RESUMEN
Sporotrichosis diagnosis involves a series of analyses, including culture and antibody detection in serum samples. Serologic methods may sometimes yield false-negative or false-positive results, leading to inaccurate diagnoses. This study assessed specific patient groups in which antibody detection of different isotypes and subclasses may lack sensitivity. An enzyme-linked immunosorbent assay (ELISA) with Sporothrix brasiliensis exoantigens was used to investigate IgM, IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgA1 and IgA2 antibodies in human serum samples. Eighty serum samples from patients with different sporotrichosis clinical manifestations, including cutaneous forms with and without hypersensitivity manifestations, extracutaneous forms (bone, ocular, meningeal and pulmonary), disseminated cutaneous forms and disseminated forms in individuals living with HIV/AIDS, diabetics and alcoholics, were evaluated. The ELISA sensitivities in the detection of different antibodies ranged from 0.85 to 0.60 for the detection of IgG2 and IgG3, respectively. The antibodies with higher area under ROC curves were IgG2, IgG, IgA and IgA1. There were no significant differences in the immunological reactivity of the tested antibodies among different clinical forms of sporotrichosis. The data revealed a higher likelihood of a false-negative outcome in patients with lesions in the nasal mucosa regarding the detection of IgM and a lower likelihood in patients with lymphocutaneous sporotrichosis regarding the detection of IgG3. Patients with hypersensitivity manifestations had a 3.71 odds ratio to yield negative results in total IgG detection. In conclusion, we identified specific patient groups in which antibody detection may lack sensitivity, thus contributing to a better understanding of the diagnostic challenges associated with this condition.
Asunto(s)
Anticuerpos Antifúngicos , Ensayo de Inmunoadsorción Enzimática , Sensibilidad y Especificidad , Sporothrix , Esporotricosis , Humanos , Esporotricosis/inmunología , Esporotricosis/diagnóstico , Anticuerpos Antifúngicos/sangre , Sporothrix/inmunología , Sporothrix/clasificación , Masculino , Femenino , Adulto , Persona de Mediana Edad , Isotipos de Inmunoglobulinas/sangre , Isotipos de Inmunoglobulinas/inmunología , Inmunoglobulina G/sangre , Anciano , Adulto Joven , Antígenos Fúngicos/inmunología , Antígenos Fúngicos/sangre , Inmunoglobulina A/sangre , Inmunoglobulina M/sangreRESUMEN
OBJECTIVES: The aim of the present study was to evaluate minimally invasive diagnostic techniques, such as the semi-quantitative indirect IgG antibody enzyme immunoassay (EIA) using blood serum and the urinary lateral flow assay (LFA), for the detection of Histoplasma capsulatum in cats with histoplasmosis. METHODS: Eight client-owned domestic cats diagnosed with histoplasmosis were selected based on cytological, histopathological, mycological, molecular or antigenic techniques. The blood serum of these animals was tested in a semi-quantitative indirect IgG antibody EIA for the detection of H capsulatum. Urine samples were tested for H capsulatum antigen using LFA. RESULTS: Five cats were seropositive on IgG EIA (5/8, with diagnostic sensitivity equal to 62.5%; 95% confidence interval [CI] 24.5-91.5) and five cats were positive on H capsulatum antigen LFA (5/7, with diagnostic sensitivity equal to 71.4%; 95% CI 29.0-96.3). The combined diagnostic sensitivity when interpreted in parallel was 87.5% (7/8, 95% CI 47.3-99.7). The specificity for the anti-Histoplasma IgG EIA was 100% (95% CI 71.5-100) and for the H capsulatum antigen LFA it was also 100% (95% CI 71.5-100). CONCLUSIONS AND RELEVANCE: The semi-quantitative indirect IgG antibody EIA for the detection of H capsulatum in blood serum and the urinary LFA for the detection of the same agent emerge as new minimally invasive diagnostic techniques that can assist in the approach to disseminated and pulmonary feline histoplasmosis, especially when both techniques are considered together.
