Pitfalls of a Multiplex PCR Panel for Identification of Bloodstream Pathogens.

BACKGROUND: We evaluated the diagnostic performance of the FilmArray Blood Culture Identification Panel (BCID; bioMerieux) for the detection of bloodstream pathogens. METHODS: From May to August 2022, up to 67 samples from positive blood cultures previously processed with BACTEC FX (BD) were collected and submitted to the BCID panel. BCID panel results were compared with traditional culture results. RESULTS: We tested 67 positive blood culture samples; 13 samples were from pediatric bottles of BACTEC Peds Plus/F media (BD). The overall sensitivity of the BCID panel was 89.9% (62/69; 95% CI, 80.2 - 95.3%). For blood-stream pathogens targeted by the BCID panel, sensitivity was 98.4% (62/63; 95% CI, 90.7 - > 99.9%). Interestingly, Proteus species were additionally detected in 6 samples from pediatric blood culture bottles. CONCLUSIONS: BCID demonstrated high clinical sensitivity for target pathogens, but positive findings for unexpected multiple targets or Proteus species require cautious interpretation to avoid false positives.

Hemocultivos contaminados: bundle para lograr proporciones aceptables / Blood cultures contaminated: bundle to achieve acceptable proportions

Introducción. El problema de la contaminación de los hemocultivos es muy frecuente en establecimientos de atención hospitalaria, da lugar a la administración de antibióticos innecesarios y prolonga la hospitalización. Objetivo principal. Aplicar un bundle para reducir la proporción de contaminación de hemocultivos. Objetivo secundario. Realizar una encuesta anónima para detectar oportunidades de mejora en la técnica de extracción de hemocultivos. Metodología. Diseño del estudio: Estudio cuasi experimental que evaluó la proporción de contaminación de hemocultivos antes y después de implementar un bundle propio. Se determinó la proporción basal de contaminación de hemocultivos (ene-jul 2022), se realizó la intervención (agosto 2022) y se estableció la proporción de contaminación post intervención (sep.-abril 2023). Intervención: Se analizó la estructura, procedimiento y conocimiento del personal mediante una encuesta propia para detectar áreas de mejora. Se capacitó, a los técnicos de laboratorio, sobre el procedimiento de la toma de muestra mediante una simulación utilizando un brazo artificial. Se diseñó un bundle de seis medidas, se adaptó el procedimiento de toma de hemocultivo y se capacitó al personal. Análisis estadístico. Se analizó la proporción de hemocultivos contaminados entre los periodos pre y post utilizando Chi2 y la relación entre la proporción del periodo pre y post vs la literatura (3.00% contaminación aceptable) utilizando test Z para una proporción. Se consideró un p<0.05 como estadísticamente significativa. Se utilizo el software Stata 8. Resultados. Durante el estudio se analizaron un total de 3,965 hemocultivos. De estos, 1,978 corresponden al periodo pre-intervención y 1,987 corresponden al periodo post intervención. Durante la pre-intervención se detectaron 61 hemocultivos contaminados (3.08% vs 3.00% bibliografía, p:0.5866) mientras que en la etapa post intervención fue de 30 hemocultivos contaminados (1.51% vs 3.00% bibliografía, p:0.0000). La proporción de hemocultivos contaminados se redujo a la mitad, 3.08% vs 1.51%, p: 0.001. Se realizó una encuesta anónima pre y post intervención logrando mejoras en la técnica de toma de hemocultivos. Conclusión. La implementación del bundle propio para la extracción de hemocultivos, permitió reducir la proporción de contaminación a la mitad. El análisis de la encuesta nos permitió identificar oportunidades de mejora en la técnica de recolección de muestra de hemocultivos

