Evaluation of the utility and cost of secondary confirmatory testing for Neisseria gonorrhoeae identification from culture.

Current guideline recommends the use of two identification methods for Neisseria gonorrhoeae. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is now used for primary identification and may be sufficient for definitive identification of N. gonorrhoeae. The performance of three secondary tests (BactiCard, RapID NH and NET test) were compared using 45 bacterial isolates, including 37 Neisseria species. These secondary tests demonstrated diminished specificity (67% - 88%) for N. gonorrhoeae compared with MALDI-TOF. Additionally, data from six clinical microbiology laboratories was used to compare confirmatory test costs and the agreement of results with MALDI-TOF. Discrepancies were documented for 9.4% of isolates, though all isolates (n= 288) identified by MALDI-TOF as N. gonorrhoeae were confirmed by the reference laboratory. These data demonstrate that MALDI-TOF alone is sufficient for N. gonorrhoeae identification, as secondary did not add diagnostic value but do add costs to the testing process.

Evaluation of 5 Polymerase Chain Reaction Assays for the Detection of Mpox Virus.

BACKGROUND: In 2022, the global dissemination of mpox virus (MPXV) outside endemic regions prompted the expansion of diagnostic testing worldwide. This study assesses the performance characteristics of 5 real-time polymerase chain reaction (PCR) assays in detecting MPXV during the 2022 outbreak. METHODS: Clinical specimens collected from patients across Ontario, Canada, were tested on the following assays: RealStar Orthopoxyvirus PCR and FlexStar Monkeypox virus PCR (Altona Diagnostics), Novaplex MPXV (Seegene), VIASURE Monkeypox virus Real Time PCR Reagents (CerTest Biotec), and a laboratory-developed test. Positive percent agreement (PPA), negative percent agreement (NPA), relative limit of detection (LOD), and precision were evaluated and MPXV lineages were determined using an amplicon-based whole-genome sequencing (WGS) assay. RESULTS: Swabs were collected from various anatomic sites (65 positive and 30 negative). All assays demonstrated 100% NPA (95% confidence interval, 88.4%/88.1%-100.0%), with PPA ranging from 92.2% (82.7%-97.4%) to 96.9% (89.3%-99.6%). LOD and precision were comparable across assays, with coefficient of variations <3%. WGS analysis identified 6 lineages, all belonging to subclade IIb. CONCLUSIONS: The assays exhibited excellent PPA, NPA, LOD, and precision. Ongoing performance monitoring is essential to detect assay escape mutants and ensure universal detection of evolving MPXV strains.

Swish and gargle saliva sampling is a patient-friendly and comparable alternative to nasopharyngeal swabs to detect SARS-CoV-2 in outpatient settings for adults and children.

IMPORTANCE: Widespread and frequent testing for COVID-19 was an important strategy to identify infected patients to isolate and control the spread of the disease during the pandemic. The nasopharyngeal swab (NPS) global supply chain and access to trained healthcare professionals for standard NPS collection were often compromised. Patient discomfort and limited access challenged health systems to reach large numbers for testing in adult and pediatric populations. Our study revealed that swish and gargle saliva (SGS) was comparable to NPS in detecting SARS-CoV-2 and more patient-friendly than NPS. Patients were more likely to repeat the test with SGS. SGS was amenable to self-collection instead of relying on skilled professionals. This comprehensive evaluation highlights the challenges of comparing the accuracy of new methods to imperfect gold standards and identifies additional patient-centric factors that should be considered when defining such standards. Thus, SGS is an advantageous alternative specimen collection for outpatient en masse testing.

Wound swab quality grading is dependent on Gram smear screening approach.

Superficial skin swab collections are inherently low-quality and may be of little clinical value due to their poor sensitivity and specificity. Clinical microbiology laboratories can use Gram smears to screen and differentiate higher and lower quality specimens to direct the extent of potential pathogen work up, including antimicrobial susceptibility testing (AST). We compared the impact of two different smear grading approaches to our current reporting practices for superficial wound swab cultures. Two variations of the Q score methodology (low power under 10X (QS10) and high power under 100X (QS100) were compared to our existing oil immersion method (OM100) (100X). We further evaluated the QS100 method by scoring superficial swab smears previously screened by OM100 from cultures submitted between November 2018 and December 2019. No significant difference in the number of low-quality specimens (N = 50) was identified by QS10 or QS100 grading (N = 9; 18%; N = 8; 16% respectively). Among 968 additional QS100 screened smears, 67 (6.9%) low quality swabs were identified and 7.4% fewer organisms (76/1020 organisms) would require reporting with AST. Implementing the Q score for superficial wound swab cultures would provide minimal improvements in their clinical relevance, laboratory quality and efficiency in our laboratory due to the low number of poor-quality swabs received.

