterase , Parasitemia , GalantaminaRESUMO
Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 × 106 cells, the recombinant baculoviruses titer obtained with modified method was about 2 × 107 pfu/ mL in a total volume of 12 mL, which is scalable to 24 liters of 1 × 108 pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages.
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Leishmaniasis comprises a group of zoonotic diseases caused by protozoa belonging to the Leishmania genus, noting that the visceral form is the most severe and lethal, if untreated. Nowadays visceral leishmaniasis is widespread in Brazil and the Adamantina microregion, located in the west of Sao Paulo State, has been affected by Human American Visceral Leishmaniasis (HAVL) since 2004. We evaluated the epidemiological profile of HAVL in the Adamantina microregion through a Geographic Information Systems (GIS) and established its incidence rate by location and time. Notified cases were provided by the Sao Paulo State Epidemiological Surveillance Center. Home addresses of patients who tested positive to HAVL were converted into geographic coordinates through the Google Geocoding Application Programming Interface submitted to ArcMap 10.5 System for georeferencing. Kernel spatial analyses were performed to obtain the incidence distribution and the total area involvement rate. From 2004 to 2018, 325 cases of HAVL were diagnosed in 11 of the 12 municipalities belonging to the of Adamantina microregion. The disease has disseminated to the Northwest and East-Southeast directions, taking place along the Comandante Joao Ribeiro de Barros highway, with higher incidences rates in the municipalities where the highway passes. HAVL incidence was higher in children aged between 0 to 9 years and in the elderly; there was no difference in relation to sex and the majority of cases were located in urban areas. The determination of the epidemiological profile and the the spread of disease patterns can indicate possible areas of vulnerability, in order to contribute to the management and prevention of the disease through a strategic resources optimization.
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Leishmaniose Cutânea , Leishmaniose Visceral , Brasil/epidemiologia , Criança , Pré-Escolar , Cidades , Humanos , Lactente , Recém-Nascido , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologiaRESUMO
The data presented here is related to negative results obtained with the recombinant expression of chitinase from four species of Leishmania parasites in two expression systems, performed in order to investigate the molecular characteristics of the Leishmania chitinase and its possible application in leishmaniasis diagnosis. Thus, heterologous Leishmania sp chitinase proteins were expressed in bacteria using the prokaryotic expression vector pET28a and Escherichia coli Mach-T1, and in Spodoptera frugiperda (Sf9) insect cells, using the eukaryotic bac-to-bac expression system (Thermo Fisher Scientific) to produce recombinant baculoviruses to infect Sf9. Biochemical and cellular analysis of the various recombinant forms of the Leishmania sp chitinase produced in prokaryotic and eukaryotic expression systems were performed through SDS-PAGE and Western blotting. Chitinase produced and purified from bacteria presented low yield and formed inactive aggregates. Heterologous chitinase obtained after infection of Sf9 insect cells with all the four Leishmania species recombinant baculoviruses presented high yield of insoluble proteins. Dot-blot serological tests presented inconclusive results against the recombinant Leishmania sp chitinases produced in both expression systems. The experiments described in this paper can help researchers to avoid errors when choosing a recombinant expression systems to produce Leishmania parasites proteins for biotechnological purposes.
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Ecotin, a serine peptidase inhibitor (ISP), discovered in Escherichia coli, inhibit a wide range of trypsin-like serine peptidases, protecting microorganisms from the host's immune response. In eukaryotes, ISPs encoding genes were found only in Trypanosomatidae protozoa, including the genus Trypanosoma, which harbors Trypanosoma cruzi, the ethiological agent of Chagas' disease. T. cruzi encodes the ISP2 Trypanosomatidae orthologous, which in Leishmania species present inhibitory activity on mammalian proteases from S1A family suggesting its role in vertebrate-host-parasite interactions. In this study, the structural and biochemical characterization of the recombinant T. cruzi ISP2 (rTcISP2), produced in E. coli was purified in soluble form and analyzed by circular dichroism, fluorescence spectroscopy, native electrophoresis, dynamic light scattering, low X-ray scattering and homology modeling. The obtained data revealed that rTcISP2 was biologically active and forms homodimers in solution. Furthermore, inhibitory activity of rTcISP2 against human neutrophil elastase (HNE) is the highest among ISP2 orthologous from bacteria and trypanosomatids. The role of NE to control T. cruzi parasites through modulation of cellular and humoral innate immune responses in vertebrate hosts, make TcISP2 a key molecular component for parasite infection efficiency, providing a useful basis for investigation of host-parasite interactions and the potential of TcISP2 for biotechnological applications.
