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1.
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
2.
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
3.
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
4.
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
5.
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
6.
Acta Vet Scand ; 61(1): 38, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391084

RESUMO

Canine leishmaniosis is a disease caused by Leishmania infantum, a vector-borne parasite. Due to the zoonotic potential of canine leishmaniosis, infected dogs must be identified. Serological assays are the most common methods for the detection of L. infantum infection in dogs used in veterinary practice. The aim of the study was to assess the performance of a rapid immunochromatographic test (FASTest LEISH®, MEGACOR Diagnostik) for the detection of specific antibodies to that of the L. infantum in dog sera. The results were simultaneously compared using a commercial brand of indirect immunofluorescence antibody test and an in-house enzyme-linked immunosorbent assay as references. Between the two reference tests, 232 serum samples out of 244, produced concordant results while 12 exhibited discordant results. Of the 232 concordant samples, 121 were classified as L. infantum seropositive, and 111 samples were previously classified as L. infantum seronegative by a combination of the reference assays. All samples that were seropositive by the reference tests were also positive according to the rapid test, and only one sample that was seronegative according to the two reference assays was positive according to the rapid test. Compared with the reference tests, the rapid test sensitivity was 100%, specificity was 99.1%, accuracy was 99.6%, Cohen's kappa coefficient was 0.99, and the area under receiver operating characteristic curve was 0.995. The FASTest LEISH® is a rapid, qualitative in-clinic test with high sensitivity and specificity.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças do Cão/diagnóstico , Ensaio de Imunoadsorção Enzimática/veterinária , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Imunoensaio/veterinária , Leishmaniose Visceral/veterinária , Animais , Doenças do Cão/sangue , Cães , Leishmania infantum , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Sensibilidade e Especificidade
7.
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
8.
Biomed Res Int ; 2019: 8240784, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428648

RESUMO

Visceral leishmaniasis (VL) is considered a major public health concern in Brazil and several regions of the world. A recent advance in the diagnosis of infectious diseases was the development of loop-mediated isothermal amplification (LAMP). The aim of this study was to develop and evaluate a new LAMP assay for detection of K26 antigen-coding gene of L. donovani complex. A total of 219 blood samples of immunocompetent patients, including 114 VL cases and 105 non-VL cases, were analyzed for the diagnosis of VL in the present study. Diagnostic accuracy was calculated against a combination of parasitological and/or serological tests as a reference standard. The results were compared to those of kDNA Leishmania-PCR. The detection limit for the K26-Lamp assay was 1fg L. infantum purified DNA and 100 parasites/mL within 60 min of amplification time with visual detection for turbidity. The assay was specific for L. donovani complex. Sensitivity, specificity, and accuracy were 98.2%, 98.1%, and 98.2%, respectively, for K26-LAMP and 100%, 100%, and 100%, respectively, for kDNA Leishmania-PCR. Excellent agreement was observed between K26-LAMP and kDNA Leishmania-PCR assays (K = 0.96). A highly sensitive and specific LAMP assay targeting K26 antigen-coding gene of L. donovani complex was developed for diagnosis in peripheral blood samples of VL patients.


Assuntos
DNA de Protozoário , Leishmania donovani/genética , Leishmaniose Visceral , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Brasil , DNA de Protozoário/sangue , DNA de Protozoário/genética , Feminino , Humanos , Leishmania donovani/metabolismo , Leishmaniose Visceral/sangue , Leishmaniose Visceral/genética , Masculino
9.
PLoS Negl Trop Dis ; 13(7): e0007493, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31260440

