Laboratory approaches to determining blood culture contamination rates: an ASM Laboratory Practices Subcommittee report.

Blood culture contamination (BCC) is the presence of specific commensal and environmental organisms cultivated from a single blood culture set out of a blood culture series and that do not represent true bacteremia. BCC can impact quality of care and lead to negative outcomes, unnecessary antibiotic exposure, prolonged hospital stays, and substantial costs. As part of the laboratory's quality management plan, microbiology laboratory personnel are tasked with monitoring BCC rates, preparing BCC rate reports, and providing feedback to the appropriate committees within their healthcare system. The BCC rate is calculated by the laboratory using pre-set criteria. However, pre-set criteria are not universally defined and depend on the individual institution's patient population and practices. This mini-review provides practical recommendations on elaborating BCC rate reports, the parameters to define for the pre-set criteria, how to collect and interpret the data, and additional analysis to include in a BCC report.

Rapid diagnostic work-up of Streptococcus dysgalactiae endophthalmitis by a novel culture processing system.

The diagnosis of infective endophthalmitis is supported by microbiological work-up. Rapid work-up is critical to confirm clinical suspicion and appropriate antimicrobial therapy. We report the novel use of an automated liquid culture processing system (FAST system, Qvella, ON, Canada) in a vitreous fluid culture. A 59-year-old patient with post-operative endophthalmitis presented with acute right eye pain and blurred vision. Vitreous fluid collected for microbiology culture was of limited quantity and only inoculated to thioglycolate broth. The broth recovered beta-haemolytic, group G Streptococcus dysgalactiae susceptible to penicillin and vancomycin. Experimental application of the FAST system to purify the organism from broth culture yielded the same identification and susceptibility test results but 1 day sooner. Despite prompt treatment with appropriate antibiotics, including vancomycin and ceftazidime, disease progressed rapidly and required enucleation to achieve a stable therapeutic outcome. Use of automated processing of monomicrobial broth cultures has thus far focused on positive blood culture broths, but could potentially include other liquid-based cultures such as for sterile body fluids of critical nature.

Clinical Significance of Ascitic Fluid Polymorphonuclear Leukocyte Percentage in Patients With Cirrhosis Without Spontaneous Bacterial Peritonitis.

INTRODUCTION: Absolute polymorphonuclear leukocyte (PMN) count (PMN-C) ≥250 cells/mm 3 in ascites is the diagnostic hallmark of spontaneous bacterial peritonitis (SBP) and is associated with high morbidity and mortality. However, the clinical significance of ascitic PMN percentage (PMN-%) and PMN-C in the absence of SBP as additional biomarkers for mortality and future incidence of SBP has not been determined. METHODS: This retrospective cohort included adults with cirrhosis undergoing first-recorded paracentesis with initial PMN-C < 250 cells/mm 3 at 2 tertiary medical centers between 2015 and 2020. Patients with prior SBP were excluded. Outcomes were death and SBP development. Cox regression estimated hazard ratios (HRs) for risk of death and SBP development and Akaike information criterion to compare model fit. RESULTS: Three hundred eighty-four adults (73% male, median age 58 years, 67% with alcohol-associated cirrhosis, median PMN-C 14 cells/mm 3 [interquartile range 5-34], and median PMN-% 10% [interquartile range 4-20]) were included in this study. Univariate risk of death increased 10% per 25-unit increase in PMN-C (95% confidence interval 1.01-1.21, P = 0.03) and 19% per 10-unit increase in PMN-% (95% confidence interval 1.06-1.33, P = 0.003) with PMN-% demonstrating better model fit in assessing mortality risk (Akaike information criterion: 1,044 vs 1,048, respectively). In models adjusted for age, chronic hepatitis C virus infection, and Model for End-Stage Liver Disease-Sodium, PMN-% was associated with risk of death (PMN-% 10%-29%, HR 1.17, P = 0.50; PMN-% ≥ 30% group, HR 1.94, P = 0.03; vs PMN-% < 10%) and SBP development (PMN-% 10%-29%, HR 1.68, P = 0.07; PMN-% ≥ 30%, HR 3.48, P < 0.001; vs PMN-% < 10%). DISCUSSION: Our results suggest PMN-% at first paracentesis represents a better biomarker compared with PMN-C for assessing risk of death and future SBP development in patients with PMN-C < 250 cells/mm 3 .

