Serology of Paracoccidioidomycosis Due to Paracoccidioides lutzii

Paracoccidioides lutzii is a new agent of paracoccidioidomycosis (PCM) and has its epicenter localized to the Central-West region of Brazil. Serological diagnosis of PCM caused by P. lutzii has not been established. This study aimed to develop new antigenic preparations from P. lutzii and to apply them in serological techniques to improve the diagnosis of PCM due to P. lutzii. Paracoccidioides lutzii exoantigens, cell free antigen (CFA), and a TCA-precipitated antigen were evaluated in immunodiffusion (ID) tests using a total of 89 patient sera from the Central-West region of Brazil. Seventy-two sera were defined as reactive for P. brasiliensis using traditional antigens (AgPbB339 and gp43). Non-reactive sera for traditional antigens (n = 17) were tested with different P. lutzii preparations and P. lutzii CFA showed 100% reactivity. ELISA was found to be a very useful test to titer anti-P. lutzii antibodies using P. lutzii-CFA preparations. Sera from patients with PCM due to P. lutzii presented with higher antibody titers than PCM due to P. brasiliensis and heterologous sera. in western blot, sera from patients with PCM due to P. lutzii were able to recognize antigenic molecules from the P. lutzii-CFA antigen, but sera from patients with PCM due to P. brasiliensis could not recognize any P. lutzii molecules. Due to the facility of preparing P. lutzii CFA antigens we recommend its use in immunodiffusion tests for the diagnosis of PCM due to P. lutzii. ELISA and western blot can be used as complementary tests.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo UNIFESP, Dept Microbiol Imunol & Parasitol, Disciplina Biol Celular, São Paulo, BrazilUniv Fed Mato Grosso do Sul UFMS, Fac Med FAMED, Campo Grande, MS, BrazilUniv Fed Mato Grosso UFMT, Nucleo Doencas Infecciosas & Trop, Cuiaba, Mato Grosso, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Microbiol Imunol & Parasitol, Disciplina Biol Celular, São Paulo, BrazilFAPESP: 2012/06593-0FAPESP: 2009/54024-2Web of Scienc

Immunodiagnosis of Paracoccidioidomycosis due to Paracoccidioides brasiliensis Using a Latex Test: Detection of Specific Antibody Anti-gp43 and Specific Antigen gp43

BackgroundParacoccidioidomycosis (PCM) is a life-threatening systemic disease and is a neglected public health problem in many endemic regions of Latin America. Though several diagnostic methods are available, almost all of them present with some limitations.Method/Principle FindingsA latex immunoassay using sensitized latex particles (SLPs) with gp43 antigen, the immunodominant antigen of Paracoccidioides brasiliensis, or the monoclonal antibody mAb17c (anti-gp43) was evaluated for antibody or antigen detection in sera, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL) from patients with PCM due to P. brasiliensis. the gp43-SLPs performed optimally to detect specific antibodies with high levels of sensitivity (98.46%, 95% CI 91.7-100.0), specificity (93.94%, 95% CI 87.3-97.7), and positive (91.4%) and negative (98.9%) predictive values. in addition, we propose the use of mAb17c-SLPs to detect circulating gp43, which would be particularly important in patients with immune deficiencies who fail to produce normal levels of immunoglobulins, achieving good levels of sensitivity (96.92%, 95% CI 89.3-99.6), specificity (88.89%, 95% CI 81.0-94.3), and positive (85.1%) and negative (97.8%) predictive values. Very good agreement between latex tests and double immune diffusion was observed for gp43-SLPs (k = 0.924) and mAb17c-SLPs (k = 0.850), which reinforces the usefulness of our tests for the rapid diagnosis of PCM in less than 10 minutes. Minor cross-reactivity occurred with sera from patients with other fungal infections. We successfully detected antigens and antibodies from CSF and BAL samples. in addition, the latex test was useful for monitoring PCM patients receiving therapy.Conclusions/SignificanceThe high diagnostic accuracy, low cost, reduced assay time, and simplicity of this new latex test offer the potential to be commercialized and makes it an attractive diagnostic assay for use not only in clinics and medical mycology laboratories, but mainly in remote locations with limited laboratory infrastructure and/or minimally trained community health workers.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Div Cell Biol, São Paulo, BrazilEvandro Chagas Inst, Bacteriol & Mycol Div, Ananindeua, Para, BrazilUniv Fed Alfenas, Inst Biomed Sci, Alfenas, MG, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Div Cell Biol, São Paulo, BrazilFAPESP: FAPESP 2011/07350-1FAPESP: 2011/01628-8FAPESP: 2009/54181-0FAPESP: 2009/54024-2Web of Scienc

