Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis.

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.

HTLV-1 and HTLV-2 infections in patients with endemic mycoses in São Paulo, Brazil: A cross-sectional, observational study.

Background: Brazil is a country endemic for human T-lymphotropic virus 1 and 2 (HTLV-1 and HTLV-2), systemic mycoses such as paracoccidioidomycosis (PCM) and histoplasmosis (HP), and aspergillosis (AP). The prevalence of HTLV-1/-2 infections in individuals with endemic mycoses in Latin America is unknown; however, an association between HTLV-1 and severe PCM and HP has been observed in Peru. Addressing this knowledge gap, we searched for HTLV-1/-2 antibodies in serum samples sent to the Instituto Adolfo Lutz, São Paulo, Brazil, for systemic mycosis diagnosis. Methods: We used 387 sera from a biorepository that had seropositive results for Paracoccidioides spp. (G1, n=212), Histoplasma capsulatum (G2, n=95), Aspergillus spp. (G3, n=61), and at least two of these fungi (G4, n=19). We searched for the presence of HTLV-1/-2 antibodies using commercial immunoassays: enzyme immunoassay (HTLV-I+II Murex, Diasorin), western blotting (HTLV Blot 2.4, MP Biomedicals), and line immunoassay (INNO-LIA HTLV I/II, Fujirebio). Demographic characteristics were evaluated in each group. Findings: Different regions in São Paulo were sampled. Most samples were from males (76.2%; p=0.001), except for G3, in which no sex bias was detected. Mean age differences were observed between groups: patients with PCM and HP had a similar mean age (42.8 and 42.0 years, respectively), while those with AP and co-fungal infection were older (55.1 and 52.8 years, respectively, (p<0.001). Noteworthy, males were older than females in G1 (p=0.005). Screening detected HTLV-1/2 antibodies in five samples (1.30%; 95% CI: 0.8-1.8%), with two borderline results. HTLV-1/2 was confirmed in two samples: 2/387 (0.52%; 0.063-1.85%): one HTLV-2, male, 42 years, from G1: 1/212 (0.47%; 0.012-2.60%), and one HTLV-1, male, 51 years, from G3: 1/61 (1.64%; 0.042-8.80%). Interpretation: In the state of São Paulo, HTLV-1 and HTLV-2 seem to circulate in male patients with systemic mycoses, and since HTLV-1 could impact fungal disease severity, the identification of co-infection is important regardless of prevalence. Funding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), and Instituto Adolfo Lutz.

Evaluation of IFN-γ secretion after stimulation with C. neoformans and C. gattii antigens in individuals with frequent exposure to the fungus.

In this study we produced antigenic extracts from prototypical strains of C. neoformans (VNI-VNIV) and C. gattii (VGI-VGIV) and tested IFN-γ secretion by Elispot. Antigens from the eight Cryptococcus molecular types (VNI -VNIV and VGI - VGIV) were obtained after capsule reduction. IFN-γ secretion by Elispot method were stimulated with C. neoformans and C. gattii antigens. Peripheral blood mononuclear cells of fourteen healthy control subjects, being: five ecotourists, two mycologists, three poultry keepers, and four individuals without reports of exposure to the fungus. We observed a significant increase in IFN-γ secretion in the group of ecotourists, mycologists and bird keepers in relation to the group of individuals without reports of occupational exposures to these agents. Our results suggest the significant increase in IFN-γ secretion may be related to the continuous exposure of these groups of individuals to the fungus, as well as to the specific antigen memory immune response developed during exposure to Cryptococcus.

Metabolomics and Machine Learning Approaches Combined in Pursuit for More Accurate Paracoccidioidomycosis Diagnoses.