Asunto(s)
Enfermedades de los Gatos , Histoplasma , Histoplasmosis , Sensibilidad y Especificidad , Gatos , Animales , Histoplasmosis/veterinaria , Histoplasmosis/diagnóstico , Enfermedades de los Gatos/diagnóstico , Enfermedades de los Gatos/microbiología , Histoplasma/aislamiento & purificación , Histoplasma/inmunología , Masculino , Femenino , Anticuerpos Antifúngicos/sangre , Técnicas para Inmunoenzimas/veterinaria , Inmunoglobulina G/sangreRESUMEN
Brazil-grown outdoor-cultivated Agaricus brasiliensis KA21 fruiting body (KA21) significantly increases the production of serum anti-beta-glucan antibody. Therefore, KA21 ingestion may be useful for the prevention and alleviation of fungal infections. This study aimed to determine the effects of KA21 in fungal infections in animals. KA21 was administered to nine dogs infected with Malassezia. Notably, the anti-beta-glucan antibody titer remained unchanged or tended to decrease in the oral steroid arm, whereas in the non-steroid arm, antibody titer increased in almost all animals after KA21 ingestion. Dogs showing improved clinical symptoms exhibited increased anti-beta-glucan antibody titers. The results of this study suggest that KA21 ingestion may alleviate the symptoms of Malassezia and other fungal infections and that continuous ingestion may help prolong recurrence-free intervals. Additionally, the ingestion of KA21 during oral steroid dosage reduction or discontinuation may enable smoother steroid withdrawal.
Asunto(s)
Agaricus , Enfermedades de los Perros , Cuerpos Fructíferos de los Hongos , Malassezia , Animales , Perros , Agaricus/química , Cuerpos Fructíferos de los Hongos/química , Malassezia/efectos de los fármacos , Enfermedades de los Perros/microbiología , Enfermedades de los Perros/tratamiento farmacológico , Dermatomicosis/veterinaria , Dermatomicosis/prevención & control , Dermatomicosis/tratamiento farmacológico , Dermatomicosis/microbiología , beta-Glucanos/administración & dosificación , beta-Glucanos/farmacología , Masculino , Brasil , Dermatitis/tratamiento farmacológico , Dermatitis/veterinaria , Dermatitis/microbiología , Dermatitis/prevención & control , Femenino , Anticuerpos Antifúngicos/sangreRESUMEN
The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.
Asunto(s)
Anticuerpos Antifúngicos , Anticuerpos Monoclonales , Cryptococcus neoformans , Hibridomas , Cryptococcus neoformans/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Humanos , Hibridomas/inmunología , Anticuerpos Antifúngicos/inmunología , Anticuerpos Antifúngicos/aislamiento & purificación , Ratones , Linfocitos B/inmunología , Criptococosis/inmunología , Criptococosis/diagnóstico , Antígenos Fúngicos/inmunología , InmunizaciónRESUMEN
BACKGROUND: Accurate diagnosis of paracoccidioidomycosis is crucial for improving patient outcomes. Paracoccidioides antibody detection by double immunodiffusion (DID) is a convenient diagnostic tool, but testing performance can vary based on certain factors. METHODS: We assessed DID performance using a commercially prepared Paracoccidioides reagents (IMMY, USA), involving 40 serum specimens, including 20 from patients with proven paracoccidioidomycosis and 20 from patients without the disease. The DID test demonstrated a sensitivity of 90% (95% CI=68%-99%) and a specificity of 100% (95% CI=83%-100%). CONCLUSIONS: Our findings suggest that DID using commercial reagents may provide a feasible tool with satisfactory testing performance for anti-Paracoccidioides antibody detection.
