medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Development of a duplex real-time PCR assay for the differentiation of Blastomyces

dermatitidis and B. gilchristii and a retrospective study of in New York

(2005-2019)

Mitchell Kaplan1, #, YanChun Zhu1,#, Julianne V Kus2, 3, Lisa McTaggart2, Vishnu

Chaturvedi1,4, and Sudha Chaturvedi1,4*

1Mycology Laboratory, Wadsworth Center, New York State Department of Health,

Albany, NY 12208

2 Public Health Ontario, Canada

3 Dept. of Laboratory Medicine and Pathobiology, University of Toronto, Canada

4 Dept. of Biomedical Sciences, University at Albany, Albany, NY

Corresponding author: Sudha Chaturvedi; email: [email protected];

phone: 518-474-7563

Running title: Duplex real-time PCR assay for Blastomyces

#Mitchell Kaplan and YanChun Zhu contributed equally

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 1 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

ABSTRACT

Blastomycosis due to Blastomyces dermatitidis and B. gilchristii is a notable cause of

respiratory mycoses in North America with recurrent outbreaks. We developed a

highly sensitive, specific, and reproducible Taqman duplex real-time PCR assay for the

differentiation of B. dermatitidis and B. gilchristii. The new assay permitted retrospective

analysis of Blastomyces cultures (2005 to 2019), and primary clinical specimens (2013-

2019) from NY patients. Blastomyces dermatitidis was the causal agent for the majority of

38 cases while B. gilchristii was implicated in five cases; a rare finding reported from

New York. The duplex real-time PCR assay will be useful for further understanding of

ecology and epidemiology of blastomycosis caused by B. dermatitidis and B. gilchristii.

2 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

INTRODUCTION

For the longest time, Blastomyces dermatitidis was considered a single species within the

Blastomyces genus as the etiologic agent of pulmonary and disseminated blastomycosis

(1). This knowledge was used to develop several diagnostic tests. With the

advancement of molecular tools, several distinct genetic populations were recognized

within the Blastomyces genus, and that led to the creation of new species including B.

gilchristii (a cryptic species phenotypically indistinguishable from B. dermatitidis) (2, 3),

B. percursus, and B. silverae (4, 5). A rare cause of blastomycosis from B. helicus in

western part of North America and B. percursus in Africa was also reported (6, 7).

Additionally, Emmonsia parva (now Blastomyces parvus) and Emmonsia helica (now

Blastomyces helicus) were assigned to the Blastomyces genus (5). Blastomyces dermatitidis is

the predominant species in North America, but B. gilchristii predominates in the

hyperendemic regions of Northwestern Ontario and northern Wisconsin including

large outbreaks (6). The ecological niche (s) of these two closely related species remains

undefined. Similarly, the role of remaining rare Blastomyces species in the overall

incidence and outcome of blastomycosis cases requires further investigations.

All Blastomyces spp. are thermally dimorphic fungi, exist as a mold form at

ambient temperature in the environment but convert to the pathogenic yeast form in

vitro at 37oC or when susceptible mammalian host inhales the conidia of the mold form.

The yeast form of these pathogens can be microscopically differentiated with B.

dermatitidis and B. gilchrtstii producing abundant large (8-20 mm) broad-based budding

yeasts; B. percursus producing large yeast-like cells from fragmented swollen hyphal

3 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

cells and B. helicus producing variably shaped yeast cells (4 to 5 µm) in short chain,

while thin walled giant cells and occasional broad-based budding yeast like-cells are

seen in B. parva and B. silverae (8). The distinct yeast morphology might be helpful in the

species differentiation from the primary specimens; but this approach would require

significant mycology expertise, which is not always available in diagnostic laboratories.

Although culture and histopathology are useful techniques in the diagnosis of

blastomycosis, the importance of molecular methods cannot be emphasized enough for

the accurate identification of newer Blastomyces species. There are currently no

molecular or serologic diagnostic tests for rapid and accurate identification of different

species within Blastomyces genus. An available commercial test (Gen-Probe, Inc., San

Diego, CA) is rather limited as it can be used only with pure cultures of B. dermatitidis.

Previously, we developed a rapid, and specific real-time PCR assay for the

identification and detection of four known haplotypes of B. dermatitidis because at the

time methods for distinct species level determination of Blastomyces genus were not

available (9).

Outdoor exposure and proximity to water have been associated with blastomycosis

(10). Blastomycosis is reportable in only five states in the US, namely Arkansas,

Louisiana, Michigan, Minnesota, and Wisconsin (6) . In New York State, Blastomycosis

is considered an emerging disease [http://www.nycasm.org/Alerts/Notification_101674.pdf].

Despite several blastomycosis cases reported from NY in the last decade, the disease

endemicity is difficult to discern as it is not reportable in NY (11-14). Likewise, in the

absence of a skin test, it is difficult to assess the geographic distribution of the exposure.

