RESUMEN
The co-infection between visceral leishmaniasis (VL) and human immunodeficiency virus (HIV) has increased in several countries in the world. The current serological tests are not suitable since they present low sensitivity to detect the most of VL/HIV cases, and a more precise diagnosis should be performed. In this context, in the present study, an immunoproteomics approach was performed using Leishmania infantum antigenic extracts and VL, HIV and VL/HIV patients sera, besides healthy subjects samples; aiming to identify antigenic markers for these clinical conditions. Results showed that 43 spots were recognized by antibodies in VL and VL/HIV sera, and 26 proteins were identified by mass spectrometry. Between them, ß-tubulin was expressed, purified and tested in ELISA experiments as a proof of concept for validation of our immunoproteomics findings and results showed high sensitivity and specificity values to detect VL and VL/HIV patients. In conclusion, the identified proteins in the present work could be considered as candidates for future studies aiming to improvement of the diagnosis of VL and VL/HIV co-infection.
Asunto(s)
Coinfección/diagnóstico , Infecciones por VIH/diagnóstico , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/diagnóstico , Proteómica/métodos , Proteínas Protozoarias/análisis , Adulto , Brasil , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.
Asunto(s)
Antígenos de Protozoos/aislamiento & purificación , Enfermedades de los Perros/parasitología , Leishmania infantum/inmunología , Leishmaniasis Visceral/diagnóstico , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Médula Ósea/parasitología , Biología Computacional , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Enfermedades de los Perros/diagnóstico , Perros , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito B/química , Femenino , Humanos , Inmunoglobulina G/sangre , Leishmania infantum/genética , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/veterinaria , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Protozoarias/química , Sensibilidad y Especificidad , Alineación de Secuencia , Pruebas Serológicas , Bazo/parasitología , Adulto JovenRESUMEN
The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.
Asunto(s)
Enfermedades de los Perros/parasitología , Factor 1 Eucariótico de Iniciación/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/veterinaria , Proteínas Protozoarias/inmunología , Adulto , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Reacciones Cruzadas , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática , Factor 1 Eucariótico de Iniciación/genética , Femenino , Humanos , Leishmania infantum/genética , Leishmania infantum/aislamiento & purificación , Leishmaniasis/diagnóstico , Leishmaniasis/inmunología , Leishmaniasis/parasitología , Leishmaniasis/veterinaria , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/inmunología , Masculino , Persona de Mediana Edad , Proteínas Protozoarias/genética , Pruebas SerológicasRESUMEN
There is no suitable vaccine against human visceral leishmaniasis (VL) and available drugs are toxic and/or present high cost. In this context, diagnostic tools should be improved for clinical management and epidemiological evaluation of disease. However, the variable sensitivity and/or specificity of the used antigens are limitations, showing the necessity to identify new molecules to be tested in a more sensitive and specific serology. In the present study, an immunoproteomics approach was performed in Leishmania infantum promastigotes and amastigotes employing sera samples from VL patients. Aiming to avoid undesired cross-reactivity in the serological assays, sera from Chagas disease patients and healthy subjects living in the endemic region of disease were also used in immunoblottings. The most reactive spots for VL samples were selected, and 29 and 21 proteins were identified in the promastigote and amastigote extracts, respectively. Two of them, endonuclease III and GTP-binding protein, were cloned, expressed, purified and tested in ELISA experiments against a large serological panel, and results showed high sensitivity and specificity values for the diagnosis of disease. In conclusion, the identified proteins could be considered in future studies as candidate antigens for the serodiagnosis of human VL.