Asunto(s)
Anticuerpos Antifúngicos , Inmunodifusión , Paracoccidioides , Paracoccidioidomicosis , Sensibilidad y Especificidad , Humanos , Anticuerpos Antifúngicos/sangre , Paracoccidioidomicosis/diagnóstico , Paracoccidioidomicosis/inmunología , Paracoccidioides/inmunología , Juego de Reactivos para Diagnóstico , Femenino , MasculinoRESUMEN
Definitive diagnosis of histoplasmosis relies on culture and/or cytology/histopathology; however, these procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has low sensitivity in immunocompromised individuals and uses histoplasmin (HMN), a crude antigenic extract, as reagent. Novel protein antigen candidates have been recently identified and produced by DNA-recombinant techniques to obtain standardized and specific reagents for diagnosing histoplasmosis. To compare the analytical performance of novel enzyme-linked immunosorbent assays (ELISAs) for antibodies testing for diagnosing histoplasmosis using different Histoplasma capsulatum antigens as reagents. The H. capsulatum 100 kDa protein (Hcp100), the M antigen and its immunoreactive fragment F1 were produced by DNA-recombinant techniques. Galactomannan was purified from both the yeast and mycelial cell walls (yGM and mGM, respectively). The analytical performance of the ELISA tests for the serological detection of antibodies against these antigens was evaluated and compared with those obtained using HMN as reagent. Antibodies detection by the Hcp100 ELISA demonstrated 90.0% sensitivity and 92.0% specificity, versus 43.3% sensitivity and 95.0% specificity of the M ELISA, 33.3% sensitivity and 84.0% specificity of the F1 ELISA, 96.7% sensitivity and 94.0% specificity of the yGM ELISA, 83.3% sensitivity and 88.0% specificity of the mGM ELISA, and 70.0% sensitivity and 86.0% specificity for the HMN ELISA. In summary, Hcp100 is proposed as the most promising candidate for the serodiagnosis of histoplasmosis. The primary immunoreactive element in HMN proved to be GM rather than the M antigen. Nevertheless, a higher incidence of cross-reactions was noted with GM compared to M.
Hcp100 is a promising serodiagnostic candidate for histoplasmosis, boasting high sensitivity and specificity. Notably, GM, rather than M antigen, emerged as the primary immunoreactive element in HMN, despite a higher incidence of cross-reactions with GM compared to M.
Asunto(s)
Histoplasmosis , Humanos , Histoplasmosis/diagnóstico , Histoplasmosis/veterinaria , Histoplasma/genética , Anticuerpos Antifúngicos , Técnicas para Inmunoenzimas , Antígenos Fúngicos , Anticuerpos , Inmunodifusión/veterinaria , Saccharomyces cerevisiae , ADNRESUMEN
Patients with severe COVID-19 are at increased risk for invasive fungal infections, which are underestimated. Histoplasmosis reactivation in endemic areas should not be overlooked in this population. In a previous study, seroconversion to anti-histoplasmin antibodies by ELISA was detected in 6/39 (15.4%) patients with severe COVID-19. In this work, samples were further investigated to detect seroconversion to antibodies against the Histoplasma capsulatum 100-kDa antigen (Hcp100) by ELISA. Seroconversion to anti-Hcp100 antibodies was detected in 7/39 patients, of whom 6 also seroconverted anti-histoplasmin antibodies. These results reinforce previous findings that show histoplasmosis as an underdiagnosed fungal entity complicating COVID-19.
This study verifies that patients with severe COVID-19 at intensive care units are at risk for histoplasmosis reactivation in endemic areas. Accurate diagnosis of this deadly fungal disease among critically ill patients with COVID-19 living in endemic areas for histoplasmosis is needed.
Asunto(s)
COVID-19 , Histoplasmosis , Animales , Histoplasmosis/diagnóstico , Histoplasmosis/epidemiología , Histoplasmosis/microbiología , Histoplasmosis/veterinaria , Histoplasmina , Histoplasma , Enfermedad Crítica , Anticuerpos Antifúngicos , COVID-19/veterinaria , Antígenos FúngicosRESUMEN
Fungal infections usually occur in immunocompromised patients. Intravenous immunoglobulin (IVIG) has been used as therapeutic interventions for many infectious diseases, but seldom applied in mycosis due to unknown antifungal specificity. This study aims to determine the presence of antifungal antibodies in IVIG. Binding reactivity of IVIG with crude and recombinant antigens of Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Talaromyces marneffei were observed in a dose-dependent manner, similar with mixed normal human sera. The antifungal specificity was further confirmed by competitive enzyme-linked immunosorbent assays (ELISA) inhibited by rabbit specific antifungal polyclonal antibodies (PAbs) and homogenous crude antigens with inhibitions of 65.5-87.2% and 73.1-94.2%, respectively. Moreover, IVIG also reacted with fungal glycoproteins (Csa2, Cpl1 and Mp1p) in a dose-dependent way, which was inhibited by specific rabbit PAbs and homogenous antigens with different inhibitions and pulled down 72.8-83.8% of specific antibodies if preabsorption IVIG with Dynabeads® coupled with homogenous glycoproteins. These results furthermore verified the antifungal specificity of IVIG. Among four brands of IVIG, there was different antifungal IgG against C. albicans (P < 0.05) and C. neoformans (P < 0.05), while no difference for A. fumigatus (P = 0.086) and T. marneffei (P = 0.057). IVIG contained a significantly higher level of specific IgG for C. albicans than other three fungi (P <0.001). In conclusion, we proved antifungal IgG against C. albicans, A. fumigatus, C. neoformans and T. marneffei present in IVIG, which might be expected to provide a possible immunoregulation choice for mycosis and an evaluation to humoral immunity against fungi.