Introduction: Contamination of blood cultures is very common in hospital care settings and results in the administration of unnecessary antibiotics and prolongs hospitalization. Main goal: Apply a bundle to reduce the rate of contamination of blood cultures. Secondary objective: Conduct an anonymous survey to detect opportunities for improvement in the blood culture extraction technique. Methodology: Study design: Quasi-experimental study that evaluated the proportion of blood culture contamination before and after implementing its own bundle. The baseline proportion of blood culture contamination was determined (Jan-July 2022), the intervention was performed (August 2022) and the post-intervention contamination proportion was established (September-April 2023). Intervention: The structure, procedure and knowledge of the staff was analyzed through an own survey to detect areas for improvement. Laboratory technicians were trained on the sample collection procedure through a simulation using an artificial arm. A bundle of six measures was designed: (hand hygiene with alcohol gel, use of common gloves and sterile gloves during extraction, antisepsis with alcoholic chlorhexidine gluconate, marking of the blood culture bottle up to the filling level, disinfection of the bottle cap). blood culture bottle with 70% alcohol, safety-lok kit with vacuum extraction system). The procedure was adapted and staff trained. Statistic analysis: The proportion of contaminated blood cultures between the pre and post periods was analyzed using Chi2 and the relationship between the proportion of the pre and post period vs the literature (3.00% acceptable contamination) using Z test for a proportion. P<0.05 was considered statistically significant. Stata 8 software was used.Results: A total of 3,965 blood cultures were analyzed during the study. Of these, 1,978 correspond to the pre-intervention period and 1,987 correspond to the post-intervention period. During the pre-intervention, 61 contaminated blood cultures were detected (3.08%) while in the post-intervention stage there were 30 contaminated blood cultures (1.51%). The proportion of contaminated blood cultures was reduced by half, 3.08% vs 1.51%, p: 0.001. An anonymous survey was carried out pre and post intervention, achieving improvements in the technique of taking blood cultures. Conclusion: The implementation of the own bundle for the extraction of blood cultures allowed the contamination rate to be reduced by ha

Clinical utility of multiplex PCR in the detection of pathogens from sterile body fluids.

Rapid identification of pathogens in normally sterile body fluid (NSBF) is essential for appropriate patient management, specifically antimicrobial therapy. Limited sensitivity and increased time to detection of traditional culture prompted us to evaluate additional testing to contribute to the diagnosis of infection. The purpose of this study was to evaluate the GenMark Dx ePlex Blood Culture Identification (BCID) Panels on positive body fluids inoculated into blood culture bottles for the detection of microorganisms. A total of 88 positive body fluids from blood culture bottles were analyzed using a Gram-Positive, Gram-Negative, and/or Fungal pathogen BCID Panel based on the Gram stain result. Each result was compared to routine culture performed from the positive bottle. When using culture as a reference standard, we found the ePlex multiplex panel performed with a positive percent agreement of 96.5% and a negative percent agreement of 99.8%. The use of multiplex PCR may be a useful supplement to routine culture for NSBF in blood culture bottles. IMPORTANCE: The identification of pathogens in normally sterile body fluid (NSBF) is performed using routine culture, the current gold standard. Limitations of this method include sensitivity and increased turnaround times which could potentially delay vital patient care, especially antimicrobial therapy. Adaptations of NSBF in blood culture bottles prompted us to consider the utility of additional methods to bridge the gap in diagnostic challenges for these life-threatening infections. Multiplex molecular panels have been manufactured for use with multiple specimen types including blood, cerebral spinal fluid, stool, and respiratory. Therefore, the purpose of this study was to evaluate the off-label use of ePlex Blood Culture Identification Panels on positive body fluids grown in blood culture bottles for the detection of microorganisms for research purposes.

Evaluation of three protocols for direct susceptibility testing for Gram-negative rods from flagged positive blood culture bottles.

Bloodstream infections are associated with high mortality, which can be reduced by targeted antibiotic therapy in the early stages of infection. Direct antibiotic susceptibility testing (AST) from flagged positive blood cultures may facilitate the administration of early effective antimicrobials much before the routine AST. This study aimed to evaluate three different direct AST protocols for Gram-negative rods from flagged positive blood culture broths. Blood culture broths showing Gram-negative rods only were subjected to direct AST by Clinical and Laboratory Standards Institute-recommended direct disk diffusion (protocol A). Additionally, automated AST (protocol B) and Kirby-Bauer disk diffusion (protocol C) were performed with standard inoculum prepared from bacterial pellets obtained by centrifuging blood culture broths in serum separator vials. For comparison, conventional AST of isolates from solid media subculture was also performed with Kirby-Bauer disk diffusion (reference standard) and the automated method. Overall, categorical agreements of protocols A, B, and C were 97.6%, 95.7%, and 95.9%, respectively. Among Enterobacterales, minor error, major error, and very major error rates of protocol B were 3.5%, 0.36%, and 0.43%, respectively, whereas minor error, major error, and very major error rates of protocol C were 3.4%, 0.72%, and 0.21%, respectively, and among non-fermenters, protocol B had a minor error rate of 6.5%, and protocol C had a minor error rate of 4.1% and major error rate of 1.9%. All three direct AST protocols demonstrated excellent categorical agreements with the reference method. Performance of protocols B and C between Enterobacterales and non-fermenters was not statistically different. IMPORTANCE: Bloodstream infections are associated with high mortality that can be reduced by targeted antibiotic therapy in the early stages of infection. Direct antibiotic susceptibility testing (AST) from flagged positive blood cultures may facilitate the administration of early effective antimicrobials much before the routine AST. Clinical and Laboratory Standards Institute-recommended direct AST can be performed with a limited number of antibiotic disks only. On the other hand, using an automated system for direct AST will not only allow effective laboratory workflow with reduced turnaround time but also provide the minimum inhibitory concentration values of tested antibiotics. However, using expensive automated systems for direct AST may not be feasible for resource-limited laboratories. Therefore, in this study, we aimed to evaluate the CLSI-recommended method and two other direct AST protocols (one with an automated system and the other with disk diffusion) for Gram-negative rods from flagged positive blood cultures.

