Molecular Epidemiology of Blastomyces gilchristii Clusters, Minnesota, USA

Abstract

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.

Three pathogenic Blastomyces species, B. dermatitidis, B. gilchristii, and B. helicus, have been identified in North America. In the United States, B. dermatitidis has been found throughout areas surrounding the Great Lakes, the Ohio and Mississippi River valleys, and the St. Lawrence River (1). In contrast, B. gilchristii has a smaller geographic range in Canada and the northern United States (2), and B. helicus has been found in the northwestern United States (3). No differences in clinical manifestations have been reported among these Blastomyces species.

In the United States, previous case reports have linked blastomycosis infections to outdoor activities, especially those involving moist soil and proximity to waterways (4,5). One of the largest reported outbreaks of blastomycosis occurred in 2015 among persons who had recreated along the Little Wolf River in Wisconsin (6). In Minnesota, blastomycosis is a reportable disease; epidemiologists at the Minnesota Department of Health (MDH) routinely collect demographic and clinical information for blastomycosis cases and attempt interviews to characterize illness and exposure history. The MDH Public Health Laboratory provides fungal identification services and stores isolates submitted by clinical laboratories.

Although whole-genome sequencing has been used to investigate outbreaks involving various fungal pathogens, such as Candida auris and Coccidioides spp. (7,8), this molecular technology has not been used to investigate Blastomyces spp. outbreaks in the United States. We performed whole-genome sequencing to determine the genetic diversity and phylogenetic relationships of 2 familial clusters of B. gilchristii infections identified in Minnesota.

In August 2020, five cases of blastomycosis were identified as cluster A, which comprised a family of 2 White Hispanic parents and 3 children (Table). Four of the 5 patients were hospitalized, of which 3 had sputum cultures that were positive for Blastomyces sp. All 5 patients recovered from illness. The mother reported that the family had visited rivers in St. Croix County, Wisconsin, numerous times during the summer. No other likely exposure locations or activities were reported.

Table. Demographic and clinical data used for molecular epidemiology of 2 Blastomyces gilchristii clusters, Minnesota, USA*.

Sample no.	Age, y/sex	Race/ethnicity	Cluster	Family relationship	Diagnosis location	Exposure location†	Clinical specimen	Specimen collection date
B19405	15/F	White Hispanic	A	Sister	MN	WI	Sputum	2020 Aug 23
B19406	27/M	White Hispanic	A	Brother	MN	WI	Sputum	2020 Aug 19
B19407	3/F	White non-Hispanic	B	Daughter	MN	MN	Bronchial washing	2020 Jul 25
B19408	38/M	White non-Hispanic	B	Father	MN	MN	Subcutaneous abscess	2014 Dec 16

*Data for 2 isolates per cluster that underwent whole-genome sequencing and single-nucleotide polymorphism analyses. †Likely exposure location on the basis of interviews with family members.

In addition, 2 cases of blastomycosis were identified in White non-Hispanic sisters. Only 1 sister was hospitalized and had a positive culture for Blastomyces sp. from a bronchoalveolar lavage specimen. MDH learned that their father had blastomycosis in 2014, which was attributed to B. dermatitidis (9). The 2 patients with isolates (1 sister and the father) were classified as cluster B (Table). The family owned a cabin in Hubbard County, Minnesota, which is highly endemic for blastomycosis and was likely the exposure location for the three cases. All 3 patients recovered from illness.

Blastomyces identification is routinely performed by MDH only at the genus level. Therefore, the Centers for Disease Control and Prevention (CDC) determined the species in 4 isolates from the 2 blastomycosis clusters and performed Illumina (https://www.illumina.com) short-read sequencing (National Center for Biotechnology Information BioProject accession no. PRJNA786864). To investigate genetic diversity between strains, we performed whole-genome single-nucleotide polymorphism (SNP) analysis using the MycoSNP version 0.19 analytical workflow (https://github.com/CDCgov/mycosnp). We used publicly available sequences from B. dermatitidis isolates (NCBI run nos. SRR11849827, SRR11849828, SRR11849829) for comparison and genome assembly data for B. gilchristii strain SLH14081 from GenBank (accession no. GCA_000003855.2) as a reference. We constructed a neighbor-joining tree showing SNP differences and maximum-likelihood tree showing bootstrap values using MEGA software version 7.0, (https://www.megasoftware.net) and FastTree 2 (10).

All the isolates were B. gilchristii rather than B. dermatitidis. Phylogenetic tree analysis showed B. dermatitidis and B. gilchristii grouped into distinct clades, which were separated by 52,431 SNPs (Figure). Sequences from all 4 B. gilchristii isolates clustered with the reference genome SLH14081 and were separated by a minimum of 11,695 SNPs. Each familial cluster formed a subclade within the B. gilchristii clade; the subclades were separated by 5,214 SNPs. In cluster A, where all family members were infected at the same time and location, we found 63 SNPs separated the 2 cases. In cluster B, where exposures occurred in the same location but infections were 6 years apart, the cases differed by 120 SNPs (Figure).

Figure.

Genetic relationships and molecular epidemiology of Blastomyces gilchristii clusters, Minnesota, USA. We performed whole-genome sequencing of isolates from 4 patients in Minnesota who had Blastomyces gilchristii infections and compared the sequences with 3 publicly available B. dermatitidis isolates (National Center for Biotechnology Information run nos. SRR11849827, SRR11849828, SRR11849829). We analyzed single-nucleotide polymorphisms (SNPs) using the MycoSNP version 0.19 analytical workflow (https://github.com/CDCgov/mycosnp). We used the genome assembly data for B. gilchristii strain SLH14081 from GenBank (accession no. GCA_000003855.2) as a reference. Neighbor-joining tree shows the genetic relationships between cluster A and B, which each comprised isolates from 2 patients, the B. gilchristii reference strain, and B. dermatitidis isolates. Numbers represent the SNPs for each strain. Ref., reference.

Both B. dermatitidis and B. gilchristii have been reported in Minnesota (2). We used whole-genome sequencing and SNP analysis to evaluate clusters of blastomycosis infections caused by B. gilchristii in Minnesota. The genomic data showed that cases within cluster A or B were closely related genetically, whereas clusters A and B were genetically distinct. B. gilchristii is likely responsible for a higher proportion of blastomycosis clusters than is currently known. Therefore, pairing genomic data with clinical information and geographic location can be used to monitor blastomycosis infections and determine whether they are clusters, outbreaks, or sporadic occurrences. Our findings demonstrate the utility of genomic analyses for investigating blastomycosis outbreaks, determining genetic diversity of B. dermatitidis and B. gilchristii, and identifying common sources of environmental exposures among cases.

Biography

Dr. Bagal is a bioinformatician with the Mycotic Diseases Branch, Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA. Her research interests are genomics and evolutionary biology, metagenomics, and data science.