Asunto(s)
Cryptococcus neoformans , Micosis , Animales , Humanos , Conejos , Antifúngicos/farmacología , Inmunoglobulinas Intravenosas , Micosis/microbiología , Candida albicans , Aspergillus fumigatus , Anticuerpos AntifúngicosRESUMEN
The patients with severe COVID-19 are at increased risk for invasive fungal infections, such as invasive pulmonary aspergillosis and candidiasis, which increase morbidity and mortality. However, clinicians should also consider the possibility of reactivating latent Histoplasma capsulatum in patients with severe COVID-19 living within areas of endemicity who have worsening respiratory function or sepsis, even if they do not have classical risk factors for histoplasmosis (e.g., HIV/AIDS). Bearing in mind this scenario, serum samples of 39 non-HIV/AIDS patients from Buenos Aires hospitalized due to severe COVID-19 pneumonia were analyzed for anti-H. capsulatum-specific IgG antibodies by an in-house ELISA. Antibodies against H. capsulatum were detected in the sera of 8/39 patients (20.51%). To exclude the possibility that these antibodies arose from past exposure of these patients to the fungus, paired serum samples obtained after an interval of at least 10 days were evaluated. Of them, five patients (62.5%) with negative anti-H. capsulatum antibodies at baseline became seropositive 7-10 days later. Three patients (37.5%) had positive anti-H. capsulatum antibodies at baseline, but at time point 2, one of them became seronegative and the other one diminished the antibody titers (4000 vs. 16000 at baseline). The remaining patients displayed higher antibody titers at time point 2 (4000 vs. 1000 at baseline) and died immediately thereafter. In conclusion, awareness of the possibility of fungal co-infections is essential to reduce delays in diagnosis and treatment in order to help prevent severe illness and death from these infections. LAY SUMMARY: This study verifies that patients with severe COVID-19 at ICU are at risk for histoplasmosis reactivation in endemic areas. Accurate diagnosis of this deadly fungal disease among critically ill patients with COVID-19 living in endemic areas for histoplasmosis is needed.
Asunto(s)
Anticuerpos Antifúngicos/sangre , COVID-19 , Histoplasmosis , COVID-19/diagnóstico , COVID-19/epidemiología , Enfermedad Crítica , Histoplasma/inmunología , Histoplasmosis/diagnóstico , Histoplasmosis/epidemiología , Humanos , SARS-CoV-2 , SeroconversiónRESUMEN
Paracoccidioidomycosis (PCM) is a systemic disease caused by Paracoccidioides spp. PCM is endemic in Latin America and most cases are registered in Brazil. This mycosis affects mainly the lungs, but can also spread to other tissues and organs, including the liver. Several approaches have been investigated to improve treatment effectiveness and protection against the disease. Extracellular vesicles (EVs) are good antigen delivery vehicles. The present work aims to investigate the use of EVs derived from Paracoccidioides brasiliensis as an immunization tool in a murine model of PCM. For this, male C57BL/6 were immunized with two doses of EVs plus adjuvant and then infected with P. brasiliensis. EV immunization induced IgM and IgG in vivo and cytokine production by splenocytes ex vivo. Further, immunization with EVs had a positive effect on mice infected with P. brasiliensis, as it induced activated T lymphocytes and NKT cell mobilization to the infected lungs, improved production of proinflammatory cytokines and the histopathological profile, and reduced fungal burden. Therefore, the present study shows a new role for P. brasiliensis EVs in the presence of adjuvant as modulators of the host immune system, suggesting their utility as immunizing agents.
