Evaluation of the virulence and immunogenicity in the emerging species sporothrix brasiliensis

Sporotrichosis is a polimorfic chronic granulomatous fungal infection that occurs in human and animals. Sporothrix schenckii complex that contain etiologic agents that causes this mycosis and is composed for species S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa and S. luriei. This fungus presents thermal dimorphism and infects the host via traumatic inoculation by contact with plants, soil and animals, among them cats and armadillos. This mycosis has a worldwide distribution, with endemic areas in countries with subtropical and tropical weather. Over the last decades outbreaks this disease were reported in Brazil, mainly due to S. brasiliensis which were intimately related with zoonotic transmission by cats. The aims of this study was to evaluate the virulence profile of representative isolates of S. brasiliensis, using S. schenckii s. str. and S. globosa as controls. We evaluate the profile of secretion of protein/glycoprotein and the immunogenicity of these molecules. The pathogenicity study was conducted using a murine model of subcutaneous infection in order to simulate the major route of infection. Thus, mice were infected subcutaneously in the left paw with 5x106 of each isolate of S. brasiliensis and controls species. S. brasiliensis isolates showed different virulence profiles being classified as low, medium or high virulence based on the fungal burden in the organs evaluated, weight loss, and induction of death of infected animals. The histopathological analysis revealed tissue damage especially when most virulent isolates were inoculated, confirming the high invasiveness of S. brasiliensis. The protein profiles of isolates were heterogeneous. Most exoantigens present proteins/glycoproteins of 30- and 35-kDa. Different proteins of the exoantigens were recognized by IgG in the sera of infected mice, with a 70-kD molecule being frequently recognized. Sera from infected mice mainly recognized the molecules of 100-kDa and 70-kDa present in total extracts of Ss54 (S. brasiliensis) and SS118 (S. schenckii). The characterization of the species of major relevance in the epidemiological scenario in Brazil contributed to a better understanding of the pathogenesis of sporotrichosis.A esporotricose é uma infecção fúngica crônica granulomatosa subcutânea e polimórfica que afeta homens e animais. O complexo Sporothrix schenckii que agrega os agentes etiológicos causadores desta micose é composto pelas espécies S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa e S. luriei. Estes fungos apresentam dimorfismo térmico e alcançam o hospedeiro via inoculação traumática pelo contato com vegetais, solo e animais infectados, entre eles, felinos e tatus. Esta micose apresenta distribuição mundial, com focos endêmicos em países de clima tropical e subtropical. Nas últimas décadas, foram relatados surtos da doença no Brasil por S. brasiliensis intimamente relacionados à transmissão zoonótica por gatos infectados. Este estudo tem como principais objetivos, avaliar o perfil de virulência de isolados representativos de S. brasiliensis, utilizando S. schenckii s. str. e S. globosa como controles. Avaliou-se o perfil de secreção de proteínas/glicoproteínas e a imunogenicidade de tais moléculas. O estudo de patogenicidade foi conduzido através de infecção subcutânea em modelo murino com o intuito de mimetizar a principal rota de infecção. Desta forma, camundongos foram infectados por via subcutânea na pata esquerda com 5x106 leveduras de cada isolado de S. brasiliensis e controles. Os isolados de S. brasiliensis apresentaram diferentes perfis de virulência sendo classificados como pouco, médio e altamente virulento através da análise de carga fúngica presente nos órgãos, perda de peso, indução à morte dos animais infectados. A análise histopatológica revelou lesões teciduais principalmente quando isolados mais virulentos foram inoculados, confirmando a alta capacidade invasiva do S. brasiliensis. Os perfis proteicos dos isolados foram heterogênios. A maioria dos exoantígenos apresentaram proteínas/glicoproteínas de 30 e 35 kDa. Diferentes proteínas dos exoantígenos foram reconhecidas por IgG nos soros de camundongos infectados, sendo a molécula de 70 kDa a mais reconhecida. Os soros dos camundongos infectados com os isolados estudados reconheceram principalmente as moléculas de 100 kDa e 70 kDa presentes no extrato total do isolado Ss54 (S. brasiliensis) e Ss118 (S. schenckii). A caracterização da espécie de relevância epidemiológica no Brasil realizada neste estudo contribuiu para a melhor compreensão da patogênese da esporotricose.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Perfis de virulência, imunogenicidade e diagnóstico dos principais patógenos do gênero Sporothrix

