BACKGROUND:Cytomegalovirus (CMV) is a major opportunistic infection in solid organ transplant (SOT) recipients. Although valganciclovir is first-line for CMV treatment, its use may be limited by resistance and myelotoxicity. Letermovir (LET) and maribavir (MBV) are increasingly used as alternative therapies, though real-world outcomes remain incompletely defined. This study evaluated real-world outcomes of LET and MBV in SOT recipients for whom standard CMV therapy was inadequate or not feasible. Viral clearance, breakthrough infection, refractory infection, and resistance emergence were assessed. METHODS:We conducted a retrospective cohort analysis of adult SOT patients receiving LET (2018-2025) and/or MBV (2019-2025) at two transplant centers. Primary outcomes included CMV breakthrough (≥ 500 IU/mL) during LET prophylaxis and CMV clearance (< 200 IU/mL) at the end of MBV treatment. Secondary outcomes included resistant, refractory, and recurrent CMV infection within 3 months post-therapy. RESULTS:Among 47 LET courses (41 patients), breakthrough CMV occurred in 14/32 (44%) secondary prophylaxis (SP) courses (median viral load [VL]: 1037 IU/mL, interquartile range [IQR] 673-3427) but in none of the 15 primary prophylaxis (PP) courses. No resistance to LET was detected during or after prophylaxis. MBV was administered in 21 courses (17 patients); MBV-refractory/resistant (R/R) infection occurred in 8 courses (38%), and 11 (52%) courses achieved CMV clearance. Within 3 months post-MBV, 14 (67%) courses had CMV VL ≥ 1000 IU/mL, and two (10%) courses experienced recurrence. MBV resistance emerged in 6/17 (35%) patients during or after treatment. CONCLUSIONS:LET was effective for PP but associated with frequent breakthroughs in SP patients. MBV was limited by incomplete viral suppression and emergent resistance. These findings highlight the variable effectiveness of LET and MBV in clinical practice and support the need for further research to define optimal antiviral use in SP and refractory CMV treatment.

Introduction. In cases where sputum is non-diagnostic or unavailable, bronchoscopy can yield high-value respiratory samples for tuberculosis (TB) diagnosis. Whilst Mycobacterium tuberculosis (MTB) complex culture remains the gold standard, molecular assays such as Xpert MTB/RIF Ultra (Xpert Ultra) are increasingly being used for rapid diagnosis. Gap Statement. Xpert Ultra is increasingly used for TB diagnosis and has been extensively evaluated on sputum specimens, but assessment of performance on bronchoscopy samples is more limited. Aim. To retrospectively evaluate the performance of Xpert Ultra on bronchoscopy specimens in comparison to culture in a low-incidence, high-resource setting. Methodology. Patients with a clinical suspicion of TB, who had non-diagnostic sputum or limited sputum production and underwent bronchoscopy between March 2019 and October 2023, were included in the study. Bronchoscopy specimens comprised bronchoalveolar lavages, bronchial washings and endobronchial ultrasound lymph node tissue biopsies. All included specimens underwent acid-fast bacilli (AFB) smear, mycobacterial culture and Xpert Ultra. Positive MTB culture was considered the reference standard for TB diagnosis. Results. One hundred thirty-five bronchoscopy samples from 126 patients were included. Cultures were positive for MTB in 47 out of 126 (37.3%) of included patients. Overall, positive percent agreement (PPA) and negative percent agreement (NPA) of Xpert Ultra to MTB culture were 93.6% and 98.7%, respectively. In 19 AFB smear-positive cases, Xpert Ultra had 100% PPA and NPA, whilst in 28 smear-negative cases, PPA and NPA were 89.3% and 98.6%, respectively. On average, positive culture results were available after 15.2 days of incubation (range, 5–42 days) versus 24 h for Xpert Ultra. Xpert Ultra PCR cycle threshold values correlated strongly with AFB-smear grade and time-to-culture positivity. Conclusion. Xpert Ultra performed on specimens collected via bronchoscopy demonstrated excellent agreement with culture, even in smear-negative cases. Our results support the use of the Ultra on bronchoscopy specimens for accurate and rapid TB diagnosis in a low-incidence setting.