[Does a rapid antibiotic susceptibility test optimize antibiotic therapy of patients with bacteremia ?] / Un antibiogramme rapide optimise-t-il la prise en charge thérapeutique des patients présentant une bactériémie ?

BACKGROUND: We evaluated the contribution of a rapid antibiotic susceptibility test performed directly from a positive blood culture (PBC), the dRAST™, in the management of patients with bacteremia. METHODS: We retrospectively compared the time from sampling to availability of antibiotic susceptibility test (AST) results («time-to-result¼, TTR) between dRAST™ and classic AST (Vitek®2), in 150 patients with bacteremia. The antibiotic treatment of these 150 patients was classified into three categories (optimal, suboptimal, ineffective) according to the time of availability of AST results. RESULTS: Adaptation of antibiotic treatment to optimal therapy following AST results occurred in 46/100 (46 %) of Gram-negative PBC and in 4/50 (2 %) of Gram-positive HP. TTR was significantly lower with dRAST™ compared with classic AST (29:35 (± 08:48) hours versus 50:55 (± 12:45) hours, p < 0.001). CONCLUSION: For patients with bacteremia requiring adjustment of empirical antibiotic therapy based on AST, dRAST™ could allow a faster administration of optimal therapy.

CONTEXTE: Nous avons évalué la contribution d'un antibiogramme rapide réalisé directement à partir d'une hémoculture positive (HP), le dRAST™, dans la prise en charge des patients présentant une bactériémie. Méthodes: Nous avons comparé, rétrospectivement, le délai entre le prélèvement et la disponibilité des résultats d'antibiogramme («temps-pour-résultats¼, TPR) entre le dRAST™ et l'antibiogramme classique (Vitek®2), auprès de 150 patients présentant une bactériémie. Les antibiothérapies de ces 150 patients ont été classés en trois catégories (optimale, suboptimale, inefficace) en fonction du moment d'obtention des résultats de l'antibiogramme. Résultats : L'adaptation du traitement antibiotique en thérapie optimale suite au résultat de l'antibiogramme est survenue chez 46/100 (46 %) des HP à Gram négatif et chez 4/50 (2 %) des HP à Gram positif. Le TPR était significativement plus faible avec le dRAST™ par rapport à l'antibiogramme classique (29:35 (± 08:48) heures versus 50:55 (± 12:45) heures, p < 0,001). CONCLUSION: Pour les patients avec bactériémie nécessitant une adaptation de l'antibiothérapie empirique basée sur l'antibiogramme, le dRAST™ permettrait une administration plus rapide du traitement optimal.

Rapid nanopore sequencing and predictive susceptibility testing of positive blood cultures from intensive care patients with sepsis.

We aimed to evaluate the performance of Oxford Nanopore Technologies (ONT) sequencing from positive blood culture (BC) broths for bacterial identification and antimicrobial susceptibility prediction. Patients with suspected sepsis in four intensive care units were prospectively enrolled. Human-depleted DNA was extracted from positive BC broths and sequenced using ONT (MinION). Species abundance was estimated using Kraken2, and a cloud-based system (AREScloud) provided in silico predictive antimicrobial susceptibility testing (AST) from assembled contigs. Results were compared to conventional identification and phenotypic AST. Species-level agreement between conventional methods and AST predicted from sequencing was 94.2% (49/52), increasing to 100% in monomicrobial infections. In 262 high-quality AREScloud AST predictions across 24 samples, categorical agreement (CA) was 89.3%, with major error (ME) and very major error (VME) rates of 10.5% and 12.1%, respectively. Over 90% CA was achieved for some taxa (e.g., Staphylococcus aureus) but was suboptimal for Pseudomonas aeruginosa. In 470 AST predictions across 42 samples, with both high quality and exploratory-only predictions, overall CA, ME, and VME rates were 87.7%, 8.3%, and 28.4%. VME rates were inflated by false susceptibility calls in a small number of species/antibiotic combinations with few representative resistant isolates. Time to reporting from sequencing could be achieved within 8-16 h from BC positivity. Direct sequencing from positive BC broths is feasible and can provide accurate predictive AST for some species. ONT-based approaches may be faster but significant improvements in accuracy are required before it can be considered for clinical use.IMPORTANCESepsis and bloodstream infections carry a high risk of morbidity and mortality. Rapid identification and susceptibility prediction of causative pathogens, using Nanopore sequencing direct from blood cultures, may offer clinical benefit. We assessed this approach in comparison to conventional phenotypic methods and determined the accuracy of species identification and susceptibility prediction from genomic data. While this workflow holds promise, and performed well for some common bacterial species, improvements in sequencing accuracy and more robust predictive algorithms across a diverse range of organisms are required before this can be considered for clinical use. However, results could be achieved in timeframes that are faster than conventional phenotypic methods.