Sporotrichosis is a chronic cutaneous and subcutaneous mycosis that affects humans and animals, classified as an implantation disease due to the traumatic inoculum of material contaminated with Sporothrix propagules or scratches and bites from contaminated cats. Sporothrix brasiliensis, S. schenckii, and S. globosa are the main human and animal pathogens and are considered more virulent than environmental species. Brazil and China harbor current outbreaks of S. brasiliensis and S. globosa, and it is relevant to increase our knowledge about virulence and the identification of Sporothrix species. This thesis is divided into three chapters, the first addressing virulence in S. globosa, the second a diagnosis by quantitative realtime PCR (qPCR) from Sporothrix, and the third to experimental pulmonary sporotrichosis. Our first objective was to evaluate the virulence and immunogenicity profiles of S. globosa distributed throughout South America, Europe, and Asia, compared with isolates of S. brasiliensis, S. schenckii, S. globosa, and S. chilensis, using a disseminated murine model and subcutaneous. To characterize virulence, BALB/c mice were divided into groups and were infected with 5x106 yeast/animal cells intravenously (7 days of infection) or subcutaneously (20 days of infection). After the challenge, isolates of S. globosa were classified as non-virulent, with low or medium virulence, based on the induction of death in infected animals, the fungal load of evaluated organs, and weight loss. CFU showed different results in each infection pathway. In the subcutaneous group, isolates Ss126, Ss226 and Ss545 developed lesions characteristic of sporotrichosis in the animals' footpad. Among histopathological features are granuloma, inflammatory infiltrate, suppurative inflammation, necrosis, hypertrophic tubules (in the kidneys), and thick pulmonary alveoli. Arthritis lesions affected almost 100% of the animals and caused irreversible sequelae in the intravenous group. In the tail, some animals showed a wound, nodule, or sinuosity. Sera from animals infected with Sporothrix spp. by the intravenous route recognized in greater number the molecules of 22, 26, and 28 kDa of the total extract of Ss226 (S. brasiliensis); 50 kDa of the extract of Ss126 (S. schenckii) and 100 kDa of the extract of Ss06 (S. globosa). The sera of animals that had an infection by the subcutaneous route recognized in prevalence the molecules of 38, 40, and 48 kDa of the total extract of Ss226 (S. brasiliensis); 30, 38, and 40 kDa of the total extract of Ss126 (S. schenckii); 38 and 40 kDa of the total extract of Ss06 (S. globosa). The subcutaneous route showed a higher level of antibodies in the animals' serum than the intravenous route due to the longer time of infection, which occurred with Western blotting. Chapter 2: The real-time PCR technique, based on fluorescent probes, is faster to identify the main species of pathogens from Sporothrix spp. The variation between the BT2 gene was explored to design a qPCR assay with triplex probes to identify the species S. brasiliensis, S. schenckii, and S.iv globosa in a multiplex qPCR assay. A panel of 84 Sporothrix revealed 100% specificity (AUC = 1,000, 95% CI = 0.971–1,000, P <0.0001) without cross-reacting with other clinically relevant fungi, human, feline, or murine DNA. We used swab samples from 70 naturally infected cats, and in our test, 69 samples were positive for S. brasiliensis (sensitivity = 98.57%; 95% CI: 92.3–100.0 and specificity = 100%; 95% CI = 78.2-100), confirming that our trial helps to detect and differentiate pathogenic Sporothrix, providing clinical laboratories with an essential tool to be applied routinely. Our trial is likely to benefit thousands of infected patients every year worldwide. Chapter 3: Pulmonary sporotrichosis occurs by inoculation through inhalation of Sporothrix conidia. It is a rare type of infection, usually affecting people who work in the field, with gardening, and usually occurs in hypertensive patients or patients with some involvement in the lungs, despite reports of immunocompetence. We performed a model of pulmonary sporotrichosis so that we could understand a little more about this disease, which has led some patients to death despite occurring at low frequency. We used the isolates of S. brasiliensis, S. schenckii, and S. globosa, classified as high, medium, and low virulence, in an experimental murine model using the intratracheal route of BALB/c mice with inoculum in groups of animals that received either conidia or yeast. The isolate of S. brasiliensis obtained greater virulence than the other isolates, as it has a high CFU and induces animals to death by the second week after infection, both by the yeast inoculum and the conidia inoculum. The isolates of S. schenckii and S. globosa were classified with low virulence by this route of infection for the CFU study and avirulent for not inducing animals to death in the survival study.A esporotricose é micose cutânea e subcutânea crônica que afeta o homem e animais, classificada como doença de implantação