Brazil and many other Latin American countries are areas of endemicity for different neglected diseases, and the fungal infection paracoccidioidomycosis (PCM) is one of them. Among the clinical manifestations, pneumopathy associated with skin and mucosal lesions is the most frequent. PCM definitive diagnosis depends on yeast microscopic visualization and immunological tests, but both present ambiguous results and difficulty in differentiating PCM from other fungal infections. This research has employed metabolomics analysis through high-resolution mass spectrometry to identify PCM biomarkers in serum samples in order to improve diagnosis for this debilitating disease. To upgrade the biomarker selection, machine learning approaches, using Random Forest classifiers, were combined with metabolomics data analysis. The proposed combination of these two analytical methods resulted in the identification of a set of 19 PCM biomarkers that show accuracy of 97.1%, specificity of 100%, and sensitivity of 94.1%. The obtained results are promising and present great potential to improve PCM definitive diagnosis and adequate pharmacological treatment, reducing the incidence of PCM sequelae and resulting in a better quality of life.IMPORTANCE Paracoccidioidomycosis (PCM) is a fungal infection typically found in Latin American countries, especially in Brazil. The identification of this disease is based on techniques that may fail sometimes. Intending to improve PCM detection in patient samples, this study used the combination of two of the newest technologies, artificial intelligence and metabolomics. This combination allowed PCM detection, independently of disease form, through identification of a set of molecules present in patients' blood. The great difference in this research was the ability to detect disease with better confidence than the routine methods employed today. Another important point is that among the molecules, it was possible to identify some indicators of contamination and other infection that might worsen patients' condition. Thus, the present work shows a great potential to improve PCM diagnosis and even disease management, considering the possibility to identify concomitant harmful factors.

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

BACKGROUND: Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility. AIMS: In this study, we sought to optimize the methodology for producing H. capsulatum antigens, and to evaluate its applicability. METHODS: Antigenic preparations obtained from 12 H. capsulatum isolates were evaluated by double immunodiffusion and immunoblotting assays against homologous and heterologous sera. RESULTS: The evaluated and optimized protocol allowed a more stable production, as well as repeatable, reproducible, with shorter culture time and less costly. By double immunodiffusion and immunoblotting assays, the best pattern of reactivity was observed for antigens obtained with 33 days of culture from the isolates 200 and 406 against the M antigen and for the isolate 200 with 15 days against H antigen. The SDS-PAGE presented antigenic components of molecular masses between 17 and 119kDa. The immunoblotting sensitivity was 95.5% and 100% with histoplasmosis sera from ill patients and sera from H. capsulatum infected but otherwise healthy patients, respectively, to the antigen derived from isolates 200 and 406. CONCLUSIONS: We suggest the employment of the antigen from isolate 200, with 15 or 30 days of culture, in the double immunodiffusion and immunoblotting assays due to its good ability to discriminate both sera from patients with histoplasmosis illness and histoplasmosis infection, in addition to its high specificity against heterologous sera.

Factors interfering with the production of Histoplasma capsulatum antigens / Fatores que interferem na produção de antígenos de Histoplasma capsulatum

Histoplasma capsulatum causes systemic mycosis that depends on host susceptibility, fungal virulence, and factors associated with the infectious process. We evaluated the possible interference of the phenotype of 12 samples of H. capsulatum isolated from HIV-positive and negative patients in obtaining antigens, aiming at the serological diagnosis through the gender-specific recognition of the H and M fractions. The antigens were evaluated by double immunodiffusion against H. capsulatum anti-antigen polyclonal antibody and serum samples from patients with histoplasmosis.The phenotypic evaluation revealed differences in the identification of the fungal agent and in the expression of H and M antigens, considered serological markers of the disease, associated with pigmentation and the production of conidia. It was found that antigenic preparations obtained from H. capsulatum isolated from HIV-positive patients may have satisfactory antigenic capacity. The patient's immune status does not seem to interfere with the expression of antigenic proteins secreted by H. capsulatum. However, we suggest that prolonged use of antiretrovirals drugs or steroids can cause important phenotypic alterations. We showed that some fungal samples from patients with a long history of immunosuppressive drugs produced atypical cellular elements and low reactivity against the H and M fractions. (AU)

