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Paracoccidioidomycosis (PCM) defines a broad spectrum of human and animal diseases caused by Paracoccidioides species (Onygenales). In the twenty-first century, Paracoccidioides advanced from a monotypic taxon to a genus that harbors seven species, including P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, P. lutzii, P. loboi, and P. cetii. Classic PCM, acquired upon inhalation of propagules from P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii, affects the human lungs and may progress to systemic granulomatous disease with tegumentary and visceral involvement. On the other hand, PCM loboi and PCM ceti caused by the unculturable P. loboi and P. cetii are subcutaneous mycoses, typically observed as keloid lesions in humans and dolphins. Such heterogeneity highlights the importance of recognizing species boundaries in Paracoccidioides to gain insights into the ecology, evolution, clinical features, and mitigation strategies to tackle the advance of PCM., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Introduction: Paracoccidioidomycosis (PCM) is caused by several species of the Paracoccidioides genus which can be differentiated by interspecific genetic variations, morphology and geographic distribution. Intraspecific variability correlation with clinical and epidemiological aspects of these species still remains unclear. This study aimed to sequence the loci GP43, exon 2 and ARF of 23 clinical isolates of Paracoccidioides spp. from patients in the Southeast Region of Brazil., Methodology and Main Findings: GenBank was used to compare the present (23) with previous described sequences (151) that included ARF and GP43. It was identified a high polymorphism rate among the 23 isolates in comparison to the other 151. Among the isolates, 22 (95.66%) were S1/P. brasiliensis and 1 (4.34%) was identified as PS2/P. americana. A total of 45 haplotypes were found as follows: 19 from S1/P. brasiliensis (13 from the present study), 15 from P. lutzii, 6 from PS2/P. americana (1 from the present study), 3 from PS3/P. restrepiensis and 2 from PS4/P. venezuelensis. Moreover, exclusive haplotypes according to clinical origin and geographical area were found. S1/P. brasiliensis (HD = 0.655 and K = 4.613) and P. lutzii (HD = 0.649 and K = 2.906) presented the highest rate of polymorphism among all species, from which 12 isolates of the present study were clustered within S1b/P. brasiliensis. The GP43 locus showed a higher variability and was found to be the main reason for the species differentiation., Conclusions: The results herein decribed show a high intraspecific genetic variability among S1/P. brasiliensis isolates and confirm the predominance of this species in the Southeast region of Brazil. The finding of exclusive haplotypes according to clinical origin and geographical area would suggest correlation between the molecular profile with the clinical form and geographic origin of patients with PCM., Competing Interests: The authors have declared that no competing interests exist.

Background: Paracoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii., Methodology and Main Findings: This study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome., Conclusions: This study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented., Competing Interests: The authors have declared that no competing interests exist.

Introduction. Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. As the disease is known to affect mostly men over 40 years old who previously worked handling soil, some cities of agricultural economy in endemic regions may have more cases of paracoccidioidal infection. Gap statement. The true frequency of PCM cannot be established in Brazil because it is not a disease of mandatory reporting. The detection of paracoccidioidal infection may assist in the planning of health services, in order to provide early detection of the disease and to prevent its worsening or even progression to death. In addition, little is described about sera reactivity with antigens from different species of Paracoccidiodes , especially P. lutzii . Aim. Current research was conducted in an inland municipality of southern Brazil, in order to assess infection rate within this endemic region of PCM disease. Methodology. ELISA was employed to evaluate 359 sera from random volunteers from Guarapuava, Paraná, Brazil, to detect IgG against cell-free antigens (CFA) from P. restrepiensis B339, P. americana LDR3 and P. lutzii LDR2. Confirmatory ELISA employed gp43 from B339. Reduction of cross-reactions was sought by treatment with sodium metaperiodate (SMP-CFA, SMP-gp43). Immunoblot was performed with 37 selected sera among those reactive in ELISA. Epidemiological profile was assessed by questionnaire. Results. ELISA reactivity was: CFA/SMP-CFA in general 37.3/17.8 %, B339 25.3/14.5 %, LDR3 24.5/1.4 %, LDR2 8.3/5.8 %; gp43/SMP-gp43 7.2/4.7 %. There were sera reactive with multiple CFAs. In immunoblot, five sera showed the same reaction profile with P. lutzii 's antigens as PCM disease sera. Rural residence and soil-related professions were risk factors for paracoccidioidal infection. Conclusion. The low prevalence is in accordance with previous reports of lower PCM disease endemicity in Guarapuava than in other areas of Paraná. Although P. brasiliensis seems to be the prevalent strain of the region, 21 sera from people who only lived in Guarapuava reacted with P. lutzii LDR2. CFA-ELISA with whole antigens seems a good option for serological screening in epidemiological surveys.

Paracoccidioidomycosis caused by Paracoccidioides lutzii is endemic in the Midwest of Brazil and its clinical spectrum is still little known due to the recent identification of this fungal species. A patient resident in Southeast Brazil, but who had lived for many years in the Midwest region, presented with skin injuries, chronic cough and bilateral adrenal involvement. Paracoccidioides spp. was isolated in culture from a skin lesion biopsy. This isolate was later identified as P. lutzii using gene sequencing. A favorable initial response to treatment with itraconazole was observed, but a few weeks later, the patient developed respiratory failure and worsening of lung lesions. Evaluation by computed tomography and echocardiography were suggestive of pulmonary arterial hypertension, and a bronchoscopic biopsy showed peribronchial remodeling. The patient completed the antifungal treatment but maintained the respiratory dysfunction. The reported case shows that P. lutzii can be isolated from patients in a geographic area far from the place of infection acquisition and that, as P. brasiliensis , it can cause adrenal injury and cardio-respiratory complications as a consequence of excessive necrosis and fibrosis.

