Anaerobic Blood Culture Bottles Research Articles

Utility of Anaerobic Blood Cultures in Neonatal Sepsis Evaluation.

Clinicians variably obtain anaerobic blood cultures as part of sepsis evaluations in the neonatal intensive care unit (NICU). Our objective was to determine if anaerobic blood culture bottles yielded clinically relevant information by either recovering pathogens exclusively or more rapidly than the concurrently obtained aerobic culture bottle in the NICU. A retrospective cohort study of blood cultures obtained from infants admitted to the NICU from 08/01/2015-08/31/2023. Standard practice was to inoculate 2mL of blood divided equally between an aerobic and an anaerobic culture bottle. We analyzed positive blood cultures where both aerobic and anaerobic bottles were obtained and compared pathogen recovery and time to positivity between the bottles. During the study period, 4599 blood cultures were obtained from 3665 infants, and 265 (5.8%) were positive. Of these, 182 cultures were sent as aerobic-anaerobic pairs and recovered pathogenic organisms. Organisms were recovered exclusively from the anaerobic bottle in 32 (17.6%) cultures. Three organisms were obligate anaerobes; the rest were facultative anaerobes including Coagulase-negative staphylococci (40.6%), Escherichia coli (15.6%), and Staphylococcus aureus (15.6%). Cultures with exclusive recovery in the anaerobic bottle were more frequently obtained ≤3 days after birth, compared to other cultures (31.3% vs. 15.3%, p=0.03). When both bottles recovered the pathogen (n=113), the anaerobic bottle had a shorter time to positivity in 76 (67.3%) cultures. Including anaerobic culture bottles could lead to identification of pathogens not recovered in the aerobic bottle, as well as earlier identification of pathogens.

172. Impact of a quality improvement re-training program on the blood culture volume, yield and contamination rates; experience from South India

Abstract Background Blood cultures help to detect causative agents in bacteremia and are important for targeted therapy. Adequate blood culture volume is considered as 8-10ml in each bottle and at least 2 aerobic and 2 anaerobic blood culture bottles are recommended. Inadequate blood culture fills are common and may affect yield. Figure 1 Methods The study was done in our healthcare facility-KIMSHEALTH, Trivandrum, India. Pre-intervention period was between May 2021 and June 2021; post-intervention was between May 2022 and June 2022. The intervention was through plan/do/study/act (PDSA) cycles and included multiple training sessions to the phlebotomy team regarding adequate fill volume which is 8-10 ml in each blood culture bottle. Training for maintaining asepsis during collection was reinforced. All blood culture samples received in the hospital microbiology laboratory during the above period were included in this study and compared between the pre-intervention and post-intervention period. Pediatric blood culture samples were excluded. Results Blood culture samples studied during pre and post intervention period were 1428 and 1716 respectively. In the pre-intervention phase adequate fill volume was noted in 30.81% bottles (figure 1), while in the post intervention adequate fill volume was noted in 68.06% bottles (p < 0.01). Blood culture contamination rate was 3.01% in the pre-intervention period which improved to 2% in the post-intervention period and the difference was significant (p< 0.01). Blood culture yield of a pathogenic organism did not show significant difference after the intervention (11.2% in pre-intervention group Vs 11.45% in post intervention group). Conclusion In this study, an improvement was noted in the blood culture fills by 38% and contamination rate reduced by 1%. Though there was no significant increase in blood culture yield with intervention, the reduction in contamination was noted which will reduce unnecessary antibiotic initiation. Disclosures All Authors: No reported disclosures

587. Research and Development of Image Recognition AI to Estimate Bacterial Species using Gram Stain Findings in Aerobic and Anaerobic Blood Culture Bottle

Abstract Background Background In the highly fatal infectious disease of bacteremia, the ability to select antimicrobial agents at an earlier time is critical not only for early patient recovery, but also for reducing the development of resistant organisms. Gram staining of blood cultures is a useful test for early selection of antimicrobial agents, but it requires some skill in deciphering. Therefore, we developed an image analysis system for Gram stain images of blood cultures and have verified whether it is possible to estimate the bacterial species to facilitate the initial selection of antimicrobial agents, regardless of the level of proficiency. Methods Method Slides and bacterial species-identification information from an anonymized Gram stain registry at two medical institutions, National Center for Global health and Medicine (NCGM) and Kobe University Hospital (KUH), were used for the study. 1113 cases of aerobic bottles and 1060 cases of anaerobic bottles were included. Mock-specimens were used for the rare bacteria. A total of 23,947 images were generated by capturing the observation field of view of an optical microscope with a smartphone. Table 1 shows the bacterial classification of aerobic bottles and Table 2 shows the bacterial classification of anaerobic bottles. The data were divided into training data and test data at a ratio of 8 to 2 for each category.Table 1.A total of 16 categories of bacteria were used for classification of aerobic bottlesTable 2.A total of 16 categories of bacteria were used for classification of anaerobic bottles Results The macro-average recall (sensitivity) and accuracy of the test data were 64% and 82% for aerobic bottles and 68% and 86% for anaerobic bottles, respectively. The results showed the possibility of classifying the performing bacterial species with an accuracy of over 70% using only Gram stained images via image recognition AI. For aerobic bottles in particular, the seven categories of GNR, GNC, GPR, GPC, yeast, No organism, and multiple bacteria are predicted to have 97% accuracy, and 90% macro average recall. Conclusion The research has shown that it is possible to classify bacterial species to a certain extent only by observing the Gram stain images. Our goal is to narrow down and focus more on the clinically-meaningful bacterial species, and to improve the accuracy of our classification. Also, we are planning to make comparisons with specialists in the future. Disclosures Kei Yamamoto, MD, Canon medical systems: Grant/Research Support|CarbGeM: Grant/Research Support|CarbGeM: 7090302|Fujirebio: Grant/Research Support|Sanyo Chemical Industries: Grant/Research Support|VisGene: Grant/Research Support Goh Ohji, MD, PhD, DTMH, CarbGeM: Advisor/Consultant