Asunto(s)
Antígenos de Protozoos/inmunología , Leishmania infantum/fisiología , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias/inmunología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , ProteómicaRESUMEN
Visceral leishmaniasis (VL) represents a serious public health problem, as Leishmania infantum is one of main disease causative agents in the Americas. In a previous immunoproteomic study, the prohibitin (PHB) protein was identified in L. infantum promastigote and amastigote extracts by antibodies in asymptomatic and symptomatic VL dog sera. This protein was found to be highly conserved between different Leishmania spp., but it presented a low identity with amino acid sequences of other organisms. The aim of the present study was to evaluate the cellular response induced by the recombinant PHB (rPHB) protein in BALB/c mice, as well as in PBMCs purified from untreated and treated VL patients, as well as to evaluate its protective efficacy against an infection by L. infantum promastigotes. Our data showed that there was a Th1 cellular response to rPHB, based on high levels of IFN-γ, IL-12, and GM-CSF in the immunized animals, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from individuals who had recovered from the disease. The protection was represented by significant reductions in the parasite load in the animals' spleen, liver, bone marrow, and draining lymph nodes, as compared to results found in the control groups. In addition, an anti-rPHB serology, using a canine and human serological panel, showed a high performance of this protein when diagnosing VL based on high sensitivity and specificity values, as compared to results found for the rA2 antigen and the soluble Leishmania antigenic extract. Our data suggest that PHB has a potential application for the diagnosis of canine and human VL through antibody detection, as well as an application as a vaccine candidate to protect against disease.
Asunto(s)
Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/inmunología , Proteínas Represoras/inmunología , Animales , Antígenos de Protozoos/inmunología , Perros , Humanos , Leishmania infantum/inmunología , Leishmania infantum/metabolismo , Leishmaniasis Visceral/metabolismo , Ratones , Ratones Endogámicos BALB C , Prohibitinas , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Células TH1/inmunología , Vacunas/metabolismoRESUMEN
Secondary infections are one of the complications in COVID-19 patients. We aimed to analyze the antimicrobial prescriptions and their influence on drug resistance in fungi and bacteria isolated from severely ill COVID-19 patients. Seventy-nine severely ill COVID-19 hospitalized patients with secondary bacterial or fungal infections were included. We analyzed the prescribed antimicrobial regimen for these patients and the resistance profiles of bacterial and fungal isolates. In addition, the association between drug resistance and patients' outcome was analyzed using correlation tests. The most prescribed antibacterial were ceftriaxone (90.7% of patients), vancomycin (86.0%), polymyxin B (74.4%), azithromycin (69.8%), and meropenem (67.4%). Micafungin and fluconazole were used by 22.2 and 11.1% of patients, respectively. Multidrug-resistant (MDR) infections were a common complication in severely ill COVID-19 patients in our cohort since resistant bacteria strains were isolated from 76.7% of the patients. Oxacillin resistance was observed in most Gram-positive bacteria, whereas carbapenem and cephalosporin resistance was detected in most Gram-negative strains. Azole resistance was identified among C. glabrata and C. tropicalis isolates. Patients who used more antimicrobials stayed hospitalized longer than the others. The patient's age and the number of antibacterial agents used were associated with the resistance phenotype. The susceptibility profile of isolates obtained from severely ill COVID-19 patients highlighted the importance of taking microbial resistance into account when managing these patients. The continuous surveillance of resistant/MDR infection and the rational use of antimicrobials are of utmost importance, especially for long-term hospitalized patients with COVID-19.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacterias , Hongos , Prescripciones , Resistencia a MedicamentosRESUMEN
One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia , Palicourea tetraphylla , Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis , Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2%) exhibited at least one biological activity. Seventeen extracts (14%) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8%) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60% and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9%) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria , Arthrinium , Cochliobolus , Colletotrichum , Penicillium , Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.
RESUMEN
The serodiagnosis of visceral leishmaniasis (VL) presents problems related to the sensitivity and/or specificity of the tests. In this context, more refined antigens should be identified and applied for the improvement of disease diagnosis. In the present study, DNA with an encoding of a Leishmania infantum hypothetical protein, LiHyC, was cloned, and the recombinant protein was expressed, purified, and evaluated for the serodiagnosis of canine and human VL. In addition, a specific B-cell epitope present in the LiHyC sequence was predicted; the peptide was both synthetized and evaluated in the ELISA experiments. For comparison, commercial diagnostic kits were used against positive (VL hosts) and negative (healthy hosts) samples. Results showed that the recombinant protein (rLiHyC) and synthetic peptide (PeptC) were highly sensitive and specific to diagnose canine and human VL, with 100% sensitivity and specificity, while no false-positive or false-negative result was detected. When the DPP® CVL kit was used to identify canine samples, 44 and 52 of the 60 L. infantum-infected animals, without or with clinical signals of disease, respectively, were identified, while eight and four samples were considered as false-negatives, respectively. For human VL, an IT LEISH® kit was used, and 33 of the 40 VL patients were identified, while seven samples were considered to be false-negatives. Post-therapeutic serological follow-up testing sera samples from treated and untreated VL patients showed a significant drop in the anti-PeptC and anti-rLiHyC antibody levels, thus suggesting the feasibility to use the recombinant protein and/or synthetic peptide in future studies as diagnostic and/or prognostic markers for VL.