Multicenter Evaluation of the BIOFIRE Blood Culture Identification 2 Panel for Detection of Bacteria, Yeasts, and Antimicrobial Resistance Genes in Positive Blood Culture Samples.

Diagnostic tools that can rapidly identify and characterize microbes growing in blood cultures are important components of clinical microbiology practice because they help to provide timely information that can be used to optimize patient management. This publication describes the bioMérieux BIOFIRE Blood Culture Identification 2 (BCID2) Panel clinical study that was submitted to the U.S. Food & Drug Administration. Results obtained with the BIOFIRE BCID2 Panel were compared to standard-of-care (SoC) results, sequencing results, PCR results, and reference laboratory antimicrobial susceptibility testing results to evaluate the accuracy of its performance. Results for 1,093 retrospectively and prospectively collected positive blood culture samples were initially enrolled, and 1,074 samples met the study criteria and were included in the final analyses. The BIOFIRE BCID2 Panel demonstrated an overall sensitivity of 98.9% (1,712/1,731) and an overall specificity of 99.6% (33,592/33,711) for Gram-positive bacteria, Gram-negative bacteria and yeast targets which the panel is designed to detect. One hundred eighteen off-panel organisms, which the BIOFIRE BCID2 Panel is not designed to detect, were identified by SoC in 10.6% (114/1,074) of samples. The BIOFIRE BCID2 Panel also demonstrated an overall positive percent agreement (PPA) of 97.9% (325/332) and an overall negative percent agreement (NPA) of 99.9% (2,465/2,767) for antimicrobial resistance determinants which the panel is designed to detect. The presence or absence of resistance markers in Enterobacterales correlated closely with phenotypic susceptibility and resistance. We conclude that the BIOFIRE BCID2 Panel produced accurate results in this clinical trial.

Impact of Bacteria Types on the Clinical Outcomes of Spontaneous Bacterial Peritonitis.

BACKGROUND AND AIMS: Cirrhotic patients presenting with spontaneous bacterial peritonitis (SBP) have elevated risk of short-term mortality. While high Model for End-Stage Liver Disease-Sodium score (MELD-Na) and ascites culture yielding multi-drug resistance (MDR) bacteria are well established risk factors for further aggravating mortality, the impact of individual, causative microorganisms and their respective pathogenesis have not been previously investigated. METHODS: This is a retrospective study of 267 cirrhotic patients at two tertiary care hospitals undergoing paracentesis from January 2015 to January 2021 who presented with ascitic PMN count > 250 cells/mm3. The primary outcome was SBP progression defined as death or liver transplantation within 1-month of paracentesis stratified by microorganism type. RESULTS: Of 267 patients with SBP, the ascitic culture yielded causative microorganism in 88 cases [median age 57 years (IQR 52-64)]; 68% male; median MELD-Na 29 (IQR 23-35). The microbes isolated were E. coli (33%), Streptococcus (15%), Klebsiella (13%), Enterococcus (13%), Staphylococcus (9%) and others (18%); 41% were MDR. Cumulative incidence of SBP progression within 1-month was 91% (95% CI 67-100) for Klebsiella, 59% (95% CI 42-76) for E. coli, and 16% (95% CI 4-51) for Streptococcus. After adjusting for MELD-Na and MDR, risk of SBP progression remained elevated for Klebsiella (HR 2.07; 95% CI 0.98-4.24; p-value = 0.06) and decreased for Streptococcus (HR 0.28; 95% CI 0.06-1.21; p-value = 0.09) compared to all other bacteria. CONCLUSION: Our study found Klebsiella-associated SBP had worse clinical outcomes while Streptococcus-associated SBP had the most favorable outcomes after accounting for MDR and MELD-Na. Thus, identification of the causative microorganism is crucial not only for optimizing the treatment but for prognostication.

