Empiric antibiotics for peripartum bacteremia: A chart review from a quaternary Canadian centre.

OBJECTIVE: To evaluate the effectiveness of empiric antibiotic protocols for peripartum bacteremia at a quaternary institution by describing incidence, microbial epidemiology, clinical source of infection, susceptibility patterns, and maternal and neonatal outcomes. METHODS: Retrospective chart review of peripartum patients with positive blood cultures between 2010 and 2018. RESULTS: The incidence of peripartum bacteremia was 0.3%. The most cultured organisms were Escherichia coli (51, 26.7%), Streptococcus spp. (52, 27.2%), and anaerobic spp. (35, 18.3%). Of the E. coli cases, 54.9% (28), 19.6% (10), and 19.6% (10) were resistant to ampicillin, first- and third-generation cephalosporins, respectively. Clinical sources of infection included intra-amniotic infection/endometritis (115, 67.6%), upper and/or lower urinary tract infection (23, 13.5%), and soft tissue infection (8, 4.7%). Appropriate empiric antibiotics were prescribed in 137 (83.0%) cases. There were 7 ICU admissions (4.2%), 18 pregnancy losses (9.9%), 9 neonatal deaths (5.5%), and 6 cases of neonatal bacteremia (3.7%). CONCLUSION: Peripartum bacteremia remains uncommon but associated with maternal morbidity and neonatal morbidity and mortality. Current empiric antimicrobial protocols at our site remain appropriate, but continuous monitoring of antimicrobial resistance patterns is critical given the presence of pathogens resistant to first-line antibiotics.

Extraction-free clinical detection of SARS-CoV-2 virus from saline gargle samples using Hamilton STARlet liquid handler.

As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput SARS-CoV-2 multiplex polymerase chain reaction (PCR) testing in order to minimize cost and technologist time. This was achieved through elimination of nucleic acid extraction and automation of sample handling on a widely available robotic liquid handler, Hamilton STARlet. A customized barcode scanning script for reading the sample ID by the Hamilton STARlet's software system was developed to allow primary tube sampling. Use of pre-frozen SARS-CoV-2 assay reaction mixtures reduced assay setup time. In both validation and live testing, the assay produced no false positive or false negative results. Of the 1060 samples tested during validation, 3.6% (39/1060) of samples required retesting as they were either single gene positive, had internal control failure or liquid aspiration error. Although the overall turnaround time was only slightly faster in the automated workflow (185 min vs 200 min), there was a 76% reduction in hands-on time, potentially reducing staff fatigue and burnout. This described process from sample self-collection to automated direct PCR testing significantly reduces the total burden on healthcare systems in terms of human resources and reagent requirements.

Comprehensive human amniotic fluid metagenomics supports the sterile womb hypothesis.

As metagenomic approaches for detecting infectious agents have improved, each tissue that was once thought to be sterile has been found to harbor a variety of microorganisms. Controversy still exists over the status of amniotic fluid, which is part of an immunologically privileged zone that is required to prevent maternal immune system rejection of the fetus. Due to this privilege, the exclusion of microbes has been proposed to be mandatory, leading to the sterile womb hypothesis. Since nucleic acid yields from amniotic fluid are very low, contaminating nucleic acid found in water, reagents and the laboratory environment frequently confound attempts to address this hypothesis. Here we present metagenomic criteria for microorganism detection and a metagenomic method able to be performed with small volumes of starting material, while controlling for exogenous contamination, to circumvent these and other pitfalls. We use this method to show that human mid-gestational amniotic fluid has no detectable virome or microbiome, supporting the sterile womb hypothesis.

Evaluation of observed and unobserved self-collection of saline gargle samples for the detection of SARS-CoV-2 in outpatients.

The diagnostic sensitivity of observed and unobserved self-collected saline gargle samples for the molecular detection of SARS-CoV-2 in adults and school-aged children was evaluated against a reference standard of health care worker collected nasopharyngeal flocked swab. A total of 46 participants had a positive nasopharyngeal swab sample; of these, 10 were in the observed phase and 36 were in the unobserved phase. Only one matching saline gargle sample tested negative and this was in the unobserved phase, giving an overall sensitivity of 98%. Average viral target Ct values were higher in the saline gargle samples. RNaseP Ct values were lower in unobserved collected samples compared to observed collected samples. Unobserved self-collection of saline gargle samples is a promising outpatient testing method for COVID-19 diagnosis. The self-collection method has potential to simplify the diagnostic cycle and facilitate implementation of COVID-19 testing, particularly in settings with limited access to health care workers.

