Microbiological characterization of Pneumocystis jirovecii pneumonia using quantitative PCR from nasopharyngeal specimens: a retrospective study in a Canadian province from 2019 to 2023.

Bronchoalveolar lavage is usually employed for molecular diagnosis of Pneumocystis jirovecii but requires a specialized procedure. By contrast, nasopharyngeal (NP) specimens are easily obtained. In this retrospective study of 35 patients with paired NP and bronchoscopy specimens, NP specimens had a 100% negative percent agreement (95% CI 80.5-100) but only 72.2% positive percent agreement (95% CI 46.5-90.3).

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.

Multicenter Evaluation of Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy-Based Method for Rapid Identification of Clinically Relevant Yeasts.

Fourier transform infrared (FTIR) spectroscopy has demonstrated applicability as a reagent-free whole-organism fingerprinting technique for both microbial identification and strain typing. For routine application of this technique in microbiology laboratories, acquisition of FTIR spectra in the attenuated total reflectance (ATR) mode simplifies the FTIR spectroscopy workflow, providing results within minutes after initial culture without prior sample preparation. In our previous central work, 99.7% correct species identification of clinically relevant yeasts was achieved by employing an ATR-FTIR-based method and spectral database developed by our group. In this study, ATR-FTIR spectrometers were placed in 6 clinical microbiology laboratories over a 16-month period and were used to collect spectra of routine yeast isolates for on-site identification to the species level. The identification results were compared to those obtained from conventional biochemical tests and/or matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Isolates producing discordant results were reanalyzed by routine identification methods, ATR-FTIR spectroscopy, and PCR gene sequencing of the D1/D2 and internal transcribed spacer (ITS) regions. Among the 573 routine clinical yeast isolates collected and identified by the ATR-FTIR-based method, 564 isolates (98.4%) were correctly identified at the species level, while the remaining isolates were inconclusive with no misidentifications. Due to the low prevalence of Candida auris in routine isolates, additional randomly selected C. auris (n = 24) isolates were obtained for evaluation and resulted in 100% correct identification. Overall, the data obtained in our multicenter evaluation study using multiple spectrometers and system operators indicate that ATR-FTIR spectroscopy is a reliable, cost-effective yeast identification technique that provides accurate and timely (∼3 min/sample) species identification promptly after the initial culture.

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.

Changing Patterns of Disease Severity in Blastomyces dermatitidis Infection, Quebec, Canada.

This retrospective multicenter cohort study assessed temporal changes in the severity and mortality rate of blastomycosis in Quebec, Canada, and identified risk factors for death in patients with blastomycosis in 1988-2016. The primary outcome was 90-day all-cause deaths. Among 185 patients, 122 (66%) needed hospitalization and 30 (16%) died. We noted increases in the proportion of severe cases, in age at diagnosis and in the proportion of diabetic and immunocompromised patients over time. Independent risk factors for death were age (adjusted odds ratio [aOR] 1.04, 95% CI 1.00-1.07), immunosuppression (aOR 4.2, 95% CI 1.5-11.6), and involvement of >2 lung lobes (aOR 5.3, 95% CI 1.9-14.3). There was no association between the Blastomyces genotype group and all-cause mortality. The proportion of severe cases of blastomycosis has increased in Quebec over the past 30 years, partially explained by the higher number of immunosuppressed patients.

Prevalence and mechanisms of azole resistance in clinical isolates of Aspergillus section Fumigati species in a Canadian tertiary care centre, 2000 to 2013.

OBJECTIVES: Azole resistance among Aspergillus fumigatus isolates is a growing concern worldwide. Induction of mutations during azole therapy, environment-acquired mutations caused by azole fungicides and intrinsic resistance of cryptic Fumigati species all contribute to the burden of resistance. However, there is a lack of data in Canada on this emerging threat. METHODS: To gain insights into the magnitude and mechanisms of resistance, a 14 year collection of Aspergillus section Fumigati comprising 999 isolates from 807 patients at a Montreal hospital was screened for azole resistance, and resistance mechanisms were investigated with the combined use of genome sequencing, 3D modelling and phenotypic efflux pump assays. RESULTS: Overall azole resistance was low (4/807 patients; 0.5%). A single azole-resistant A. fumigatus sensu stricto strain, isolated from a patient with pulmonary aspergillosis, displayed efflux-pump-mediated resistance. Three patients were colonized or infected with azole-resistant cryptic Fumigati species (one Aspergillus thermomutatus, one Aspergillus lentulus and one Aspergillus turcosus). Evidence is presented that azole resistance is efflux-pump-mediated in the A. turcosus isolate, but not in the A. lentulus and A. thermomutatus isolates. CONCLUSIONS: Azole resistance is rare in our geographic area and currently driven by cryptic Fumigati species. Continued surveillance of emergence of resistance is warranted.

