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.

Multiplex polymerase chain reaction as an improved method for screening Histoplasma capsulatum mating types.

Histoplasmosis is a systemic mycosis infection caused by Histoplasma capsulatum, a heterothallic ascomycete. The sexual reproduction of this fungus is regulated by the mating type (MAT1) locus that contains MAT1-1 and MAT1-2 idiomorphs, which were identified by uniplex polymerase chain reaction (PCR). This study aimed to optimise single-step multiplex PCR for the accurate detection of the distinct mating types of H. capsulatum. Among the 26 isolates tested, 20 had MAT1-1 genotype, while six showed MAT1-2 genotype, in agreement with the uniplex PCR results. These results suggest that multiplex PCR is a fast and specific tool for screening H. capsulatum mating types.

Comparison of four molecular approaches to identify Candida parapsilosis complex species.

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.

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.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA.

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Trends in the Epidemiological and Clinical Profile of Paracoccidioidomycosis in the Endemic Area of Rio de Janeiro, Brazil.

Paracoccidioidomycosis (PCM) is a neglected endemic mycosis in Latin America. Most cases occur in Brazil. It is classified as PCM infection and PCM disease and is subdivided into chronic (adult type) or acute (juvenile type) disease, with the latter being less frequent and more severe. In 2016, we reported an increase in the numbers of patients diagnosed with acute PCM after a highway's construction. We conducted a study at INI-Fiocruz, a reference center for infectious diseases, including endemic mycoses, in Rio de Janeiro, Brazil, aiming to deepen the analysis of this new clinical and epidemiological profile of PCM. The authors developed a retrospective study including 170 patients diagnosed with PCM between 2010 and 2019. There was an increase in the number of atypical and severe forms, starting in 2014. In subsequent years, we detected a higher incidence of adverse outcomes with patients requiring more hospitalizations and an increased mortality rate. We estimate that PCM has become more severe throughout the Rio de Janeiro state, affecting a greater number of young individuals and leading to a greater number of and longer hospitalizations. Surveillance measures and close monitoring of future notification data in the state, with emphasis on children, adolescents, and young adults are necessary for a better understanding of the perpetuation of this public health challenge.

Prevalence of pulmonary mycoses in smear-negative patients with suspected tuberculosis in the Brazilian Amazon.

BACKGROUND: Pulmonary mycoses resemble clinically and radiologically chronic pulmonary tuberculosis. Studies describing the prevalence, etiology and clinical features of pulmonary mycosis are of crucial importance in the Brazilian Amazon. AIMS: To estimate the frequency of pulmonary mycoses in smear-negative tuberculosis patients; to describe their demographic, epidemiological, and clinical characteristics; and to evaluate diagnostic methods. METHODS: A cross-sectional study was conducted at two tuberculosis reference institutions in Amazonas, Brazil. We included 213 patients and collected clinical data, blood and induced sputum to perform serological, direct microscopy, microbiologic culture and PCR-based assays to identify infections caused by Aspergillus fumigatus, Paracoccidioides brasiliensis, Histoplasma capsulatum, Cryptococcus, and HIV. Chest computed tomography was also performed. RESULTS: Pulmonary mycoses were diagnosed in 7% (15/213) of the cases, comprising ten aspergillosis cases, three cases of paracoccidioidomycosis and one case each of histoplasmosis and cryptococcosis. Among the patients with pulmonary mycoses, 86.7% were former tuberculosis patients. The most significant clinical characteristics associated with pulmonary mycoses were cavity-shaped lung injuries, prolonged chronic cough and hemoptysis. CONCLUSIONS: Our study confirmed the high prevalence of pulmonary mycoses in smear-negative tuberculosis patients in the Brazilian Amazon.

Comparison of different DNA-based methods for molecular typing of Histoplasma capsulatum.

Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited.

Postpartum histoplasmosis in an HIV-negative woman: a case report and phylogenetic characterization by internal transcribed spacer region analysis.

The present report describes the first case of postpartum disseminated histoplasmosis in a 24-year-old HIV-negative woman. On the tenth day after vaginal delivery, the patient presented with dyspnea, fever, hypotension, tachycardia, and painful hepatomegaly. Yeast-like Histoplasma capsulatum features were isolated in the buffy coat. The phylogenetic analysis demonstrated that the fungal isolate was similar to other H. capsulatum isolates identified in HIV patients from Ceará and Latin America. Thus, histoplasmosis development in individuals with transitory immunosuppression or during the period of immunological recovery should be carefully examined.

Neuroparacoccidioidomycosis: A 13-Year Cohort Study, Rio de Janeiro, Brazil.

