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Background: Sepsis and septic shock have high mortality rates and often require a prolonged hospital stay. Patient outcomes may vary according to multiple factors. We aim to determine the prevalence of antimicrobial resistance and factors associated with mortality and hospital stay. Methods: Clinical and microbiological data of patients with sepsis or septic shock were retrospectively collected for 15 months. Patients with negative blood cultures and patients that did not meet the SEPSIS 3 criteria were excluded. Results: We included 48 septic shock and 28 septic patients (mean APACHE II 20.32 ± 5.61 and mean SOFA 9.41 ± 3.17), with a mean age of 60.5 ± 16.8 years and 56.6% males. WBCs, neutrophils, INR, and fibrinogen levels were significantly associated with mortality. 59.5% of the cultured bacteria were gram-negative (most common E. coli) and 27.8% were gram-positive (most common S. aureus), while 7.6% were other types of bacteria and 5.1% were fungi. Resistance patterns to gram-negative were varying, and resistance to piperacillin/tazobactam, carbapenems, and aminoglycosides were from 60% to 100% (A. baumanii), while they were highly sensitive to Colistin. E. coli was also resistant to ceftriaxone (77.8%) and sulbactam/cefoperazone (44.4%). Resistance rates for Gram-positives were high, from 86% to 100% for oxacillin, while for vancomycin, teicoplanin, and linezolid, they were often low but arrived up to 42.8%. According to our logistic regression analysis, patients over 65 year-old and those who received corticosteroids had a significantly increased risk of in-hospital mortality (OR: 4.0; OR: 4.8). Conclusion: Sepsis still poses a significant threat to patients’ health, even when positive blood culture results allow the administration of specific antibiotic treatment., Hospital Practice, 50(5), pp.407-415; 2022

Background Brucellosis causes a disabling human disease and loss of animals' lives. The clinical significance of Brucella bacteremia is still unclear and Brucella identification in blood culture is suboptimal. Methods This was a retrospective study conducted in Medina in Saudi Arabia from August 2016 to May 2019. We included cases with brucellosis symptoms and a positive culture or serological evidence for brucellosis, comparing bacteremic with non-bacteremic brucellosis cases for the rates of complications, infection relapses and brucellosis development. Also, we estimated blood culture positivity rates and the time to detect Brucella in an automatic blood culture instrument. Results Of the total number of 147 cases, 62 (42%) had a positive blood culture for Brucella, and the blood culture instrument (BACT/ALERT 3D) detected all positive blood cultures within 3 d of incubation. We found higher rates of chronic brucellosis in bacteremia than non-bacteremia cases (OR 7.25, 95% CI 1.41 to 37.23; p=0.018). Patients aged Conclusion Brucella bacteremia is an independent predictor for the development of chronic infection. Brucella bacteremia cases may need long follow-up periods and a more thorough evaluation to exclude deep-seated infection.

Introduction Bacteraemia in pregnancy and the post-partum period can lead to maternal and newborn morbidly. The purpose of this study was to use machine learning tools to identify if bacteraemia in pregnant or post-partum women could be predicted by full blood count (FBC) parameters other than the white cell count. Methods The study was performed on 129 women with a positive blood culture (BC) for a clinically significant organism, who had a FBC taken at the same time. They were matched with controls who had a negative BC taken at the same time as a FBC. The data were split in to a training (70%) and test (30%) data set. Machine learning techniques such as recursive partitioning and classification and regression trees were used. Results A neutrophil/lymphocyte ratio (NLR) of >20 was found to be the most clinically relevant and interpretable construct of the FBC result to predict bacteraemia. The diagnostic accuracy of NLR >20 to predict bacteraemia was then examined. Thirty-six of the 129 bacteraemia patients had a NLR >20, while only 223 of the 3830 controls had a NLR >20. This gave a sensitivity of 27.9% (95% CI 20.3-36.4), specificity of 94.1% (93.3-94.8), positive predictive value of 13.9% (10.6-17.9) and a negative predictive value (NPV) of 97.4% (97.2-97.7) when the prevalence of bacteraemia was 3%. Conclusion The NLR should be considered for use in routine clinical practice when assessing the FBC result in patients with suspected bacteraemia during pregnancy or in the post-partum period.

The optimal care of septic patients depends on the successful recovery of clinically relevant microorganisms from blood cultures and the timely reporting of organism identification and antimicrobial susceptibility testing (AST) results. Many preanalytic factors play a critical role in culturing microorganisms, and advancements in blood culture instrument technology have reduced the time to positive results. Additionally, rapid organism identification and AST results directly from positive blood culture broth via new methods help to further shorten the time from empiric to targeted treatment. This article summarizes the current state of blood culture methods, including preanalytic, analytical, and postanalytic factors that are available to clinical microbiology laboratories.

Introduction. Rapid identification of the causative agent of sepsis is crucial for patient outcomes. Aim. The Sepsityper sample preparation method enables direct microbial identification of positive blood culture samples via matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS). Hypothesis/Gap statement. The implementation of the Sepsityper method in the routine practice could represent a fundamental tool to achieve a prompt identification of the causative agent of bloodstream infections, and therefore accelerate the adoption of the proper antibiotic treatment. Methodology. In this study, the novel rapid workflow of the MALDI Biotypr Sepsityper kit (Bruker Daltonik GmbH, Germany) was evaluated using routine samples from a 2-year period (n=6918), and dedicated optimized protocols for the microbial groups that were more difficult to identify were developed. Moreover, the use of the residual bacterial pellet to perform susceptibility testing using different methods (commercial broth microdilution, disc diffusion, gradient diffusion) was investigated. Results. The rapid Sepsityper protocol allowed the identification of 5470/6338 (86.3 %) monomicrobial samples at species level, with very good performance for all of the clinically most significant pathogens (2510/2592 enterobacteria, 631/669 Staphylococcus aureus and 223/246 enterococci were identified). Streptococcus pneumoniae , Bacteroides fragilis and yeasts were the most troublesome to identify, but the application of specific optimized protocols significantly improved their rate of identification (from 14.7–71.5 %, 47.8–89.7 % and 37.1–89.5 %, respectively). Specificity was 100 % (no identification was made for the false-positive samples). Further, the residual pellet proved to be suitable to investigate susceptibility to antimicrobials, enabling us to simplify the workflow and shorten the time to report. Conclusion. The Rapid Sepsityper workflow proved to be a reliable sample preparation method for identification and susceptibility testing directly from positive blood cultures, providing novel approaches for accelerated diagnostics of bloodstream infections.

Neonatal sepsis is a major cause of worldwide morbidity and mortality. Blood cultures are considered the gold standard for diagnosis, but results are often delayed for 24 to 48 hours, and sensitivity, although improved by modern techniques, such as automated blood cultures, is variable and affected by the bacterial load. For these reasons, empiric antibiotics are frequently administered to avoid potential devastating consequences of untreated sepsis. Unnecessary antibiotic treatment has been associated with increased mortality and other adverse outcomes; therefore, antibiotics should be discontinued as soon as sepsis has been ruled out. Negative cultures pose a challenge to clinicians, who must distinguish between real sepsis and sepsis-like conditions (noninfectious or viral) which do not require antibiotics. Focal infections with negative blood cultures do require antibiotic treatment. Ultra-low bacteremia, primary or secondary to recent antibiotic exposure, is often associated with negative cultures, and some consider a short course of empiric antibiotics sufficient for clearing of bacteremia. Biomarkers and molecular methods based on polymerase chain reaction are important add-ons to clinical signs or symptoms for establishing the diagnosis of sepsis. Other promising future potential adjuvants are metabolomics. Antibiotic stewardship should be implemented to avoid or discontinue unnecessary treatment. Prevention of infection still remains the most important step for dealing with neonatal sepsis. KEY POINTS: · Blood cultures are the gold standard diagnosis of neonatal sepsis but sometimes may be negative.. · Other bacterial, viral, and noninfectious conditions may mimic sepsis, prompting initiation of empiric antibiotic treatments.. · Since a definition of neonatal sepsis is lacking, recognizing real septic episodes may be challenging..

