Case report: Misleading serological diagnosis of paracoccidioidomycosis in a young patient with the cute form disease: Paracoccidioides brasiliensis or paracoccidioides lutzii?

Negative results in serological routine screening of patients with microbiologically proven aracoccidioidomycosis (PCM) are occasionally reported. Failure in detecting anti-Paracoccidioides antibodies has been ascribed to factors either related to serological techniques or to the status of the host immune reactivity. Recently, this issue has been renewed by the recognition that the Paracoccidioides genera comprises two species, lutzii and brasiliensis, which have distinct antigenic profiles and, therefore, may elicit different host antibody responses. We describe a patient with the acute form PCM due to Paracoccidioides brasiliensis with negative results on two reference centers' routine screening for P. brasiliensis antibodies, but positive results with Paracoccidioides lutzii antigens. The present case report suggests that antibodies elicited during P. brasiliensis infection recognize antigenic fractions shared by both species, highlighting the difficulties in distinguishing the two infections by means of the currently available routine serological assays

Case Report: Misleading Serological Diagnosis of Paracoccidioidomycosis in a Young Patient with the Acute Form Disease: Paracoccidioides brasiliensis or Paracoccidioides lutzii?

Negative results in serological routine screening of patients with microbiologically proven Paracoccidioidomycosis (PCM) are occasionally reported. Failure in detecting anti-Paracoccidioides antibodies has been ascribed to factors either related to serological techniques or to the status of the host immune reactivity. Recently, this issue has been renewed by the recognition that the Paracoccidioides genera comprises two species, lutzii and brasiliensis, which have distinct antigenic profiles and, therefore, may elicit different host antibody responses. We describe a patient with the acute form PCM due to Paracoccidioides brasiliensis with negative results on two reference centers' routine screening for P. brasiliensis antibodies, but positive results with Paracoccidioides lutzii antigens. The present case report suggests that antibodies elicited during P. brasiliensis infection recognize antigenic fractions shared by both species, highlighting the difficulties in distinguishing the two infections by means of the currently available routine serological assays.

Serological diagnosis of paracoccidioidomycosis: high rate of inter-laboratorial variability among medical mycology reference centers.

BACKGROUND: Serological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and antigen preparations are used and the few attempts to standardize the routine serological methods have not succeeded. METHODOLOGY/PRINCIPAL FINDINGS: We compared the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the "reference" titers. Each center then applied its own antigen preparation and serological routine test, either semiquantitative double immunodifusion or counterimmmunoelectrophoresis, in the 150 sera from the other five centers blindly as regard to the "reference" titers. Titers were transformed into scores: 0 (negative), 1 (healing titers), 2 (active disease, low titers) and 3 (active disease, high titers) according to each center's criteria. Major discordances were considered between scores indicating active disease and scores indicating negative or healing titers; such discordance when associated with proper clinical and other laboratorial data, may correspond to different approaches to the patient's treatment. Surprisingly, all centers exhibited a high rate of "major" discordances with a mean of 31 (20%) discordant scores. Alternatively, when the scores given by one center to their own sera were compared with the scores given to their sera by the remaining five other centers, a high rate of major discordances was also found, with a mean number of 14.8 sera in 30 presenting a discordance with at least one other center. The data also suggest that centers that used CIE and pool of isolates for antigen preparation performed better. CONCLUSION: There are inconsistencies among the laboratories that are strong enough to result in conflicting information regarding the patients' treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.

Viability and molecular authentication of Coccidioides spp. isolates from the Instituto de Medicina Tropical de São Paulo culture collection, Brazil / Viabilidade e autenticação molecular de isolados de Coccidioides spp. da coleção de culturas do Instituto de Medicina Tropical de São Paulo, Brasil

Coccidioidomycosis is an emerging fungal disease in Brazil; adequate maintenance and authentication of Coccidioides isolates are essential for research into genetic diversity of the environmental organisms, as well as for understanding the human disease. Seventeen Coccidioides isolates maintained under mineral oil since 1975 in the Instituto de Medicina Tropical de São Paulo (IMTSP) culture collection, Brazil, were evaluated with respect to their viability, morphological characteristics and genetic features in order to authenticate these fungal cultures. Only five isolates were viable after almost 30 years, showing typical morphological characteristics, and sequencing analysis using Coi-F and Coi-R primers revealed 99% identity with Coccidioides genera. These five isolates were then preserved in liquid nitrogen and sterile water, and remained viable after two years of storage under these conditions, maintaining the same features.

Coccidioidomicose é uma doença emergente no Brasil; a manutenção adequada e autenticação de isolados de Coccidioides spp são essenciais para a pesquisa em diversidade genética de micro-organismos, bem como para a compreensão da doença em humanos. Dezessete isolados de Coccidioides preservados em óleo mineral desde 1975 na coleção de culturas do Instituto de Medicina Tropical de São Paulo (IMTSP) foram avaliados com relação à viabilidade, características morfológicas e genéticas, com o objetivo de autenticação das culturas fúngicas. Dos 17 isolados, apenas cinco foram viáveis após quase 30 anos mantidos em óleo mineral, apresentando características morfológicas e moleculares típicas do gênero, o sequenciamento utilizando os oligonucleotídeos Coi-F e Coi-R revelou identidade de 99% com isolados de Coccidioides. Estes cinco isolados foram preservados em nitrogênio líquido e água destilada esterilizada, e permaneceram viáveis após dois anos de armazenamento sob estas condições, mantendo as mesmas características.