Background Next-generation sequencing (NGS) for Hepatitis B virus (HBV) antiviral resistance (AVR) testing is a highly sensitive diagnostic method, able to detect low-level mutant subpopulations. Our clinical virology laboratory previously transitioned from DNA hybridization (INNO-LiPA) to NGS, initially with the GS Junior System and subsequently the MiSeq. The Oxford Nanopore Technology (ONT) sequencing system was evaluated for HBV resistance testing, with regards to sequencing accuracy and turn-around time. Methods We performed amplicon sequencing of the HBV polymerase gene from patient plasma and external quality assessment (EQA) samples on the MiSeq Reagent Nano Kit v2 and GridION ONT with R10.4.1 flowcells. Mutational analysis and genotyping were performed by DeepChek®Assay-HBV (version 2.0). Results A total of 49 patient samples and 15 EQA samples were tested on both the MiSeq and ONT. There was high agreement for both patient and EQA samples between the MiSeq and ONT systems, with regards to total drug resistance mutations detected and total patient sample agreement, 68/70 (97%) and 47/49 (96%), respectively. Conclusion The ONT NGS platform provided accurate HBV AVR results, with improved turn-around times. Sequencing error rates at AVR codons were below 1%.

BACKGROUND:Cytomegalovirus (CMV) gastrointestinal (GI) disease is traditionally diagnosed via histopathology of endoscopic tissue biopsies. The utility of tissue PCR for predicting CMV GI disease remains unclear. We conducted a 10-year retrospective single-center study comparing tissue PCR performance to histopathology for the diagnosis of CMV GI disease. METHODS:Adult patients with GI tissue biopsy between February 2014 and January 2024 were included. Qualitative tissue PCR was compared to histopathology (gold standard) to determine sensitivity, specificity, and receiver operating characteristic (ROC) curves. Log-binomial regression models were performed to evaluate potential predictors of CMV GI disease. RESULTS:Our study comprised 533 patients with 635 endoscopies. Underlying diagnoses included solid organ transplant, hematopoietic stem cell transplantation (HSCT), inflammatory bowel disease, and others. Histopathologic evidence of CMV disease was found in 41/635 biopsies and in 37/533 patients. Compared to histopathology, tissue PCR sensitivity was 100 % (95 % CI 91.4-100 %), and specificity was 71.7 % (67.9-75.3 %). Area under ROC curve (AUC) was 0.86 (0.84-0.88). Exclusion of specimens with cycle threshold >32 increased specificity to 82.7 % (79.4-85.6 %) but at the cost of decreased sensitivity (87.8 %, 73.8-95.9 %). Multivariable analyses demonstrated that plasma CMV DNAemia >1000 IU/mL increased risk of GI disease (risk ratio 10.9, 95 % CI 5.32-22.49) while HSCT was negatively associated (risk ratio 0.18, 95 % 0.05-0.78). CONCLUSION:CMV tissue PCR correctly identified CMV GI disease in 100 % of histology-proven cases. Specificity was poor, likely reflecting detection of viral shedding. Overall, our study suggests that CMV tissue PCR should be reserved to rule out CMV GI disease in high pre-test probability settings (e.g. patients with known risk factors and compatible symptoms).

