Emergence of zoonotic sporotrichosis in Brazil: a genomic epidemiology study.

BACKGROUND: Zoonotic sporotrichosis is a neglected fungal disease, whereby outbreaks are primarily driven by Sporothrix brasiliensis and linked to cat-to-human transmission. To understand the emergence and spread of sporotrichosis in Brazil, the epicentre of the current epidemic in South America, we aimed to conduct whole-genome sequencing (WGS) to describe the genomic epidemiology. METHODS: In this genomic epidemiology study, we included Sporothrix spp isolates from sporotrichosis cases from Brazil, Colombia, and the USA. We conducted WGS using Illumina NovaSeq on isolates collected by three laboratories in Brazil from humans and cats with sporotrichosis between 2013 and 2022. All isolates that were confirmed to be Sporothrix genus by internal transcribed spacer or beta-tubulin PCR sequencing were included in this study. We downloaded eight Sporothrix genome sequences from the National Center for Biotechnology Information (six from Brazil, two from Colombia). Three Sporothrix spp genome sequences from the USA were generated by the US Centers for Disease Control and Prevention as part of this study. We did phylogenetic analyses and correlated geographical and temporal case distribution with genotypic features of Sporothrix spp isolates. FINDINGS: 72 Sporothrix spp isolates from 55 human and 17 animal sporotrichosis cases were included: 67 (93%) were from Brazil, two (3%) from Colombia, and three (4%) from the USA. Cases spanned from 1999 to 2022. Most (61 [85%]) isolates were S brasiliensis, and all were reported from Brazil. Ten (14%) were Sporothrix schenckii and were reported from Brazil, USA, and Colombia. For S schenckii isolates, two distinct clades were observed wherein isolates clustered by geography. For S brasiliensis isolates, five clades separated by more than 100 000 single-nucleotide polymorphisms were observed. Among the five S brasiliensis clades, clades A and C contained isolates from both human and cat cases, and clade A contained isolates from six different states in Brazil. Compared with S brasiliensis isolates, larger genetic diversity was observed among S schenckii isolates from animal and human cases within a clade. INTERPRETATION: Our results suggest that the ongoing epidemic driven by S brasiliensis in Brazil represents several, independent emergence events followed by animal-to-animal and animal-to human transmission within and between Brazilian states. These results describe how S brasiliensis can emerge and spread within a country. FUNDING: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil; the São Paulo Research Foundation; Productivity in Research fellowships by the National Council for Scientific and Technological Development, and Ministry of Science and Technology of Brazil.

Bimodal distribution of azole susceptibility in Sporothrix brasiliensis isolates in Brazil.

Sporothrix brasiliensis is an emerging zoonotic fungal pathogen that can be difficult to treat. Antifungal susceptibility testing was performed on the mold phase of a convenience sample of 61 Sporothrix spp. isolates from human and cat sporotrichosis cases in Brazil using the Clinical and Laboratory Standards Institute standard M38. A bimodal distribution of azole susceptibility was observed with 50% (28/56) of S. brasiliensis isolates showing elevated itraconazole minimum inhibitory concentrations ≥16 µg/mL. Phylogenetic analysis found the in vitro resistant isolates were not clonal and were distributed across three different S. brasiliensis clades. Single nucleotide polymorphism (SNP) analysis was performed to identify potential mechanisms of in vitro resistance. Two of the 28 resistant isolates (MIC ≥16 mg/L) had a polymorphism in the cytochrome P450 gene, cyp51, corresponding to the well-known G448S substitution inducing azole resistance in Aspergillus fumigatus. SNPs corresponding to other known mechanisms of azole resistance were not identified in the remaining 26 in vitro resistant isolates.

Identificação e perfil de sensibilidade de aspergillus spp. presente no ar atmosférico / Identification and sensitivity profile of aspergillus spp. present in atmospheric air

