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Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the P. brasiliensis complex and P. lutzii in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972–1.000, p &lt, 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the P. brasiliensis complex (n = 7) or P. lutzii (n = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient’s organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas.

Made available in DSpace on 2022-04-29T08:46:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Paracoccidioidomycosis (PCM) is a systemic mycosis caused by a group of cryptic species embedded in the Paracoccidioides brasiliensis complex and Paracoccidioides lutzii. Four species were recently inferred to belong to the P. brasiliensis complex, but the high genetic diversity found in both human and environmental samples have suggested that the number of lineages may be higher. This study aimed to assess the 43-kilodalton glycoprotein genotypes (PbGP43) in paraffin-embedded samples from PCM patients to infer the phylogenetic lineages of the P. brasiliensis complex responsible for causing the infection. Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with histopathological diagnosis of PCM were analyzed. DNAs were extracted and amplified for a region of the second exon of the PbGP43 gene. Products were sequenced and aligned with other PbGP43 sequences available. A haplotype network and the phylogenetic relationships among sequences were inferred. Amino acid substitutions were investigated regarding the potential to modify physicochemical properties in the proteins. Six phylogenetic lineages were identified as belonging to the P. brasiliensis complex. Two lineages did not group with any of the four recognized species of the complex, and, interestingly, one of them comprised only FFPE samples. A coinfection involving two lineages was found. Five parsimony-informative sites were identified and three of them showed radical non-synonymous substitutions with the potential to promote changes in the protein. This study expands the knowledge regarding the genetic diversity existing in the P. brasiliensis complex and shows the potential of FFPE samples in species identification and in detecting coinfections. Centro de Diagnóstico e Pesquisa em Biologia Molecular Dr Ivo Ricci Departamento de Patologia Escola Paulista de Medicina Universidade Federal de São Paulo Departamento de Genética e Evolução Universidade Federal de São Carlos Disciplina de Biologia Celular Departamento de Microbiologia Imunologia e Parasitologia Universidade Federal de São Paulo Faculdade de Medicina de Botucatu Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP) LADR GmbH MVZ Dr, Kramer & Kollegen, Lauenburger Straße 67 Departamento de Ciências Farmacêuticas Universidade Federal de São Paulo, Campus Diadema Faculdade de Medicina de Botucatu Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)

Malassezia spp in skin microbiome scalp has been implicated in seborrheic dermatitis pathogenesis. Thus, treatment based in antifungal combined to topical keratolitic agents have been indicated as well as oral isotretinoin as it reduces the sebum production, glandular's size and possesses anti-inflammatory properties. This randomized, comparative and therapeutic trial aimed toper form the genotypic identification of Malassezia species before and after low-dose oral isotretinoin or topical antifungal treatments for moderate to severe seborrhea and/or seborrheic dermatitis on scalp. Scales and sebum of the scalp were seeded in the middle of modified Dixon and incubated at 32°C. For genotypic identification polymerase chain reaction primers for the ITS and D1/D2 ribossomal DNA were used and followed by samples sequencing. The procedure was conducted before and after therapeutic and randomized intervention for moderate to severe seborrhea/seborrheic dermatitis on the scalp, including oral isotretinoin, 10 mg, every other day and anti-seborrheic shampoo (piroctone olamine), over six months. The M. globosa and M. restricta were the most frequent species isolated on the scalp before and after both treatments. Other non-Malassezia species were also identified. The Malassezia spp. were maintained in the scalp after both treatments that were equally effective for the control of seborrhea/seborrheic dermatitis clinical signs.