Transfusion of platelet concentrates (PCs) is associated with several adverse patient reactions, the most common of which are febrile non-hemolytic transfusion reactions (FNHTRs) and transfusion-associated bacterial-infection/transfusion-associated sepsis (T-ABI/TA-S). Diagnosis of T-ABI/T-AS requires a positive blood culture (BC) result from the transfusion recipient and also a positive identification of bacterial contamination within a test aliquot of the transfused PC. In a significant number of cases, clinical symptoms post-transfusion are reported by the clinician, yet the BCs from the patient and/or PC are negative. The topic of 'missed bacterial detection' has therefore been the focus of several primary research studies and review articles, suggesting that biofilm formation in the blood bag and the presence of viable but non-culturable (VBNC) pathogens are the major causes of this missed detection. However, platelets are emerging as key players in early host responses to infection and as such, the aforementioned biofilm formation could elicit 'platelet priming', which could lead to significant immunological reactions in the host, in the absence of planktonic bacteria in the host bloodstream. This review reflects on what is known about missed detection and relates this to the emerging understanding of the effect of bacterial contamination on the platelets themselves and the significant role played by platelets in exacerbation of an immune response to infection within the transfusion setting.

Introduction In the era of shortage of organs for donation, transplantation from suboptimal donors is an expanding alternative to minimize waitlist mortality. In that sense, the safety of using organs from bacteremic donors has been a recurrent matter of discussion. We aimed to evaluate the influence of donor positive blood culture in the recipient and graft outcomes after liver transplantation from deceased donors. Material and Methods Blood culture results from 255 deceased liver donors were retrospectively reviewed. Patients were categorized into 2 groups based on the recipients who obtained a graft from a donor with negative or positive blood culture. Graft and recipient outcomes were compared between the 2 groups using univariate survival analysis and multivariate regression models. Transmission of bloodstream infection from donor to recipient was assessed by reviewing recipients’ microbiologic status when there was evidence of infection. Results Positive blood culture in donors was not associated with negative outcomes after transplantation. Death within 30 days after transplantation and overall recipient and graft survival did not differ between the 2 groups. Only Child-Pugh score ≥10 and retransplantation status were considered independent predictors of recipient death and graft failure. We identified 1 potential case of bacteremia transmission from donor to recipient. Conclusion Donor positive blood culture was not associated with negative outcomes after liver transplantation. Transmission of infection from donor to recipient is possible, but rare. The results support the usage of bacteremic donors as a safe alternative to the scarcity of optimal donors.

Previous studies showed that red blood cell distribution width (RDW) can be used as a prognostic and diagnostic index in various non-hematological diseases, including severe infections and sepsis. Here, we provide a narrative review to summarize the findings of available studies investigating the relationship between RDW and sepsis. Current evidence suggests that increased RDW on admission, both in adults and neonates, may be associated with unfavorable outcomes on the short- and long-term. In patients with suspected sepsis, RDW has modest value for predicting positive blood culture. Accordingly, its diagnostic value for sepsis seems limited, whilts dynamic changes of RDW are associated with outcome of sepsis. Taken together, these results suggest that RDW could be used as a prognostic index in septic patients.

ObjectivesWith increasing antimicrobial resistance, rapid antimicrobial susceptibility testing (RAST) becomes important, especially in patients with bloodstream infections. EUCAST decided to develop a standardized rapid method, based on EUCAST disc diffusion, to offer susceptibility reports within 4–8 h of a positive blood culture (BC).MethodsBC bottles were spiked with clinical isolates (n = 332) of the seven most relevant sepsis pathogens with a variety of resistance mechanisms. RAST was performed directly from the bottle and zones read after 4, 6 and 8 h. Several variables were investigated, including the effect of using different BC bottles and of a 0–18 h delay between a positive signal and the performance of RAST.ResultsFor five species, most inhibition zones could be read after 4 h. The proportion of results that could be interpreted increased from 75% at 4 h to 84% after 8 h. Categorical agreement against the reference method was good, with error rates of false susceptibility of 0.2%, 0.2% and 0.2% at 4, 6 and 8 h and false resistance of 1.2%, 0.2% and 0.1% at 4, 6 and 8 h, respectively.ConclusionsWith the EUCAST RAST method, reliable AST results can be delivered within 4–8 h of positivity of BC bottles for seven important bloodstream infection pathogens. To reduce the occurrence of errors and to absorb the variability caused by using a non-standardized inoculum, material from different manufacturers and workflow-related delays, we have introduced an area in which interpretation is not permitted, the Area of Technical Uncertainty.

Many studies have suggested that procalcitonin can predict bloodstream infection and also distinguish between Gram-negative, Gram-positive and fungal infections after burn. However, up to now, there is no literature on serum procalcitonin level of multidrug-resistant pathogens and non-multidrug-resistant pathogens among Gram-negative bloodstream infections after burn. The purpose of this study is to explore the value of serum procalcitonin in identifying Gram-negative bloodstream infection in patients with febrile critical burn and then to investigate the difference of serum procalcitonin level between multidrug-resistant pathogens and non-multidrug-resistant pathogens among Gram-negative bloodstream infections after burn.Patients with febrile critical burn admitted to the burn department of our hospital from 1 January 2014 to 1 August 2018 were retrospectively analysed. Patients with positive blood culture whose blood samples were collected for simultaneous blood culture and procalcitonin testing were enrolled. All strains were identified by an automatic microorganism analyser, and procalcitonin was analysed by an automatic electrochemiluminescence immunoassay.Overall, a total of 119 patients with positive blood culture met the inclusion criteria. There were 64 Gram-negative bacilli, 38 Gram-positive bacteria, 8 C. albicans and 9 polymicrobial bloodstream infections. The median procalcitonin value in Gram-negative bloodstream infections (2.67 ng/mL, interquartile range (IQR) 1.58-6.08) was significantly higher than that in Gram-positive bloodstream infections (1.04 ng/mL, IQR 0.35-1.60, P 0.01), or C. albicans bloodstream infections (1.09 ng/mL, IQR 0.82-2.30, P 0.05). Receiver operating characteristic curve (ROC) analysis showed that in addition to polymicrobial bloodstream infections, the area of procalcitonin under the curve distinguishing Gram-negative bloodstream infections from all other blood culture-positive bloodstream infections was 0.761, the best critical value was 1.73 ng/mL, the sensitivity was 73%, the specificity was 74%, the positive predictive value was 80%, the negative predictive value was 67%, The level of procalcitonin was significantly higher in multidrug-resistant Gram-negative bacilli (A. baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) (2.76 ng/mL, IQR 2.01-7.76) than in non-multidrug-resistant bacilli (1.01 ng/mL, IQR 0.58-1.56, P 0.01).Elevated serum procalcitonin can identify Gram-negative bloodstream infections in patients with febrile critical burn. In Gram-negative bloodstream infections, high procalcitonin levels may be associated with multidrug-resistant Gram-negative bacilli (A. baumannii, K. pneumoniae and P. aeruginosa).