Viability and molecular authentication of Coccidioides spp. isolates from the Instituto de Medicina Tropical de São Paulo culture collection, Brazil.

Coccidioidomycosis is an emerging fungal disease in Brazil; adequate maintenance and authentication of Coccidioides isolates are essential for research into genetic diversity of the environmental organisms, as well as for understanding the human disease. Seventeen Coccidioides isolates maintained under mineral oil since 1975 in the Instituto de Medicina Tropical de São Paulo (IMTSP) culture collection, Brazil, were evaluated with respect to their viability, morphological characteristics and genetic features in order to authenticate these fungal cultures. Only five isolates were viable after almost 30 years, showing typical morphological characteristics, and sequencing analysis using Coi-F and Coi-R primers revealed 99% identity with Coccidioides genera. These five isolates were then preserved in liquid nitrogen and sterile water, and remained viable after two years of storage under these conditions, maintaining the same features.

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains.

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance--that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance - that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.

Immunoprecipitation techniques and Elisa in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), counterimmunoelectrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53 percent sensitivity and 96 percent specificity for DID, while CIE presented 68 percent sensitivity and 90.5 percent specificity. ELISA demonstrated 78 percent sensibility and 83 percent specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

Immunoprecipitation techniques and Elisa in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), counterimmunoelectrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

Atypical disseminated cutaneous histoplasmosis in an immunocompetent child, caused by an aberrant variant of Histoplasma capsulatum var. capsulatum

O presente trabalho registra caso de histoplasmose em paciente de 5 anos, HIV negativo, natural e procedente da cidade da Sao Paulo, com lesoes cutaneas nao diagnosticadas clinicamente. Exame histopatologico negativo para infeccao fungica. Cultivos em duas ocasioes, positivos para Histoplasma capsulatum var. capsulatum (amostras 361 e 387). Sorologia negativa para anticorpos anti-Histoplasma capsulatum e Paracoccidioides brasiliensis pelas provas de Imunodifusao dupla e ...

Paracoccidioides brasiliensis: a mycologic and immunochemical study of two strains

Os autores estudaram do ponto de vista micologico, imunoquimico e de sua biologia molecular, duas amostras de Paracoccidioides brasiliensis, uma isolada do solo, no municipio de IBIA (MG) por Silva-Vergara et al (1996,1998) denominada IBIA e outra, BAT, cultivada de um caso humano de paracoccidioidomicose em Ribeirao Preto (SP) por Freitas da Silva (1996). Tais amostras apresentam colonias cotonosa (M) e leveduriforme (L ou Y), sendo patogenicas para cobaios inoculados por via testicular, produzindo orquite granulomatosa e/ou supurativa. Do ponto de vista imunoquimico, atraves de provas de Imunodifusao dupla, Imunoeletroforese e Western Blotting, foi demostrada a presenca da gp43

Paracoccidioides cerebriformis Moore, 1935: mycologic and immunochemical study

O presente estudo trata dos resultados obtidos, do ponto de vista micologico e imunoquimico, de duas amostras de Paracoccidioides consideradas como pertencentes a espécie cerebriformis, criada por MOORE em 1935 e mantidas desde aquela época, através de repiques em agar-Sabouraud, na Micoteca do Instituto de Medicina Tropical de Säo Paulo. Após cerca de 60 anos, tais amostras conservavam as mesmas características descritas por MOORE (1935). Näo foram registradas lesöes experimentais em cobaias inoculadas por via intratesticular, näo se demonstrando também, pelas técnicas SDS PAGE e Western blotting...

Reactivity of anti-gp43 antibodies from Paracoccidioides brasiliensis antiserum with extracts from cutaneous lesions of Lobo's disease: preliminary note

Atraves de varias provas imunoquimicas foi demonstrada a presenca da gp43 em extratos de lesoes cutaneas da doenca de Jorge Lobo. A glicoproteina de 43 kDa e um dos antigenos dominantes do Paracoccidioides brasiliensis. permitindo a identificacao desta especie fungica

Paracoccidioides brasiliensis: a mycologic and immunochemical study of a sample isolated from a armadillo (Dasipus novencinctus)

Amostra de Paracoccidioides brasiliensis isolada de visceras (baco e figado) de um tatu (Dasipus novencintus)foi estudada no ponto de vista micologico e imunoquimico. O tatu havia sido capturado em area da usina hidroeletrica de Tucurui (Estado do Para). Este ja havia sido considerado como reservatorio enzootico do Paracoccidioides brasiliensis naquela regiao. Esta amostra, conservada na Micoteca do Instituto de Medicina Tropical de Sao Paulo sob o numero 135, apresenta todas as caracteristicas de Paracoccidioides brasiliensis, com elevado poder antigenico e baixa virulencia para cobaios e ratos Wistar. A demonstracao do exo-antigeno especifico do P. brasiliensis, representado pela glicoproteina de peso molecular 43 KDa, foi evidente atraves das tecnicas de Imunodifusao Dupla, Imunoeletroforese, SDS-PAGE e Imunoblotting.