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

Changes in clinical parameters and inflammatory markers after blood culture collection facilitate early identification of positive culture in adult patients with COVID-19 and clinically suspected bloodstream infection.

OBJECTIVE: We explored whether changes in clinical parameters and inflammatory markers can facilitate early identification of positive blood culture in adult patients with COVID-19 and clinically suspected bloodstream infection (BSI). METHODS: This single-center retrospective study enrolled 20 adult patients with COVID-19 admitted to the intensive care unit who underwent blood culture for clinically suspected BSI (February 2020-November 2021). We divided patients into positive (Pos) and negative blood culture groups. Clinical parameters and inflammatory markers were obtained from medical records between blood culture collection and the first positive or negative result and compared between groups on different days. RESULTS: Patients in the positive culture group had significantly older age and higher D-dimer, immunoglobulin 6 (IL-6), and Sequential Organ Failure Assessment score as well as lower albumin (ALB). The area under the receiver operating characteristic curve (AUC) was 0.865 for IL-6, D-dimer and ALB on the first day after blood culture collection; the AUC was 0.979 for IL-6, IL-10, D-dimer, and C-reactive protein on the second day after blood culture collection. CONCLUSION: Changes in clinical parameters and inflammatory markers after blood culture collection may facilitate early identification of positive culture in adult patients with COVID-19 and clinically suspected BSI.

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.

Rapid antibiotic susceptibility testing (RAST) of blood cultures in enterobacteria with inducible chromosomal AmpC-type ß-lactamase.

INTRODUCTION: Early and adequate treatment of bloodstream infections decreases patient morbidity and mortality. The objective is to develop a preliminary method for rapid antibiotic susceptibility testing (RAST) in enterobacteria with inducible chromosomal AmpC. METHODS: RAST was performed directly on spiked blood cultures of 49 enterobacteria with inducible chromosomal AmpC. Results were read at 4, 6 and 8h of incubation. Commercial broth microdilution was considered the reference method. Disks of 10 antibiotics were evaluated. RESULTS: The proportion of readable tests at 4h was 85%. All RAST could be read at 6 and 8h. For most antibiotics, the S or R result at 4, 6 and 8h was greater than 80% after tentative breakpoints were established and Area of Technical Uncertainty was defined. CONCLUSIONS: This preliminary method seems to be of practical use, although it should be extended to adjust the breakpoints and differentiate them by species.

Analysis of Blood Cultures from Major Burns Patients in a Tertiary Care Burn Unit in Oman.

Objectives: This study aimed to review the blood stream infections of major burn patients in a tertiary care burn unit to determine the most prevalent infecting organisms in order to have a better empirical therapy protocol. Methods: This retrospective study analysed the blood stream infection of 155 major burn (>20% Total Body Surface Area [TBSA]) patients in Khoula Hospital, Muscat, Oman between January 2014 to December 2019. Results: The median age was 33 years and 57.42% of patients were male. The median TBSA was 38%, mortality was 25.16% and 50.9% of patients had positive blood cultures. The expired patients had higher TBSAs, Abbreviated Burns Severity Index scores and earlier first positive blood cultures. Candida was commonly grown in all the blood cultures, but the most prevalent organisms were Acinetobacter, Staphylococci, Klebsiella, Enterococcus and Pseudomonas. All Acinetobacter species are multidrug resistant. Of the 17 patients who had Kelbsiella grown in the blood culture, 8 grew multidrug-resistant Klebsiella. Only 4 patients' blood cultures grew methicillin-resistant Staphylococcus aureus. The number of blood culture samples taken ranged between 1-28 (median = 6). The first positive blood culture showed that Staphylococcus epidermidis and Acinetobacter were the most common infecting organisms. Conclusions: Multidrug-resistant Acinetobacter was the most predominant microorganism grown from the blood cultures of major burn patients in a tertiary care burn unit. Empirical therapy should include antibiotics that are effective against this organism to reduce the mortality.

Time to positivity of Klebsiella pneumoniae in blood cultures as prognostic marker in patients with intra-abdominal infection: A retrospective study.

