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.

A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way.

Pulmonary Nontuberculous Mycobacteria, Ontario, Canada, 2020.

We measured annual prevalence of microbiologically defined nontuberculous mycobacterial lung disease in Ontario, Canada. Mycobacterium avium prevalence was 13 cases/100,000 persons in 2020, a 2.5-fold increase from 2010, indicating a large increase in true M. avium lung disease. During the same period, M. xenopi decreased nearly 50%, to 0.84 cases/100,000 persons.

Genomic investigation of the most common Streptococcus pneumoniae serotypes causing invasive infections in Canada: the SAVE study, 2011-2020.

OBJECTIVES: To investigate the lineages and genomic antimicrobial resistance (AMR) determinants of the 10 most common pneumococcal serotypes identified in Canada during the five most recent years of the SAVE study, in the context of the 10-year post-PCV13 period in Canada. METHODS: The 10 most common invasive Streptococcus pneumoniae serotypes collected by the SAVE study from 2016 to 2020 were 3, 22F, 9N, 8, 4, 12F, 19A, 33F, 23A and 15A. A random sample comprising ∼5% of each of these serotypes collected during each year of the full SAVE study (2011-2020) were selected for whole-genome sequencing (WGS) using the Illumina NextSeq platform. Phylogenomic analysis was performed using the SNVPhyl pipeline. WGS data were used to identify virulence genes of interest, sequence types, global pneumococcal sequence clusters (GPSC) and AMR determinants. RESULTS: Of the 10 serotypes analysed in this study, six increased significantly in prevalence from 2011 to 2020: 3, 4, 8, 9N, 23A and 33F (P ≤ 0.0201). Serotypes 12F and 15A remained stable in prevalence over time, while serotype 19A decreased in prevalence (P < 0.0001). The investigated serotypes represented four of the most prevalent international lineages causing non-vaccine serotype pneumococcal disease in the PCV13 era: GPSC3 (serotypes 8/33F), GPSC19 (22F), GPSC5 (23A) and GPSC26 (12F). Of these lineages, GPSC5 isolates were found to consistently possess the most AMR determinants. Commonly collected vaccine serotypes 3 and 4 were associated with GPSC12 and GPSC27, respectively. However, a more recently collected lineage of serotype 4 (GPSC192) was highly clonal and possessed AMR determinants. CONCLUSIONS: Continued genomic surveillance of S. pneumoniae in Canada is essential to monitor for the appearance of new and evolving lineages, including antimicrobial-resistant GPSC5 and GPSC162.

Post-traumatic Fungal Keratitis and Endophthalmitis Caused by Coniochaeta Hoffmannii with Late Recurrence following Therapeutic Full-thickness Penetrating Keratoplasty.

BACKGROUND: To report a rare case of fungal keratitis and endophthalmitis due to Coniochaeta hoffmannii. METHODS: Case report. RESULTS: A 71-year-old immunocompetent male sustained a corneal laceration, traumatic cataract, and retinal detachment due to penetrating injury from a nail pulled from a wooden deck. The patient's postoperative course was complicated by infectious keratitis. Fungal cultures, DNA sequencing and analysis of the internal transcribed spacer sequence confirmed Coniochaeta hoffmannii. Topical and oral voriconazole treatments were initiated; however, due to impending perforation, a therapeutic corneal transplant was required. One year later, the patient developed a new corneal infiltrate at the graft-host junction: Corneal scrapings were culture positive for Coniochaeta hoffmannii. This was treated with topical and intrastromal voriconazole along with oral itraconazole 200 mg once daily for 8 months. CONCLUSIONS: Coniochaeta hoffmannii may cause recalcitrant keratitis and endophthalmitis, which required longstanding antifungal treatment.

A Practical Workflow for the Identification of Aspergillus, Fusarium, Mucorales by MALDI-TOF MS: Database, Medium, and Incubation Optimization.