Neuroparacoccidioidomycosis (NPCM) is a rare and severe clinical presentation of paracoccidioidomycosis (PCM). We performed a retrospective cohort study at the Evandro Chagas National Institute of Infectious Diseases (INI/Fiocruz), a reference center for PCM in the state of Rio de Janeiro, Brazil. All cases of PCM admitted to the INI/Fiocruz from January 2007 to December 2019 were reviewed. Eight (3.9%) among 207 patients met the diagnostic criteria for NPCM. The mean age was 44.6 years and the male:female ratio was 7:1. All cases presented multifocal disease, 5 (62.5%) the chronic form and 3 (37.5%) the acute/subacute form. All patients presented the pseudotumoral pattern and 6 (75.0%) had multiple lesions in the cerebral hemispheres. Seizures and motor symptoms were the most frequent clinical manifestations (50.0%, each). The treatment of choice was sulfamethoxazole/trimethoprim (SMZ-TMP) and fluconazole, in association (87.5%). Most patients responded well to the treatment. Sequela and death occurred in one (12.5%) patient, each.

Molecular characterisation of Sporothrix schenckii isolates from humans and cats involved in the sporotrichosis epidemic in Rio de Janeiro, Brazil.

An epidemic of sporotrichosis, a subcutaneous mycosis caused by the fungus Sporothrix schenckii, is ongoing in Rio de Janeiro, Brazil, in which cases of human infection are related to exposure to cats. In an attempt to demonstrate the zoonotic character of this epidemic using molecular methodology, we characterised by DNA-based typing methods 19 human and 25 animal S. schenckii isolates from the epidemic, as well as two control strains. To analyse the isolates, the random amplified polymorphic DNA (RAPD) technique was performed using three different primers, together with DNA fingerprinting using the minisatellite derived from the wild-type phage M13 core-sequence. The analyses generated amplicons with considerable polymorphism. Although isolates exhibited high levels of genetic relatedness, they could be clustered into 5-10 genotypes. The RAPD profiles of epidemic S. schenckii isolates could be distinguished from that of the United States isolate, displaying 20% similarity to each primer and 60% when amplified with the M13 primer. DNA fingerprinting of S. schenckii isolated from the nails (42.8%) and the oral cavities (66%) of cats were identical to related human samples, suggesting that there is a common infection source for animals and humans in this epidemic. It is clear that cats act as a vehicle for dissemination of S. schenckii.

Mixed infection by Histoplasma capsulatum isolates with different mating types in Brazilian AIDS-patients.

Mixed infection by Histoplasma capsulatum isolates with different mating types, in AIDS-patients are described in this study. Morphological, mating type-specific PCR assay and multilocus sequencing type analysis of H. capsulatum isolates recovered from two Brazilian AIDS-patients were performed. Five H. capsulatum isolates were recovered at different times from the two patients. Three isolates were obtained from bone marrow (day 1 - CE0411) and buffy coat cultures (day 1 - CE0311; day 2 - CE0511) of patient 1, and two isolates were isolated from buffy coat cultures (day 3 - CE2813; day 12 - CE2513) of patient 2. The mycelial colonies depicted different textures and pigmentation features. Dimorphic conversion to the yeast-phase in ML-Gema medium was achieved in all isolates. MAT1-1 idiomorph was identified in CE0311, CE0411 and CE2813 isolates; MAT1-2 idiomorph was found in CE0511 and CE2513 isolates. These H. capsulatum isolates were grouped within LAm A clade, highlighting that CE0311 and CE0411 isolates formed a subgroup supported by a high bootstrap value. The CE0511, CE2513, and CE2813 isolates clustered together with a Brazilian H151 isolate. This research reports mixed infections caused by H. capsulatum isolates with different mating types in Brazilian AIDS-patients for the first time in the literature.

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.

Chronic disseminated histoplasmosis with lesions restricted to the mouth: case report.

We report a rare case of chronic disseminated histoplasmosis with several ulcerated lesions in the oral cavity in an alcoholic patient without human immunodeficiency virus infection, with no detectable signs and symptoms of systemic disease or extraoral manifestations. Histopathological analysis revealed chronic inflammatory process with granulomas containing Histoplasma-like organisms. The isolation of Histoplasma capsulatum provided the definitive diagnosis. Treatment with itraconazole resulted in complete remission of oral lesions. As far we aware, this is the second case report of oral histoplasmosis in an HIV negative patient described in Brazil.

Multiple opportunistic fungal infections in an individual with severe HIV disease: A case report.