Klebsiella pneumoniae is a common causative pathogen of intra-abdominal infection with concomitant bacteraemia, leading to a significant mortality risk. The time to positivity (TTP) of blood culture is postulated to be a prognostic factor in bacteraemia caused by other species. Therefore, this study aimed to investigate the prognostic value of TTP in these patients. The single-centred, retrospective, observational cohort study was conducted between 1 July 2016 and 30 June 2021. All adult emergency department patients with diagnosis of intra-abdominal infection and underwent blood culture collection which yield K. pneumoniae during this period were enrolled. A total of 196 patients were included in the study. The overall 30-day mortality rate was 12.2% (24/196), and the median TTP of the studied cohort was 12.3 h (10.5-15.8 h). TTP revealed a moderate 30-day mortality discriminative ability (area under the curve 0.73, p < 0.001). Compared with the late TTP group (>12 h, N = 109), patients in the early TTP (≤12 h, N = 87) group had a significantly higher risk of 30-day morality (21.8% vs. 4.6%, p < 0.01) and other adverse outcomes. Furthermore, TTP (odds ratio [OR] = 0.79, p = 0.02), Pitt bacteraemia score (OR = 1.30, p = 0.03), and implementation of source control (OR = 0.06, p < 0.01) were identified as independent factors related to 30-day mortality risk in patients with intra-abdominal infection and K. pneumoniae bacteraemia. Therefore, physicians can use TTP for prognosis stratification in these patients.

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.

Isolation and identification of Wickerhamiella tropicalis from blood culture by MALDI-MS.

Wickerhamiella is a genus of budding yeast that is mainly isolated from environmental samples, and 40 species have been detected. The yeast isolated from human clinical samples usually only contain three species: W. infanticola, W. pararugosa and W. sorbophila. In this study, we isolated W. tropicalis from a blood sample of a six-year-old female with a history of B-cell precursor lymphoblastic leukemia in Japan in 2022. Though the strain was morphologically identified as Candida species by routine microbiological examinations, it was subsequently identified as W. tropicalis by sequencing the internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The isolate had amino acid substitutions in ERG11 and FKS1 associated with azole and echinocandin resistance, respectively, in Candida species and showed intermediate-resistant to fluconazole and micafungin. The patient was successfully treated with micafungin. Furthermore, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detected three novel peaks that are specific for W. tropicalis, indicating that MALDI-MS analysis is useful for rapid detection of Wickerhamiella species in routine microbiological examinations.

Analysis of antibiotic resistance of KPC-2-positive Klebsiella pneumoniae strains isolated from blood cultures in Van, Turkey.

OBJECTIVE: Recently, Klebsiella pneumoniae strains causing bacteremia and showing significant antibiotic resistance have raised serious health concerns. Especially carbapenem-resistant K. pneumoniae has spread worldwide and caused an increase in mortality rates. In this study, we aimed to present information about KPC-2 carriage and molecular characteristics of K. pneumoniae strains showing multiple antibiotic resistance among patients in our hospital. PATIENTS AND METHODS: Blood samples were collected from patients hospitalized in the intensive care units (ICU) of Van Regional Training and Research Hospital between 2020-2021. Culture, biochemical tests, antibiotic susceptibility tests, and molecular tests were performed to identify K. pneumoniae strains isolated from blood culture samples. RESULTS: Two hundred and sixteen K. pneumoniae were isolated from patients with positive blood cultures. Twenty-seven of these isolates showed multidrug resistance. Carbapenem, ß-lactam, and quinolone resistance were particularly high. On the contrary, almost all of these isolates were susceptible to Amikacin (AK), Gentamicin (CN), Colistin (CT) and Tigecycline (TGC). Molecular analysis revealed that all of these isolates were KPC-2-positive and ST11 variants. CONCLUSIONS: It was observed that KPC-2- positive K. pneumoniae strains with multi-drug resistance may pose a serious risk in patients hospitalized in ICU in our hospital. It was concluded that surveillance and personnel training regarding the hospital and community-acquired infections due to these isolates that show pandemic spread would be important.

Performance evaluation of machine-assisted interpretation of Gram stains from positive blood cultures.