Long-lasting cluster of nosocomial pneumonia with a single Pneumocystis jirovecii genotype involving different organ allograft recipients.

Pneumocystis pneumonia (PCP) outbreaks may occur in solid organ transplant (SOT) patients. Transmissibility of Pneumocystis jirovecii among SOT and non-SOT patients has not been investigated. Ten SOT (ie, 4 heart, 4 kidney, 2 liver allograft recipients) and 11 non-SOT (ie, 7 HIV-infected, 3 hematologic malignancies, and 1 stem cell transplant) patients with PCP were admitted to London Health Sciences Center (LHSC) from October 2014 to August 2016. We investigated the course of illness and outcome of PCP in SOT and non-SOT patients. Post-transplant PCP was frequently an acute-onset disease (90% vs. 18.2%, p = .01) requiring ICU admission (70% vs. 20%, p = .03) and hemodialysis (60% vs. 0, p = .003). Mortality was more frequent in SOT patients (40% vs. 18.1%, p = .36). Multilocus sequence typing (MLST) demonstrated circulation of a single genotype of P. jirovecii among SOT patients. However, 8 different genotypes were detected from non-SOT patients. Reinstitution of prophylaxis successfully controlled post-transplant cluster until end of observation period in October 2019. No transmission was detected from non-SOT patients to SOT recipients. Detection of a single P. jirovecii genotype from all SOT recipients highlights the likelihood of nosocomial transmission. No source control method is recommended by current guidelines. Improvement of preventive strategies is required.

Reagent-Free Identification of Clinical Yeasts by Use of Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy.

Invasive fungal infections by opportunistic yeasts have increased concomitantly with the growth of an immunocompromised patient population. Misidentification of yeasts can lead to inappropriate antifungal treatment and complications. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy is a promising method for rapid and accurate identification of microorganisms. ATR-FTIR spectroscopy is a standalone, inexpensive, reagent-free technique that provides results within minutes after initial culture. In this study, a comprehensive spectral reference database of 65 clinically relevant yeast species was constructed and tested prospectively on spectra recorded (from colonies taken from culture plates) for 318 routine yeasts isolated from various body fluids and specimens received from 38 microbiology laboratories over a 4-month period in our clinical laboratory. ATR-FTIR spectroscopy attained comparable identification performance with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In a preliminary validation of the ATR-FTIR method, correct identification rates of 100% and 95.6% at the genus and species levels, respectively, were achieved, with 3.5% unidentified and 0.9% misidentified. By expanding the number of spectra in the spectral reference database for species for which isolates could not be identified or had been misidentified, we were able to improve identification at the species level to 99.7%. Thus, ATR-FTIR spectroscopy provides a new standalone method that can rival MALDI-TOF MS for the accurate identification of a broad range of medically important yeasts. The simplicity of the ATR-FTIR spectroscopy workflow favors its use in clinical laboratories for timely and low-cost identification of life-threatening yeast strains for appropriate treatment.

Identification of Candida auris by Use of the Updated Vitek 2 Yeast Identification System, Version 8.01: a Multilaboratory Evaluation Study.