BACKGROUND: Fungal infections have been commonly diagnosed in individuals with advanced HIV disease. Cryptococcosis, pneumocystosis, and histoplasmosis are the most frequent systemic mycoses in people suffering from HIV/AIDS. CASE REPORT: We report a case of multiple fungal infections in an advanced AIDS-patient. A 33-year-old HIV-positive man from Brazil was hospitalized due to diarrhea, dyspnea, emaciation, hypoxemia, extensive oral thrush, and a CD4+ T lymphocyte count of 20cells/mm(3). Honeycombed-structures consistent with Pneumocystis jirovecii were observed by direct immunofluorescence in induced sputum. Cryptococcus neoformans was recovered from respiratory secretion and cerebrospinal fluid cultures. Histopathology of the bone marrow also revealed the presence of Histoplasma capsulatum. Molecular assays were performed in a sputum sample. Nested-PCR confirmed the presence of P. jirovecii and H. capsulatum; qPCR multiplex was positive for C. neoformans and H. capsulatum. With the treatment of antifungal drugs the patient progressed satisfactorily. CONCLUSIONS: The diagnosis of several systemic mycoses demonstrates the vulnerability of advanced AIDS-patients. Thus, the detection of AIDS cases in the early stages of infection is necessary for a prompt and adequate introduction of HAART therapy, and the use of prophylaxis to control opportunistic infections.

Investigação de surto de Histoplasmose pulmonar aguda entre bombeiros em Brazlândia, Distrito Federal, 2017 / Investigation of acute histoplasmosis outbreak among firefighters, Distrito Federal, 2017

Objetivos: Investigar o surto de histoplasmose em bombeiros no Distrito federal/DF, ocorrido em junho de 2017. Métodos: Realizou-se um estudo de coorte por meio das entrevistas realizadas com os bombeiros mediante um questionário semiestruturado. Considerou-se infectado o bombeiro que apresentou tomografia de tórax sugestiva de histoplasmose ou reagente nos testes de imunodifusão e/ou Western blot. Coletou-se amostra ambiental e realizou-se Nested PCR específico para Histoplasma capsulatum. Resultados: Entre 35 bombeiros, 94,3% eram homens; com a mediana de idade de 37 (24-45) anos, 28 foram classificados como infectados. A média de permanência dentro da caverna foi 25 minutos. O fator de risco associado à infecção foi o ato de entrar na caverna (RR=3,86; RA=71,6; p<0,02). Entre 14 amostras ambientais, 50% foram positivas para H. capsulatum. Conclusão: Confirmou-se o surto de histoplasmose de bombeiros em Brazlândia-DF, e foram tomadas ações como a interdição da caverna e o tratamento dos bombeiros.

Objectives: To nvestigate the outbreak of histoplasmosis in firefighters in Federal District/DF, which occurred in June 2017. Methods: A cohort study was conducted through interviews with firefighters by means of a semi-structured questionnaire. Firefighters who presented chest tomography suggestive of histoplasmosis or reacted to immunodiffusion and/or Western blot tests were considered infected. Environmental samples were collected and Nested PCR specific for Histoplasma capsulatum was performed. Results: Among 35 firefighters, 94.3% were men; with a median age of 37 (24-45) years, 28 were classified as infected. The average length of stay inside the cave was 25 minutes. The risk factor associated with infection was the act of entering the cave (RR = 3.86, RA = 71.6, p <0.02). Among 14 environmental samples, 50% were positive for H. capsulatum. Conclusion: The outbreak of histoplasmosis in firefighters in Brazlândia-DF was confirmed, and actions were taken such as banning the cave and treating the firefighters

Genetic diversity of Histoplasma capsulatum strains in Brazil.

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.

Different scenarios for Candida parapsilosis fungaemia reveal high numbers of mixed C. parapsilosis and Candida orthopsilosis infections.

Nosocomial fungal bloodstream infections (BSI) are increasing significantly in hospitalized patients and Candida parapsilosis has emerged as an important pathogen responsible for numerous outbreaks. The objective of this study was to evaluate C. parapsilosis sensu lato infection scenarios, regarding species distribution and strain relatedness. One hundred isolates of C. parapsilosis sensu lato derived from blood cultures and catheter tips were analysed by multiplex microsatellite typing and by sequencing D1/D2 regions of the ribosomal DNA. Our results indicate that 9.5 % of patients presented infections due to C. parapsilosis and Candida orthopsilosis, 57.1 % due to C. parapsilosis, 28.3 % due to C. orthopsilosis and 4.8 % due to Candida metapsilosis. Eighty per cent of the C. parapsilosis BSIs were due to a single strain that was also identified in the catheter, but in 10 % of the cases C. parasilosis was identified in the catheter but the BSI was due to C. orthopsilosis. There is a significant probability that C. parapsilosis isolates collected from the same patient at more than 3 months interval are of different strains (P = 0.0179). Moreover, several isolates were identified persistently in the same hospital, infecting six different patients. The incidence of polyfungal BSI infections with C. parapsilosis and C. orthopsilosis is reported herein for the first time, emphasizing the fact that the species identified in the catheter is not always responsible for the BSI, thus impacting the treatment strategy. The observation that strains can remain in the hospital environment for years highlights the possible existence of reservoirs and reinforces the need for accurate genotyping tools, such as the markers used for elucidating epidemiological associations and detecting outbreaks.