Histoplasma capsulatum causa micose sistêmica endêmica que depende da suscetibilidade do hospedeiro, da virulência fúngica e de fatores associados ao processo infeccioso. Avaliamos a possível interferência do fenótipo de 12 amostras de H. capsulatumisolados de pacientes HIV positivos e negativos na obtenção de antígenos, visando o diagnóstico sorológico por meio do reconhecimento gênero-específico das frações H e M. Os antígenos foram avaliados por imunodifusão dupla, frente a anticorpo policlonal anti-antígeno de H. capsulatum e frente a amostras de soro de pacientes com histoplasmose. A avaliação fenotípica revelou diferenças, não só na identificação do agente fúngico, mas também na expressão dos antígenos H e M, considerados marcadores sorológicos da doença, associados à pigmentação e produção de conídios. Verificou-se que preparações antigênicas obtidas de H. capsulatum isoladas de pacientes HIV positivos podem ter capacidade antigênica satisfatória. O estado imunológico do paciente parece não interferir na expressão de proteínas antigênicas secretadas por H. capsulatum. No entanto, sugerimos que o uso prolongado de antirretrovirais e/ou esteróides pode causar alterações fenotípicas importantes. Verificou-se que algumas amostras fúngicas isoladas de pacientes com longo histórico de uso de imunossupressores produziram elementos celulares atípicos e baixa reatividade sorológica contra as frações H e M de H. capsulatum. (AU)

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.

Comparison of diagnostic methods to detect Histoplasma capsulatum in serum and blood samples from AIDS patients.

BACKGROUND: Although early and rapid detection of histoplasmosis is essential to prevent morbidity and mortality, few diagnostic tools are available in resource-limited areas, especially where it is endemic and HIV/AIDS is also epidemic. Thus, we compared conventional and molecular methods to detect Histoplasma capsulatum in sera and blood from HIV/AIDS patients. METHODOLOGY: We collected a total of 40 samples from control volunteers and patients suspected of histoplasmosis, some of whom were also infected with other pathogens. Samples were then analyzed by mycological, serological, and molecular methods, and stratified as histoplasmostic with (group I) or without AIDS (group II), uninfected (group III), and infected with HIV and other pathogens only (group IV). All patients were receiving treatment for histoplasmosis and other infections at the time of sample collection. RESULTS: Comparison of conventional methods with nested PCR using primers against H. capsulatum 18S rRNA (HC18S), 5.8S rRNA ITS (HC5.8S-ITS), and a 100 kDa protein (HC100) revealed that sensitivity against sera was highest for PCR with HC5.8S-ITS, followed by immunoblotting, double immunodiffusion, PCR with HC18S, and PCR with HC100. Specificity was equally high for double immunodiffusion, immunoblotting and PCR with HC100, followed for PCR with HC18S and HC5.8-ITS. Against blood, sensitivity was highest for PCR with HC5.8S-ITS, followed by PCR with HC18S, Giemsa staining, and PCR with HC100. Specificity was highest for Giemsa staining and PCR with HC100, followed by PCR with HC18S and HC5.8S-ITS. PCR was less efficient in patients with immunodeficiency due to HIV/AIDS and/or related diseases. CONCLUSION: Molecular techniques may detect histoplasmosis even in cases with negative serology and mycology, potentially enabling early diagnosis.

Multiparametric assay of screening for the diagnosis of mycoses of interest in Public Health: standardization of methodology / Ensaio multiparamétrico de triagem para diagnóstico de micoses de interesse em Saúde Pública: padronização da metodologia