Manual microscopy of Gram stains from positive blood cultures (PBCs) is crucial for diagnosing bloodstream infections but remains labor intensive, time consuming, and subjective. This study aimed to evaluate a scan and analysis system that combines fully automated digital microscopy with deep convolutional neural networks (CNNs) to assist the interpretation of Gram stains from PBCs for routine laboratory use. The CNN was trained to classify images of Gram stains based on staining and morphology into seven different classes: background/false-positive, Gram-positive cocci in clusters (GPCCL), Gram-positive cocci in pairs (GPCP), Gram-positive cocci in chains (GPCC), rod-shaped bacilli (RSB), yeasts, and polymicrobial specimens. A total of 1,555 Gram-stained slides of PBCs were scanned, pre-classified, and reviewed by medical professionals. The results of assisted Gram stain interpretation were compared to those of manual microscopy and cultural species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparison of assisted Gram stain interpretation and manual microscopy yielded positive/negative percent agreement values of 95.8%/98.0% (GPCCL), 87.6%/99.3% (GPCP/GPCC), 97.4%/97.8% (RSB), 83.3%/99.3% (yeasts), and 87.0%/98.5% (negative/false positive). The assisted Gram stain interpretation, when compared to MALDI-TOF MS species identification, also yielded similar results. During the analytical performance study, assisted interpretation showed excellent reproducibility and repeatability. Any microorganism in PBCs should be detectable at the determined limit of detection of 105 CFU/mL. Although the CNN-based interpretation of Gram stains from PBCs is not yet ready for clinical implementation, it has potential for future integration and advancement.

Streptococcus taonis sp. nov., a novel bacterial species isolated from a blood culture of a patient.

One Gram stain-positive, catalase-negative, α-hemolytic, chain-forming or paired cocci, designated ST22-14T, was isolated from a blood culture of a child with suspected infection. The results of 16S rRNA gene sequences analyses showed that the most closely related species to strain ST22-14T were "Streptococcus vulneris" DM3B3T (99.2%), Streptococcus mitis NCTC 12261T (99.0%), "Streptococcus gwangjuense" ChDC B345T, (99.0%), Streptococcus oralis subsp. dentisani 7747T (99.0%), Streptococcus downii CECT 9732T (99.0%), and Streptococcus infantis ATCC 700779T (98.9%). The genome of strain ST22-14T consists of 2,053,261 bp with a G + C content of 39.4%. Average nucleotide identity values between strain ST22-14T and Streptococcus mitis NCTC 12261T or other five species were from 82.2 to 88.0%. In silico DNA-DNA hybridization of ST22-14T showed an estimated DNA reassociation value of 34.6% with the closest species. The main cellular fatty acids of strain ST22-14T were 16:0, 18:0, 14:0, 18:1ω7c and 18:1ω6c. Based on these results, strain ST22-14T should be classified as a novel species of genus Streptococcus, for which the name Streptococcus taonis sp. nov. is proposed (type strain ST22-14T = NBRC 116002T = BCRC 81402T).

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.

Plasma Microbial Cell-Free DNA Sequencing for Pathogen Detection and Quantification in Children With Musculoskeletal Infections.

BACKGROUND: Nearly half of all pediatric musculoskeletal infections (MSKIs) are culture negative. Plasma microbial cell-free DNA (mcfDNA) sequencing is noninvasive and not prone to the barriers of culture. We evaluated the performance of plasma mcfDNA sequencing in identifying a pathogen, and examined the duration of pathogen detection in children with MSKIs. METHODS: We conducted a prospective study of children, aged 6 months to 18 years, hospitalized from July 2019 to May 2022 with MSKIs, in whom we obtained serial plasma mcfDNA sequencing samples and compared the results with cultures. RESULTS: A pathogen was recovered by culture in 23 of 34 (68%) participants, and by initial mcfDNA sequencing in 25 of 31 (81%) participants. Multiple pathogens were detected in the majority (56%) of positive initial samples. Complete concordance with culture (all organisms accounted for by both methods) was 32%, partial concordance (at least one of the same organism(s) identified by both methods) was 36%, and discordance was 32%. mcfDNA sequencing was more likely to show concordance (complete or partial) if obtained prior to a surgical procedure (82%), compared with after (20%), (RR 4.12 [95% CI 1.25, 22.93], p = .02). There was no difference in concordance based on timing of antibiotics (presample antibiotics 60% vs no antibiotics 75%, RR 0.8 [95% CI 0.40, 1.46], p = .65]). mcfDNA sequencing was positive in 67% of culture-negative infections and detected a pathogen for a longer interval than blood culture (median 2 days [IQR 1, 6 days] vs 1 day [1, 1 day], p < .01). CONCLUSIONS: Plasma mcfDNA sequencing may be useful in culture-negative pediatric MSKIs if the sample is obtained prior to surgery. However, results must be interpreted in the appropriate clinical context as multiple pathogens are frequently detected supporting the need for diagnostic stewardship.

