Immunologic Diagnosis of Endemic Mycoses.

The endemic mycoses blastomycosis, coccidioidomycosis, histoplasmosis, paracoccidioidomycosis, cryptococcosis, sporotrichosis, talaromycosis, adiaspiromycosis, and emergomycosis are mostly caused by geographically limited thermally dimorphic fungi (except for cryptococcosis), and their diagnoses can be challenging. Usual laboratory methods involved in endemic mycoses diagnosis include microscopic examination and culture of biological samples; however, serologic, histopathologic, and molecular techniques have been implemented in the last few years for the diagnosis of these mycoses since the recovery and identification of their etiologic agents is time-consuming and lacks in sensitivity. In this review, we focus on the immunologic diagnostic methods related to antibody and antigen detection since their evidence is presumptive diagnosis, and in some mycoses, such as cryptococcosis, it is definitive diagnosis.

Histoplasma capsulatum Glycans From Distinct Genotypes Share Structural and Serological Similarities to Cryptococcus neoformans Glucuronoxylomannan.

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans (Cn), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides, and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo. In this work, we characterized the ability of Hc to produce cellular-attached (C-gly-Hc) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly-Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly-Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly-Hc and E-gly-Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly-Hc and E-gly-Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly-Hc and E-gly-Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella. These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis.

Novel clinical and dual infection by Histoplasma capsulatum genotypes in HIV patients from Northeastern, Brazil.

Histoplasmosis is a worldwide-distributed deep mycosis that affects healthy and immunocompromised hosts. Severe and disseminated disease is especially common in HIV-infected patients. At least 11 phylogenetic species are recognized and the majority of diversity is found in Latin America. The northeastern region of Brazil has one of the highest HIV/AIDS prevalence in Latin America and Ceará State has one of the highest death rates due to histoplasmosis in the world, where the mortality rate varies between 33-42%. The phylogenetic distribution and population genetic structure of 51 clinical isolates from Northeast Brazil was studied. For that morphological characteristics, exoantigens profile, and fungal mating types were evaluated. The genotypes were deduced by a MSLT in order to define local population structure of this fungal pathogen. In addition, the relationships of H. capsulatum genotypes with clinically relevant phenotypes and clinical aspects were investigated. The results suggest two cryptic species, herein named population Northeast BR1 and population Northeast BR2. These populations are recombining, exhibit a high level of haplotype diversity, and contain different ratios of mating types MAT1-1 and MAT1-2. However, differences in phenotypes or clinical aspects were not observed within these new cryptic species. A HIV patient can be co-infected by two or more genotypes from Northeast BR1 and/or Northeast BR2, which may have significant impact on disease progression due to the impaired immune response. We hypothesize that co-infections could be the result of multiple exposure events and may indicate higher risk of disseminated histoplasmosis, especially in HIV infected patients.

Diagnostic performance of mycologic and serologic methods in a cohort of patients with suspected sporotrichosis / Resultados diagnósticos de los métodos micológicos y serológicos en una cohorte de pacientes con sospecha de esporotricosis

Background: The gold standard for the sporotrichosis diagnosis is culture; however, serologic approaches have been recently implemented to aid in the sporotrichosis diagnosis. Nevertheless, the clinical consequences of the introduction of serologic tests are poorly addressed. Aims: To correlate the results of culture and serology of patients with suspected sporotrichosis. Methods: A retrospective study of 198 patients with suspected sporotrichosis was conducted. Information about culture isolation of Sporothrix from clinical samples and antibody detection by an enzyme-linked immunosorbent assay (ELISA) were obtained from the medical records of the patients. Results: Positive culture and antibody detection was observed in the samples of 84 patients (42.4%). Forty-one samples (20.7%) showed negative results with both techniques and divergent results were obtained in the samples of 73 patients (36.9%). False negative results in the ELISA were observed with 23 patients (31.5%), 78.3% of them with less than 30 days of infection (p = 0.0045). Among the initial false positive ELISA in the sera of 50 patients, four samples in culture yielded the growth of Sporothrix, and 27 improved with itraconazole. At the end of follow-up, a diagnosis of proven or probable sporotrichosis was established in 139 patients, and possible sporotrichosis in 11 patients. The treatment of the patients with probable sporotrichosis with antifungal drugs resulted in clinical cure for these individuals. Conclusions: These two techniques are complementary in the diagnosis of sporotrichosis, making diagnosis and clinical decision more precise