RESUMO

BACKGROUND: Visceral Leishmaniasis in humans presents with fever, anemia, and splenomegaly and can be lethal if not treated. Nevertheless, the majority of Leishmania infantum-infected individuals does not manifest symptoms and remain so provided they are not immunosuppressed. In this work, the performance of different tests was evaluated to detect asymptomatic individuals who were living in Teresina, Piauí state, Brazil, an endemic area for VL. METHODOLOGY: L. infantum-specific antibodies were detected by ELISA and two different rapid immunochromatographic (IC) diagnostic tests, Kalazar Detect and OnSite, and parasitic loads were detected by real time PCR [qPCR]. Additionally, we measured levels of the biomarkers monokine induced by IFN-γ (MIG) and IFN-γ-induced protein 10 (IP-10) before and after stimulation of whole blood with soluble Leishmania antigen [SLA]. PRINCIPAL FINDINGS: Kalazar Detect and OnSite detected, respectively, 76% and 64% of patients presenting with active Visceral Leishmaniasis; 50% and 57% of patients remained positive in these tests, respectively, after treatment. Of the healthy participants in the study who were living in the endemic area, only 1.7% were positive with both of the IC tests. On the other hand, reactivity in ELISA tests revealed that 13% of these individuals presented asymptomatic infections; among VL patients, 84% presenting with active disease were reactive in ELISA, and after treatment, 55.5% were seropositive. L. infantum DNA was present in the blood of 37.9% of infected individuals living in the endemic area, while IP-10 and MIG biomarkers were detected in 26.7% of them. The greatest concordance of positivity occurred between ELISA and qPCR. CONCLUSION: The association of different techniques can detect asymptomatic infections, however, more research is necessary to develop ideal biomarkers that are simple to use in the clinic and in field studies in areas endemic for Visceral Leishmaniasis.


Assuntos
Anticorpos Antiprotozoários/sangue , Infecções Assintomáticas , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Adulto , Biomarcadores/sangue , Brasil , Quimiocina CXCL10/sangue , DNA de Protozoário/genética , Doenças Endêmicas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoensaio , Interferon gama/sangue , Leishmania infantum , Masculino , Pessoa de Meia-Idade , Carga Parasitária
10.
Artigo em Inglês | MEDLINE | ID: mdl-31316919

RESUMO

Concurrently, leishmaniasis and AIDS are global public health issues and the overlap between these diseases adds additional treats to the management of co-infected patients. Lopinavir (LPV) has a well characterized anti-HIV and leishmanicidal action, and to analyze its combined action with miltefosine (MFS) could help to envisage strategies to the management of co-infected patients. Here, we evaluate the interaction between LPV and MFS against Leishmania infantum infection by in vitro and in vivo approaches. The effect of the compounds alone or in association was assessed for 72 h in mouse peritoneal macrophages infected with L. infantum by the determination of the IC50s and FICIs. Subsequently, mice were orally treated twice daily during 5 days with the compounds alone or in association and evaluated after 30 days. The in vitro assays revealed an IC50 of 0.24 µM and 9.89 µM of MFS and LPV, respectively, and an additive effect of the compounds (FICI 1.28). The in vivo assays revealed that LPV alone reduced the parasite load in the spleen and liver by 52 and 40%, respectively. The combined treatment of infected BALB/c mice revealed that the compounds alone required at least two times higher doses than when administered in association to virtually eliminate the parasite. Mice plasma biochemical parameters assessed revealed that the combined therapy did not present any relevant hepatotoxicity. In conclusion, the association of MFS with LPV allowed a reduction in each compound concentration to achieve the same outcome in the treatment of visceral leishmaniasis. Although a pronounced synergistic effect was not evidenced, it does not discard that such combination could be useful in humans co-infected with HIV and Leishmania parasites.


Assuntos
Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/complicações , Leishmaniose Visceral/tratamento farmacológico , Lopinavir/uso terapêutico , Fosforilcolina/análogos & derivados , Administração Oral , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/uso terapêutico , Coinfecção/tratamento farmacológico , Modelos Animais de Doenças , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Concentração Inibidora 50 , Leishmaniose Visceral/sangue , Fígado/parasitologia , Lopinavir/administração & dosagem , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Fosforilcolina/administração & dosagem , Fosforilcolina/uso terapêutico , Baço/parasitologia , Resultado do Tratamento
11.
Parasitol Res ; 118(9): 2715-2721, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286264