Comparison of new and old BacT/ALERT aerobic bottles for detection of Candida species.

PURPOSE: A new version of aerobic blood culture media has been developed for the BacT/ALERT (bioMérieux) blood culture system. We evaluated the time to detection and yeast cell counts in positive blood cultures for each Candida spp. according to changes in media. METHODS: Isolates from defibrinated horse blood were inoculated into three types of bottles: the old version of aerobic bottle, new version of aerobic bottle, and anaerobic bottle. All bottles were incubated in the BacT/ALERT Virtuo blood culture system. The time to detection was monitored for each bottle, and yeast cell counts were performed immediately after testing positive, determined via the plate count method. Clinical retrospective data of the candidemia samples before and after aerobic bottle change also were analyzed. RESULTS: The median time to detection was 52.47 hours in the old aerobic bottles versus 19.92 hours in the new aerobic bottles (P < 0.001) for Candida glabrata, and standard and clinical strains showed similar results. C. albicans (27.6 to 24.95 hours) and C. guilliermondii (28.92 to 26.9 hours) had shorter time to detection. However, C. auris (25.43 to 28.25 hours) had a longer time to detection in the new aerobic bottle. The retrospective clinical analysis showed a significant decrease in time to detection (45.0 to 19.4 hours) for C. glabrata, which is consistent with our simulated study result for C. glabrata. As a result of analysis including all blood specimens, C. tropicalis showed a significant delay in time to detection in new aerobic bottles. In an analysis limited to peripheral blood specimens, the time to detection of C. parapsilosis was longer in new aerobic bottles than in old aerobic bottles. CONCLUSION: Most Candida species did not show remarkable TTD differences, but TTD of C. glabrata was markedly reduced in the New FA Plus bottle. The reduction of time to detection enables faster detection and therapeutic approach for C. glabrata infections.

Blood culture identification (BCID) performance in polymicrobial bacteremia.

The rapid multiplex PCR (rmPCR)-based FilmArray® blood culture identification (BCID) assay reduces time from positive blood culture to organism identification. Polymicrobial bacteremia (PMB) is a known area of reduced diagnostic fidelity for BCID and remains incompletely characterized. All cases of clinically confirmed PMB at a large academic single center from a 23-month period were evaluated in a retrospective cohort analysis. A total of 207 samples were identified and studied. Overall, 49.3% (N = 102) of polymicrobial cultures were incompletely identified by FilmArray® result. There were no significant between-group differences in comorbidity status, length of stay, mortality, or source between patients with PMB who had complete versus incomplete BCID identification. Our results suggest that rmPCR-based assays frequently miss organisms in PMB and should be interpreted accordingly.

Optimizing Blood Culture Collection Volumes.

ABSTRACT: Infection in an immunocompromised person can be a life-threatening emergency. Collection of blood culture specimens is an important method for detecting organisms when infection is suspected. One aspect of proper blood culture collection is obtaining an accurate blood volume in the sample in accordance with the blood culture bottle manufacturer's recommendation. Underfilling the culture bottle can lead to false-negative results, while overfilling it can lead to false-positive results. At our institution, the Department of Laboratory Medicine (DLM) routinely monitors blood culture bottle volumes and notifies the nursing department of underfill/overfill events, which the department then reviews. Over several years, the DLM and the nursing department noted an increase in these events. A clinical nurse specialist and three staff nurses in the oncology/critical care services area partnered with the DLM to determine why. Upon investigation, two key issues-imprecise weighing of bottles and staff misunderstanding of the proper blood culture collection technique-were discovered. In response, the nursing standard of practice and guidance on the DLM webpage were updated and nursing education was performed. As a result, underfill/overfill events decreased by 71% in a little over a year.