Purpose . The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long-read and real-time sequencing. In this study, we validated this technology for the identification of pathogens from positive blood culture (BC) bottles. Methodology . A total of 38 positive BC bottles were collected from patients with bloodstream infections, and 18 isolates of Gram-negative (GN) bacteria and 20 isolates of Gram-positive (GP) bacteria were identified from these using 16S rRNA sequencing and then used in this study. DNA was extracted from each aliquot using an extraction protocol that combined glass bead beating and chemical lysis. Up to 200 ng of each purified DNA sample was processed for library preparation and whole-genome sequencing was performed on up to 12 samples through a single MinION flow cell. Results . All GN bacteria identifications made by MinION sequencing for 30 min using the What’s In My Pot? (WIMP) workflow via EPI2ME on the basis of the most frequent classified reads were consistent with those made by 16S rRNA sequencing. On the other hand, for GP bacteria specimens, the identification results for 16S rRNA sequencing and MinION were only in agreement in 12 out of 20 (60.0 %) cases. ARMA analysis was able to detect extended-spectrum β-lactamase (ESBL)-associated genes among various antimicrobial resistance-related genes. Conclusion . We demonstrated the potential of the MinION sequencer for the identification of GN bacteria from positive BC bottles and the confirmation of an ESBL phenotype. This innovative sequence technology and its application could lead to a breakthrough in the diagnosis of infectious diseases.

Neonatologists usually wait 48 hours for blood culture results before deciding to discontinue antibiotics. The objective of the study was to analyze time to positive blood culture in rule out sepsis and estimate the minimum duration of antibiotics.Retrospective analysis of blood culture at the Neonatal Intensive Care Unit, McMaster Children's Hospital (January 2004 to December 2013) using BacT/Alert® 3D microbial system was conducted. We calculated average time taken for blood culture samples to emit a positive signal and compared it between Gram-positive and Gram-negative organisms. Kaplan-Meier curves for time to detect positive culture were generated. A Cox proportional hazard regression model with the outcome variable "time to detect positive blood culture" and predictor variables "early-onset sepsis (EOS) versus late-onset sepsis (LOS)", "Gram-positive versus Gram-negative" and "definite versus possible pathogen versus contaminant" was generated.Of 7,480 blood cultures performed in 9,254 neonates, 885 samples grew microorganisms. 845 culture reports from 627 neonates were analyzed. Definite or opportunistic pathogens caused 815 (96%) infections (54 EOS and 791 LOS) and the rest were contaminants. Gram-negative organisms grew significantly faster than Gram-positive (P0.001). Cultures from EOS were positive significantly earlier than LOS (P = 0.032). Gram-negative status was an independent predictor of early detection of a positive culture (hazard ratio 3.5 [95% CI, 2.7-4.5] P0.001).The probability of positive blood culture beyond 24 hours for a Gram-negative organism is small. Empiric antimicrobial treatment can be reduced after 24 hours to target only Gram-positive organisms in LOS and can be stopped in EOS unless clinical or laboratory parameters strongly suggest sepsis.

In this study, we identified specific carbapenemase-producing isolates applying an easy and rapid protocol for the detection of mature KPC-2 β-lactamase by MALDI-TOF MS from colony and positive blood culture bottles. In addition, we evaluated the correlation of the ~11,109 Da signal as a biomarker associated with KPC-2 production. A collection of 126 well-characterized clinical isolates were evaluated (including 60 KPC-2-producing strains). Presence of KPC-2 was assessed by MALDI-TOF MS on protein extracts. Samples were prepared using the double layer sinapinic acid technique. In order to identify mature KPC-2, raw spectra were analyzed focusing on the range between m/z 25,000–30,000 Da. A single distinctive peak, at approximately m/z 28,544 Da was found in all clinical and control KPC-2-producing strains, and consistently absent in the control groups (ESBL producers and susceptible strains). This peak was detected in all species independently of where the gene bla KPC-2 was embedded. Statistical results showed 100% sensitivity, CI95%: [94.0%; 100%] and 100% specificity, CI95%: [94.6%; 100%], indicating a promising test with a high discriminative power. KPC-2 β-lactamase could be directly detected from both colonies and blood culture bottles. On the other hand, the m/z 11,109 Da signal determinant was only associated with 32% of Klebsiella pneumoniae and Escherichia coli KPC positive isolates. This MALDI-TOF MS methodology has the potential to detect directly the widespread and clinically relevant carbapenemase, KPC-2, in Enterobacterales with a straightforward, low cost process, assuming MALDI-TOF MS is already adopted as the main identification tool, with clear clinical implications on antibiotic stewardship for early infection treatment. Fil: Figueroa Espinosa, Roque Arnulfo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Costa, Agustina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Cejas, Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Barrios, Rubén. No especifíca; Fil: Vay, Carlos Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

We studied the performance of an algorithm combining multiplex polymerase chain reaction with phenotypic detection of extended-spectrum β-lactamases and carbapenemases directly from positive blood culture bottles in patients with gram-negative bacteremia and found good concordance with routine cultures. Such an algorithm may be a tool to improve time to optimal therapy in patients with gram-negative bacteremia.

Direct use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) on positive blood culture (PBC) bottles may accelerate the identification of bloodstream infection (BSI) causing organisms (1), particularly anaerobic bacteria for which BSI-associated mortality rates are high (2).….

Background Rapid and accurate microbial identification and antimicrobial susceptibility testing (AST) are essential for timely use of appropriate antimicrobial agents for bloodstream infection. To shorten the time for isolating colonies from the positive blood culture, various preparation methods for direct identification using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) system were developed. Here, we evaluated the SepsiPrep kit (ASTA Corp.) for direct identification of microorganisms and AST from positive blood cultures using MicroIDSys Elite MALDI‐TOF MS system (ASTA Corp.) and VITEK‐2 system (bioMérieux). Methods For direct identification, a total of 124 prospective monomicrobial positive blood culture bottles were included. For direct identification, the pellet was prepared by centrifugation and washing twice. For direct AST, the pellet was suspended in 0.45% saline and adjusted to McFarland 0.5. The results from the direct identification and AST using MicroIDSys Elite and VITEK‐2 system were compared to those from the conventional method performed with pure colony subcultured on agar plate. Results Compared to the conventional method using pure colony, correct direct identification rate was 96.5% and 98.5% for 57 gram‐positive isolates and 67 gram‐negative isolates, respectively. For direct AST, among the 55 gram‐positive isolates, the categorical agreement (CA) for staphylococci, streptococci, and enterococci was 96.7%, 98.4%, and 94.1%, respectively. For 66 gram‐negative isolates, the CA for Enterobacterales and non‐fermentative gram‐negative rods was 99.0% and 96.6%, respectively. Conclusions The SepsiPrep kit was easy to use combined with MicroIDSys Elite and VITEK‐2 system and also, the correct identification and AST rate were very high.

Direct susceptibility testing from blood cultures has been reported to reduce the time interval between a positive blood culture to preliminary reporting of susceptibility and can underpin timely appropriate treatment of candidemia. The aim of this study was to evaluate direct susceptibility testing of Candida glabrata to fluconazole using disk diffusion compared to the Sensititre YeastOne broth microdilution-based method. We tested 83 isolates recovered from 93 spiked and prospective blood culture bottles. Comparison of the two methods showed excellent agreement, with no very major errors and only two major errors (2.4%). The accuracy of the fluconazole disk method was 97.6% (95% confidence interval [CI], 91.6 to 99.7), with a sensitivity of 100% (95% CI, 82.3 to 100) and a specificity of 96.9% (95% CI, 89.2 to 99.6). Direct antifungal disk susceptibility testing from blood cultures is a rapid and easy-to-perform method to determine fluconazole susceptibility of C. glabrata isolates and can be used safely to reduce susceptibility report time and improve clinical decision making regarding appropriate treatment.

