Repeat virological and serological profiles in hospitalized patients initially tested by nasopharyngeal RT-PCR for SARS-CoV-2.

Real-time polymerase chain reaction (PCR) for SARS-CoV-2 is the mainstay of COVID-19 diagnosis, yet there are conflicting reports on its diagnostic performance. Wide ranges of false-negative PCR tests have been reported depending on clinical presentation, the timing of testing, specimens tested, testing method, and reference standard used. We aimed to estimate the frequency of discordance between initial nasopharyngeal (NP) PCR and repeat NP sampling PCR and serology in acutely ill patients admitted to the hospital. Panel diagnosis of COVID-19 infection is further utilized in discordance analysis. Included in the study were 160 patients initially tested by NP PCR with repeat NP sampling PCR and/or serology performed. The percent agreement between initial and repeat PCR was 96.7%, while the percent agreement between initial PCR and serology was 98.9%. There were 5 (3.1%) cases with discordance on repeat testing. After discordance analysis, 2 (1.4%) true cases tested negative on initial PCR. Using available diagnostic methods, discordance on repeat NP sampling PCR and/or serology is a rare occurrence.

Practical challenges to the clinical implementation of saliva for SARS-CoV-2 detection.

Due to global shortages of flocked nasopharyngeal swabs and appropriate viral transport media during the COVID-19 pandemic, alternate diagnostic specimens for SARS-CoV-2 detection are sought. The accuracy and feasibility of saliva samples collected and transported without specialized collection devices or media were evaluated. Saliva demonstrated good concordance with paired nasopharyngeal swabs for SARS-CoV-2 detection in 67/74 cases (90.5%), though barriers to saliva collection were observed in long-term care residents and outbreak settings. SARS-CoV-2 RNA was stable in human saliva at room temperature for up to 48 h after initial specimen collection, informing appropriate transport time and conditions.

Phenotypic and Genotypic Correlates of Penicillin Susceptibility in Nontoxigenic Corynebacterium diphtheriae, British Columbia, Canada, 2015-2018.

In 2015, the Clinical and Laboratory Standards Institute (CLSI) updated its breakpoints for penicillin susceptibility in Corynebacterium species from <1 mg/L to <0.12 mg/L. We assessed the effect of this change on C. diphtheriae susceptibility reported at an inner city, tertiary care center in Vancouver, British Columbia, Canada, during 2015-2018 and performed whole-genome sequencing to investigate phenotypic and genotypic resistance to penicillin. We identified 44/45 isolates that were intermediately susceptible to penicillin by the 2015 breakpoint, despite meeting previous CLSI criteria for susceptibility. Sequencing did not reveal ß-lactam resistance genes. Multilocus sequence typing revealed a notable predominance of sequence type 76. Overall, we saw no evidence of penicillin nonsusceptibility at the phenotypic or genotypic level in C. diphtheriae isolates from our institution. The 2015 CLSI breakpoint change could cause misclassification of penicillin susceptibility in C. diphtheriae isolates, potentially leading to suboptimal antimicrobial treatment selection.

HIV serology signal-to-cutoff ratio as a rapid method to predict confirmation of HIV infection.

Early and rapid detection of patients with HIV is a key to preventing further transmission. The purpose of this study was to assess the ability of signal-to-cutoff (S/CO) ratio from initial screening fourth-generation HIV serology to predict subsequent confirmation of HIV. Patients with a first-time positive HIV serology (S/CO ratio ≥ 1) from 2012 to 2016 were included. Ratios were compared to the results of confirmatory testing. Predictive probabilities (PPs) of a positive confirmatory result were calculated based on a logistic regression model. A total of 45,138 HIV serology tests were performed; 250 patients met inclusion criteria, comprising 84 (34%) HIV negative patients, 136 (54%) chronic infections, and 30 (12%) acute infections. The PP of a confirmed positive result increased with higher S/CO ratios, with a PP of 100% for a S/CO of 55 (95% CI 95-100). This study enables a more informed discussion of the probability of HIV infection, based on HIV serology S/CO thresholds, prior to a confirmatory result.

Clinical heterogeneity of patients with stool samples testing PCR+/Tox- from a two-step Clostridium difficile diagnostic algorithm.