pelo inóculo traumático de material contaminado com propágulos de Sporothrix ou arranhões e mordidas de felinos contaminados. Sporothrix brasiliensis, S. schenckii e S. globosa são os principais patógenos humanos e animais e são considerados com maior virulência em comparação com espécies ambientais. Brasil e China abrigam surtos atuais de S. brasiliensis e S. globosa, sendo relevante aumentar nossos conhecimentos sobre a virulência e a identificação das espécies de Sporothrix. Esta tese se divide em 3 capítulos, o primeiro aborda a virulência em S. globosa, o segundo em diagnóstico por PCR quantitativa em tempo real (qPCR) de Sporothrix e o terceiro a esporotricose pulmonar experimental. Nosso primeiro objetivo foi avaliar os perfis de virulência e imunogenicidade de S. globosa distribuídos pela América do Sul, Europa e Ásia, comparado com isolados de S. brasiliensis, S. schenckii, S. globosa e S. chilensis, utilizando modelo murino disseminado e subcutâneo. Para a caracterização da virulência, camundongos BALB/c foram divididos em grupos e foram infectados com 5x106 células de levedura/animal por via intravenosa (7 dias de infecção) ou subcutânea (20 dias de infecção). Após o desafio, os isolados de S. globosa foram classificados como não virulentos, com baixa ou média virulência, com base na indução de morte de animais infectados, carga fúngica dos órgãos avaliados e perda de peso. As UFC mostraram resultados diferentes em cada via de infecção. No grupo subcutâneo, os isolados Ss126, Ss226 e Ss545 desenvolveram lesões características da esporotricose na pata dos animais. Dentre as características histopatológicas encontramos granuloma, infiltrado inflamatório, inflamação supurativa, necrose, túbulos hipertróficos (nos rins) e alvéolos pulmonares espessos. As lesões de artrite na pata dianteira e para traseira acometeram quase 100% dos animais e causaram sequelas irreversíveis no grupo intravenoso. Na cauda alguns animais apresentaram ferida, nódulo ou sinuosidade. Os soros dos animais infectados com os isolados de Sporothrix spp. pela via intravenosa reconheceram em maior número as moléculas de 22, 26 e 28 kDa do extrato total de Ss226 (S. brasiliensis); 50 kDa do extrato de Ss126 (S. schenckii) e 100 kDa do extrato de Ss06 (S. globosa). Os soros dos animais que tiveram infecção pela via subcutânea reconheceram em prevalência as moléculas de 38, 40 e 48 kDa do extrato total de Ss226 (S. brasiliensis); 30, 38 e 40 kDa do extrato total de Ss126 (S. schenckii); 38 e 40 kDa do extrato total de Ss06 (S. globosa). A via subcutânea apresentou um maior nível de anticorpos no soro dos animais do que a via intravenosa, devido ao tempo maior de infecção, o mesmo que ocorreu com o Western blotting. Capítulo 2: A técnica de PCR em tempo real, baseada em sondas fluorescentes é mais rápida para identificação das principais espécies de patógenos de Sporothrix spp. A variação entre o gene BT2 foi explorada para projetar um ensaio qPCR com sondas triplex para identificar as espécies S. brasiliensis, S. schenckii e S. globosa em um ensaio qPCR multiplex. Um painel de 84 Sporothrix revelou 100% deii especificidade (AUC = 1,000, 95% CI = 0,971–1,000, P <0,0001) sem reação cruzada com outros fungos clinicamente relevantes, DNA humano, felino ou murino. Utilizamos amostras de esfregaços de lesões de 70 gatos naturalmente infectados e em nosso teste 69 amostras foram positivas para S. brasiliensis (sensibilidade = 98,57%; IC 95%: 92,3–100,0 e especificidade = 100%; IC95% = 78,2-100), confirmando que nosso ensaio ajuda a detectar e diferenciar Sporothrix patogênico, fornecendo aos laboratórios clínicos uma ferramenta essencial para ser aplicada rotineiramente. Nosso ensaio provavelmente beneficiará milhares de pacientes infectados todos os anos em todo o mundo. Capítulo 3: A esporotricose pulmonar ocorre por inoculação através da inalação de conídios de Sporothrix. É um tipo de infecção rara, geralmente acomete pessoas que trabalham no campo, com jardinagem e geralmente ocorre em hipertensos ou pacientes com algum acometimento nos pulmões, apesar de haver relatos de imunocompetentes. Realizamos um modelo de esporotricose pulmonar para que pudéssemos entender um pouco mais sobre esta doença que apesar de ocorrer em baixa frequência, tem levado alguns pacientes à óbito. Utilizamos os isolados de S. brasiliensis, S. schenckii e S. globosa, classificados com alta, média/baixa e baixa virulência em modelo murino experimental pela via intratraqueal de camundongos BALB/c com inóculos em grupos de animais que receberam ou conídios ou leveduras. O isolado de S. brasiliensis obteve uma maior virulência em comparação aos outros isolados, por possuir uma alta UFC e induzir os animais à morte até a segunda semana após a infecção, tanto pelo inóculo de levedura, quanto o inóculo de conídios. Os isolados de S. schenckii e S. globosa foram classificados com uma baixa virulência por esta via de infecção para o estudo de UFC e avirulentos por não induzirem os animais à morte no estudo de sobrevida.Dados abertos - Sucupira - Teses e dissertações (2021