The Accelerate Pheno system is approved for rapid identification and phenotypic antimicrobial susceptibility testing (AST) of microorganisms grown from positive blood cultures inoculated with blood from septic patients. We evaluated the performance of the system for identification and AST from positive blood culture bottles inoculated with primary sterile nonblood specimens from patients with suspected severe infections. One hundred positive blood culture bottles with primary sterile specimens (63 cerebrospinal fluids, 16 ascites, 7 pleural fluids, 4 vitreous fluids, 5 joint aspirates, and 5 other aspirates) from 100 patients were included. Pathogen identification was in agreement with conventional methods for 72 of 100 cultures (72%) and for 81 of 112 (72%) pathogens when considering all pathogens and for 72 of 92 (78%) cultures and 81 of 104 (78%) pathogens when considering on-panel pathogens only. Eight of 31 isolates (26%) not identified by APS were pathogens not included in the APS panel. APS and conventional methods accordingly identified all pathogens from two of nine polymicrobial cultures (22%). APS generated antimicrobial resistance results for 57 pathogens of 57 cultures. The overall category agreement between APS and culture-based AST was 91.2%; and the rate for minor errors was 6.9%, for major was 1.7%, and for very major errors was 0.2%. APS may accelerate pathogen identification and phenotypic AST from positive blood culture bottles inoculated with primary sterile specimens from patients with serious infections, especially for hospitals without an on-site microbiology laboratory. However, the inclusion of nonblood specimens with a high likelihood of polymicrobial infections may result in an inferior performance.

BackgroundBlood cultures are one of the most important tests performed by microbiology laboratories. Many hospitals, particularly in low and middle-income countries, lack either microbiology services or staff to provide 24 h services resulting in delays to blood culture incubation. There is insufficient guidance on how to transport/store blood cultures if delays before incubation are unavoidable, particularly if ambient temperatures are high. This study set out to address this knowledge gap.MethodsIn three South East Asian countries, four different blood culture systems (two manual and two automated) were used to test blood cultures spiked with five common bacterial pathogens. Prior to incubation the spiked blood culture bottles were stored at different temperatures (25 °C, in a cool-box at ambient temperature, or at 40 °C) for different lengths of time (0 h, 6 h, 12 h or 24 h). The impacts of these different storage conditions on positive blood culture yield and on time to positivity were examined.ResultsThere was no significant loss in yield when blood cultures were stored ConclusionBlood cultures should be incubated with minimal delay to maximize pathogen recovery and timely result reporting, however, this study provides some reassurance that unavoidable delays can be managed to minimize negative impacts. If delays to incubation ≥ 12 h are unavoidable, transportation at a temperature not exceeding 25 °C, and blind sub-cultures prior to incubation should be considered.

Third generation cephalosporins are frequently used in the first-line treatment of Gram-negative rod (GNR) bacteremia but are unsuitable in the case of extended-spectrum-beta-lactamase-producing Enterobacterales (ESBL-E) or non-fermenting GNR infections. The aim of this study was to develop and evaluate a simple and rapid two-test protocol involving oxidase and β-Lacta tests performed directly on positive blood culture broth as a preliminary screen for non-fermenting or third generation cephalosporins-resistant GNR. The diagnostic performance of this approach was evaluated on 294 bottles for the oxidase test and 267 bottles for the β-Lacta Test. The sensitivity and specificity of the oxidase test were respectively 93.1% and 100%, and the sensitivity of the β-Lacta Test for ESBL-E was 100% and the specificity 99.5%. This simple protocol, which can be implemented in all laboratories and performed in only 20 min, may be a valuable tool to optimize first-line antibiotic therapy for bacteremia.

Rapid identification of extended-spectrum-β-lactamase (ESBL)-, serine carbapenemase-, and metallo-β-lactamase (MBL)-producing Enterobacterales directly from positive blood culture (BC) bottles is of paramount importance to early optimize antimicrobial management and infection control measures. In this study, we describe and evaluate an improved variant of direct β-lactam inactivation method, named direct ethylenediaminetetraaceticacid-modified-β-lactam inactivation method (deBLIM). The deBLIM test is designed to detect ESBL or carbapenemase activity and to differentiate serine-carbapenemases from MBLs directly from Enterobacterale-positive BCs. The deBLIM was evaluated on both aerobic and anaerobic BCs spiked with 167 characterized Enterobacterale isolates. The deBLIM showed 100% sensitivity in detecting KPC and OXA-48-like serine carbapenemase, CTX-M, SHV variants, and TEM-10 ESBLs both in aerobic and anaerobic conditions. In contrast, a significant discrepancy between aerobic and anaerobic BCs was observed in detecting MBLs. The sensitivity rate in aerobic BCs was of 100% for all metalloenzyme types, whereas only 56.1% and 80% of VIM and NDM producers were detected in anaerobic bottles, respectively. IMP-producing Escherichia coli NCTC 13476 was also not detected in the anaerobic BC. No false positive result was observed among ESBL producers and broad-spectrum-β-lactamase nonproducers, demonstrating an overall specificity of 100%. The deBLIM could be a cost-effective screening method for the identification of ESBLs, serine carbapenemases, and MBLs directly from Enterobacterale-positive BCs on the same day of bottle positivity detection. Nevertheless, it must be considered its poor performance in detecting MBLs in the anaerobic condition.

A 24-year-old woman referred to our radiology department with the diagnosis of new positive blood culture brucellosis and severe aortic regurgitation for evaluation of periaortic thickening. Cardiac magnetic resonance imaging elegantly revealed the presence of active aortitis, most probably caused by brucellosis.

Introduction. Empirical vancomycin (VAN) treatment failure for methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia, with significantly higher mortality, has been reported for MRSA strains with reduced VAN susceptibility. Aim. Our goal was to study the effect of sub-culture on VAN minimum inhibitory concentration (MIC) values compared to direct susceptibility of MRSA-positive blood cultures. Methodology. Using 19 MRSA-positive blood cultures and 19 seeded MRSA-positive blood cultures, we compared the VAN MICs from direct susceptibility testing of MRSA-positive blood cultures and MRSA sub-cultured from positive blood cultures. Results. In comparing direct VAN MICs from MRSA-positive blood cultures and standard agar dilution, nearly half of the MICs from agar dilution were lower, with one sample decreasing from 1.5 to 0.75 µg ml−1. Furthermore, in seeded blood cultures, 80 % or more showed lower values from standard agar dilution compared to direct VAN MICs. Conclusion. Our results reveal a trend towards lower MICs after positive blood culture isolates are sub-cultured. Some clinical failures among MRSA infections treated with VAN may result from this phenomenon.

Using conventional methods, organism identification (ID) and antibiotic susceptibility testing (AST) results are available ∼1.5–3 days after positive blood culture. New technologies can reduce this time to 8–12 h, allowing therapy to be optimized substantially sooner. To make full use of fast ID and AST results requires overcoming various hurdles to effective implementation, including restructuring laboratory workflows to optimize timeliness of results and modifying clinical pathways to respond more quickly when results are available. Efficient laboratory procedures and clinical interventions coupled with fast and accurate identification and AST results have the potential to substantially reduce overall costs and provide more-sophisticated and effective patient management.

The introduction of dedicated phlebotomy teams certified for blood collection has been reported to be highly cost-effective by reducing contamination rates. However, data on their effects on blood volume and true positive rate are limited. Therefore, we investigated the effect of replacing interns with a phlebotomy team on blood culture results. We performed a 24-month retrospective, quasi-experimental study before and after the introduction of a phlebotomy team dedicated to collecting blood cultures in a 2700-bed tertiary-care hospital. The microbiology laboratory database was used to identify adult patients with positive blood culture results. During the study period, there were no changes in blood collection method, blood culture tubes, and the application of antiseptic measures. Blood volume was measured by the BACTEC™ FX system based on red blood cell metabolism. A total of 162,207 blood cultures from 23,563 patients were analyzed, comprising 78,673 blood cultures during the intern period and 83,534 during the phlebotomy team period. Blood volume increased from a mean of 2.1 ml in the intern period to a mean of 5.6 ml in the phlebotomy team period (p

Purpose. Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles. Methodology. 16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION. Results. The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcusand Escherichia coli. The ‘pass’ reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D2 chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing. Conclusion. We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.