The clinical significance of indeterminate (PCR+/Tox-) results for patients tested with a two-step algorithm for Clostridium difficile infection (CDI) is uncertain. We aimed to evaluate the clinical presentation and 8-week outcomes of patients with indeterminate test results. Patients with stool samples testing positive by PCR and negative by toxin A/B immunoassay between February 1, 2017, and April 30, 2018, were assessed by antimicrobial stewardship program (ASP) clinicians and classified as colonized or infected. Retrospective chart review was performed to obtain outcomes occurring within 8 weeks of testing, including recurrent C. difficile diarrhea, subsequent treatment for CDI, follow-up C. difficile testing, all-cause mortality, and CDI-related complications. In total, 110 PCR+/Tox- patients were evaluated. ASP classified 54% of patients as infected and 46% as colonized. Patients assessed and classified as colonized did not have increased adverse outcomes by 8 weeks compared to those assessed as infected, despite not receiving treatment for CDI. We conclude that PCR+/Tox- patients are heterogeneous with respect to clinical presentation. Negative toxin A/B immunoassay in a two-step algorithm should not be interpreted in isolation to distinguish colonization from infection as many PCR+/Tox- results may be clinically significant for CDI.

Epidemiologic and Genotypic Review of Carbapenemase-Producing Organisms in British Columbia, Canada, between 2008 and 2014.

Carbapenemase-producing organisms (CPOs) are a serious emerging problem for health care facilities worldwide. Owing to their resistance to most antimicrobial therapies, CPOs are difficult to treat and pose a challenge for infection prevention and control. Since 2010, lab-based surveillance for CPOs and PCR-based testing were implemented in British Columbia (BC), Canada. A review of CPOs in BC from 2008 to March 2014 was done to characterize the resistance mechanisms and possible clonal strain transmission and to compare pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and plasmid restriction fragment length polymorphism (RFLP) as molecular typing tools. During this study period, a total of 177 CPO cases were identified. Patient demographics and travel history were reviewed, and a descriptive analysis was carried out. PFGE profiles, MLST, and plasmid RFLP analysis for a subset of Escherichia coli, Klebsiella pneumoniae, and Enterobacter species isolates were obtained and analyzed. Our findings demonstrate that CPOs have been increasing in number in BC over time, from 1 isolate/year retrospectively identified in 2008 and 2009 to 82 isolates in 2013 and 30 isolates in the first quarter of 2014. Overall, K. pneumoniae isolates lack clonality, although some seemingly related clusters have been found. Plasmid analysis showed evidence of the spread of plasmids carrying carbapenemase-encoding genes between the examined isolates. Analysis of Enterobacter cloacae isolates revealed a more clonal nature of these CPOs in BC. The presence of related clusters provides evidence of interpatient organism transmission both within and between institutions. Although in our study, NDM-harboring E. cloacae isolates appeared to spread clonally, the spread of carbapenem resistance in K. pneumoniae seems to be plasmid mediated.

Characterization of Clostridium difficile Strains in British Columbia, Canada: A Shift from NAP1 Majority (2008) to Novel Strain Types (2013) in One Region.

Background. Clostridium difficile is a major cause of gastrointestinal illness. Epidemic NAP1 strains contain toxins A and B, a deletion in repressor tcdC, and a binary toxin. Objectives. To determine the molecular epidemiology of C. difficile in British Columbia and compare between two time points in one region. Methods. C. difficile isolates from hospital and community laboratories (2008) and one Island Health hospital laboratory (2013) were characterized by pulsed-field gel electrophoresis, PCR-ribotyping, toxin possession, tcdC genotype, and antimicrobial susceptibility. Results. In 2008, 42.7% of isolates had NAP1 designation. Hospital-collected isolates were associated with older patients and more NAP1 types. Unlike other isolates, most NAP1 isolates possessed binary toxin and a 19 bp loss in tcdC. All isolates were susceptible to metronidazole and vancomycin. A 2013 follow-up revealed a 28.9% decrease in NAP1 isolates and 20.0% increase in isolates without NAP designation in one region. Then, community-associated cases were seen in younger patients, while NAP types were evenly distributed. Isolates without NAP designation did not cluster with a PFGE pattern or ribotype. Conclusions. Evaluation of C. difficile infections within British Columbia revealed demographic associations, epidemiological shifts, and characteristics of strain types. Continuous surveillance of C. difficile will enable detection of emerging strains.

Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia.

BACKGROUND: Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP. METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ-]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP. RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%. CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients.

