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Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the "steering wheel" or "Mickey Mouse" shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964-1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 ± 0.05, 95% CI = 0.756-0.979, p < 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872-1.000, p < 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas.

RESUMO

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.

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BACKGROUND: Melanized fungi are a distinct group of pathogens that cause infections like chromoblastomycosis and phaeohyphomycosis, especially in a state of immunosuppression including solid organ transplant recipients. Guidelines for the treatment of these infections are lacking, and there is no available standard recommendation. OBJECTIVE: To evaluate the therapeutic aspects of subcutaneous melanized fungal infections in kidney transplant recipients. METHODS: A retrospective medical record was performed for kidney transplant recipients with melanized fungal infection evaluated in a single institution from January 1996 to December 2018. RESULTS: Eighty-two episodes were noticed in our series. The treatment of subcutaneous phaeohyphomycosis was managed by surgical excision without antifungal therapy in 34 cases (34/68; 50%), and the association of both methods occurred in 18 cases (18/68; 26.5%). A complete surgical excision without antifungal therapy was observed in six (6/14; 42.9%) episodes of chromoblastomycosis, and combined treatment was possible in three (3/14; 21.4%) cases. Five episodes of chromoblastomycosis and 16 episodes of phaeohyphomycosis were managed only with antifungal therapy. CONCLUSION: Management depends on the dermatologic lesion, immunosuppression condition, and anatomical cleavage plane. The sample size is still small in order to dictate a guideline, but it can be hard to execute a larger study given the rarity of this group of infections.

Assuntos

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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 < 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.

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BACKGROUND: Diseases caused by melanized fungi include mycetoma, chromoblastomycosis and phaeohyphomycosis. This broad clinical spectrum depends on the dynamic interactions between etiologic agent and host. The immune status of the host influences on the development of the disease, as, an exemple. phaeohyphomicosis is more frequently observed in immunocompromised patients. OBJECTIVES: Examine the histological inflammatory response induced by Fonsecaea pedrosoi in several different strains of mice (BALB/c, C57BL/6, Nude and SCID, and reconstituted Nude). METHODS: Fonsecaea pedrosoi was cultivated on agar gel and a fragment of this gel was implanted subcutaneously in the abdominal region of female adult mice. After infection has been obtained, tissue fragment was studied histopathologically. RESULTS: There were significant changes across the strains, with the nodular lesion more persistent in Nude and SCID mice, whereas in immunocompetent mice the lesion progressed to ulceration and healing. The histopathological analysis showed a significant acute inflammatory reaction which consisted mainly of neutrophils in the initial phase that was subsequently followed by a tuberculoid type granuloma in immunocompetent mice. STUDY LIMITATIONS: There is no a suitable animal model for chromoblastomycosis. CONCLUSIONS: The neutrophilic infiltration had an important role in the containment of infection to prevent fungal spreading, including in immunodeficient mice. The fungal elimination was dependent on T lymphocytes. The re-exposure of C57BL/6 mice to Fonsecaea pedrosoi caused a delay in resolving the infection, and appearance of muriform cells, which may indicate that re-exposure to fungi, might lead to chronicity of infection.

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Abstract: Background: Diseases caused by melanized fungi include mycetoma, chromoblastomycosis and phaeohyphomycosis. This broad clinical spectrum depends on the dynamic interactions between etiologic agent and host. The immune status of the host influences on the development of the disease, as, an exemple. phaeohyphomicosis is more frequently observed in immunocompromised patients. Objectives: Examine the histological inflammatory response induced by Fonsecaea pedrosoi in several different strains of mice (BALB/c, C57BL/6, Nude and SCID, and reconstituted Nude). Methods: Fonsecaea pedrosoi was cultivated on agar gel and a fragment of this gel was implanted subcutaneously in the abdominal region of female adult mice. After infection has been obtained, tissue fragment was studied histopathologically. Results: There were significant changes across the strains, with the nodular lesion more persistent in Nude and SCID mice, whereas in immunocompetent mice the lesion progressed to ulceration and healing. The histopathological analysis showed a significant acute inflammatory reaction which consisted mainly of neutrophils in the initial phase that was subsequently followed by a tuberculoid type granuloma in immunocompetent mice. Study limitations: There is no a suitable animal model for chromoblastomycosis. Conclusions: The neutrophilic infiltration had an important role in the containment of infection to prevent fungal spreading, including in immunodeficient mice. The fungal elimination was dependent on T lymphocytes. The re-exposure of C57BL/6 mice to Fonsecaea pedrosoi caused a delay in resolving the infection, and appearance of muriform cells, which may indicate that re-exposure to fungi, might lead to chronicity of infection.

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Pneumocystis jirovecii can cause severe potentially life-threatening pneumonia (PCP) in kidney transplant patients. Prophylaxis of patients against PCP in this setting is usually performed during 6 months after transplantation. The aim of this study is to describe the molecular epidemiology of a cluster of PCP in renal transplant recipients in Brazil. Renal transplant patients who developed PCP between May and December 2011 had their formalin-fixed paraffin-embedded (FFPE) lung biopsy samples analysed. Pneumocystis jirovecii 23S mitochondrial large subunit of ribosomal RNA (23S mtLSU-rRNA), 26S rRNA, and dihydropteroate synthase (DHPS) genes were amplified by polymerase chain reaction (PCR), sequenced, and analysed for genetic variation. During the study period, 17 patients developed PCP (only four infections were documented within the first year after transplantation) and six (35.3%) died. Thirty FFPE samples from 11 patients, including one external control HIV-infected patient, had fungal DNA successfully extracted for further amplification and sequencing for all three genes. A total of five genotypes were identified among the 10 infected patients. Of note, four patients were infected by more than one genotype and seven patients were infected by the same genotype. DNA extracted from FFPE samples can be used for genotyping; this approach allowed us to demonstrate that multiple P. jirovecii strains were responsible for this cluster, and one genotype was found infecting seven patients. The knowledge of the causative agents of PCP may help to develop new initiatives for control and prevention of PCP among patients undergoing renal transplant and improve routine PCP prophylaxis.

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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.

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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.

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Candidemia remains a major cause of morbidity and mortality in the health care environment. The epidemiology of Candida infection is changing, mainly in relation to the number of episodes caused by species C. non-albicans. The overall objective of this study was to evaluate the frequency of yeasts of the genus Candida, in a four-year period, isolated from blood of pediatric patients hospitalized in a public hospital of the city of São Paulo, Brazil. In this period, yeasts from blood of 104 patients were isolated and, the identified species of Candida by phenotypic and genotypic methods were: C. albicans (39/104), C. tropicalis (25/104), C. parapsilosis (23/104), Pichia anomala (6/104), C. guilliermondii (5/104), C. krusei (3/104), C. glabrata (2/104) and C. pararugosa (1/104). During the period of the study, a higher frequency of isolates of C. non-albicans (63.55%) (p = 0.0286) was verified. In this study we verified the increase of the non-albicans species throughout the years (mainly in 2009 and 2010). Thus, considering the peculiarities presented by Candida species, a correct identification of species is recommended to lead to a faster diagnosis and an efficient treatment.

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