In vitro activity of sulopenem against 1880 bacterial pathogens isolated from Canadian patients with urinary tract infections (CANWARD, 2014-21).

INTRODUCTION: There are limited oral antimicrobial options for the treatment of urinary infections caused by ESBL-producing and MDR Enterobacterales. Sulopenem is an investigational thiopenem antimicrobial that is being developed as both an oral and IV formulation. The purpose of this study was to evaluate the in vitro activity of sulopenem versus bacterial pathogens recovered from the urine of patients admitted to or assessed at hospitals across Canada (CANWARD). MATERIALS AND METHODS: The in vitro activity of sulopenem and clinically relevant comparators was determined for 1880 Gram-negative and Gram-positive urinary isolates obtained as part of the CANWARD study (2014 to 2021) using the CLSI broth microdilution method. RESULTS: Sulopenem demonstrated excellent in vitro activity versus members of the Enterobacterales, with MIC90 values ranging from 0.06 to 0.5 mg/L for all species tested. Over 90% of ESBL-producing, AmpC-producing and MDR (not susceptible to ≥1 antimicrobial from ≥3 classes) Escherichia coli were inhibited by ≤0.25 mg/L of sulopenem. Sulopenem had an identical MIC90 to meropenem for ESBL-producing and MDR E. coli. The MIC90 of sulopenem and meropenem versus MSSA was 0.25 mg/L. Sulopenem was not active in vitro versus Pseudomonas aeruginosa (similar to ertapenem), and it demonstrated poor activity versus Enterococcus faecalis (similar to meropenem). CONCLUSIONS: Sulopenem demonstrated excellent in vitro activity versus bacterial pathogens recovered from the urine of Canadian patients. These data suggest that sulopenem may have a role in the treatment of urinary infections caused by antimicrobial-resistant Enterobacterales, but additional clinical studies are required.

In vitro activity of fosfomycin against bacterial pathogens isolated from urine specimens in Canada from 2007 to 2020: CANWARD surveillance study.

BACKGROUND: Multiple susceptible breakpoints are published to interpret fosfomycin MICs: ≤64 mg/L for Escherichia coli and Enterococcus faecalis grown from urine (CLSI M100); ≤32 mg/L for Enterobacterales and staphylococci when parenteral fosfomycin is prescribed (EUCAST); and ≤8 mg/L for uncomplicated urinary tract infection with E. coli when oral fosfomycin is used (EUCAST). Clinical laboratories are frequently requested to test fosfomycin against antimicrobial-resistant urinary isolates not included in standard documents. METHODS: The in vitro activity of fosfomycin was determined using the CLSI agar dilution method for a 2007-20 collection of clinically significant Gram-negative (3656 Enterobacterales; 140 Pseudomonas aeruginosa) and Gram-positive (346 E. faecalis; 94 Staphylococcus aureus) urinary isolates from the CANWARD surveillance study. Comparator agents were tested using CLSI broth microdilution. RESULTS: Using the CLSI MIC breakpoint (≤64 mg/L), 99.2% of E. coli (n = 2871; MIC90, 4 mg/L), including 96.7% of ESBL-positive isolates, were fosfomycin susceptible. Similarly, 95.8% of E. coli, including 95.2% of ESBL-positive isolates, were fosfomycin susceptible at ≤8 mg/L (EUCAST oral susceptible MIC breakpoint). All other species of Enterobacterales (except Citrobacter freundii) and P. aeruginosa had higher fosfomycin MICs (MIC90s, 64 to >512 mg/L) than E. coli. Using published breakpoints, 88.4% of E. faecalis (MIC ≤64 mg/L) and 97.9% of S. aureus (MIC ≤32 mg/L) isolates were fosfomycin susceptible. CONCLUSIONS: Fosfomycin demonstrated in vitro activity against frequently encountered Gram-positive and Gram-negative urinary pathogens; however, the extrapolation of current CLSI and EUCAST MIC breakpoints to pathogens not specified by standard methods requires further study and is currently not recommended.

Effect of clinical versus administrative data definitions on the epidemiology of C. difficile among hospitalized individuals with IBD: a population-based cohort study.