Limited canine blastomycosis data suggest that it is endemic in the Saint Lawrence

4 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

River Valleys on the New York-Canada border (11). While pneumonia is the most

common manifestation of blastomycosis, approximately half of all cases are

asymptomatic (15). Infection however can lead to severe and fatal disease, often as a

result of respiratory failure (16). Disseminated infection can involve any organ, often

including cutaneous abscesses and osteomyelitis, and is frequently accompanied by

fever, weight loss, and night sweats (17).

The BAD-1 is an important conserved adhesion-promoting protein and virulence

factor of B. dermatitidis (18). Two large insertions were noted in the B. gilchristii BAD1

(19), while this gene was absent in African strains of Blastomyces spp. (20). Several

conventional PCR assays were designed using BAD1 gene (21) for the detection of B.

dermatitidis DNA from clinical and soil samples (22, 23). Sidamonidze et al. (2012)

developed a real-time PCR assay targeting BAD1 gene to identify B. dermatitidis in

culture and primary specimens. In the present investigation, we exploited the sequence

variations observed within BAD1 gene to develop a duplex real-time PCR assay for the

differentiation of B. dermatitidis and B. gilchristii. We present data on the sensitivity,

specificity, and reproducibility of the assay and retrospective analysis of blastomycosis

cases from 2005 to 2019 in New York. Our results highlight the preponderance of B.

dermatitidis and a minor population of B. gilchristii in New York cases.

MATERIALS AND METODS

Strains, primary specimens, and DNA: Seventy-nine isolates of Blastomyces spp.

were evaluated in this study. These included 48 clinical isolates from the

sporadic cases of blastomycosis obtained between 2005 to 2019 from New York;

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canine (6), feline (1), sea lion (2), bat (1), polar bear (1), soil (4), human (2)

obtained from Dr. Gene M. Scalrone, Dept. of Biological Sciences, Idaho State

University; American Type Culture Collection (ATCC) strains MYA2586 (soil),

MYA2585 (dog), 56214 (human, Africa), and 56216 (human, Africa) and five each

well characterized strains of B. dermatitidis and B. gilchristii from Public Health

Ontario Laboratory, Toronto, Canada were part of this investigation. A total of 33

primary clinical specimens from New York patients including 13 paraffin tissues,

6 bronchial washes, 5 lung tissues, 3 cerebrospinal fluids, 2 skin lesion tissues, 2

bone marrows, one each blood, brain lesion tissue, and sputum submitted for B.

dermatitidis identification, was also part of this investigation. Additionally, DNA

from closely and distantly related fungal pathogens were included in this

investigation (9).

DNA extraction. DNA from Blastomyces spp. was extracted using Qiagen DNA

mini kit on the Qiacube automated extractor. In brief, fungal growth

(approximately 5x5 mm in size) was removed from the agar surface using a

sterile loop and added into a lysing reagent in the BSL3 laboratory. The fungal

suspension was incubated at 90o C for one hour and then brought to the BSL2

laboratory for further processing. The heat-killed fungal suspension was

homogenized in the Precellys homogenizer at 6,500 RPM for 15 second each at

three times (Program #5; 6500-3x60-015). The homogenized suspension was

transferred to a 2 ml screw cap tube leaving behind the beads. DNA from

samples were extracted using the Qiagen DNA mini kit in the QiaCube semi-

automated DNA extractor resulting in 50 µL of eluted DNA. All extracted DNAs

6 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

were stored at -80o C. DNA of fungal species (yeasts and molds) other than

Blastomyces spp. were procured from Wadsworth Center Mycology Laboragtory

(WCML) DNA Collection Repository.

DNA from paraffin fixed tissue was first sectioned by the Wadsworth

Center Histopathology Core, then using 1 mL of xylene, the paraffin was

dissolved, followed by two washes using 1 mL of 100% ethanol. The sample was

then dried and extracted using the Qiagen DNA mini kit as described above with

an incubation temperature of 70°C instead of 90°C. For non-fixed tissues,

approximately 5x5 mm of tissue was used and extraction was also done using the

Qiagen DNA mini kit as described for the isolates with a incubation temperature

of 70°C instead of 90°C.

DNA from soil samples were manually extracted using the DNeasy

PowerSoil Kit as described previously (24). In brief, about 0.25 g of soil sample

was added to the Powerbead tube with 60 µL C1 solution followed by

homogenizing in a vortex genie for 20 minutes. Sample was then processed

through multiple spin column washes and DNA was eluted in 100 µL of elution

buffer.

