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1.
Mem Inst Oswaldo Cruz ; 116: e200428, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33729396

RESUMO

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças do Cão/diagnóstico , Ensaio de Imunoadsorção Enzimática/normas , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Animais , Antígenos de Protozoários/biossíntese , Brasil , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/sangue , Leishmaniose Visceral/veterinária , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos
2.
Exp Parasitol ; 218: 107970, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32828829

RESUMO

Visceral leishmaniasis (VL) is an anthropozoonosis endemic in Brazil. We included 20 patients with confirmed diagnosis of VL and 20 healthy individuals to evaluate the expression levels of complement receptor 1 (CR1)/CD35 and CR3/CD11b on leukocytes in the peripheral blood and determine their correlation with the clinical state of patients. CR1/CD35 expression increased on CD11b+CD35+granulocytes of patients, while CR1/CD35 and CR3/CD11b expression levels increased on CD14+CD11b+CD35+ monocytes. Among patients, those with severe clinical state had higher expression of CR3/CD11b on CD14+monocytes. The count of CD19+CD35+B lymphocytes reduced in the blood samples from patients. These observed changes may indicate the modulation in CR1/CD35 and CR3/CD11b complement receptor expressionlevels on granulocyte and monocyte populations in response to Leishmania sp.


Assuntos
Antígeno CD11b/metabolismo , Leishmaniose Visceral/imunologia , Leucócitos/metabolismo , Antígeno de Macrófago 1/metabolismo , Receptores de Complemento 3b/metabolismo , Adolescente , Adulto , Idoso , Brasil/epidemiologia , Doenças Endêmicas/estatística & dados numéricos , Feminino , Citometria de Fluxo , Granulócitos/imunologia , Granulócitos/patologia , Humanos , Hipertrofia , Leishmaniose Visceral/sangue , Leishmaniose Visceral/epidemiologia , Leucócitos/imunologia , Fígado/patologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/patologia , Pancitopenia , População Rural , Baço/patologia , População Urbana , Adulto Jovem
3.
PLoS Negl Trop Dis ; 14(7): e0008488, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716931

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis. CONCLUSION: The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.


Assuntos
Doenças do Cão/diagnóstico , Leishmaniose Visceral/veterinária , Testes Sorológicos/veterinária , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Doenças do Cão/sangue , Cães , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Humanos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Transcriptoma
4.
Int J Infect Dis ; 98: 470-477, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32645376

RESUMO

OBJECTIVES: Visceral leishmaniasis (VL) is a progressive disease that, left untreated, is typically fatal. The purpose of this investigation was to detect Leishmania sp. infection in hemodialysis patients who had received multiple blood transfusions at a private clinic in Campo Grande, Mato Grosso do Sul state, Midwest Brazil. METHODS: Fifty randomly selected volunteers were interviewed for collection of demographic, socioeconomic, and epidemiological data. Indirect immunofluorescence (titers positive when ≥1:40) and rK39 immunochromatographic tests were employed for serological investigation. RESULTS: Males predominated (60%). Age ranged from 20 to 77 years. Most subjects reported being on hemodialysis for at least one year (94%) and 84% were candidates for kidney transplantation, 67% of whom were on the waiting list. Leishmania sp. infection was detected in 32%. Contact with infected dogs was the only variable associated with infection. CONCLUSIONS: Under immunocompromised conditions, VL is opportunistic and potentially fatal. Despite existing risks, screening for VL is not performed in asymptomatic donors and recipients. The detection of anti-Leishmania antibodies in these patients reinforces the need for infection screening before immunosuppressive treatment is initiated to reduce not only the risks of VL development and severity, but also mortality rates in cases of reactivation of latent infection.