HISTORIQUE: Le Pneumocystis jirovecii (PJ), un champignon pathogène, provoque une grave pneumonie à Pneumocystis interstitielle (PPC) chez les patients immunodéprimés. Les chercheurs ont validé un test de réaction en chaîne par polymérase (PCR) en temps réel pour détecter le PJ et ainsi améliorer le diagnostic de PPC. MÉTHODOLOGIE: Les chercheurs ont d'abord vérifié 40 prélèvements de liquide bronchoalvéolaire (LBA) entreposés (20 cas positifs connus au PJ [PJ+] et 20 cas négatifs connus au PJ [PJ−]) au moyen du test moléculaire. Ils ont ensuite analysé 92 prélèvements séquentiels de LBA au moyen d'un test par immunofluorescence (IFA), puis d'un test de PCR en temps réel du PJ. Ils ont résolu les résultats divergents au moyen d'un nouveau test par PCR en temps réel des prélèvements de LBA axée sur une autre cible. Ils ont comparé le résultat du test de PCR en temps réel du PJ à la référence absolue (l'IFA) et à une référence modifiée, dans laquelle un véritable cas positif désignait un prélèvement positif par deux méthodes sur trois chez un patient atteint d'une PPC présumée. RÉSULTATS: Quatre-vingt-dix prélèvements de LBA (68 %) sur 132 provenaient de patients immunodéprimés. Treize prélèvements de LBA (14 %) sur 92 étaient PJ+ d'après l'IFA. Dans la présente étude, 40 prélèvements de LBA étaient PJ+, y compris tous les prélèvements positifs à l'IFA (n=13), tous les prélèvements de LBA PJ+ aiguillés (n=20) et sept autres prélèvements de LBA négatifs à l'IFA, mais positifs selon la référence modifiée. Par rapport à l'IFA, la PCR en temps réel du PJ avait une sensibilité, une spécificité et des valeurs prédictives positive et négative de 100 %, 91 %, 65 % et 100 %, respectivement. Par rapport à la référence modifiée, la PCR en temps réel du PJ avait une sensibilité, une spécificité et des valeurs prédictives positive et négative de 100 %. CONCLUSION: La PCR en temps réel du PJ en améliore la détection chez les patients immunodéprimés.

Automated molecular testing of saliva for SARS-CoV-2 detection.

With surging global demand for SARS-CoV-2 testing capacity, laboratories seek automated, high-throughput molecular solutions, particularly for specimens not requiring specialized collection devices or viral transport media. Saliva specimens submitted from patients under investigation for COVID-19 from March to July 2020 were processed in the laboratory with sterile phosphate-buffered saline in a 1:2 dilution and tested using manual extraction and a commercial assay for detection of the SARS-CoV-2 E gene (LightMix®) in comparison to the Roche cobas® SARS-CoV-2 Test on the cobas® 6800 instrument. 34.4% (22/64) of saliva samples were positive for SARS-CoV-2. Positive and negative concordance between the LightMix® and cobas® assays were 100%. The overall invalid rate for saliva on the cobas® 6800 (1/128, 0.78%) was similar to the baseline invalid rate observed for nasopharyngeal swabs/viral transport media. Saliva is a feasible specimen type for SARS-CoV-2 testing on the cobas® 6800 platform, with potential to improve turnaround time and enhance testing capacity.

Level of patients' knowledge, confidence, and acceptance regarding the role of residents in a family medicine teaching clinic.

BACKGROUND: Although participation of patients is essential for completing the training of medical residents, little is known about the relationships among patients' level of knowledge about the role and responsibilities of medical residents, their confidence in residents' abilities, and their acceptance toward receiving care from residents. The study sought to clarify if and how these three patient-resident relationship components are interrelated. METHODS: This is a cross-sectional study using a self-administered questionnaire distributed in 2016 to a convenience sample of adult patients (≥ 18 years old) visiting a family medicine teaching clinic. Proportions and chi-square statistics were used to describe and compare groups, respectively. RESULTS: Of the 471 patients who answered the questionnaire, only 28% were found to be knowledgeable about the role of family medicine residents. Between 54% and 83% of patients reported being highly confident in the ability of residents to perform five routine tasks. Of the patients surveyed, 69% agreed to see a resident during their next appointments. Patients with a high level of confidence in residents' abilities were more likely to agree to see a resident during future appointments (p <0.0001). There was no significant association between level of knowledge and either confidence or acceptance. CONCLUSIONS: Although the majority of patients had poor knowledge about the role of residents, this was not related to their acceptance of being cared for by residents. A higher level of confidence in residents' ability to perform certain tasks was associated with greater acceptance toward seeing a resident during future appointments.