Development and validation of a new triplex real-time quantitative reverse Transcriptase-PCR assay for the clinical detection of SARS-CoV-2.

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.

Self-Collected Saline Gargle Samples as an Alternative to Health Care Worker-Collected Nasopharyngeal Swabs for COVID-19 Diagnosis in Outpatients.

We assessed the performance, stability, and user acceptability of swab-independent self-collected saliva and saline mouth rinse/gargle sample types for the molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in adults and school-aged children. Outpatients who had recently been diagnosed with COVID-19 or were presenting with suspected COVID-19 were asked to have a nasopharyngeal (NP) swab collected and provide at least one self-collected sample type. Participants were also asked about sample acceptability using a five-point Likert scale. For those previously diagnosed with COVID-19, all samples underwent real-time PCR testing using a lab-developed assay, and the majority were also tested using an FDA-authorized assay. For those presenting with suspected COVID-19, only those with a positive nasopharyngeal swab sample went on to have other samples tested. Saline mouth rinse/gargle and saliva samples were tested daily at time zero, day 1, and day 2 to assess nucleic acid stability at room temperature. Fifty participants (aged 4 to 71 years) were included; of these, 40 had at least one positive sample and were included in the primary sample yield analysis. Saline mouth rinse/gargle samples had a sensitivity of 98% (39/40), while saliva samples had a sensitivity of 79% (26/33). Both saline mouth rinse/gargle and saliva samples showed stable viral RNA detection after 2 days of room temperature storage. Mouth rinse/gargle samples had the highest (mean, 4.9) and health care worker (HCW)-collected NP swabs had the lowest acceptability scores (mean, 3.1). In conclusion, saline mouth rinse/gargle samples demonstrated higher combined user acceptability ratings and analytical performance than saliva and HCW-collected NP swabs. This sample type is a promising swab-independent option, particularly for outpatient self-collection in adults and school-aged children.

Development of a national neonatal intensive care unit-specific antimicrobial stewardship programme in Canada: protocol for a cohort study.

INTRODUCTION: Early empiric treatment with broad-spectrum antimicrobials is common in neonatal intensive care units (NICU) due to the non-specific clinical presentation of infection. However, excessive and inappropriate antimicrobial use can lead to the emergence of drug-resistant organisms and adverse neonatal outcomes. This study aims to develop and implement a nationwide NICU-specific antimicrobial stewardship programme (ASP) to promote judicious antimicrobial use and control the emergence of multidrug-resistant organisms (MDROs) in Canada. METHODS AND ANALYSIS: Our study population will include all very low-birth-weight neonates admitted to participating tertiary NICU in Canada. Based on the existing limited literature, we will develop consensus on NICU antimicrobial stewardship interventions to enhance best practices. Using an expanded Canadian Neonatal Network (CNN) platform, we will collect data on antimicrobial use and the susceptibility of organisms identified in clinical samples from blood and cerebrospinal fluid over a period of 2 years. These data will be used to provide all NICU stakeholders with benchmarked centre-adjusted antimicrobial use and MDRO prevalence reports. An ASP plan will be developed at both individual unit and national levels in the subsequent years. Knowledge translation strategies will be implemented through the well-established Evidence-based Practice for Improving Quality methodology. ETHICS AND DISSEMINATION: Ethics for the study has been granted by the University of British Columbia Children's & Women's Research Ethics Board (H19-02490) and supported by CNN Executive Committee. The study results will be disseminated through national organisations and open access peer-reviewed publications. TRIAL REGISTRATION NUMBER: NCT04388293.

A metagenomics-based diagnostic approach for central nervous system infections in hospital acute care setting.