Asunto(s)
Epítopos de Linfocito B/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/diagnóstico , Adulto , Animales , Antígenos de Protozoos/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Pruebas Serológicas/métodosRESUMEN
The detection of Leishmania spp. in skin lesion aspirates, using a puncture technique, was evaluated in 76 patients with cutaneous leishmaniasis (CL) who were referred to a Leishmaniasis Reference Centre in Brazil. CL was defined based on skin lesions suggestive of the disease and on a positive result of the Montenegro skin test or Giemsa-stained imprints of biopsy fragments. The aspirates were cultured using a vacuum tube device containing culture medium and evaluated for the presence of Leishmania spp. The biphasic medium culture was examined once a week for three weeks. Promastigotes were observed in 53/76 (69.7%) cultures. Stained smears from 60 of the 76 patients were evaluated using PCR-RFLP to detect the conserved minicircle region of Leishmania spp. and to classify the parasite. Of these patients, 45 (75%) showed positive results in aspirate culture and 15 presented negative results. The PCR was positive in 80% (53/60) samples. The PCR-RFLP profile was determined in 49 samples, of which 45 (92%) showed a pattern compatible with Leishmania (Viannia) braziliensis. The aspirate culture is a sensitive and feasible method for diagnosing CL and may be routinely adopted by health services for L. (V.) braziliensis isolation and identification.
Asunto(s)
Leishmania braziliensis/aislamiento & purificación , Leishmania mexicana/aislamiento & purificación , Leishmaniasis Cutánea/diagnóstico , Adulto , Animales , Técnicas de Cultivo , ADN Protozoario/análisis , Femenino , Humanos , Leishmania braziliensis/genética , Leishmania braziliensis/crecimiento & desarrollo , Leishmania mexicana/genética , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Cutánea/parasitología , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Pruebas CutáneasRESUMEN
Serological tests are important tools for the diagnosis of Leishmania infection. However, they are not effective markers to diagnose asymptomatic cases of visceral leishmaniasis (VL) and patients developing tegumentary leishmaniasis (TL), since antileishmanial antibodies can be encountered in low levels resulting in false-negative results in the serological trials. In this context, antigens able to be recognized by antibodies in sera from both VL and TL patients will be desirable to be employed in a more sensitivity and specific diagnosis of disease. In the present study, a conserved Leishmania protein, small myristoylated protein-3 (SMP-3), which was showed to be conserved in different Leishmania species and an effective vaccine candidate against Leishmania infantum infection in a murine model, was cloned and the recombinant protein was evaluated as a serological marker for the diagnosis of human TL and canine VL. In addition, a linear B cell-specific epitope (MQKDEESGEFKCEL) was identified, synthetized and also investigated as a serological marker. As antigen controls, rA2 protein and antigenic Leishmania extracts (SLA) were used. Results showed that ELISA-rSMP-3 and ELISA-Peptide presented sensitivity and specificity values higher than 90% in both diseases in humans and canids, having identified all asymptomatic cases and did not present cross-reaction with cross-reactivity diseases in both mammalian hosts. On the other hand, sensitivity and specificity values were worst when rA2 or SLA were used as antigens in humans and dogs. In conclusion, results showed the efficacy and Leishmania SMP-3 protein, employed as a recombinant antigen or a B cell epitope, for the improvement of the serodiagnosis of human TL and canine VL. This candidate can be tested in other diagnostic platforms, such as rapid immunochromatographic dipstick tests, aiming its use in epidemiological studies in remote areas where laboratories are not readily accessible for conventional assays.
