Positive Blood Culture Bottles Research Articles

A Fast Preliminary Identification Test in Fungemia: Evaluation of Germ Tube Test Directly from Positive Blood Culture Bottles

A Fast Preliminary Identification Test in Fungemia: Evaluation of Germ Tube Test Directly from Positive Blood Culture Bottles

Direct Identification and Antimicrobial Susceptibility Testing of Bacteria From Positive Blood Culture Bottles by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and the Vitek 2 System.

BackgroundWe 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.MethodsDirect 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.ResultsThe 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%.ConclusionsThis 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.

Agreement of Direct Antifungal Susceptibility Testing from Positive Blood Culture Bottles with the Conventional Method for Candida Species.

Early availability of antifungal susceptibilities can ensure timely institution of targeted therapy in candidemia, which can improve patient outcomes. This study prospectively determines the agreement between the results of direct testing of antifungal susceptibilities from blood culture bottles by disk diffusion and Etest and the results of standardized susceptibility testing methods; direct testing would allow susceptibility results to be available 1 to 2 days earlier. A total of 104 blood cultures with different Candida species (28% C. albicans, 27% C. parapsilosis, 26% C. tropicalis, etc.) were evaluated between January 2012 and May 2013 for agreement of fluconazole, voriconazole, and amphotericin B susceptibility results by disk diffusion. Agreement in MICs obtained by Etest was determined for fluconazole (21 isolates), voriconazole (28 isolates), amphotericin (29 isolates), and caspofungin (29 isolates). The kappa scores for categorical agreement were highest for fluconazole by disk diffusion (0.902, standard error [SE] = 0.076) and Etest (1.00, SE = 0.218) and for amphotericin B by disk diffusion (1.00, SE = 0.098). The Pearson correlation (r) of zone diameters was strongest for fluconazole (0.69) and amphotericin (0.70) and moderate for voriconazole (0.60), and the Pearson correlation of MICs was strongest for fluconazole (0.94) and caspofungin (0.88). However, the moderate correlation of amphotericin MICs with zone diameters (-0.42) precludes the use of amphotericin B disk diffusion for susceptibility testing. There were no very major errors; however, there were 1 (1%) major and 5 (4.8%) minor errors with disk diffusion and 4 (13.3%) minor errors with Etest. Thus, antifungal disk diffusion directly from blood culture bottles is a rapid and easy method for fluconazole and voriconazole susceptibility testing for timely tailoring of candidemia therapy.

Microbial identification and automated antibiotic susceptibility testing directly from positive blood cultures using MALDI-TOF MS and VITEK 2

The study addresses the utility of Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight mass spectrometry (MALDI-TOF MS) using VITEK MS and the VITEK 2 antimicrobial susceptibility testing (AST) system for direct identification (ID) and timely AST from positive blood culture bottles using a lysis-filtration method (LFM). Between July and December 2014, a total of 140 non-duplicate mono-microbial blood cultures were processed. An aliquot of positive blood culture broth was incubated with lysis buffer before the bacteria were filtered and washed. Micro-organisms recovered from the filter were first identified using VITEK MS and its suspension was used for direct AST by VITEK 2 once the ID was known. Direct ID and AST results were compared with classical methods using solid growth. Out of the 140 bottles tested, VITEK MS resulted in 70.7 % correct identification to the genus and/ or species level. For the 103 bottles where identification was possible, there was agreement in 97 samples (94.17 %) with classical culture. Compared to the routine method, the direct AST resulted in category agreement in 860 (96.5 %) of 891 bacteria-antimicrobial agent combinations tested. The results of direct ID and AST were available 16.1 hours before those of the standard approach on average. The combined use of VITEK MS and VITEK 2 directly on samples from positive blood culture bottles using a LFM technique can result in rapid and reliable ID and AST results in blood stream infections to result in early institution of targeted treatment. The combination of LFM and AST using VITEK 2 was found to expedite AST more reliably.

Rapid identification of gram-negative bacteria and corresponding resistance determinants from blood cultures by BC-GN assay