Assessment of the impact of centralized bioMérieux BACT/ALERT® VIRTUO® blood culture system (VIRTUO) implementation on outcomes in patients with gram-negative bacteremia.

BACKGROUND: We evaluated pre- and postimplementation of Virtuo on outcome in patients with gram-negative bacteremia using a quasiexperimental time-in-motion design. METHODS: Becton Dickinson BACTEC™ 9000 series (Bactec) (2018) and Virtuo system (2020) were utilized in a decentralized and centralized process, respectively. Data collected in August-December in 2018 and 2020 were analyzed with SPSS (ver 28). RESULTS: For 185 patients in each time period, patient age, gender, length of hospitalization were not different. However, blood culture (BC) volume was significantly lower in 2020 (7.1 ± 2.6 mL) compared to 2018 (8.9 ± 1.9 mL). Time from BC draw and time from pathogen identification (ID) to treatment change were both significantly faster in 2020 (52.9 ± 38.3 hours; 15.1 ± 27.4 hours), compared to 2018 (65.0 ± 46.3 hours; 23.8 ± 33.8), respectively. CONCLUSIONS: Replacement of decentralized Bactec with centralized Virtuo, resulted in significant improvement in management of patients gram-negative bacteremia.

Prediction of methicillin-resistant Staphylococcus aureus and carbapenem-resistant Klebsiella pneumoniae from flagged blood cultures by combining rapid Sepsityper MALDI-TOF mass spectrometry with machine learning.

This study investigated combination of the Rapid Sepsityper Kit and a machine learning (ML)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) approach for rapid prediction of methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Klebsiella pneumoniae (CRKP) from positive blood culture bottles. The study involved 461 patients with monomicrobial bloodstream infections. Species identification was performed using the conventional MALDI-TOF MS Biotyper system and the Rapid Sepsityper protocol. The data underwent preprocessing steps, and ML models were trained using preprocessed MALDI-TOF data and corresponding labels. The interpretability of the model was enhanced using SHapely Additive exPlanations values to identify significant features. In total, 44 S. aureus isolates comprising 406 MALDI-TOF MS files and 126 K. pneumoniae isolates comprising 1249 MALDI-TOF MS files were evaluated. This study demonstrated the feasibility of predicting MRSA among S. aureus and CRKP among K. pneumoniae isolates using MALDI-TOF MS and Sepsityper. Accuracy, area under the receiver operating characteristic curve, and F1 score for MRSA/methicillin-susceptible S. aureus were 0.875, 0.898 and 0.904, respectively; for CRKP/carbapenem-susceptible K. pneumoniae, these values were 0.766, 0.828 and 0.795, respectively. In conclusion, the novel ML-based MALDI-TOF MS approach enables rapid identification of MRSA and CRKP from flagged blood cultures within 1 h. This enables earlier initiation of targeted antimicrobial therapy, reducing deaths due to sepsis. The favourable performance and reduced turnaround time of this method suggest its potential as a rapid detection strategy in clinical microbiology laboratories, ultimately improving patient outcomes.

Rapid phenotypic antimicrobial susceptibility testing of Gram-negative rods directly from positive blood cultures using the novel Q-linea ASTar system.

Adequate and timely antibiotic therapy is crucial for the treatment of sepsis. Innovative systems, like the Q-linea ASTar, have been developed to perform rapid antimicrobial susceptibility testing (AST) directly from positive blood cultures (BCs). We conducted a prospective study to evaluate ASTar under real-life conditions with a focus on time-to-result and impact on antimicrobial therapy. Over 2 months, all positive BCs that showed Gram-negative rods upon microscopy were tested with the ASTar and our standard procedure (VITEK 2 from short-term culture). Additionally, we included multidrug-resistant Gram-negative bacteria from our archive. Both methods were compared to broth microdilution. In total, 78 bacterial strains (51 prospective and 27 archived) were tested. ASTar covered 94% of the species encountered. The categorical and essential agreement was 95.6% and 90.7%, respectively. ASTar caused 2.4% minor, 2.0% major, and 2.4% very major errors. The categorical agreement was similar to standard procedure. The average time between BC sampling and the availability of the antibiogram for the attending physician was 28 h 49 min for ASTar and 44 h 18 min for standard procedure. ASTar correctly identified all patients who required an escalation of antimicrobial therapy and 75% of those who were eligible for de-escalation. In conclusion, ASTar provided reliable AST results and significantly shortened the time to obtain an antibiogram. However, the percentage of patients that will profit from ASTar in a low-resistance setting is limited, and it is currently unclear if a change of therapy 29 h after BC sampling will have a significant impact on the patient's prognosis.