The etiology of central nervous system (CNS) infections such as meningitis and encephalitis remains unknown in a large proportion of cases partly because the diversity of pathogens that may cause CNS infections greatly outnumber available test methods. We developed a metagenomic next generation sequencing (mNGS)-based approach for broad-range detection of pathogens associated with CNS infections suitable for application in the acute care hospital setting. The analytical sensitivity of mNGS performed on an Illumina MiSeq was assessed using simulated cerebrospinal fluid (CSF) specimens (n = 9). mNGS data were then used as a training dataset to optimize a bioinformatics workflow based on the IDseq pipeline. For clinical validation, residual CSF specimens (n = 74) from patients with suspected CNS infections previously tested by culture and/or PCR, were analyzed by mNGS. In simulated specimens, the NGS reads aligned to pathogen genomes in IDseq were correlated to qPCR CT values for the respective pathogens (R = 0.96; p < 0.0001), and the results were highly specific for the spiked pathogens. In clinical samples, the diagnostic accuracy, sensitivity and specificity of the mNGS with reference to conventional methods were 100%, 95% and 96%, respectively. The clinical application of mNGS holds promise to benefit patients with CNS infections of unknown etiology.

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.

Third-generation cephalosporin-resistant urinary tract infections in children presenting to the paediatric emergency department.

BACKGROUND: The incidence of antibiotic-resistant urinary tract infections (UTIs) in children is increasing. The purpose of this study was to describe the incidence, clinical characteristics, and risk factors for third-generation cephalosporin-resistant UTIs presenting to the paediatric emergency department (ED). METHODS: This was a retrospective cohort study conducted at British Columbia Children's Hospital. Children aged 0 to 18 years old presenting to the ED between July 1, 2013 and June 30, 2014 and were found to have UTI due to Enterobacteriaceae and Pseudomonas species were included. Patient demographics, clinical features, laboratory findings, and outcomes were compared using standard statistical analyses. Risk factors for resistant UTIs were analyzed using multiple logistic regression analysis. RESULTS: There were 294 eligible patients. The median age was 27.4 months. A third-generation cephalosporin-resistant organism was identified in 36 patients (12%). Patients with resistant UTI had lower rates of appropriate empiric antibiotic therapy (25% versus 95.3%, P<0.05), higher rates of hospitalization (38.9% versus 21.3%, P<0.05), higher rates of undergoing a voiding cystourethrogram (19.4% versus 5.0%, P<0.05), and higher rates of UTI recurrence within 30 days (13.9% versus 4.7%, P<0.05). In multivariate analysis, recent hospitalization (odds ratio [OR] 4.3, confidence interval [CI] 1.2 to 16) and antibiotic therapy (OR 3.5, CI 1.5 to 8.5) within the previous 30 days were risk factors for resistant UTI. CONCLUSIONS: Third-generation cephalosporin-resistant organisms account for a significant proportion of community-acquired paediatric UTIs. Recent hospitalization and antibiotic use are associated with increased risk of resistant UTI.

Asymptomatic Pharyngeal Carriage of Kingella kingae Among Young Children in Vancouver, British Columbia, Canada.

BACKGROUND: Kingella kingae has emerged as a significant cause of osteoarticular infections in young children. Pharyngeal colonization is considered a prerequisite for invasive K. kingae infection. We conducted a prospective study to estimate the prevalence of pharyngeal carriage of K. kingae among healthy young children in Vancouver. METHODS: From March 2016 to May 2017, children between 6 and 48 months of age visiting British Columbia Children's Hospital outpatient clinics for noninfectious causes were included in the study. Another set of participants was enrolled from a day-care center located at British Columbia Children's Hospital. A single-throat swab was collected after obtaining consent from parent/guardian. The samples were stored at -70°C and tested using an in-house developed real-time polymerase chain reaction assay. Epidemiologic characteristics and risk factors for K. kingae colonization were collected via a study questionnaire. RESULTS: A total of 179 children were enrolled in the study, but only 174 samples were eligible for testing. Of the 174 samples, 5 had indeterminate results and the remaining 169 samples were negative by K. kingae polymerase chain reaction. The median age of participants was 23 months. About 36% of children were attending day care and had another sibling <5 years of age. Previous history of cold symptoms and antibiotic use was reported in 42% and 12%, respectively. CONCLUSIONS: The results of our study showed no prevalence of asymptomatic pharyngeal carriage of K. kingae in young children in Vancouver. Additional multicenter studies may help to understand the differences in pharyngeal carriage rate among healthy children.

