First Canadian report of transmission of fluconazole-resistant Candida parapsilosis within two hospital networks confirmed by genomic analysis.

Candida parapsilosis is a common cause of non-albicans candidemia. It can be transmitted in healthcare settings resulting in serious healthcare-associated infections and can develop drug resistance to commonly used antifungal agents. Following a significant increase in the percentage of fluconazole (FLU)-nonsusceptible isolates from sterile site specimens of patients in two Ontario acute care hospital networks, we used whole genome sequence (WGS) analysis to retrospectively investigate the genetic relatedness of isolates and to assess potential in-hospital spread. Phylogenomic analysis was conducted on all 19 FLU-resistant and seven susceptible-dose dependent (SDD) isolates from the two hospital networks, as well as 13 FLU susceptible C. parapsilosis isolates from the same facilities and 20 isolates from patients not related to the investigation. Twenty-five of 26 FLU-nonsusceptible isolates (resistant or SDD) and two susceptible isolates from the two hospital networks formed a phylogenomic cluster that was highly similar genetically and distinct from other isolates. The results suggest the presence of a persistent strain of FLU-nonsusceptible C. parapsilosis causing infections over a 5.5-year period. Results from WGS were largely comparable to microsatellite typing. Twenty-seven of 28 cluster isolates had a K143R substitution in lanosterol 14-α-demethylase (ERG11) associated with azole resistance. As the first report of a healthcare-associated outbreak of FLU-nonsusceptible C. parapsilosis in Canada, this study underscores the importance of monitoring local antimicrobial resistance trends and demonstrates the value of WGS analysis to detect and characterize clusters and outbreaks. Timely access to genomic epidemiological information can inform targeted infection control measures.

Increased Incidence of Invasive Haemophilus influenzae Disease Driven by Non-Type B Isolates in Ontario, Canada, 2014 to 2018.

Haemophilus influenzae can cause serious invasive disease. We report the epidemiology and antimicrobial susceptibility of invasive H. influenzae in Ontario, Canada, from 2014 to 2018 from laboratory-based data. Blood was the most common specimen source (89.5%). Consistent with widespread vaccination against serotype b (Hib), the incidence of Hib in Ontario remained low (0.04 cases per 100,000 population). H. influenzae disease primarily afflicted those <1 and ≥65 years of age. From 2014 to 2018, cases of invasive H. influenzae increased 5.6%, from 1.67 to 2.06 cases per 100,000 population, the majority of which were attributed to a 7.6% increase in the incidence of H. influenzae in those ≥65 years old. H. influenzae disease was primarily caused by nontypeable H. influenzae (NTHi) (74.2%) and, to a much lesser extent, serotype a (Hia) (8.9%) and serotype f (Hif) (10.2%). Serotype-dependent trends in antimicrobial susceptibility were observed. Hia and Hif isolates were predominantly susceptible to all antibiotics tested, while 27.2% of NTHi isolates were nonsusceptible to ampicillin. Resistance to ceftriaxone and meropenem, first-line antibiotics for invasive disease treatment, was nonexistent. The incidence of invasive H. influenzae in Ontario is increasing. The incidence and antimicrobial susceptibility of all serotypes and nontypeable H. influenzae should be monitored. IMPORTANCE H. influenzae can cause serious invasive, life-threatening disease and is considered 1 of 12 priority pathogens by the World Health Organization. Widespread vaccination against H. influenzae serotype b (Hib) has resulted in very low incidence of Hib in Ontario and other regions that have vaccination programs. However, the epidemiology of non-Hib serotypes and nontypeable H. influenzae (NTHi) remains poorly understood. Here, we describe the epidemiology of all invasive H. influenzae isolates (N = 1,338) received by our laboratory over the 5-year period and report on the antimicrobial susceptibility patterns by serotype. Overall, we observed an increase in the incidence of invasive disease over the study period, primarily driven by NTHi. Serotype-dependent trends in antimicrobial susceptibility were also observed. This work contributes to the global understanding of H. influenzae epidemiology and antimicrobial resistance and is additionally important for further vaccine planning initiatives.