Assuntos
Anticorpos Antiprotozoários/sangue , Leishmania/imunologia , Leishmaniose Visceral/sangue , Adulto , Idoso , Animais , Brasil , Cães , Seleção do Doador , Feminino , Humanos , Transplante de Rim , Leishmania/fisiologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
5.
Ann Parasitol ; 66(2): 143-156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32531102

RESUMO

Recently, Leishmania infantum has increasingly been detected in stray cats in endemic regions of the world. Cats have been considered playing a role in the epidemiology of visceral leishmaniosis, an endemic zoonosis in Iran. The studies concerning feline leishmaniosis (FeL) allow the hypothesis that cats can be considered as potential reservoirs. The investigations on Leishmania infection in cats are very few in Iran and therefore we aimed to assess the L. infantum infection in stray cats and its possible role in transmission of the disease to human by direct agglutination test (DAT), ELISA, nested-PCR and confirmation via sequencing and phylogenetic analysis in Fars province, Iran. Whole blood samples were obtained from 174 stray cats. Anti-Leishmania antibodies were detected in the sera using DAT and ELISA. DNA was extracted from the buffy coat of each subject and PCR amplified, targeting Leishmania kDNA gene. PCR results were confirmed by sequence analysis. Prevalence of clinical signs in positive cats was 19.0%. Anti-Leishmania antibodies with different titers were detected in 48 (27.59%) and leishmanial DNA in 36 (20.69%) of the cats. The sequencing of PCR-positive cats revealed the parasite as L. infantum. A high seroprevalence of L. infantum was revealed, with higher levels in males, adult cats, and those living in rural districts and southern zones. Despite the reservoir task of cats in nature is still ambiguous, the high serological and molecular detection of L. infantum in stray cats indicates that cats are regularly bitten by infected sand flies in Fars province, southern Iran, and may have a potential reservoir role in the maintenance of L. infantum in the endemic areas of zoonotic visceral leishmaniosis in Iran. Anyway, Leishmania infection must be appraised in the differential diagnosis of cutaneous or systemic clinical signs in cats.


Assuntos
Anticorpos Antiprotozoários/sangue , Leishmania infantum , Leishmaniose Visceral/veterinária , Animais , Gatos , DNA de Protozoário/sangue , Feminino , Humanos , Irã (Geográfico)/epidemiologia , Leishmaniose Visceral/sangue , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/transmissão , Masculino , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Estudos Soroepidemiológicos
6.
Rev Bras Parasitol Vet ; 29(2): e016919, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428183

RESUMO

Dogs have been implicated as main reservoirs for visceral leishmaniasis (VL) in urban areas. Therefore, studies on this species provide important data for public health. Thus, the objective of the present study was to ascertain the seroprevalence of canine VL (CVL) and the associated factors in the Brejo Paraibano microregion, northeastern Brazil. A total of 409 dogs were sampled from the eight municipalities of the microregion: Alagoa Grande, Alagoa Nova, Areia, Bananeiras, Borborema, Matinhas, Pilões and Serraria. The diagnosis of CVL was made using an enzyme-linked immunosorbent assay (ELISA S7®), through which the prevalence observed was 29.3%. By robust Poisson regression analysis two factors were associated with seroprevalence: illiterate/incomplete elementary owner's education level (prevalence ratio = 1.57; 95% CI = 1.05-2.34; p-value = 0.027) and tick infestation (prevalence ratio = 1.82; 95% CI = 1.27-2.61; p-value = 0.001). It is concluded that the seroprevalence of CVL in the Brejo Paraibano microregion is high. The factors associated with seroprevalence indicated the importance to develop socioeducational actions on the population, and the finding that tick infestation was associated with seroprevalence shows that there is a need for investigation regarding the role of ticks in the epidemiology of CVL.


Assuntos
Doenças do Cão/epidemiologia , Leishmaniose Visceral/epidemiologia , Distribuição Animal , Animais , Brasil/epidemiologia , Reservatórios de Doenças/parasitologia , Reservatórios de Doenças/veterinária , Doenças do Cão/sangue , Doenças do Cão/parasitologia , Cães , Escolaridade , Feminino , Humanos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/veterinária , Masculino , Prevalência , Fatores de Risco , Estudos Soroepidemiológicos , Infestações por Carrapato/veterinária
7.
PLoS One ; 15(4): e0230610, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240188