Asunto(s)
Antígenos Fúngicos/inmunología , Vesículas Extracelulares/microbiología , Paracoccidioides/inmunología , Paracoccidioidomicosis/inmunología , Paracoccidioidomicosis/microbiología , Sustancias Protectoras/farmacología , Animales , Anticuerpos Antifúngicos/inmunología , Movimiento Celular , Citocinas/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Inmunización , Memoria Inmunológica , Pulmón/microbiología , Pulmón/patología , Activación de Linfocitos/inmunología , Masculino , Ratones Endogámicos C57BL , Células T Asesinas Naturales/inmunología , Estándares de ReferenciaRESUMEN
BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii. METHODOLOGY AND MAIN FINDINGS: This study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome. CONCLUSIONS: This study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented.
Asunto(s)
Antígenos Fúngicos/inmunología , Paracoccidioides/inmunología , Paracoccidioidomicosis/inmunología , Paracoccidioidomicosis/microbiología , Adolescente , Adulto , Anticuerpos Antifúngicos/inmunología , Brasil/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Paracoccidioides/clasificación , Paracoccidioides/aislamiento & purificación , Paracoccidioidomicosis/diagnóstico , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Serotipificación , Adulto JovenRESUMEN
The genus Scedosporium is composed of clinically relevant fungal species, such as Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium boydii. Surface molecules have been described that play crucial roles in fungi-macrophage interaction, and many of them are pathogen-associated molecular patterns (PAMPs). The present study aims to characterize peptidoglycans obtained from Scedosporium aurantiacum and Scedosporium minutisporum, a clinical and an environmental isolate, respectively, and compare their roles in pathogen-host interaction. Both molecules were characterized as peptidorhamnomannans (PRMs), similar to what has been already described for other Scedosporium species. Rabbit immune sera obtained by injecting whole cells from each species recognized both fungal cells and purified PRMs, suggesting that a cross-reaction occur between both fungi. Immunofluorescent microscopy revealed that PRMs are exposed on fungal surface. Prior incubation of purified molecules with immune sera before adding to cells led to loss of fluorescent, indicating that PRM is a major molecule recognized by immune sera. Fungi-macrophage interaction revealed that S. aurantiacum is able to survive more inside phagocytic cells than S. minutisporum, and PRM from both fungi plays a role in phagocytosis when the purified molecule is pre-incubated with macrophage. In addition, PRM induce nitric oxide release by macrophages. Our data indicate that PRM is an important PAMP exposed on fungal surface with the potential of immune modulation.
In this work, peptidorhamnomannans from Scedosporium aurantiacum and Scedosporium minutisporum have been characterized. These molecules play important roles in phagocytosis and oxidative burst in peritoneal macrophages and are recognized by immune sera.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/fisiología , Macrófagos/metabolismo , Macrófagos/microbiología , Scedosporium/metabolismo , Animales , Anticuerpos Antifúngicos/química , Anticuerpos Antifúngicos/inmunología , Femenino , Interacciones Microbiota-Huesped , Humanos , Infecciones Fúngicas Invasoras/inmunología , Infecciones Fúngicas Invasoras/microbiología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Fagocitosis , ConejosRESUMEN
Sporotrichosis zoonotic transmission by cats has obtained hyperendemic magnitude in Rio de Janeiro, Brazil. Atypical cases, relapses, and reinfections as well as reduced diagnostic sensitivity of conventional methods have been reported. Previously, the anti-SsCBF enzyme-linked immunosorbent assay (ELISA) test was shown to be useful as a diagnostic tool for human sporotrichosis. Effective diagnosis and treatment are critical to interrupt the chain of transmission of this major pathogen in Brazilian Public Health. To evaluate its applicability for feline sporotrichosis diagnosis and/or therapeutic follow-up, 15 domestic cats from Rio de Janeiro were clinically and laboratory monitored by cytopathology, culture, Sporothrix genotyping, and anti-SsCBF IgG levels. Subsequently, animals were divided into satisfactory and non-satisfactory therapeutic responders. Averages of antibody serum levels obtained for diagnosis (first consultation) compared with the levels found after follow-up (last consultation) were significantly different in both groups (p = 0.0002 and p = 0.038, respectively). We conclude that the SsCBF ELISA test can predict feline sporotrichosis therapeutic responses even for animals with distinct clinical evolutions.