Development and evaluation of a multiplex qPCR assay for rapid diagnostics of emerging sporotrichosis

Sporothrix schenckii and related species are the agents of human and animal sporotrichosis. Routine diagnoses using classical mycological approaches are unspecific due to overlapping phenotypes. As the frequency and prevalence of sporotrichosis increases worldwide, developing specific, sensitive and cost-effective diagnostic tools is essential to understand the distribution patterns, map-affected areas and promote specific public health strategies to mitigate future outbreaks. Polymorphisms among the β-tubulin gene were exploited to speciate S. brasiliensis, S. schenckii and S. globosa in a one-tube multiplex probe-based qPCR assay. A panel of 84 Sporothrix revealed 100% specificity (AUC = 1.000, 95% CI = 0.971-1.000, p < .0001) without cross-reacting with other medically relevant fungi, human, feline or murine DNA. Speciation via multiplex qPCR matched phylogenetic identification (Kappa = 1.0; 95% CI = 1.0-1.0; very good agreement), supporting its use as a reliable alternative to DNA sequencing. Remarkably, the lower limit of detection was 3 copies of the target for all species. As a proof of concept, we used swabs of wound exudate of 70 cats suspected of sporotrichosis to reveal an overwhelming occurrence of S. brasiliensis in 69 specimens (sensitivity = 98.57%; 95%CI: 92.3-100.0 and specificity = 100%; 95% CI = 78.2-100). In comparison to culture, qPCR showed a larger area under the curve (AUC = 0.993±0.007; 95% CI = 0.944-1.000; p < .0001; Youden's index = 0.9857), supporting that qPCR is an essential tool for accurately detect Sporothrix DNA directly from clinical samples, thus accelerating the diagnosis of sporotrichosis. Moreover, our multiplex qPCR system has the potential to increase diagnostic capacity in Sporothrix-affected areas, helping the local animal health agent or veterinarian to quickly identify and isolate new cases, which will likely benefit thousands of patients infected every year worldwide