Primers and probes. Primers and probes for the real-time PCR assay were designed

from the promoter region of the BAD1 gene using Geneious R9 9.1.6 software. The

choice of BAD1 promoter was based upon our earlier successful utilization for this

region for B. dermatitidis identification by real-time PCR assay (9). Multiple alignment of

7 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

BAD1 promoter sequences revealed unique sequences for B. dermatitidis and B.

gilchristii, which were used for primers and probes design (Supplementary Figure 1).

Oligonucleotide sequences of the primers and probes for B. gilchristii, and B. dermatitidis

are as follows: V2556 (Bg; forward) 5'-ATGGGTGCAAAATCCGCCTA-3', V2558 (Bg;

reverse) 5'AATCTAGAAGCTGGAGCGCC -3', V2557 (Bg; probe) 5'-/6-FAM-

CCGTACTCCCTCCCCGGTACTCC-ZEN/3' IB FQ. V2559 (Bd; forward) 5'-

GCAAAATCCGCCTACTACTA-3', V2561 (Bd; reverse) 5'

AGCTGAACCTGGAAGTATTG -3', V2560 (Bd; probe) 5'-/6-Cy5-

TCCCTACCCCTGGCTACTTTTCT-TAO/3' IB FQ.

Real-time PCR Assay. Each sample DNA (isolate or primary specimen) was tested in

duplicate in 20 l reaction volumes using an optical 96-well plate. Each reaction mixture

contained 1× PerfeCTa Multiplex qPCR ToughMix (Quanta Biosciences), a 1,000 nM

concentration of each B. gilchristii (V2556 & V2558) and B. dermatitidis (V2559 & V2561)

primers, a 250 nM concentration of each B. gilchristii (V2557) and B. dermatitidis (V2560)

probe, and 2 l of DNA extracted from Blastomyces spp. or 5 l of DNA extracted from

primary clinical specimens. Each PCR run also included 2 l of positive extraction

control (M808; B. dermatitidis), 2 l of positive amplification controls (M808; B.

dermatitidis and MB97; B. gilchristii) and 2 l of negative extraction (extraction reagents

only) and negative amplification (sterilized nuclease-free water) controls. To prevent

any cross-contamination, a unidirectional workflow was followed by keeping reagent

preparation, specimen preparation, and amplification/detection areas separate. Cycling

conditions on the ABI 7500 FAST were initial denaturation at 95°C for 20 s, followed by

45 cycles of 95°C for 3 s and 60°C for 30 s. Based on limit of detection (LOD), a cycle

threshold (Ct) value of ≤38 was reported as positive and >38 was reported as negative.

8 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Specimens were reported as inconclusive if PCR inhibition was observed for the

primary specimens.

RESULTS

Assay sensitivity, specificity, and reproducibility-The duplex real-time PCR assay was

highly sensitive with linearity over five logs, a correlation coefficient of >0.99, and

amplification efficiency of >92%. The limit of detection (LOD) of the assay was one pg

gDNA per PCR reaction, which was within the linear range of the standard curve for B.

dermatitidis and B. gilchristii (Fig. 1 and Table 1). These results indicated that the duplex

real-time PCR assay targeting the BAD1 gene is highly sensitive for the culture

identification of B. dermatitidis and B. gilchristii. The assay was highly specific as it did

not cross-react with other closely and distantly related fungal pathogens

(Supplementary Table 2). Ten blinded genomic DNA received from Ontario, Canada,

including five each of B. dermatitidis and B. gilchristii, identified both species accurately,

further confirming the assay validity (Table 2). Additionally, DNA from B. dermatitidis

and B. gilchristii strains run on three different days and within the same day in

triplicates yielded consistent Ct values with cofficient of variance <5.0, confirming assay

reproducibility (Supplementary Table 3 A and 3 B).

Retrospective analysis of Blastomyces isolates and primary specimens. The

retrospective analysis of 79 isolates of Blastomyces spp. revealed 62 to be B. dermatitidis,

and 15 to be B. gilchristii (Table 3 A). One isolate (ATCC 56214), which was identified by

an older single-plex real-time PCR assay as Blastomyces spp., was neither B. dermatitidis

9 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

nor B. gilchristii by the new duplex real-time PCR assay. It was later confirmed to be B.

percursus by sequencing of the ribosomal genes and BLAST search. One strain of B.

emazntsi (ATCC 21516), identified by sequencing, was neither picked up by the old

single-plex real-time PCR nor by the new duplex real-time PCR assay further

confirming the specificity of the duplex real-time PCR assay. Of 15 B. gilchristii isolates

identified, 5 were well- characterized strains of B. gilchristii from Canada, 4 were from

the Eagle River Wisconsin outbreak involving human, dog, and soil, and 6 were from

five patients from NY. All six NY isolates of B. gilchristii confirmed by duplex real-time

PCR assay, were also confirmed by sequencing of the BAD1 and ribosomal genes

(GenBank Accession No for BAD1: MT822768 – MT822773; GenBank Accession Number

for ITS: MT822762 – MT822767 for ITS). Of 62 B. dermatitidis isolates identified, 5 were

well- characterized strains of B. dermatitidis from Canada, 15 were from Illinois,

Kentucky, Minnesota, Tennessee, and Wisconsin involving animals or soil, and 42

isolates were from 38 patients from NY collected from 2005 to 2019. Of 33 primary

specimens analyzed from 2013 to 2019, five specimens (one skin lesion, one bronchial

wash, two tissue blocks of nasal mass, and one tissue block of unknown origin) from

five patients were positive for B. dermatitidis DNA while none were positive for B.

gilchristii DNA (Table 3 B).