Paracoccidioidomycosis (PCM) is a life-threatening systemic mycosis widely reported in the Gran Chaco ecosystem. The disease is caused by different species from the genus Paracoccidioides, which are all endemic to South and Central America. Here, we sequenced and analyzed 31 isolates of Paracoccidioides across South America, with particular focus on isolates from Argentina and Paraguay. The de novo sequenced isolates were compared with publicly available genomes. Phylogenetics and population genomics revealed that PCM in Argentina and Paraguay is caused by three distinct Paracoccidioides genotypes, P. brasiliensis (S1a and S1b) and P. restrepiensis (PS3). P. brasiliensis S1a isolates from Argentina are frequently associated with chronic forms of the disease. Our results suggest the existence of extensive molecular polymorphism among Paracoccidioides species, and provide a framework to begin to dissect the connection between genotypic differences in the pathogen and the clinical outcomes of the disease., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier Inc.)

Background: The fungus Paracoccidioides lutzii was recently included as a new causative species of paracoccidioidomycosis (PCM) and most cases have been reported from Brazil. According to available epidemiological information, P. lutzii is concentrated in the Middle-West region in Brazil, mainly in the state of Mato Grosso. However, clinical and laboratorial data available on patients infected with P. lutzii remain extremely limited., Methodology/main Findings: This work describes the clinical manifestations of 34 patients suffering from PCM caused by P. lutzii, treated along 5 years (2011-2017) at a reference service center for systemic mycoses in Mato Grosso, Brazil. Adult rural workers (men), aged between 28 and 67 predominated. All patients had the chronic form of the disease, and the oral mucosa (n = 19; 55.9%), lymph nodes (n = 23; 67.7%), skin (n = 16; 47.1%) and lung (n = 28; 82.4%) were the most affected sites. Alcohol intake (n = 19; 55.9%) and smoking (n = 29; 85.3%) were frequent habits among the patients. No patient suffered from any other life-threatening disease, such as tuberculosis, cancer or other inflammatory or infectious parasitic diseases. The positivity in culture examination (97.1%) was higher than that found for the direct mycological examination (88.2%). Particularly, one patient presented fungemia at diagnosis, which lead to his death. The time elapsed between the initial symptoms and the initiation of treatment of PCM caused by P. lutzii was 19.7 (31.5) months, with most patients diagnosed 7 months after the symptoms' onset., Conclusions/significance: Compared with the classical clinical-epidemiological profile of PCM caused by P. brasiliensis, the results of this descriptive study did not show significant clinical or epidemiological differences that could be attributed to the species P. lutzii. Future studies may confirm or refute the existence of clinical differences between the two fungal species., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The authors have declared that no competing interests exist.

Introduction: The agents of paracoccidioidomycosis, historically identified as Paracoccidioides brasiliensis, are in fact different phylogenetic species. This study aims to evaluate associations between Paracoccidioides phylogenetic species and corresponding clinical data., Methods: Paracoccidioides strains from INI/Fiocruz patients (1998-2016) were recovered. Socio-demographic, epidemiological, clinical, serological, therapeutic and prognostic data of the patients were collected to evaluate possible associations of these variables with the fungal species identified through partial sequencing of the ADP-ribosylation factor (arf) and the 43-kDa-glycoprotein (gp43) genes., Results: Fifty-four fungal strains were recovered from 47 patients, most (72.3%) infected in Rio de Janeiro state, Brazil. Forty-one cases were caused by Paracoccidioides brasiliensis and six by Paracoccidioides americana (former PS2). P. brasiliensis was responsible for severe lymph abdominal forms, whereas patients infected with P. americana presented a high rate of adrenal involvement. However, no statistically significant associations were found for all variables studied. P. americana presented 100% reactivity to immunodiffusion, even when tested against antigens from other species, while negative results were observed in 9 (20%) cases caused by P. brasiliensis, despite being tested against a homologous antigen., Conclusions: P. brasiliensis and P. americana are sympatric and share similar clinical features and habitat, where they may compete for similar hosts., Competing Interests: The authors have declared that no competing interests exist.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides , is a systemic mycosis with granulomatous character and a restricted therapeutic arsenal. The aim of this work was to search for new alternatives to treat largely neglected tropical mycosis, such as PCM. In this context, the enzymes of the shikimate pathway constitute excellent drug targets for conferring selective toxicity because this pathway is absent in humans but essential for the fungus. In this work, we have used a homology model of the chorismate synthase (EC 4.2.3.5) from Paracoccidioides brasiliensis ( Pb CS) and performed a combination of virtual screening and molecular dynamics testing to identify new potential inhibitors. The best hit, CP1, successfully adhered to pharmacological criteria (adsorption, distribution, metabolism, excretion, and toxicity) and was therefore used in in vitro experiments. Here we demonstrate that CP1 binds with a dissociation constant of 64 ± 1 μM to recombinant chorismate synthase from P. brasiliensis and inhibits enzymatic activity, with a 50% inhibitory concentration (IC 50 ) of 47 ± 5 μM. As expected, CP1 showed no toxicity in three cell lines. On the other hand, CP1 reduced the fungal burden in lungs from treated mice, similar to itraconazole. In addition, histopathological analysis showed that animals treated with CP1 displayed less lung tissue infiltration, fewer yeast cells, and large areas with preserved architecture. Therefore, CP1 was able to control PCM in mice with a lower inflammatory response and is thus a promising candidate and lead structure for the development of drugs useful in PCM treatment., (Copyright © 2018 American Society for Microbiology.)

Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well-defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS-rDNA and the gp43-exon-2 region, and by PCR-RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast-to-mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil-related condition.

Molecular studies have shown more than one species of the genus Paracoccidioides to be the causal agent of paracoccidioidomycosis. Efforts have been made to correlate the identified species with epidemiological and clinical data of patients, aiming to determine the real meaning and impact of new species. Bearing this objective in mind, the authors report a clinical case of paracoccidioidomycosis, from São Paulo state, Brazil, that manifested as uncommon sarcoid-like cutaneous lesions and was caused by Paracoccidioides brasiliensis sensu stricto (S1a). The patient was treated with itraconazole 200mg/day for 12 months, with complete clinical remission.

Since the new species Paracoccidioides lutzii emerged in 2009, much has been researched on strains previously considered atypical. Still, there is no consensus about recognition of antigens from P. lutzii by antibodies directed to other Paracoccidioides species, which can have great impact on Paracoccidioidomycosis (PCM) diagnosis. Current research investigated soluble protein/carbohydrate epitopes from P. lutzii LDR2, Paracoccidioides restrepiensis B339 and Paracoccidioides americana LDR3 recognised by IgG directed to Paracoccidioides brasiliensis. Cell free antigens (CFA) were analysed by: (a)silver and periodic acid-Schiff staining of SDS-PAGE; (b)immunoblot (IB) with rabbit IgG anti-P. brasiliensis Pb18; (c)IB and ELISA with a pool of PCM patients' sera before and after treatment with sodium metaperiodate (SMP) to oxidise carbohydrate epitopes. Both rabbit and human immune sera recognised several antigens of P. lutzii LDR2, P. restrepiensis B339 and P. americana LDR3. P. lutzii's gp43 was not observed in IB or silver/PAS staining. SMP treatment affected reactions with all 3 CFAs, but more intensely with antigens from P. lutzii LDR2. In conclusion, antibodies directed to P. brasiliensis recognised antigens from P. lutzii LDR2. The use of any of the recognised antigens in a broad spectrum diagnostic model for Paracoccidioides species complex needs to be further investigated., (© 2018 Blackwell Verlag GmbH.)

Paracoccidioidomycosis (PCM) is among the systemic mycoses which are endemic only in Latin America. In Argentina, the vast majority of the cases are reported at north of latitude 34.5° S. The disease is produced by thermodimorphic fungi of the genus Paracoccidoides: P. brasiliensis (S1), P. americana (PS2), P. restrepiensis (PS3), P. venezuelensis (PS4) y P. lutzii. The natural habitat of members of this genus is the soil, where they produce infectious conidia. Little is known, however, about their specific ecologic niche(s), and this knowledge gap hampers the design of measures to control the infection. Rural male workers are the group most at risk of developing PCM. Infection occurs by inhalation of aerosolized conidia and may either be asymptomatic or cause mild respiratory symptoms. In turn, this primary infection may be self-limited or progress to severe pulmonary or disseminated disease. The disease has two clinical presentations: (i) acute or subacute (juvenile), frequent in children, adolescents and people with immunodeficiencies; and (ii) chronic progressive, in adults. Active lesions often resolve into fibrotic scars which can cause dysphagia, dysphonia, adrenal insufficiency, and intestinal obstruction. Although efficient tools are available for diagnosis and treatment, the nonspecific nature of PCM clinical manifestations frequently delay the diagnosis. In addition, the poor adherence to long antifungal treatments allows the advance of the disease and the development of extensive fibrosis compromising severely and permanently respiratory and adrenal functions, thus altering the patient"s quality of life and even causing his/her death.

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis endemic to Latin America caused by dimorphic fungi of the genus Paracoccidioides. The acute juvenile PCM is a severe type of presentation that usually affects young vulnerable patients and rarely progresses to portal hypertension. Here, two cases of liver disease and portal hypertension as complications of acute juvenile PCM are reported. Diagnosis of PCM was performed by isolation of the fungus and molecular identification of the strains provided through partial sequencing of two protein encoding genes, arf and gp43. Genotypic analysis revealed that Paracoccidioides brasiliensis S1 was the phylogenic species involved in both cases. Patients presented a good clinical response to amphotericin B and sulfamethoxazole-trimethoprim. These results highlight the importance of the interdisciplinary approach in patients with severe forms of PCM to avoid and treat complications, and the necessity of further investigations focusing on host-pathogen interaction in order to explain the broad clinical spectrum in PCM as well as the severity and poor outcome in some clinical cases.

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.