BACKGROUND: Hospitalization admissions and discharge databases (DAD) using the International Classification of Diseases (ICD) codes are often used to describe the epidemiology of Clostridioides difficile infections (CDI) among those with Inflammatory bowel disease (IBD), even though DAD CDI definition can miss many cases of CDI. There are no data comparing the assessment of the epidemiology of CDI among those with IBD by DAD versus laboratory diagnosis. We used a population-based dataset to determine the effect of using DAD versus laboratory CDI diagnosis on CDI assessment among those with IBD. METHODS: We linked the University of Manitoba IBD Epidemiology Database to the provincial CDI laboratory dataset for the years 2005-2014. Time trends of CDI were assessed using joinpoint analyses. We used stratified logistic regression analysis to assess factors associated with CDI among individuals with IBD. RESULTS: Time trends of CDI among hospitalized individuals with IBD were similar when using DAD or the laboratory CDI diagnosis. Prior hospital admission and antibiotic exposure were associated with CDI using either of the CDI definitions, 5-ASA use was associated with CDI using DAD but not laboratory diagnosis, whereas corticosteroid exposure was associated with laboratory-based CDI diagnosis. Using laboratory results as gold standard, DAD had a sensitivity and specificity of 75.4% and 99.6% for CDI among those with IBD. CONCLUSIONS: Using ICD codes in the DAD for CDI provides similar epidemiological time trend patterns as identifying CDI in the laboratory dataset. Hence, ICD codes are reliable to determine CDI epidemiology among hospitalized individuals with IBD.

Use of Fosfomycin Etest To Determine In Vitro Susceptibility of Clinical Isolates of Enterobacterales Other than Escherichia coli, Nonfermenting Gram-Negative Bacilli, and Gram-Positive Cocci.

Clinical isolates of Enterobacterales other than Escherichia coli (EOTEC), nonfermenting Gram-negative bacilli, and Gram-positive cocci were tested for susceptibility to fosfomycin using Etest and reference agar dilution. Applying EUCAST (v. 11.0, 2021) intravenous fosfomycin breakpoints, Etest MICs for EOTEC showed essential agreement (EA), categorical agreement (CA), major error (ME), and very major error (VME) rates of 70.4%, 88.4%, 4.1%, and 32.1%, respectively. No species of EOTEC tested with acceptable rates for all of EA (≥90%), CA (≥90%), ME (≤3%), and VME (≤3%). Etest MICs for Enterococcus faecalis, interpreted using CLSI oral/urine criteria (M100, 2021) showed EA, CA, minor error, ME, and VME rates of 98.5%, 81.2%, 18.8%, 0%, and 0%. Against Staphylococcus aureus, EA, CA, and ME rates were 84.1%, 98.7%, and 1.3% (EUCAST intravenous criteria). S. aureus isolates with fosfomycin MICs of >32 µg/ml (resistant) were not identified by agar dilution. We conclude that performing fosfomycin Etest on isolates of S. aureus will reliably identify fosfomycin-susceptible isolates with low, acceptable rates of MEs and VMEs. Testing of urinary isolates of E. faecalis by Etest is associated with an unacceptably high rate of minor errors (18.8%) but low, acceptable rates of MEs and VMEs when results are interpreted using CLSI criteria. Isolates of EOTEC tested by Etest with resulting MICs interpreted by EUCAST criteria were associated with an unacceptably high VME rate (32.1%). In vitro testing of clinical isolates beyond E. coli, E. faecalis, and S. aureus to determine susceptibility to fosfomycin is problematic with current methods and breakpoints.

ESBL-positive Escherichia coli and Klebsiella pneumoniae isolates from across Canada: CANWARD surveillance study, 2007-18.

OBJECTIVES: ESBL-producing Escherichia coli and Klebsiella pneumoniae are pathogens of increasing importance in Canada and elsewhere in the world. The purpose of this study was to phenotypically and molecularly characterize ESBL-producing E. coli and K. pneumoniae clinical isolates obtained from patients attending Canadian hospitals over a 12 year period. METHODS: Isolates were collected between January 2007 and December 2018 as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the CLSI (M100) phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent WGS. RESULTS: In total, 671 E. coli and 141 K. pneumoniae were confirmed to be ESBL producers. The annual proportion of ESBL-producing isolates increased for both E. coli (from 3.3% in 2007 to 11.2% in 2018; P < 0.0001) and K. pneumoniae (from 1.3% in 2007 to 9.3% in 2018; P < 0.0001). The most frequent STs were ST131 for E. coli [62.4% (419/671) of isolates] and ST11 [7.8% (11/141)] and ST147 [7.8% (11/141)] for K. pneumoniae. Overall, 97.2% of ESBL-producing E. coli and K. pneumoniae isolates were MDR. blaCTX-M-15 predominated in both ESBL-producing E. coli (62.3% of isolates) and ESBL-producing K. pneumoniae (48.9% of isolates). CONCLUSIONS: The proportion of ESBL-producing E. coli, especially ST131, and K. pneumoniae, especially ST11 and ST147, in Canada increased significantly from 2007 to 2018. Continued prospective surveillance of these evolving MDR and at times XDR pathogens is imperative.