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Enfermedades de los Gatos/tratamiento farmacológico , Monitoreo de Drogas/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Sporothrix/efectos de los fármacos , Esporotricosis/veterinaria , Animales , Anticuerpos Antifúngicos/sangre , Brasil/epidemiología , Enfermedades de los Gatos/sangre , Enfermedades de los Gatos/epidemiología , Enfermedades de los Gatos/microbiología , Gatos , Sporothrix/clasificación , Sporothrix/genética , Sporothrix/fisiología , Esporotricosis/tratamiento farmacológico , Esporotricosis/epidemiología , Esporotricosis/microbiologíaRESUMEN
Sporotrichosis is a subcutaneous mycosis caused by traumatic inoculation into the skin by fungi species of the genus Sporothrix. The disease has different clinical manifestations (cutaneous, lymphocutaneous, and disseminated), and can also progress to a systemic infection. Despite having a worldwide distribution, sporotrichosis is most prevalent in tropical and subtropical countries. In Brazil, reports of the disease are higher frequent, where cases of the disease were found in Rio de Janeiro, Sao Paulo, Curitiba, Pernambuco, and Paraiba, among others. Certain groups of people may be more exposed to the causative agent of disease, such as residents of rural areas. Thus, this work aimed to carry out a seroepidemiological survey of the prevalence of sporotrichosis in four rural locations in the south of Minas Gerais State, Brazil. In this study, we used an indirect ELISA test in the survey on the prevalence of sporotrichosis. Data obtained in this study evaluated a population of 631 individuals and showed a prevalence of 44.69%. The distribution of seroprevalence of sporotrichosis with respect to age groups and gender showed no significant statistical difference. Thus, we found a high seroprevalence of sporotrichosis-infection in rural regions of southern Minas Gerais State, Brazil, with no difference in prevalence in relation to gender and age.
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Anticuerpos Antifúngicos/sangre , Sporothrix/inmunología , Esporotricosis/sangre , Esporotricosis/epidemiología , Adolescente , Adulto , Distribución por Edad , Anciano , Anciano de 80 o más Años , Animales , Brasil/epidemiología , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Población Rural/estadística & datos numéricos , Estudios Seroepidemiológicos , Sporothrix/genética , Esporotricosis/microbiología , Adulto JovenRESUMEN
Introduction. Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. As the disease is known to affect mostly men over 40 years old who previously worked handling soil, some cities of agricultural economy in endemic regions may have more cases of paracoccidioidal infection.Gap statement. The true frequency of PCM cannot be established in Brazil because it is not a disease of mandatory reporting. The detection of paracoccidioidal infection may assist in the planning of health services, in order to provide early detection of the disease and to prevent its worsening or even progression to death. In addition, little is described about sera reactivity with antigens from different species of Paracoccidiodes, especially P. lutzii.Aim. Current research was conducted in an inland municipality of southern Brazil, in order to assess infection rate within this endemic region of PCM disease.Methodology. ELISA was employed to evaluate 359 sera from random volunteers from Guarapuava, Paraná, Brazil, to detect IgG against cell-free antigens (CFA) from P. restrepiensis B339, P. americana LDR3 and P. lutzii LDR2. Confirmatory ELISA employed gp43 from B339. Reduction of cross-reactions was sought by treatment with sodium metaperiodate (SMP-CFA, SMP-gp43). Immunoblot was performed with 37 selected sera among those reactive in ELISA. Epidemiological profile was assessed by questionnaire.Results. ELISA reactivity was: CFA/SMP-CFA in general 37.3/17.8â%, B339 25.3/14.5â%, LDR3 24.5/1.4â%, LDR2 8.3/5.8â%; gp43/SMP-gp43 7.2/4.7â%. There were sera reactive with multiple CFAs. In immunoblot, five sera showed the same reaction profile with P. lutzii's antigens as PCM disease sera. Rural residence and soil-related professions were risk factors for paracoccidioidal infection.Conclusion. The low prevalence is in accordance with previous reports of lower PCM disease endemicity in Guarapuava than in other areas of Paraná. Although P. brasiliensis seems to be the prevalent strain of the region, 21 sera from people who only lived in Guarapuava reacted with P. lutzii LDR2. CFA-ELISA with whole antigens seems a good option for serological screening in epidemiological surveys.