Antimicrobial Susceptibility Testing Performed in RPMI 1640 Reveals Azithromycin Efficacy against Carbapenem-Resistant Acinetobacter baumannii and Predicts In Vivo Outcomes in Galleria mellonella.

Antimicrobial susceptibility testing (AST) in RPMI 1640, a more physiologically relevant culture medium, revealed that a substantial proportion of carbapenem-resistant Acinetobacter baumannii isolates were susceptible to azithromycin, a macrolide antibiotic not currently considered effective against A. baumannii. Experiments using Galleria mellonella validated these in vitro data. Our finding that RPMI 1640's predictive accuracy for in vivo outcomes is superior to that of Mueller-Hinton II broth also supports the use of more physiologically relevant AST culturing conditions.

Direct MALDI-TOF MS and Antimicrobial Susceptibility Testing of Positive Blood Cultures Using the FASTTM System and FAST-PBC Prep Cartridges-Performance Evaluation in a Clinical Microbiology Laboratory Serving High-Risk Patients.

Bloodstream infections are a leading cause of morbidity and mortality. The rapid diagnostic testing of positive blood cultures (PBCs) shortens times to effective therapy and the de-escalation of broad-spectrum empiric therapy. This is the first study examining the Qvella FASTTM System for the rapid (~20 min) purification of microorganisms directly from PBCs using BacT/Alert® FA/FAN bottles in the bioMérieux Virtuo instrument. We compared the performance of the FASTTM System Liquid ColonyTM (LC), for immediate downstream ID and phenotypic AST, to standard workflow involving colonies obtained by overnight subculture. The LC yielded a concordant species ID by VITEK MS in 121/138 (87.7%) samples, identifying 32 different Gram-positive and Gram-negative species with 3/123 (2.6%) discordances. Compared to standard workflow, direct AST of the LC using VITEK® 2 yielded 98.4% categorical agreement and 98.0% essential agreement. Very major error, major error, and minor error rates were 1.0%, 0.0%, and 1.8%, respectively, for Gram-negative organisms; and 1.9%, 0.2%, and 1.2%, respectively, for Gram-positive organisms. The median times from positive blood culture flag to results by FASTTM System for ID and AST were 7.8 h and 15.7 h, respectively, versus 22.4 h and 36.6 h for standard workflow, respectively. In conclusion, the FASTTM System provides reliable results for direct ID and AST from PBCs with significantly decreased turnaround times.

Clostridioides difficile Toxin B PCR Cycle Threshold as a Predictor of Toxin Testing in Stool Specimens from Hospitalized Adults.

Rapid, accurate detection of Clostridioides difficile toxin may potentially be predicted by toxin B PCR cycle threshold (tcdB Ct). We investigated the validity of this approach in an inpatient adult population. Patients who tested positive by C. difficile PCR (Cepheid GeneXpert) from December 2016 to October 2020 (n = 368) at a tertiary medical center were included. All stool samples were further tested by rapid glutamate dehydrogenase (GDH)/toxin B EIA and cell cytotoxin neutralization assay (CCNA). Receiver operating characteristic curves were analyzed. The area under the curve for tcdB Ct predicting toxin result by EIA was 0.795 (95% confidence interval (CI) 0.747−0.843) and by CCNA was 0.771 (95% CI 0.720−0.822). The Youden Ct cutoff for CCNA was ≤27.8 cycles (sensitivity 65.0%, specificity 77.2%). For specimens with Ct ≤ 25.0 cycles (n = 115), CCNA toxin was positive in >90%. The negative predictive value of tcdB Ct for CCNA was no greater than 80% regardless of cutoff chosen. In summary, very low Ct values (≤25.0) could have limited value as a rapid indicator of positive toxin status by CCNA in our patient population. A broad distribution of Ct values for toxin-negative and toxin-positive specimens precluded more robust prediction. Additional data are needed before broader application of Ct values from qualitatively designed assays to clinical laboratory reporting.

Raising the Bar: Improving Antimicrobial Resistance Detection by Clinical Laboratories by Ensuring Use of Current Breakpoints.