RESUMO

BACKGROUND: The development of rK39-based immunochromatographic rapid diagnostic tests represents an important advance for serodiagnosis of visceral leishmaniasis, being cheap and easy to use at the point of care (POC). Although the use of rK39 have considerably improved the sensitivity and specificity of serological tests compared with total antigens, great variability in sensitivity and specificity was reported. This study aimed at the evaluation of "Kalazar Detect™ Rapid Test, Whole Blood" (Kalazar Detect RDT) for Visceral Leishmaniasis (VL) diagnosis using oral fluid, whole blood and serum specimens collected at different endemic areas of VL of Brazil. METHODOLOGY: To evaluate Kalazar Detect RDT, oral fluid, whole blood and serum specimens from 128 VL patients, 85 healthy individuals, 22 patients with possible cross-reactivity diseases and 20 VL/aids coinfected patients were collected and assayed at the POC. PRINCIPAL FINDINGS AND CONCLUSIONS: The performance of Kalazar Detect RDT in whole blood and serum was similar; however, using oral fluid, the sensitivity was low. Particularly in samples from the city of Natal, Rio Grande do Norte state in Northeastern Brazil, we observed low sensitivity, 80.0% (95% CI: 62.7-90.5), using whole blood and serum, and poor sensitivity, 43.3% (95% CI: 27.4-60.8) with oral fluid. Those values were much lower than in the other regions, where sensitivity ranged from 92.7-96.3% in whole blood and serum, and 80.0-88.9% in oral fluid. Besides, in VL/aids coinfected patients, lower sensitivity was achieved compared with VL patients. In samples from Natal, the sensitivity was 0.0% (95% CI: 0.0-49.0) and 25.0% (95% CI: 4.6-69.9), using oral fluid and serum/whole blood, respectively; in samples from the other regions, the sensitivity ranged from 40.0-63.6% and 80.0-81.8%, respectively. As for specificity, high values were observed across the fluids, 100.0% (95% CI: 96.5-100.0) in whole blood, 96.3% (95% CI: 90.8-98.5) in serum, and 95.3% (95% CI: 89.5-98.0) in oral fluid; across localities, specificity ranged from 85.7-100.0%. Serum samples sent by the collaborating centers to Instituto de Medicina Tropical (n = 250) were tested by Kalazar Detect RDT, Direct Agglutination Test, Indirect immunofluorescence assay, Enzyme-linked immunosorbent assay, and IT-Leish® RDT. The regional difference in the performance of rK39-based RDT and lower sensitivity in Leishmania/HIV coinfected patients raise concern on the routine use of these products for the diagnosis of VL.


Assuntos
Líquidos Corporais/química , Cromatografia de Afinidade/métodos , Testes Diagnósticos de Rotina/métodos , Leishmania/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/imunologia , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Brasil/epidemiologia , Criança , Pré-Escolar , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lactente , Leishmaniose Visceral/sangue , Leishmaniose Visceral/epidemiologia , Masculino , Pessoa de Meia-Idade , Sistemas Automatizados de Assistência Junto ao Leito , Adulto Jovem
8.
J Immunol Methods ; 480: 112765, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32119888

RESUMO

The performance of distinct serological tests (rK39-ICT, IFAT, DAT-LPC, FC-Simplex IgG1) was assessed and a laboratorial algorithm was proposed for accurate diagnosis of VL. DAT-LPC and FC-Simplex IgG1 showed outstanding accuracy (AUC = 0.93) to identify VL patients. The use of a sequential serological algorithm (rK39-ICT screening followed by DAT-LPC or FC-Simplex IgG1) improved the global accuracy for VL (97.2%) diagnosis. An alternative approach for diagnosis of VL has been also assessed for interchangeable use of serum/whole blood lysate samples in DAT-LPC and FC-Simplex IgG1. Our data showed an outstanding agreement for the results obtained with whole blood lysate samples as compared to serum samples (DAT-LPC =100%; FC-Simplex IgG1 = 99%). Together, these findings provide insights to improve the current overall accuracy of VL diagnosis and present innovative laboratorial tests and alternative samples from use in public health services.