Clinical usefulness of anaerobic blood culture in pediatric patients with bacteremia

ObjectivesThe purpose of this study is to evaluate the usefulness of anaerobic blood culture in pediatric patients by comparing the detection rate and distribution of bacteria between aerobic and anaerobic blood culture bottles. MethodsA retrospective analysis was conducted on 11,664 blood cultures obtained from children under the age of 14 between January 2013 and June 2020. The positive rate of total, aerobic, and anaerobic blood culture, as well as the species distribution of each blood culture bottle, were investigated. ResultsThe positive rate of blood culture was 2.4 % (N = 281). Among them, 67 (23.8 %), 85 (30.3 %) and 129 (45.9 %) organisms were grown in only aerobic, only anaerobic, and both blood culture bottles, respectively. Gram-positive cocci were cultured on both, only aerobic, and only anaerobic blood culture bottles in proportions of 46.4 %, 23.4 %, and 30.2 %, respectively. Gram-negative bacilli were cultured on both, only aerobic, and only anaerobic blood culture bottles in proportions of 58.5 %, 12,3 %, and 29.2 %, respectively. Gram-positive bacilli grew best in aerobic bottle only. There were seven strains of obligate anaerobes. ConclusionBecause many facultative anaerobic bacteria are recognized primarily from anaerobic blood culture bottles, combining aerobic and anaerobic blood culture bottles might be beneficial in pediatric patients with suspected blood stream infection.

Validation of the Accuracy of Automatic Measurement of Blood Volume in Culture Bottles for Blood Culture.

Several manufacturers have developed systems that automatically measure the amount of blood in culture bottles. We compared the volumes measured automatically by the Virtuo instrument (bioMérieux, France) (height-based volumes) and those calculated by weighing the bottles. In all, 150 pairs of aerobic and anaerobic blood culture bottles (BacT/ALERT FA/FN Plus, bioMérieux) were randomly selected over two periods to compare the height- and weight-based volumes and analyze the effect of foam. We also estimated the limit of detection (LOD) and the cut-off value for 5 mL equine blood. The mean height-based volume was approximately 0.67 mL greater than the weight-based volume, particularly in anaerobic culture bottles. Foam did not have a significant effect. The LOD for the automatic height-based volume of equine blood was 0.2-0.4 mL. The 5 mL cut-off was 4-4.2 mL. Therefore, when reporting or monitoring blood volume within culture bottles in the laboratory, these performance characteristics should be adequately considered.

Vertebral osteomyelitis caused by Fusobacterium nucleatum with an associated asymptomatic liver abscess in an immunocompetent adult: A case report and literature review

Fusobacterium nucleatum rarely causes vertebral osteomyelitis or liver abscesses, and no reports exist of it concurrently causing vertebral osteomyelitis and pyogenic liver abscess. A 58-year-old woman with a history of periodontitis presented with worsening lumbago, left lower leg pain, numbness, and fever for a week. Physical examination indicated knocking pain at the L2-L3 levels with a psoas sign on the left side. A magnetic resonance image showed L2-S1 vertebral osteomyelitis and intervertebral discitis, with a left psoas major muscle abscess. Vertebral osteomyelitis caused by Staphylococcus aureus was suspected; blood cultures were obtained, and intravenous cefazolin was administered. Computed tomography, which was performed to detect disseminated foci, revealed a multilocular liver abscess. On day 4 of incubation, the anaerobic blood culture bottles were positive for characteristic filamentous gram-negative rods. The empiric antimicrobial therapy was changed to ampicillin/sulbactam. The isolate was identified as F. nucleatum based on 16S rRNA gene sequencing. The liver abscess was drained on day 12. Based on the antimicrobial susceptibility test results, the patient was treated with intravenous ampicillin/sulbactam for 4 weeks followed by oral amoxicillin/clavulanate for an additional 8 weeks and remained disease-free at the 1-year follow-up. Clinicians should consider F. nucleatum as the causative organism for vertebral osteomyelitis presenting with asymptomatic pyogenic liver abscess. The gold standard for identifying and diagnosing F. nucleatum infections is 16S rRNA gene sequencing, and gram staining helps determine appropriate antimicrobials.