Affordable blood culture systems from China: in vitro evaluation for use in resource-limited settings.

BACKGROUND: Bloodstream infections (BSI) pose a significant threat due to high mortality rates and the challenges posed by antimicrobial resistance (AMR). In 2019, an estimated 4.95 million deaths were linked to bacterial AMR. The highest impact was seen in resource-limited settings (RLS). For diagnosis of BSI, performant continuously-monitoring blood culture systems (CMBCS) have been optimized. However, in RLS, the implementation of CMBCS is hindered by budget constraints and unsuitable environmental conditions. Manufacturers from growing economies are currently producing affordable in vitro diagnostics, which could fill the gap in capacity, but so far these are not established outside their domestic markets. METHODS: This study evaluated the performance, usability, and interchangeability of Chinese CMBCS in a laboratory setting using simulated blood cultures with a panel of 20 BSI-associated strains. Four systems were selected for the assessment: Autobio BC60, Mindray TDR60, Scenker Labstar50, and DL-biotech DL-60. FINDINGS: Overall, all evaluated CMBCS demonstrated good performance with high yield (96.7-100%) and specificity (97.5-100%), comparable to the reference system (bioMérieux 3D). In addition, when used as "manual" blood cultures in a conventional incubator with visual growth detection, performance was also satisfactory: yield was between 90 and 100% and specificity was 100% for all BCBs. Both the CMBCS and the BCBs were easy to use and lot-to-lot variability in BCBs was minimal. The interchangeability testing indicated that the BCBs from different brands (all except Scenker) were compatible with the various automates, further highlighting the potential for a harmonized "universal BCB." INTERPRETATION: Based on this in vitro study, we recommend the use of these systems in settings with challenging environments and limited resources. The Autobio system performed best for automatic detection and DL-Biotech BCBs for manual cultures respectively (combination of performance, price, usability). The appropriateness for use in RLS should still be confirmed in a field study. FUNDING: The study was funded by FIND.

Comparison of metagenomic next-generation sequencing and blood culture for diagnosis of bloodstream infections.

Objectives: This study aimed to evaluate the clinical performance of plasma cell-free DNA (cfDNA) next-generation sequencing (NGS) for pathogen detection in patients with sepsis. Methods: A total of 43 pairs of blood and plasma samples form 33 blood culture-positive patients were used as testing samples in metagenomic NGS (mNGS) and NGS of 16S ribosomal RNA gene amplicons (16S rRNA NGS). The results of routine tests, including microbial culture, complete blood count, and biochemical tests, were collected from electronic medical records. Results: Using blood as an mNGS testing sample, the proportion of host DNA was 99.9%, with only three bacteria and no fungi detected. When using plasma in mNGS, the proportion of host DNA was approximately 97%, with 84 bacteria and two fungi detected. Notably, 16S rRNA NGS detected 15 and 16 bacteria in 43 pairs of blood and plasma samples, respectively. Blood culture detected 49 bacteria (23 gram-negative bacilli and 26 gram-positive cocci) and four fungi, with 14 bacteria considered contaminants by clinical microbiologists. For all blood cultures, plasma cfDNA mNGS detected 78.26% (19/23) gram-negative rods, 17% (2/12) gram-positive cocci, and no fungi. Compared to blood cultures, the sensitivity and specificity of plasma cfDNA mNGS for detecting bacteria and fungi were 62.07% and 57.14%, respectively. Conclusion: Compared to blood, plasma is more suitable for the detection of bloodstream infections using mNGS and is less affected by host DNA. The positive detection rate of plasma cfDNA mNGS for bloodstream infections caused by gram-negative bacteria was higher than that caused by gram-positive cocci.

