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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.

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.

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

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.

Cases of meningoencephalitis have increased in the zoonotic hyperendemia of sporotrichosis in the state of Rio de Janeiro, Brazil. The gold standard for the diagnosis of sporotrichosis is the isolation of Sporothrix spp. from clinical specimens, not always possible from the cerebrospinal fluid (CSF) of these patients, since fungus recovery in this specimen is difficult in most cases. This limitation led us to pursue a new approach on central nervous system (CNS) sporotrichosis diagnosis based on existing molecular methodologies for the detection of Sporothrix spp. in skin samples. Kano and colleagues (2003) designed species-specific primers for polymerase chain reaction (PCR) based on Sporothrix schenckii sensu lato chitin synthase 1 (CHS1) gene sequence and applied it in skin biopsy paraffin block. Hu and colleagues (2003) used a nested PCR in human clinical samples and samples from infected mice, with the 18S rRNA gene sequence as target. The assay was successfully used to detect S. schenckii DNA from strains from different areas of the world. However, Mendoza and colleagues compared the previously described nested PCR with conventional diagnostic methods, and the molecular methodology presented lower efficacy. Liu and colleagues, using the primer pair S2-R2 targeting the CHS1 gene in the PCR of biopsy tissue, verified positive results in 25 out of 30 cases (83.3%). The nested PCR targeting the partial sequence of the 18S rRNA gene was the best choice in terms of sensitivity due to a low fungal burden in CSF.

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.

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.

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.

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.

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.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by pathogenic dimorphic fungi of the Paracoccidioides brasiliensis complex. It is the most important systemic mycosis in Latin America, mainly in Brazil. Despite its severity and high mortality rates, it is considered a neglected disease. Species within the genus Paracoccidioides present genetics and morphological variations with probable clinical, diagnostic and therapeutic consequences. In fact, there are a very small number of detailed case reports with molecular identification of these fungal agents. Here, it is reported a case of PCM due to Paracoccidioides brasiliensis PS2. Molecular identification of the isolate was performed by amplification and sequencing of the arf and gp43 genes. Clinical cases and strain reports with molecular identification in the literature are also reviewed. The case herein presented is the first autochthonous report of PCM due to Paracoccidioides brasiliensis PS2 species in the state of Rio de Janeiro, Brazil, an important endemic area. The patient presented a chronic pulmonary form of PCM and had a satisfactory response to sulfamethoxazole/trimethoprim although sequelae such as adrenal insufficiency and dysphonia were observed. This study may contribute to improve the knowledge about this severe disease, its causative cryptic species and their consequences to patients.

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 20 cells/mm3. 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.

A delay in laboratory diagnosis is related to sequelae and death. Thus, early diagnosis is the key to decrease the high lethality rate due to cryptococcosis. The cryptococcal antigen lateral flow assay (CrAg LFA) Immy test is standardized for a fast screening point-of-care test for cryptococcosis, detecting cryptococcal antigen in serum and cerebrospinal fluid (CSF). Urine screening would be ideal as a noninvasive approach, but previous studies have shown that fresh urine present false positive results. For this reason, we introduced just one simple physical procedure (i.e, heating of fresh urine prior to CrAg LFA Immy testing) to increase the specificity without compromising test sensitivity.

The gold standard for the diagnosis of paracoccidioidomycosis is direct examination and culture; however, serologic, histopathologic, and molecular approaches have been recently adopted for the diagnosis of this mycosis. Few molecular methods have been applied to the diagnosis of paracoccidioidomycosis to detect Paracoccidioides spp. DNA from clinical specimens, and to identify the same fungi in culture. In this review, we focus on the current diagnosis of the paracoccidioidomycosis, and discuss the current molecular tools applied to the diagnosis and identification of the Paracoccidioides complex species.

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.

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.