Assuntos
Algoritmos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Imunoglobulina G/sangue , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Kit de Reagentes para Diagnóstico , Testes Sorológicos , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Brasil , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Interações Hospedeiro-Parasita , Humanos , Lactente , Leishmaniose Visceral/sangue , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Adulto Jovem
9.
PLoS One ; 15(3): e0230381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176727

RESUMO

Visceral leishmaniasis (VL) is the most fatal form of leishmaniasis if left untreated and 50,000 to 90,000 new cases of VL occur worldwide each year. Although various vaccines had been studied in animal models, none of them was eligible to prevent human from infections. In this study, according to the silico analysis of Leishmania Amastin, Kmp-11 and Gp63 protein, dominant epitope sequences of these proteins were selected and linked to construct dominant multi-epitopes DNA and protein vaccines (Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63) against VL. BALB/c mice were immunized with a DNA prime-protein boost immunization strategy and challenged with a new Leishmania parasite strain isolated from a VL patient. After immunization, the results including specific antibody titers, IL-4 and TNF-α levels, and CD4 and CD8 T cell proportion suggested the potent immunogenicity of the three vaccines. After infection, the results of spleen parasite burdens in the three vaccine groups were significantly lower than those of control groups, and the parasite reduction rates of Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63 groups were 89.38%, 91.01% and 88.42%, respectively. Spleen smear observation and liver histopathological changes showed that all vaccine groups could produce significant immunoprotection against VL and Amastin-Gp63 vaccine was the best. In conclusion, our work demonstrated that the three dominant multi-epitopes Amastin-Kmp-11, Amastin-Gp63 and Kmp-11-Gp63 DNA prime-protein boost vaccines might be new vaccine candidates for VL, and the Amastin-Gp63 vaccine have best efficacy.


Assuntos
Epitopos/imunologia , Imunidade , Imunização Secundária , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Proteínas de Protozoários/imunologia , Vacinas de DNA/imunologia , Animais , Sítios de Ligação , Citocinas/sangue , Epitopos/química , Feminino , Imunoglobulina G/sangue , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Fígado/patologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Parasitos/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/imunologia , Baço/parasitologia
10.
Comp Immunol Microbiol Infect Dis ; 69: 101431, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32059125

RESUMO

Leishmania is an intracellular protozoan parasite, mainly infects macrophages of mammalian tissues. Inflammatory related cytokines have a crucial role in the pathogenesis of leishmaniasis. The aim of the present study was to evaluate serum concentrations of IL-1ß and IL-17 in patients with active visceral leishmaniasis (VL) and control group. Serum concentrations of both IL-1ß and IL-17 cytokines were assessed by ELISA in Leishmania infantum infected patients (n = 25) and healthy individuals (n = 25) from Meshkin-Shahr, northwest of Iran. Mean serum concentrations of IL-1ß in the patients and control groups were 47.34 ± 23.82, and 20.49 ± 9.38, respectively, which was statistically significant (p < 0.001). Furthermore, mean IL-17 concentration in patients with VL (243.96 ± 73.46) was twice higher comparing to control group (106.38 ± 129.06) (p < 0.001). Several cytokines are involved in the regulation of immunity against VL. The present data has shown that, increased serum concentrations of IL-1ß and IL-17 are present in the patients with VL. Further investigations are needed to enhance our knowledge about the regulatory role of these cytokines in leishmaniasis.


Assuntos
Interleucina-17/sangue , Interleucina-1beta/sangue , Leishmania donovani , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lactente , Mediadores da Inflamação/sangue , Masculino
11.
Parasite Immunol ; 42(4): e12701, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31990371

RESUMO

AIMS: The aim of the present study was to assess serum cytokine and miRNA expression in visceral leishmaniasis-HIV (VL-HIV) co-infection and HIV mono-infection. METHODS AND RESULTS: We analysed 113 serum samples from HIV patients in areas endemic for leishmaniasis. The diagnosis of VL was confirmed in 65 of these 113 samples. The VL-HIV and HIV groups presented significant differences regarding haemoglobin level (P < .0001), lymphocyte count (P = .0444), white blood cell count (P = .0108), weight loss (P = .0310), HIV load (P < .0001) and CD4+ T-lymphocytes count (P = .0003). Levels of IL-6 and IL-10, IFN-γ and IL-6, IFN-γ and IL-10, TNF and IL-2 were positively correlated in VL-HIV co-infection, indicating higher serum levels of TNF and IL-4 (P < .0001). In addition, miR-182 expression was found to be significantly higher in HIV (P = .009), miR-210 exhibited no statistically significant difference between groups, and nonexpression of miR-122 was found in both groups. CONCLUSION: Together, TNF, IL-4 and miR-182 may represent circulatory biomarkers of VL-HIV co-infection.