Candida auris is an emerging multidrug-resistant yeast that has been systematically incorrectly identified by phenotypic methods in clinical microbiology laboratories. The Vitek 2 automated identification system (bioMérieux) recently included C. auris in its database (version 8.01). We evaluated the performance of the Vitek 2 YST ID card to identify C. auris and related species. A panel of 44 isolates of Candida species (C. auris, n = 35; Candida haemulonii, n = 5; Candida duobushaemulonii, n = 4) were tested by three different hospital-based microbiology laboratories. Among 35 isolates of C. auris, Vitek 2 yielded correct identification in an average of 52% of tested samples. Low-discrimination (LD) results with an inability to distinguish between C. auris, C. duobushaemulonii, and Candida famata were obtained in an average of 27% of samples. Incorrect identification results were obtained in an average of 21% of samples, the majority (91%) of which were reported as C. duobushaemulonii and the remaining 9% of which were reported as Candida lusitaniae/C. duobushaemulonii. The proportion of correct identification was not statistically different across different centers (P = 0.78). Stratification by genetic clades demonstrated that 100% (n = 8) of the strains of the South American clade were correctly identified compared to 7% (n = 10) and 0% (n = 4) from the African and East Asian clades, respectively. None of the non-auris Candida strains (n = 9) were incorrectly identified as C. auris Our results show that the Vitek 2 (version 8.01) yeast identification system has a limited ability to correctly identify C. auris These data suggest that an identification result for C. duobushaemulonii should warrant further testing to rule out C. auris The overall performance of the Vitek 2 seems to differ according to C. auris genetic clade, with the South American isolates yielding the most accurate results.

Case Series: Report of the First Two Human Indigenous Cases of Cryptococcus gattii Infection in Eastern Canada.

We report the two first cases of human C. gattii meningoencephalitis acquired on the Canadian east coast, from the province of Quebec. Unlike C. neoformans, C. gattii is not known to have an established ecological niche on the North American east coast. C. gattii has recently been responsible for major outbreaks in British Columbia, Canada, and in the American pacific northwest. However, no human cases acquired in other Canadian provinces have been reported to our knowledge. The source of acquisition remains unclear for both patients but since neither had traveled outside of the province of Quebec, we discuss the possibilities of environmental and animal-associated acquisition, as well as the possible established endemicity in new areas. These cases add to the growing reported human and animal cases in areas previously not thought to be endemic for C. gattii.

High frequency of pathogenic Aspergillus species among nonsporulating moulds from respiratory tract samples.

Nonsporulating moulds (NSM) represent an identification challenge for clinical laboratories. Data on the prevalence of pathogenic species among NSM are lacking. We prospectively investigated consecutive thermotolerant (36°C) clinical NSM isolates from respiratory tract samples. A total of 123 isolates were identified by DNA sequencing and phenotypically characterized. Of those, 13 (11%) were pathogenic species (Aspergillus fumigatus, n = 10; A. flavus, n = 1; A. hiratsukae, n = 1; Schizophyllum commune, n = 1). Presumptive identification of Aspergillus species among NSM can be achieved by simple phenotypic testing.

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular "machinery" required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

A Disseminated Cryptococcus gattii VGIIa Infection in a Citron-Crested Cockatoo (Cacatua sulphurea citrinocristata) in Québec, Canada.

Cryptococcus gattii infection in mammals and birds has been confined historically to tropical and subtropical regions in Australia, Southeast Asia, Africa, and South America. Since the early 2000s, numerous reports describe the emergence of C. gattii on the Pacific Coast of North America. We report on a C. gattii infection in an 8-year-old male citron-crested cockatoo (Cacatua sulphurea citrinocristata) hatched on the Canadian Pacific Coast and raised in the province of Québec, Canada. The bird developed a slow growing ulcerated, fleshy, crusty, and hemorrhagic mass infiltrating the left lower rhamphotheca. Cryptococcus gattii infection was confirmed by cytologic examination of a fine needle aspirate of the mass, and results of fungal culture and sequencing. The genotype of the strain was determined to be VGIIa sequence type 20, the strongly overrepresented subgroup found on the Canadian Pacific coast. Minimum inhibitory concentrations for multiple antifungal drugs were determined. The bird received fluconazole but died acutely 55 days after initial presentation. Postmortem examination revealed a disseminated infection, with involvement of the beak, lungs, spleen, and brain.

Quick versus Quantitative: Evaluation of Two Commercial Real-Time PCR Assays for the Detection of Pneumocystis jirovecii from Bronchoalveolar Lavage Fluids.