Histoplasmosis is a systemic mycosis caused by Histoplasma capsulatum, a dimorphic fungal pathogen that can infect both humans and animals. This disease has worldwide distribution and affects mainly immunocompromised individuals. In the environment, H. capsulatum grows as mold but undergoes a morphologic transition to the yeast morphotype under special conditions. Molecular techniques are important tools to conduct epidemiologic investigations for fungal detection, identification of infection sources, and determination of different fungal genotypes associated to a particular disease symptom. In this study, we performed a systematic review in the PubMed database to improve the understanding about the molecular epidemiology of histoplasmosis. This search was restricted to English and Spanish articles. We included a combination of specific keywords: molecular typing [OR] genetic diversity [OR] polymorphism [AND] H. capsulatum; molecular epidemiology [AND] histoplasmosis; and molecular epidemiology [AND] Histoplasma. In addition, we used the specific terms: histoplasmosis [AND] outbreaks. Non-English or non-Spanish articles, dead links, and duplicate results were excluded from the review. The results reached show that the main methods used for molecular typing of H. capsulatum were: restriction fragment length polymorphism, random amplified polymorphic DNA, microsatellites polymorphism, sequencing of internal transcribed spacers region, and multilocus sequence typing. Different genetic profiles were identified among H. capsulatum isolates, which can be grouped according to their source, geographical origin, and clinical manifestations.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens., This study was supported by an National Health and Medical Research Council of Australia (NH&MRC) grant [#APP1031952] to W Meyer, S Chen, V Robert, and D Ellis; CNPq [350338/2000-0] and FAPERJ [E-26/103.157/2011] grants to RM Zancope-Oliveira; CNPq [308011/2010-4] and FAPESP [2007/08575-1] Fundacao de Amparo Pesquisa do Estado de So Paulo (FAPESP) grants to AL Colombo; PEst-OE/BIA/UI4050/2014 from Fundacao para a Ciencia e Tecnologia (FCT) to C Pais; the Belgian Science Policy Office (Belspo) to BCCM/IHEM; the MEXBOL program of CONACyT-Mexico, [ref. number: 1228961 to ML Taylor and [122481] to C Toriello; the Institut Pasteur and Institut de Veil le Sanitaire to F Dromer and D Garcia-Hermoso; and the grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado de Goias (FAPEG) to CM de Almeida Soares and JA Parente Rocha. I Arthur would like to thank G Cherian, A Higgins and the staff of the Molecular Diagnostics Laboratory, Division of Microbiology and Infectious Diseases, Path West, QEII Medial Centre. Dromer would like to thank for the technical help of the sequencing facility and specifically that of I, Diancourt, A-S Delannoy-Vieillard, J-M Thiberge (Genotyping of Pathogens and Public Health, Institut Pasteur). RM Zancope-Oliveira would like to thank the Genomic/DNA Sequencing Platform at Fundacao Oswaldo Cruz-PDTIS/FIOCRUZ [RPT01A], Brazil for the sequencing. B Robbertse and CL Schoch acknowledge support from the Intramural Research Program of the NIH, National Library of Medicine. T Sorrell's work is funded by the NH&MRC of Australia; she is a Sydney Medical School Foundation Fellow., info:eu-repo/semantics/publishedVersion

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. Reportamos um caso raro de histoplasmose crônica disseminada com diversas lesões ulceradas na cavidade oral de um paciente alcoólatra, sem o vírus da imunodeficiência humana, com nenhum outro sinal detectável ou sintomas de doença sistêmica ou manifestações extra-orais. Análise histopatológica revelou processo inflamatório crônico com granulomas contendo organismos fúngicos. O isolamento do Histoplasma capsulatum forneceu o diagnóstico definitivo. Tratamento com itraconazol resultou numa remissão completa das lesões orais. De acordo com nosso conhecimento, este é o segundo caso reportado de histoplasmose oral em um paciente HIV negativo descrito no Brasil.

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 Clinica 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.

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.

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 .