Burden of Severe Illness Associated With Laboratory-Confirmed Influenza in Adults Aged 50-64 Years, 2010-2011 to 2016-2017.

Background: Understanding the burden of influenza is necessary to optimize recommendations for influenza vaccination. We describe the epidemiology of severe influenza in 50- to 64-year-old residents of metropolitan Toronto and Peel region, Canada, over 7 influenza seasons. Methods: Prospective population-based surveillance for hospitalization associated with laboratory-confirmed influenza was conducted from September 2010 to August 2017. Conditions increasing risk of influenza complications were as defined by Canada's National Advisory Committee on Immunization. Age-specific prevalence of medical conditions was estimated using Ontario health administrative data. Population rates were estimated using Statistics Canada data. Results: Over 7 seasons, 1228 hospitalizations occurred in patients aged 50-64 years: 40% due to A(H3N2), 30% A(H1N1), and 22% influenza B. The average annual hospitalization rate was 15.6, 20.9, and 33.2 per 100 000 in patients aged 50-54, 55-59, and 60-64 years, respectively; average annual mortality was 0.9/100 000. Overall, 33% of patients had received current season influenza vaccine; 963 (86%) had ≥1 underlying condition increasing influenza complication risk. The most common underlying medical conditions were chronic lung disease (38%) and diabetes mellitus (31%); 25% of patients were immunocompromised. The average annual hospitalization rate was 6.1/100 000 in those without and 41/100 000 in those with any underlying condition, and highest in those with renal disease or immunocompromise (138 and 281 per 100 000, respectively). The case fatality rate in hospitalized patients was 4.4%; median length of stay was 4 days (interquartile range, 2-8 days). Conclusions: The burden of severe influenza in 50- to 64-year-olds remains significant despite our universal publicly funded vaccination program. These data may assist in improving estimates of the cost-effectiveness of new strategies to reduce this burden.

Emergence of a mutation in the nucleocapsid gene of SARS-CoV-2 interferes with PCR detection in Canada.

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) was met with rapid development of robust molecular-based detection assays. Many SARS-CoV-2 molecular tests target multiple genetic regions of the virus to maximize detection and protect against diagnostic escape. Despite the relatively moderate mutational rate of SARS-CoV-2, numerous mutations with known negative impact on diagnostic assays have been identified. In early 2021, we identified four samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing revealed a single common mutation in the N gene C29200T. Spatiotemporal analysis showed that the mutation was found in at least six different Canadian provinces from May 2020 until May 2021. Phylogenetic analysis showed that this mutation arose multiple times in Canadian samples and is present in six different variants of interest and of concern. The Cepheid testing platform is commonly used in Canada including in remote regions. As such, the existence of N gene mutation dropouts required further investigation. While commercial SARS-CoV-2 molecular detection assays have contributed immensely to the response effort, many vendors are reluctant to make primer/probe sequences publicly available. Proprietary primer/probe sequences create diagnostic 'blind spots' for global SARS-CoV-2 sequence monitoring and limits the ability to detect and track the presence and prevalence of diagnostic escape mutations. We hope that our industry partners will seriously consider making primer/probe sequences available, so that diagnostic escape mutants can be identified promptly and responded to appropriately to maintain diagnostic accuracy.

Fourier Transform Infrared Spectroscopy for Typing Burkholderia cenocepacia ET12 Isolates.