The standardization and validation of a multiplex assay requires the combination of important parameters such as sensitivity and specificity, acceptable levels of performance, robustness, and reproducibility. We standardized a multiparametric Dot-blot aimed at the serological screening of paracoccidioidomycosis, histoplasmosis, and aspergillosis. A total of 148 serum were evaluated: 10 from healthy subjects, 36 from patients with paracoccidioidomycosis, 62 from patients with histoplasmosis, and 40 from patients with aspergillosis. It was found that the multiparametric Dot-blot showed a high percentage of cross-reactivity. However, when evaluated individually, in the serological screening of histoplasmosis, a good performance was observed when compared to the double immunodiffusion assay, considered the gold standard test, with 100% co-positivity and 83.3% co-negativity. The performance of serological screening for aspergillosis was not satisfactory when compared to double immunodiffusion, showing 71.4% co-positivity and 100% co-negativity. The evaluation of the stability of nitrocellulose membranes showed that membranes sensitized with H. capsulatum antigen remained stable for 90 days and those sensitized with A. fumigatus antigen for 30 days. We conclude that the use of crude antigens was not suitable for the standardization of the multiparametric Dot-blot assay, due to the high cross-reactivity, and that further tests should be performed with purified proteins (AU).

A padronização e validação de um ensaio multiplex requer a combinação de parâmetros importantes, como sensibilidade e especificidade, níveis aceitáveis de desempenho, robustez e reprodutibilidade. Este trabalho padronizou um Dot-blot multiparamétrico visando a triagem sorológica da paracoccidioidomicose, histoplasmose e aspergilose. Foram avaliadas 148 amostras de soro: 10 de indivíduos saudáveis, 36 de pacientes com paracoccidioidomicose, 62 de pacientes com histoplasmose e 40 de pacientes com aspergilose. Verificou-se que o Dot-blot multiparamétrico apresentou elevado percentual de reatividade cruzada. Entretanto, quando avaliado individualmente, na triagem sorológica da histoplasmose observou-se bom desempenho quando comparado ao ensaio de imunodifusão dupla, considerado o teste padrão ouro, com 100% de co-positividade e 83,3% de co-negatividade. O desempenho da triagem sorológica da aspergilose não foi satisfatório quando comparado a imunodifusão dupla, apresentando 71,4% de co-positividade e 100% de co-negatividade. A avaliação da estabilidade das membranas de nitrocelulose mostrou que membranas sensibilizadas com antígeno de H. capsulatum permaneceram estáveis por 90 dias e as sensibilizadas com antígeno de A. fumigatus, por 30 dias. Concluímos que o uso de antígenos brutos não foi adequado para a padronização do ensaio de Dot-blot multiparamétrico, devido ao alto índice de reatividade cruzada, e que novos testes devem ser realizados com proteínas purificadas (AU).

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection.

BACKGROUND: Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). METHODS: In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. RESULTS: The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. CONCLUSION: Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life - membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories - and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.

Immunological assays employed for the elucidation of an histoplasmosis outbreak in São Paulo, SP.

Several reports showed outbreaks of histoplasmosis acquired while bat-inhabited caves were visited by tourists, miners or researchers. We evaluated the performance of double immunodifusion (DI) and immunoblotting (IB) assays, employed for the histoplasmosis outbreak elucidation occurred in Vale do Paraíba, São Paulo. The existence of epidemiologic link, four patients with clinical signs suggestive of histoplasmosis and mycological confirmation has made that all 35 individuals involved to the cave visit were subjected to serological evaluation. By DI, we observed reactivity against H. capsulatum antigen in a single serum examined nearly 20 days after exposure to fungal propagules. On the other hand, IB showed reactivity against H and M fractions in 50% of samples evaluated. The analysis of the second sample batch, collected two months after the exposure showed that 96.7% were reactive by DI with antibodies titers ranging from 1 to 16 and 100% of reactivity against H and M fractions, by IB, suggesting an acute infection. The analysis of the overall agreement between the methods showed to be reasonable (κ = 0.37). This study confirms the importance and efficacy of more sensitive methodologies, such as IB assay, to early elucidation of disease, especially in cases of patients without mycological information.