Simultaneous infection of human host with genetically distinct isolates of Paracoccidioides brasiliensis

This study is the first report on genetic differences between isolates of Paracoccidioides brasiliensis from a single patient. We describe a simultaneous infection with genetically distinct isolates of P. brasiliensis in a patient with chronic paracoccidioidomycosis. The clinical isolates were obtained from lesions in different anatomical sites and were characterised by random amplified polymorphic DNA (RAPD) analysis. The RAPD technique can be helpful for distinguishing between clinical isolates. Different random primers were used to characterise these clinical isolates. The RAPD patterns allowed for differentiation between isolates and the construction of a phenetic tree, which showed more than 28% genetic variability in this fungal species, opening new possibilities for clinical studies of P. brasiliensis. Based on these results and preliminary clinical findings, we suggest that different genotypes of P. brasiliensis might infect the same patient, inducing the active form of the disease.Universidade Federal de Mato Grosso Faculdade de Ciências Médicas Laboratório de MicologiaUniversidade Federal de São Paulo (UNIFESP) Departamento de Microbiologia, Imunologia e ParasitologiaUniversidade de Cuiabá Hospital Geral Universitário Laboratório de MicologiaUNIFESP, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Rapid diagnosis of coccidioidomycosis by nested PCR assay of sputum

Coccidioidomycosis is a deep infection caused by two dimorphic fungi, Coccidioides immitis and Coccidioides posadasii. Diagnosis of the disease requires culture of suspicious clinical samples on mycological media. However, as these species are virulent pathogens, handling of their cultures is a high-risk activity, and is limited to Biosafety Level 3 laboratories. This study describes the direct detection of C. posadasii DNA in an inappropriate sputum sample by PCR amplification of the highly specific Ag2/PRA antigen gene. the results obtained suggest that direct detection of the Ag2/PRA sequence in sputum is an excellent method for rapid and specific diagnosis of coccidioidomycosis.Fed Univ Ceara, Med Mycol Specialized Ctr, Fortaleza, Ceara, BrazilFed Univ Ceara, Postgrad Program Med Sci, Fortaleza, Ceara, BrazilState Univ Ceara, Postgrad Program Vet Sci, Fortaleza, Ceara, BrazilState Univ Ceara, Dept Sci Biol, Fortaleza, Ceara, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilWeb of Scienc

Characteristics of 151 Brazilian Sporothrix schenckii Isolates from 5 Different Geographic Regions of Brazil: A Forgotten and Re-Emergent Pathogen

Abstract: We used RAPD (random amplification of polymorphic DNA) to analyze the phenotypic and genotypic characteristics of Sporothrix schenckii isolated from five geographic regions of Brazil, from clinical, animal, and environmental sources. Our results yielded a significant difference (P<0.01) in the mean conidial area of S. schenckii animal isolates (2.96 ± 1.07) compared with those of clinical isolates (fixed form, 2.33 ± 0.53; lymphocutaneous form, 2.37 ± 0.43). There was no association among S. schenckii clinical isolates and geographic region. Isolates from the Northeast region exhibited the lowest thermotolerance (% growth inhibition) at 35ºC ( x = 49.23% ± 17.25) and at 37ºC (70.43% ± 10.93%). Northern isolates exhibited the highest thermotolerance at 35ºC (12.82% ± 5.73%) and at 37ºC (23.81% ± 8.27%). RAPD with a 10-mer primer OPD-18 generated 67 PCR fingerprint patterns. The 151 S. schenckii isolates fell into seven major clusters with such great genetic diversity that an association of isolates with clinical forms or geographic areas could not be determined, even with investigations focused on more restricted geographic areas. The main physiological characteristics of Brazilian S. schenckii isolates were also characterized, including osmophilia, halophilia, pH tolerance, urease activity, casein hydrolysis, and gelatinase, proteinase, and DNAase production