Comparison of phenotypic antimicrobial susceptibility testing results and WGS-derived genotypic resistance profiles for a cohort of ESBL-producing Escherichia coli collected from Canadian hospitals: CANWARD 2007-18.

OBJECTIVES: To determine whether the genotypic resistance profile inferred from WGS could accurately predict phenotypic resistance for ESBL-producing Escherichia coli isolated from patient samples in Canadian hospital laboratories. METHODS: As part of the ongoing CANWARD study, 671 E. coli were collected and phenotypically confirmed as ESBL producers using CLSI M100 disc testing criteria. Isolates were sequenced using the Illumina MiSeq platform, resulting in 636 high-quality genomes for comparison. Using a rules-based approach, the genotypic resistance profile was compared with the phenotypic resistance interpretation generated using the CLSI broth microdilution method for ceftriaxone, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole. RESULTS: The most common genes associated with non-susceptibility to ceftriaxone, gentamicin and trimethoprim/sulfamethoxazole were CTX-M-15 (n = 391), aac(3)-IIa + aac(6')-Ib-cr (n = 121) and dfrA17 + sul1 (n = 169), respectively. Ciprofloxacin non-susceptibility was most commonly attributed to alterations in both gyrA (S83L + D87N) and parC (S80I + E84V), with (n = 187) or without (n = 197) aac(6')-Ib-cr. Categorical agreement (susceptible or non-susceptible) between actual and predicted phenotype was 95.6%, 98.9%, 97.6% and 88.8% for ceftriaxone, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole, respectively. Only ciprofloxacin results (susceptible or non-susceptible) were predicted with major error (ME) and very major error (VME) rates of <3%: ciprofloxacin (ME, 1.5%; VME, 1.1%); gentamicin (ME, 0.8%-31.7%; VME, 4.8%); ceftriaxone (ME, 81.8%; VME, 3.0%); and trimethoprim/sulfamethoxazole (ME, 0.9%-23.0%; VME, 5.2%-8.5%). CONCLUSIONS: Our rules-based approach for predicting a resistance phenotype from WGS performed well for ciprofloxacin, with categorical agreement of 98.9%, an ME rate of 1.5% and a VME rate of 1.1%. Although high categorical agreements were also obtained for gentamicin, ceftriaxone and trimethoprim/sulfamethoxazole, ME and/or VME rates were ≥3%.

Identification and Characterization of a Novel FosA7 Member from Fosfomycin-Resistant Escherichia coli Clinical Isolates from Canadian Hospitals.

Here, we characterize the fosA genes from three Escherichia coli clinical isolates recovered from Canadian patients. Each fosA sequence was individually overexpressed in E. coli BW25113, and antimicrobial susceptibility testing was performed to assess their role in fosfomycin resistance. The findings from this study identify and functionally characterize FosA3, FosA8, and novel FosA7 members and highlight the importance of phenotypic characterization of fosA genes.

In Vitro Activity of Plazomicin against Gram-Negative and Gram-Positive Bacterial Pathogens Isolated from Patients in Canadian Hospitals from 2013 to 2017 as Part of the CANWARD Surveillance Study.

The Clinical and Laboratory Standards Institute (CLSI) broth microdilution method was used to evaluate the in vitro activities of plazomicin and comparator antimicrobial agents against 7,712 Gram-negative and 4,481 Gram-positive bacterial pathogens obtained from 2013 to 2017 from patients in Canadian hospitals as part of the CANWARD Surveillance Study. Plazomicin demonstrated potent in vitro activity against Enterobacteriaceae (MIC90 ≤ 1 µg/ml for all species tested except Proteus mirabilis and Morganella morganii), including aminoglycoside-nonsusceptible, extended-spectrum ß-lactamase (ESBL)-positive, and multidrug-resistant (MDR) isolates. Plazomicin was equally active against methicillin-susceptible and methicillin-resistant isolates of Staphylococcus aureus.

In Vitro Activity of Sulopenem, an Oral Penem, against Urinary Isolates of Escherichia coli.