Antecedentes: El método de referencia en el diagnóstico de la esporotricosis es el cultivo, aunque las técnicas serológicas pueden complementar el diagnóstico. Sin embargo, la interpretación de las pruebas serológicas en la práctica clínica y en el diagnóstico de la enfermedad necesitan un abordaje más eficiente. Objetivos: Correlacionar los resultados del cultivo y la serología en pacientes con posibles síntomas de esporotricosis. Métodos: Se realizó un estudio retrospectivo de 198 pacientes con posibles síntomas de esporotricosis. Para establecer el diagnóstico se tuvieron en cuenta el aislamiento de Sporothrix a partir de las muestras clínicas y la detección de anticuerpos anti-Sporothrix realizados por un análisis de inmunoabsorción enzimática (ELISA), datos todos ellos registrados en las respectivas historias clínicas. Resultados: Los cultivos y la detección de anticuerpos fueron positivos en 84 pacientes (42,4%). Las muestras de 41 pacientes (20,7%) resultaron negativas con ambas técnicas y en 73 pacientes (36,9%) los resultados fueron divergentes. Se obtuvieron resultados falsos negativos en el ELISA en 23 pacientes (31,5%), el 78,3% de ellos con menos de 30días de infección (p = 0,0045). De los 50 pacientes con un resultado falso positivo en el ELISA, en 4 de ellos se obtuvo cultivo positivo de Sporothrix y 27 mejoraron con itraconazol. Al finalizar el estudio se estableció un diagnóstico de esporotricosis, que fue probada o probable en 139 pacientes y posible en 11 pacientes. El tratamiento de pacientes con esporotricosis probable con fármacos antifúngicos produjo la cura clínica de estos individuos. Conclusiones: Estos dos métodos son complementarios en el diagnóstico de la esporotricosis y ayudan a la toma de las decisiones clínicas más acertadas

Diagnostic performance of mycologic and serologic methods in a cohort of patients with suspected sporotrichosis.

BACKGROUND: The gold standard for the sporotrichosis diagnosis is culture; however, serologic approaches have been recently implemented to aid in the sporotrichosis diagnosis. Nevertheless, the clinical consequences of the introduction of serologic tests are poorly addressed. AIMS: To correlate the results of culture and serology of patients with suspected sporotrichosis. METHODS: A retrospective study of 198 patients with suspected sporotrichosis was conducted. Information about culture isolation of Sporothrix from clinical samples and antibody detection by an enzyme-linked immunosorbent assay (ELISA) were obtained from the medical records of the patients. RESULTS: Positive culture and antibody detection was observed in the samples of 84 patients (42.4%). Forty-one samples (20.7%) showed negative results with both techniques and divergent results were obtained in the samples of 73 patients (36.9%). False negative results in the ELISA were observed with 23 patients (31.5%), 78.3% of them with less than 30 days of infection (p=0.0045). Among the initial false positive ELISA in the sera of 50 patients, four samples in culture yielded the growth of Sporothrix, and 27 improved with itraconazole. At the end of follow-up, a diagnosis of proven or probable sporotrichosis was established in 139 patients, and possible sporotrichosis in 11 patients. The treatment of the patients with probable sporotrichosis with antifungal drugs resulted in clinical cure for these individuals. CONCLUSIONS: These two techniques are complementary in the diagnosis of sporotrichosis, making diagnosis and clinical decision more precise.

Role of western blot assay for the diagnosis of histoplasmosis in AIDS patients from a National Institute of Infectious Diseases in Rio de Janeiro, Brazil.

BACKGROUND: Histoplasmosis is a frequent fungal infection in HIV/AIDS patients, with high morbimortality rates when diagnosis and treatment are delayed. Antibody detection, which is faster than the gold standard culture test, hastens the laboratory investigation. OBJECTIVES: To evaluate the role of WB for antibody detection in the diagnosis of histoplasmosis among HIV/AIDS patients. PATIENTS AND METHODS: Fifty patients with proven or probable histoplasmosis were included. Clinical, epidemiological and laboratory data were described in the same population after a review of their medical records. WB was performed using deglycosylated histoplasmin. RESULTS: About 82% of patients were adult males and the mean age was 39.3 years. CD4+ T lymphocyte count less than 150 cells/mm3 was observed in 62% patients. Antibodies against Histoplasma capsulatum M antigen were detected in 62% of patients, and against both M and H antigens in 28% of individuals. Sera from 10% of patients were nonreactive. Histoplasmosis was the first opportunistic infection in 38% of the cases. Disseminated and pulmonary histoplasmosis occurred in 84% and 16% of patients, respectively. The overall mortality was 16%. CONCLUSION: WB could be useful for the histoplasmosis diagnosis in HIV/AIDS patients because of its easefulness and good sensitivity in a population where antibody production is hampered.