Helicobacter pylori is associated with gastrointestinal diseases including gastritis and peptic ulcers. Despite its significance, there is a scarcity of antimicrobial susceptibility testing (AST) data available for this organism in North America. The aim of this study was to assess the AST profile and identify factors associated with H. pylori culture positivity in a cohort of patients with refractory H. pylori undergoing gastric biopsies. We retrospectively reviewed gastric biopsy specimens received for culture between July 2009 and February 2023. We analyzed specimen transport time, Gram smear results, direct urease test findings, culture positivity and AST profiles. Using gradient strip methodology and European Committee on Antimicrobial Susceptibility Testing breakpoints, AST was conducted for amoxicillin, clarithromycin, metronidazole, levofloxacin and tetracycline. Of 579 biopsy samples received for H. pylori culture, 228 (39.4%) tested positive. Samples transported within <1 h had significantly higher odds (1.81 times, P < 0.015) of being culture positive compared to those with longer transport times. Smear-positive samples had substantially higher odds (18.8 times, P < 0.001) of culture positivity compared to smear-negative. Urease-positive samples demonstrated notably higher odds (7.7 times, P < 0.001) of culture positivity compared to urease-negative samples. The collection of isolates from gastric biopsies showed susceptibility rates of 97.3% to amoxicillin, 99.1% to tetracycline, 50.4% to levofloxacin, 25.9% to metronidazole and 12.9% to clarithromycin. Short sample transport time was associated with improved H. pylori recovery rates. In this cohort of refractory H. pylori cases, susceptibility rates were high for amoxicillin and tetracycline and low for clarithromycin, metronidazole and levofloxacin. Susceptibility rates remained stable over time.

We performed phenotypic susceptibility testing and whole-genome sequencing to investigate in vitro mecillinam activity against multidrug-resistant (MDR) Shigella. Ninety-five isolates (53 [56%] Shigella sonnei and 42 [44%] Shigella flexneri) were included. The MIC50 and MIC90 were 1 and 2 µg/mL, respectively, by agar dilution, and all zone diameters were ≥15 mm by disc diffusion testing. No known clinically relevant mecillinam resistance mutations (including cysB) were detected. The favorable MIC profile and absence of resistance markers make pivmecillinam an agent worth considering for the treatment of infections caused by MDR Shigella. Clinically feasible susceptibility testing methods and standardized interpretations for mecillinam and Shigella spp. are needed to guide therapy. IMPORTANCE Rapidly emerging resistance in Shigella species leaves few antibiotic treatment options. The World Health Organization recommends pivmecillinam, a prodrug of mecillinam, for the treatment of Shigella infections. However, little is known about the susceptibility of Shigella spp. to mecillinam in North America. We performed mecillinam susceptibility testing on our collection of resistant Shigella isolates and investigated genetic mechanisms of resistance using whole-genome sequencing. We observed a favorable mecillinam susceptibility profile and a lack of known genetic mechanisms of resistance. However, in the absence of standardized laboratory guidelines for mecillinam susceptibility testing, interpreting susceptibility test results is challenging. We propose that further studies are needed to correlate susceptibility testing data with clinical outcomes, with the aim of establishing standardized clinical breakpoints for Shigella spp. and mecillinam.

BACKGROUND:Next-generation sequencing for hepatitis B virus (HBV) and cytomegalovirus (CMV) antiviral drug resistance (AVDR) testing improves the sensitivity of variant detection, but availability of bioinformatics and analytical pipelines are key barriers to implementation. METHODS:Plasma was extracted on MagNA Pure 24 (Roche Diagnostics) and next-generation sequencing performed on GridION (Oxford Nanopore Technologies) with R.10.4.1 flowcells. An in-house bioinformatics pipeline was developed using Nextflow and deployed on Microsoft Azure to process FASTQ files and automate reporting of HBV genotype and AVDR, as well as CMV AVDR (UL97/54). RESULTS:A total of 71 samples for HBV genotyping and AVDR testing and 56 samples for CMV AVDR testing were analyzed and compared to reference pipelines (DeepChek® HBV and CMV). All HBV genotypes and resistant mutations were concordant. For CMV, 74 mutations were identified in the UL97/54 region by both pipelines. However, our in-house developed method identified an additional UL97 drug resistant mutation (del598-603) in one sample. CONCLUSIONS:A custom bioinformatics pipeline was developed for HBV and CMV genotyping and AVDR sequencing, which could be adapted to other targets to enable our clinical laboratory to expand clinical testing using next-generation sequencing.