Pediatric oncology and stem cell transplant patients with healthcare-associated Clostridium difficile infection were already colonized on admission.

Clostridium difficile is the leading cause of healthcare-associated infections worldwide. The diagnosis of C. difficile infection (CDI) in pediatric oncology patients is complex as diarrhea is common, and there is a high rate of colonization in infants and young children. This study was conducted to assess the accuracy of the surveillance definitions of healthcare-associated CDI (HA-CDI) and to determine the prevalence of toxigenic C. difficile colonization among pediatric oncology and stem cell transplant patients. METHODS: A prospective cohort study was conducted over a three-year period in an inpatient pediatric oncology and stem cell transplant setting. Baseline stool samples were collected within three days of admission and were genotypically compared with clinically indicated samples submitted after three days of admission. RESULTS: A total of 175 patients were recruited with a total of 536 admissions. The adjusted prevalence of baseline toxigenic C. difficile colonization among admissions was 32.8%. Seventy-eight percent of positive admissions did not have history of CDI. Colonization with a toxigenic strain on admission was predictive of CDI (OR = 28.6; 95% CI, 6.58-124.39; P < 0.001). Nearly all clinical isolates (8/9) shared identical pulsed-field gel electrophoresis patterns with baseline isolates or were closely related (1/9). Only one of the 11 cases that were considered HA-CDI was potentially nosocomially acquired. CONCLUSION: The prevalence of colonization with toxigenic C. difficile in our cohort is high. Unfortunately, the current CDI surveillance definitions overestimate the incidence of HA-CDI in pediatric oncology and stem cell transplantation settings.

A novel real-time PCR assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format.

Commercial multiplex assays, built on different chemistries and platforms are widely available for simultaneous detection of pathogens that cause respiratory infections. However, these tests are often difficult to implement in a resource limited setting because of high cost. In this study, we developed and validated a method for simultaneous testing of common respiratory pathogens (Respanel) by real-time PCR in a convenient, strip-tube array format. Primers and probes for sixteen PCR assays were selected from the literature or newly designed. Following optimization of individual PCR assays, strip-tube arrays were prepared by dispensing primer-probe mixes (PPM) into two sets of 8-tube strips. Nucleic acid extracts from specimens were mixed with PCR master mix, and dispensed column-wise into 2 × 8-wells of a 96-well plate. PPMs from strip-tubes were then added to the wells using a multichannel pipette for real-time PCR. Individual PCR assays were optimized using previously known specimens (n = 394) with 91%-100% concordance with culture, DFA or PCR results. Respanel was then tested in a routine manner at two different sites using specimens (n = 147) previously tested by Qiagen Resplex I&II or Fast-Track Diagnostics Respiratory Pathogens 21 assays. The sensitivity, specificity and accuracy of Respanel were 94%, 95% and 95%, respectively, against Resplex and 88%, 100% and 99%, respectively, against FTDRP21. Respanel detected more pathogens (p < 0.05) than Resplex but the rate of pathogen detection was not significantly different from FTDRP21. Respanel is a convenient and inexpensive assay that is more sensitive than Resplex and comparable to FTDRP21 for the detection of common respiratory pathogens.

Reduction of Inappropriate Antimicrobial Prescriptions in a Tertiary Neonatal Intensive Care Unit After Antimicrobial Stewardship Care Bundle Implementation.