RESUMO

The occurrence of infection or exposure to Toxoplasma gondii, Neospora caninum, and Leishmania infantum was investigated in European brown hares (Lepus europaeus, EBH) hunter-harvested over two consecutive hunting seasons in northern and central Greece. Geographical information system was used along with the ecological niche model to define the geographical distribution of seropositive hares relative to environmental parameters and to identify high-risk areas for hare exposure. Molecular analysis showed that 3.8% and 9.6% of the examined hares were infected with N. caninum and L. infantum, respectively, while, 5.7%, 0.95%, and 12.4% of the hares tested positive for the presence of antibodies against T. gondii, N. caninum, and L. infantum respectively. None of the examined hares was polymerase chain reaction-positive for T. gondii. Mixed exposure against both T. gondii and L. infantum was found in 2.9% of the hares examined. Rainfall indices and land uses significantly influenced the exposure of hares to T. gondii and L. infantum. This is the first molecular and serological survey of protozoan pathogens in EBH in Greece. Furthermore, we report the environmental parameters related to hare seropositivity and present a risk map for hare exposure to T. gondii and L. infantum in northern and central Greece. The ecological niches of T. gondii and L. infantum in the hares presented herein could be applied to other regions with similar environmental and climatic conditions.


Assuntos
Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Feminino , Grécia/epidemiologia , Lebres/parasitologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Masculino , Estudos Soroepidemiológicos , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose Animal/sangue
12.
Vet Parasitol ; 271: 87-97, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31303211

RESUMO

The natural history of canine visceral leishmaniasis (CVL) has been well described, particularly with respect to the parasite load in different tissues and immunopathological changes according to the progression of clinical forms. The biomarkers evaluated in these studies provide support for the improvement of the tools used in developing vaccines against CVL. Thus, we describe the major studies using the dog model that supplies the rationale for including different biomarkers (tissue parasitism, histopathology, hematological changes, leucocytes immunophenotyping, cytokines patterns, and in vitroco-culture systems using purified T-cells subsets and macrophages infected with L. infantum) for immunogenicity and protection evaluations in phases I and II applied to pre-clinical and clinical vaccine trials against CVL. The search for biomarkers related to resistance or susceptibility has revealed a mixed cytokine profile with a prominent proinflammatory immune response as relevant for Leishmania replication at low levels as observed in asymptomatic dogs (highlighted by high levels of IFN-γ and TNF-α and decreased levels in IL-4, TGF-ß and IL-10). Furthermore, increased levels in CD4+ and CD8+ T-cell subsets, presenting intracytoplasmic proinflammatory cytokine balance, have been associated with a resistance profile against CVL. In contrast, a polyclonal B-cell expansion towards plasma cell differentiation contributes to high antibody production, which is the hallmark of symptomatic dogs associated with high susceptibility in CVL. Finally, the different studies used to analyze biomarkers have been incorporated into vaccine immunogenicity and protection evaluations. Those biomarkers identified as resistance or susceptibility markers in CVL have been used to evaluate the vaccine performance against L. infantum in a kennel trial conducted before the field trial in an area known to be endemic for visceral leishmaniasis. This rationale has been a guiding force in the testing and selection of the best vaccine candidates against CVL and provides a way for the veterinary industry to register commercial immunobiological products.


Assuntos
Biomarcadores/sangue , Doenças do Cão/sangue , Leishmaniose Visceral/veterinária , Animais , Biomarcadores/análise , Suscetibilidade a Doenças/metabolismo , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/sangue , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Vacinas Protozoárias/imunologia
13.
Diagn Microbiol Infect Dis ; 95(2): 134-143, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31155395

RESUMO

The diagnosis of visceral leishmaniasis (VL) presents problems due to the toxicity and/or high cost of drugs. In addition, no vaccine exists to protect against human disease. In this study, the antigenicity and immunogenicity of amastin protein were evaluated in L. infantum-infected dogs and humans. For the diagnosis, besides the recombinant protein, 1 linear B-cell epitope was synthetized and evaluated in serological assays. Results showed high sensitivity and specificity values to detect the disease when both antigens were employed against a canine and human serological panel. By contrast, when using rA2 and a soluble Leishmania antigenic preparation, sensitivity and specificity values proved to be lower. A preliminary immunogenicity study showed that the amastin protein induced high IFN-γ and low IL-10 production in stimulated PBMC derived from treated VL patients and healthy subjects, thus suggesting a potential use of this protein as an immunogen to protect against human disease.