BACKGROUND: Antimicrobial resistance (AMR) is a pressing global challenge detected by antimicrobial susceptibility testing (AST) performed by clinical laboratories. AST results are interpreted using clinical breakpoints, which are updated to enable accurate detection of new and emerging AMR. Laboratories that do not apply up-to-date breakpoints impede global efforts to address the AMR crisis, but the extent of this practice is poorly understood. METHODS: A total of 1490 clinical laboratories participating in a College of American Pathologists proficiency testing survey for bacterial cultures were queried to determine use of obsolete breakpoints. RESULTS: Between 37.9% and 70.5% of US laboratories reported using obsolete breakpoints for the antimicrobials that were queried. In contrast, only 17.7%-43.7% of international laboratories reported using obsolete breakpoints (P < .001 for all comparisons). Use of current breakpoints varied by AST system, with more laboratories reporting use of current breakpoints in the US if the system had achieved US Food and Drug Administration clearance with current breakpoints. Among laboratories that indicated use of obsolete breakpoints, 55.9% had no plans to update to current standards. The most common reason cited was manufacturer-related issues (51.3%) and lack of internal resources to perform analytical validation studies to make the update (23.4%). Thirteen percent of laboratories indicated they were unaware of breakpoint changes or the need to update breakpoints. CONCLUSIONS: These data demonstrate a significant gap in the ability to detect AMR in the US, and to a lesser extent internationally. Improved application of current breakpoints by clinical laboratories will require combined action from regulatory agencies, laboratory accreditation groups, and device manufacturers.

Distance Between Home and the Admitting Hospital and Its Effect on Survival of Low Socioeconomic Status Population With Staphylococcus aureus Bacteremia.

OBJECTIVE: Bacteremia is the presence of bacteria in the bloodstream. The objective of this study was to determine the relationship between low socioeconomic status (SES) and the epidemiology, process of care, and outcomes of patients with Staphylococcus aureus bacteremia (SAB). METHODS: We conducted a multicenter, retrospective, cohort study that evaluated adult patients with SAB in 3 Los Angeles County hospitals from July 15, 2012, through May 31, 2018. We determined SES (low SES, intermediate SES, and high SES) for each patient and compared sociodemographic and epidemiologic characteristics, management of care received by patients with SAB (ie, process of care), and outcomes. We used a Cox proportional hazards model to determine predictors of 30-day mortality for each SES group. RESULTS: Of 915 patients included in the sample, 369 (40%) were in the low-SES group, 294 (32%) in the intermediate-SES group, and 252 (28%) in the high-SES group. Most significant predictors of 30-day mortality in the Cox proportional hazards model were admission to an intensive care unit (hazard ratio [HR] = 9.04; 95% CI, 4.26-19.14), Pitt bacteremia score ≥4 indicating critical illness (HR = 4.30; 95% CI, 2.49-7.44), having ≥3 comorbidities (HR = 2.05; 95% CI, 1.09-3.85), and advanced age (HR = 1.03; 95% CI, 1.01-1.05). Distance between home and admitting hospital affected mortality only in the low-SES group (HR = 1.02; 95% CI, 1.00-1.02). CONCLUSIONS: SES did not independently affect the outcome of SAB; however, the farther the patient's residence from the hospital, the greater the negative effect on survival in a low-SES population. Our findings underscore the need to develop multipronged, targeted public health efforts for populations that have transportation barriers to health care.

The clinical significance of staphylococcus pettenkoferi: a retrospective review at a tertiary care medical center.

Staphylococcus pettenkoferi is a recently described coagulase-negative staphylococcal pathogen. We retrospectively reviewed 25 cases in which S. pettenkoferi was identified in routine cultures (12 blood, 13 other). Most were found with commensal flora and considered clinically insignificant, but its significance was uncertain in two cases from non-healing, deep foot wounds.