There is an increasing body of literature on the utility of MALDI-TOF MS in the identification of filamentous fungi. However, the process still lacks standardization. In this study, we attempted to establish a practical workflow for the identification of three clinically important molds: Aspergillus, Fusarium, and Mucorales using MALDI-TOF MS. We evaluated the performance of Bruker Filamentous Fungi database v3.0 for the identification of these fungi, highlighting when there would be a benefit of using an additional database, the MSI-2 for further identification. We also examined two other variables, namely, medium effect and incubation time on the accuracy of fungal identification. The Bruker database achieved correct species level identification in 85.7% of Aspergillus and 90% of Mucorales, and correct species-complex level in 94.4% of Fusarium. Analysis of spectra using the MSI-2 database would also offer additional value for species identification of Aspergillus species, especially when suspecting species with known identification limits within the Bruker database. This issue would only be of importance in selected cases where species-level identification would impact therapeutic options. Id-Fungi plates (IDFP) had almost equivalent performance to Sabouraud dextrose agar (SDA) for species-level identification of isolates and enabled an easier harvest of the isolates with occasional faster identification. Our study showed accurate identification at 24 h for Fusarium and Mucorales species, but not for Aspergillus species, which generally required 48 h.

Molecular identification of Aspergillus fumigatus complex from lung transplant recipients using multilocus sequencing analysis (MLSA).

BACKGROUND: Aspergillus infection causes significant morbidity and mortality among lung transplant recipients (LTRs). It is primarily caused by Aspergillus fumigatus. Other closely related species belonging to the section Fumigati have also been found. These cryptic species are often misidentified as A. fumigatus. Thus, we used multilocus sequencing analysis (MLSA) of the calmodulin, ß-tubulin, and hydrophobin gene sequences to identify these species and to determine the frequency with which they occur among LTRs. METHODS: A total of 81 A. fumigatus isolates were initially isolated from bronchoalveolar lavage fluid or sputum specimens collected from lung transplant patients. These isolates were then sub-cultured and genotyped using MLSA. Of these isolates, 53, 17, and 11 were isolated from double LTRs, single LTRs, and pre-LTRs, respectively. RESULTS: All isolates (100%) carried DNA sequences identical to those of A. fumigatus reference strains and thus clustered in the same clade with A. fumigatus. Analysis of the MLSA data revealed that A. fumigatus species were the only species recovered in this population of LTRs. The MLSA results were consistent with those routinely obtained by conventional mycological procedures in the microbiology laboratory. CONCLUSIONS: A. fumigatus appears to be the primary causative agent of colonization or invasive aspergillosis among LTRs. No cryptic species were identified.

HISTORIQUE: L'infection à Aspergillus est responsable d'une morbidité et d'une mortalité importantes chez les transplantés du poumon (TP). Elle est surtout causée par l'Aspergillus fumigatus. D'autres espèces proches, de la famille des fumigati, ont également été observées. Ces espèces cryptiques sont souvent identifiées à tort comme un A. fumigatus. Les chercheurs ont fait appel à l'analyse de séquençage multilocus (ASML) des séquences géniques de la calmoduline, de la ß-tubuline et de l'hydrophobine pour repérer ces espèces et en déterminer la fréquence chez les TP. MÉTHODOLOGIE: Au total, les chercheurs ont d'abord mis en culture 81 isolats d'A. fumigatus dans le liquide de lavage bronchoalvéolaire ou les échantillons d'expectoration de TP. Ils en ont ensuite fait une sous-culture et ont procédé au génotypage par ASML. Au total, 53, 17 et 11 d'entre eux provenaient de doubles TP, de TP simples et de futurs TP, respectivement. RÉSULTATS: Tous les isolats (100 %) contenaient des séquences d'ADN pareilles à celles des souches de référence d'A. fumigatus et ont donc été groupés dans le même clade que l'A fumigatus. L'ASML a révélé que les espèces d'A. fumigatus étaient les seules à être récupérées dans cette population de TP. Les résultats de l'ASML étaient conformes à ceux obtenus systématiquement lors d'interventions classiques au laboratoire de microbiologie. CONCLUSIONS: L'A. fumigatus semble être l'agent causal primaire de colonisation ou d'aspergillose invasive chez les TP. Aucune espèce cryptique n'a été observée.

Monkeypox Virus Detection in Different Clinical Specimen Types.

A global monkeypox outbreak began in May 2022. Limited data exist on specimen type performance in associated molecular diagnostics. Consequently, a diverse range of specimen sources were collected in the initial weeks of the outbreak in Ontario, Canada. Our clinical evaluation identified skin lesions as the optimal diagnostic specimen source.

Estimating population-based incidence of community-acquired pneumonia and acute otitis media in children and adults in Ontario and British Columbia using health administrative data, 2005-2018: a Canadian Immunisation Research Network (CIRN) study.