Assuntos
Antígenos de Protozoários/imunologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Células Cultivadas , Citocinas/metabolismo , Doenças do Cão/sangue , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Epitopos de Linfócito B/imunologia , Humanos , Imunogenicidade da Vacina , Leishmania infantum/imunologia , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Leucócitos Mononucleares/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos
14.
Exp Parasitol ; 201: 78-89, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31047987

RESUMO

Hamsters are a suitable experimental model for visceral leishmaniasis (VL) because they mimic the features of the human disease. However, the infection after inoculation can only be verified after sacrifice of the animal or several months following infection, when obvious signs of the disease appear, compromising animal welfare in both cases. Unlike other studies, the present work used an inoculum of 5 × 108 promastigotes to induce Leishmania infantum infection, which are easier to produce than amastigotes, in in vitro culture. The infection in hamsters was detected using non-invasive methods such as ultrasound imaging (USG) and blood gases, in addition to alterations in hematological parameters and weight loss. USG imaging identified changes in the size and echogenicity of the spleen, liver, and kidney as early as week 9 (W9) after experimental inoculation. However, blood gases, specially lactate, was increased in response to the infection, with statistically significant differences between W9 and W0 (before infection) (p < 0.0001). The conventional hematological parameters showed progressive pancytopenia and weight loss of 15% and 10% in infected males and females respectively, at W9 versus W0 (p < 0.0001). Histological changes in the liver, kidney, and spleen correlated with changes detected by USG imaging and the number of parasites increased proportionately to the progression of infection, being higher at W24. In conclusion, USG imaging, lactate levels, hematocrit and hemoglobin parameters, along with weight loss allowed early detection of infection, which was then confirmed by the identification and quantification of parasites in the blood, liver, and spleen by qRT-PCR. In contrast, blood chemistry was not a useful tool in the early detection of VL infection because it did not correlate with alterations evident in other techniques. The use of non-invasive tools eliminates the need for animal sacrifice to confirm infection, thus reducing the number of animals required for a given study and eliminating the need to wait until the appearance of severe signs of infection, which affect animal welfare. These tools are therefore advantageous for use in preclinical studies, for studying pathogenesis as also for vaccine and drug development.


Assuntos
Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Animais , Análise Química do Sangue , Gasometria , Cricetinae , Modelos Animais de Doenças , Feminino , Testes Hematológicos , Rim/diagnóstico por imagem , Rim/patologia , Leishmania infantum/classificação , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico por imagem , Fígado/diagnóstico por imagem , Fígado/parasitologia , Fígado/patologia , Masculino , Mesocricetus , Reação em Cadeia da Polimerase em Tempo Real , Fatores Sexuais , Baço/diagnóstico por imagem , Baço/parasitologia , Baço/patologia , Ultrassonografia , Ganho de Peso
15.
Vet Parasitol Reg Stud Reports ; 15: 100267, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30929937

RESUMO

Visceral leishmaniosis (VL) remains a serious public health problem in Brazil. Dogs are the main hosts of the parasite, developing canine leishmaniosis (CanL), hence the importance of an accurate diagnosis of the animals. Recently, the application of qPCR method to non-invasive samples obtained from dogs with CanL has shown high sensitivity. Thus, we analyzed by qPCR blood, hair (from healthy zones and cutaneous lesions) and cerumen of 16 dogs with confirmed leishmaniosis from Araçatuba, a Brazilian endemic area. Cerumen-qPCR showed the highest sensitivity (87.5%), followed by hair (lesions: 78.57%, healthy skin: 62.5%), and blood (68.75%). We also analyzed blood, hair and cerumen of 5 healthy dogs from a non-endemic area, obtaining 100% of specificity in all samples. The use of cerumen and hair for qPCR analysis provides high reliability, taking into account the sensitivity and total specificity of the method. The non-invasive sampling procedure without the need of specific conditions of storage and transport support the usefulness of hair and cerumen for the diagnosis of CanL.