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Anticuerpos Antifúngicos/sangre , Antígenos Fúngicos/sangre , Portador Sano/sangre , Inmunoglobulina G/sangre , Paracoccidioides/inmunología , Paracoccidioidomicosis/sangre , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , Portador Sano/epidemiología , Portador Sano/microbiología , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Persona de Mediana Edad , Paracoccidioides/clasificación , Paracoccidioides/genética , Paracoccidioides/aislamiento & purificación , Paracoccidioidomicosis/epidemiología , Paracoccidioidomicosis/microbiología , Adulto JovenRESUMEN
Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFß, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.
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Candida albicans/inmunología , Candidiasis/inmunología , Vesículas Extracelulares/inmunología , Animales , Anticuerpos Antifúngicos/sangre , Antígenos Fúngicos/inmunología , Candidiasis/prevención & control , Frío , Citocinas/sangre , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Vacunas Fúngicas/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Interleucina-6/biosíntesis , Ratones , Ratones Endogámicos BALB C , Mariposas Nocturnas/inmunología , Mariposas Nocturnas/microbiología , VacunaciónRESUMEN
The diagnosis of histoplasmosis depends on various approaches: direct clinical examination, fungus isolation from cultures of clinical samples, histopathological evaluation, and serological testing. In serodiagnostic assays, the Histoplasma capsulatum H and M antigenic glycoproteins have been extensively used. However, both antigens showed limitations attributed mainly to their cross-reactivity with glycoproteins from other pathogenic fungi, which compromises specificity, and generates false positives, misdiagnosis, and therapeutic failure. In this work, we deglycosylated extracellular released antigens from the Venezuelan 7090 H. capsulatum clinical isolate, using chemical and enzymatic methods and evaluated their effectiveness by indirect enzyme-linked immunosorbent assay (ELISA) with sera from patients with either histoplasmosis or PCM. Prior to deglycosylation, the extracellular released antigen showed 62% of sensitivity 66% of specificity and 68% of cross-reactivity with paracoccidioidomicosis sera. The chemically deglycosylated extracellular released antigen, for 8 or 18 h showed 72 and 52% sensitivity with 98% and 92% specificity, respectively. Moreover, cross-reactivity with Paracoccidioides decreased to 4 and 16%, following deglycosylation for 8 or 18 h, respectively. The enzymatically treated antigen showed 52% of sensitivity, 92% of specificity and 8% cross-reactivity against Paracoccidioides. Deglycosylation of the H. capsulatum antigen improves its specificity and decreases its cross-reactivity against Paracoccidioides when using indirect ELISA for serodiagnosis. Therefore, it is recommended to deglycosylate the fungal extracellular released antigen for clinical serodiagnosis, and to monitor humoral immune responses during therapy of patients with the different clinical forms of histoplasmosis.
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Anticuerpos Antifúngicos/sangre , Antígenos Fúngicos , Histoplasma/aislamiento & purificación , Histoplasmosis/diagnóstico , Pruebas Serológicas/métodos , Antígenos Fúngicos/sangre , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática/métodos , Glicosilación , Humanos , Sensibilidad y Especificidad , Venezuela/epidemiologíaRESUMEN
Candida is one of the most frequent pathogens of bloodstream infections, which is associated with high morbidity and mortality rates. Rapid immunological detection methods are essential in the early diagnosis of candidemia. Anti-mannan is one of host-derived biomarkers against cell wall components of Candida. We conducted this study to evaluate the diagnostic performance of two anti-mannan assays (IgM, IgG) for candidemia through the analysis of 40 candidemia patients, 48 participants with Candida colonization and 213 participants with neither Candida colonization nor Candida infections (13 patients with other bloodstream infections, 145 hospitalized patients and 55 healthy controls). The performance of the two assays were evaluated by calculating their sensitivity and specificity. The sensitivity ranged from 0.78 to 0.80 for the IgM assay and 0.68 to 0.75 for the IgG assay. The specificity ranged from 0.97 to 0.98 for the IgM assay and 0.91 to 0.94 for the IgG assay. The diagnostic performance of the anti-mannan IgM assay was better than that of IgG, with higher sensitivity and specificity. Combining the two assays (positive results of single or both assays are both considered as positive) could improve the sensitivity up to 0.93 (0.79-0.98) and only slightly reduce the specificity (0.93(0.89-0.95)). The anti-mannan IgM, IgG assays are rapid and cost-effective assays that may be probably useful in the diagnosis of candidemia.