343. Relevance of Use of Anaerobic Blood Culture Bottle for the Diagnosis of Bacteremia

Abstract Background Blood cultures remain the cornerstone for the diagnosis of bacteremia. Although an anaerobic blood culture bottle can recover facultative and obligate anaerobes, routine use of an anaerobic bottle paired with an aerobic bottle remains controversial. We aimed to evaluate the role of anaerobic bottle for the diagnosis of bacteremia. Methods We conducted a retrospective cohort study in a tertial hospital in Japan from January 2019 to September 2021. Blood culture results were collected from microbiology laboratory records. Blood cultures for which simultaneous pairs of aerobic and anaerobic bottles were collected and from which a single organism was recovered were included. Positive blood cultures were considered as a single episode of bacteremia when obtained within 14 consecutive days. Results A total of 22677 blood cultures were collected, with 22650 bottles were paired. In 2962 paired blood cultures of which one or both bottles were positive, 1079 episodes of clinically significant monomicrobial bacteremia were observed. In these episodes, 898 had a positive aerobic bottle, while 789 had a positive anaerobic bottle, with 608 had both positive bottles. In 181 episodes (16.8%), clinically significant organisms were isolated in an anaerobic bottle without concomitant positive aerobic bottle. In these anaerobic bottles, 146 facultative and 35 obligate anaerobes were recovered. The most common facultative anaerobe was Escherichia coli (30 isolates), followed by Staphylococcus aureus (27 isolates). Bacteroides fragilis (11 isolates) was the most common obligate anaerobe. Conclusion Recovery of clinically significant facultative and obligate anaerobes from anaerobic bottle alone was observed in 17% of episodes, which supports that the routine collection of both aerobic and anaerobic bottles is relevant. Disclosures All Authors: No reported disclosures.

Routine Use of Anaerobic Blood Culture Bottles for Specimens Collected from Adults and Children Enhances Microorganism Recovery and Improves Time to Positivity.

The utility of anaerobic blood culture bottles remains controversial, especially for specimens from children. Data are limited on the inclusion of an anaerobic bottle as part of a blood culture "set" when using contemporary blood culture instruments and media. Here, we evaluated the clinical utility of anaerobic blood culture bottles (FN Plus) and aerobic bottles (FA Plus) for the BacT/Alert Virtuo blood culture system (bioMérieux). A total of 158,710 bottles collected between November 2018 and October 2019 were evaluated. There were 6,652 positive anaerobic bottles, of which 384 (5.8%) contained 403 obligate anaerobes. In patients <19 years old, there were 389 positive anaerobic bottles, with 15 (1.8%) containing 16 obligate anaerobes. If not for anaerobic bottles, all but 8 obligate anaerobes would have gone undetected. Furthermore, anaerobic bottles were advantageous for some facultative anaerobes. Staphylococcus aureus from anaerobic bottles demonstrated statistically significant increased recovery (1,992 anaerobic versus 1,901 aerobic bottles, P = 0.009) and faster mean time to positivity (1,138 versus 1,174 min, P = 0.027). Only 25 microorganisms had statistically significant improved recovery and/or faster time to positivity from aerobic versus anaerobic bottles, suggesting anaerobic bottles offer comparable growth for most species. Finally, if only an aerobic bottle had been collected, 2,027 fewer positive cultures would have been detected and 7,452 fewer isolates would have been reported, including cultures with S. aureus (413 isolates, 10.6% less), Pseudomonas aeruginosa (9 isolates, 3.1% less) and Escherichia coli (193 isolates, 14.0% less). Taken together, these findings support the practice of routinely including an anaerobic bottle for blood culture collection.

An Improvement in Diagnostic Blood Culture Conditions Allows for the Rapid Detection and Isolation of the Slow Growing Pathogen Yersinia pestis.

Plague, caused by the human pathogen Yersinia pestis, is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing countries. Owing to the possibility of its malicious use as a bio-threat agent, Y. pestis is classified as a tier-1 select agent. The prompt administration of an effective antimicrobial therapy, essential for a favorable patient prognosis, requires early pathogen detection, identification and isolation. Although the disease rapidly progresses and the pathogen replicates at high rates within the host, Y. pestis exhibits a slow growth in vitro under routinely employed clinical culturing conditions, complicating the diagnosis and isolation. In the current study, the in vitro bacterial growth in blood cultures was accelerated by the addition of nutritional supplements. We report the ability of calcium (Ca+2)- and iron (Fe+2)-enriched aerobic blood culture media to expedite the growth of various virulent Y. pestis strains. Using a supplemented blood culture, a shortening of the doubling time from ~110 min to ~45 min could be achieved, resulting in increase of 5 order of magnitude in the bacterial loads within 24 h of incubation, consequently allowing the rapid detection and isolation of the slow growing Y. pestis bacteria. In addition, the aerobic and anaerobic blood culture bottles used in clinical set-up were compared for a Y. pestis culture in the presence of Ca+2 and Fe+2. The comparison established the superiority of the supplemented aerobic cultures for an early detection and achieved a significant increase in the yields of the pathogen. In line with the accelerated bacterial growth rates, the specific diagnostic markers F1 and LcrV (V) antigens could be directly detected significantly earlier. Downstream identification employing MALDI-TOF and immunofluorescence assays were performed directly from the inoculated supplemented blood culture, resulting in an increased sensitivity and without any detectable compromise of the accuracy of the antibiotic susceptibility testing (E-test), critical for subsequent successful therapeutic interventions.