CONTEXTE: Tandis que la participation des patients est essentielle pour la formation de résidents en médecine, on en sait peu sur le rapport entre le niveau de connaissance qu'ont les patients du rôle et des responsabilités des résidents, leur confiance dans les compétences des résidents et leur acceptation de recevoir des soins de leur part. La présente étude visait à clarifier si et de quelle manière ces trois composantes du rapport patient-résident sont interreliées. MÉTHODES: Il s'agit d'une étude transversale réalisée au moyen d'un questionnaire auto-administré distribué en 2016 à un échantillon de convenance de patients adultes (≥ 18 ans) ayant fréquenté une clinique universitaire de médecine familiale. La proportion et le test du khi carré ont été utilisés respectivement pour décrire et pour comparer les groupes. RÉSULTATS: Parmi les 471 patients qui ont répondu au questionnaire, à peine 28 % connaissaient bien le rôle des résidents en médecine familiale. Entre 54 % et 83 % des patients ont déclaré avoir une grande confiance dans la capacité des résidents à effectuer cinq tâches de routine. Parmi les patients interrogés, 69 % ont accepté de voir un résident lors de leurs prochains rendez-vous. Les patients ayant un niveau de confiance élevé dans les capacités des résidents étaient plus susceptibles d'accepter de voir un résident lors de leurs prochains rendez-vous (p <0,0001). Il n'y avait pas d'association significative entre le niveau de connaissance des patients et leur confiance dans les résidents ou leur acceptation d'être traités par ces derniers. CONCLUSIONS: Bien que la majorité des patients aient une mauvaise connaissance du rôle des résidents, celle-ci n'a pas d'incidence sur leur acceptation d'être soignés par de résidents. Un niveau de confiance plus élevé dans la capacité des résidents à effectuer certaines tâches était associé à une plus grande acceptation de voir un résident à l'avenir.

Detection of low levels of SARS-CoV-2 RNA from nasopharyngeal swabs using three commercial molecular assays.

In response to the COVID-19 pandemic, commercial molecular assays for SARS-CoV-2 testing have been rapidly developed and broadly deployed in laboratories worldwide. Although these assays have been reported to correlate well, we sought to compare the Xpert® Xpress SARS-CoV-2 to the cobas® SARS-CoV-2 or the Lightmix® Modular SARS and Wuhan CoV E-gene assay for nasopharyngeal (NP) swabs with low levels of SARS-CoV-2 RNA. Thirty-seven NP swabs were studied, including 10 samples with a moderate cycle threshold (Ct) between 30-33.9, and 22 with Ct≥34, and 5 negative for SARS-CoV-2. Overall concordance on initial comparison was 86.5 % (32/37), which was 100 % concordance for samples with Ct values ranging between 30-33.9. Discordance amongst samples showing a Ct ≥34 was 22.7 % (5/22). Endpoint value analysis on the Xpress SARS-CoV-2 within the discordant samples noted two with an endpoint value >5, which were detected by the cobas® or Lightmix®. Testing of SARS-CoV-2 on the three commercial assays was comparable for NP swabs with moderate Ct values, while high Ct values were less concordant. Importantly, analysis of Xpert® endpoint values improved interpretation of discrepant results.

Matrix-assisted laser desorption/ionization time-of-flight MS for the accurate identification of Burkholderia cepacia complex and Burkholderia gladioli in the clinical microbiology laboratory.

Introduction. Burkholderia cepacia complex (Bcc) bacteria, currently consisting of 23 closely related species, and Burkholderia gladioli, can cause serious and difficult-to-treat infections in people with cystic fibrosis. Identifying Burkholderia bacteria to the species level is considered important for understanding epidemiology and infection control, and predicting clinical outcomes. Matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF) is a rapid method recently introduced in clinical laboratories for bacterial species-level identification. However, reports on the ability of MALDI-TOF to accurately identify Bcc to the species level are mixed.Aim. The aim of this project was to evaluate the accuracy of MALDI-TOF using the Biotyper and VITEK MS systems in identifying isolates from 22 different Bcc species and B. gladioli compared to recA gene sequencing, which is considered the current gold standard for Bcc.Methodology. To capture maximum intra-species variation, phylogenetic trees were constructed from concatenated multi-locus sequence typing alleles and clustered with a novel k-medoids approach. One hundred isolates representing 22 Bcc species, plus B. gladioli, were assessed for bacterial identifications using the two MALDI-TOF systems.Results. At the genus level, 100 and 97.0 % of isolates were confidently identified as Burkholderia by the Biotyper and VITEK MS systems, respectively; moreover, 26.0 and 67.0 % of the isolates were correctly identified to the species level, respectively. In many, but not all, cases of species misidentification or failed identification, a representative library for that species was lacking.Conclusion. Currently available MALDI-TOF systems frequently do not accurately identify Bcc bacteria to the species level.

Prospective Review of Clostridioides difficile Testing Indications to Inform Local Laboratory Stewardship Initiatives.