Background We evaluated the two in-house sample preparation methods (saponin method (SAP) and [saponin + Sputazyme] method (SSPZ)) for direct identification of microorganisms using MALDI-TOF MS from positive blood culture bottles. Also, we evaluated the [saponin + Sputazyme] method for direct antimicrobial susceptibility testing (AST) using Vitek 2 system. Methods For direct identification, 163 prospective, monomicrobial positive blood culture bottles and 25 contrived blood culture bottles spiked with 25 infrequently isolated bacterial strains were included. For direct AST, pellets obtained by SSPZ method from 102 prospective blood culture bottles were tested. The results from the direct identification and AST were compared with those from the routine diagnostic method performed with colonies sub cultured on solid media. Results In 163 prospective specimens, SAP method correctly identified 132/163 (81.0%) isolates and SSPZ method correctly identified 148/163 (90.8%) isolates (P = .018). Among the 92 Gram-positive isolates, the correct identification rate was significantly higher with the SSPZ method than the SAP method (92.4% vs. 81.5%), respectively (P = .041). However, the SSPZ method failed to identify Streptococcus pneumoniae . Among the 64 Gram-negative isolates, the correct identification rate was 82.8% (53/64) and 87.5% (56/64) for the former and the latter method, respectively (P = .491). Compared with standard methods direct AST showed 98.5% (1523/1547) agreement. Conclusion The addition of Sputazyme improved the identification of commonly isolated bacteria, especially for Gram-positive isolates and yeasts and can be applied for direct antimicrobial susceptibility testing of bacteria. Although SAP method showed better results for Campylobacter spp. and anaerobic bacteria , considering their very low incidence, routine use of SSPZ will be more practical.

[Introduction] The rapid identification of bacteraemia-causing pathogens could assist clinicians in the timely prescription of targeted therapy, thereby reducing the morbidity and mortality of this infection. In recent years, numerous techniques that rapidly and directly identify positive blood cultures have been marketed, with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) being one of the most commonly used., [Methods] The aim of this systematic review and meta-analysis was to evaluate the accuracy of MALDI-TOF (Bruker) for the direct identification of positive blood culture bottles., [Results] A meta-analysis was performed to summarize the results of the 32 studies evaluated. The overall quality of the studies was moderate. For Gram-positive bacteria, overall rates of correct identification of the species ranged from 0.17 to 0.98, with a cumulative rate (random-effects model) of 0.72 (95% CI: 0.64–0.80). For Gram-negative bacteria, correct identification rates ranged from 0.66 to 1.00, with a cumulative effect of 0.92 (95% CI: 0.88–0.95). For Enterobacteriaceae, the rate was 0.96 (95% CI: 0.94–0.97)., [Conclusion] MALDI-TOF mass spectrometry shows high accuracy for the correct identification of Gram-negative bacteria, particularly Enterobacteriaceae, directly from positive blood culture bottles, and moderate accuracy for the identification of Gram-positive bacteria (low for some species)., [Introducción] La identificación rápida de los patógenos causantes de bacteriemia orienta a los clínicos a prescribir con mayor celeridad un tratamiento dirigido y reducir así la morbimortalidad de dicha infección. Durante los últimos años, han aparecido en el mercado numerosas técnicas con la intención de cubrir esta necesidad, que logran una identificación rápida y directa a partir de los frascos de hemocultivos positivos. La espectrometría de masas matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) es una de las tecnologías más utilizadas en este campo., [Métodos] El objetivo de ese estudio es realizar una revisión sistemática y metaanálisis que evalúe la precisión de MALDI-TOF MS (Bruker) para la identificación directa a partir de frascos de hemocultivos positivos., [Resultados] El metaanálisis fue realizado para sintetizar los resultados de los 32 estudios evaluados. La calidad total de los estudios fue moderada. Para las bacterias grampositivas, el ratio total de identificaciones correctas a nivel de especie fue del 0,17 al 0,98 con un ratio acumulativo (modelo de efectos aleatorios) de 0,72 (IC 95%: 0,64-0,80). Para las bacterias gramnegativas, el rango de identificaciones correctas fue del 0,66 al 1,00 con un efecto acumulativo de 0,92 (IC 95%: 0,88-0,95), llegando a un 0,96 (IC 95%: 0,94-0,97) en Enterobacteriaceae., [Conclusiones] La espectrometría de masas MALDI-TOF muestra una alta precisión para la correcta identificación de bacterias gramnegativas realizada directamente a partir de los frascos de hemocultivos positivos, siendo mayor en el grupo de las enterobacterias. Para grampositivas, la precisión es moderada, llegando a ser baja para alguna especie.

To improve time to identification of pathogens and detection of resistance genes, we evaluated the BioFire FilmArray Blood Culture Identification Panel (BCID) as compared to: (1) direct MALDI-TOF MS (DM) and (2) standardized culture-based identification (ID) with antibiotic susceptibility testing (AST). BCID gave an accurate identification in 102/112 (91 %) of cases (102/103 for on-panel organisms). DM gave an accurate identification in 91/112 (81 %) of cases, with 13/91 (14 %) requiring repeat testing from the residual pellet. The mean time to an identification result was 2.4 and 2.9 h for BCID and DM, respectively. Standardized ID and AST results were available at a mean time of 26.5 and 33 h, respectively. There were 44 BCID/DM results that had an antimicrobial treatment change made based on rapid identification and resistant gene detection of pathogens. Both BCID and DM are accurate and rapid methods for the identification of new positive blood culture pathogens.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial components and selectively collect microorganisms are a prerequisite for successful identification, and a variety of home-brew and commercial protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We recently developed a method to collect bacteria from positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing. This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). The operation time required for processing using this novel kit is approximately 10 min, and the entire procedure can be completed within a biosafety cabinet. Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit as compared with the Sepsityper® kit. Performance of the rapid BACpro® II kit was evaluated using a total of 193 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the rapid BACpro® II kit or the Sepsityper® Kit, and isolated cells were subjected to direct identification by MS fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with >1.7 score and >2.0 score obtained using the rapid BACpro® II kit were 99.5% and 80.8%, respectively, whereas those obtained using the Sepsityper® Kit were 89.1% and 68.4%, respectively (P 1.7 and P 2.0 by Pearsons's chi-square). In Gram-positive cases, the rapid BACpro® II kit gave identification rate of 100% with >1.7 score and 69.4% with >2.0 score, whereas there were 84.7% and 56.8%, respectively by the Sepsityper® Kit (P 1.7). These results are preliminary, but considering that this new kit is easy to perform and the identification rates are promising, the rapid BACpro® II kit deserves assessment in a larger-scale study with a variety of platforms for MS-based bacterial identification .

Rapid molecular blood culture Gram-positive (BC-GP) assay can promptly identify vancomycin-resistant enterococcal (VRE) bloodstream infections (BSIs). We sought to evaluate patients with VRE BSI following the pre (N = 44) and post (N = 20) implementation of Verigene BC-GP assay. The average time to detection of VRE was 25.9 ± 4.1h (95% confidence interval (CI): 17.6-34.1; P 0.001) earlier with Verigene BC-GP assay. Compared to patients in the pre-Verigene BC-GP period, the mean adjusted difference in time to administration of anti-VRE therapy was 18.2 ± 7.8h (95% CI: 2.5-33.8; P = 0.024) earlier among patients in the post-Verigene BC-GP period.

Catabacter hongkongensis, an increasingly recognized bacteria in clinical samples, was identified by direct metagenomic sequencing of positive blood culture fluid from a 55-year-old patient with colonic perforation. The bacteremia was cleared by both antibiotic treatment and surgical intervention. This is the first case report of C. hongkongensis infection in the US.