BACKGROUND: There is a paucity of data on the burden of the full spectrum of community-acquired pneumonia (CAP) and acute otitis media (AOM) from outpatient and inpatient settings across the age spectrum. METHODS: We conducted a population-based retrospective study in Ontario and British Columbia (BC), Canada, to estimate the incidence rate of CAP and AOM in children and adults over a 14-year period using health administrative databases. CAP and AOM cases were identified from outpatient physician consultation and hospitalisation data in both provinces, and from emergency department visit data in Ontario. RESULTS: During 2005-2018, Ontario had 3 607 124 CAP, 172 290 bacterial CAP, 7814 pneumococcal pneumonia, and 8 026 971 AOM cases. The incidence rate of CAP declined from 3077/100 000 in 2005 to 2604/100 000 in 2010 before increasing to 2843/100 000 in 2018; bacterial CAP incidence rate also declined from 178/100 000 in 2005 to 112/100 000 in 2010 before increasing to 149/100 000 in 2018. The incidence rate of AOM decreased from 4192/100 000 in 2005 to 3178/100 000 in 2018. BC had 970 455 CAP, 317 913 bacterial CAP, 35 287 pneumococcal pneumonia and 2 022 871 AOM cases. The incidence rate of CAP in BC decreased from 2214/100 000 in 2005 to 1964/100 000 in 2010 before increasing to 2176/100 000 in 2018; bacterial CAP incidence rate increased from 442/100 000 in 2005 to 981/100 000 in 2018. The incidence rate of AOM decreased from 3684/100 000 in 2005 to 2398/100 000 in 2018. The incidence rate of bacterial CAP increased with age in older adults (≥65 years) with the highest burden in the oldest cohort aged ≥85 years both before and after 13-valent pneumococcal conjugate vaccine (PCV13) programme in both provinces. Hospitalised pneumococcal pneumonia decreased slightly but non-hospitalised pneumococcal pneumonia increased in BC during PCV13 period. No consistent direct benefit of PCV13 on CAP was observed in the paediatric population. CONCLUSIONS: There is a substantial burden of CAP and AOM in Ontario and BC. Indirect benefits from childhood PCV vaccination and polysaccharide vaccination of older adults have not substantially decreased the burden of pneumococcal pneumonia in older adults.

First reported case of Robinsoniella peoriensis pyometra and bloodstream infection: A case report and review of the literature.

We report on a 47-year-old woman with jejunal adenocarcinoma and concurrent endometrial cancer, admitted with sepsis. Uterine fluid and blood cultures were positive for Robinsoniella peoriensis. This is the first case report of Robinsoniella peoriensis in Canada. We encourage clinicians to publish their experience treating gynecologic infections caused by Robinsoniella peoriensis. Failure to recognize this pathogen as causative for pyometra, may result in insufficient antimicrobial treatment, and death.

Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern.

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole-genome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times.

Linear Regression Equations To Predict ß-Lactam, Macrolide, Lincosamide, and Fluoroquinolone MICs from Molecular Antimicrobial Resistance Determinants in Streptococcus pneumoniae.

Antimicrobial resistance in Streptococcus pneumoniae represents a threat to public health, and monitoring the dissemination of resistant strains is essential to guiding health policy. Multiple-variable linear regression modeling was used to determine the contributions of molecular antimicrobial resistance determinants to antimicrobial MICs for penicillin, ceftriaxone, erythromycin, clarithromycin, clindamycin, levofloxacin, and trimethoprim-sulfamethoxazole. Training data sets consisting of Canadian S. pneumoniae isolates obtained from 1995 to 2019 were used to generate multiple-variable linear regression equations for each antimicrobial. The regression equations were then applied to validation data sets of Canadian (n = 439) and U.S. (n = 607 and n = 747) isolates. The MICs for ß-lactam antimicrobials were fully explained by amino acid substitutions in motif regions of the penicillin binding proteins PBP1a, PPB2b, and PBP2x. Accuracies of predicted MICs within 1 doubling dilution to phenotypically determined MICs were 97.4% for penicillin, 98.2% for ceftriaxone, 94.8% for erythromycin, 96.6% for clarithromycin, 98.2% for clindamycin, 100% for levofloxacin, and 98.8% for trimethoprim-sulfamethoxazole, with an overall sensitivity of 95.8% and specificity of 98.0%. Accuracies of predicted MICs to the phenotypically determined MICs were similar to those of phenotype-only MIC comparison studies. The ability to acquire detailed antimicrobial resistance information directly from molecular determinants will facilitate the transition from routine phenotypic testing to whole-genome sequencing analysis and can fill the surveillance gap in an era of increased reliance on nucleic acid assay diagnostics to better monitor the dynamics of S. pneumoniae.