Aspergillus spp. são fungos saprófitos distribuídos mundialmente, sendo agentes de aspergilose, micose causada por via aérea. Em meio ambiente agrícola, fungos podem estar expostos a compostos azólicos usados como agrotóxicos e sofrerem pressão seletiva para surgimento de cepas resistentes. Se inaladas, principalmente, por indivíduos imunosuprimidos, tais cepas podem desencadear infecção refratária a tratamentos com fármacos da mesma classe química, como itraconazol e voriconazol. A busca da resistência ambiental contribui para o desvendamento da epidemiologia molecular da aspergilose. O objetivo deste estudo foi identificar as espécies de Aspergillus e determinar os respectivos perfis de sensibilidade a azóis, fármacos e fungicidas agrícolas, de amostras de regiões de cinco estados do Brasil. Foram estudados 128 isolados de Aspergillus spp., provenientes de 114 amostras de ar atmosférico. Os isolados foram classificados em seções e sequenciados para identificação da espécie. As seções mais frequentes foram: Nigri (50,8%), Flavi (29,7%), Circumdati (9,3%), além de Fumigati, Clavati, Terrei, Restricti e Nidulantes. Valores altos de concentração inibitória mínima (MIC), situados acima dos pontos de corte epidemiológicos (ECOFF) que caracterizam isolados não-selvagens para itraconazol e posaconazol, foram observados para um isolado da seção Nigri, identificado como Aspergillus welwitschiae. Para seção Restricti (n=2), apesar de não haver ECOFF, os valores de MIC foram mais elevados para posaconazol, voriconazol, itraconazol, anfotericina B e para o fungicida agrícola difenoconazol, quando comparados às demais seções. Para isolados das seções Flavi, Circumdati e Clavati, foram encontrados valores altos de MIC dos agrotóxicos tebuconazol e metconazol. Não foi constatada ocorrência de resistência cruzada a mais de...(AU)

Aspergillus spp. are saprophytic fungi distributed worldwide, being agents of aspergillosis, a mycosis caused by the airborne route. In an agricultural environment, fungi may be exposed to azole compounds used as pesticides and undergo selective pressure for the emergence of resistant strains. If inhaled, mainly by immunosuppressed individuals, these strains can trigger an infection that is refractory to treatments with drugs of the same chemical class, such as itraconazole and voriconazole. The search for environmental resistance contributes to the unveiling of the molecular epidemiology of aspergillosis. The aim of this study was to identify Aspergillus species and determine the respective profiles of sensitivity to azoles, pharmaceuticals and agricultural fungicides, from samples isolated from regions of five states of Brazil A total of 128 Aspergillus spp. isolates from 114 atmospheric air samples were studied. The isolates were classified into sections and sequenced to identify the species. The most frequent sections were Nigri (50.8%), Flavi (29.7%), Circumdati (9.3%), in addition to Fumigati, Clavati, Terrei, Restricti and Nidulantes. High values of minimum inhibitory concentration (MIC) above the epidemiological cut-off points (ECOFF), characterizing non-wild isolates, for itraconazole and posaconazole, were observed for an isolate from the Nigri section, identified as Aspergillus welwitschiae. For the Restricti section (n=2), despite the absence of ECOFF, the MIC were higher for posaconazole, voriconazole, itraconazole, amphotericin B and for agricultural fungicide difenoconazole, when compared to the other sections. For isolates from Flavi, Circumdati, and Clavati sections, high MIC values of the pesticides tebuconazole and metconazole were found. Cross-resistance to more than one azole drug was not found. It is concluded that in the atmospheric air of the cities of Itu, São Paulo, Azenha, and Porto Alegre there are non-wild isolates with the possibility of presenting resistance mechanisms. Complementary studies to this one, using gene sequencing methods, are necessary to elucidate the underlying possible resistance mechanism of mutation. This is the first study in Brazil to demonstrate non-wild isolates from urban atmospheric air, harboring possible mechanisms of environmental resistance. (AU)

Identificação fenotípica e perfil de sensibilidade de isolados de aspergillus / Phenotypic identification and sensitivity profile of aspergillus isolates

Os fungos do gênero Aspergillus são fungos anemófilos, cosmopolistas e possuem grande impacto socioeconômico, pois produzem micotoxinas e degradam alimentos, causam infecções em animais, plantas e seres humanos. Isolados desse agente estão presentes no meio ambiente e são responsáveis por infectar seres humanos e causar os mais variáveis quadros clínicos podendo ser letais na maioria dos casos. Esse trabalho objetivou identificar cepas ambientais e observar o seus perfis de sensibilidade, para melhor conhecer a epidemiologia desse agente. Para a identificação fenotípica foi utilizado a chave dicotômica de Klich, 2002 em ágar Czapek e os isolados foram então divididos em seções e espécies. Os testes de sensibilidade a antifúngicos foram realizados de acordo com o documento europeu AFST-EUCAST 9.3 ( 2015 ) para os antifúngicos triazólicos, anfotericina B e equinocandinas. Foram isolados quatorze espécies de Aspergillus de diferentes amostras ambientais, totalizando seis espécies de cinco Seções. A espécie mais encontrada foi Aspergillus fumigatus seguido de Aspergillus niger. Espécies menos usuais também foram observadas como A. parasiticus e A. ochraceus . A maioria dos isolados de A. fumigatus apresentaram MICs ( minimum inhibitory concentration )