AimsAlthough treatment for candidaemia is time critical, culture-based tests prolong turnaround times and may promote underdiagnosis. Non-culture-based tests have the potential to overcome these difficulties but are in limited clinical use. The aim of this work was to undertake an initial evaluation of two non-culture-based tests for diagnosis of candidaemia.MethodsPatients with candidaemia were identified prospectively over a 4-month period. Sera drawn from case (candidaemic) and control (non-candidaemic) patients on the same day as the positive blood culture were tested with both the Renishaw RenDx Fungiplex test and a commercial β-d-glucan (BDG) assay (Fungitell, Associates of Cape Cod). Sensitivity and specificity were calculated independently and in combination, using paired blood culture as the reference standard.ResultsThere were 10 eligible case patients and 39 negative controls. PCR sensitivity and specificity were found to be 44.4% (95% CI 18.9% to 73.3%) and 87.2% (72.8% to 94.8%), respectively. BDG sensitivity and specificity were 80% (47.9% to 95.4%) and 89.7% (75.9% to 96.5%), respectively. When combining PCR and BDG, sensitivity was 90% (95% CI 57.4% to 100%) and specificity was 79.5% (64.2% to 89.5%). When two sequential specimens were tested, PCR sensitivity increased to 60% (95% CI 31.2% to 83.3%) and BDG sensitivity to 90% (54.7% to 100%).ConclusionA combination of tests, or a single test at multiple time points, may be preferable to relying on one test at a single time point. This should be accounted for in design of future diagnostic accuracy studies of tests for invasive candidosis.

The Nanosphere Verigene Blood Culture Nucleic Acid Tests allow pathogen and antimicrobial resistance marker identification within 2.5 hours of a positive blood culture. This study assessed the sensitivity of the Verigene among Burn Intensive Care Unit (BICU) isolates and acceleration to targeted antibiotic therapy.Bacterial identifications from BICU patients with positive blood cultures over 8 months were compared using 2 different platforms, the Verigene Gram-positive and Gram-negative blood culture tests vs. the bioMerieux Vitek2 automated system. Turnaround times were compared, and Verigene sensitivity for identification and resistance marker detection was calculated. Antimicrobial stewardship was assessed by comparing date/time for empiric and targeted therapy to the Verigene result time.Forty-four isolates (29 target and 15 nontarget) from 17 patients were included. The Verigene correctly identified 15 of 17 Gram-negative (sensitivity 88.2%; 95% confidence interval [CI] [87.9, 88.9]) and 8 of 12 Gram-positive target organisms (sensitivity 66.7%; 95% CI [66.3, 67.5]). None of the nontarget isolates were identified. There were no discordant identifications. Resistance marker identification by the Verigene was 100% concordant with confirmatory testing. For 11 isolates with complete laboratory and clinical data, the median time between Verigene and final culture results was 59.3 hours (37.3, 102.2) and from Verigene results to targeted therapy was 62.2 hours (43.6, 66.2).Reasons for lower sensitivity than previously reported are unclear and, on the basis of this limited retrospective review, further study in the BICU population is needed. The Verigene appears useful for antimicrobial stewardship by accelerating the identification of blood isolates.

Blood cultures are the gold standard in the detection of bloodstream infections, an important cause of morbidity and mortality. Blood cultures are one of the highest volume tests and the most important test performed in the clinical microbiology laboratory despite a low, 8%–10%, positivity rate. Most blood cultures are incubated in continuously monitored blood culture systems for 5 days. It is recommended that blood cultures are taken before antibiotics are given. Antimicrobial agents, usually broad spectrum, are started after blood culture collection and are continued until antimicrobial susceptibilities are reported, 48–72 h after the report of a positive blood culture. At this time, deescalation to targeted antimicrobials can occur. Prompt effective antimicrobial therapy has been shown to …

Background: Microbial invasion of the bloodstream is associated with a major burden of illness. Despite its importance, there is inconsistency in utilization of terms used to define it. Objective: To characterize the contemporary use of terms to define microbial invasion of the bloodstream for surveillance and research purposes. Methods: Structured review of publications reported from 2000 to 2019. Results: The search strategy retrieved 10,095 citations of which bloodstream infection, bacteraemia and fungaemia were included in 2813, 6900 and 1054 articles, respectively. There was a tripling of the number of annual citations during the study and although bacteraemia was most frequent, there was a progressive increase in the use of the term bloodstream infection. Among the 100 reports randomly selected for detailed review, the terms bacteraemia, bloodstream infection and fungaemia were used in 57, 51 and 19 publications, respectively. Explicit definitions for bloodstream infection (26/51; 51%), bacteraemia (13/57; 23%) and fungaemia (7/19; 37%) were included in reports where these terms were used. Although nearly all (95%) of the studies indicated a positive blood culture as an inclusion criteria and/or definition, only a minority indicated means to exclude contaminants (33%) or specific attributes to support clinical significance (38%). Use of explicit definitions was more common among reports that exclusively used the term bloodstream infection as compared to bacteraemia. Conclusions: Terms have been inconsistently defined and imprecisely used to refer to microbial invasion of the bloodstream. Clinically relevant and objective definitions that are widely acceptable are needed for surveillance and research purposes.

Objective To evaluate a direct antibiotic susceptibility testing method for blood culture.. Methods The cross-sectional comparative study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from December 2016 to October 2017. Direct antimicrobial susceptibility testing was performed from positive blood culture bottles. Bacterial identification was done by using API 10S. Different antimicrobial panels were employed for Gram-negative rods (GNRs), gram-positive cocci (like suspected Staphylococci and Enterococci). Results were compared with conventional disk diffusion testing and very major, major and minor errors were calculated. Result agreement and kappa coefficient scores were generated for categorical agreement. SPSS 24 was used for data analysis. Results Of the 101 bacterial isolates, 82(81.2%) were Gram negative rods and 19(18.8%) were Grampositive cocci. Among 781 bacteria-antibiotic comparisons, the number of very major errors was 3(0.4%), major errors were 7(0.9%) and minor errors were 12(1.5%), while, 759(97.2%) comparisons yielded the same results. The kappa coefficient was 0.946, showing almost perfect agreement. Direct identification of Gram negative rods was successful in 53(64.6%) cases. Conclusions Direct susceptibility testing of blood culture produced reliable results for majority of the antimicrobials.

ObjectivesThe role of follow-up blood cultures (FUBCs) in the management of Gram-negative bacteraemia (GNB) is poorly understood. We aimed to determine the utility of FUBCs in identifying patients with increased mortality risk.MethodsAn observational study with a prospectively enrolled cohort of adult inpatients with GNB was conducted at Duke University Health System from 2002 to 2015. FUBCs were defined as blood cultures performed from 24hours to 7days from initial positive blood culture.ResultsAmong 1702 patients with GNB, 1164 (68%) had FUBCs performed. When performed, FUBCs were positive in 20% (228/1113) of cases. FUBC acquisition was associated with lower all-cause in-hospital mortality (108/538, 20%, vs. 176/1164, 15%; p 0.01) and attributable in-hospital mortality (78/538, 15%, vs. 98/1164, 8%; p&nbsp

We directly tested 484 organisms from clinical (n = 310) and simulated (n = 174) positive blood cultures using the NG-Test Carba 5 assay for carbapenemase-producing Enterobacterales detection. The assay identified all but 4 of the KPC (170/171), OXA-48-like (22/22), VIM (19/21), and NDM (14/15) producers with no false positives. Among the clinical Klebsiella pneumoniae organisms tested, 122 of 123 KPC, 1 of 1 OXA-48-like, and 1 of 2 VIM producers were detected by the assay. Some VIM and NDM producers yielded scant but still-readable bands with the assay. No organisms produced the IMPs that the assay was designed to detect.