Blastomycosis in New York. Analysis of 43 cases of blastomycosis from NY

(Supplementary Table 1) revealed one to four cases per year from 2005 until 2016, but

that number increased to 7 in 2017, and cases remained high with 9 identified in 2018

and 8 identified in 2019 (Fig. 2). Men were more frequently infected than women with

B. dermatitidis, while number of cases was too small to derive this conclusion for B.

gilchristii. Older people (over age 40) were found to be more prone to symptomatic

10 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

infection irrespective of B. dermatitidis or B. gilchristii. B. dermatitidis was most

commonly isolated from respiratory specimens, followed by

skin/wounds/subcutaneous tissue, and bone, while all six isolates of B. gilchristii were

isolated from respiratory specimens (Table 4). Geographic distribution of blastomycosis

revealed that the majority of cases presented with symptoms lived in Mohawk Valley,

Capital District and Finger Lakes. Few cases were also reported from other regions in

NY (Supplementary Figure 2).

Analysis of soil samples. Two field studies were conducted to find the environmental

source of B. dermatitidis and B. gilchristii. The first extensive collection focused on a

variety of sites in and around the city of Amsterdam (Mohawk Valley), NY where a

number of blastomycosis case patients lived or visited. The second more limited

sampling took place in the Lake George (Capital District), NY area where one patient of

blastomycosis resided. None of the sample yielded any positive DNA for Blastomyces

spp. by our earlier developed single-plex real-time PCR assay (9) or newly developed

duplex real-time PCR assay. None of the soil samples yielded any positive growth of

Blastomyces spp. in culture (details not shown).

DISCUSSION

In this study, we have developed a duplex real-time PCR assay to differentiate B.

dermatitidis from B. gilchristii as these two closely related species possibly overlap in

their endemicity in North America. The duplex real-time PCR assay targeting the

putative promoter region of the BAD1 gene was highly sensitive, specific, and

reproducible. BAD1 gene and its promoter have been extensively used for the

11 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

differentiation of Blastomyces species using restriction fragment length polymorphism

(RFLP), PCR and real-time PCR assays (9, 19, 23). The polymorphism in BAD1 gene and

markedly different sizes including 363-bp in B. dermatitidis, 663-bp in B. gilchrstii and

absence of this gene in African isolates of Blastomyces (8, 9, 19, 20), proved this target as

highly specific for the identification of B. dermatitidis, and B. gilchristii in the present

investigation.

The retrospective analysis of Blastomyces cultures and primary specimens confirmed 43

cases of blastomycosis from NY from 2005 to 2019. These results further confirmed the

endemicity of blastomycosis in New York (14). Of interest was the identification of five

of 43 blastomycosis cases due to B. gilchristii. Interestingly, patients infected with B.

gilchristii resided in different parts of NY, indicating broader geographic distribution of

this species in NY. Additional studies are needed to determine if these cases are due to

patients traveling between regions or if there is a focal niche of B. gilchristii in

NY. Preponderance of blastomycosis cases due to B. dermatitidis with concentration in

Mohawk Valley and Capital District followed by Finger Lakes, and sporadic cases from

other regions of NY was noted. These results indicate that there is a broader

distribution of B. dermatitidis compared to B. gilchristii in NY. These results are in

agreement with earlier studies where B. dermatitidis has shown to have broader

geographic range of blastomycosis cases from Canada to the southeastern United States

while B. gilchristii appears limited to regions of Ontario, Saskatchewan, Alberta,

Minnesota, and Wisconsin (25). The limited soil samples investigated in the present

study from Mohawk Valley and Capital District failed to yield any positive DNA or

culture of B. dermatitidis or B. gilchristii. These results are not surprising as the precise

ecological niche(s) of B. dermatitidis or B. gilchristii is far from clear except in a few

12 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

instance such as the Eagle River outbreak (26). An understanding of the ecological

factors that favors growth, reproduction, and dispersal would potentially provide a

means of predicting and controlling human acquisition of infection. This is particularly

critical as in recent years, cases of blastomycosis in NY have increased and majority of

patients reported no travel to regions of known endemicity (14). Given the seriousness

of blastomycosis and consistently elevated incidence in NY, there is a proposal to make

blastomycosis a reportable disease in NY.