Antifungal Susceptibility of Clinical Yeast Isolates from a Large Canadian Reference Laboratory and Application of Whole-Genome Sequence Analysis To Elucidate Mechanisms of Acquired Resistance.

To understand the epidemiology and susceptibility patterns of yeast infections in Ontario, Canada, we examined 4,715 clinical yeast isolates submitted to our laboratory for antifungal susceptibility testing from 2014 to 2018. Candida albicans was the most frequently submitted species (43.0%), followed by C. glabrata (21.1%), C. parapsilosis (15.0%), and C. tropicalis (6.2%). Twenty-three other Candida spp. (11.6%) and 4 non-Candida species (3.1%) were also identified. Few changes in species distribution were observed from 2014 to 2018, but the total numbers of yeast isolates sent for testing increased, with an annual 7.4% change. According to CLSI clinical breakpoints, resistance rates remained low overall. Moderate fluconazole resistance was noted among C. glabrata (9%), C. parapsilosis (9%), and C. tropicalis (12%) isolates. Only 1% of C. glabrata isolates were resistant to caspofungin, micafungin, and anidulafungin. Whole-genome sequence analysis confirmed 11 cases of acquired resistance to azoles or echinocandins via in-host evolution. There were mutations in the gene for the catalytic subunit of 1,3-beta-glucan synthase-mediated echinocandin resistance in 3 of 3 C. albicans strains, 3 of 4 C. glabrata strains, and 1 strain of C. tropicalis Azole resistance was likely caused by a homozygous ERG3 mutation in 1 C. albicans strain and a previously undescribed chromosomal-duplication event involving ERG11 and TAC1 orthologs in 1 C. tropicalis strain. While antifungal resistance rates remain low among yeast isolates in Ontario, ongoing surveillance is necessary to inform empirical therapy for optimal patient management and to guide antifungal stewardship.

Mycobiome Sequencing and Analysis Applied to Fungal Community Profiling of the Lower Respiratory Tract During Fungal Pathogenesis.

Invasive fungal infections are an increasingly important cause of human morbidity and mortality. We generated a next-generation sequencing (NGS)-based method designed to detect a wide range of fungi and applied it to analysis of the fungal microbiome (mycobiome) of the lung during fungal infection. Internal transcribed spacer 1 (ITS1) amplicon sequencing and a custom analysis pipeline detected 96% of species from three mock communities comprised of potential fungal lung pathogens with good recapitulation of the expected species distributions (Pearson correlation coefficients r = 0.63, p = 0.004; r = 0.71, p < 0.001; r = 0.62, p = 0.002). We used this pipeline to analyze mycobiomes of bronchoalveolar lavage (BAL) specimens classified as culture-negative (n = 50) or culture-positive (n = 39) for Blastomyces dermatitidis/gilchristii, the causative agent of North America blastomycosis. Detected in 91.4% of the culture-positive samples, Blastomyces dominated (>50% relative abundance) the mycobiome in 68.6% of these culture-positive samples but was absent in culture-negative samples. To overcome any bias in relative abundance due to between-sample variation in fungal biomass, an abundance-weighting calculation was used to normalize the data by accounting for sample-specific PCR cycle number and PCR product concentration data utilized during sample preparation. After normalization, there was a statistically significant greater overall abundance of ITS1 amplicon in the Blastomyces-culture-positive samples versus culture-negative samples. Moreover, the normalization revealed a greater biomass of yeast and environmental fungi in several Blastomyces-culture-positive samples than in the culture-negative samples. Successful detection of Coccidioides, Scedosporium, Phaeoacremonium, and Aspergillus in 6 additional culture-positive BALs by ITS1 amplicon sequencing demonstrates the ability of this method to detect a broad range of fungi from clinical specimens, suggesting that it may be a potentially useful adjunct to traditional fungal microbiological testing for the diagnosis of respiratory mycoses.

Application of whole genome sequencing to query a potential outbreak of Elizabethkingia anophelis in Ontario, Canada.