Assuntos
Infecções por HIV/sangue , Leishmaniose Visceral/sangue , MicroRNAs/sangue , Adulto , Biomarcadores/sangue , Linfócitos T CD4-Positivos/imunologia , Coinfecção , Citocinas/sangue , Feminino , Infecções por HIV/complicações , Humanos , Interleucina-10/sangue , Interleucina-4/sangue , Leishmaniose Visceral/complicações , Contagem de Leucócitos , Masculino
12.
Transbound Emerg Dis ; 67(1): 318-327, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31512804

RESUMO

The objective of this study was to identify changes in serum proteome in dogs that may occur after an experimental infection at subclinical and clinical stages of canine leishmaniosis (CanL). For this purpose, canine pre- and post-infection with Leishmania infantum serum proteomes in the same dogs were analysed by a high-throughput label-based quantitative LC-MS/MS proteomic approach. A total of 169 proteins were identified, and 74 of them including complement C8 alpha chain, adiponectin, transferrin, sphingomyelin phosphodiesterase acid-like 3A and immunoglobulins showed different modulation between the different stages of CanL. These proteins could be considered as potential serum biomarkers of early diagnostic or disease progression in CanL. Additionally, biological pathways modulated during CanL such as blood coagulation or gonadotropin-releasing hormone receptor were revealed, which could help to understand the pathological mechanisms of the disease.


Assuntos
Biomarcadores/sangue , Doenças do Cão/sangue , Leishmania infantum/metabolismo , Leishmaniose Visceral/veterinária , Proteoma , Animais , Cromatografia Líquida/veterinária , Doenças do Cão/fisiopatologia , Doenças do Cão/virologia , Cães , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/sangue , Leishmaniose Visceral/fisiopatologia , Leishmaniose Visceral/virologia , Proteômica , Espectrometria de Massas em Tandem/veterinária
13.
Parasit Vectors ; 12(1): 593, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852505

RESUMO

BACKGROUND: Kinesin-related gene diversity among strains and species of Leishmania may impact the sensitivity and specificity of serodiagnostic tests for visceral leishmaniasis (VL). METHODS: In this study, we report on the recombinant expression of this novel Iranian Leishmania infantum (MCAN14/47) homologue of rK39 (Li-rK39), in L. tarentolae. The diagnostic potential of the Li-rK39 antigen was evaluated in an ELISA, using sera from 100 VL patients, 190 healthy endemic controls, 46 non-endemic healthy controls and 47 patients with other infections. RESULTS: The results showed a sensitivity of 96% and a specificity of 93.8%. A commercial rK39 immunochromatographic test (ICT) was 90% sensitive and 100% specific on the same cohort. CONCLUSIONS: Here, we show that the K39 gene from an Iranian L. infantum isolate is heterozygous as compared to the sequence of the Brazilian L. infantum (former L. chagasi), whose antigen is incorporated in most rK39-based immunochromatographic tests. Therefore, Li-rK39 has the potential to be used as an alternative for VL diagnosis in Iran.