L-tyrosine induces the production of a pyomelanin-like pigment by the parasitic yeast-form of Histoplasma capsulatum.

Melanization of Histoplasma capsulatum remains poorly described, particularly in regards to the forms of melanin produced. In the present study, 30 clinical and environmental H. capsulatum strains were grown in culture media with or without L-tyrosine under conditions that produced either mycelial or yeast forms. Mycelial cultures were not melanized under the studied conditions. However, all strains cultivated under yeast conditions produced a brownish to black soluble pigment compatible with pyomelanin when grew in presence of L-tyrosine. Sulcotrione inhibited pigment production in yeast cultures, strengthening the hyphothesis that H. capsulatum yeast forms produce pyomelanin. Since pyomelanin is produced by the fungal parasitic form, this pigment may be involved in H. capsulatum virulence.

Acute juvenile Paracoccidioidomycosis: A 9-year cohort study in the endemic area of Rio de Janeiro, Brazil.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by pathogenic dimorphic fungi of the genus Paracoccidioides. It is the most important systemic mycosis in Latin America and the leading cause of hospitalizations and death among them in Brazil. Acute PCM is less frequent but relevant because vulnerable young patients are affected and the severity is usually higher than that of the chronic type. METHODS: The authors performed a retrospective cohort study from 2001 to 2009 including acute juvenile PCM patients from a reference center in Rio de Janeiro, Brazil. Clinical, epidemiological, diagnostic, therapeutic, and prognostic data were reported. RESULTS: Twenty-nine patients were included. The average age was 23 years old and the male to female ratio was 1:1.07. All cases were referred from 3 of 9 existing health areas in the state of Rio de Janeiro, predominantly from urban areas (96.5%). Lymph nodes were the most affected organs (100%), followed by the skin and the spleen (31% each). Twenty-eight patients completed treatment (median 25 months) and progressed to clinical and serological cure; 1 death occurred. Twenty-four patients completed 48-month median follow-up. Four patients abandoned follow-up after the end of treatment. The most frequent sequela was low adrenal reserve. Paracoccidioides brasiliensis S1 was identified by partial sequencing of the arf and gp43 genes from 4 patients who presented a viable fungal culture. CONCLUSION: Acute juvenile PCM is a severe disease with a high rate of complications. There are few cohort clinical studies of acute PCM in the literature. More studies should be developed to promote improvement in patients' healthcare.

Validation of western blot for Histoplasma capsulatum antibody detection assay.

BACKGROUND: Histoplasmosis is worldwide systemic mycoses caused by the dimorphic fungus Histoplasma capsulatum. The isolation and identification of H. capsulatum in culture is the reference test for histoplasmosis diagnosis confirmation. However, in the absence of it, serology has been used as a presumptive diagnosis through antibody and antigen detection. The purpose of the present study was to validate an immunoassay method (western blot) for antibodies detection in the diagnosis of histoplasmosis. METHODS: To validate the western blot (WB) a study was conducted using 118 serum samples from patients with histoplasmosis and 118 serum controls collected from January 2000 to December 2013 in residents of the Rio de Janeiro State, Brazil. Diagnostic validation parameters were calculated based on the categorization of results obtained in a 2 × 2 table and subjected to statistical analysis. In addition, the viability of deglycosylated histoplasmin antigen (ptHMIN) onto nitrocellulose membranes previously sensitized was evaluated during the same period. RESULTS: The WB test showed sensitivity of 94.9 %, specificity of 94.1 %, positive predictive value of 94.1 %, negative predictive value of 94.9 %, accuracy of 94.5 %, and almost perfect precision. Besides, the strips have proved to be viable for using at least 5 years after ptHMIN antigen sensitization. CONCLUSION: Western blot test using ptHMIN provides sensitive, specific, and faster results. Therefore, could be considered a useful tool in the diagnosis of histoplasmosis being used by public health system, even in situations where laboratory facilities are relatively limited.

Sexual variability in Histoplasma capsulatum and its possible distribution: what is going on?