Cutaneous granulomas in dolphins were believed to be caused by Lacazia loboi, which also causes a similar disease in humans. This hypothesis was recently challenged by reports that fungal DNA sequences from dolphins grouped this pathogen with Paracoccidioides brasiliensis. We conducted phylogenetic analysis of fungi from 6 bottlenose dolphins (Tursiops truncatus) with cutaneous granulomas and chains of yeast cells in infected tissues. Kex gene sequences of P. brasiliensis from dolphins showed 100% homology with sequences from cultivated P. brasiliensis, 73% with those of L. loboi, and 93% with those of P. lutzii. Parsimony analysis placed DNA sequences from dolphins within a cluster with human P. brasiliensis strains. This cluster was the sister taxon to P. lutzii and L. loboi. Our molecular data support previous findings and suggest that a novel uncultivated strain of P. brasiliensis restricted to cutaneous lesions in dolphins is probably the cause of lacaziosis/lobomycosis, herein referred to as paracoccidioidomycosis ceti.

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.

Paracoccidioidomycosis is a systemic mycosis endemic to Latin America, with Paracoccidioides brasiliensis and P. lutzii being the causal agents of this disorder. Several issues have been raised in the 100 years since its discovery and in this article we discuss features of this fascinating fungal pathogen, including its biology, eco-epidemiology and aspects of its pathogenicity. We also consider some of its virulence determinants, the most recent advances in the study of its metabolic pathways and the molecular and genetic research tools developed for this research. We also review the animal models used to study host-fungal interactions and how the host defence mechanisms against this pathogen work., (Copyright © 2015 John Wiley & Sons, Ltd.)

Paracoccidioidomycosis is an important systemic fungal infection that occurs throughout Latin America. The etiological agents comprise a species complex that includes two major groups: P. brasiliensis (including subgroups S1, PS2, and PS3) and P. lutzii. A great number of phenotypes may overlap, especially among closely related groups, discouraging the use of morphology alone for species recognition. To overcome this problem, here we propose identifying cryptic Paracoccidioides spp. using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the alpha-tubulin (TUB1) gene. In silico analysis of 90 TUB1 sequences led to the identification of two restriction enzymes with the potential to identify Paracoccidioides: Bcl I and Msp I. A portion of the TUB1 gene was amplified and double digested in vitro with the Bcl I and Msp I endonucleases, which generated four different electrophoretic patterns corresponding to the four main genetic groups: S1, PS2, and PS3 of P. brasiliensis and P. lutzii. The major P. brasiliensis group recognized was S1 (n = 17; 42.5%), followed by PS2 (n = 9; 22.5%) and PS3 (n = 6; 15%). A total of eight (20%) P. lutzii isolates were identified, mainly from midwestern Brazil. Our data revealed that TUB1-RFLP is an efficient, fast, and inexpensive tool for identifying Paracoccidioides spp., which may be directly applied to the molecular epidemiological studies of paracoccidioidomycosis., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Understanding the possible methodologies for the rapid and inexpensive identification of fungal infections is essential for disease diagnosis, but there are some limitations. To help with this problem, serological methods that detect antigens or antibodies are widely used and are useful for the diagnosis of paracoccidioidomycosis (PCM) through the detection of gp43, which is the main antigen employed for the immunodiagnosis of this disease caused by Paracoccidioides brasiliensis. However, the use of gp43 has become restricted because it was recently found that this marker is not identified in the infections caused by Paracoccidioides lutzii. Therefore, it is necessary to identify new antigens in both species or antigens specific for P. lutzii to decrease the morbidity and/or mortality associated with PCM. This review provides a discussion of new diagnostic challenges after the recent discoveries regarding the taxonomy of the Paracoccidioides genus., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Studies comparing Paracoccidioides brasiliensis and Paracoccidioides lutzii have shown that these fungi have significant genomic differences that may have implications in the clinical manifestation, diagnosis, and treatment of paracoccidioidomycosis caused by them. Thus, molecular typing methods are required that can distinguish between various species of Paracoccidioides. The aim of this study was to explore the potential use as molecular markers of the transposable elements Trem A-H recently identified and characterized in the genus Paracoccidioides as a means of differentiating the species. We take advantage of the abundance and distribution of these transposons in the Paracoccidioides genomes to develop a simple and highly reproducible polymerase chain reaction (PCR)-based technique. Furthermore we compare the performance of this test with two other molecular markers already in use to identify these fungi., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Clinical Paracoccidioides spp. isolates from patients with paracoccidioidomycosis (PCM) in Mato Grosso, Brazil exhibit different patterns of serologic reactivity. The results observed for reactions of radial immunodiffusion against the commonly used exoantigens containing a 43-kDa glycoprotein (gp43) suggest that this fungus exhibits major antigenic variability by geographic region. There is a phylogenetic gap between Paracoccidioides spp. isolates among different regions of Latin America. In particular, those from the central region of Brazil (i.e. Mato Grosso state) exhibit a lower rate of genetic similarity. We aimed at investigating the phylogenetic classification of clinical isolates of Paracoccidioides spp. in Central Brazil and the different antigenic profiles that produce. Exoantigens were obtained from five clinical isolates: two P. brasiliensis (Pb166 and Pb2880) and three P. lutzii (PL2875, PL9840, and PL2912). The protein/glycoprotein profiles of P. lutzii exoantigens were different from each other. Isolate PL9840 exhibited the most distinct bands, and isolates PL2875 and PL2912 exhibited more diffuse bands and a very intense band between 50 and 60 kDa. P. brasiliensis isolates had similar protein profiles, exhibiting a low-intensity band at 220 kDa and a diffuse band between 50 and 60 kDa. P. lutzii isolates exhibit high species-specific antigen variability, which we have already been assessed in proteomic studies., (© 2014 Blackwell Verlag GmbH.)