Asunto(s)
Antígenos de Protozoos/inmunología , Enfermedades de los Perros/diagnóstico , Epítopos de Linfocito B/inmunología , Leishmania/fisiología , Leishmaniasis Visceral/diagnóstico , Proteínas Recombinantes/inmunología , Zoonosis/diagnóstico , Adulto , Anciano , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Reacciones Cruzadas , Perros , Epítopos de Linfocito B/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/genética , Sensibilidad y Especificidad , Pruebas Serológicas , Adulto JovenRESUMEN
The measures for leishmaniasis control include the precise diagnosis of disease. However, although several recombinant antigens have been tested with this biotechnological purpose, no effective product exists, which could detects patients with the active disease, as well as differentiates them from cured and treated patients. In this study, a conserved Leishmania hypothetical protein, which was identified in Leishmania infantum parasites, but evaluated to presents high homology in the amino acid sequences between distinct parasite species, was evaluated for the diagnosis of tegumentary and visceral leishmaniasis. In addition, PBMCs collected from treated and untreated mucosal leishmaniasis (ML) and visceral leishmaniasis (VL) patients, as well as in healthy subjects living in endemic region of disease, were in vitro stimulated, when IFN-γ, IL-4 and IL-10 levels were evaluated in the cell supernatant. Regarding the serological analyses, ELISA experiments using the recombinant protein (rLiHyL) and a human serological panel revealed high sensitivity and specificity values to detect both diseases, while control antigens showed worst results. Regarding the cellular response, results showed that rLiHyL-stimulated cells produced higher IFN-γ and lower IL-4 and IL-10 levels in the supernatants. Also, the anti-protein antibody production was evaluated in these patients, and data showed higher IgG2 and lower IgG1 levels found in the treated patients and healthy controls, demonstrating the stimulation of a Th1-type response induced by the rLiHyL protein. In conclusion, this hypothetical protein can be considered as antigenic in TL and VL, as well as a vaccine candidate to be tested in future studies to protect against disease.
Asunto(s)
Antígenos de Protozoos/inmunología , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes , Adulto , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/genética , Biomarcadores , Estudios de Casos y Controles , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/parasitología , Masculino , Persona de Mediana Edad , Proteínas Protozoarias/genética , Pruebas SerológicasRESUMEN
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.
Asunto(s)
Antígenos de Protozoos/inmunología , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/veterinaria , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Células Cultivadas , Citocinas/metabolismo , Enfermedades de los Perros/sangre , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Perros , Epítopos de Linfocito B/inmunología , Humanos , Inmunogenicidad Vacunal , Leishmania infantum/inmunología , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/parasitología , Leucocitos Mononucleares/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
Visceral leishmaniasis (VL) is a potentially fatal disease, in which the treatment based on chemotherapy is considered toxic. The cure of disease is associated with the life-long Th1-type immunity against the infection. The Th1-related cytokines production by peripheral blood mononuclear cells (PBMCs) seems to be crucial for host control of parasite load and clinical cure. In the current study, we used five proteins (IgE-dependent histamine-releasing factor [HRF], LiHyD, LiHyV, LiHyT and LiHyp6) recently shown to be antigenic and/or immunogenic in the canine VL, aiming to evaluate the antigen-specific antibody levels and cytokine production in PBMCs culture supernatants collected from VL patients before and after anti-VL treatment. In the results, when PBMCs were exposed to rHRF, rLiHyD and rLiHyT, higher IFN-γ and lower IL-10 levels were observed in all patients that were treated and clinically cured. Analysis of specific antibody subclasses was in line with in vitro cellular response, since a higher IgG2 production was found in the treated and cured patients, when compared to the IgG1 subclass levels. In addition, evaluating the diagnostic efficacy of the recombinant molecules, the rHRF, rLiHyD and rLiHyT proteins showed the best results in the serology assays to identify all VL patients, as well as these antigens were not recognized by antibodies in sera from non-infected subjects or those with leishmaniasis-related diseases. Our results corroborate the view that clinical cure of VL is associated with a sustained Th1-related response, and indicate the potential use of rHRF, rLiHyD and rLiHyT as immune biomarkers of VL treatment.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Biomarcadores Farmacológicos/sangre , Leishmania infantum/fisiología , Leishmaniasis Visceral/diagnóstico , Leucocitos Mononucleares/inmunología , Células TH1/inmunología , Adulto , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Células Cultivadas , Progresión de la Enfermedad , Perros , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Leishmaniasis Visceral/terapia , Leucocitos Mononucleares/parasitología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Adulto JovenRESUMEN
In the present study, a conserved Leishmania hypothetical protein, LiHyE, was evaluated for the serodiagnosis of leishmaniasis. Results showed that it presented high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) to serologically identify visceral leishmaniasis (VL) dogs when 40 positive sera and 95 cross-reactive samples were used. rLiHyE also showed the best results of sensitivity, specificity, PPV, and NPV to identify tegumentary leishmaniasis (TL) and VL patients when 45 leishmaniasis patients' sera and 90 cross-reactive samples were used. Results were better in comparison to those obtained when rA2 or Leishmania antigenic extract was employed as controls. The posttreatment follow-up showed that rLiHyE-specific antibodies declined significantly after the end of treatments, and a predominance of the IgG2 subclass was found in comparison to IgG1 levels in both TL and VL patients. In conclusion, rLiHyE can be considered a candidate for the serodiagnosis of canine and human leishmaniasis.