In Vitro Activity of Rifabutin and Rifampin against Antibiotic-Resistant Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

We recently reported that the antimicrobial activity of rifabutin against Acinetobacter baumannii is best modeled by the use of RPMI for in vitro susceptibility testing. Here, we define the effects of medium on the susceptibility and frequency of resistance emergence in a panel of A. baumannii, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa clinical isolates. Only A. baumannii was hypersusceptible to rifabutin in vitro and in vivo using a Galleria mellonella infection model. In vitro, the frequency of resistance emergence was greater when the bacteria were selected on RPMI versus tryptic soy agar (TSA) or Mueller-Hinton II (MHII) agar plates. However, the frequency of resistance emergence was lower in vivo than in the RPMI in vitro condition. IMPORTANCE Rifabutin has been recently described as a potential adjunctive therapy for antibiotic-resistant A. baumannii infections due to hypersensitivity in iron-depleted media, which may more closely mimic an in vivo environment. Here, we report that this hyperactivity is specific for A. baumannii, rather than being a general effect for other pathogens.

A Longitudinal Study of Assisted Infant Toilet Training During the First Year of Life.

OBJECTIVE: Previous retrospective studies have examined elimination signals, stool toileting refusal, and completion age in Assisted Infant Toilet Training (AITT). The aim of this longitudinal cohort study was to describe the practice of AITT and caregiver satisfaction in a primarily Western setting during the first year of life. METHODS: Families who started AITT before 4 months of age were recruited. Standardized interviews of caregivers were conducted at 1- to 2-month intervals. To identify trends over time, data were fitted to a linear mixed-effect model. Data were analyzed according to five 2-month blocks, starting at 3 to 4 months. RESULTS: Of 85 participating families, 87 children started AITT at a mean age of 2.5 months. At all age intervals, 88% to 94% of caregivers could identify elimination signals. Toileting attempts decreased from 10/day at 3 to 4 months to 7/day at 11 to 12 months (p < 0.001). Many families (45%-53%) practiced AITT on a part-time basis. Daytime dryness was noted in 12% to 14% of infants throughout the first year. Although more than 63% of families used cloth or disposable diapers throughout this study, use of trainers and underwear increased significantly by 2- to 3-fold (p < 0.01 for both). Caregiver satisfaction was high overall. Although negatively associated with potty refusal, it was positively associated with daytime and nighttime dryness, perceived elimination signals, and a better understanding of their infant's needs (p < 0.001 for all). CONCLUSION: This study demonstrates that AITT is a worthy viable alternative to the use of diapers even in Western settings. Better understanding of AITT provides a new perspective to properly meet infants' basic needs.

Considerations from the College of American Pathologists for Implementation of an Assay for SARS-CoV-2 Testing after a Change in Regulatory Status.

The U.S. Food & Drug Administration (FDA) regulates the marketing of manufacturers' in vitro diagnostic tests (IVDs), including assays for the detection of SARS-CoV-2. The U.S. government's Clinical Laboratory Improvement Amendments (CLIA) of 1988 regulates the studies that a clinical diagnostic laboratory needs to perform for an IVD before placing it into use. Until recently, the FDA has authorized the marketing of SARS-CoV-2 IVDs exclusively through the Emergency Use Authorization (EUA) pathway. The regulatory landscape continues to evolve, and IVDs will eventually be required to pass through conventional non-EUA FDA review pathways once the emergency declaration is terminated, in order to continue to be marketed as an IVD in the United States. When FDA regulatory status of an IVD changes or is anticipated to change, the laboratory should review manufacturer information and previously performed internal verification studies to determine what, if any, additional studies are needed before implementing the non-EUA version of the IVD in accordance with CLIA regulations. Herein, the College of American Pathologists' Microbiology Committee provides guidance for how to approach regulatory considerations when an IVD is converted from EUA to non-EUA status.

Microbiological Evaluation of Opened Saline Bottles for Scleral Lens Use and Hygiene Habits of Scleral Lens Patients.