The IR Biotyper and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using ClinProTools software (MALDI-TOF MS-ClinProTools) are two novel typing methods that rely on the analysis of carbohydrate and peptide residues in intact bacterial cells. These two methods have shown promising results in the rapid and accurate typing of bacteria. In this study, we evaluated these novel typing methods in comparison with genotypic typing for cluster analysis of Burkholderia cenocepacia epidemic strain ET12, isolated from adult cystic fibrosis patients. Sixty-six isolates of B. cenocepacia were used in this study, 35 of which were identified as the ET12 strain and 31 as non-ET12 strains by repetitive-element PCR (rep-PCR). Twelve isolates were used for the creation of typing models using IR Biotyper and MALDI-TOF MS-ClinProTools, and 54 isolates were used for external validation of the typing models. The IR Biotyper linear discriminant analysis (LDA) model had a diagnostic sensitivity of 84.6% for typing the epidemic strain, ET12. At a cutoff of 70%, MALDI-TOF MS-ClinProTools had 87.5% diagnostic sensitivity in detecting the ET12 strain (P = 1.00). Both methods had a diagnostic specificity of ≥80% for detecting the ET12 strain. In conclusion, IR Biotyper and MALDI-TOF MS-ClinProTools offer rapid typing using proteomics and analysis of small cellular molecules with a low running cost. Our pilot study showed suboptimal accuracy of both methods for typing outbreak strains of B. cenocepacia. Extending the spectral region analyzed by the IR Biotyper can improve the accuracy and has the potential of improving the generalizability of this technique for typing other organisms. IMPORTANCE Respiratory infections due to Burkholderia cenocepacia, particularly the ET12 epidemic strain, are considered sentinel events for persons with cystic fibrosis, as they are often associated with person-to-person transmission and accelerated decline in lung function and early mortality. Current typing methods are generally only available at reference centers, with long turn-around-times, which can affect the identification of outbreaks and critical patient triage. This pilot study aims to add to the growing literature illustrating the potential utility of Fourier transform infrared spectroscopy (FTIR), a novel rapid method, for the successful typing of clinically significant bacteria. In this study, we evaluated its utility to discriminate between the ET12 clone and non-ET12 isolates of B. cenocepacia and compared it to proteomics cluster analysis using MALDI-TOF MS and ClinProTools software. Both methods had encouraging but suboptimal accuracy (≥85% sensitivity and ≥83% specificity), which will likely be improved by extending the spectral region analyzed by the IR Biotyper with updated software.

The Benefits and Harms of Antibiotic Prophylaxis for Urinary Tract Infection in Older Adults.

BACKGROUND: The role of antibiotics in preventing urinary tract infection (UTI) in older adults is unknown. We sought to quantify the benefits and risks of antibiotic prophylaxis among older adults. METHODS: We conducted a matched cohort study comparing older adults (≥66 years) receiving antibiotic prophylaxis, defined as antibiotic treatment for ≥30 days starting within 30 days of a positive culture, with patients with positive urine cultures who received antibiotic treatment but did not receive prophylaxis. We matched each prophylaxis recipient to 10 nonrecipients based on organism, number of positive cultures, and propensity score. Outcomes included (1) emergency department (ED) visit or hospitalization for UTI, sepsis, or bloodstream infection within 1 year; (2) acquisition of antibiotic resistance in urinary tract pathogens; and (3) antibiotic-related complications. RESULTS: Overall, 4.7% (151/3190) of UTI prophylaxis patients and 3.6% (n = 1092/30 542) of controls required an ED visit or hospitalization for UTI, sepsis, or bloodstream infection (hazard ratio [HR], 1.33; 95% confidence interval [CI], 1.12-1.57). Acquisition of antibiotic resistance to any urinary antibiotic (HR, 1.31; 95% CI, 1.18-1.44) and to the specific prophylaxis agent (HR, 2.01; 95% CI, 1.80-2.24) was higher in patients receiving prophylaxis. While the overall risk of antibiotic-related complications was similar between groups (HR, 1.08; 95% CI, .94-1.22), the risk of Clostridioidesdifficile and general medication adverse events was higher in prophylaxis recipients (HR [95% CI], 1.56 [1.05-2.23] and 1.62 [1.11-2.29], respectively). CONCLUSIONS: Among older adults with UTI, the harms of long-term antibiotic prophylaxis may outweigh their benefits.

Antibiotic susceptibility reporting and association with antibiotic prescribing: a cohort study.