Difference in virulence have long been observed among Blastomyces spp.(20, 27). It has

been demonstrated previously that African strains of B. dermatitidis differ from North

American strains in their growth and morphology and are thought to cause less severe

disease (20). BAD1 has been implicated as the major virulence factor of B. dermatitidis

(28). The significant size difference in BAD1 promoter identified in B. gilchristii and

sequence analysis revealed that it was due to two large insertion (19). Whether these

insertions have any influence on BAD1 gene expression and virulence, need further

investigation. Additionally, to the best of our knowledge, no studies are available to

demonstrate the comparative virulence of B. dermatitidis and B. gilchristii. A number of

cases of blastomycosis due to B. gilchristii including the fatal case of acute respiratory

distress syndrome indicate that B. gilchristii pathogenic potential is comparable to that

of B. dermatitidis (29). A highly sensitive and specific assay would allow more

comprehensive surveillance to monitor the disease incidence. Mandatory disease

reporting and surveillance will also aid in the diagnosis of unknown cases, enable

prompt initiation of treatment to decrease illness and death, and provide support for

targeted public health interventions, such as public awareness campaigns (e.g., health

13 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

advisories for blastomycosis in New York). In summary, the newly developed duplex

real-time PCR assay would help understand the ecology and epidemiology of

blastomycosis caused by B. dermatitidis and B. gilchristii in New York and North

America.

ACKNOWLEDGMENTS

We thank Drs. Robert McDonald and Elizabeth Dufort, Division of Epidemiology,

NYSDOH, and Elis Tobin, Albany Medical Center, Albany for providing soil samples

and clinical specimens. We thank Wadsworth Center, Tissue Culture & Media,

Histopathology, and Applied Genomic Technologies Cores for providing media, fixed

tissue sectioning, and Sanger sequencing, respectively. This work was supported partly

by the funds from the Wadsworth Center, New York State Department of Health

(NYSDOH), and Centers for the Disease Control and Prevention (CDC) grant number

NU50CK000516. In addition, Mitchell Kaplan was partially supported with National

Science Foundation funding in the Wadsworth Center’s Research Experience for

Undergraduates (REU) program (grant DBI1757170). Its contents are solely the

responsibility of the authors and do not necessarily represent the official views of the

Department of Health and Human Services or the CDC.

AUTHOR CONTRIBUTIONS

SC conceived the study, supervised experiments and wrote the manuscript. MK

designed primers and probes under YZ’s supervision and performed majority of the

experiments, prepared graphs and tables. YZ supervised MK’s work, performed few

key experiments, and prepared graphs and tables.VC edited the draft manuscript, JVK,

14 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

and LM provided strains of Blastomyces dermatitidis and B. gilchristii and edited the draft

manuscript.

FIGURE LIGENDS

Figure 1. Sensitivity of duplex real-time PCR assay. Serial 10-fold dilution series of

gDNA of B. dermatitidis and B. gilchristii were prepared and PCR was run in duplicate.

The slope (s) and correlation coefficient (R) are shown. The limit of detection of the

assay was 1 gDNA/PCR reaction for both Bd and Bg.

Figure. 2. Blastomycosis cases in New York from 2005 to 2019. Blastomycosis cases

earlier confirmed by single plex real-time PCR assay for isolates and primary specimens

were analyzed retrospectively by the newly developed duplex real-time PCR assay. Of

43 cases, 38 were confirmed as B. dermatitidis and 5 as B. gilchristii. There was marked

increase in blastomycosis cases from 2017 onwards.

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Table 1. Sensitivity study of B. dermatitidis and B. gilchristii

DNA B. dermatitidis B. gilchristii Con. Ct 1 Ct 2 Mean Ct Ct 1 Ct 2 Mean Ct (ng/µL) 10 20.73 20.70 20.72 19.85 19.76 19.80 1 23.88 23.87 23.88 23.10 23.08 23.09 0.1 27.25 27.51 27.38 26.54 26.44 26.49 0.01 32.96 32.60 32.78 30.59 30.15 30.37 0.001 35.96 35.27 35.62 33.68 33.64 33.66 0.0001 36.91 37.48 37.20 36.22 37.74 36.98 0.00001 Undet Undet Undet Undet Undet Undet

Table 2. Blinded panel of DNA of B. dermatitidis and B. gilchristii*

Sample Single plex Duplex real-timePCR Assay Final ID No. real-time B. dermatitidis B. gilchristii PCR Assay Mean Ct (Cy5) Mean Ct (FAM) Mean Ct 1 23.02 23.48 Undet B. dermatitidis 2 22.4 Undet 21.74 B. gilchristii 3 18.52 20.04 Undet B. dermatitidis 4 22.31 22.64 Undet B. dermatitidis 5 19.86 Undet 19.35 B. gilchristii 6 22.12 Undet 21.54 B. gilchristii 7 23.28 Undet 22.46 B. gilchristii 8 19.75 20.15 Undet B. dermatitidis 9 22.23 22.39 Undet B. dermatitidis 10 21.45 Undet 20.39 B. gilchristii * DNA was provided by Dr. Julianne V. Kus, University of Toronto, Canada, in a blinded fashion

16 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Table 3 A. Validity of duplex real-time PCR assay for culture identification of Blastomyces spp.