Assuntos
Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Concentração de Íons de Hidrogênio , Proteínas Recombinantes , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Relação Estrutura-Atividade , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genéticaRESUMO
BACKGROUND: Leishmania infantum, the etiological agent of visceral leishmaniasis, is a neglected zoonosis that requires validation and standardization of satisfactory diagnostic methodologies. Thus, the aim of the present study was to evaluate the effectiveness of cathepsin L-like protease as a target for making molecular diagnoses and as a phylogenetic marker enabling to understand the intraspecies variations and evolutionary history of L. infantum in Brazil. METHODS: We used 44 isolates of L. infantum. The cathepsin L-like gene fragments were amplified, sequenced, manually aligned and analyzed using inference methods. The sequences generated were used to search and design oligonucleotide primers to be used in reactions specific to the target parasite. RESULTS: The cathepsin L-like gene did not show any intraspecies variability among the isolates analyzed. The pair of primers proposed amplified the target deoxyribonucleic acid (DNA) of L. infantum isolates and were effective for DNA amplification at concentrations of as low as 10- 11 ng/µl. The proposed marker did not present cross-reactions with other hemoparasites. When used for making the diagnosis in a panel of clinical samples from dogs, a positivity rate of 49.03% (102/208) was obtained, versus 14.42% (30/208) for a ribosomal internal transcribed spacer (ITS) marker. In samples from sandflies, the rate was 6.25% and from humans, 14.28%. CONCLUSIONS: The results described in this work allow us to infer that CatLeish-PCR is a sensitive and specific marker for use in diagnostic trials of L. infantum and in clinical and epidemiological surveys.
Assuntos
Catepsinas/genética , Leishmania infantum/enzimologia , Leishmaniose Visceral/diagnóstico , Filogenia , Animais , Sequência de Bases , Biomarcadores , Brasil , Ensaios Enzimáticos Clínicos/normas , Reações Cruzadas/imunologia , Primers do DNA/genética , DNA de Protozoário/genética , Doenças do Cão/parasitologia , Cães , Humanos , Leishmania infantum/classificação , Doenças Negligenciadas , Reação em Cadeia da Polimerase , Psychodidae/parasitologia , Padrões de Referência , Zoonoses/parasitologiaRESUMO
Visceral leishmaniasis is a zoonotic disease caused by Leishmania infantum for which dogs are the main reservoir. In South America, presence of this disease is expanding along with increasing dispersion of its principal vector, the sand-fly Lutzomyia longipalpis. Feline leishmaniasis is an emerging disease in domestic cats, but epidemiological studies in endemic areas of the Amazon region of Brazil are scarce and the role of cats as reservoirs of L. infantum has been debated. The aim of this study was to investigate L. infantum infection in cats living in the Amazon biome region, using serological and molecular methods. A total of 105 cats were subjected to clinical examination and blood samples were taken for immunofluorescent-antibody (IFAT) serological evaluation, to determine anti-Leishmania antibody titers. Conventional PCR and Sanger's sequencing targeting L. infantum chitinase and Leishmania species ribosomal internal transcribed spacer (ITS-1) encoding genes were performed on conjunctival swabs from these cats. Seropositivity was detected in 32 animals (30.48%), thus confirming that contact between these cats and the parasite was occurring. PCR followed by amplicon sequencing showed that three samples (2.86%) were positive for a chitinase gene and six (5.71%) were positive for the ITS-1 gene. Parasite-positive diagnoses presented a statistically significant association with free access to the streets (pâ¯=â¯0.0111), cohabitation with dogs affected previously by VL (pâ¯=â¯0.0006) and absence of backyard cleaning and garbage collection (pâ¯=â¯0.00003). These results emphasize that cats should be included in epidemiological surveys of leishmaniasis, especially in endemic areas, if not as the reservoir host (unproven), at least as a "sentinel host" that is useful for revealing situations of endemic circulation of L. infantum. Moreover, in these areas, feline leishmaniasis needs to be considered in the differential diagnosis among domestic cats presenting alopecia, rarefied hair, lacerations and ulcerative dermatitis.