Histoplasma capsulatum is a dimorphic fungal pathogen naturally found in the soil. Inhalation of conidia can result in pulmonary histoplasmosis and, in some cases, causes severe disseminated disease and death. This fungus is an ascomycete that has an anamorphic or asexual stage and a teleomorphic or sexual stage, known as Ajellomyces capsulatus, which results from (+) and (-) mating types. Sexual reproduction is regulated by a specialized genomic region known as the mating-type (MAT1) locus. The mating process in this heterothallic species is represented by isolates that contain only one of the two different MAT1 locus idiomorphs (MAT1-1 or MAT1-2) that have unrelated sequences encoding different transcription factors. In medically important dimorphic pathogens and in most ascomycete molds, one MAT locus idiomorph encodes a high-mobility-group (HMG) box-domain transcription factor, and the other idiomorph encodes an alpha-box domain transcription factor. There is scarce molecular information about H. capsulatum mating type although recombinant population structures have been reported that could occur in nature and this process has been documented in distinct models such as parasites and other fungi. In this review, we shall focus on published studies on H. capsulatum sexuality, and outline the distribution of the two H. capsulatum mating types in Latin America. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Ferramentas proteômicas na identificação de novos alvos antigênicos na proteína M do Histoplasma capsulatum e aplicação em ensaios imunoenzimáticos / Proteomic tools to identify new targets in the M protein antigen of Histoplasma capsulatum and application to immunoassays

A histoplasmose é uma infecção que apresenta amplo espectro clínico, variando desde forma leves, a graves e disseminadas. O diagnóstico da histoplasmose baseia-se nos aspectos clínicos, radiológicos e epidemiológicos. A confirmação se dá pelo isolamento e identificação do Histoplasma capsulatum através de procedimentos microbiológicos. [...] O antígeno M obtido do extrato antigênico histoplasmina é considerado um antígeno imunodominante para produção de anticorpos, sendo reconhecido em cerca de 90 porcento dos soros dos pacientes com histoplasmose, sendo assim nosso grupo vem trabalhando a vários anos em estudos para um melhor conhecimento desta molécula e aplicação no diagnóstico.Um Modelo molecular do antigeno M foi desenvolvido através de sua sequência, tendo então confirmada sua natureza biológica como catalase, sendo observado também que esta molécula apresentava regiões comuns bem como especificas quando comparadas a catalases de organismos eucariotas. No presente estudo procuramos determinar a presença de possíveis epitopos antigênicos na proteína M empregando ferramentas proteômicas, para posterior emprego em ensaios imunoenzimáticos. Para tal foi utilizada a combinação da técnica de coimunoprecipitação com espectometria de massas e posteriormente a técnica de Spot synthesis. Com o emprego do anticorpo monoclonal (mAb 1A7) produzido contra a proteína M recombinante foi possível detectar uma sequência que foi comum as duas metodologias empregadas (PTKIIPEELVPFTP). Esta sequência encontra-se localizada na região onde em estudos anteriores por análise in silico foi apontada como a região mais antigênica desta molécula. Foi realizada a síntese desta sequência, e diferentes desenhos foram utilizados, extensão de resíduos de lisina e adição da molécula de biotina em ambas as extremidades, carboxi e amino terminal, bem como a síntese da sequência sem adição de outras moléculas. Diferentes desenhos de ensaios imunoenzimáticos foram realizados, um ELISA empregando microesferas carboxiladas, ELISA indireto empregando placas de microtitulação revestidas com estreptoavidina e um ELISA sandwich. A sequência não apresentou resultados satisfatórios nos diferentes ensaios. Observamos que apenas no ensaio onde utilizamos o peptídeo ligado a molécula de biotina 1-a e 1-b, foi possível obter um poder discriminatório entre o grupos de pacientes com histoplasmose e o grupos de indivíduos hígidos, o ponto de corte foi obtido pela media das DOs das amostras de indivíduos hígidos mais duas vezes o desvio padrão, onde este teste apresentou uma boa sensibilidade(100 - 95 porcento), porém a especificidade encontrada não foi satisfatória (27 - 20 porcento). Estes resultados não foram concordantes com a análise feita in silico da sequência sintetizada. O antígeno M recombinante foi testado em um ELISA de captura de antígeno, onde os resultados preliminares apresentaram-se promissores necessitando de novos testes para avaliarmos parâmetros como sensibilidade e especificidade.