Assuntos
Antígenos de Protozoários/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Leishmania infantum/genética , Leishmania/genética , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/sangue , Adolescente , Adulto , Animais , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Expressão Gênica , Humanos , Lactente , Irã (Geográfico) , Leishmania/metabolismo , Leishmania infantum/imunologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Masculino , Proteínas de Protozoários/genética , Testes Sorológicos/métodos , Adulto Jovem
14.
Iran J Immunol ; 16(4): 311-320, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31885008

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) can lead to death in more than 95% of cases if left untreated. Accurate and early diagnosis has an important role in reducing mortality rate of this disease. OBJECTIVE: To express recombinant H2B antigen from an Iranian isolate of Leishmania Infantum and evaluate its efficacy in the diagnosis of VL. METHODS: The recombinant H2B antigen was produced in a prokaryotic system, and its efficacy for VL diagnosis was evaluated by ELISA. The serum samples from 80 VL patients, 100 individuals from endemic and non-endemic regions of VL, and 58 non-VL patients were collected. VL cases were confirmed based on the clinical sign, positive IFAT (>64), real time PCR, and response to treatment. RESULTS: The H2B gene sequence of the Iranian L. infantum isolate had about 4% diversity in comparison with the H2B gene of the L. infantum counterpart. ELISA, using the produced H2B recombinant antigen, showed sensitivity of 71.25% (95% CI: 60.05%-80.82%) and specificity of 69.62% (95% CI: 61.81%-76.68%) regarding VL diagnosis. CONCLUSION: Recombinant H2B antigen expressed in the prokaryotic system had suboptimal performance for the serological diagnosis of VL. It seems that the production and expression of recombinant H2B antigen in a eukaryotic system may enhance the performance of this antigen in the diagnosis of VL in Iran.


Assuntos
Anticorpos Antiprotozoários/sangue , Leishmania infantum/química , Proteínas de Protozoários/química , Testes Sorológicos , Adolescente , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lactente , Irã (Geográfico) , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Masculino , Proteínas Recombinantes/química
15.
Vet J ; 253: 105387, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31685139

RESUMO

Diagnosing canine visceral leishmaniasis (CVL) is difficult because clinical signs of the disease are non-specific and a many infected animals in endemic areas, as in Brazil, are asymptomatic. Serological tests are the most common diagnostic methods employed, but most have limitations. For this reason, the implementation of a rapid, sensitive, and specific diagnostic test for CVL has become increasingly important. In this study, we adapted a chemiluminescent enzyme-linked immunosorbent assay (CL ELISA), using two multi-epitope recombinant proteins (PQ10 and PQ20) and a crude Leishmania antigen produced using promastigotes of L. infantum, as antigens to detect CVL infection in animals from Belo Horizonte. To investigate cross-reactions, samples from dogs with other infections (babesiosis, ehrlichiosis and Trypanosoma cruzi) were tested. Assay performance validations were conducted to analyse parameters such as variability, reproducibility, and stability. CL ELISA sensitivity/specificity with PQ10 antigen was 93.1%/80.0%; with the PQ20 protein 93.1%/96.6%; and with the crude antigen 75%/73.3%. Inter-assay variability and inter-operator coefficient of variation were <7% and <15%, with PQ10 and PQ20, respectively. The accuracy of the CL ELISA was classified as excellent for PQ10 (AUC = 0.95) and PQ20 (AUC = 0.98) and moderate for the crude antigen (AUC = 0.77). The kappa score for qualitative agreement between two plate lots was excellent for PQ10 (0.89) and good for PQ20 (0.65). PQ20 remained more stable than PQ10. The CL ELISA with recombinant proteins is a promising tool to diagnose CVL.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doenças do Cão/diagnóstico , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Animais , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Medições Luminescentes/veterinária , Masculino , Sensibilidade e Especificidade , Testes Sorológicos/veterinária
16.
J Appl Lab Med ; 4(3): 404-409, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659077