OBJECTIVE: Selective reporting of antibiotic susceptibility test results may help guide appropriate antibiotic prescribing, particularly for urinary tract infections. Our objective was to describe laboratory urine culture susceptibility reporting practices and to estimate their impact on antibiotic prescribing in outpatients. METHODS: We examined all positive urine cultures with Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis associated with an antibiotic prescription among outpatients over 65 years of age in Ontario, Canada from 2014 through 2017. We evaluated antibiotic prescribing in the empirical window (1-3 days before culture result) and in the directed window (0-5 days after culture result). Unadjusted and adjusted odds ratios were reported to estimate the association between reporting and prescribing. RESULTS: In total 113 780 eligible urine cultures from 48 laboratories were included in the study cohort. Susceptibility reporting practices were highly variable between laboratories, with a range across antibiotics from norfloxacin (n = 5/48, 10.4% reporting) to nitrofurantoin (n = 40/48, 83.3% reporting). Reporting antibiotic susceptibility was associated with increased odds of prescribing that antibiotic in the directed window (aOR 2.98, 95%CI 2.07-4.28). At the laboratory level, the proportion of urine cultures reporting specific antibiotic susceptibility results was also associated with an increase in prescribing of that antibiotic in the empirical window (adjusted OR 1.23, 95%CI 1.13-1.33, per 25% increase in reporting). CONCLUSIONS: Laboratory reporting of antibiotic susceptibility results for urine cultures is associated with empirical and directed prescribing of the reported antibiotics. Laboratories can play an important role in guiding appropriate antibiotic selection for urinary indications.

COVID-19 Outbreak in an Urban Hemodialysis Unit.

RATIONALE & OBJECTIVE: Hemodialysis patients are at increased risk for coronavirus disease 2019 (COVID-19) transmission due in part to difficulty maintaining physical distancing. Our hemodialysis unit experienced a COVID-19 outbreak despite following symptom-based screening guidelines. We describe the course of the COVID-19 outbreak and the infection control measures taken for mitigation. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: 237 maintenance hemodialysis patients and 93 hemodialysis staff at a single hemodialysis center in Toronto, Canada. EXPOSURE: Universal screening of patients and staff for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OUTCOMES: The primary outcome was detection of SARS-CoV-2 in nasopharyngeal samples from patients and staff using reverse transcriptase-polymerase chain reaction (RT-PCR). ANALYTICAL APPROACH: Descriptive statistics were used for clinical characteristics and the primary outcome. RESULTS: 11 of 237 (4.6%) hemodialysis patients and 11 of 93 (12%) staff members had a positive RT-PCR test result for SARS-CoV-2. Among individuals testing positive, 12 of 22 (55%) were asymptomatic at time of testing and 7 of 22 (32%) were asymptomatic for the duration of follow-up. One patient was hospitalized at the time of SARS-CoV-2 infection and 4 additional patients with positive test results were subsequently hospitalized. 2 (18%) patients required admission to the intensive care unit. After 30 days' follow-up, no patients had died or required mechanical ventilation. No hemodialysis staff required hospitalization. Universal droplet and contact precautions were implemented during the outbreak. Hemodialysis staff with SARS-CoV-2 infection were placed on home quarantine regardless of symptom status. Patients with SARS-CoV-2 infection, including asymptomatic individuals, were treated with droplet and contact precautions until confirmation of negative SARS-CoV-2 RT-PCR test results. Analysis of the outbreak identified 2 index cases with subsequent nosocomial transmission within the dialysis unit and in shared shuttle buses to the hemodialysis unit. LIMITATIONS: Single-center study. CONCLUSIONS: Universal SARS-CoV-2 testing and universal droplet and contact precautions in the setting of an outbreak appeared to be effective in preventing further transmission.

Nudging In MicroBiology Laboratory Evaluation (NIMBLE): A scoping review.

BACKGROUND: Nudging in microbiology is an antimicrobial stewardship strategy to influence decision making through the strategic reporting of microbiology results while preserving prescriber autonomy. The purpose of this scoping review was to identify the evidence that demonstrates the effectiveness of nudging strategies in susceptibility result reporting to improve antimicrobial use. METHODS: A search for studies in Ovid MEDLINE, Embase, PsycINFO, and All EBM Reviews was conducted. All simulated and vignette studies were excluded. Two independent reviewers were used throughout screening and data extraction. RESULTS: Of a total of 1,346 citations screened, 15 relevant studies were identified. Study types included pre- and postintervention (n = 10), retrospective cohort (n = 4), and a randomized controlled trial (n = 1). Most studies were performed in acute-care settings (n = 13), and the remainder were in primary care (n = 2). Most studies used a strategy to alter the default antibiotic choices on the antibiotic report. All studies reported at least 1 outcome of antimicrobial use: utilization (n = 9), appropriateness (n = 7), de-escalation (n = 2), and cost (n = 1). Moreover, 12 studies reported an overall benefit in antimicrobial use outcomes associated with nudging, and 4 studies evaluated the association of nudging strategy with subsequent antimicrobial resistance, with 2 studies noting overall improvement. CONCLUSIONS: The number of heterogeneous studies evaluating the impact of applying nudging strategies to susceptibility result reports is small; however, most strategies do show promise in altering prescriber's antibiotic selection. Selective and cascade reporting of targeted agents in a hospital setting represent the majority of current research. Gaps and opportunities for future research identified from our scoping review include performing prospective randomized controlled trials and evaluating other approaches aside from selective reporting.