Bacteremia due to obligate anaerobes following large bowel surgery in a tertiary care hospital in South India

In view of the rising incidence of Anaerobic bacteremia(AB), the use of anaerobic blood culture bottles have been recommended in addition to the aerobic blood culture bottles. The need to perform antimicrobial susceptibility testing(AST) for anaerobes has become mandatory owing to increasing metronidazole resistance. The frequency of AB following large bowel surgery and the metronidazole susceptibility for members of the Bacteroides fragilis group were determined. The incidence of AB was found to be 16%. Seventeen obligate anaerobes were isolated in total, of which B. fragilis was the most common. Two of twelve isolates of B. fragilis were resistant to metronidazole.

Prevalence and Risk Factors for Unsuspected Spontaneous Ascitic Fluid Infection in Cirrhotics Undergoing Therapeutic Paracentesis in an Outpatient Clinic

Background and Aim: Ascites is a common complication of liver cirrhosis, making patients more vulnerable to infectious diseases such as spontaneous bacterial peritonitis. There hasn't been much research done on infectious ascitic fluid in asymptomatic patients. The purpose of the study was to find out the infectious asymptomatic ascitic fluid incidence and risk factors in liver cirrhotic patients. Materials and Methods: This cross-sectional study was conducted on 76 cirrhotic patients who underwent therapeutic paracentesis between September 2020 and February 2021 in an outpatient department of Jinnah Medical College, Peshawar. An 18-G catheter was used to collect ascitic fluid under strict aseptic conditions. Total and differential leucocyte counts, as well as total protein and albumin levels, were measured. The fluid was injected for bacterial culture of aerobic type and anaerobic blood culture bottles (10 mL each) under strict aseptic conditions. Individuals with abdominal pain, recent gastrointestinal bleeding, fever, SBP previous history, hepatic encephalopathy, impaired renal function, and treatment with antibodies were excluded. Written informed consent and ethical approval were taken prior to study conduction. Demographic details, liver disease severity, and etiology were noted along with laboratory technique-based biochemical tests, ascitic fluid count, and culture. SPSS version 20 was used for data analysis. Results: A total of 192 paracenteses were done on 76 liver cirrhosis patients with an average of 2.53 per patient. The overall mean age was 43.65±8.7 years. Of the total 76 patients, 55 (72.4%) were male and 21 (27.6%) were female. The ascites duration for study inclusion was 3 to12 months. Hepatitis B, fatty liver disease, hepatitis C, and drugs were the major causes of cirrhosis among study patients. The prevalence of Hepatitis B, fatty liver disease, hepatitis C, and drugs was 27 (35.5), 23 (30.3%), 11 (14.5%), and 15 (19.7%) respectively. The hepatic encephalopathy and variceal bleeding history were present in 16 (33.3%) and 32 (66.7%) respectively in a total of 48 (63.2%) cirrhosis patients. The class C and child Pugh class had 23 (30.3%) and 53 (69.7%) respectively. Null mortality was found in patients due to infection caused by spontaneous ascitic fluid. Conclusion: Our study found that hepatitis B, fatty liver disease, hepatitis C, and drugs were the major causes of cirrhosis. Asymptomatic ascitic fluid infection was extremely rare in cirrhotic patients who attended an outpatient clinic and underwent therapeutic paracentesis. Additionally, our study found that the peritoneal fluid asymptomatic spontaneous infection is rare among cirrhotic patients undergoing outpatient base therapeutic paracentesis. Further investigation for ascitic fluid analysis's role in such infection without treatment is to be carried out. Keywords: Ascitic fluid; Cirrhosis; Infection; Therapeutic paracentesis; Spontaneous bacterial peritonitis

BLOOD CULTURES IN PATIENTS WITH ACUTE COVID-19 PNEUMONITIS: CONTAMINATION OR BACTERIAL CO-INFECTION?