RESUMO

BACKGROUND: Hypergammaglobulinemia is present in visceral leishmaniasis (VL), inducing the formation of immune complexes (ICs), which interferes in conventional serology. Parasitic haptens block antibodies, making it difficult to identify and detect them. ICs could be determined indirectly by acid dissociative ELISA (DE) seroconversion in natural and experimental VL. METHODS: We determined the frequency of samples that seroconverted in DE or presented a 10% increase in DE (ΔDE) in 3 types of VLs-hamster, canine, and human samples-with larger antigen determination by direct antigen capture in experimental samples. RESULTS: The ΔDE frequency is increased in all VL models: human (34%), canine (27%), and hamster (25%) samples. Seroconversion was present in hamster (14%), dog (1%), and human (6%) samples. During experimental infection, higher frequencies (28%) of circulating antigens were observed at the 30th day, associated with higher ΔDE (47%) and seroconversion (22%), with lower frequencies in other periods. CONCLUSIONS: The frequency of ΔED and seroconversion samples found in natural and experimental infection suggests that specific antibodies can be blocked by low molecular weight antigens that interfere qualitatively (seroconversion) or quantitatively (ΔDE) in serology. Several antigen types may be involved, as high molecular weight proteins and low molecular weight glycoconjugates. The higher frequency of those indirect demonstrations of antibody-blocking antigen or haptens that could be acid-removed in VL has implications for the development of assays for detection of circulating or antibody-bound 1- to 3-kDa antigens, which could interfere in diagnosis and also in the immune response of the host.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Antígenos de Protozoários/imunologia , Doenças do Cão/diagnóstico , Haptenos/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Complexo Antígeno-Anticorpo/sangue , Antígenos de Protozoários/sangue , Cricetinae , Doenças do Cão/sangue , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Testes Imunológicos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Soroconversão
17.
BMC Res Notes ; 12(1): 566, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511056

RESUMO

OBJECTIVES: Infection with the causative agent of visceral leishmaniasis (VL) may be either symptomatic or asymptomatic. In this study we aimed at investigating the prevalence of asymptomatic infections of leishmania in non-endemic villages in Gedaref state, Sudan. A descriptive cross-sectional study conducted during September and October 2014. Blood samples were collected for serological and molecular analysis. Sticky-traps, knockdown spray and CDC-miniature light traps were used for the collection of sandflies. RESULTS: Ninety-Five participants were included; 52 from Abukishma, 15 Algadamblia Tirfa, 25 Abualnaja and 3 were from Algadamblia Aljabal. Females constituted 56 (58.9%) of the study participants while males were 39 (41.1%). The most frequent age group was > 40-years (54.7%). Balanites/Acacia trees were the most planted tree inside the houses; 78 (82.1%). Also, 85 (89.5%) of the participants breed animals inside the house. DAT test revealed 5 positive participants (5.2%). 4/5 DAT positive were past VL infection. PCR detected 35 (36.8%) positive participants. A total of 31/35 was considered asymptomatic infections based on PCR. Households planted Balanites/Acacia trees or breed domestic animals were found in high percentages with L. donovani PCR positive participants (60.1%, 91.4%). No statistically significant was found for VL associated risk factors and VL asymptomatic participants.


Assuntos
Infecções Assintomáticas/epidemiologia , Leishmania donovani/genética , Leishmaniose Visceral/epidemiologia , Acacia/parasitologia , Adulto , Animais , Estudos Transversais , DNA de Protozoário/genética , Feminino , Geografia , Humanos , Leishmania donovani/fisiologia , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Masculino , Phlebotomus/parasitologia , Reação em Cadeia da Polimerase , Prevalência , Sudão/epidemiologia
18.
J Immunol Methods ; 474: 112664, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31521674

RESUMO

Recently, a novel Enzyme-Linked Immunosorbent Assay (ELISA) strategy has emerged, known as "plasmonic ELISA" (pELISA), which enables the detection of disease biomarkers at low concentrations with the naked eye. For the first time, this research has developed a signal-generation mechanism for the detection of anti-Leishmania sp. IgG antibodies with the naked eye using pELISA. The immunoassay incorporates an indirect ELISA with successive growth of gold nanoparticles to obtain blue or red-colored solutions in the presence or absence of anti-Leishmania sp. IgG antibodies in canine serum, respectively. The technique we developed was successfully tested in canine serum positive and negative for canine leishmaniasis (CanL), and was shown to be an effective method that could be used as an additional tool for CanL diagnosis. It will be particularly useful in resource-constrained countries, because it does not require sophisticated instruments to read the results, increasing the practicality of CanL detection in these areas.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças do Cão/diagnóstico , Ensaio de Imunoadsorção Enzimática/veterinária , Imunoglobulina G/sangue , Leishmania donovani/imunologia , Leishmaniose Visceral/veterinária , Animais , Biomarcadores/sangue , Doenças do Cão/sangue , Doenças do Cão/imunologia , Cães , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes
19.
Vet Parasitol ; 274: 108920, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31493694