Objectives: The aim of this study was to elaborate on and validate a score for the early diagnosis of mediastinitis after cardiothoracic surgery. Methods: Between 2007 and 2017, patients who experienced thoracic surgical-site infection after cardiothoracic surgery were enrolled. Laboratory, clinical, and chest CT findings were retrospectively analyzed. Patients were followed up until hospital discharge or intra-hospital death. Univariate and multivariate regression analyses were performed. Results: 950 surgical-site infections were found and analyzed (131 mediastinitis, 819 superficial/deep infections). Of the 131 mediastinitis episodes, 88% required surgical thoracic debridement,Staphylococcus aureus was identified in 43%, and overall mortality was 42%. The following variables were related to mediastinitis diagnosis: sternal diastasis (OR = 2.5; 95% confidence interval [95%CI]: 1.2–5.3; P = 0.012), bilateral pleural effusion (OR = 1.9; 95%CI: 1.0–3.6; P = 0.04), leukocyte count ≥14,000cells/mm3 (OR = 2.5; 95%CI: 1.3–4.7; P = 0.006), male sex (OR = 2; 95%CI: 1.11–4; P = 0.022), and positive blood culture (OR = 3.0; 95%CI: 1.6–5.6; P = 0.001). The score predicted with reasonable accuracy mediastinitis in the derivation cohort (AUC-ROC, 0.7476) and the validation cohort (AUC-ROC, 0.7149). Groups with high (31%) and low (5%) risk of mediastinitis were identified. Conclusions: An early diagnostic score in patients with surgical-site infection after cardiothoracic surgery identified groups with a low and high risk for mediastinitis. Keywords: Mediastinitis, Diagnosis, Surgical site infections

Background The rate of bloodstream infections (BSIs) is disproportionately high in hemodialysis (HD) patients with central venous catheters (CVCs) versus those with permanent accesses, contributing to poorer outcomes, such as increased rates of death and hospitalizations. Study Design 12-month, prospective, cluster-randomized, multicenter, open-label trial. Setting & Participants 40 Fresenius Medical Care North America dialysis facilities were matched and paired by positive blood culture rate and number of patients with CVCs and then cluster-randomized with 20 in each study group. 2,470 patients participated in the study (1,245, intervention group; 1,225, control group), accruing approximately 350,000 CVC-days. Intervention Use of ClearGuard HD Antimicrobial Barrier Caps versus use of standard CVC caps; assigned at the facility level. Outcome Primary end point was positive blood culture rate as an indicator of BSI rate. Measurements Positive blood cultures, hospital admissions for BSI, hospitalization-days for BSI, intravenous antibiotic starts, and CVC-days. Results Baseline positive blood culture rates were similar ( P =0.8) between groups. Use of ClearGuard HD caps for 12 months was associated with a 56% lower BSI rate versus use of standard CVC caps (0.26 vs 0.59/1,000 CVC-days, respectively; P =0.01). When considering sustained use (defined as last 6 months of the study), the intervention versus the control was associated with a 69% lower BSI rate (0.22 vs 0.72/1,000 CVC-days, respectively; P =0.01), 43% fewer hospital admissions for BSI (0.28 vs 0.48/1,000 CVC-days, respectively; P =0.04), and 51% fewer hospitalization days for BSI (2.42 vs 4.94/1,000 CVC-days, respectively; P =0.04). No device-related adverse events were reported. Limitations Study was open label; patients occasionally received HD at nonresearch facilities; patients did not receive the intervention when hospitalized. Conclusions The findings show that use of ClearGuard HD Antimicrobial Barrier Caps, when compared with standard CVC caps, significantly lowers rates of catheter-related BSIs and hospital admissions for BSI in HD patients using CVCs.

In cases of fungal infection of the bloodstream, rapid species identification is crucial to provide adapted therapy and thereby ameliorate patient outcome. Currently, the commercial Sepsityper kit and the sodium-dodecyl sulfate (SDS) method coupled with MALDI-TOF mass spectrometry are the most commonly reported lysis protocols for direct identification of fungi from positive blood culture vials. However, the performance of these two protocols has never been compared on clinical samples. Accordingly, we performed a two-step survey on two distinct panels of clinical positive blood culture vials to identify the most efficient protocol, establish an appropriate log score (LS) cut-off, and validate the best method. We first compared the performance of the Sepsityper and the SDS protocols on 71 clinical samples. For 69 monomicrobial samples, mass spectrometry LS values were significantly higher with the SDS protocol than with the Sepsityper method (P < .0001), especially when the best score of four deposited spots was considered. Next, we established the LS cut-off for accurate identification at 1.7, based on specimen DNA sequence data. Using this LS cut-off, 66 (95.6%) and 46 (66.6%) isolates were correctly identified at the species level with the SDS and the Sepsityper protocols, respectively. In the second arm of the survey, we validated the SDS protocol on an additional panel of 94 clinical samples. Ninety-two (98.9%) of 93 monomicrobial samples were correctly identified at the species level (median LS = 2.061). Overall, our data suggest that the SDS method yields more accurate species identification of yeasts, than the Sepsityper protocol.

Background We evaluated the reliability and accuracy of the combined use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial identification and Vitek 2 antimicrobial susceptibility testing (AST) for bacteria from positive blood culture bottles. Methods Direct identification and AST were performed in parallel to the standard methods in monomicrobial positive blood culture bottles. In total, 254 isolates grown on aerobic and/or anaerobic bottles were identified with MALDI-TOF Vitek MS (bioMérieux, France), and 1,978 microorganism/antimicrobial agent combinations were assessed. For isolates from anaerobic bottles, an aliquot of the culture broth was centrifuged, washed, and filtered through a nylon mesh. For isolates from aerobic/pediatric bottles, a lysis step using 9.26% ammonium chloride solution and 2% saponin solution was included. Results The overall correct identification rate was 81.8% (208/254) and that for gram-positive/gram-negative isolates was 73.9%/92.6%, respectively, and it was 81.8%, 87.6%, and 57.9% for isolates from aerobic, anaerobic, and pediatric bottles, respectively. Identification was not possible in 45 cases, and most of these isolates were streptococci (N=14) and coagulase-negative staphylococci (N=11). Misidentification occurred only in one case. Compared with standard methods, direct AST showed 97.9% (1,936/1,978) agreement with very major error of 0.25%, major error of 0.05%, and minor error of 1.8%. Conclusions This simple and cost-effective sample preparation method gives reliable results for the direct identification and AST of bacteria. For the identification of streptococci and coagulase-negative staphylococci, the method should be further improved.