Perfil sorológico dos pacientes com suspeita clínica de paracoccidiodomicose avaliados pelo Instituto Adolfo Lutz no ano de 2016 / Serological profile of patients with clinical suspicion of paracoccidiodomycosis evaluated by Instituto Adolfo Lutz in 2016

Oitenta porcento dos casos de paracoccidioidomicose (PMC) ocorrem no Brasil. As regiões brasileiras com maior número de casos são: sul, sudeste e centro-oeste, sendo emergente no norte e nordeste. A imunodifusão dupla em gel de agarose assume grande importância no diagnóstico, por permitir o monitoramento da doença e por oferecer subsídios para levantamentos soroepidemiológicos. O objetivo deste trabalho foi de avaliar e caracterizar os pacientes atendidos no Laboratório de Imunodiagnóstico das Micoses do Instituto Adolfo Lutz de São Paulo, em 2016. Trata-se de um estudo retrospectivo realizado utilizando-se dados secundários e avaliando-se as seguintes informações: idade, sexo, procedência do pedido médico, resultado e histórico sorológico dos pacientes. Dos 1.408 pacientes, 12,8% apresentaram reatividade sorológica para Paracoccidioides brasiliensis. Destes, 42,5% não possuiam histórico sorológico, sendo considerados como casos novos da doença. A classificação dos pacientes reagentes por gênero demonstrou que 83,4% eram do sexo masculino, com razão de masculinidade de 5:1. A faixa etária variou de um (1) a 92 anos, e aproximadamente 40% dos pacientes eram da faixa etária de 41 a 60 anos. Este estudo demonstra e reforça a importância da implementação dos estudos soroepidemiológicos como ferramenta auxiliar para nortear as ações de vigilância e políticas em saúde na PCM. (AU)

Eighty percent of paracoccidioidomycosis (PMC) cases occur in Brazil. The highest numbers occur in south, southeast and center-west region, being emergent in the north and northeast areas. The double immunodiffusion in agarose gel is valuable for its diagnosis, as it allows the monitoring of the disease and offers subsidies for the seroepidemiological surveys. This study evaluated and characterized the patients attended in 2016 at the Mycoses Immunodiagnosis Laboratory of Adolfo Lutz Institute of São Paulo. This retrospective study, based on the secondary data, evaluated the information: age, sex, medical request origin, result and serological history of the patients. Of 1,408 patients, 12.8% presented positive serological reactivity for Paracoccidioides brasiliensis. Of them, 42.5% had no serological history and they were considered as new cases. The classification of reactive patients by gender showed that 83.4% were males, being the masculinity ratio of 5:1. The age range was one (1) to 92 years old, and ±40% of the patients were of age ranging from 41-60 years old. This study reinforces the importance of the implementation of the seroepidemiological studies as to guide the surveillance actions and the public health politics in PCM. (AU)

Perfil sorológico dos pacientes com suspeita clínica de paracoccidiodomicose avaliados pelo Instituto Adolfo Lutz no ano de 2016 / Serological profile of patients with clinical suspicion of paracoccidiodomycosis evaluated by Instituto Adolfo Lutz in 2016

Oitenta porcento dos casos de paracoccidioidomicose (PMC) ocorrem no Brasil. As regiões brasileiras com maior número de casos são: sul, sudeste e centro-oeste, sendo emergente no norte e nordeste. A imunodifusão dupla em gel de agarose assume grande importância no diagnóstico, por permitir o monitoramento da doença e por oferecer subsídios para levantamentos soroepidemiológicos. O objetivo deste trabalho foi de avaliar e caracterizar os pacientes atendidos no Laboratório de Imunodiagnóstico das Micoses do Instituto Adolfo Lutz de São Paulo, em 2016. Trata-se de um estudo retrospectivo realizado utilizando-se dados secundários e avaliando-se as seguintes informações: idade, sexo, procedência do pedido médico, resultado e histórico sorológico dos pacientes. Dos 1.408 pacientes, 12,8% apresentaram reatividade sorológica para Paracoccidioides brasiliensis. Destes, 42,5% não possuiam histórico sorológico, sendo considerados como casos novos da doença. A classificação dos pacientes reagentes por gênero demonstrou que 83,4% eram do sexo masculino, com razão de masculinidade de 5:1. A faixa etária variou de um (1) a 92 anos, e aproximadamente 40% dos pacientes eram da faixa etária de 41 a 60 anos. Este estudo demonstra e reforça a importância da implementação dos estudos soroepidemiológicos como ferramenta auxiliar para nortear as ações de vigilância e políticas em saúde na PCM.