RESUMO

Visceral leishmaniasis (VL) is a zoonosis caused by the parasite Leishmania infantum and the dog is its main reservoir in rural and urban areas. The diagnosis of infection is mainly based on the presence of anti-Leishmania IgG antibodies in the serum of infected dogs. In this study, the sensitivity and specificity of qualitative rapid tests (RTs) dual path platform (DPP) Bio-Manguinhos, rapid enzyme-linked immunosorbent assay (ELISA) IDEXX, Kalazar Detect and ALERE, as well as quantitative ELISA Bio-Manguinhos and in-house indirect immunofluorescence assay (IFA) tests were analyzed in sera from infected and uninfected dogs. Serial dilutions of the in-house IFA were compared with RTs and ELISA Bio-Manguinhos. The results showed that none of the tests reached 100% sensitivity and specificity. There was no statistical difference between the analyzed RTs. The most sensitive test was the DPP Bio-Manguinhos (97.9%), while the rapid ELISA IDEXX showed higher specificity (100%). In the treatment setting of infected and/or diseased animals, quantitative tests for monitoring the evolution of antibody titers are required, which indicates the maintenance of in-house IFA in animal handling. Furthermore, we demonstrate that the RTs present higher sensitivity in serum samples with superior antibody titers obtained in the in-house IFA. However, the RTs exhibited false negatives in samples with low titers of antibodies. Among the RTs, only the DPP Bio-Manguinhos presented better performance in this situation. Therefore, the use of RTs for the diagnosis of VL in dogs with low titers of antibodies, such as asymptomatic, should be carefully evaluated.


Assuntos
Doenças do Cão/sangue , Leishmania infantum , Leishmaniose Visceral/veterinária , Testes Sorológicos/veterinária , Animais , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Sensibilidade e Especificidade
20.
Indian J Dermatol Venereol Leprol ; 85(6): 578-589, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31397399

RESUMO

Background: Elimination of kala azar from India is challenging as there are potential reservoirs of Leishmania donovani in patients with post-kala-azar dermal leishmaniasis (PKDL). The vast repertoire of carbohydrate moieties on L. donovani is known to elicit specific and strong humoral responses in patients with kala azar. Aim: The present study was undertaken to evaluate the diagnostic performances of anti-gal antibodies using enzyme-linked immunosorbent assay for successful serological diagnosis of PKDL in Indian patients and to differentiate cases of past cured visceral leishmaniasis infections. Methods: We developed Gal enzyme-linked immunosorbent assay to measure specific anti-gal IgG isotype in the sera of 71 Indian patients with PKDL. The diagnostic efficacy of the newly developed assay was evaluated for precision, sensitivity and accuracy. Results: Gal2 enzyme-linked immunosorbent assay revealed three-fold increased anti-gal titers in 71 patients with active PKDL compared to controls. Subclass enzyme-linked immunosorbent assay analysis further revealed enhanced IgG2 and IgG3 anti-gal titers in patients with PKDL compared to control subjects. The rank order for specificity and sensitivity for IgG subclasses was IgG3>IgG2>IgG4>IgG1. The area under the curve values of 0.98 and 0.99 were obtained for IgG and IgG3 Gal2 enzyme-linked immunosorbent assays respectively. Overall sensitivity and specificity were 95.7% (95% CI: 88.1-99.1) and 98.1% (95% confidence interval: 90.1-99.9), and 98.5% (95% CI: 92.4-99.9) and 98.1% (95% CI: 90.1-99.9), respectively. Intra-assay coefficient of variation was 1.5% and inter-assay coefficient of variation was 11.7%. Limitations: The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. Conclusion: Taken together, anti-gal titers detected through Gal2 enzyme-linked immunosorbent assay can serve as an effective diagnostic tool in disease elimination setting and help in better case management in endemic districts.


Assuntos
Leishmania donovani/isolamento & purificação , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/diagnóstico , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Testes Diagnósticos de Rotina/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Suínos , Adulto Jovem
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