We report the first case of fungemia caused by Paracoccidioides lutzii in a 51-year-old male farm worker from the central-west region of Brazil. The fungus was isolated from blood cultures and the species was confirmed by phylogenetic identification. Despite specific treatment and intensive care, the patient died 39 days after admission., (© The American Society of Tropical Medicine and Hygiene.)

Paracoccidioides brasiliensis, a dimorphic pathogenic fungus, causes the principal form of systemic mycosis in Brazil. The literature furnishes only limited data on the ecology of this fungus in the state of Rio Grande do Sul, the southernmost state of Brazil. The purpose of this study was to evaluate the prevalence of fungal infection in wild animals, using serological tests and using the animals as sentinels of the presence of P. brasiliensis in three specified mesoregions of Rio Grande do Sul. A total of 128 wild animals from the three mesoregions were included in the study. The serum samples were evaluated by immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) technique to detect anti-gp43 antibodies from P. brasiliensis. Two conjugates were tested and compared with the ELISA technique. Although no positive samples were detected by immunodiffusion, 26 animals (20%), belonging to 13 distinct species, were found to be seropositive by the ELISA technique. The seropositive animals were from two mesoregions of the state. The results were similar according to the gender, age, and family of the animals, but differed significantly according to the conjugate used (p < 0.001), showing more sensitivity to protein A-peroxidase than to protein G-peroxidase. The finding that wild animals from the state of Rio Grande do Sul are exposed to P. brasiliensis suggests that the fungus can be found in this region despite the often-rigorous winters, which frequently include below-freezing temperatures.

The genus Paracoccidioides comprises a complex of phylogenetic species of dimorphic pathogenic fungi, the etiologic agents of paracoccidioidomycosis (PCM), a disease confined to Latin America and of marked relevance in its endemic areas due to its high frequency and severity. The members of the Paracoccidioides genus are distributed in distinct phylogenetic species (S1, PS2, PS3 and 01-like) that potentially differ in their biochemical and molecular characteristics. In this work, we performed the proteomic characterization of different members of the genus Paracoccidioides. We compared the proteomic profiles of Pb01 (01-like), Pb2 (PS2), Pb339 (S1) and PbEPM83 (PS3) using 2D electrophoresis and mass spectrometry. The proteins/isoforms were selected based on the staining intensity of the spots as determined by image analysis. The proteins/isoforms were in-gel digested and identified by peptide mass fingerprinting and ion fragmentation. A total of 714 spots were detected, of which 343 were analyzed. From these spots, 301 represented differentially expressed proteins/isoforms among the four analyzed isolates, as determined by ANOVA. After applying the FDR correction, a total of 267 spots were determined to be differentially expressed. From the total, 193 proteins/isoforms were identified by PMF and confirmed by ion fragmentation. Comparing the expression profiles of the isolates, the proteins/isoforms that were related to glycolysis/gluconeogenesis and to alcohol fermentation were more abundant in Pb01 than in other representatives of the genus Paracoccidioides, indicating ahigher use of anaerobic pathways for energy production. Those enzymes related to the oxidative stress response were more abundant in Pb01, Pb2 and Pb339, indicating a better response to ROS in these members of the Paracoccidioides complex. The enzymes of the pentose phosphate pathway were abundant in Pb2. Antigenic proteins, such as GP43 and a 27-kDa antigenic protein, were less abundant in Pb01 and Pb2. The proteomic profile indicates metabolic differences among the analyzed members of the Paracoccidioides genus., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Acquired by inhalation of the thermal dimorphic fungi Paracoccidioides spp. conidia, paracoccidioidomycosis ranges from symptomatic to severe and potentially fatal disseminated disease. The main focus of this review is to highlight clinical aspects of paracoccidioidomycosis and, its pathogens' diversity ecology and particularities. In addition, we present strategies for therapy, including DNA vaccines and nanostructured drugs. Molecular and morphological data supported the split of the Paracoccidioides genus into two species, Paracoccidioides brasiliensis and Paracoccidioides lutzii. An acute form of the disease affects approximately 5% of cases and involves the phagocytic mononuclear system, resulting in progressive lymphadenopathy. The chronic form affects adult men and frequently involves lungs, skin and mucous membranes, lymph nodes, and adrenal glands. The clinical manifestations depend on the ability of the host to control the fungal multiplication and dissemination. The long survival time of the fungus in the host tissues allows it to evade immune responses; therefore, successful treatment often requires long-time therapy. The consensus for treatment must consider the severity of the disease and includes sulfone derivatives, amphotericin B and azoles. Novel strategies for therapy, based on DNA vaccines and nanostructured drugs are also presented and discussed in this review.