Rapid phenotypic testing for detection of carbapenemase- or extended-spectrum ß-lactamase-producing Enterobacterales directly from blood cultures: a systematic review and meta-analysis.

BACKGROUND: Early identification of extended-spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacterales (CP-CRE) is critical for timely therapy. Rapid phenotypic tests identifying these resistance mechanisms from pure bacterial colonies have been developed. OBJECTIVES: To determine the operating characteristics of available rapid phenotypic tests when applied directly to positive blood cultures. METHODS OF DATA SYNTHESIS: Bivariate random effects models were used unless convergence was not achieved where we used separate univariate models for sensitivity and specificity. DATA SOURCES: MEDLINE, CENTRAL, Embase, BIOSIS, and Scopus from inception to 16 March 2021. STUDY ELIGIBILITY CRITERIA: Studies using any rapid phenotypic assay for detection of ESBL or CP-CRE directly from blood cultures positive for Enterobacterales, including those utilizing spiked blood cultures. Case reports/series, posters, abstracts, review articles, those with ≤5 resistant isolates, and studies lacking data or without full text were excluded. PARTICIPANTS: Consecutive patient samples (main analysis) or spiked blood cultures (sensitivity analysis). TESTS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assays (MALDI-TOF) and commercially available chromogenic or immunogenic assays. REFERENCE STANDARD: Conventional laboratory methods and/or polymerase chain reaction (PCR). ASSESSMENT OF RISK OF BIAS: Quality Assessment of Diagnostic Accuracy Studies Version 2 (QUADAS-2). RESULTS: For detection of the ESBL phenotype the respective pooled sensitivities and specificities for consecutive clinical samples were as follows: 94% (95% CI 93-99%) and 97% (95% CI 95-100%) for MALDI-TOF/mass spectrometry (n = 1); and 98% (95% CI 92-100%) and 100% (95% CI 96-100%) for chromogenic assays (n = 7). For the CP-CRE phenotype the respective pooled sensitivity and specificities for consecutive clinical samples were as follows: 100% (95% CI 99-100%) and 100% (95% CI 100-100%) for MALDI-TOF (n = 2); 96% (95% CI 77-99%) and 100% (95% CI 81-100%) for chromogenic assays (n = 4); and 98% (95% CI 96-100%) and 100% (95% CI 100-100%) for immunogenic testing (n = 2). CONCLUSIONS: Rapid phenotypic assays that can be directly applied to positive blood cultures to detect ESBL and carbapenemase production from Enterobacterales exist and, although clinical studies are limited, they appear to have high sensitivity and specificity. Their potential to facilitate patient care through timely identification of bacterial resistance should be further explored.

Evaluation of the Qvella FAST System and the FAST-PBC cartridge for rapid species identification and antimicrobial resistance testing directly from positive blood cultures.

Blood culture diagnostics require rapid and accurate identification (ID) of pathogens and antimicrobial susceptibility testing (AST). Standard procedures, involving conventional cultivation on agar plates, may take up to 48 hours or more until AST completion. Recent approaches aim to shorten the processing time of positive blood cultures (PBC). The FAST System is a new technology, capable of purifying and concentrating bacterial/fungal pathogens from positive blood culture media and producing a bacterial suspension called "liquid colony" (LC), which can be further used in downstream analyses (e.g., ID and AST). Here, we evaluated the performance of the FAST System LC generated from PBC in comparison to our routine workflow including ID by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using Sepsityper, AST by automatized MicroScan WalkAway plus and directly inoculated disk diffusion (DD), and MICRONAUT-AM for yeast/fungi. A total of 261 samples were analyzed, of which 86.6% (226/261) were eligible for the comparative ID and AST analyses. In comparison to the reference technique (culture-grown colonies), ID concordance of the FAST System LC and Sepsityper was 150/154 (97.4%) and 123/154 (79.9%), respectively, for Gram positive; 67/70 (95.7%) and 64/70 (91.4%), respectively, for Gram negative. For AST, categorical agreement (CA) of the FAST System LC in comparison to the routine workflow for Gram-positive bacteria was 96.1% and 98.7% for MicroScan and DD, respectively. Similar results were obtained for Gram-negative bacteria with 96.6% and 97.5% of CA for MicroScan and DD, respectively. Taken together, the FAST System LC allowed the laboratory to significantly reduce the time to obtain correct ID and AST (automated MicroScan) results 1 day earlier and represents a promising tool to expedite the processing of PBC.