The in vitro activity of sulopenem was assessed against a collection from 2014 to 2016 of 539 urinary isolates of Escherichia coli from Canadian patients by using CLSI-defined broth microdilution methodology. A concentration of sulopenem 0.03 µg/ml inhibited both 50% (MIC50) and 90% (MIC90) of isolates tested; sulopenem MICs ranged from 0.015 to 0.25 µg/ml. The in vitro activity of sulopenem was unaffected by nonsusceptibility to trimethoprim-sulfamethoxazole and/or ciprofloxacin, multidrug-resistant phenotypes, extended-spectrum ß-lactamases, or AmpC ß-lactamases.

Characterization of carbapenem-resistant and XDR Pseudomonas aeruginosa in Canada: results of the CANWARD 2007-16 study.

OBJECTIVES: Carbapenem-resistant Pseudomonas aeruginosa are emerging worldwide with increasing reports of carbapenemase-producing isolates. Carbapenem-resistant isolates may also be XDR. This study characterized carbapenem-resistant and XDR P. aeruginosa isolated from patients receiving care at Canadian hospitals from 2007 to 2016. METHODS: Antimicrobial susceptibility testing was performed using CLSI broth microdilution methods. PCR was used to detect carbapenemases (GES, KPC, NDM, IMP, VIM, OXA-48) and other resistance markers; specific carbapenemase gene variants were identified by DNA sequencing. Genetic relatedness was assessed by MLST and PFGE. RESULTS: From 2007 to 2016, 3864 isolates of P. aeruginosa were collected; 466 (12.1%) isolates were carbapenem resistant. The prevalence of carbapenem-resistant P. aeruginosa reached a peak of 17.3% in 2014. Colistin (94% susceptible) and ceftolozane/tazobactam (92.5%) were the most active agents against carbapenem-resistant P. aeruginosa. XDR P. aeruginosa comprised 4.5% of isolates; they were found to be genetically diverse and remained susceptible to colistin and ceftolozane/tazobactam. Only 4.3% (n = 20) of carbapenem-resistant P. aeruginosa harboured a carbapenemase; most were blaGES-5 (35%, n = 7). Wide genetic diversity was observed among carbapenem-resistant P. aeruginosa with >200 different sequence types identified. CONCLUSIONS: Although the prevalence of carbapenem-resistant P. aeruginosa in Canada spiked in 2014 and 2015, carbapenemase-producing P. aeruginosa remain rare with only 20 (4.3%) isolates identified over a 10 year period. Broad genetic diversity was observed among both carbapenem-resistant and XDR phenotypes of P. aeruginosa. Pan-drug-resistant P. aeruginosa have not yet been identified in Canada.

42936 pathogens from Canadian hospitals: 10 years of results (2007-16) from the CANWARD surveillance study.

OBJECTIVES: The CANWARD surveillance study was established in 2007 to annually assess the in vitro susceptibilities of a variety of antimicrobial agents against bacterial pathogens isolated from patients receiving care in Canadian hospitals. METHODS: 42 936 pathogens were received and CLSI broth microdilution testing was performed on 37 355 bacterial isolates. Limited patient demographic data submitted with each isolate were collated and analysed. RESULTS: Of the isolates tested, 43.5%, 33.1%, 13.2% and 10.2% were from blood, respiratory, urine and wound specimens, respectively; 29.9%, 24.8%, 19.0%, 18.1% and 8.2% of isolates were from patients in medical wards, emergency rooms, ICUs, hospital clinics and surgical wards. Patient demographics associated with the isolates were: 54.6% male/45.4% female; 13.1% patients aged ≤17 years, 44.3% 18-64 years and 42.7% ≥65 years. The three most common pathogens were Staphylococcus aureus (21.2%, both methicillin-susceptible and MRSA), Escherichia coli (19.6%) and Pseudomonas aeruginosa (9.0%). E. coli were most susceptible to meropenem and tigecycline (99.9%), ertapenem and colistin (99.8%), amikacin (99.7%) and ceftolozane/tazobactam and plazomicin (99.6%). Twenty-three percent of S. aureus were MRSA. MRSA were most susceptible to ceftobiprole, linezolid and telavancin (100%), daptomycin (99.9%), vancomycin (99.8%) and tigecycline (99.2%). P. aeruginosa were most susceptible to ceftolozane/tazobactam (98.3%) and colistin (95.0%). CONCLUSIONS: The CANWARD surveillance study has provided 10 years of reference antimicrobial susceptibility testing data on pathogens commonly causing infections in patients attending Canadian hospitals.