Vibrio cholerae is a motile, gram-negative bacterium typically found in brackish, coastal waters and most often associated with gastroenteritis. This case details a rare presentation of non-O1/O139 V. cholerae cystitis in an immunocompetent 27-year-old male with no urogenital tract abnormalities following exposure to geothermal hot springs in California. We present the diagnostic considerations and challenges in clinical management for this case, and the relevance of climate change for understanding epidemiology and exposure risk relevant to clinical practice.

BACKGROUND:Timely identification of extended-spectrum beta-lactamase (ESBL)-producing bacteria supports early antimicrobial optimization, especially for patients with invasive infections. NG-Test® CTX-M MULTI (CTX-M LFA) is a rapid immunochromatographic assay for detection of Cefotaximase-Munich (CTX-M)-type ESBL. We evaluated its performance against existing molecular platforms. METHODS:A sampling of archived Gram-negative bacteria (including Enterobacterales and non-fermenters) was included in the study. All isolates underwent testing for CTX-M ESBL by CTX-M LFA. CTX-M LFA results were compared to results from molecular methods, laboratory-developed (LD)-PCR (2013-2019) or BCID2 (2022-2023). Discordances were resolved with a combination of ESBL phenotypic testing and an alternative PCR platform, or whole-genome sequencing. RESULTS:Seventy-eight isolates were included in the study, all of which underwent CTX-M LFA and molecular testing (n = 47 by LD-PCR, n = 31 by BCID2). Post-discordance analysis showed positive percent agreement of 89.7 % (95 %CI 71.5-97.2 %), negative percent agreement 100 % (95 %CI 90.9-100 %) and overall categorical agreement 96.2 %, κ=0.92 (95 %CI 88.4-99.0 %). One E. coli was negative by CTX-M LFA but positive for blaCTX-M by BCID2. Whole-genome sequencing identified a non-functional blaCTX-M-27 gene with adenine insertion at position 70 in codon 24, causing a frameshift mutation resulting in a premature stop codon (codon 58). CONCLUSION:CTX-M LFA improved performance in comparison with molecular methods by accurately identifying an isolate with a prematurely truncated protein as a true-negative for CTX-M. This highlights potential limitations of molecular-based resistance detection. CTX-M LFA was simple to perform and yielded results within 15 minutes.

Hepatitis B; Tenofovir resistance; Virological response.

BACKGROUND:Shigella sonnei has caused sexually transmitted enteric infections in men who have sex with men (MSM) in Vancouver. We recently observed a high rate of multidrug-resistant (MDR) S. sonnei bacteremia among persons experiencing homelessness (PEH). We aimed to describe the wider epidemiology, clinical outcomes, and genomics of S. sonnei infections over time. METHODS:A retrospective review of 163 patients with S. sonnei infections was undertaken from 2015 to 2022. We collected demographic, clinical, and microbiological data over 2 time periods: historical (2015-2020) and recent (2021-2022). Severe shigellosis definition included hospitalization, bacteremia, or death. Whole-genome sequencing was performed to identify genotype, infer relatedness, and predict antimicrobial resistance. RESULTS:S. sonnei infections increased from 8.3 (historical period) to 56.5 (recent period) cases/year. Over time, the primary population characteristics associated with shigellosis shifted from MSM (45; 98%) to PEH (86; 77%). The population intersection between MSM and PEH historically and recently was similar and occurred in 3 (6%) and 10 (9%) of patients, respectively. Severe shigellosis was significantly higher in the recent versus historical period (69 [61%] vs 7 [14%]; P < .001). A dominant clone of MDR S. sonnei, 3.6.1.1.2 (CipR.MSM5), emerged with resistance to all first- and second-line agents, yet with susceptibility to ceftriaxone. CONCLUSIONS:We observed a substantial increase in severe shigellosis and shift from sexually transmitted S. sonnei infections in MSM to likely environmental transmission among PEH. More severe disease associated with the 3.6.1.1.2 clone of MDR S. sonnei in PEH could be a result of underlying vulnerabilities of the affected population.