Cytokine expression in subjects with Mycobacterium avium ssp. paratuberculosis positive blood cultures and a meta-analysis of cytokine expression in Crohn's disease.

Objectives: 1) Culture Mycobacterium avium ssp. paratuberculosis (MAP)from blood, 2) assess infection persistence, 3) determine Crohn's disease (CD) cytokine expression, 4) compare CD cytokine expression to tuberculosis, and 5) perform a meta-analysis of cytokine expression in CD. Methods: The Temple University/Abilene Christian University (TU/ACU) study had a prospective case control design with 201 subjects including 61 CD patients and 140 non-CD controls. The culture methods included MGIT, TiKa and Pozzato broths, and were deemed MAP positive, if IS900 PCR positive. A phage amplification assay was also performed to detect MAP. Cytokine analysis of the TU/ACU samples was performed using Simple Plex cytokine reagents on the Ella ELISA system. Statistical analyses were done after log transformation using the R software package. The meta-analysis combined three studies. Results: Most subjects had MAP positive blood cultures by one or more methods in 3 laboratories. In our cytokine study comparing CD to non-CD controls, IL-17, IFNγ and TNFα were significantly increased in CD, but IL-2, IL-5, IL-10 and GM-CSF were not increased. In the meta-analysis, IL-6, IL-8 and IL-12 were significantly increased in the CD patients. Conclusion: Most subjects in our sample had MAP infection and 8 of 9 subjects remained MAP positive one year later indicating persistent infection. While not identical, cytokine expression patterns in MAP culture positive CD patients in the TU/ACU study showed similarities (increased IL-17, IFNγ and TNFα) to patterns of patients with Tuberculosis in other studies, indicating the possibilities of similar mechanisms of pathogen infection and potential strategies for treatment.

Rapid determination of antimicrobial susceptibility of Gram-negative bacteria from clinical blood cultures using a scattered light-integrated collection device.

Background. A bloodstream infection (BSI) presents a complex and serious health problem, a problem that is being exacerbated by increasing antimicrobial resistance (AMR).Gap Statement. The current turnaround times (TATs) for most antimicrobial susceptibility testing (AST) methods offer results retrospective of treatment decisions, and this limits the impact AST can have on antibiotic prescribing and patient care. Progress must be made towards rapid BSI diagnosis and AST to improve antimicrobial stewardship and reduce preventable deaths from BSIs. To support the successful implementation of rapid AST (rAST) in hospital settings, a rAST method that is affordable, is sustainable and offers comprehensive AMR detection is needed.Aim. To evaluate a scattered light-integrated collection (SLIC) device against standard of care (SOC) to determine whether SLIC could accelerate the current TATs with actionable, accurate rAST results for Gram-negative BSIs.Methods. Positive blood cultures from a tertiary referral hospital were studied prospectively. Flagged positive Gram-negative blood cultures were confirmed by Gram staining and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Vitek 2, disc diffusion (ceftriaxone susceptibility only) and an SLIC device. Susceptibility to a panel of five antibiotics, as defined by European Committee on Antimicrobial Susceptibility Testing breakpoints, was examined using SLIC.Results. A total of 505 bacterial-antimicrobial combinations were analysed. A categorical agreement of 95.5 % (482/505) was achieved between SLIC and SOC. The 23 discrepancies that occurred were further investigated by the broth microdilution method, with 10 AST results in agreement with SLIC and 13 in agreement with SOC. The mean time for AST was 10.53±0.46 h and 1.94±0.02 h for Vitek 2 and SLIC, respectively. SLIC saved 23.96±1.47 h from positive blood culture to AST result.Conclusion. SLIC has the capacity to provide accurate AST 1 day earlier from flagged positive blood cultures than SOC. This significant time saving could accelerate time to optimal antimicrobial therapy, improving antimicrobial stewardship and management of BSIs.