Histoplasmosis is a worldwide distribution infection with several clinical spectrum, from asymptomatic to severe and disseminated disease. The diagnosis of histoplasmosis is based onclinical, radiological and epidemiological findings. The laboratory diagnosis of histoplasmosis is based on fungus isolation by culture, direct examination in tissue or other clinical specimens. However, such procedures have limitations and are time-consuming. For these reasons serological tests play an important role on presumptive diagnosis. [...] The M antigen is obtain by the antigenic extract histoplasmin and is considered as an immunodominant antigen recognized by 90 percent of sera from patients with histoplasmosis, For a better understanding of the molecule biological nature and its application in diagnosis methodologies our group has been working for several years. The molecular analysis of the M antigen was based on the sequence protein and confirmed as a catalase. It was also observed that this molecule showed specific and common polypeptide regions when compared to catalases from others eukaryotic organisms. In this study we evaluated the possible presence of antigenic epitopes in the M protein sequence that could represent potential candidate as diagnostic markers for histoplasmosis. For this reason we used the combining co-immunoprecipitations and mass spectrometry and spot synthesis technique. The application of a monoclonal antibody against to the M antigen (mAb 1A7) produced by our group allowed the detection of a same sequence in both employed methodologies (PTKIIPEELVPFTP). This was synthesized with different conformations, addition lysine residues and biotin molecules in both amino and carboxy terminal regions. Different immunoassays were performed, carboxylated microspheres, ELISA indireta with microplate coated with streptoavidin and ELISA sandwich. The sequence did not show good specificity and sensibility in different tests. A good discriminatory power was possible when the peptide biotin molecule bind (P1-a and P1-b) was used in serum samples from groups of histoplasmosis patients and healthy controls. This test showed a high sensitivity (100-95 percent), however the specificity was not satisfactory (27-20 percent) respectevily. The ELISA´s cut-off points were established as the mean of absorbances plus two standard deviation of the healthy controls. The immunoassay´s results were discordant when compared on in silico analysis using as antigen the synthesized sequence. In another approach, the M antigen was tested in an antigen-capture enzyme-linked immunosorbent assay (ELISAs) and promising results were observed, but further studies must be done in order to evaluate parameters such as sensitivity and specificity.

Cell-free antigens of Sporothrix brasiliensis: antigenic diversity and application in an immunoblot assay.

Sporotrichosis is a subcutaneous mycosis diagnosed by isolation of the fungus in culture. Serological tests for help in diagnosis in general do not use purified or recombinant antigens, because there is a paucity of described immunoreactive proteins, especially for the new described Sporothrix species, such as Sporothrix brasiliensis. This study aims to characterise antigens from S. brasiliensis and verify their application in serodiagnosis of sporotrichosis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in a cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis.

Evaluation of an enzyme-linked immunosorbent assay using purified, deglycosylated histoplasmin for different clinical manifestations of histoplasmosis.

Diagnosis of invasive fungal diseases remains problematic, especially in undeveloped countries. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Histoplasma capsulatum using metaperiodate treated purified histoplasmin (ptHMIN). Our ELISA was validated comparing sera from patients with histoplasmosis, related mycoses, and healthy individuals. The overall test specificity was 96%, with sensitivities of 100% (8/8) in acute disease, 90% (9/10) in chronic disease, 89% (8/9) in disseminated infection in individuals without HIV infection, 86% (12/14) in disseminated disease in the setting of HIV infection and 100% (3/3) in mediastinal histoplasmosis. These parameters are superior to the use of untreated histoplasmin in diagnostic ELISAs. The high specificities, sensitivities, and simplicity of our ELISA support further development of a deglycosylated HMIN ELISA for clinical use and for monitoring the humoral immune response during therapy in patients with chronic and disseminated histoplasmosis.

Use of mycelial-phase Sporothrix schenckii exoantigens in an enzyme-linked immunosorbent assay for diagnosis of sporotrichosis by antibody detection.

An enzyme-linked immunosorbent assay (ELISA) was developed for specific antibody detection in serum specimens of patients with sporotrichosis. The assay was made with mycelial-phase Sporothrix schenckii exoantigens and was tested against 90 sera from patients with different clinical forms of sporotrichosis. Potential cross-reactions were analyzed with 72 heterologous sera from patients with paracoccidioidomycosis, cryptococcosis, aspergillosis, histoplasmosis, tuberculosis, and American tegumentary leishmaniasis, as well as 76 sera from healthy controls. We found a sensitivity of 97% and a specificity of 89% in this assay. Some cross-reactions were seen, as observed in other immunoassays for the diagnosis of sporotrichosis. The ELISA appears to be especially useful for cutaneous forms of disease, since these are not promptly diagnosed with available immunoprecipitation or agglutination techniques. These results suggest that the ELISA using mycelial-phase S. schenckii exoantigens is a very sensitive diagnostic tool for the serodiagnosis of sporotrichosis and can be used in conjunction with conventional methods of diagnosis, particularly in cases where cross-reactions or false-positive results are experienced with the serodiagnosis.