Eighty percent of paracoccidioidomycosis (PMC) cases occur in Brazil. The highest numbers occur in south, southeast and center-west region, being emergent in the north and northeast areas. The double immunodiffusion in agarose gel is valuable for its diagnosis, as it allows the monitoring of the disease and offers subsidies for the seroepidemiological surveys. This study evaluated and characterized the patients attended in 2016 at the Mycoses Immunodiagnosis Laboratory of Adolfo Lutz Institute of São Paulo. This retrospective study, based on the secondary data, evaluated the information: age, sex, medical request origin, result and serological history of the patients. Of 1,408 patients, 12.8% presented positive serological reactivity for Paracoccidioides brasiliensis. Of them, 42.5% had no serological history and they were considered as new cases. The classification of reactive patients by gender showed that 83.4% were males, being the masculinity ratio of 5:1. The age range was one (1) to 92 years old, and ±40% of the patients were of age ranging from 41-60 years old. This study reinforces the importance of the implementation of the seroepidemiological studies as to guide the surveillance actions and the public health politics in PCM.

Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptoccocus neoformans infection: a case report.

This case reports an immunocompetent 29-year-old woman with suspected pneumonia, suggestive of fungal infection. Immunoblotting analysis reactivity against Histoplasma capsulatum and Paracoccidioides brasiliensis were observed. Nested-PCR in blood employing species-specific primers was positive for H. capsulatum and Cryptococcus neoformans. The evaluation of paucisymptomatic patients with positive results for H. capsulatum and C. neoformans could be relevant for the prevention as well as the possible evaluation of the reactivated quiescent foci. In conclusion, the associated methodology may have contributed to the monitoring endogenous reactivation of these diseases.

Prevalence and serological diagnosis of relapse in paracoccidioidomycosis patients.

A review of 400 clinical records of paracoccidioidomycosis (PCM) patients, 93 with the acute/subacute (AF) and 307 with the chronic form (CF), attended from 1977 to 2011, selected as to the schedule of release for study by the Office of Medical Records at the University Hospital of the Faculdade de Medicina de Botucatu-São Paulo State University--UNESP, was performed to detect cases in relapse. The control of cure was performed by clinical and serological evaluation using the double agar gel immunodiffusion test (DID). In the diagnosis of relapse, DID, enzyme-linked immunosorbent assay (ELISA) and immunoblotting assay (IBgp70 and IBgp43) were evaluated. Out of 400 patients, 21 (5.2%) went through relapse, 18 of them were male and 3 were female, 6∶1 male/female ratio. Out of the 21 patients in relapse, 15 (4.8%) showed the CF, and 6 (6.4%) the AF (p>0.05). The sensitivity of DID and ELISA before treatment was the same (76.1%). DID presented higher sensitivity in pre-treatment (80%) than at relapse (45%; p = 0.017), while ELISA showed the same sensitivity (80% vs 65%; p = 0.125). The serological methods for identifying PCM patients in relapse showed low rates of sensitivity, from 12.5% in IBgp70 to 65.0% in IBgp43 identification and 68.8% in ELISA. The sensitivity of ELISA in diagnosing PCM relapse showed a strong tendency to be higher than DID (p = 0.06) and is equal to IBgp43 (p = 0.11). In sum, prevalence of relapse was not high in PCM patients whose treatment duration was based on immunological parameters. However, the used methods for serological diagnosis present low sensitivity. While more accurate serological methods are not available, we pay special attention to the mycological and histopathological diagnosis of PCM relapse. Hence, direct mycological, cytopathological, and histopathological examinations and isolation in culture for P. brasiliensis must be appropriately and routinely performed when the hypothesis of relapse is considered.

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.

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

Abstract Background Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.