Bioinformatic analysis of whole genome sequence (WGS) data is emerging as a tool to provide powerful insights for clinical microbiology. We used WGS data to investigate the genetic diversity of clinical isolates of the bacterial pathogen Elizabethkingia anophelis to query the existence of a single-strain outbreak in Ontario, Canada. The Public Health Ontario Laboratory (PHOL) provides reference identification of clinical isolates of bacteria for Ontario and prior to 2016 had not identified E. anophelis . In the wake of the Wisconsin outbreak of 2015-2016 for which a source was never elucidated, the identification of E. anophelis from clinical specimens from five Ontario patients gave reason to question the presence of an outbreak. Genomic comparisons based on core genome multi-locus sequence typing conclusively refuted the existence of an outbreak, since the 5 Ontario isolates were genetically dissimilar, representing at least 3 distinct sub-lineages scattered among a set of 39 previously characterized isolates. Further interrogation of the genomic data revealed multiple antimicrobial resistance genes. Retrospective reidentification via rpoB sequence analysis of 22 clinical isolates of Elizabethkingia spp. collected by PHOL from 2010 to 2018 demonstrated that E. anophelis was isolated from clinical specimens as early as 2010. The uptick in E. anophelis in Ontario was not due to an outbreak or increased incidence of the pathogen, but rather enhanced laboratory identification techniques and improved sequence databases. This study demonstrates the usefulness of WGS analysis as a public health tool to quickly rule out the existence of clonally related case clusters of bacterial pathogens indicative of single-strain outbreaks.

Incubation time and culture media impact success of identification of Nocardia spp. by MALDI-ToF mass spectrometry.

Identification of Nocardia can be challenging, even by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS). We examined the impact of incubation duration, culture media and bead-beading on identification success. When isolates were grown for 3 days on SAB (Saboround dextrose agar), then extracted using a bead-beating, ethanol/formic acid (EtOH/FA) procedure, MALDI-ToF MS correctly identified 36.4% to species level. By contrast, 80.9% of isolates were correctly identified to species level when processed with the standard EtOH/FA extraction from Columbia blood agar (CBA) cultures incubated until colonies first appeared. Excluding rare species, the latter approach correctly identified 93.0% of isolates of commonly-encountered Nocardia species when additional database entries were included to expand representation of select species. We demonstrate that while bead-beating has little impact on identification success by MALDI-ToF MS, analysis of early growth is crucial. Additionally, culture media that promotes quick growth of Nocardia (e.g. CBA) is recommended.

Epidemiology and Geographic Distribution of Blastomycosis, Histoplasmosis, and Coccidioidomycosis, Ontario, Canada, 1990-2015.

Endemic mycoses represent a growing public health challenge in North America. We describe the epidemiology of 1,392 microbiology laboratory-confirmed cases of blastomycosis, histoplasmosis, and coccidioidomycosis in Ontario during 1990-2015. Blastomycosis was the most common infection (1,092 cases; incidence of 0.41 cases/100,000 population), followed by histoplasmosis (211 cases) and coccidioidomycosis (89 cases). Incidence of blastomycosis increased from 1995 to 2001 and has remained elevated, especially in the northwest region, incorporating several localized hotspots where disease incidence (10.9 cases/100,000 population) is 12.6 times greater than in any other region of the province. This retrospective study substantially increases the number of known endemic fungal infections reported in Canada, confirms Ontario as an important region of endemicity for blastomycosis and histoplasmosis, and provides an epidemiologic baseline for future disease surveillance. Clinicians should include blastomycosis and histoplasmosis in the differential diagnosis of antibiotic-refractory pneumonia in patients traveling to or residing in Ontario.

Correction: Phylogenetic Analysis Reveals a Cryptic Species Blastomyces gilchristii, sp. nov. within the Human Pathogenic Fungus Blastomyces dermatitidis.

[This corrects the article DOI: 10.1371/journal.pone.0059237.].