Colistin resistance testing methods such as broth microdilution (BMD) are time-consuming and labour intensive for clinical laboratories. MBT Lipid Xtract Kit on MALDI Biotyper Sirius System (Bruker, Billerica, MA, USA) utilizes lipidomic analysis to identify specific cell wall modifications associated with colistin resistance. We compared MBT to BMD (ComASP Colistin, Liofilchem) across 36 Gram-negative isolates (non-resistant MIC <= 2 mu g ml(-1), resistant MIC >= 4 mu g ml(-1)). All samples were tested twice on MBT with discrepant results repeated before assessing categorical agreement between MBT and BMD. 44.4% (16/36) of isolates were colistin resistant via BMD. MBT Lipid Xtract had 80.6% agreement (29/36) with BMD, with 5/7 discrepancies corrected to match upon repeat testing. There was 100% agreement for Escherichia coli isolates (n=16). The whole-genome sequencing was completed on the two discrepant Klebsiella pneumoniae isolates, with variants within colistin resistance-associated loci identified (MIC 0.5 mu g ml(-1): arnC S30T, pmrB T246A, lapB N212T, lpxM S253G, crrB Q287K and MIC >16 mu g ml(-1): arnC S30T, pmrB R90insRN, pmrB T246A, pmrA E57G, lpxM S253G). Further evaluation, particularly for non-E. coli, of MBT is required prior to implementation in clinical laboratories.

Background:Multiplex real-time RT-PCR assays for respiratory pathogens are valuable tools to optimize laboratory workflow and turnaround time. At a time when resurgence of influenza and respiratory syncytial virus (RSV) cases have been widely observed along with continued transmission of SARS-CoV-2, timely identification of all circulating respiratory viruses is crucial. This study evaluates the detection of low viral loads of SARS-CoV-2 by four multiplex molecular assays: Roche cobas 6800/8800 SARS-CoV-2 & Influenza A/B Test, Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV, cobas Liat SARS-CoV-2 & Influenza A/B, and a laboratory-developed test (LDT). Methods:Retrospective upper respiratory tract specimens positive for various respiratory viruses at a range of cycle threshold (Ct) values (18-40) were tested by four multiplex assays. Positive and negative percent agreement (PPA and NPA) with validated RT-PCR assays were calculated. Results:A total of 82 samples were assessed, with discordant results observed in a portion of the samples (10/82, 12.2%) where Ct values were >33. The majority of the discordant results (6/10, 60%) were false negatives. Overall, PPA was 100% (58/58) for cobas 6800, 97.4% (38/39) for GeneXpert, 100% (17/17) for Liat, and 90.5% (57/63) for the LDT. PPA for the LDT increased to 92.1% after manual review of amplification curves. Conclusions:Commercial multiplex respiratory virus assays have good performance for samples with medium to high viral loads (Ct values <33). Laboratories should consider appropriate test result review and confirmation protocols to optimize sensitivity, and may consider reporting samples with additional interpretive comments when low viral loads are detected.

Introduction. BK polyomavirus (BKPyV) quantitative testing is an important screening tool post-transplantation, although interpretation can be challenging due to lack of standardization, assay heterogeneity and variability of BKPyV DNA over time (in urine). Methods. Remnant clinical EDTA plasma and urine samples were tested by the cobas BKV test and a validated laboratory-developed test (LDT). Accuracy [positive and negative percent agreement (PPA and NPA), Pearson’s correlation, Bland–Altman analysis] and reproducibility were evaluated. To assess BKPyV DNA stability in urine, prospective urine samples were maintained at two different storage temperatures and tested in triplicate over 7 days. Results. Overall PPA was 95.6 % (43/45) and NPA was 94.4 % (170/180). For plasma, Pearson’s correlation (0.950) and Bland–Altman analysis (0.113±0.22 log 10 IU ml –1 ) showed high agreement. For neat urine, Pearson’s correlation (0.842) and Bland–Altman analysis (0.326±0.80 log 10 IU ml –1 ) showed somewhat higher variability. Reproducibility was high for the cobas BKV versus the LDT. BKPyV DNA levels in neat urine remained relatively stable over 7 days at both storage temperatures, although outlier results were intermittently detected. Conclusion. The cobas BKV test showed high agreement and reproducibility compared to the reference LDT. BKPyV viral load testing in urine has known limitations, but neat urine can be processed by the cobas BKV.

