Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Freitas, Roseli Santos de; Kamikawa, Camila Mika; Vicentini, Adriana Pardini

doi:10.1016/j.riam.2017.04.004

Cited by 12 publications

(13 citation statements)

References 33 publications

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“…The lower specificity of the anti‐HMN ELISA might be related to the nature of the reagent used (i.e. a mixture of crude antigens, whose polysaccharides are related to the different cross‐reactions reported in the literature) 25–27 . For both assays, cross‐reactivity was noted with paracoccidioidomycosis, coccidioidomycosis, cryptococcosis and tuberculosis.…”

Section: Discussionmentioning

confidence: 99%

“…HMN is composed predominantly by the C‐antigen (a galactomannan, GM), the H‐antigen (a β‐glucosidase), and the M‐antigen (a catalase) 22–24 . However, this serological reagent is not ideal due to the presence of cross‐reactive carbohydrates (i.e., GM and glycoconjugates), and because its production is problematic and time‐consuming 25–27 …”

Section: Introductionmentioning

confidence: 99%

“…[22][23][24] However, this serological reagent is not ideal due to the presence of cross-reactive carbohydrates (i.e., GM and glycoconjugates), and because its production is problematic and time-consuming. [25][26][27] Despite the fact that urinary Histoplasma antigens assays have proven highly sensitive (67.3%-95%) for diagnosing PDH and that some commercial kits are available in many regions of Latin America, they are not yet routinely used, mainly, due to their high cost. 10,[28][29][30] In Argentina, the only commercially available antigen assays are the Clarus Histoplasma Galactomannan Enzyme Immunoassay (HGM) (IMMY, USA) and the Histoplasma Urine Antigen Lateral Flow Assay (LFA) (MiraVista Diagnostics, USA).…”

Section: Introductionmentioning

confidence: 99%

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Role of recombinant Histoplasma capsulatum 100‐kilodalton antigen in the diagnosis of histoplasmosis among HIV/AIDS patients: Antigenuria and antibodies detection

Toscanini

Laboccetta

Garrido

et al. 2023

Mycoses

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Background: Diagnosing progressive disseminated histoplasmosis (PDH) is still challenging in many countries where this disease is highly endemic. Definitive diagnosis is established by culture and/or by cytology/histopathology but both procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has a low sensitivity in immunocompromised individuals. Commercially available antigen detection assays have high sensitivity in PDH cases; however, they are expensive and only performed in few laboratories.Aims: To describe the potential use of a novel ELISA for antibodies testing and a dot blot assay for antigen testing for diagnosing PDH using the recombinant 100 kDa protein of Histoplasma capsulatum (Hcp100) and their polyclonal antibodies as novel reagents, respectively.Methods: Serum and urine samples from a cohort of patients with HIV/AIDS and proven PDH were studied for the detection of anti-Hcp100 antibodies by ELISA and Hcp100 antigen by dot blot, respectively. Sensitivity, specificity and cross-reactions with other diseases were estimated for each assay and compared with those obtained using histoplasmin (HMN) as a reagent for antibodies detection by ELISA and immunodiffusion, and using a commercial antigenuria test.Results: Antibodies detection by the Hcp100 ELISA demonstrated 78.6% sensitivity and 88.4% specificity, versus 85.7% sensitivity and 81.0% specificity for the HMN ELISA and 26.1% sensitivity and 100% specificity for the immunodiffusion assay.Antigen detection by the Hcp100 dot blot demonstrated 89.3% sensitivity and 97.0% specificity versus 82.1% sensitivity and 90.9% specificity for the commercial test. Conclusion:The immunoassays described herein based on Hcp100 would be a valuable screening tool for diagnosing PDH.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Role of recombinant Histoplasma capsulatum 100‐kilodalton antigen in the diagnosis of histoplasmosis among HIV/AIDS patients: Antigenuria and antibodies detection

Toscanini

Laboccetta

Garrido

et al. 2023

Mycoses

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“…The antigens were obtained from Paracoccidioides lutzii isolates (01, 66, and 8334), Paracoccidioides brasiliensis sensu stricto (113), and Paracoccidioides restripiensis (B-339). These fungi were grown at 36 °C ± 1 °C, on modified Fava-Netto agar, according to Freitas et al (2018). Paracoccidioides lutzii antigens were obtained after 5, 10, and 20 days of culture, whereas P. brasiliensis and P. restripiensis antigens were obtained after 10 days.…”

mentioning

confidence: 99%

“…antigens are problematic in terms of standardization, specificity, stability, repeatability, and reproducibility (10)(11)(12). Thus, in this study, we sought to optimize the methodology for antigen production described by Freitas et al, evaluating its applicability in paracoccidioidomycosis immunodiagnosis using a double immunodiffusion assay (13).…”

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confidence: 99%

Fast and cost-effective protocol to produce Paracoccidioides spp. antigens

Fernandes-Beraldo,

Santos de Freitas-Xavier,

Pardini-Vicentini

2023

biomedica

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Introduction. The existing methods for Paracoccidioides spp. antigen production are problematic in terms of standardization, specificity, stability, repeatability, and reproducibility.Objective. To optimize the methodology for Paracoccidioides spp. antigen production and evaluate its applicability in paracoccidioidomycosis immunodiagnosis.Materials and methods. The antigens were obtained from Paracoccidioides lutzii isolates (01, 66, and 8334), Paracoccidioides brasiliensis sensu stricto (113), and Paracoccidioides restripiensis (B-339). These fungi were grown at 36 °C ± 1 °C, on modified Fava-Netto agar, according to Freitas et al. (2018). Paracoccidioides lutzii antigens were obtained after , 10, and 20 days of culture, whereas P. brasiliensis and P. restripiensis antigens were obtained after 10 days. Antigens were evaluated in natura, 10 and 20 times concentrated. Antigenic capacity was evaluated using a double immunodiffusion assay against serum samples from patients with paracoccidioidomycosis, histoplasmosis, and aspergillosis, and random blood donors.Results. Cross-reactivity between Paracoccidioides spp. antigens was observed when P. brasiliensis, P. restrepiensis antigens, and P. lutzii antigens were evaluated with the polyclonal antibodies against P. lutzii and P. brasiliensis, respectively. No cross-reactivity was obtained for polyclonal antibodies against Histoplasma capsulatum, Aspergillus fumigatus, and random blood donors. The proposed protocol allowed stable, repeatable, and reproducible genus-specific antigen production at a low cost and in a short cultivation time.Conclusion. The proposed protocol allowed us to obtain genus-specific antigens that can be developed and reproduced in all laboratories in Brazil and South America, where paracoccidioidomycosis is a neglected disease, contributing to an early diagnosis, especially in endemic regions, regardless of the species.

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Histoplasma capsulatum 100-kilodalton antigen: recombinant production, characterization, and evaluation of its possible application in the diagnosis of histoplasmosis

Toscanini

Maglio

Capece

et al. 2020

Appl Microbiol Biotechnol

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Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Cited by 12 publications

References 33 publications

Role of recombinant Histoplasma capsulatum 100‐kilodalton antigen in the diagnosis of histoplasmosis among HIV/AIDS patients: Antigenuria and antibodies detection

Role of recombinant Histoplasma capsulatum 100‐kilodalton antigen in the diagnosis of histoplasmosis among HIV/AIDS patients: Antigenuria and antibodies detection

Fast and cost-effective protocol to produce Paracoccidioides spp. antigens

Histoplasma capsulatum 100-kilodalton antigen: recombinant production, characterization, and evaluation of its possible application in the diagnosis of histoplasmosis

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