Trends in antimicrobial resistance over 10 years among key bacterial pathogens from Canadian hospitals: results of the CANWARD study 2007-16.

OBJECTIVES: We sought to analyse 10 years of longitudinal surveillance data (2007-16) from the CANWARD study and describe emerging trends in antimicrobial resistance for key bacterial pathogens across Canada. METHODS: Longitudinal data from CANWARD study sites that contributed isolates every year from 2007 to 2016 were analysed to identify trends in antimicrobial resistance over time using univariate tests of trend and multivariate regression models to account for the effects of patient demographics. RESULTS: Statistically significant increases occurred in the proportion of Escherichia coli isolates resistant to extended-spectrum cephalosporins, amoxicillin/clavulanate, trimethoprim/sulfamethoxazole and ciprofloxacin. Similarly, the proportion of Klebsiella pneumoniae isolates resistant to extended-spectrum cephalosporins, amoxicillin/clavulanate, trimethoprim/sulfamethoxazole, ciprofloxacin and carbapenems increased during the study. The proportion of Enterobacter cloacae isolates resistant to ceftazidime and trimethoprim/sulfamethoxazole increased. The proportion of both ESBL-positive E. coli and K. pneumoniae (including bloodstream isolates) increased significantly between 2007 and 2016. A reduction in the proportion of Pseudomonas aeruginosa that were ciprofloxacin, cefepime, colistin, amikacin and gentamicin resistant and an increase in the proportion of P. aeruginosa isolates non-susceptible to meropenem were observed. The proportion of isolates of Staphylococcus aureus non-susceptible to clarithromycin, clindamycin and trimethoprim/sulfamethoxazole decreased over time while an increase in the proportion of isolates of Streptococcus pneumoniae non-susceptible to clarithromycin, clindamycin and doxycycline was observed. CONCLUSIONS: Increases in Enterobacteriaceae resistance to multiple classes of antimicrobials, increases in ESBL-positive E. coli and K. pneumoniae, and the small but significant increase in carbapenem-resistant K. pneumoniae were the most remarkable changes in antimicrobial resistance observed from 2007 to 2016 in Canada.

Antimicrobial-resistant pathogens in Canadian ICUs: results of the CANWARD 2007 to 2016 study.

OBJECTIVES: To describe the microbiology and antimicrobial resistance patterns of cultured samples acquired from Canadian ICUs. METHODS: From 2007 to 2016, tertiary care centres from across Canada submitted 42938 bacterial/fungal isolates as part of the CANWARD surveillance study. Of these, 8130 (18.9%) were from patients on ICUs. Susceptibility testing guidelines and MIC interpretive criteria were defined by CLSI. RESULTS: Of the 8130 pathogens collected in this study, 58.2%, 36.3%, 3.1% and 2.4% were from respiratory, blood, wound and urine specimens, respectively. The top five organisms collected from Canadian ICUs accounted for 55.4% of all isolates and included Staphylococcus aureus (21.5%), Pseudomonas aeruginosa (10.6%), Escherichia coli (10.4%), Streptococcus pneumoniae (6.5%) and Klebsiella pneumoniae (6.4%). MRSA accounted for 20.7% of S. aureus collected, with community-associated (CA) MRSA genotypes increasing in prevalence over time (P < 0.001). The highest susceptibility rates among MRSA were 100% for vancomycin, 100% for ceftobiprole, 100% for linezolid, 99.7% for ceftaroline, 99.7% for daptomycin and 99.7% for tigecycline. The highest susceptibility rates among E. coli were 100% for tigecycline, 99.9% for meropenem, 99.7% for colistin and 94.2% for piperacillin/tazobactam. MDR was identified in 26.3% of E. coli isolates, with 10.1% producing an ESBL. The highest susceptibility rates among P. aeruginosa were 97.5% for ceftolozane/tazobactam, 96.1% for amikacin, 94.7% for colistin and 93.3% for tobramycin. CONCLUSIONS: The most active agents against Gram-negative bacilli were the carbapenems, tigecycline and piperacillin/tazobactam. Against Gram-positive cocci, the most active agents were vancomycin, daptomycin and linezolid. The prevalence of CA-MRSA genotypes and ESBL-producing E. coli collected from ICUs increased significantly over time.

In vitro activity of ceftolozane/tazobactam versus antimicrobial non-susceptible Pseudomonas aeruginosa clinical isolates including MDR and XDR isolates obtained from across Canada as part of the CANWARD study, 2008-16.