TOPIC: Chest Infections TYPE: Original Investigations PURPOSE: INTRODUCTION: Research shows that 90% of blood cultures show no growth and a third of the remainder who test positive are identified as false positives [Garcia RA et al. Am J Infect Control 2015]. Although blood culture contamination rates of <1% are achievable, historical rates at <3% are industry accepted standards[Wayne PA. Clinical and laboratory Standards Institute (CLSI) document M47-A;2007];contaminants from skin flora are the most common, but 20% are from microbes deep in the dermis layer which may be drawn into blood specimens. Evidence for early use of antibiotics managing patients with COVID-19 pneumonitis is lacking but there are anecdotal concerns that more blood cultures than usual have identified organisms usually considered contaminants in sampling. Objectives were to quantify our local findings and relate these to outcome at discharge and during follow up. METHODS: Computer based retrospective review of 228 patients, mean age 71.8 (SD 8.7, range 29-87) years admitted at this hospital between March-May 2020 during the UK COVID-19 (SARS-Cov-2 RNA) peak and surge. Blood cultures reported here correspond to initial presentation with COVID-19 following a sepsis protocol. Comparative analysis by chi square (X2). RESULTS: 137/228 (60%) of patients had blood cultures at admission. 21/137 (15.3%) identified organisms from either one (n=13) or both (n=8) aerobic and anaerobic blood culture bottles. 12/21 (57.1%) (8 died) were identified as coagulase negative staphylococci (CoNS), traditionally considered contaminants at sampling;others included coagulase positive staph aureus (2), Klebsiella (2), E coli (2), and one each for Diptheroids, Proteus Miribalis and Aerococcus Viridans. The remaining 116 reported no growth from initial samples but 3 had positive results later in the admission (2 with CoNS, 1 with E Coli). 7/21 (33.3%) of those with any growth had died during the admission and this was proportionately similar to the 38/116 (32.8%) with no growth on blood cultures [X20.0027, p=.9588, not significant]. At 6 month follow up however, 15/21 (71.4%) of those who had positive findings on original cultures had died compared with 48/116 (41.4%) that had shown no growth [X26.4639, p=.0110, statistically significant]. CONCLUSIONS: Although death rates during admission did not differ, comparing those with and without positive findings on initial blood cultures, a large percentage with positive initial findings then died during follow up. Despite several organisms traditionally considered contaminants, the higher (15.3%) reporting and potential false positive rates requires further study;this should address sampling errors but also revisit bacterial co-infection in COVID-19. CLINICAL IMPLICATIONS: Improving sampling for blood cultures, but research is also needed to make sure this is not a signal for underlying bacterial co-infection. DISCLOSURES: No relevant relationships by Nawaid Ahmad, source=Web Response No relevant relationships by Emma-Jane Crawford, source=Web Response No relevant relationships by Annabel Makan, source=Web Response No relevant relationships by Nikhita Moudgil, source=Web Response No relevant relationships by Harmesh Moudgil, source=Web Response No relevant relationships by Afrah Riaz, source=Web Response No relevant relationships by Koottalai Srinivasan, source=Web Response

Epidemiology and antibiotic susceptibility in anaerobic bacteraemia: a 15-year retrospective study in South-Eastern Hungary

Introduction Bloodstream infections are considered as important factors of morbidity and represent a remarkable number of death, especially in developed countries. Methods In a 15-year retrospective study in the South-Eastern region of Hungary, the microbiological results of anaerobic blood culture (BC) bottles were analysed. The incubation of BCs was performed in the automated incubation system, and the isolates were cultured in an anaerobic chamber. The identification of the isolates was based on their biochemical profile (2004–2012) and mass spectrometry analyses (2013–2018). Antibiotic susceptibility testing was performed with minimal inhibitory concentration gradient diffusion method. Results The majority of the samples were submitted by the Department of Internal Medicine (37.7%), Intensive Care Unit (31.6%) and the Emergency Department (16.4%), and the number of strict anaerobic isolates per year showed an increasing tendency throughout the study. More than half of the isolates were Cutibacterium acnes (54.4%), but this bacterium was considered as a contaminant. The most frequent species upon Cutibacterium acnes were Bacteroides fragilis (9.2%), Clostridium perfringens (6.4%), other Clostridium species (4.8%), other Cutibacterium species (3.5%) and other Bacteroides species (2.5%). Penicillin, amoxicillin/clavulanic acid, imipenem, clindamycin, metronidazole and tigecyclin were very effective against Bacteroides fragilis, Clostridium perfringens, Fusobacterium nucleatum, Gemella morbillorum, Parvimonas micra and Eggerthella lenta strains. Conclusion We performed a 15-year period, retrospective investigation in South-Eastern Hungary with the participation of more than 1000 patients. The results of antibiotic susceptibility tests are in line with previous reports in the literature, and the results from the different geographical regions of the world suggest similar resistance mechanisms.