Asunto(s)
Biomarcadores/sangre , Enfermedades de los Perros/diagnóstico , Leishmaniasis Visceral/diagnóstico , Leishmaniasis/diagnóstico , Proteínas Protozoarias , Pruebas Serológicas , Animales , Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/parasitología , Perros , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Seguimiento , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/parasitología , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas/métodosRESUMEN
New candidates for serological markers against leishmaniasis are required to be identified, since the presence of high titers of anti-Leishmania antibodies remain detected in sera of treated and cured patients, when current antigens have being employed. In this study, the diagnostic performance of a conserved Leishmania hypothetical protein was evaluated against a human and canine serological panel. The serological follow-up of the patients was also evaluated, using this recombinant antigen (rLiHyS) in ELISA assays. In the results, high sensitivity and specificity values were found when rLiHyS was used in the serological tests, while when the recombinant A2 (rA2) protein or an antigenic Leishmania preparation were used as controls, low sensitivity and specificity were found. Regarding the serological follow-up of the patients, significant reductions in the anti-rLiHyS antibody levels were found and, one year after the treatments, the anti-protein IgG production was similar to this found in the non-infected groups, reflecting a drop of the anti-rLiHyS antibody production. In conclusion, the present study shows for the first time a new recombinant antigen used to identify tegumentary and visceral leishmaniasis, as well as being able to serologically distinguish treated and cured patients from those developing active disease.
Asunto(s)
Leishmania braziliensis/inmunología , Leishmania infantum/inmunología , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Visceral/diagnóstico , Proteínas Protozoarias/inmunología , Adulto , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Biomarcadores/sangre , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/inmunología , Enfermedades de los Perros/sangre , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/parasitología , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Leishmania braziliensis/química , Leishmania infantum/química , Leishmaniasis Cutánea/inmunología , Leishmaniasis Visceral/dietoterapia , Leishmaniasis Visceral/inmunología , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Protozoarias/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas/métodos , Adulto JovenRESUMEN
BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. OBJECTIVE: The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. METHODS: To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. RESULTS: The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. CONCLUSION: The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological samples in order to validate this method and then generate a diagnostic kit for Paracoccidioidomycosis.
Asunto(s)
Proteínas Fúngicas/genética , Paracoccidioides/aislamiento & purificación , Paracoccidioidomicosis/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Brasil , Simulación por Computador , Genoma Fúngico , Humanos , Paracoccidioides/genética , Paracoccidioidomicosis/microbiología , Patentes como Asunto , Reacción en Cadena en Tiempo Real de la Polimerasa/economía , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
BACKGROUND: Subcutaneous phaeohyphomycosis is an infection caused by melanized fungi and is increasingly reported among immunosuppressive patients. The most commonly cited etiologic agent is Exophiala jeanselmei, followed by Alternaria spp. We present a case of subcutaneous phaeohyphomycosis in a 48-yearold woman, with a history of lepromatous leprosy, using corticosteroid in immunosuppressive doses due to a type 2 repetitive reaction leprosy outbreak. RESULT AND DISCUSSION: The diagnosis was confirmed by fine-needle aspiration of the secretion, with subsequent direct mycological observations, culture and molecular analysis. The species agent was identified by culture and nucleotide sequences of ribosomal DNA as Exophiala dermatitidis.