Shortening identification times: comparative observational study of three early blood culture testing protocols.

Background: While early appropriate antibiotic therapy is a proven means of limiting the progression of infections, especially bacteremia, empirical antibiotic therapy in sepsis is ineffective up to 30%. The aim of this study was to compare early blood culture testing protocols in terms of their ability to shorten the delay between blood sampling and appropriate antibiotic therapy. Methods: In this french observational study, we compared three blood culture testing protocols. Positive blood cultures were tested using either GenMark ePlex panels (multiplex PCR period), a combination of MRSA/SA PCR, ß-Lacta and oxidase tests (multitest period), or conventional identification and susceptibility tests only (reference period). Conventional identification and susceptibility tests were performed in parallel for all samples, as the gold standard. Results: Among the 270 patients with positive blood cultures included, early and conventional results were in good agreement, especially for the multitest period. The delay between a blood culture positivity and initial results was 3.8 (2.9-6.9) h in the multiplex PCR period, 2.6 (1.3-4.5) h in the multitest period and 3.7 (1.8-8.2) h in the reference period (p<0.01). Antibiotic therapy was initiated or adjusted in 68 patients based on early analysis results. The proportion of patients receiving appropriate antibiotic therapy within 48 h of blood sampling was higher in the multiplex PCR and multitest periods, (respectively 90% and 88%) than in the reference period (71%). Conclusion: These results suggest rapid bacterial identification and antibiotic resistance tests are feasible, efficient and can expedite appropriate antibiotic therapy.

The Yield, Safety, and Cost-effectiveness of Decreasing Repeat Blood Cultures Beyond 48 Hours in a Pediatric Hematology-Oncology Unit.

Clear recommendations are needed on when repeat blood cultures (BCxs) in hospitalized children with cancer should be obtained. We reviewed all BCx obtained on the Hematology-Oncology Unit at Riley Hospital for Children, regardless of reason for patient admission or neutropenia status, between January 2015 and February 2021. Patients with positive BCx within 48 hours of initial cultures, history of stem cell transplant, or admitted to the intensive care unit were excluded. Medical records of patients with new positive BCx drawn >48 hours after initial BCx were reviewed. Seven (1.2%) hospitalization episodes grew new pathogens, or commensals treated as pathogens, on cultures beyond 48 hours. All patients with new, true pathogens were hemodynamically unstable or had recurrent fever when the new positive BCx was obtained. Twenty-three (4.0%) hospitalization episodes had contaminant cultures beyond 48 hours, with 74 (5.4%) of 1362 BCx collected beyond 48 hours being contaminated, resulting in an additional cost of $210,519 from increased length of stay. In conclusion, repeat BCx beyond 48 hours in pediatric hematology-oncology patients with negative initial cultures are low yield and costly. Repeat BCx can be safely and cost-effectively ceased after 48 hours of negative cultures in hemodynamically and clinically stable patients.

Evaluation of the liquid colony for identification and antimicrobial susceptibility testing directly from positive blood cultures.

BACKGROUND: Sepsis represents a time-sensitive disease requiring early therapeutical intervention to avoid adverse patient outcomes. Rapid microbiological diagnosis is essential to investigate sepsis aetiological agents. The FAST™ system (Qvella, ON, Canada) provides a concentrated microbial suspension, known as a Liquid Colony™ (LC), directly from positive blood samples (PBCs) in 30-40 min to perform rapid identification (ID) and antimicrobial susceptibility testing (AST). METHODS: Qvella's FAST™ System and FAST PBC Prep cartridges were tested on PBCs from the Policlinico Hospital of Catania during a six-month study. Two millilitres of PBC were converted into an LC for rapid ID and AST using Bruker Biotyper Sirius MALDI and BD Phoenix systems. Standard of care (SOC) methods were used as a reference, requiring 48-72 h. Agreement between the innovative technology and the standard method was calculated. RESULTS: FAST System processing was performed on 100 monomicrobial PBCs. Median turnaround times from blood cultures flagging positive to ID and AST completion were 2 and 26 h respectively. Therefore, the LC procedure was 24 h faster than the median turnaround times for SOC methods. 100% ID identification concordance was observed across 48 Gram-negative bacteria, 42 Gram-positive bacteria and 11 yeast for the genus level. 78% of Gram-negative and 95% of Gram-positive bacteria were resistant to ≥ 2 antimicrobial agents, including 45% (15/33) carbapenem-resistant enteric Gram-negative bacteria and 90% (28/31) oxacillin-resistant staphylococci. An AST essential agreement of 100% was observed due to the absence of MIC discrepancies > 1-fold dilution. Categorical errors were not observed due to the absence of MIC categorization discordances. Yeast AST was not performed. CONCLUSIONS: The Qvella FAST System produces an LC that reliably reflects the MALDI spectra and phenotypic antimicrobial susceptibility profile of microbial cells growing in the blood culture. Timely processing of PBCs with the Qvella FAST System enables sepsis diagnostic confirmation 1 day sooner than the standard methods. In line with these results, it is vital now to focus attention on establishing best practices for incorporating this strategic tool into the clinical microbiology laboratory workflow.