A Validated and Licensed Method of Bone Marrow Aspirate Procurement to Support Advanced Therapy Investigational Medicinal Product (ATIMP) Manufacture

The Blood & Tissue Establishment (BTE) of Galway University Hospitals supplies sterile bone marrow (BM) aspirate to European cellular manufacturing facilities as a starting material in ATIMP manufacture. We have developed a validated, sterile bone marrow procurement method in accordance with the European Union Tissue & Cells Directive (EUTCD) and Good Manufacturing Practice (GMP). The BTE achieved the requisite procurement licence, from the Irish competent authority, the Health Products Regulatory Authority (HPRA).Under the BTE Quality Management System all standard operating procedures and forms are document controlled, all consumable items are batch accepted, all staff involved undergo training and competency assessment (including adequate sanitisation of the iliac crest), and all issues are recorded and reported appropriately to the HPRA.The prospective donor must first undergo a two part ‘donor assessment’ procedure which evaluates the donor's ‘fitness’ to undergo the procedure, while also having due regard for any potential recipient.Part One of the assessment process requires that the entire procedure is explained by a trained clinician / nurse; a patient information leaflet is provided and written consent obtained. A full blood count, coagulation screen, glucose test, chemistry screen, blood group and virology screen is performed. The virology screen must, at minimum, include the viruses listed by the EUTCD and the donation must take place within 30 days of sample collection. The patient will then undergo a ‘lifestyle questionnaire’ to assess donor medical, travel, sexual and other risk history. Guidance for allowing the donation to proceed will depend on a number of factors, including, whether it is an allogeneic or autologous donation. Consideration is given to the ‘Bone Marrow and Peripheral Blood Stem Cell Donor Selection Guidelines for Unrelated Donors’ of the ‘Guidelines for the Blood Transfusion Services in the United Kingdom’. HPRA and European Centre for Disease Prevention and Control alerts are also incorporated into selection criteria.The Responsible Person (RP) of the BTE (also a Consultant Haematologist) must review the answers to the questionnaire, together with the blood test results and assess whether the donor is fit to proceed to aspiration.Part Two of the assessment process takes place immediately prior to the scheduled aspiration. The donor will repeat the lifestyle questionnaire once more and the answers will be reviewed and compared with the previous questionnaire. The Consultant Haematologist will carry out a clinical examination and assessment of the donor. Upon completion, final donor eligibility is signed off and the aspiration can proceed. The procedure takes place in an operating theatre by a scrubbed Consultant Haematologist. A sterile field is created around the aspiration site. The aspiration site is cleansed with Chlorhexadine. Local anaesthetic (lidocaine 2%) is administered. 11 Gauge BM needles are used to puncture. Syringes are flushed with heparin sodium flushing solution. The flushed syringes are used to withdraw the BM, handed to a sterile assistant and transferred into 10ml lithium heparin vacutainers. The volume of 30ml of BM is aspirated from the iliac crest of both healthy donors (allogeneic) and patients (autologous) enrolled in clinical trials.These vacutainers are capped and inverted several times to prevent clotting, labelled and packaged for transport.The puncture site is dressed, donor vitals are observed and noted post procedure and the donor may be discharged after 2 hours.BM is transported to the manufacturing facility at monitored ambient temperature. Within the Grade A controlled environment, the BM is pooled and 1ml is used to inoculate one aerobic and one anaerobic blood culture bottle (validated for 0.5ml in each, incubated for 14 days, automated detection system).As of July 2017, we have performed 24 collections, 11 of which are allogeneic donations from healthy volunteers. The average white cell count in the aspirate from healthy donors is 43.1 x 106/ml. The sterility is satisfactory, with only one instance of contamination over the 24 collections, where, on the final day of incubation (day 14), we detected propionibacterium spp, a notoriously slow-growing commensal skin flora.Our regulatory approved protocol provides a steady supply of GMP grade bone marrow aspirates for the purpose of clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Detection of Carbapenem-Resistant Enterobacterales in Simulated Blood Culture in 15 Minutes.

Bacteremia leading to sepsis and organ dysfunction is a life-threatening situation, leading to death of up to one fourth of the infected individuals around the world. One major challenge in the treatment of sepsis is the rising prevalence of antibiotic resistant bacteria, such as carbapenem-resistant Enterobacterales (CRE). In recent years, several molecular assays have been developed for the detection of CRE mechanisms, enabling rapid results reporting. We evaluated the performance of the NG-Test CARBA 5 (NG Biotech) kit in detection of CRE in simulated blood cultures. Carbapenemase-producing (CP) CRE isolates (n = 38) and non-carbapenemase CRE (Non-CP) isolates (n = 10), previously identified using the routine methods practiced at the clinical microbiology laboratory of the Baruch Padeh Medical Center, Israel, were used in this analysis. Variable concentrations of the bacterial isolates were added to a suspension composed of human blood and saline, simulating the composition of a blood culture. Samples were then transferred to an anaerobic blood culture bottle and later tested with the NG-Test CARBA 5 (NG Biotech) kit, that identifies the CRE mechanism within 15 min. The NG-Test CARBA 5 kit correctly identified 43 samples (89.5%). The sensitivity and specificity of the kits were 86.8% and 100%, respectively. In conclusion, the NG-Test CARBA 5 kit is a reliable and accessible tool for the rapid diagnosis of CRE bloodstream infections.