Single plex real-time Duplex real-time PCR PCR (Blastomyces spp.) B. dermatitidis B. gilchristii

Positive Negative Positive Negative

Positive (n=78) 62 16 15 63

Negative (n=60) 0 60 0 60

Table 3 B. Validity of duplex real-time PCR assay for the direct detection of Blastomyces spp. DNA from primary specimens

Single plex real-time PCR (Blastomyces spp.) Duplex real-time PCR

B. dermatitidis B. gilchristii

Positive Negative Positive Negative

Positive (n=5) 5 0 0 5

Negative (n=28) 0 28 0 28

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Table 4. Characteristics of Mycology Laboratory confirmed blastomycosis cases in New York from 2005 to 2019

Characteristic Blastomycosis B. dermatitidis B. gilchristii

Patient Sex n = 43 n = 38 n = 5 M 29 26 3 F 14 12 2

Patient Age (Y) n = 43 20 - 29 4 4 0 30-39 4 4 0 40-49 8 7 1 50-59 11 10 1 60-69 13 10 3 >69 3 3 0

Source of Specimen Isolation n = 48 Bronchial wash 28 22 6 Skin, wound, subcutaneous tissue 15 15 Bone 4 4 Brain 1 1

Specimen positive for DNA 5 Bronchial wash 1 1 Tissue block - Nasal mass 2 2 Tissue block-source not provided 1 1 Skin wound fluid 1 1

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Figure 1.

45 40 R² = 0.98 35 s= -3.5152 30 E= 92.5% 25 20 B. dermatitidis 15 R² = 0.9994 B. gilchristii s = -3.4705 Mean Ct Values Ct Mean 10 E= 94.2% 5 0 -1 0 1 2 3 4 5

Log10 gDNA (ng)/µL

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Figure 2.

9 8 B. dermatitidis 7 B. gilchristii 6 5 4 3 No. Cases No. 2 1 0 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019

Year of Confirmation

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Supplementary Figure 1. Multiple alignment of BAD1 gene of B. dermatitidis and B.

gilchristii. The sequences of BAD1 gene from B. dermatitidis (Bd) and B. gilchristii (Bg)

were aligned using MUSCLE alignment program in Geneious. The position of primers

and probes sequences used in this study are shown.

21 medRxiv preprint doi: https://doi.org/10.1101/2020.08.11.20172932; this version posted August 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

Spplementary Fig. 2. Geographic distribution of blastomycosis cases in New York from

2005 to 2019. Patient residence (zip code) was utilized to map blastomycosis cases due

to B. dermatitidis (Bd) and B. gilchristii (Bg). Most of the cases were clustered in the

Mohawk Valley and Capital District (A) followed by Finger Lake (B) with solitary cases

reported from other regions of NY.

A

C

B

Supplementary Figure 2. Geographic distribution of blastomycosis cases in New York

from 2005 to 2019. (A) Patient’s residence (Zip Code) was utilized to map cases of

blastomycosis due to B. dermatitidis (Bd) and B. gilchristii (Bg) in New York. (B)

Clustering of cases in Mohawk Valley and Capital District, and (C) Finger Lakes, are

shown.

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Supplementary Table 1. Details of duplex real-time PCR assay of Blastomycosis cases from New York (2005-2019)