A prospective study evaluating the effect of a 'Diagnostic Stewardship Care-Bundle' for automated blood culture diagnostics.

OBJECTIVES: We prospectively implemented a diagnostic stewardship care-bundle checklist, 'Sepsis-48 DSB', with the aim of reducing intervening duration of key steps of automated blood culture diagnostics (aBCD). METHODS: Sepsis-48 DSB was implemented for automated blood culture bottles (BCBs) received from adult intensive care units (AICUs) during the intervention period (P2; July 2020-June 2021) and intervening durations were compared with those during the retrospective, pre-intervention period (P1; March-June 2020). During both periods, provisional blood culture reports (pBCR) were issued wherein direct microbial identification (dID) was performed in BCBs with Gram-negatives by directly inoculating conventional biochemical tests and direct antimicrobial susceptibility testing (dAST) using EUCAST RAST method. The results were compared with the standard of care (SoC) method (i.e. full incubation followed by identification and AST by VITEKⓇ-2 Compact). RESULTS: During P2, significant reductions in loading time (LT) [median: 63.5 vs. 32 minutes, P < 0.001], time to dID+dAST performance (TTD) [186 vs. 115 minutes, P = 0.0018] and an increase in compliance to bundle targets [LT ≤45: 44% vs. 66%, P = 0.006 and TTD ≤120: 34% vs. 51.7%, P = 0.03] were observed. Using dID+dAST method, results were read 694 minutes earlier than SoC method. Of 176 pBCR, 165 (94%) were concordant with SoC in microbial identification of species. Categorical agreement for any drug-bug combination was 92.7% (1079/1164) and corresponding major, very major, and minor error rates were 8.8% (19/216), 4.9% (45/921), and 1.8% (21/1164), respectively. CONCLUSION: The 'diagnostic stewardship care-bundle' strategy was successfully implemented with considerable diagnostic accuracy leading to significant reductions in duration of targeted steps of aBCD.

Comparison of 3 diagnostic platforms for identification of bacteria and yeast from positive blood culture bottles.

Managing bloodstream infections requires fast and accurate diagnostics. Culture-based diagnostic methods for identification from positive blood culture require 24-hour subculture, potentially delaying time to appropriate therapy. Positive blood cultures were collected (n = 301) from September 2021 to August 2022 at the University of Maryland Medical Center. Platforms compared were BioFire® BCID2, Sepsityper®, and short-term culture. For monomicrobial cultures, FilmArray® BCID2 identified 88.3% (241/273) of pathogens. Rapid Sepsityper® identified 76.9% (210/273) of pathogens. Sepsityper® extraction identified 82.4% (225/273) of pathogens. Short-term culture identified 83.5% (228/273) of pathogens. For polymicrobial cultures, Sepsityper®, short-term culture, and BioFire® BCID2 had complete identifications at 10.7% (3/28), 0%, and 92.9% (26/28), respectively. Time-to-results for Rapid Sepsityper®, Sepsityper® extraction, BioFire® BCID2, and Short-term culture were 35, 52, 65, and 306 minutes, respectively. Performance of these platforms can reduce time-to-results and may help effectively treat bloodstream infections faster. Accuracy, time-to-result, and hands-on time are important factors when evaluation diagnostic platforms.

[Application and Prospect of Nanopore Sequencing Technology in Etiological Diagnosis of Blood Stream Infection].

Blood stream infection (BSI),a blood-borne disease caused by microorganisms such as bacteria,fungi,and viruses,can lead to bacteremia,sepsis,and infectious shock,posing a serious threat to human life and health.Identifying the pathogen is central to the precise treatment of BSI.Traditional blood culture is the gold standard for pathogen identification,while it has limitations in clinical practice due to the long time consumption,production of false negative results,etc.Nanopore sequencing,as a new generation of sequencing technology,can rapidly detect pathogens,drug resistance genes,and virulence genes for the optimization of clinical treatment.This paper reviews the current status of nanopore sequencing technology in the diagnosis of BSI.