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

Paracoccidioidomycosis (PCM), the most important human systemic mycosis in Latin America, is known to be caused by at least four different phylogenetic lineages within the Paracoccidioides brasiliensis complex, including S1, PS2, PS3, and Pb01-like group. Herein, we describe two cases of PCM in patients native from the Amazon region. The disease was originally thought to have been caused by P. brasiliensis. Despite the severity of the cases, sera from the patients were negative in immunodiffusion tests using the standard exoantigen from P. brasiliensis B-339. However, a positive response was recorded with an autologous preparation of Paracoccidioides lutzii exoantigen. A phylogenetic approach based on the gp43 and ARF loci revealed high similarity between our clinical isolates and the Pb01-like group. The occurrence of PCM caused by P. lutzii in the Brazilian Amazon (Pará State) was thus proven. The incidence of PCM caused by P. lutzii may be underestimated in northern Brazil.

This study is the first report on genetic differences between isolates of Paracoccidioides brasiliensis from a single patient. We describe a simultaneous infection with genetically distinct isolates of P. brasiliensis in a patient with chronic paracoccidioidomycosis. The clinical isolates were obtained from lesions in different anatomical sites and were characterised by random amplified polymorphic DNA (RAPD) analysis. The RAPD technique can be helpful for distinguishing between clinical isolates. Different random primers were used to characterise these clinical isolates. The RAPD patterns allowed for differentiation between isolates and the construction of a phenetic tree, which showed more than 28% genetic variability in this fungal species, opening new possibilities for clinical studies of P. brasiliensis. Based on these results and preliminary clinical findings, we suggest that different genotypes of P. brasiliensis might infect the same patient, inducing the active form of the disease.

An atypical isolate of Paracoccidioides brasiliensis (IFM54648), recovered from the sputum of a Brazilian man, was not detected in immunodiffusion tests for paracoccidioidomycosis and in species-specific PCR for the major antigen 43-kDa glycoprotein coding gene (gp43). The mycological characteristics of the isolate were similar to those of a typical P. brasiliensis. A total of 8 genes were sequenced from IFM54648, and the sequences were compared between the new isolate and other reference isolates and database sequences. We analyzed fragments of the gene sequences that code for gp43, the internal transcribed spacer regions of ribosomal RNA, the D1/D2 domains of the large subunit ribosomal RNA, glucan synthase, chitin synthase, glyoxalase I mRNA, 70-kDa heat-shock protein mRNA and urease. The gene sequences were 98.9-100% identical between IFM54648 and Pb01 (another atypical isolate). When compared to the other typical isolates, the identities were generally lower than 98%. A phylogenetic tree constructed using gp43 sequences showed that IFM54648 clustered with Pb01 at a considerable distance from other isolates. Therefore, this isolate is likely related to Pb01, which has recently been shown to be genetically distinct from other isolates of this species.

Paracoccidioides brasiliensis is a dimorphic fungus that is the causative agent of paracoccidioidomycosis, the most important prevalent systemic mycosis in Latin America. Recently, the existence of three genetically isolated groups in P. brasiliensis was demonstrated, enabling comparative studies of molecular evolution among P. brasiliensis lineages. Thirty-two gene sequences coding for putative virulence factors were analyzed to determine whether they were under positive selection. Our maximum likelihood-based approach yielded evidence for selection in 12 genes that are involved in different cellular processes. An in-depth analysis of four of these genes showed them to be either antigenic or involved in pathogenesis. Here, we present evidence indicating that several replacement mutations in gp43 are under positive balancing selection. The other three genes (fks, cdc42 and p27) show very little variation among the P. brasiliensis lineages and appear to be under positive directional selection. Our results are consistent with the more general observations that selective constraints are variable across the genome, and that even in the genes under positive selection, only a few sites are altered. We present our results within an evolutionary framework that may be applicable for studying adaptation and pathogenesis in P. brasiliensis and other pathogenic fungi.

A recent species status investigation of the pathogenic fungus Paracoccidioides brasiliensis suggested the existence of three cryptic species. In the present study, the sequences of the PRP8 intein from P. brasiliensis isolates belonging to the three described genetic groups and two unidentified isolates were determined and analyzed in order to check their functionality and usefulness for species identification. All the isolates presented a full-length intein, although the Endonuclease domain seems to be inactive due to substitutions in the second essential aspartic acid residue. Phylogenetic analysis by Maximum-Parsimony, Maximum Likelihood, and Bayesian analysis clearly separated the isolates from the three species and revealed a significant difference between the Pb01 isolate and the remaining ones. The Pb01 isolate does not belong to any of the groups previously described since it presented a high divergence level compared to the three different genetic groups, corroborating some previous studies that suggested this isolate represents a new species of Paracoccidioides.

Paracoccidioides brasiliensis is a dimorphic fungus, a causative agent of paracoccidioidomycosis, one of the most frequent systemic mycoses that affect the rural population in Latin America, only geographical region in which this fungus is to be found. In this work, we discuss matters related to (a) cell wall studies based on the cloning and analysis of genes involved in the synthesis of cell wall components, and their possible roles in virulence and dimorphism in P. brasiliensis, (b) molecular taxonomy and the molecular classification of P. brasiliensis as an Ascomycete belonging in the Order Onygenales, (c) phylogeny of P. brasiliensis and the possible existence of cryptic species within the genus Paracoccidioides, and (d) new experimental antifungal drugs such as azasterols or sterol hydrazones, compounds that affect the activity of delta 24(28) sterol methyl reductase (SMR) and/or delta (24)-sterol methyl transferase (SMT), and (e) specific primers for the molecular detection of P. brasiliensis in vitro and in clinical samples.