Assuntos
Cerume/parasitologia , Doenças do Cão/diagnóstico , Cabelo/parasitologia , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase em Tempo Real , Animais , Anticorpos Antiprotozoários/sangue , Brasil , Doenças do Cão/parasitologia , Cães/parasitologia , Leishmania infantum/genética , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
Internist (Berl) ; 60(8): 867-870, 2019 Aug.
Artigo em Alemão | MEDLINE | ID: mdl-30969356

RESUMO

A 52-year-old patient developed pancytopenia of unknown origin 1.5 years after allogeneic stem cell transplantation. The bone marrow aspirate showed visceral leishmaniasis (VL). Although VL is distributed world-wide, the incidence in patients after allogeneic stem cell transplantation is rare.


Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Pancitopenia/etiologia , Exame de Medula Óssea , Doença Enxerto-Hospedeiro , Humanos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Pessoa de Meia-Idade , Infecções Oportunistas/sangue , Infecções Oportunistas/diagnóstico , Infecções Oportunistas/parasitologia
17.
Vet J ; 245: 22-28, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30819422

RESUMO

The objective of this study was to compare changes in serum concentrations of acute phase proteins (APPs) and paraoxanase (PON-1) in response to two treatments in dogs with leishmaniosis (CanL). For this purpose, 20 dogs with CanL were assigned to two treatment groups: antimonial plus allopurinol (Group G, n=12) and miltefosine plus allopurinol (Group M, n=8). Serum concentrations of PON-1 and APPs including C-reactive protein, haptoglobin (Hp), ferritin (Ft) and albumin were monitored over a period of 3 months after treatment. At the beginning of the study (day 0), most of the dogs had APP abnormalities. None of the variables differed significantly between groups in the first or subsequent visits. There was a significantly higher reduction in serum Ft in Group G than in Group M from day 0 to day 30 (P=0.0085), and also from day 0 to day 90 (P=0.0214). There was a higher increase in serum PON-1 in Group G than in than Group M from day 0 to day 30 (P=0.0039), and also from day 0 to day 90 (P=0.0404). This is the first report of APPs in dogs with natural clinical leishmaniosis treated with miltefosine. There was faster resolution of serum APP concentrations in dogs treated with antimonials (P<0.05).


Assuntos
Proteínas da Fase Aguda/análise , Antiprotozoários/uso terapêutico , Doenças do Cão/sangue , Doenças do Cão/tratamento farmacológico , Leishmaniose Visceral/veterinária , Alopurinol/uso terapêutico , Animais , Arildialquilfosfatase/análise , Cães , Leishmania infantum , Leishmaniose Visceral/sangue , Leishmaniose Visceral/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Fosforilcolina/análogos & derivados , Fosforilcolina/uso terapêutico
18.
Am J Trop Med Hyg ; 100(4): 808-815, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30761980

RESUMO

We analyzed the association between insulin-like growth factor-I (IGF-I) and the pathogenesis of anemia during active visceral leishmaniasis (VL). Serum levels of IGF-I, IGF-binding protein 3 (IGFBP3), and cytokines were measured in samples from individuals with active VL and cured VL, asymptomatic Leishmania-infected, and noninfected individuals. Then, we extended our analysis to VL dogs to evaluate hematimetric parameters, bone marrow alterations, and cytokine and IGF-I expression. We identified a positive correlation between lower IGF-I and IGFBP3 levels in active VL patients and lower hemoglobin levels. In infected dogs, there was a positive correlation between lower IGF-I expression in the bone marrow and lower peripheral blood hematocrit and hemoglobin levels. There was no correlation between decreased IGF-I level/expression and any measured cytokine serum levels in either host. The data suggest that low IGF-I expression is associated with pathogenesis of anemia in active VL, primarily in severe cases, by mechanisms other than alterations in cytokine production.