We conducted patient chart reviews and whole-genome sequencing of wound specimens containing presumptive Corynebacterium ulcerans from Vancouver, British Columbia, Canada, during July 2019-July 2023. Sequencing confirmed 8/14 isolates were C. ramonii and identified 2 distinct clusters. Molecular methods should be used to clinically differentiate potential toxin-producing Corynebacterium spp.

BackgroundMolecular syndromic panels can improve rapidity of results and ease clinical laboratory workflow, although caution has been raised for potential false-positive results. Upon implementation of a new panel for infectious diarrhea (BioFire® FilmArray® Gastrointestinal [GI] Panel, bioMérieux) in our clinical laboratory, a higher than expected number of stool samples with norovirus were detected.ObjectivesThe goal of this study was to investigate positive percent agreement and the false-positive rate of norovirus detected by the multiplex BioFire GI panel compared to a singleplex commercial assay.Study designFrom October 2023 to January 2024, all prospective stool samples with a positive norovirus result by BioFire had melting curves reviewed manually using the BioFire FilmArray Torch System. Stool samples further underwent testing by a supplementary real-time RT-PCR assay (Xpert® Norovirus, Cepheid) for comparative analysis.ResultsOf the 50 stool samples with norovirus detected by BioFire, 18 (36%) tested negative by Xpert (deemed "false-positives"). Furthermore, melting curve analysis revealed nearly all of these samples had atypical melting curve morphologies for the "Noro-1" target on BioFire (16/18, 89%), which was statistically significant (Odds Ratio 173.2, 95% CI [22.2, 5326.9], p<0.0001). Stool samples with multiple pathogens detected by BioFire including norovirus were not more likely to produce false-positive norovirus results (Odds Ratio 1, 95% CI [0.3, 3.3], p=1).ConclusionsAlthough not described in the manufacturer's Instructions for Use, we propose routine manual review of melting curves for the BioFire GI panel prior to reporting, to mitigate potential false-positive norovirus results.

Background In vitro data suggested reduced neutralizing capacity of sotrovimab, a monoclonal antibody, against Omicron BA.2 subvariant. However, limited in vivo data exist regarding clinical effectiveness of sotrovimab for coronavirus disease 2019 (COVID-19) due to Omicron BA.2. Methods A multicentre, retrospective cohort study was conducted at three Canadian academic tertiary centres. Electronic medical records were reviewed for patients ≥ 18 years with mild COVID-19 (sequencing-confirmed Omicron BA.1 or BA.2) treated with sotrovimab between February 1 to April 1, 2022. Thirty-day co-primary outcomes included hospitalization due to moderate or severe COVID-19; all-cause intensive care unit (ICU) admission, and all-cause mortality. Risk differences (BA.2 minus BA.1 group) for co-primary outcomes were adjusted with propensity score matching (e.g., age, sex, vaccination, immunocompromised status). Results Eighty-five patients were included (15 BA.2, 70 BA.1) with similar baseline characteristics between groups. Adjusted risk differences were non-statistically significant between groups for 30-day hospitalization (− 14.3%; 95% confidence interval (CI): − 32.6 to 4.0%), ICU admission (− 7.1%; 95%CI: − 20.6 to 6.3%), and mortality (− 7.1%; 95%CI: − 20.6 to 6.3%). Conclusions No differences were demonstrated in hospitalization, ICU admission, or mortality rates within 30 days between sotrovimab-treated patients with BA.1 versus BA.2 infection. More real-world data may be helpful to properly assess sotrovimab’s effectiveness against infections due to specific emerging COVID-19 variants.