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Anticuerpos Antifúngicos/sangre , Candidemia/diagnóstico , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Mananos/sangre , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Adulto JovenRESUMEN
The goal of the present work was to develop a novel reagent with potential for histoplasmosis diagnosis. For this purpose, the genetic sequence of the 100 kDa protein of Histoplasma capsulatum (Hcp100) was cloned and expressed as a secretory protein in Pichia pastoris. After optimizing the culture conditions and purifying by immobilized metal ion affinity chromatography, the highest yield of Hcp100 reached approximately 1.3 mg/l with > 90% purity in shake flasks using basal salt medium supplemented with casamino acids after 72 h of methanol induction. To investigate its potential for diagnosis, its detection in urine samples using specific polyclonal antibodies as reagent was evaluated by dot blot in 6 patients with progressive disseminated histoplasmosis (PDH), of whom all had AIDS. Antigen was detected in urine from all 6 (100%) PDH patients. Urine samples from a pool of 20 healthy individuals did not react with the anti-Hcp100 antibodies. The dot blot assay performed in this study provides preliminary data of a simple technology that can be performed in medical institutions with limited resources to facilitate the rapid diagnosis of histoplasmosis, particularly the disseminated forms. Hence, use of these assays may provide a rapid diagnostic tool of PDH in endemic areas for histoplasmosis where PDH-related mortality is high, hastening treatment and improving patient survival. Finally, this novel antigen and its specific antibodies may provide an alternative diagnostic reagent to the largely unknown and poorly characterized polysaccharide antigens (HPA, galactomannan, histoplasmin) frequently used in the diagnostic tests. KEY POINTS: Few antigens are used as laboratory tools for the immunodiagnosis of histoplasmosis. P. pastoris was an excellent system for recombinant Hcp100 expression. Maximum expression levels of rHcp100 were achieved in BSM with 1% casamino acids. Dot blot assays with anti-rHcp100 antisera can be successfully used for diagnosing PHD.
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Antígenos Fúngicos/metabolismo , Proteínas Fúngicas/metabolismo , Histoplasma/aislamiento & purificación , Histoplasmosis/diagnóstico , Animales , Anticuerpos Antifúngicos/sangre , Antígenos Fúngicos/genética , Antígenos Fúngicos/inmunología , Antígenos Fúngicos/aislamiento & purificación , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/aislamiento & purificación , Histoplasma/inmunología , Histoplasmosis/orina , Humanos , Pruebas Inmunológicas , Ratones , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismoRESUMEN
The thermodimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), a deep mycosis endemic in Latin American countries that affects mainly male rural workers. Infection by P. brasiliensis has also been reported in several species of terrestrial animals; however, the capacity of the fungus to infect aquatic organisms is poorly known. The aim of this study was to detect P. brasiliensis in a fish species, Nile tilapia (Oreochromis niloticus), the most farmed and widely distributed fish in endemic areas for human PCM in Brazil. As a first step, the humoral immune response against the fungus was evaluated in an experimental group of three fish immunized with inactivated P. brasiliensis yeast cells. For the seroepidemiological study, serum samples of Nile tilapia raised in cages (n = 109) and in ponds (n = 105), collected from a fish slaughterhouse, were analyzed for P. brasiliensis antibodies by ELISA using gp43 as antigen. All the inoculated fish produced antibodies against the fungus. The seropositivity observed in fish raised in cages and ponds was 17.4 and 5.7%, respectively. Due to the higher seropositivity observed in caged fish, 100 tissue samples (encephalon, liver, and kidney), from another group of tilapia raised in cages, were analyzed by polymerase chain reaction (PCR; Pb-ITSR and Pb-ITSE). Three tissue samples (liver n = 1, kidney n = 1, and enchepahlon n = 1) from three different fish resulted positive to PCR. This is the first report to show serological and molecular evidence of P. brasiliensis infection in a fish species.