Case Primary Source Single plex Duplex real-time PCR Final ID No. Sample Blastomyces real-time Bd Mean Bg Mean Ct Collection recovered in PCR Ct (Cy5) (FAM) Date culture (Blastomyces spp.) Ct (FAM) Case 1 7/14/2005 Bone 22 22.03 Undet B. dermatitidis Case 2 2/23/2006 BAL 20.7 20.71 Undet B. dermatitidis Case 3 8/10/2006 Tissue 20.7 22.57 Undet B. dermatitidis Case 4 1/17/2008 Tissue 22.5 22.57 Undet B. dermatitidis Case 4 1/17/2008 Tissue 21.5 20.51 Undet B. dermatitidis Case 4 1/17/2008 Tissue 21.2 20.46 Undet B. dermatitidis Case 5 4/23/2008 BAL 20.19 Undet 20.67 B. gilchristii Case 6 8/17/2009 Tissue 22.01 21.31 Undet B. dermatitidis Case 7 11/18/2009 Tissue Culture** 24.17 Undet B. dermatitidis Case 8 2/10/2010 LUNG 22.29 Undet 22.75 B. gilchristii Case 9 3/1/2012 BAL 23.33 22.43 Undet B. dermatitidis Case 10 9/26/2012 Lung 20.97 19.47 Undet B. dermatitidis Case 11 6/6/2013 Aspirate 20.94 18.57 Undet B. dermatitidis Case 12 10/25/2013 Bone 18.8 23.58 Undet B. dermatitidis Case 13 11/19/2013 BAL 20.54 22.12 Undet B. dermatitidis Case 14 2/11/2014 Skin lesion fluid* 28.25 20.23 Undet B. dermatitidis Case 15 2/17/2014 Sputum 20.52 22.47 Undet B. dermatitidis Case 15 2/18/2014 BAL 24.34 21.46 Undet B. dermatitidis Case 16 5/29/2014 BAL 27.63 20.73 Undet B. dermatitidis Case 16 6/12/2014 BAL 21.73 18.99 Undet B. dermatitidis Case 17 8/8/2014 Brain 28.99 27.95 Undet B. dermatitidis Case 18 9/29/2015 Cyst 26.94 24.04 Undet B. dermatitidis Case 19 10/20/2016 Hand 27.7 20.68 Undet B. dermatitidis Case 20 1/13/2017 Lung 24.36 20.14 Undet B. dermatitidis Case 21 1/16/2017 BAL 26.33 Undet 19.26 B. gilchristii Case 22 3/1/2017 Lung 26.79 23.74 Undet B. dermatitidis Case 23 5/2/2017 Sputum 21.73 21.07 Undet B. dermatitidis Case 24 5/17/2017 BAL 22.01 20.37 Undet B. dermatitidis Case 25 6/28/2017 Tissue 23.22 23.06 Undet B. dermatitidis Case 26 10/26/2017 BAL 21.19 23.49 Undet B. dermatitidis Case 26 10/26/2017 BAL 21.34 24.37 Undet B. dermatitidis Case 26 10/27/2017 Sputum 32.47 36.56 Undet B. dermatitidis Case 26 10/27/2017 Sputum 29.71 32.02 Undet B. dermatitidis Case 26 10/27/2017 Sputum 26.8 29.58 Undet B. dermatitidis Case 27 10/3/2018 Bone - Left Tibia 19.11 21.54 Undet B. dermatitidis Case 28 2/13/2018 Bone (Tibia) 20.05 22.71 Undet B. dermatitidis Case 29 3/6/2018 Tissue block - 37.4 39.63 Undet B. dermatitidis Left nasal Mass* Case 29 3/6/2018 Tissue block - 33.29 37.24 Undet B. dermatitidis Left nasal Mass* Case 30 2/28/2018 Tissue - Left 20.47 23.58 Undet B. dermatitidis Thigh

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Case 31 8/5/2018 BAL 22.63 27.74 Undet B. dermatitidis Case 32 7/2/2018 BAL 22.89 27.36 Undet B. dermatitidis Case 33 9/27/2018 Tissue block* 36.02 40.29 Undet B. dermatitidis Case 34 10/2/2018 Bone (Femur) 19.09 22.77 Undet B. dermatitidis Case 35 12/17/2018 BAL 19.72 Undet 22.49 B. gilchristii Case 36 1/20/2019 Tissue 20.87 24.81 Undet B. dermatitidis Case 37 4/25/2019 BAL 21.01 24.54 Undet B. dermatitidis Case 38 5/5/2019 BAL* 23.54 27.13 Undet B. dermatitidis Case 39 5/9/2019 BAL 23.8 24.52 Undet B. dermatitidis Case 40 5/23/2019 Wound fluid 20.16 23.26 Undet B. dermatitidis Case 41 7/2/2019 BAL 18.74 23.06 Undet B. dermatitidis Case 42 7/19/2019 BAL 19.45 24.28 Undet B. dermatitidis Case 43 10/22/2019 BAL 18.88 Undet 17.73 B. gilchristii Case 43 10/22/2019 BAL 18.74 Undet 17.73 B. gilchristii