Clinical Features of Cases with Anaerobic Infection, Distribution of Anaerobic Bacteria and Fatality in Anaerobic Bacteremia

Introduction: This study aimed to determine the distribution of anaerobic bacteria species, which are the causative agents of infection, in order to investigate the epidemiological and clinical characteristics of the cases and evaluate fatality in cases with an aerobic bacteremia. Materials and Methods: One hundred and twenty-five cases who were determined with 148 isolates of anaerobic bacteria were included into this study at Ankara Numune Training and Research Hospital Medical Microbiology Laboratory between January 2014-February 2018. The specimens requested to undergo anaerobic evaluation and transported to the laboratory under anaerobic suitable conditions were prepared with Gram staining and inoculated in the Schaedler agar. After appropriate incubation, the identification of isolated anaerobic bacteria at the level of species was performed by using matrix-assisted laser desorption time-of-flight (MALDI-TOF MS). Results: Of the 125 cases who had an anaerobic infection, 60% were males, and mean age was 52.69 ± 18.5 years. Intra-abdominal and skin and soft tissue infections were the most common source of infection. One hundred and twenty-eight clinical specimens (78 abscesses, 50 blood cultures) which were sent from cases were identified with 148 anaerobic bacteria species. We detected the most common isolated species as Bacteriodes spp., Actinomyces spp. and Clostridium spp., respectively. The most common anaerobic bacteria species isolated from blood cultures of 50 cases with anaerobic bacteremia were Bacteroides spp., Clostridium spp., and intra-abdominal infection was the most common source for anaerobic bacteremia. Fusobacterium nucleatum (5/6; 83.3%) was the most frequent species in Fusobacterium bacteremia. F. nucleatum bacteremia was detected in an older population and was often associated with underlying malignancy. F. necrophorum bacteremia occurred in a younger population without underlying comorbidities and was diagnosed as Lemierre’s syndrome. Fatality rate was 51% in anaerobic bacteremia. Fatality was significantly higher in patients who were followed up in the intensive care unit and had high Charlson index (p&lt; 0.05). Conclusion: MALDI-TOF MS has become an important method for the identification of anaerobic bacteria with its reliable results in a very short time. The most common anaerobic microorganism was Bacteroides spp., and the two most frequent sources of infection were intraabdominal and skin and soft tissue infections. We observed that most of the cases with F. nucleatum bacteremia had malignancy and an older population. The case with F. necrophorum bacteremia was young and had no underlying comorbidities. In this study, fatality rate (51%) was detected high in anaerobic bacteremia. Therefore, the routine use of anaerobic blood culture bottles is important for the identification and for the effective treatment of cases with anaerobic bacteremia.

Anaerobes Direct from Blood Culture Bottles Can Be Identified by Early Matrix-Assisted Laser Desorption Ionization/ Time-of-Flight Mass Spectrometry (MALDI-TOF MS) at 24 Hours or Less

Abstract Introduction/Objective Matrix-assisted laser desorption ionization/ time-of-flight mass spectrometry (MALDI-TOF MS) direct from positive blood culture bottles has facilitated drastic drops in turn-around times for microorganism identification but has been poorly studied for anaerobes. We investigated the ability of MALDI-TOF to provide early anaerobe identification at 4 hours and 18-24 hours of growth on agar from anaerobic blood culture bottles. Additionally, we reviewed medical records of such patients to ascertain impact of early identification on antimicrobial treatment. Methods Over 9 months, we ran MALDI-TOF on early growth from blood cultures positive for growth in BACTEC™ Lytic/ 10 Anaerobic/F bottles. Broth from each bottle was subbed to sheep blood agar (4 hours, 5% CO2) and 2 CDC (BD-BBL™) anaerobic blood agar plates (examined 18-24 hours and 48 hours). Bruker Biotyper® RUO v7854 and Mayo Clinic Custom MALDI-TOF MS libraries were used for identification. Results 144/184 (78%) bottles resulted in growth of aerobic bacteria. Of the remaining bottles with growth of anaerobes, 38 were assessed by early MALDI-TOF. Early MALDI-TOF at 4 hours identified 3 Clostridium perfringens (8%) and an additional 26/38 (68%) isolates at 18-24 hours (both Gram-positive and –negative). Routine 48 hour identification was required for 9 (24%) isolates. In 7 cases, early MALDI-TOF resulted in a change to more appropriate antimicrobial therapy, most often for Bacteroides. Conclusion 29/38 (76%) of anaerobes from blood culture bottles were identified by early MALDI-TOF and reported to the clinician at least 24 hours before routine review of anaerobic sub plates for growth. All C. perfringens and Bacteroides were identified by 4 and 24 hours, respectively. Although early MALDI-TOF resulted in antimicrobial therapy adjustments in a minority of cases, it may allow for more targeted and earlier antimicrobial therapy. Early MALDI-TOF from anaerobic blood culture bottles should be considered for improved patient care and antimicrobial stewardship.

