Fast and cost-effective protocol to produce Paracoccidioides spp. antigens
Abstract
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.
Downloads
References
Gonzalez A, Hernandez O. New insights into a complex fungal pathogen: the case of Paracoccidioides spp. Yeast. 2016;33:113-28. https://doi.org/10.1002/yea.3147
Queiroz-Telles F, Fahal AH, Falci DR, Caceres DH, Chiller T, Pasqualotto AC. Neglected endemic mycoses. Lancet Infect Dis. 2017;17:e377. https://doi.org/10.1016/S1473-3099(17)30306-7
Shikanai-Yasuda MA, Mendes RP, Colombo AL, Queiroz-Telles F, Kono ASG, Paniago AMM, et al. Brazilian guidelines for the clinical management of paracoccidioidomycosis. Rev Soc Bras Med Trop. 2017;50:715-40. https://doi.org/10.1590/0037-8682-0230-2017
Martinez, R. New trends in Paracoccidioidomycosis epidemiology. J Fungi. 2017;3:1. https://doi.org/10.3390/jof3010001
Mendes RP, Cavalcante RS, Marques SA, Marques MEA, Venturini J, Sylvestre TF. Paracoccidioidomycosis: Current perspectives from Brazil. Open Microbiol J. 2017;31:224-82. https://doi.org/10.2174/1874285801711010224
da Silva JF, de Oliveira HC, Marcos CM, Assato PA, Fusco-Almeida AM, Mendes-Giannini MJ. Advances and challenges in paracoccidioidomycosis serology caused by Paracoccidioides species complex: an update. Diagn Microbiol Infect Dis. 2016;8487-94. https://doi.org/10.1016/j.diagmicrobio.2015.06.004
Ferreira CS, Ribeiro EMC, Goes AM, Silva BM. Current strategies for diagnosis of paracoccidioidomycosis and prospects of methods based on gold nanoparticles. Future Microbiol. 2016;11:973-85. https://doi.org/10.2217/fmb-2016-0062
Almeida-Paes R, Bernardes-Engemann AR, da Silva Motta B, Pizzini CV, de Abreu Almeida M, de Medeiros Muniz M, et al. Immunologic diagnosis of endemic mycoses. J Fungi (Basel). 2022;8:993. https://doi.org/10.3390/jof8100993
Peçanha PM, Peçanha-Pietrobom PM, Grão-Velloso TR, Rosa Júnior M, Falqueto A, Gonçalves SS. Paracoccidioidomycosis: What we know and what is new in epidemiology, diagnosis, and treatment. J Fungi (Basel). 2022;8:1098. https://doi.org/10.3390/jof8101098
Kamikawa CM, Mendes RP, Vicentini AP. Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection. J Venom Anim Toxins Incl Trop Dis. 2017;23:11. https://doi.org/10.1186/s40409-017-0101-3
Vidal MS, Del Negro GM, Vicentini AP, Svidzinski TI, Mendes-Giannini MJ, Almeida AM, et al. Serological diagnosis of paracoccidioidomycosis: high rate of inter-laboratorial variability among medical mycology reference centers. PLoS Negl Trop Dis. 2014;8:e3174. https://doi.org/10.1371/journal.pntd.0003174
de Camargo ZP. Serology of paracoccidioidomycosis. Mycopathologia. 2008;165:289-302. https://doi.org/10.1007/s11046-007-9060-5
de Freitas RS, Kamikawa CM, Vicentini AP. Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis. Rev Iberoam Micol. 2018;35:27-31. https://doi.org/10.1016/j.riam.2017.04.004
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. https://doi.org/10.1006/abio.1976.9999
Ouchterlony O. Antigen-antibody reactions in gels. Acta Pathol Microbiol Scand. 1949;26:507-15. https://doi.org/10.1111/j.1699-0463.1949.tb00751.x
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature.1970;227:680-5. https://doi.org/10.1038/227680a0
Ansorge W. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J Biochem Biophys Methods. 1985;11:13-20. https://doi.org/10.1016/0165-022x(85)90037-5
Vicentini-Moreira AP. Paracoccidioidomicose: histórico, etiologia, epidemiologia, patogênese, formas clínicas, diagnóstico laboratorial e antígenos. Bol Epidemiol Paul. 2008;5:11-24.