Impact of coronavirus disease 2019 (COVID-19) pre-test probability on positive predictive value of high cycle threshold severe acute respiratory coronavirus virus 2 (SARS-CoV-2) real-time reverse transcription polymerase chain reaction (RT-PCR) test results.

OBJECTIVES: Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent presymptomatic or asymptomatic infection, resolved infection with persistent RNA shedding, or a false-positive test. This study assessed the positive predictive value of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from 5 pre-test probability groups ranging from high to low with an alternate assay. METHODS: In total, 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. RESULTS: Significantly fewer (15.6%) positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in each of the 4 groups with higher pre-test probability (individual group range, 50.0%-85.0%). CONCLUSIONS: Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false-positive results and consequent potential for harm at the individual and population level.

Four genomic clades of Candida auris identified in Canada, 2012-2019.

Candida auris is an emerging yeast that is associated with antifungal resistance and healthcare-associated outbreaks. From 2012 to 2019, there were 24 known cases of C. auris colonization or infection in Canada. Isolates were from axilla/groin (n = 6), ear (n = 5), blood (n = 4), toe (n = 2), and a variety of other sites (n = 7). Canadian isolates belonged to the four main genomic clades: Clade I (formerly called South Asian clade, n = 12), Clade II (East Asian, n = 3), Clade III (African, n = 4), and Clade IV (South American, n = 5). Isolates within each clade were clonal; however, whole genome sequencing may be helpful in identifying clusters within healthcare facilities. LAY SUMMARY: The fungal pathogen Candida auris has caused many hospital outbreaks and is often multidrug resistant. All four major strains of C. auris were identified in Canada from 2012 to 2019. Genomic epidemiology may be useful for identifying and reducing transmission of C. auris within hospitals.

Invasive pneumococcal disease surveillance in Canada, 2020.

Background: Invasive pneumococcal disease (IPD), which is caused by Streptococcus pneumoniae, has been a nationally notifiable disease in Canada since 2000. The use of conjugate vaccines has markedly decreased the incidence of IPD in Canada; however, the distribution of serotypes has shifted in favour of non-vaccine types. This report summarizes the demographics, serotypes and antimicrobial resistance of IPD infections in Canada in 2020. Methods: The Public Health Agency of Canada's National Microbiology Laboratory (Winnipeg, Manitoba) collaborates with provincial and territorial public health laboratories to conduct national surveillance of IPD. A total of 2,108 IPD isolates were reported in 2020. Serotyping was performed by Quellung reaction and antimicrobial susceptibilities were determined in collaboration with the University of Manitoba/Canadian Antimicrobial Resistance Alliance. Population-based IPD incidence rates were obtained through the Canadian Notifiable Disease Surveillance System. Results: Overall incidence of IPD in Canada decreased significantly from 11.5 (95% confidence interval [CI]: 10.1-13.1) to 6.0 (95% CI: 5.0-7.2), and from 10.0 (95% CI: 9.7-10.3) to 5.9 (95% CI: 5.7-6.2) cases per 100,000 from 2019 to 2020; in those younger than five years and those five years and older, respectively. The most common serotypes overall were 4 (11.2%, n=237), 3 (10.9%, n=229) and 8 (7.2%, n=151). From 2016 to 2020, serotypes with increasing trends (p<0.05) included 4 (6.4%-11.2%), 3 (9.5%-10.9%), 8 (5.2%-7.2%) and 12F (3.6%-5.7%). The overall prevalence of PCV13 serotypes increased over the same period (30.3%-34.9%, p<0.05). Antimicrobial resistance rates in 2020 included 23.0% clarithromycin and 9.9% penicillin (IV meningitis breakpoints). Multidrug-resistant IPD has significantly increased since 2016 (4.2%-9.5%, p<0.05). Conclusion: Though the incidence of IPD decreased in 2020 in comparison to previous years across all age groups, disease due to PCV13 serotypes 3 and 4, as well as non-PCV13 serotypes such as 8 and 12F, increased in prevalence. Continued surveillance of IPD is imperative to monitor shifts in serotype distribution and antimicrobial resistance.