Lacazia loboi, the aetiological agent of lacaziosis (Jorge Lobo's disease), is an uncultivated anomalous fungal microbe closely related to Paracoccidioides brasiliensis, both restricted Latin American Pathogens. Early reports suggesting that L. loboi had been isolated in pure culture from cases of lacaziosis, only added more confusion to the already confusing aetiology of this disease. These strains were later identified as unusual contaminants and some of them as P. brasiliensis. Recent phylogenetic analysis grouped L. loboi as the sister taxon to P. brasiliensis, thus it was postulated that the original P. brasiliensis strains recovered from cases of lacaziosis, could be the aetiological agent of the disease. Using molecular methodologies, we investigated the archival P. brasiliensis isolate No. 525 from a case of lacaziosis, as well as other archival isolates, identified earlier as common contaminants, all recovered from similar cases of the disease. Phylogenetic analysis, using the 18S small subunit rDNA sequences of these isolates showed that strain No. 525 was a typical P. brasiliensis isolate and the other studied strains were indeed contaminants. This study unequivocally indicates that the aetiological agent of lacaziosis is yet to be cultured.

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis (PCM). Here, we describe the microsatellite patterns observed in a collection of P. brasiliensis random sequence tags. We identified 1,117 microsatellite patterns in about 3.8 Mb of unique sequences (0.47% of the total DNA used in the analysis). The majority of these microsatellites (87.5%) are found in noncoding sequences. We used two polymorphic microsatellites located on noncoding and coding sequences, as well as two microsatellites located on introns, as molecular markers to discriminate P. brasiliensis isolates, to look for relationships between the genetic background of the strains and the types of human disease they cause. We did not observe any correlation between the clinical form of human PCM and four simple sequence repeat patterns analyzed.

Paracoccidioidomycosis (PCM) is a severe disease caused by the dimorphic fungus Paracoccidioides brasiliensis, which is characterized by granulomatous pulmonary and systemic lesions, affecting mainly men between 20 and 60 years of age. Reports of PCM disease in animals are rare, but the disease has been described in armadillos. On the other hand, PCM infection of domestic and wild animals detected by serological or cutaneous tests in the absence of apparent disease has been frequently reported. We present here the case of a female adult Doberman that developed cervical lymphadenomegaly. Histopathological examination of a cervical biopsy specimen revealed active PCM, with an epithelioid, granulomatous inflammation containing numerous yeast-like, multiple budding fungal forms. The diagnosis of PCM was confirmed by immunohistochemistry using a specific antibody anti-gp43 and by nested PCR using primers for the amplification of the gp43 gene region. This is the first report of PCM disease occurring in a dog, an animal that has been shown to play an important role in the natural history of North American blastomycosis.

Randomly amplified polymorphic DNA (RAPD) has been successfully used to detect genetic variations among isolates of Paracoccidioides brasiliensis. However, the usefulness of this technique for assessing important parasitic properties is still unconfirmed. In the present work we further investigated the applicability of RAPD in revealing important intrinsic and extrinsic features of this fungus associated with geographical origin, time of isolation, source of clinical specimen, clinical forms of human disease and also in vitro and in vivo susceptibility to antimicrobial and antifungal drugs. The RAPD patterns allowed us to distinguish all of the analyzed strains, which included 26 clinical isolates, 2 animal isolates, and 1 environmental isolate of P. brasiliensis obtained from different geographic regions, confirming the strong discriminating power of this technique. A phenetic tree, build from the RAPD data, showed that although the two nonclinical Brazilian strains were set together the majority of the clinical Brazilian strains were randomly distributed through different sub-branches of a major cluster without any correlation to any of the parameters analyzed. A second major cluster, however, has grouped isolates from Mato Grosso and Roraima (Brazil) that not only were susceptible in vitro to trimethoprim-sulfamethoxazole but also produced a good in vivo response. These results open new vistas for epidemiological and clinical studies of P. brasiliensis.

Paracoccidioides brasiliensis is an amenable model to study the molecular and biochemical events that lead to morphological transition in fungi, because temperature seems to be the only factor regulating this process. It is the causative agent of paracoccidioidomycosis, a systemic mycosis that affects humans and that is geographically confined to Latin America, where it constitutes one of the most prevalent deep mycoses. With the help of molecular tools, events leading to the morphological transition have been traced to genes that control cell wall glucan and chitin syntheses, and other metabolic processes such as production of heat shock proteins and ornithine decarboxylase activity. Molecular diagnosis and epidemiology of paracoccidioidomycosis are also the focus of intensive research, with several primers being proposed as specific probes for clinical and field uses. Although P. brasiliensis is refractory to cytogenetic analysis, electrophoretic methods have allowed an approximation of its genomic organization and ploidy. Finally, the recognition of P. brasiliensis as an anamorph in the phylum Ascomycota, order Onygenales, family Onygenaceae, has been accomplished by means of molecular tools. This phylogenetic placement has revised the taxonomic position of this fungus, which was traditionally included within now-abandoned higher anamorph taxa, the phylum Deuteromycota and the class Hyphomycetes.