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., Financial support was provided by CAPES Foundation (BEX 19194/12-9), Ministry of Education of Brazil, Brasilia (DF 70.040-020), by FEDER through POFC-COMPETE and by Portuguese funds from FCT (PEst-OE/BIA/UI4050/2014). R.M.Z.-O. is supported in part by CNPq (350338/2000-0) and FAPERJ E (26/103.157/2011). We are grateful to Ronaldo Rozembaun from HSE and SAM and Andrea Pussenti Derossi from HUPE for providing the Candida isolates and technical assistance in sampling. Automated sequencing was done using the genomic platform/DNA sequencing platform at Fundacao Oswaldo Cruz, PDTIS/FIOCRUZ (RPT01A), Brazil. The authors declare that they have no conflict of interest., info:eu-repo/semantics/publishedVersion

In order to correlate the findings of two serological tests, double immunodiffusion (IDD) and immunoblotting (IB), with the clinical diagnosis and follow-up of paracoccidioidomycosis (PCM), 325 serum samples from PCM patients were tested at the beginning of specific therapy and after its completion. Group I included 245 PCM patients at the onset of symptoms without treatment. In 221 cases (90.2%) the IDD showed positive reactions and in 24 (9.8%) the results were negative. Of the 24 IDD negative samples, 23 were investigated by IB and were positive. Group II included 80 PCM patients under follow-up after treatment. There were four cases of relapse in which the IDD and IB tests were positive (100%). Among the 76 cases with inactive mycotic infection, the IDD was negative in 71.2% and positive in 28.8%; the IB was positive in all cases (100%). The control group (Group III) included 27 samples from patients with other mycoses, tuberculosis and from healthy individuals. All showed negative IDD tests but positive reactions with IB, which could be abolished by serum dilutions without altering the PCM reactivity. Therefore, the utilization of the IB, an immunoenzymatic method for the diagnosis of PCM, raised the sensitivity to 100%.

The present study used two molecular typing methods to investigate a cluster of eight cases of Candida parapsilosis fungemia in a hospital in Rio de Janeiro, Brazil. Candida parapsilosis is an important opportunistic pathogen that is frequently involved in outbreaks of nosocomial fungemia. Identification of a common source of infection and determination of genetic relatedness among the strains involved in outbreaks are important for infection control. Candida parapsilosis strains were isolated from the bloodstream of patients housed in an intensive-care unit (n=5) and in individual rooms (n=3). An additional strain of Candida parapsilosis was isolated from a hyperalimentation infusion flask, which was implicated by molecular typing to be the source of infection. All strains were identified using morphological and biochemical methods. The genetic relationship between patients' strains and the hyperalimentation infusion strain was assessed by electrophoretic karyotype (EK) analysis and random amplification of polymorphic DNA (RAPD). Both methods resulted in patterns that allowed differentiation of the isolates. Candida parapsilosis fungemia, in three of the eight patients, resulted from a common source of infection, as demonstrated by molecular typing methods. Image analysis of EK patterns indicated that these strains were closest to Candida parapsilosis Group II, a grouping that is a less frequent clinical isolate than the major Group I strains.

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).

The MAT1-1 and MAT1-2 idiomorphs associated with the MAT1 locus of Histoplasma capsulatum were identified by PCR. A total of 28 fungal isolates, 6 isolates from human clinical samples and 22 isolates from environmental (infected bat and contaminated soil) samples, were studied. Among the 14 isolates from Mexico, 71.4% (95% confidence interval [95% CI], 48.3% to 94.5%) were of the MAT1-2 genotype, whereas 100% of the isolates from Brazil were of the MAT1-1 genotype. Each MAT1 idiomorphic region was sequenced and aligned, using the sequences of the G-217B (+ mating type) and G-186AR (− mating type) strains as references. BLASTn analyses of the MAT1-1 and MAT1-2 sequences studied correlated with their respective + and − mating type genotypes. Trees were generated by the maximum likelihood (ML) method to search for similarity among isolates of each MAT1 idiomorph. All MAT1-1 isolates originated from Brazilian bats formed a well-defined group; three isolates from Mexico, the G-217B strain, and a subgroup encompassing all soil-derived isolates and two clinical isolates from Brazil formed a second group; last, one isolate (EH-696P) from a migratory bat captured in Mexico formed a third group of the MAT1-1 genotype. The MAT1-2 idiomorph formed two groups, one of which included two H. capsulatum isolates from infected bats that were closely related to the G-186AR strain. The other group was formed by two human isolates and six isolates from infected bats. Concatenated ML trees, with internal transcribed spacer 1 (ITS1) -5.8S-ITS2 and MAT1-1 or MAT1-2 sequences, support the relatedness of MAT1-1 or MAT1-2 isolates. H. capsulatum mating types were associated with the geographical origin of the isolates, and all isolates from Brazil correlated with their environmental sources.