Gegembauer G, Araujo LM, Pereira EF, Rodrigues AM, Paniago AM, Hahn RC, et al. Serology of paracoccidioidomycosis due to Paracoccidioides lutzii. PLoS Negl Trop Dis 2014;8:e2986. https://doi.org/10.1371/journal.pntd.0002986
Queiroz Junior LDP, de Camargo ZP, Tadano T, Rodrigues AM, Takarara DT, Gegembauer G, et al. Serological and antigenic profiles of clinical isolates of Paracoccidioides spp. from Central Western Brazil. Mycoses. 2014;57:466-72. https://doi.org/10.1111/myc.12183
de Camargo ZP, de Franco MF. Current knowledge on pathogenesis and immunodiagnosis of paracoccidioidomycosis. Rev Iberoam Micol. 2000;17:41-8.
Camargo ZP, Berzaghi R, Amaral CC, Silva SH. Simplified method for producing Paracoccidioides brasiliensis exoantigens for use in immunodiffusion tests. Med Mycol. 2003;41:539-42. https://doi.org/10.1080/13693780310001615358
Leitão NP Jr, Vallejo MC, Conceição PM, Camargo ZP, Hahn R, Puccia R. Paracoccidioides lutzii Plp43 is an active glucanase with partial antigenic identity with P. brasiliensis gp43. PLoS Negl Trop Dis. 2014;8:e3111. https://doi.org/10.1371/journal.pntd.0003111
Arantes TD, Theodoro RC, Teixeira M de M, Bosco S de M, Bagagli E. Environmental mapping of Paracoccidioides spp. in Brazil reveals new clues into genetic diversity, biogeography and wild host association. PLoS Negl Trop Dis. 2016;10:e0004606. https://doi.org/10.1371/journal.pntd.0004606
Mendes JF, Klafke GB, Albano APN, Cabana AL, Teles AJ, de Camargo ZP, et al. Paracoccidioidomycosis infection in domestic and wild mammals by Paracoccidioides lutzii. Mycoses. 2017;60:402-6. https://doi.org/10.1111/myc.12608
Buccheri R, Morais VDS, Kamikawa CM, Vidal MSM, Naves G, Del Negro GMB, et al. Case Report: misleading serological diagnosis of paracoccidioidomycosis in a young patient with the acute form disease: Paracoccidioides brasiliensis or Paracoccidioides lutzii? Am J Trop Med Hyg. 2018;98:1082-5. https://doi.org/10.4269/ajtmh.17-0812
Lenhard-Vidal A, Assolini JP, Chiyoda FAS, Ono MA, Sano A, Itano EN. Polyclonal antibodies to Paracoccidioides brasiliensis are able to recognise antigens from different strains from Paracoccidioides species complex, including Paracoccidioides lutzii LDR2. Mycoses. 2018;61:826-32. https://doi.org/10.1111/myc.12819
Do Valle ACF, Costa RLB, Fialho Monteiro PC, Von Helder J, Muniz MM, Zancopé-Oliveira RM. Interpretation and clinical correlation of serological tests in paracoccidioidomycosis. Med Mycol. 2001;39:373-7. https://doi.org/10.1080/mmy.39.4.373.37
Torres Esteche V, Arteta Z, Torres G, Vaucher A, Gezuele E, Balleste R. Concomitant pulmonary paracoccidioidomycosis and pulmonary histoplasmosis: a rare case. J Bras Pneumol. 2012;38:264-8. https://doi.org/10.1590/s1806-37132012000200017
Fernandes VC, Coitinho JB, Veloso JM, Araújo SA, Pedroso EP, Goes AM. Combined use of Paracoccidioides brasiliensis recombinant rPb27 and rPb40 antigens in an enzyme-linke immunosorbent assay for immunodiagnosis of paracoccidioidomycosis. J Immunol Methods. 2011;367:78-84. https://doi.org/10.1016/j.jim.2011.02.006
Kamikawa CM, Vicentini AP. Multiparametric assay of screening for the diagnosis of mycoses of interest in Public Health: standardization of methodology. Rev Inst Adolfo Lutz. 2022;81:1-11,e37165. https://doi.org/10.53393/rial.2022.v.81.37165
