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.

Candida auris is a newly emerged multidrug-resistant fungus capable of causing invasive infections with high mortality. Despite intense efforts to understand how this pathogen rapidly emerged and spread worldwide, its environmental reservoirs are poorly understood. Here, we present a collaborative effort between the U.S. Centers for Disease Control and Prevention, the National Center for Biotechnology Information, and GridRepublic (a volunteer computing platform) to identify C. auris sequences in publicly available metagenomic datasets. We developed the MetaNISH pipeline that uses SRPRISM to align sequences to a set of reference genomes and computes a score for each reference genome. We used MetaNISH to scan ~300,000 SRA metagenomic runs from 2010 onwards and identified five datasets containing C. auris reads. Finally, GridRepublic has implemented a prospective C. auris molecular monitoring system using MetaNISH and volunteer computing.

Histoplasmosis is one of the most under-diagnosed and under-reported endemic mycoses in the United States. Histoplasma capsulatum is the causative agent of this disease. To date, molecular epidemiologic studies detailing the phylogeographic structure of H. capsulatum in the United States have been limited. We conducted genomic sequencing using isolates from histoplasmosis cases reported in the United States. We identified North American Clade 2 (NAm2) as the most prevalent clade in the country. Despite high intra-clade diversity, isolates from Minnesota and Michigan cases were predominately clustered by state. Future work incorporating environmental sampling and veterinary surveillance may further elucidate the molecular epidemiology of H. capsulatum in the United States and how genomic sequencing can be applied to the surveillance and outbreak investigation of histoplasmosis.

Working overnight at a large swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, nanopore-sequenced 13 IAV genomes from samples collected, and in real-time, determined that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current pre-pandemic candidate vaccine viruses (CVV). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 hours after unpacking the mobile lab. Swine are important animal IAV reservoirs that have given rise to pandemic viruses via zoonotic transmission. Genomic analyses of IAV in swine are critical to understanding pandemic risk of viruses in this reservoir, and characterization of viruses circulating in exhibition swine enables rapid comparison to current seasonal influenza vaccines and CVVs. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes: A(H1N1), A(H3N2) and A(H1N2). Additional analysis of the HA protein sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 portion of the hemagglutinin of these viruses and the most closely related pre-2009 CVV. All virus sequences were emailed to colleagues at CDC who initiated development of a synthetically derived CVV designed to provide an optimal antigenic match with the viruses detected in the exhibition. In subsequent months, this virus caused 13 infections in humans, and was the dominant variant virus in the US detected in 2018. Had this virus caused a severe outbreak or pandemic, our proactive surveillance efforts and CVV derivation would have provided an approximate 8 week time advantage for vaccine manufacturing. This is the first report of the use of field-derived nanopore sequencing data to initiate a real-time, actionable public health countermeasure.

Madurella mycetomatis is one of the main causative agents of mycetoma, a debilitating neglected tropical disease. Improved understanding of the genomic diversity of the fungal and bacterial causes of mycetoma is essential to advances in diagnosis and treatment. Here, we describe a high-quality genome assembly of M. mycetomatis and results of the whole genome sequence analysis of 26 isolates from Sudan. We demonstrate evidence of at least seven genetically diverse lineages and extreme clonality among isolates within these lineages. We also performed shotgun metagenomic analysis of DNA extracted from mycetoma grains and showed that M. mycetomatis reads were detected in all sequenced samples with the average of 11,317 reads (s.d. +/- 21,269) per sample. In addition, 10 (12%) of the 81 tested grain samples contained bacterial reads including Streptococcus sp., Staphylococcus sp. and others.

We characterized 2 clusters of blastomycosis cases in Minnesota, USA, using whole-genome sequencing and single-nucleotide polymorphism analyses. Blastomyces gilchristii was confirmed as the cause of infection. Genomic analyses corresponded with epidemiologic findings for cases of B. gilchristii infections, demonstrating the utility of genomic methods for future blastomycosis outbreak investigations.

Candida auris is an urgent public health threat characterized by high drug-resistant rates and rapid spread in healthcare settings worldwide. As part of the C. auris response, molecular surveillance has helped public health officials track the global spread and investigate local outbreaks. Here, we describe whole-genome sequencing analysis methods used for routine C. auris molecular surveillance in the United States; methods include reference selection, reference preparation, quality assessment and control of sequencing reads, read alignment, and single-nucleotide polymorphism calling and filtration. We also describe the newly developed pipeline MycoSNP, a portable workflow for performing whole-genome sequencing analysis of fungal organisms including C. auris.

While working overnight at a swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, Nanopore sequenced 13 IAV genomes from samples we collected, and predicted in real time that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current prepandemic candidate vaccine viruses (CVVs). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 h after unpacking the mobile lab. Exhibition swine are a known source for zoonotic transmission of IAV to humans and pose a potential pandemic risk. Genomic analyses of IAV in swine are critical to understanding this risk, the types of viruses circulating in swine, and whether current vaccines developed for use in humans would be predicted to provide immune protection. Nanopore sequencing technology has enabled genome sequencing in the field at the source of viral outbreaks or at the bedside or pen-side of infected humans and animals. The acquired data, however, have not yet demonstrated real-time, actionable public health responses. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes, A(H1N1), A(H3N2), and A(H1N2). Analysis of the hemagglutinin (HA) sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 of these viruses and the most closely related CVV. As an exercise in pandemic preparedness, all sequences were emailed to CDC collaborators who initiated the development of a synthetically derived CVV.IMPORTANCE Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018.