Asunto(s)
Exophiala/aislamiento & purificación , Lepra Lepromatosa/complicaciones , Feohifomicosis/complicaciones , Feohifomicosis/microbiología , Corticoesteroides/uso terapéutico , Biopsia con Aguja Fina , ADN Ribosómico , Exophiala/genética , Femenino , Humanos , Huésped Inmunocomprometido , Lepra Lepromatosa/microbiología , Persona de Mediana Edad , Feohifomicosis/diagnósticoRESUMEN
BACKGROUND: Propolis has been used as a natural health product mainly due to the presence of polyphenols, flavonoids, phenolic aldehydes, amino acids, vitamins and others bioactive constituents. To this natural substance are attributed different biological and pharmacological properties which are influenced by its chemical composition and organoleptic properties. The aim of this work was to evaluate the physicochemical properties and parameters of green propolis collected during a period of six years (2008-2013) in the state of Minas Gerais, located at the southeastern region of Brazil. METHODS: The methodology were in accordance with Brazilian legislation on the identity and quality standards of propolis. The evaluated parameters of hydroalcoholic from green propolis were total flavonoids, antioxidant activity - DPPH method, oxidation index, wax content, humidity and insoluble impurities. RESULTS: Propolis samples collected in different seasons during the years 2008 to 2013 presented mean values of total flavonoids (3.4 ± 0.11 mg/g), antioxidant activity DPPH (4.76 ± 0.16 µg/mL), oxidation index (3, 4 ± 0.33 seconds) and wax (15.14 ± 0.78% m/m), which are in accordance with Brazilian legislation. CONCLUSION: Green propolis did not show abrupt seasonal changes during the six years of investigation, and may be considered as an adequate functional ingredient.
Asunto(s)
Própolis/química , Antioxidantes/química , Baccharis , Compuestos de Bifenilo/química , Brasil , Flavonoides/análisis , Fenoles/análisis , Picratos/química , Estaciones del Año , Ceras/análisisRESUMEN
Different Leishmania proteins have been evaluated in order to find a potential vaccine candidate or diagnostic marker capable of providing long lasting protection against infection or helping to identify infected mammalian hosts, respectively. However, just few molecules have fulfilled all the requirements to be evaluated. In the current study, we evaluated the prophylactic and diagnostic value against visceral leishmaniasis (VL) of a small glutamine-rich tetratricopeptide repeat-containing (SGT) protein from Leishmania infantum species. In a first step, the immune response elicited by the immunization using the recombinant protein (rSGT) plus saponin was evaluated in BALB/c mice. Immunized animals had a low parasitism in all evaluated organs. They developed a specific Th1 immune response, which was based on protein-specific production of IFN-γ, IL-12 and GM-CSF, and a humoral response dominated by antibodies of the IgG2a isotype. Both CD4+ and CD8+ T cells contributed to the IFN-γ production, showing that both T cell subtypes contribute to the resistance against infection. Regarding its value as a diagnostic marker, rSGT showed maximum sensitivity and specificity to serologically identify L. infantum-infected dog and human sera. No cross-reactivity with sera from humans or dogs that had other diseases was found. Although further studies are necessary to validate these findings, data showed here suggest immunogenicity of rSGT and its protective effect against murine VL, as well as its potential for the serodiagnosis of human and canine VL.
Asunto(s)
Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/prevención & control , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Reacciones Cruzadas , Citocinas/inmunología , Perros , Femenino , Humanos , Inmunoglobulina G/inmunología , Leishmania infantum/genética , Vacunas contra la Leishmaniasis/genética , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/inmunología , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Proteínas Protozoarias/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Células TH1/inmunología , Células TH1/patologíaRESUMEN
A duplex polymerase chain reaction (PCR) assay was performed using a sense Leishmania genus-specific oligonucleotides to amplify the conserved region of 120 bp of minicircle kDNA and an additional antisense Leishmania donovani oligonucleotide that amplifies a complex-specific fragment of 90 bp. All 12 tested reference isolates of the L. donovani complex yielded a complex-specific amplification product of 90 bp concurrently with a genus-specific 120-bp product. All 17 tested reference isolates pertaining to other complexes presented only the genus-specific 120-bp product. Peripheral blood samples of twenty patients with visceral leishmaniasis positive by the genus-specific PCR presented both fragments with the duplex assay. This duplex PCR can be applied as a 1-step diagnostic tool where discrimination of L. donovani complex species is relevant for clinical and epidemiologic purposes.