Assuntos
Anemia/parasitologia , Progressão da Doença , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/análise , Leishmaniose Visceral/sangue , Leishmaniose Visceral/imunologia , Anemia/veterinária , Animais , Infecções Assintomáticas/epidemiologia , Brasil/epidemiologia , Criança , Pré-Escolar , Citocinas/sangue , Cães , Feminino , Humanos , Leishmania infantum , Leishmaniose Visceral/epidemiologia , Masculino
19.
Talanta ; 195: 327-332, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625550

RESUMO

Leishmaniasis is a disease with high impact on public health in many countries. Visceral leishmaniasis (VL) is a vectorial zoonosis, with dogs as primary reservoirs in the domestic environment. VL presents high morbidity, mortality and importance in epidemiology in the American continent. In the present study, the first label-free electrochemical impedance immunosensor using screen-printed electrodes (SPEs) for the detection of anti-Leishmania infantum antibodies was developed. The soluble antigens of L. infantum were immobilized on an SPE by a 3-mercaptopropionic acid monolayer. Electrochemical impedance spectroscopy (EIS) was used for detecting bimolecular interactions occurring at the electrode surface. The addition of real samples consisting of canine and human sera positive and negative for VL presented high sensitivity and selectivity through EIS. Based on the results, a sensitive, specific, rapid and simple immunosensor was developed successfully with potential application for the serological diagnosis of leishmaniasis disease.


Assuntos
Anticorpos Antiprotozoários/sangue , Técnicas Biossensoriais , Doenças do Cão/diagnóstico , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Espectroscopia Dielétrica , Doenças do Cão/sangue , Doenças do Cão/imunologia , Cães , Eletrodos , Ouro/química , Humanos , Imunoensaio , Leishmaniose Visceral/sangue , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/veterinária
20.
Microb Pathog ; 126: 263-268, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30419342

RESUMO

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoa of the genus Leishmania. Among the species, L. infantum and/or L. infantum (chagasi) are the most important species affecting the Americas. Domestic dogs are the main reservoir of the parasite and participate effectively in the parasite' transmission cycle. The Canine Visceral Leishmaniasis Control Program (PCLV) adopted in Brazil present as strategies the vector control, health education and serological diagnosis of CVL in dogs followed by culling of the seropositive ones. The resolution to eliminate seropositive dogs by euthanasia, when necessary, are the most controversial and least accepted by society. The diagnostic methods for canine visceral leishmaniasis, currently indicated and approved in Brazil by the Ministry of Health from Brazil are the Dual Path Platform (DPP)® as a screening test and the Enzyme immunoassay test (ELISA®). This study aimed to verify the presence of Leishmania spp. DNA in peripheral blood samples of dogs presenting positive serological results byDPP® and ELISA® tests,throughreal-time polymerase chain reaction (rt-PCR), using the pair of primers 150-152 already described. For this purpose, were collected blood samples from 185 seropositive dogs among them, 41 (22%) exhibited some clinical signal of disease, whereas 144 (78%) was asymptomatic. The animals were also analyzed according to gender, race and hair size. According to the results of rt-PCR, it was observed that among the185 seropositive dogs analyzed, only 132 (71%) presented positive results for CVL and 53 (29%) presented negative results. From this, 41/41 symptomatic dogs were positive (100%), while among the asymptomatic dogs, 91/144 were positive (63, 2%) and 53/144 were negative (36, 8%). Concerning the hair size of seropositive dogs, we found that 41 (22%) had long hair, while 144 (78%) had short hair. No statistical significance occurred between the results of rt-PCR, ELISA and DPP tests and the profile of the animals (gender, size of the dogs and hair size), probably due to the small number of samples and the sampling differences of each profile. But statistical significance occurred between the results of rt-PCR and the clinical evaluation, since the rt-PCR was positive in all symptomatic dogs. Thus, through these results, we reached at the following question, which may contribute to an important current debate: the dogs presenting CVL seropositive diagnosis confirmed by tests distributed by the Ministry of Health were in reality ill or were they seropositive by living in an endemic area of the disease? Would these asymptomatic seropositive dogs spread the disease to the inhabitants even presenting a low parasite charge circulating in the blood.


Assuntos
Doenças do Cão/diagnóstico , Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Anticorpos Antiprotozoários/sangue , Brasil , DNA de Protozoário/análise , Testes Diagnósticos de Rotina , Doenças do Cão/parasitologia , Cães , Feminino , Leishmania/patogenicidade , Leishmaniose Visceral/sangue , Masculino , Patologia Molecular , Testes Sorológicos/métodos , Testes Sorológicos/veterinária
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