The performance of the VITEK® 2 system for direct rapid identification and antimicrobial susceptibility testing of the bacteria responsible for blood infections was determined. The isolates studied included 166 Gram-negative rods and 74 Gram-positive cocci from inpatients. Specially treated monomicrobial samples from positive blood culture bottles were directly inoculated into the VITEK 2 system and the results were compared with those from cards inoculated with standardized bacterial suspensions. Compared with the standard method, 95.8% of Gram-negative rods were correctly identified by VITEK 2 and the overall level of agreement between the two methods in susceptibility testing was 92.0%. For Gram-positive bacteria, 89.2% were correctly identified by VITEK 2 and susceptibility testing revealed an overall agreement rate of 91.3%. These results suggest that VITEK 2 cards inoculated with fluids sampled directly from positive blood culture bottles are suitable for speedy identification and susceptibility testing of Gram-negative bacilli and Gram-positive cocci.دقة نظام 2 ®VITEK للاستعراف السريع والمباشر عى العصيات سلبية الجرام والمكورات إيجابية الجرام في عينات الدم واختبار حساسيتها.نبيل عوني النمر، رائدة جمال السعايدة، عمر أبو العيش.تم تحديد أداء نظام 2 ® VITEK للاستعراف السريع المباشر عى الجراثيم المسؤولة عن عداوى الدم واختبار حساسيتها للأدوية. وقد شملت المستفرَدات المدروسة 166 مستفردة لعصيت سلبية الجرام و 74 مستفردة لمكورات إيجابية الجرام من مرضى داخلين. وأخذت عينات أحادية الجرثوم - معالجة بطريقة خاصة - من قوارير مستنبَتات الدم الإيجابية، ولقِّحت مباشرة في نظام 2 ®VITEK ، وقورنت النتائج مع تلك المأخوذة من بطاقات ملقحة بمستعلقات جرثومية معرَّة. بالمقارنة مع الطريقة المعيارية تم الاستعراف الصحيح عى %95.8 من العصيات سلبية الجرام بواسطة 2 ®VITEK ، وكان مستوى التوافق الإجمالي بن الطريقتن في اختبار الحساسية %92 وبالنسبة للجراثيم إيجابية الجرام تم الاستعراف الصحيح عى %89.2 بواسطة 2 ®VITEK ، وأظهر اختبار الحساسية توافقاً إجمالياً بمعدل %91.3. تشر هذه النتائج إلى أن بطاقات 2 ®VITEK التي تلقَّح بسوائل مأخوذة مباشرة من قوارير مستنبتات الدم الإيجابية تعتر مناسبة للاستعراف السريع عى العصيات سلبية الجرام والمكورات إيجابية الجرام ولاختبار حساسيتها.Précision du système VITEK® 2 pour une identification et un test de sensibilité directs et rapides des bâtonnets à gram négatif et des cocci à gram positif dans des échantillons sanguins.La performance du système VITEK® 2 pour une identification et un test de sensibilité aux antimicrobiens rapides et directs des bactéries responsables d’infections sanguines a été mesurée. Les isolats étudiés concernaient 166 bâtonnets à gram négatif et 74 cocci à gram positif prélevés sur des patients hospitalisés. Des échantillons monomicrobiens issus de flacons d’hémoculture positifs ayant été soumis à un traitement spécial ont été directement inoculés dans le système VITEK 2 et les résultats ont été comparés avec ceux issus de cartes inoculées à l’aide de suspensions bactériennes standards. Par comparaison avec la méthode standard, 95,8 % des bâtonnets à gram négatif ont été correctement identifiés par VITEK 2 et le niveau de concordance global entre les deux méthodes en matière de test de sensibilité était de 92,0 %. Pour les bactéries à gram positif, 89,2 % ont été correctement identifiées par VITEK 2 et le test de sensibilité a révélé un taux de concordance de 91,3 %. Ces résultats suggèrent que les cartes VITEK 2 inoculées à l’aide de liquides prélevés directement dans des flacons d’hémoculture positifs sont adaptées à une identification et à un test de sensibilité rapides des bacilles à gram négatif et des cocci à gram positif.

Rapid identification of bacterial pathogens facilitates earlier optimization of antibiotic treatment and reduces morbidity and mortality in sepsis patients. The aim of this research was to design an in-house chemical-free method for direct bacterial identification in positive blood culture (BC) broths and to compare the performance of this method with that of the commercial Sepsityper® kit. The overall species identification rates for the in-house and Sepsityper methods were 88.4% and 85.8%, respectively (n = 190). Among 146 facultative anaerobes, 92.5% and 95.9% were identified to the species level using the in-house and Sepsityper methods, respectively. For 32 anaerobic bacteria, the in-house method showed a higher species identification rate (75.0%) than the Sepsityper method (53.1%). The in-house method correctly identified more Bacteroides species (100.0%) than the Sepsityper method (18.2%). Our novel in-house method and the Sepsityper method showed a high accuracy for direct bacterial identification in positive BC broths using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We evaluated the performance of OXA-48 K-SeT assay for detecting OXA-370 directly from spiked rectal swabs and blood culture vials. The limit of detection of this test was 10 4 UFC/mL for rectal swabs. Detection of the OXA-370-producing isolates was successfully achieved directly from positive blood culture vials independent of growing conditions.

Background Short-term culture followed by MALDI-TOF MS is one of the most widely used methods for fast identification of microorganisms from blood cultures. The method identifies the vast majority of bloodstream infection pathogens in 2–6 h after positive blood culture. Transport time of blood culture bottles to laboratories is a major problem affecting total turnaround time. Therefore, many central laboratories establish satellite blood culture systems in other clinics and hospitals to allow blood culture bottles to be incubated immediately after sampling. However, positive blood culture bottles still need to be transported to the clinical microbiology laboratory for analysis. The aim of this study was to investigate how delayed analysis of positive blood culture bottles would affect the short-term culture followed by MALDI-TOF MS method. Materials/methods To simulate the effect of transportation and delayed analysis of blood culture bottles, 51 simulated blood culture bottles were incubated for 0, 2, 4 and 24 h at room temperature. After each time interval, a 2 to 4 h short-term culture followed by MALDI-TOF MS was performed. In addition, 257 prospective clinical positive blood culture bottles were analysed with the same method after a 24 h incubation at room temperature. Results In simulated samples, all (120/120) Gram-negative bacteria and 77/84 (91.6%) Gram-positive bacteria were accurately identified at species-level after a 2 h short-term culture, regardless of the duration of simulated transport time. In the clinical samples, 100/116 (86.2%) Gram-negative, and 44/141 (31.2%) Gram-positive bacteria were accurately identified at species-level after a 2 h short-term culture. After contaminants were excluded, 39/71 (54.9%) Gram-positive bacteria could be identified after 2 h. After a 4 h short-term culture, 112/116 (96.6%) Gram-negative, and 108/141 (76.6%) Gram-positive bacteria were accurately identified at species-level. Of the clinically relevant Gram-positive bacteria, 68/71 (95.8%) were identified at species-level after 4 h. Conclusions Short-term culture followed by MALDI-TOF MS can provide fast and accurate results for identification of clinically relevant bacteria, despite long transportation times from satellite laboratories. The present data shows that the method can be used for identification of microorganisms from positive blood cultures transported from satellite blood culture systems.

Invasive Streptococcus pyogenes diseases represent the most severe form of infection produced by this microorganism. Early diagnosis and treatment are important, due to its potential severity. Etiological confirmation of invasive infection is performed by culture, which takes between 18 and 48 h. We tested a rapid immunochromatographic assay directly from clinical samples from normally sterile sites and positive blood culture bottles when pos-itive cocci chains were observed by Gram staining. Eighty samples were analyzed. The rapid test was positive in 35 samples: in 34 of them S. pyogenes was confirmed by culture. The immunochromatographic method showed 97.1% sensitivity and 97.8% specificity. The strept A® immunochromatographic rapid test allows to obtain reliable results in less than 10min and is accessible to any microbiology laboratory. This study demonstrates the potential use of a rapid immunochromatographic method directly from clinical samples and positive blood cultures. Resumen La enfermedad invasiva por Streptococcus pyogenes representa la forma más grave de infección producida por este microorganismo y requiere un rápido diagnóstico, a fin de instaurar un tratamiento adecuado. La confirmación etiológica de esta infección se realiza por cultivo, lo que puede llevar entre 18 y 48 h. En este estudio ensayamos una prueba inmunocromatográfica rápida directamente de muestras clínicas de sitios normalmente estériles y de botellas de hemocultivos positivos cuando la coloración de Gram evidenció cocos gram positivos en cadena. Se analizaron 80 muestras. La prueba rápida fue positiva en 35 muestras: en 34 de ellas se confirmó la presencia de S. pyogenes por cultivo. La sensibilidad y la especificidad de la prueba fueron del 97,1 y el 97,8%, respectivamente. La prueba inmunocromatográfica rápida monteBIO Strep A® permite obtener resultados confiables en menos de 10 min y es accesible para cualquier laboratorio de microbiología. Este estudio demuestra la utilidad de dicha prueba para ser practicada directamente en muestras clínicas y botellas de hemocultivos positivos.