Evaluation of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry and ClinPro Tools as a Rapid Tool for Typing Streptococcus pyogenes.

BACKGROUND: Timely strain typing of group A Streptococci (GAS) is necessary to guide outbreak recognition and investigation. We evaluated the use of (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) combined with cluster analysis software to rapidly distinguish between related and unrelated GAS isolates in real-time. METHODS: We developed and validated a typing model using 177 GAS isolates with known emm types. The typing model was created using 43 isolates, which included 8 different emm types, and then validated using 134 GAS isolates of known emm types that were not included in model generation. RESULTS: Twelve spectra were generated from each isolate during validation. The overall accuracy of the model was 74% at a cutoff value of 80%. The model performed well with emm types 4, 59, and 74 but showed poor accuracy for emm types 1, 3, 12, 28, and 101. To evaluate the ability of this tool to perform typing in an outbreak situation, we evaluated a virtual outbreak model using a "virtual outbreak strain; emm74" compared with a non-outbreak group or an "outgroup " of other emm types. External validation of this model showed an accuracy of 91.4%. CONCLUSIONS: This approach has the potential to provide meaningful information that can be used in real time to identify and manage GAS outbreaks. Choosing isolates characterized by whole genome sequencing rather than emm typing for model generation should improve the accuracy of this approach in rapidly identifying related and unrelated GAS strains.

Rapid detection of resistance to carbapenems and cephalosporins in Enterobacteriaceae using liquid chromatography tandem mass spectrometry.

Carbapenemase producing Enterobacteriaceae (CPE) are becoming a global healthcare concern. Current laboratory methods for the detection of CPE include screening followed by confirmatory phenotypic and genotypic tests. These processes would generally take ≥72 hours, which could negatively impact patient care and Infection Control practices. To this end, we developed a protocol for rapid resistance testing (RRT) to detect hydrolysis in a panel of beta lactam antibiotics consisting of ampicillin, cefazolin, cefotaxime and imipenem, using liquid chromatography tandem mass spectrometry. Ninety-nine beta lactamase producing Enterobacteriaceae isolates were used to evaluate the RRT method, 54 isolates were CPE and 45 isolates were Class A or AmpC beta lactamase producing Enterobacteriaceae but not carbapenemase producers. We also tested 10 E.coli isolates that were susceptible to ampicillin, cefazolin, cefotaxime and imipenem. Receiver Operating Characteristic (ROC) Curves analysis showed that imipenem had a sensitivity and a specificity of 100% for crabapenemase detection at hydrolysis cut off values that are greater than 50% and less than or equal to 80%. The RRT protocol can be conducted in a time frame of less than 2 hours. This preliminary study shows that the rapid resistance testing protocol might have utility for the rapid detection of CPE. Additional work with a greater number and variety of beta- lactamase producing Enterobacteriaceae isolates is required to validate these preliminary findings.

Direct identification and antimicrobial susceptibility testing of microorganisms from positive blood cultures following isolation by lysis-centrifugation.

We evaluated a direct from positive blood culture pelleting procedure that utilizes a lysis-centrifugation protocol for the identification of microorganisms by MALDI-TOF MS with subsequent antimicrobial susceptibility testing (AST) and rapid methicillin- and beta-lactam-resistance screening. The identification evaluation was performed on 125 cultures and resulted in the correct genus-level identification in 91.2% of cultures and a species-level concordance of 82.4% compared to routine subcultured growth. For the AST evaluation, susceptibility results from direct pelleting and subcultured growth for 187 cultures were compared; an average ±2-fold dilution agreement of 98.2% (1650/1681) and 98.6% (1375/1394) for Gram-negatives and -positives, respectively, was found. Major errors fell below 5% except for MRSA, which was falsely reported as oxacillin sensitive 17.2% (11/66) of the time. Lastly, the sensitivity and specificity of rapid MRSA screening were 94.7% (36/38) and 90.0% (9/10), respectively, while the ESBL screening results were 90.3% (65/72) and 100.0% (13/13) respectively.