Sporotrichosis has significantly increased in Brazil in the last decade, particularly in the state of Rio de Janeiro, with the occurrence of an epidemic related to zoonotic transmission from cats to humans. Recently, four new phylogenetic species were incorporated into the Sporothrix species complex based on the phenotypic and molecular characteristics, and a new species name (Sporothrix brasiliensis) was proposed for some of the Sporothrix isolates from this epidemic. This study describes the characterization of 246 isolates obtained from patients attending the Laboratory of Infectious Dermatology, IPEC-FIOCRUZ, between 1998 and 2008, together with one environmental sample. Two hundred and six of the isolates (83.4%) were characterized as S. brasiliensis, 15 (6.0%) as S. schenckii, and one (0.5%) as S. mexicana. Twenty-five isolates (10.1%) could not be identified according to their phenotype and were classified as Sporothrix spp. The calmodulin gene was sequenced to confirm the identity of these isolates. The molecular analysis demonstrated that 24 of the isolates were S. brasiliensis, with the remainder being a S. globosa isolate. The isolate characterized phenotypically as S. mexicana was clustered on the S. schenckii clade. The correlation between molecular data and phenotypic characteristics described in this study is fundamental to the identification of the Sporothrix complex.

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.

Little is known about the genetic strain diversity and geographical range of Histoplasma capsulatum isolated in Rio de Janeiro State, Brazil. We characterized 13 environmental, 7 animal, and 28 clinical H. capsulatum isolates by using a PCR-based random amplified polymorphic DNA (RAPD) assay. DNA fingerprinting of these soil, animal, and clinical specimens was performed with four primers (1253, 1281, D-9355, and D-10513) and generated amplicons with considerable polymorphism. Although all of the isolates exhibited more than 80% genetic relatedness, they could be clustered into four to six genotypes for each primer. The RAPD profiles of H. capsulatum isolated from Rio de Janeiro State could be distinguished from those of the U.S. strains included in this study (Downs, G222B, G-186B, and FLS1) by showing less than 70% similarity to each primer. The genetic polymorphisms between H. capsulatum strains isolated from animals and soil obtained in the same geographic areas were 100% similar, suggesting that an environmental microniche could be acting as a source of infection for animals and the local human population.

The results of 32 cases studied lead us to the conclusion that erythema nodosum's investigation routine is very important, once in our retrospective study, the percentage of cases of unknown etiology was 69.4%, and in this prospective study it is 21.8%. In 10 cases (31.2%), more than one causing agent was suspected. Infections (bacterial, helminthic, fungal, by protozoa) were diagnosed in 26 cases, streptococcal infection having predominated (12 cases). Drugs-dipirone, aspirin, anovulatory - were suspected as causing agents in 13 cases. The association of erythema nodosum and histoplasmosis capsulata is described for the first time in Brazil. We consider erythema nodosum to be a complex syndrome which should be regarded as a manifestation of underlying diseases. The fact that all 32 subjects were women, 26 of them during menacme, suggests that particular hormonal media may favor the action of various processes (infections and drugs), precipitating erythema nodosum's clinical picture. O estudo de 32 casos de eritema nodoso permite concluir que a rotina de investigação é extremamente importante já que em nosso estudo retrospectivo, o percentual de etiologia indeterminada foi de 69,4% e neste estudo prospectivo é de 21,8%. Em 10 casos (31,2%) mais de um agente causal foi suspeitado. Infecções (bacteriana, helmíntica, fúngica e por protozoário) foram diagnosticadas em 26 casos, tendo predominado a infecção estreptocócica com 12 casos. As drogas de suspeição foram dipirona, aspirina e anovulatório em 12 casos. A associação de eritema nodoso e histoplasmose capsulata é descrita pela primeira vez no Brasil. O eritema nodoso é síndrome complexa e deveria ser considerado sempre como manifestação de doença subjacente. A amostra de 32 pacientes exclusivamente do sexo feminino sendo que 26 em idade fértil sugere que vários processos (infecções e drogas) podem atuar em meio hormonal, particular e favorecedor, precipitando o quadro de eritema nodoso.