Histoplasmosis in a Brazilian center: clinical forms and laboratory tests.

Histoplasmosis, caused by the dimorphic fungus Histoplasma capsulatum, is endemic in many regions of the Americas, Asia and Africa. It has a wide spectrum of clinical manifestations, from asymptomatic infection to severe disseminated disease. A retrospective study was carried out to describe the clinical forms and assess the clinical significance of the laboratory diagnostic tests of patients with histoplasmosis during the period of July 1987 to December 2003 at Instituto de Pesquisa Clínica Evandro Chagas/ FIOCRUZ, RJ, Brazil. Seventy-four patients were included. Forty-nine percent of the cases (n = 36) occurred in HIV positive patients who presented with disseminated disease. The remaining 38 cases were classified in different clinical forms. Histoplasma capsulatum was isolated from 69.5% of the clinical specimens sent to culture. Immunodiffusion and immunoblot were positive in 72.6% and 100% of the performed tests, respectively. Histopathologic findings suggestive of H. capsulatum were found in 63.2% of the performed exams. Serology had a lower proportion of positivity amongst AIDS patients, when compared with HIV negative patients (X2 = 6.65; p lower than 0.008). Statistical differences between AIDS and non-AIDS patients were not observed with culture and histopathology. The specific role of each test varies according to the clinical form. Physicians need to know the value and limitations of the available diagnostic tests, but before that, they have to think about histoplasmosis and consider this clinical entity in their differential diagnosis.

ELISA for early diagnosis of histoplasmosis.

An ELISA was developed and evaluated as a method for detecting antibodies against glycosylated and deglycosylated histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, sporotrichosis, coccidioidomycosis, aspergillosis, cryptococcosis and healthy donors were tested by ELISA against purified, deglycosylated histoplasmin (ptHMIN) and compared with purified, native (i.e. glycosylated) histoplasmin (pHMIN). Although cross-reactivity was not abolished when ptHMIN was used in the test, it was reduced (pHMIN ELISA 93 % versus ptHMIN ELISA 96 %). However, there were statistically significant differences between the sensitivities of these two methods for the detection of antibodies (pHMIN ELISA 57 % versus ptHMIN ELISA 92 %; P < 0.001) and between the efficiency of the methods (pHMIN ELISA 83 % versus ptHMIN ELISA 95 %; P < 0.001). These parameters compare better than previously published data relating to the use of treated HMIN in diagnostic ELISAs. Some of the reactivities of serum samples were compared by immunoblotting using deglycosylated HMIN and by immunodiffusion using the crude antigen. The results demonstrated that cross-reactions with heterologous sera in both ELISAs could also be observed in immunoblotting and arose from shared protein epitopes. These data suggest that ELISA using deglycosylated HMIN is a very sensitive diagnostic method and, by using commercially available antigen, it can be easily standardized and performed faster than previous Western blot-based tests using the same antigen. It provides a useful adjunct to existing methods of diagnosis that could be applied even in situations where laboratory facilities were relatively limited.

PCR assay for identification of histoplasma capsulatum based on the nucleotide sequence of the M antigen.

The major diagnostic antigens of Histoplasma capsulatum var. capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. The gene encoding the M antigen has previously been sequenced, and its sequence has significant overall homology to those of the genes for fungal catalases. Regions of the M-antigen gene with little or no homology were used to design four oligonucleotide sequences for application in the PCR detection and identification of H. capsulatum var. capsulatum. The PCR correctly identified the 31 H. capsulatum var. capsulatum strains isolated from human, animal, and soil specimens and 1 H. capsulatum var. duboisii isolate. PCR products of 111 and 279 bp were amplified with primers Msp1F-Msp1R and Msp2F-Msp2R, respectively. No amplification product was obtained from DNA extracted from an H. capsulatum var. farciminosum isolate. The specificity of the PCR with the M-antigen-derived primers was confirmed by the total absence of amplification products when genomic DNA from Paracoccidioides brasiliensis, Candida spp., Sporothrix schenckii, Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides immitis, Aspergillus niger, and Aspergillus fumigatus were applied in the reaction. This rapid, sensitive, and specific assay provides a way to identify typical and atypical isolates of H. capsulatum var. capsulatum.