Phylogeographic Analysis of Blastomyces dermatitidis and Blastomyces gilchristii Reveals an Association with North American Freshwater Drainage Basins.

Blastomyces dermatitidis and Blastomyces gilchristii are dimorphic fungal pathogens that cause serious pulmonary and systemic infections in humans. Although their natural habitat is in the environment, little is known about their specific ecologic niche(s). Here, we analyzed 25 microsatellite loci from 169 strains collected from various regions throughout their known endemic range in North America, representing the largest and most geographically diverse collection of isolates studied to date. Genetic analysis of multilocus microsatellite data divided the strains into four populations of B. dermatitidis and four populations of B. gilchristii. B. dermatitidis isolates were recovered from areas throughout North America, while the B. gilchristii strains were restricted to Canada and some northern US states. Furthermore, the populations of both species were associated with major freshwater drainage basins. The four B. dermatitidis populations were partitioned among (1) the Nelson River drainage basin, (2) the St. Lawrence River and northeast Atlantic Ocean Seaboard drainage basins, (3) the Mississippi River System drainage basin, and (4) the Gulf of Mexico Seaboard and southeast Atlantic Ocean Seaboard drainage basins. A similar partitioning of the B. gilchristii populations was observed among the more northerly drainage basins only. These associations suggest that the ecologic niche where the sexual reproduction, growth, and dispersal of B. dermatitidis and B. gilchristii occur is intimately linked to freshwater systems. For most populations, sexual reproduction was rare enough to produce significant linkage disequilibrium among loci but frequent enough that mating-type idiomorphic ratios were not skewed from 1:1. Furthermore, the evolutionary divergence of B. dermatitidis and B. gilchristii was estimated at 1.9 MYA during the Pleistocene epoch. We suggest that repeated glaciations during the Pleistocene period and resulting biotic refugia may have provided the impetus for speciation as theorized for other species associated with temperate freshwater systems.

Antimicrobial susceptibility among clinical Nocardia species identified by multilocus sequence analysis.

Antimicrobial susceptibility patterns of 112 clinical isolates, 28 type strains, and 9 reference strains of Nocardia were determined using the Sensititre Rapmyco microdilution panel (Thermo Fisher, Inc.). Isolates were identified by highly discriminatory multilocus sequence analysis and were chosen to represent the diversity of species recovered from clinical specimens in Ontario, Canada. Susceptibility to the most commonly used drug, trimethoprim-sulfamethoxazole, was observed in 97% of isolates. Linezolid and amikacin were also highly effective; 100% and 99% of all isolates demonstrated a susceptible phenotype. For the remaining antimicrobials, resistance was species specific with isolates of Nocardia otitidiscaviarum, N. brasiliensis, N. abscessus complex, N. nova complex, N. transvalensis complex, N. farcinica, and N. cyriacigeorgica displaying the traditional characteristic drug pattern types. In addition, the antimicrobial susceptibility profiles of a variety of rarely encountered species isolated from clinical specimens are reported for the first time and were categorized into four additional drug pattern types. Finally, MICs for the control strains N. nova ATCC BAA-2227, N. asteroides ATCC 19247(T), and N. farcinica ATCC 23826 were robustly determined to demonstrate method reproducibility and suitability of the commercial Sensititre Rapmyco panel for antimicrobial susceptibility testing of Nocardia spp. isolated from clinical specimens. The reported values will facilitate quality control and standardization among laboratories.

Effective method for the heat inactivation of Blastomyces dermatitidis.

Manipulation of Blastomyces dermatitidis requires the use of containment level 3 (CL3) practices. However, access to CL3 laboratories is limited and working conditions are restrictive. We describe the validation of a "heat-killing" method to inactivate B. dermatitidis, thus allowing cellular material to be removed from the CL3 laboratory for subsequent DNA isolation that is suitable for genetic applications.

Phylogenetic analysis reveals a cryptic species Blastomyces gilchristii, sp. nov. within the human pathogenic fungus Blastomyces dermatitidis.