Objectives: Ceftolozane/tazobactam is a novel ß-lactam ß-lactamase inhibitor combination with a broad spectrum of activity that includes Pseudomonas aeruginosa. The purpose of this study was to evaluate the in vitro activity of ceftolozane/tazobactam and relevant comparators versus a large collection of antimicrobial non-susceptible P. aeruginosa clinical isolates recovered from patients across Canada (CANWARD, 2008-16). Methods: Susceptibility testing was performed on P. aeruginosa clinical isolates obtained from sentinel hospitals across Canada between January 2008 and December 2016 using broth microdilution, as described by the CLSI. MDR P. aeruginosa were defined as isolates that tested non-susceptible to at least one antimicrobial from ≥3 classes. XDR P. aeruginosa were defined as isolates that tested non-susceptible to at least one antimicrobial from ≥5 classes. Results: In total, 3229 P. aeruginosa isolates were obtained as a part of CANWARD. Ceftolozane/tazobactam was the most active antimicrobial evaluated, with 98.3% of isolates testing susceptible. The percentage of antimicrobial non-susceptible isolates that remained susceptible to ceftolozane/tazobactam ranged from 85.3% (amikacin non-susceptible subset) to 95.0% (ciprofloxacin non-susceptible subset). Four-hundred and sixty-two P. aeruginosa isolates were MDR (14.3% of all isolates tested) and 84 were XDR (2.6% of all isolates tested). Ceftolozane/tazobactam demonstrated excellent in vitro activity versus the MDR and XDR isolates, with 90.5% and 78.6% remaining susceptible, respectively. Conclusions: Ceftolozane/tazobactam demonstrated excellent in vitro activity against antimicrobial non-susceptible P. aeruginosa clinical isolates, including MDR and XDR subsets. It may prove useful in the treatment of infections caused by these organisms.

Disseminated Mycobacterium bovis infection post-kidney transplant following remote intravesical BCG therapy for bladder cancer.

Intravesical Bacillus Camlette-Guérin (BCG) is the treatment of choice for non-muscle invasive bladder cancer, and has been used successfully for over 40 years. A rare and potentially fatal complication of intravesical BCG therapy is BCG-induced sepsis. We report a rare case in which a patient with end-stage renal disease secondary to chronic granulomatous interstitial nephritis underwent remote, pre-transplant intravesical BCG treatment for high-grade non-invasive papillary bladder carcinoma. The patient subsequently received a deceased donor kidney transplant 5 years after BCG therapy, with thymoglobulin induction therapy and standard triple maintenance immunosuppression. Two years post-transplant, he developed BCG-induced sepsis confirmed by cultures from urine, blood, and left native kidney biopsy. He died from disseminated BCG-induced sepsis and failure of his renal allograft. This case highlights the potential adverse reactions associated with intravesical BCG therapy that may occur years after bladder cancer therapy is completed, and should heighten physician awareness for BCG-related infections during pre-transplant assessment and post-transplant care of solid organ transplants recipients.

Evaluation of three MALDI-TOF mass spectrometry libraries for the identification of filamentous fungi in three clinical microbiology laboratories in Manitoba, Canada.

Matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) is commonly used by clinical microbiology laboratories to identify bacterial pathogens and yeasts, but not for the identification of moulds. Recent progress in extraction protocols and the composition of comparative libraries support potential application of MALDI-TOF MS for mould identification in clinical microbiology laboratories. We evaluated the performance of the Bruker Microflex™ MALDI-TOF MS instrument (Billerica, MA, USA) to identify clinical isolates and reference strains of moulds using 3 libraries, the Bruker mould library, the National Institutes of Health (NIH) library and the Mass Spectrometry Identification (MSI) online library, and compared those results to conventional (morphological) and molecular (18S/ITS; gold standard) identification methods. All 3 libraries demonstrated greater accuracy in genus identification (≥94.9%) than conventional methods (86.4%). MALDI-TOF MS identified 73.3% of isolates to species level compared to only 31.7% by conventional methods. The MSI library demonstrated the highest rate of species-level identification (72.0%) compared to NIH (19.5%) and Bruker (13.6%) libraries. Greater than 20% of moulds remained unidentified to species level by all 3 MALDI-TOF MS libraries primarily because of library limitations or imperfect spectra. The overall identification rate of each MALDI-TOF MS library depended on the number of species and the number of spectra representing each species in the library.