SIGNIFICANCE: Scleral lenses have become a widely used treatment option for patients with irregular corneas and ocular surface disease. Successful wear entails use of a nonpreserved saline solution to fill the lens before application on the eye. PURPOSE: The purposes of this study were to evaluate solution from opened bottles of multidose preservative-free saline for microbiological growth and to better understand study participant hygiene habits while handling these bottles for scleral lens wear. METHODS: Eligible study participants in this single-center prospective study were patients who routinely used multidose preservative-free saline solution for scleral lens rinsing and filling. Study participants completed a 12-question survey regarding their scleral lens hygiene habits and donated their opened multidose preservative-free saline bottle (PuriLens Plus; The Lifestyle Company, Inc., Freehold, NJ), which was processed for bacterial and fungal cultures. RESULTS: Thirty-five participants (19 males, 16 females) with ages ranging from 6 to 81 years (mean, 47.9 years) were included. Indications for scleral lens wear included those with irregular corneas and ocular surface disease. The overall rate of microbial contamination among saline samples was 62.9% (n = 22). Twenty-one different microorganisms were identified. The survey responses did not differ significantly (P > .05) for any of the questions with regard to likelihood of positive culture. There were no significant age or sex differences between participants with positive or negative culture results. No significant differences were found between isolation of specific microorganisms and any of the survey responses. CONCLUSIONS: This study suggests that off-label multidose preservative-free saline commonly used to rinse and fill scleral lenses before application on the eye may become contaminated with microorganisms once the bottle has been opened. Eye care practitioners and scleral lens patients should be aware of these potential contaminations and prioritize lens, hand, and environmental hygiene to minimize the risk of ocular complications.

The Bactericidal Activity and Spore Inhibition Effect of Manuka Honey against Clostridioides Difficile.

Clostridioides difficile colitis overgrowth occurs when the normal gut microbiome becomes disrupted, often due to antibiotics. Effective treatment remains elusive, due partly to the persistence of its spores in the gut. Natural substances like manuka honey offer an alternative antimicrobial mechanism of action to conventional antibiotics. We investigated the antibiotic activity of manuka honey against 20 C. difficile isolates. The minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBC) of manuka honeys of methylglyoxal (MGO) grades 30+, 100+, 250+, and 400+ were determined based on broth microdilution. Sporicidal activity was assessed in a range of honey concentrations by enumerating total viable cell and spore counts at 0-96 h after organism inoculation. The MICs of C. difficile ranged from 4% to >30% (w/v). MIC50 for the four MGO grades were similar at 10-14%. MBC results for the majority of isolates were distributed bimodally at MBC/MIC ratios ≤4 or MBC >30%. Growth kinetics in honey showed total viable cell counts remaining >105 colony-forming units (CFU)/mL at all time points, whereas spore counts remained within 1-log of baseline (102 CFU/mL) in honey but steadily increased in the drug-free control to >105 CFU/mL by 96 h. Manuka honey demonstrated variable inhibitory and bactericidal activity against C. difficile. MGO grade had no noticeable impact on overall MIC distributions or bactericidal activity. Although manuka honey could inhibit spore proliferation, it did not eradicate spores completely.

Lightweight UV-C disinfection system.

UV-C exposure is an effective disinfectant for a range of bacteria and viruses. As such, UV-C treatment, in combination with a chemical wipe, is a common cleaning protocol in medical facilities. Given the increase in severe bacterial and viral agents in society, having access to environmentally friendly disinfectant methods is of increasing interest. In response, we designed, constructed, and validated a UV-C disinfection system from readily accessible components. To improve the UV-C intensity, the enclosure interior was coated with chrome paint. The system is validated using Bacillus cereus, a gram-positive endospore-forming bacteria.

A nutrient-limited screen unmasks rifabutin hyperactivity for extensively drug-resistant Acinetobacter baumannii.

Industry screens of large chemical libraries have traditionally relied on rich media to ensure rapid bacterial growth in high-throughput testing. We used eukaryotic, nutrient-limited growth media in a compound screen that unmasked a previously unknown hyperactivity of the old antibiotic, rifabutin (RBT), against highly resistant Acinetobacter baumannii. In nutrient-limited, but not rich, media, RBT was 200-fold more potent than rifampin. RBT was also substantially more effective in vivo. The mechanism of enhanced efficacy was a Trojan horse-like import of RBT, but not rifampin, through fhuE, only in nutrient-limited conditions. These results are of fundamental importance to efforts to discover antibacterial agents.