Two commercial real-time PCR assays for the detection of Pneumocystis jirovecii were compared, the quantitative RealStar P. jirovecii assay and the qualitative DiaSorin P. jirovecii assay, the latter of which can be used without nucleic acid extraction. Archived bronchoalveolar lavage (BAL) specimens (n = 66), previously tested by molecular methods, were tested by both assays, and the results were compared to the respective original result. The RealStar P. jirovecii assay demonstrated good positive percent agreement (PPA) (90% [95% confidence interval (CI), 72 to 97%]; 27/30) and negative percent agreement (NPA) (100% [95% CI, 88 to 100%]; 36/36) with the reference method. The DiaSorin P. jirovecii assay concordantly detected P. jirovecii in 19 of 24 positive BAL samples (PPA = 73% [95% CI, 52 to 88%]). All negative BAL samples gave concordant results (NPA = 100% [95% CI, 87 to 100%]; 34/34). Discordant results occurred mostly in samples with low fungal loads. In conclusion, the RealStar assay demonstrated good concordance with reference results, and the DiaSorin P. jirovecii assay performed well for negative BAL and positive BAL samples with P. jirovecii concentrations of greater than 260 copies/mL. IMPORTANCE Pneumonia, caused by the opportunistic fungus Pneumocystis jirovecii, poses a significant risk for immunocompromised individuals. Laboratory testing for P. jirovecii is progressively shifting toward the use of molecular tests such as real-time PCR; however, this is often performed at reference laboratories. Many frontline laboratories are looking into improving their service and reducing turnaround times for obtaining P. jirovecii results by bringing molecular P. jirovecii testing in-house. We evaluated and compared two commercial real-time PCR assays with different workflows for the detection of P. jirovecii from bronchoalveolar lavage specimens. The RealStar P. jirovecii assay requires nucleic acid extraction and provides a quantification of fungal load for positive samples. The DiaSorin P. jirovecii assay offers a simple workflow without nucleic extraction from patient samples and qualitative results. Results from this study provide valuable information on performance and workflow considerations for laboratories that wish to implement P. jirovecii molecular testing.

Corrigendum to 'Rhinitis due to Aspergillus pseudoviridinutans in an orange-winged Amazon parrot (Amazona amazonica)'[Med. Mycol. Case Rep. 30 (2020) 46-50].

[This corrects the article DOI: 10.1016/j.mmcr.2020.11.001.].

Natural spring water gargle and direct RT-PCR for the diagnosis of COVID-19 (COVID-SPRING study).

BACKGROUND: Nasopharyngeal swab has long been considered the specimen of choice for the diagnosis of respiratory viral infections, including SARS-CoV-2 infection, but it suffers from several drawbacks: its discomfort limits screening acceptability, and it is vulnerable to shortages in both specialized materials and trained healthcare workers in the context of a pandemic. METHODS: We prospectively compared natural spring water gargle to combined oro-nasopharyngeal swab (ONPS) for the diagnosis of coronavirus disease 2019 (COVID-19) in paired clinical specimens (1005 ONPS and 1005 gargles) collected from 987 unique early symptomatic as well as asymptomatic individuals from the community. RESULTS: Using a direct RT-PCR method with the Allplex™ 2019-nCoV Assay (Seegene), the clinical sensitivity of the gargle was 95.3% (95% confidence interval [CI], 90.2 - 98.3%), similar to the sensitivity of the ONPS (93.8%; 95% CI, 88.2 - 97.3%), despite significantly lower viral RNA concentration in gargles, as reflected by higher cycle threshold values. No single specimen type detected all COVID-19 cases. SARS-CoV-2 RNA was stable in gargles at room temperature for at least 7 days. CONCLUSION: The simplicity of this sampling method coupled with the accessibility of spring water are clear advantages in a pandemic situation where testing frequency, turnaround time and shortage of consumables and trained staff are critical elements.

Draft Genome Sequence of Azole-Resistant Aspergillus thermomutatus (Neosartorya pseudofischeri) Strain HMR-AF-39, Isolated from a Human Nasal Septum Abscess Aspirate.

Here, we present the draft genome sequence of Aspergillus thermomutatus (formerly known as Neosartorya pseudofischeri; strain HMR-AF-39/LSPQ-01276), a cryptic species of Aspergillus section Fumigati. This species is intrinsically resistant to antifungal azoles and is recognized as an agent of invasive aspergillosis among immunocompromised hosts.