BACKGROUND: Our objective was to evaluate the appropriateness of antibiotic prescriptions in the neonatal intensive care unit using standardized criteria and determine the effects of an antimicrobial stewardship program (ASP) on patterns of antibiotic usage. METHODS: A retrospective audit of antibiotic use from July 2010 to June 2013 was conducted, focusing on prescriptions of vancomycin, cefotaxime, meropenem and linezolid for >3 calendar-days. We evaluated the appropriateness of each course of antibiotic treatment based on the Centers for Disease Control and Prevention 12-Step Guidelines to Prevent Antimicrobial Resistance (steps 4, 6 and 9). An ASP was introduced in August 2014, and the same audit criteria were applied to review antimicrobial use in the subsequent 12 months. RESULTS: In the pre-ASP era, 26.3%, 12.1%, 11.4% and 0% of meropenem, cefotaxime, vancomycin and linezolid courses, respectively, were inappropriate. The most common instance of inappropriate utilization included failure to use narrow-spectrum antimicrobials when appropriate. After the introduction of ASP program, 22.2%, 7.5%, 5.4% and 0% of meropenem, cefotaxime, vancomycin and linezolid courses, respectively, were inappropriate. The numbers of inappropriate antibiotic-days/1000 days of therapy with meropenem, cefotaxime and vancomycin changed from 1.89 to 1.96 [rate ratio (RR), 1.04 (0.70-1.52)], 3.56 to 1.73 [RR, 0.49 (0.33-0.71)] and 2.70 to 1.01 [RR, 0.37 (0.22-0.60)], respectively. In subgroup analysis, very low birth weight infants (birth weight, <1500 g) showed no improvement in the rates of inappropriate antibiotic prescriptions. CONCLUSIONS: In this study, we found that ASP initiatives can be applied in neonatal populations to reduce inappropriate antimicrobial prescriptions, though a different approach may be needed among very low birth weight infants.

Cellular metabolism constrains innate immune responses in early human ontogeny.

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.

Is it safe to re-access sodium bicarbonate bottles for use in minor surgery?

BACKGROUND/PURPOSE: Sodium bicarbonate is added to lidocaine to reduce injection pain. In Canada, it is available in vials exceeding the injection volume 100-fold. These are single-use vials that should be disposed of after one access. Some surgeons re-use vials to reduce waste, potentially causing contamination. This study aims to review the safety of sodium bicarbonate and assess alternatives to current practice. METHODS: Strains of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Burkholderia cepacia were used to assess bacterial growth in vials of sodium bicarbonate. Each pathogen was inoculated into a vial for 14 days at room temperature. At several time points, 1 mL of solution was removed and diluted. One hundred microliters were transferred to blood agar plates and incubated at 35 °C. Colony counts were calculated, averaged and plotted onto a logarithmic graph. RESULTS: Colony counts of all strains fell below observational threshold after 7 days in sodium bicarbonate. CONCLUSIONS: Although all strains were reduced, bacteria can survive in sodium bicarbonate for several days, during which transmission may occur. Sodium bicarbonate vials should be treated as single-dose, as indicated by the manufacturers. To reduce waste, hospital pharmacies can repackage sodium bicarbonate into smaller vials or pre-alkalize lidocaine with sodium bicarbonate.

Recovery of cytomegalovirus DNA from newborn saliva samples by different methods.

BACKGROUND: Polymerase chain reaction (PCR) testing of saliva swabs has been shown to be an attractive method to screen for congenital cytomegalovirus (CMV) infection. However, few data are available regarding the impact of pre-analytic methods on CMV DNA recovery from saliva samples under various conditions. OBJECTIVES: The purpose of this study was to evaluate the impact of various transport media and transport conditions on the recovery of CMV DNA from spiked and clinical saliva samples from newborns using PCR testing. STUDY DESIGN: Negative control saliva samples were spiked with known concentrations of World Health Organization CMV standards. Collection was simulated using filter paper and different types of swabs which were stored either dry or in various transport media at specified temperatures. Specimens were tested by quantitative PCR at multiple time points. Results were compared with samples taken from newborns infected with CMV using the same methods. RESULTS: Nucleic acid stabilization media demonstrated superior CMV stability for up to 3 weeks. Also observed was a reduction in the quantity of spiked CMV DNA over time in swab samples stored at room temperature without media or in conventional universal transport media. Swab samples from newborns infected with CMV did not demonstrate loss of CMV DNA after a week of room temperature storage in nucleic acid stabilization media or conventional universal transport media. CONCLUSIONS: Although nucleic acid preservation media appear to enhance CMV DNA recovery from spiked control samples over prolonged transport times and diverse conditions, this differential yield appears be to less significant for actual clinical samples collected from infected newborns. Further evaluation of pre-analytic conditions using actual clinical samples is warranted.