We conducted a prospective chart review to determine the prevalence of and reasons for inappropriate Clostridioides difficile test-ordering at a tertiary care hospital. Inappropriate orders accounted for 54% of all tests. The two primary aetiologies of inappropriate test-ordering were an alternative reason for diarrhoea (34%) and an asymptomatic patient (20%). These results highlight the need to focus diagnostic stewardship of C. difficile testing on pre-analytical factors.

Large community outbreak of Streptococcus pneumoniae serotype 5 invasive infection in an impoverished, urban population.

BACKGROUND: Streptococcus pneumoniae is a common cause of sporadic invasive infections, but outbreaks of invasive pneumococcal disease are infrequent. In August 2006, a sudden increase in the number of patients presenting with invasive pneumococcal disease was noted at St. Paul's Hospital (Vancouver, Canada). Most patients with severe disease resided in an area referred to as the Downtown Eastside, a neighborhood known for its high rates of poverty and illicit drug use. METHODS: Prospective, laboratory-based surveillance for invasive pneumococcal disease was initiated, including on-site serotyping of S. pneumoniae isolates. A vaccination campaign using 23-valent polysaccharide pneumococcal vaccine was launched in the Downtown Eastside. Multiple logistic regression was used to examine the association of sociodemographic variables and medical risk factors with S. pneumoniae serotype status. RESULTS: A single S. pneumoniae serotype (serotype 5) was responsible for 78% of invasive pneumococcal disease cases (137 of 175 cases) during the outbreak period of August 2006-July 2007. The outbreak strain, although fully susceptible to penicillin, caused significant morbidity and placed considerable strain on the acute care system within the Vancouver Coastal Health region. Crack cocaine use was found to be the main independent risk factor associated with invasive pneumococcal disease due to S. pneumoniae serotype 5 (odds ratio, 12.4; 95% confidence interval, 2.22-69.5). CONCLUSIONS: A targeted vaccination campaign using polysaccharide pneumococcal vaccine appeared to help control this outbreak. In urban centers with high rates of illicit drug use, vaccination strategies for preventing invasive pneumococcal disease may need to be refined to include individuals who use crack cocaine.

Evaluation of the FilmArray Blood Culture Identification Panel compared to direct MALDI-TOF MS identification for rapid identification of pathogens.

To improve time to identification of pathogens and detection of resistance genes, we evaluated the BioFire FilmArray Blood Culture Identification Panel (BCID) as compared to: (1) direct MALDI-TOF MS (DM) and (2) standardized culture-based identification (ID) with antibiotic susceptibility testing (AST). BCID gave an accurate identification in 102/112 (91 %) of cases (102/103 for on-panel organisms). DM gave an accurate identification in 91/112 (81 %) of cases, with 13/91 (14 %) requiring repeat testing from the residual pellet. The mean time to an identification result was 2.4 and 2.9 h for BCID and DM, respectively. Standardized ID and AST results were available at a mean time of 26.5 and 33 h, respectively. There were 44 BCID/DM results that had an antimicrobial treatment change made based on rapid identification and resistant gene detection of pathogens. Both BCID and DM are accurate and rapid methods for the identification of new positive blood culture pathogens.

Reduction in hospital-associated methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus with daily chlorhexidine gluconate bathing for medical inpatients.

BACKGROUND: Daily bathing with chlorhexidine gluconate (CHG) is increasingly used in intensive care units to prevent hospital-associated infections, but limited evidence exists for noncritical care settings. METHODS: A prospective crossover study was conducted on 4 medical inpatient units in an urban, academic Canadian hospital from May 1, 2014-August 10, 2015. Intervention units used CHG over a 7-month period, including a 1-month wash-in phase, while control units used nonmedicated soap and water bathing. Rates of hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) colonization or infection were the primary end point. Hospital-associated S. aureus were investigated for CHG resistance with a qacA/B and smr polymerase chain reaction (PCR) and agar dilution. RESULTS: Compliance with daily CHG bathing was 58%. Hospital-associated MRSA and VRE was decreased by 55% (5.1 vs 11.4 cases per 10,000 inpatient days, P = .04) and 36% (23.2 vs 36.0 cases per 10,000 inpatient days, P = .03), respectively, compared with control cohorts. There was no significant difference in rates of hospital-associated Clostridium difficile. Chlorhexidine resistance testing identified 1 isolate with an elevated minimum inhibitory concentration (8 µg/mL), but it was PCR negative. CONCLUSIONS: This prospective pragmatic study to assess daily bathing for CHG on inpatient medical units was effective in reducing hospital-associated MRSA and VRE. A critical component of CHG bathing on medical units is sustained and appropriate application, which can be a challenge to accurately assess and needs to be considered before systematic implementation.