Sporotrichosis is a polymorphic chronic infection of humans and animals classically acquired after traumatic inoculation with soil and plant material contaminated with Sporothrix spp. propagules. An alternative and successful route of transmission is bites and scratches from diseased cats, through which Sporothrix yeasts are inoculated into mammalian tissue. The development of a murine model of subcutaneous sporotrichosis mimicking the alternative route of transmission is essential to understanding disease pathogenesis and the development of novel therapeutic strategies. To explore the impact of horizontal transmission in animals (e.g., cat-cat) and zoonotic transmission on Sporothrix fitness, the left hind footpads of BALB/c mice were inoculated with 5×106 yeasts (n = 11 S. brasiliensis, n = 2 S. schenckii, or n = 1 S. globosa). Twenty days post-infection, our model reproduced both the pathophysiology and symptomology of sporotrichosis with suppurating subcutaneous nodules that progressed proximally along lymphatic channels. Across the main pathogenic members of the S. schenckii clade, S. brasiliensis was usually more virulent than S. schenckii and S. globosa. However, the virulence in S. brasiliensis was strain-dependent, and we demonstrated that highly virulent isolates disseminate from the left hind footpad to the liver, spleen, kidneys, lungs, heart, and brain of infected animals, inducing significant and chronic weight loss (losing up to 15% of their body weight). The weight loss correlated with host death between 2 and 16 weeks post-infection. Histopathological features included necrosis, suppurative inflammation, and polymorphonuclear and mononuclear inflammatory infiltrates. Immunoblot using specific antisera and homologous exoantigen investigated the humoral response. Antigenic profiles were isolate-specific, supporting the hypothesis that different Sporothrix species can elicit a heterogeneous humoral response over time, but cross reaction was observed between S. brasiliensis and S. schenckii proteomes. Despite great diversity in the immunoblot profiles, antibodies were mainly derived against 3-carboxymuconate cyclase, a glycoprotein oscillating between 60 and 70 kDa (gp60-gp70) and a 100-kDa molecule in nearly 100% of the assays. Thus, our data broaden the current view of virulence and immunogenicity in the Sporothrix-sporotrichosis system, substantially expanding the possibilities for comparative genomic with isolates bearing divergent virulence traits and helping uncover the molecular mechanisms and evolutionary pressures underpinning the emergence of Sporothrix virulence., Author summary Sporotrichosis is polymorphic infection that is acquired after traumatic implantation of Sporothrix propagules into host tissues. In Brazil, large epizooties occur in domestic cats and consequently massive zoonotic transmission has been observed since the 2000s without signs of decreasing over time. In this study, we explored a subcutaneous murine model of sporotrichosis mimicking the alternative route of transmission considering animal horizontal transmission (cat-cat) and zoonotic transmission (cat-human) in which S. brasiliensis yeasts are inoculated. Surprisingly, we found that virulence in the clonal offshoot S. brasiliensis is strain-dependent where highly virulent isolates can disseminate from the site of inoculation (footpad) to vital organs of infected animals, inducing dramatic weight loss, which correlates with host death. Such variation may be regarded as a function of the host's immune response delivered against Sporothrix. Additionally, humoral immune responses produced IgG against isoforms of 3-carboxymuconate cyclase (gp60-gp70) and a 100-kDa molecule. Our study demonstrated that increased or attenuated virulence occurs in S. brasiliensis considering the route of infection, morphotype inoculated and the mammalian immune response. In addition to improving our understanding of the pathogenesis of the emerging animal-born S. brasiliensis, this study has key implications for future developments in serological diagnosis and vaccination efforts.

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

Background Genetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis. Methods We selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C® system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis. Results Among 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C® profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp. Conclusions Molecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-0793-3) contains supplementary material, which is available to authorized users.

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species. Univ Sao Paulo, Fac Odontol, Dept Estomatol, Ave Prof Lineu Prestes 2227, BR-05508000 Sao Paulo, SP, Brazil Adolfo Lutz Inst, Nucleo Ciencias Biomed, Sao Paulo, Brazil Univ Sao Paulo, Inst Med Trop Sao Paulo, Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Lab Especial Micol, Sao Paulo, SP, Brazil Univ Fed Sao Paulo, Lab Especial Micol, Sao Paulo, SP, Brazil Web of Science