Dos Santos PO, Rodrigues AM, Fernandes GF, da Silva SH, Burger E, de Camargo ZP. Immunodiagnosis of paracoccidioidomycosis due to Paracoccidioides brasiliensis using a latex test: detection of specific antibody anti-gp43 and specific antigen gp43. PLoS Negl Trop Dis. 2015;13:e0003516. https://doi.org/10.1371/journal.pntd.0003516
Ortiz BL, Díez S, Urán ME, Rivas JM, Romero M, Caicedo, et al. Use of the 27-kilodalton recombinant protein from Paracoccidioides brasiliensis in serodiagnosis of paracoccidioidomycosis. Clin Diagn Lab Immunol. 1998;5:826-30. https://doi.org/10.1128/CDLI.5.6.826-830.1998
Some similar items:
- Norma B. Fernández, Adriana Toranzo, Luciana Farias, Cristina E. Canteros, Mycological diagnosis of paracoccidioidomycosis in a hospital from a nonendemic area: classical and molecular methods , Biomedica: Vol. 43 No. Sp. 1 (2023): Agosto, Micología médica
- Lina M. Osorio-Cock, Sandra Catalina Jaramillo-Pulgarín, Alba P. Ferrín-Bastidas, Diana Y. Molina-Colorado, Óscar M. Gómez-Guzmán, Alejandra Zuluaga, Juan G. McEwen-Ochoa, Martha E. Urán-Jiménez, María del Pilar Jiménez-Alzate, Pseudoepitheliomatous hyperplasia: Squamous cell carcinoma versus oral paracoccidioidomycosis, a case from a dermatological perspective , Biomedica: Vol. 43 No. Sp. 1 (2023): Agosto, Micología médica
- Ana María García, Orville Hernández, Beatriz H. Aristizábal, Luz Elena Cano, Juan G. McEwen, Ángela Restrepo, Identification of genes associated with germination of conidia to form mycelia in the fungus Paracoccidioides brasiliensis , Biomedica: Vol. 29 No. 3 (2009)
- Edilma Torrado, Elizabeth Castañeda, Fernando de la Hoz, Angela Restrepo, Definition of paracoccidioidomycosis endemic areas in Colombia , Biomedica: Vol. 20 No. 4 (2000)
- Angel González, Luz E. Cano, Polymorphonuclear neutrophil participation in immune response to Paraccocidioides brasiliensis , Biomedica: Vol. 21 No. 3 (2001)
- José M. Oñate, Angela María Tobón, Angela Restrepo, Adrenal gland insufficiency secondary to paracoccidioidomycosis. , Biomedica: Vol. 22 No. 3 (2002)
- Jenny Fabiola Hernández, Sofía Duque, Adriana Arévalo, Rafael Guerrero, Rubén Santiago Nicholls, Identification of antigens of Colombian Giardia duodenalis isolates recognized by total IgG and subclasses. , Biomedica: Vol. 23 No. 3 (2003)
- Rosana Natalia Olmos, Sofía Duque, Myriam Consuelo López, Adriana Arévalo, Rafael Guerrero, Martha Patricia Velandia, Ruben Santiago Nicholls, Identification of cyst and trophozoite antigens from Colombian Giardia duodenalis isolates recognized by IgA. , Biomedica: Vol. 23 No. 3 (2003)
- Yenny Alviarez, María Lares, Mercedes Viettri, Cruz M. Aguilar, Leidi Herrera, Elizabeth Ferrer, Standardization of a direct agglutination test for the immunodiagnosis of Chagas disease , Biomedica: Vol. 34 No. 2 (2014)
- Angélica Ballesteros, Sandra Beltrán, Jaime Patiño, Cynthia Bernal, Rocío Orduz, Disseminated juvenile paracoccidioidomycosis diagnosed in a girl in an urban area , Biomedica: Vol. 34 No. 1 (2014)
Copyright (c) 2023 Biomedica
This work is licensed under a Creative Commons Attribution 4.0 International License.
| Article metrics | |
|---|---|
| Abstract views | |
| Galley vies | |
| PDF Views | |
| HTML views | |
| Other views | |