*Primary samples; **culture was confirmed by converting to the yeast form

Supplementary Table 2. Specificity study of duplex real-time PCR assay

Fungal ID Source Isolate DNA Duplex rt-PCR (ng/µL) Result MOLDS Alternaria alternata Nasal cavity 108.3 Negative Aspergillus flavus Sputum 60.3 Negative A. fumigatus Sputum 18.5 Negative A. nidulans BAL 54.2 Negative A. niger Nasal cavity 147.1 Negative A. A. versicolor Lung 123.4 Negative Blastobotrys adeninivorans CSF 255.8 Negative Blastomyces percursus (ATCC 56214) Human (Mozambique) 101.5 Negative B. emzantsi (ATCC 56216) Human (South Africa) 159.2 Negative Cladosporium halotolerans Nail 92.8 Negative C. ramotenellum Sputum 2.2 Negative Chaetomium globosum Nail 90.1 Negative queenslandicum BAL 21.2 Negative C. queenslandicum No source 50.1 Negative BAL 6.9 Negative C. posadasii BAL 18.2 Negative C. posadasii Sputum 120 Negative Epidermophyton floccosum Nail 34 Negative Exophiala dermatitidis BAL 110.8 Negative E. lecanii-corni Nail 102.4 Negative Fusarium oxysporum Nail 152.4 Negative Geomyces pannorum BAL 280.4 Negative Glomerella lagenaria BAL 3.6 Negative Blood 110.7 Negative H. capsulatum Lung 17.7 Negative

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Hyphopichia burtonii Mouth 302.5 Negative Lichtheimia corymbifera BAL 10.5 Negative Malbranchea circinata Nail 19.8 Negative Microsporum gypseum Nail 318.7 Negative M. canis Scalp 117.4 Negative Phoma herbarum Nail 91.2 Negative Phlebia fuscoatra Sputum 28.9 Negative oryzae Urine 116.4 Negative Scedosporium boydii Nail 53.3 Negative Sporothrix schenckii Finger 72.8 Negative Trametes versicolor BAL 43.2 Negative T. versicolor Eye 17.8 Negative Trichosporon asahii Urine 132.4 Negative Trichophyton mentagrophytes Nail 62.6 Negative T. rubrum Nail 9.3 Negative T. violaceum Scalp 33.9 Negative YEASTS Candida albicans BAL 252.4 Negative C. auris Blood 180.8 Negative C. auris Blood 108.6 Negative C. auris Blood 173.8 Negative C. duobushaemulonii Nail 112.6 Negative C. glabrata Blood 127 Negative C. haemulonii Tissue 55.7 Negative C. haemulonii Bone 149 Negative C. krusei Blood 3 Negative C. lusitaniae Blood 95.8 Negative C. orthopsilosis Abdominal fluid 52.6 Negative C. parapsilosis Blood 78.5 Negative C. tropicalis Sputum 101.5 Negative Cryptococcus albidus Nail 7.9 Negative C. diffluens No source 6.2 Negative C. gattii (VG1) CSF 54.7 Negative C. liquefaciens Right Posterior Ankle 82.7 Negative Debaryomyces hansenii Foot 48 Negative Pichia norvegensis No source 194.8 Negative Vaginal swab 100.8 Negative Pos Ctrl Bg (MB97) N/A 10.0 Positive Pos Ctrl Bd (M808) N/A 15.5 Positive

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Supplementary Table 3 A. Inter-assay reproducibility of duplex real-time PCR assay

Blastomyces B. dermatitidis (Cy5) B. gilchristii (FAM) spp. Strain Mean Mean No. Ct 1 Ct 2 Ct 3 Ct Ct 1 Ct 2 Ct 3 Ct ±SD %CV B. gilchristii MB95 Undet Undet Undet N/A 22.9 22.47 21.35 22.24 0.8 3.59 MB96 Undet Undet Undet N/A 24.21 23.68 23.2 23.7 0.51 2.15 MB97 Undet Undet Undet N/A 23.78 23.24 22.37 23.13 0.71 3.07 B. dermatitidis MB98 24.88 25.63 25.09 25.2 Undet Undet Undet N/A 0.39 1.55 MB99 24.18 24.97 24.24 24.46 Undet Undet Undet N/A 0.44 1.8 MB100 22.68 23.42 22.49 22.86 Undet Undet Undet N/A 0.49 2.14

Supplementary Table 3 B. Intra-assay reproducibility of duplex real-time PCR assay

B. dermatitidis (Cy5) B. gilchristii (FAM) Blastomyces Strain Mean Mean spp No. Ct 1 Ct 2 Ct 3 Ct Ct 1 Ct 2 Ct 3 Ct ±SD %CV B. gilchristii MB95 Undet Undet Undet N/A 22.99 22.85 22.88 22.91 0.0737 0.322 MB96 Undet Undet Undet N/A 24.24 24.15 24.23 24.21 0.0493 0.204 MB97 Undet Undet Undet N/A 23.77 23.79 23.78 23.78 0.01 0.0421 B. dermatitidis MB98 24.91 24.87 24.87 24.88 Undet Undet Undet N/A 0.0231 0.0928 MB99 24.03 24.15 24.36 24.18 Undet Undet Undet N/A 0.167 0.691 MB100 22.81 22.69 22.55 22.68 Undet Undet Undet N/A 0.13 0.573

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