Objective: To describe the main cytological findings associated with smears collected from oral lesions of paracoccidioidomycosis and to appraise the use of cytology as a diagnostic tool for the disease., Study Design: Cytological smears and biopsies were collected from 40 lesions with a clinical suspicion of paracoccidioidomycosis. Evaluation of the sensitivity, specificity, positive and negative predictive values, accuracy and the positive likeness ratio of the oral smear when compared with the histological diagnosis, was performed. The latter is considered the 'gold standard' for comparison., Results: The main morphological findings were the rounded-shaped, birefringent and multiple-budded fungi, Langhans' giant cells and epithelioid cells. The following associative measures were found: sensitivity, 67.9%; specificity, 91.7%; positive predictive value, 95.0%; negative predictive value, 55.0%; accuracy, 75.0%; and positive likeness ratio, 8.14., Conclusion: The cytological findings of paracoccidioidomycosis are characteristic and cytology is accurate in the diagnosis of the disease. Positive patients should be treated. Negative patients should be submitted to biopsy to confirm or to dismiss the diagnosis of this mycosis.

We have amplified and sequenced the 5.8S and 28S ribosomal DNA genes and intergenic regions of Paracoccidioides brasiliensis, strain Pb01. Using primers specifically designed for both ribosomal DNA regions, we were able to discriminate between P. brasiliensis and other human pathogenic fungi by PCR. The use of this molecular marker could be important for paracoccidiodomycosis diagnosis and ecological and molecular epidemiological studies of P. brasiliensis in Latin America.

Paracoccidioidomycosis is a systemic infection caused by the dimorphic fungus Paracoccidioides brasiliensis. It is commonly an endemic disease in Latin America, but several cases have been reported outside this area, particularly now in this time of world globalization. Primary pulmonary infection occurs commonly in the first and second decades of life and usually has a benign, self-limited respiratory infection course. The adult chronic manifestation of the disease is usually the result of reactivation of quiescent lesions with diffuse lung infiltrates, predominately of the interstitial pattern, with or without involvement of various other organs. The finding of this disease in a patient is an important step for the large differential diagnosis of the interstitial lung diseases group.

Five Paracoccidioides brasiliensis isolates of humans origin were analyzed using three arbitrary primers (3301, 3304 and 3307 of 10, 9 and 10 oligonucleotídes respectively) in random amplified polymorphic DNA (RAPD) analysis. The analysis of the complex RAPD profiles obtained were carried out using the Dice similarity coefficient that distinguished the isolated Pb 02 from the others (Pb 18, Pb 192, Pb 265 and Pb SN). The results revealed limited intraspecific genomic variations in these P. brasiliensis isolates and indicate that RAPD can be useful for analysis of P. brasiliensis genome for characterization or differentiation within this genus.

The new genus Lacazia P. Taborda, V. Taborda, et McGinnis is proposed to accommodate Lacazia loboi (O. M. Fonseca et Lacaz) P. Taborda, V. Taborda, et McGinnis, the obligate pathogen that causes lobomycosis in mammals. The continued placement of that fungus in the genus Paracoccidioides Almeida as Paracoccidioides loboi is taxonomically inappropriate. Loboa loboi Ciferri et al. is a synonym of Paracoccidioides brasiliensis.

Randomly amplified polymorphic DNA (RAPD) analysis of 33 Paracoccidioides brasiliensis strains from Argentina, Brazil, Colombia, Peru, and Venezuela produced reproducible amplification products which were sufficiently polymorphic to allow differentiation of the strains. Types generated with five primers (OPG 03, OPG 05, OPG 14, OPG 16, and OPG 18) resulted in a high discriminatory index (0.956). The discriminatory index was slightly reduced (0.940) when only two primers (OPG 3 and OPG 14) were used. A dendrogram based on these results showed a high degree of similarity among the strains, and genetic differences were expressed in clusters related to geographical regions but not to pathological features of the disease. With a few exceptions, strains were sorted into five groups by geographical origin as follows: group I, Venezuelan strains; group II, Brazilian strains; group III, Peruvian strains; group IV, Colombian strains; and group V, Argentinian strains. The group containing the most disparate strains was group V (discriminatory index, 0.633); the discriminatory index for the other four groups was 0.824. The use of primer OPG 18 by itself was sufficient to discriminate species specificity, and the use of primer OPG 14 by itself was sufficient to discriminate among the geographical locations of the strains in the sample. This method may be helpful for epidemiological studies of P. brasiliensis.

Paracoccidioides brasiliensis has rarely been isolated from its habitat in rural areas. In order to investigate the hypothesis that human infection with this fungus is linked to coffee plantations (Coffea arabica), material was collected monthly over a period of 1 year from farms in the town of Ibiá, State of Minas Gerais, Brazil. A total of 760 samples of soil, coffee leaves and fruits was cultured and inoculated into mice. A fungus isolated from the liver of a mouse inoculated with soil showed temperature-dependent dimorphism and in vitro mycelium and yeast phases characteristic of P. brasiliensis. Yeast cells of this fungus caused disseminated infection after intraperitoneal inoculation in Wistar rats from which the fungus was re-isolated. An antigen reacting with sera from patients with paracoccidioidomycosis was obtained from this P. brasiliensis strain; antigenic identity with strain 339 and with four other P. brasiliensis strains was detected by gel immunodiffusion. However, when the exo-antigen was submitted to SDS-PAGE, we observed low gp43 expression in this new strain, which we called Ibiá. The isolation of P. brasiliensis from the soil at a coffee plantation suggests that this is one of its habitats and supports the hypothesis of acquisition of paracoccidioidomycosis during agricultural activity in these areas.