BACKGROUND: Analysis of the population genetic structure of microbial species is of fundamental importance to many scientific disciplines because it can identify cryptic species, reveal reproductive mode, and elucidate processes that contribute to pathogen evolution. Here, we examined the population genetic structure and geographic differentiation of the sexual, dimorphic fungus Blastomyces dermatitidis, the causative agent of blastomycosis. METHODOLOGY/PRINCIPAL FINDINGS: Criteria for Genealogical Concordance Phylogenetic Species Recognition (GCPSR) applied to seven nuclear loci (arf6, chs2, drk1, fads, pyrF, tub1, and its-2) from 78 clinical and environmental isolates identified two previously unrecognized phylogenetic species. Four of seven single gene phylogenies examined (chs2, drk1, pyrF, and its-2) supported the separation of Phylogenetic Species 1 (PS1) and Phylogenetic Species 2 (PS2) which were also well differentiated in the concatenated chs2-drk1-fads-pyrF-tub1-arf6-its2 genealogy with all isolates falling into one of two evolutionarily independent lineages. Phylogenetic species were genetically distinct with interspecific divergence 4-fold greater than intraspecific divergence and a high Fst value (0.772, P<0.001) indicative of restricted gene flow between PS1 and PS2. Whereas panmixia expected of a single freely recombining population was not observed, recombination was detected when PS1 and PS2 were assessed separately, suggesting reproductive isolation. Random mating among PS1 isolates, which were distributed across North America, was only detected after partitioning isolates into six geographic regions. The PS2 population, found predominantly in the hyper-endemic regions of northwestern Ontario, Wisconsin, and Minnesota, contained a substantial clonal component with random mating detected only among unique genotypes in the population. CONCLUSIONS/SIGNIFICANCE: These analyses provide evidence for a genetically divergent clade within Blastomyces dermatitidis, which we use to describe a novel species, Blastomyces gilchristii sp. nov. In addition, we discuss the value of population genetic and phylogenetic analyses as a foundation for disease surveillance, understanding pathogen evolution, and discerning phenotypic differences between phylogenetic species.

Validation of the MycAssay Pneumocystis kit for detection of Pneumocystis jirovecii in bronchoalveolar lavage specimens by comparison to a laboratory standard of direct immunofluorescence microscopy, real-time PCR, or conventional PCR.

Pneumocystis jirovecii pneumonia is a significant cause of morbidity and mortality in AIDS patients as well as those with non-HIV immunosuppressive diseases. To aid diagnosis, the commercial MycAssay Pneumocystis real-time PCR assay (Myconostica, Ltd., Manchester, United Kingdom) targeting the mitochondrial ribosomal large subunit (mtLSU) has been developed to detect P. jirovecii in bronchoalveolar lavage (BAL) specimens. Here, we validated this assay against a laboratory standard of direct immunofluorescence microscopy, a cdc2 real-time PCR assay, or conventional PCR and sequencing of mtLSU. While more sensitive than any of these three assays analyzed individually, the MycAssay Pneumocystis assay demonstrated 100% sensitivity, 100% specificity, a 100% negative predictive value, and a 100% positive predictive value for detecting the presence of P. jirovecii in BAL specimens compared to the laboratory standard. Of note, two samples with positive cycle threshold (C(T)) values according to the MycAssay Pneumocystis assay lacked exponential amplification curves and thus were deemed negative. Also negative according to the laboratory standard, these samples highlight the importance of examining the amplification curves, in addition to noting the C(T) values, when interpreting positive results. Comparison of the MycAssay Pneumocystis assay to a laboratory standard establishes this assay to be a highly sensitive and specific method for the detection of P. jirovecii in bronchoalveolar lavage specimens. The approach may also be useful for the clinical laboratory validation of other sensitive real-time PCR assays.

Rapid identification of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Compared to DNA sequence analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) correctly identified 100% of Cryptococcus species, distinguishing the notable pathogens Cryptococcus neoformans and C. gattii. Identification was greatly enhanced by supplementing a commercial spectral library with additional entries to account for subspecies variability.