A New Duplex PCR-Assay for the Detection and Identification of <i>Paracoccidioides</i> Species

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 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

Exploring virulence and immunogenicity in the emerging pathogen <i>Sporothrix brasiliensis</i>

<div><p>Sporotrichosis is a polymorphic chronic infection of humans and animals classically acquired after traumatic inoculation with soil and plant material contaminated with <i>Sporothrix</i> spp. propagules. An alternative and successful route of transmission is bites and scratches from diseased cats, through which <i>Sporothrix</i> 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 <i>Sporothrix</i> fitness, the left hind footpads of BALB/c mice were inoculated with 5×10⁶ yeasts (n = 11 <i>S</i>. <i>brasiliensis</i>, n = 2 <i>S</i>. <i>schenckii</i>, or n = 1 <i>S</i>. <i>globosa</i>). 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 <i>S</i>. <i>schenckii</i> clade, <i>S</i>. <i>brasiliensis</i> was usually more virulent than <i>S</i>. <i>schenckii</i> and <i>S</i>. <i>globosa</i>. However, the virulence in <i>S</i>. <i>brasiliensis</i> 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 <i>Sporothrix</i> species can elicit a heterogeneous humoral response over time, but cross reaction was observed between <i>S</i>. <i>brasiliensis</i> and <i>S</i>. <i>schenckii</i> 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 <i>Sporothrix</i>-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 <i>Sporothrix</i> virulence.</p></div

Immunoreactivity of <i>S</i>. <i>brasiliensis</i> Ss54 (= CBS 132990) proteins resolved by 2DGE with mice sera reveals high cross-reactivity and isoforms of gp60 as major component recognized.

<p><i>S</i>. <i>brasiliensis</i> whole cellular extract (yeast phase) were separated by IEF at pH 4 to 7 in the first dimension, followed by 10% SDS-PAGE in the second dimension. Gels were subsequently silver stained (A) or immunoblotted (B-I) with a 1:200 dilution of infected mice sera indicated at the top. Red dot areas indicate all seroreactive protein spots identified as 3-carboxymuconate cyclase. Immunoblot assay using Ss104 antisera did not detect immunoreactivity (I), similar to the 1D assay. Acidic and basic ends are denoted at the bottom, and the positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels series.</p

Characteristics of <i>Sporothrix</i> spp. isolates.

<p>Characteristics of <i>Sporothrix</i> spp. isolates.</p

Highly virulent <i>S</i>. <i>brasiliensis</i> isolates induce critical weight loss leading to impaired development and death of infected animals.

<p>Data are expressed as percent weight loss (%) determined by measuring the animal’s weight every week post-inoculation and comparing them to the weight of the animal on the day of inoculation. (A) Significant weight loss in <i>S</i>. <i>brasiliensis</i> Ss66, Ss99, Ss174, and Ss226-infected groups, leading to the death of animals (Ss174 and Ss226) and impaired development compared to the PBS group (<i>P</i> < 0.0001). (B) Moderate development for <i>S</i>. <i>brasiliensis</i> Ss34, Ss54, Ss67, Ss104, Ss252, Ss261, and Ss265 and the <i>S</i>. <i>globosa</i> Ss06-infected group. Despite initial weight loss in early infection, mice began to regain weight between the 2^nd and 5^th week post-inoculation, but this evolution was not similar to the PBS group (<i>P</i> < 0.0001). (C) Normal development for <i>S</i>. <i>schenckii</i> Ss39 and Ss126-infected groups, similar to the PBS group, demonstrating the appearance and spontaneous resolution of <i>Sporothrix</i> infection. Statistical analysis is presented in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005903#pntd.0005903.s002" target="_blank">S2 Table</a>.</p

Virulence characteristics of <i>Sporothrix</i> isolates based on the murine model of subcutaneous sporotrichosis.

<p>Virulence characteristics of <i>Sporothrix</i> isolates based on the murine model of subcutaneous sporotrichosis.</p