Evaluation of EUCAST rapid antimicrobial susceptibility testing (RAST) for positive blood cultures in clinical practice using a total lab automation

Our objective was to evaluate EUCAST’s ‘rapid antimicrobial susceptibility testing’ (RAST) directly from positive blood culture that delivers antimicrobial results within 6 h for Staphylococcus aureus, Enterococcus spp., Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, using total lab automation. Zone diameters from RAST were compared with MIC results. Furthermore, its influence on time to report was investigated. RAST was performed to all positive aerobic and anaerobic blood culture bottles by subculturing them, i.e. onto Mueller-Hinton agar and adding six antibiotic discs covering Gram-negative and Gram-positive therapy (cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin). RAST was automatically imaged after 6 h. Zone sizes were measured using a TLA software tool and interpreted according to EUCAST clinical breakpoints. Bacteria were identified using MALDI-TOF MS and MIC results were determined using Vitek2 panels. Categorial agreement between agar diffusion and MIC results was investigated. Additionally, time to RAST and time to Vitek were compared for 100 isolates (20 per species). Between November 2018 and April 2019, 3313 positive mono-bacterial blood culture bottles were collected of which 894 bottles with RAST-validated species were investigated. Among these bottles, 2029 individual antibiotic measurements were compared with MIC results from Vitek2 and 14 very major, 28 major and 12 minor errors were found. A median reduction of 17:30 h in time to report was observed. Introduction of RAST with automatic TLA imaging function could reduce time to report by 17:30 h. Excellent accordance between zone diameter and MIC results, particularly for cefoxitin, vancomycin and meropenem, was observed, but drawbacks due to ATU were seen.

Sonicate fluid inoculated into blood culture bottles does not improve diagnosis of periprosthetic joint infection caused by anaerobes. A retrospective analysis

BackgroundIn microbiological diagnosis of periprosthetic joint infection (PJI) there is much controversial discussion about culture media and incubation time, especially if anaerobic bacteria are the causative agents. This retrospective analysis was conducted to compare the results obtained by inoculation of sonicate fluid from prosthetic components into BD Bactec blood culture bottles with those obtained by our culture method using sensitive supplemented growth media. MethodsTwenty-eight cases were included in this study. For definition of PJI, the criteria of the Musculoskeletal Infection Society (MSIS) were considered. The quantity and time to positivity of anaerobes detected in sonicate fluid were monitored both from inoculated supplemented liver thioglycollate broth and anaerobic blood culture bottles. Furthermore, phenotypic testing was performed on the antimicrobial activity within the sonicate fluid. ResultsThe most frequently isolated microbes were Cutibacterium species, followed by Finegoldia magna, Parvimonas micra, Robinsoniella peoriensis, Clostridium species, Peptoniphilus harei and Slackia exigua. In 24 cases, the microorganisms became detectable within five days (median time 3.2 days) when sonicate fluid was incubated in supplemented liver thioglycollate broth, regardless of whether the patients had taken antimicrobial agents prior to surgery. However, when sonicate fluid was inoculated into anaerobic Bactec bottles, the median time to positivity was 7.4 days and only 12 cases (43%) were correctly identified. Sixteen cases remained negative after 14 days of incubation. ConclusionDepending on the pathogen, incubation of sonicate fluid using blood culture bottles can support diagnosis of PJI but compared with our culture medium it is less efficient if anaerobes are the suspected cause of infection. Microbiological expertise is therefore indispensable to ensure reliable detection of these microorganisms in PJI until a gold standard for laboratory handling of anaerobes has been established.

Growth detection of Cutibacterium acnes from orthopaedic implant-associated infections in anaerobic bottles from BACTEC and BacT/ALERT blood culture systems and comparison with conventional culture media

Cutibacterium acnes is a major etiologic agent of orthopaedic implant-associated infections (IAIs) and requires up to 14 days of incubation in an anaerobic atmosphere for growth detection. As blood culture (BC) systems are increasingly being used to monitor the growth of IAI specimens, we compared different BC media for growth detection of C. acnes. Non-duplicate C. acnes isolates (n = 99) obtained from sonicate-fluid cultures of orthopaedic IAIs from Slovenia (n = 54), conventional tissue samples of monomicrobial orthopaedic IAIs from France (n = 43) and two reference strains were inoculated to anaerobic BC bottles of two major BC systems and 3 conventional culture media types (thioglycolate broth, Schaedler and chocolate agar). Growth and time-to-detection (TTD) were recorded. Only Lytic (BACTEC) and SN (BacT/ALERT) bottles consistently detected growth of C. acnes within 14 days with 94% (n = 93) and 92% (n = 91) detection rates, respectively (p = 0.79). Lytic was superior to Plus BACTEC medium (p < 0.001), while SN was superior to all other BacT/ALERT media (p < 0.001). Mean TTD was 128 ± 43 h (61–336 h) for Lytic and 158 ± 65 h (77–336 h) for SN medium. Among the conventional media, 99% (n = 98) of the isolates grew on Schaedler agar, 96% (n = 95) in thioglycolate broth and 74% (n = 73) on chocolate agar. Inconsistent growth of C. acnes in different BC media can critically influence the detection of this major IAI pathogen. Only Lytic (BACTEC) and SN (BacT/ALERT) BC media types were consistently able to detect C. acnes within 14 days of incubation. However, visible growth was observed faster in thioglycolate broth and Schaedler agar media.