Objective To evaluate the performance and clinical utility of Verigene gram-negative blood culture (BC-GN) assay for a rapid identification of gram-negative bacteria and resistance genes. Methods Non-repetitive blood culture samples containing gram-negative bacteria were collected from inpatients in the Second Affiliated Hospital of Zhejiang University School of Medicine from June to October, 2013. BC-GN assay was performed to identify the species and genetic resistance determinants of gram-negative bacteria directly from the positive blood culture bottles. VITEK MS and the VITEK 2 Compact were used for species identification and antimicrobial susceptibility test, the results of which were considered as gold standards. The resistance genes were further validated by PCR amplification and sequencing analysis. A comparison of the results and time between the BC-GN assay and routine methods was conducted. Results The detection range of BC-GN assay almost covered all of the common gram-negative bacteria. BC-GN assay showed an advantage of high accuracy in the identification of Escherichia coli (13/13), Klebsiella pneumoniae (19/24), Klebsiella oxytoca (9/9), Pseudomonas aeruginosa (39/39), Serratia marcescens (4/5), Enterobacter spp. (6/8), Citrobacter spp. (11/11), Proteus spp. (6/6) and Acinetobacter spp. (24/24) with an overall accuracy of 94.24% for the identification of mono-microbial blood culture samples. Moreover, BC-GN assay accurately identified all of the bacteria and resistance genes from the two multi-microbial samples. Species identification and resistance profiles could be 42 hours earlier available by using BC-GN assay than those by using routine methods. Conclusion BC-GN assay could simultaneously and accurately identify bacteria and resistance determinants from blood cultures within 2 hours. More time for clinically effective therapy could be achieved by using BC-GN assay for the reduction of mortality associated with bloodstream infection. Key words: Bloodstream infection; Gram-negative; Antimicrobial resistance; Rapid identification

Evaluation of Bloodstream Infections During Chemotherapy-Induced Febrile Neutropenia in Patients with Malignant Hematological Diseases: Single Center Experience.

From year to year, it is important to get an overview of the occurrence of causative agents in febrile neutropenic patients to determine the empiric treatment. Thus our aims were to evaluate a four-year period regarding the prevalence of bloodstream infections and the most important causative agents. During this period, 1,361 patients were treated in our hematology ward because of various hematological disorders. 812 febrile episodes were recorded in 469 patients. At that time, 3,714 blood culture (BC) bottles were sent for microbiological investigations, 759 of them gave positive signal. From the majority of positive blood culture bottles (67.1%), Gram-positive bacteria, mainly coagulase-negative staphylococci (CNS), were grown. Gram-negative bacteria were isolated from 32.9% of the positive blood culture bottles, in these cases the leading pathogen was Escherichia coli. The high prevalence of CNS was attributed to mainly contamination, while lower positivity rate for Gram-negative bacteria was associated with the use of broad-spectrum empiric antibiotic treatment.

Answer to September 2015 Photo Quiz

Answer: Community-acquired pneumonia caused by Acinetobacter baumannii. The Gram-positive rods seen on the initial Gram stain from the blood culture broth were later identified as Acinetobacter baumannii. A. baumannii is a strictly aerobic, nonfermenting, oxidase-negative, catalase-positive coccus/coccobacillus that grows well on both blood agar and MacConkey agar. In Gram-stained preparations, it typically appears as Gram-negative diplococci with an arrangement resembling that of Neisseria spp. (1). It is well recognized that some Acinetobacter isolates tend to retain crystal violet and resist decolorization (2) and may initially appear as Gram-positive cocci in direct smears prepared from positive blood culture bottles (3). This may explain the Gram-positive appearance of the original Gram-stained smear. Of note, there was a similar report of A. baumannii presenting as Gram-positive bacilli in a bronchial washing specimen from a patient with fulminant A. baumannii community-acquired pneumonia (4). However, the mechanism by which this phenomenon occurs remains unclear. The unusual bacillary appearance may be contributed to by the growth phase of the organism as well as the presence of antibiotic in the blood. It was reported that Acinetobacter spp. are plump rods measuring 0.9 to 1.6 by 1.5 to 2.5 μm during exponential growth and become coccoid after entry into the stationary phase (2). The organisms seen on the original Gram stain, however, measured up to 6 μm in length. This morphological change is likely caused by the presence of sub-MIC levels of β-lactam antibiotic in the blood, amoxicillin in this case. This effect has been described to occur in various Gram-negative bacilli in the presence of β-lactam antibiotics, which were proposed to inhibit septum formation, resulting in the elongated morphology (5, 6). The subsequent Gram-stained smear of the isolate from sheep blood agar showed Gram-negative diplococci, in contrast to the Gram-positive bacilli seen in the original smear. The isolate was identified as A. baumannii by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Bruker). The Vitek 2 system (GN ID card) and an API 20 NE strip identified the organism as a member of the Acinetobacter baumannii-Acinetobacter calcoaceticus complex. A. baumannii is ubiquitous in freshwater and soil. It is an opportunistic pathogen that plays a significant role in nosocomial infections and hospital outbreaks. It is also an uncommon cause of severe community-acquired pneumonia. While health care-associated A. baumannii is often multidrug resistant, strains of A. baumannii causing community-acquired pneumonia are usually susceptible to multiple antibiotics. The strain in this case tested susceptible to ampicillin-sulbactam, piperacillin-tazobactam, cefepime, gentamicin, ciprofloxacin, and imipenem via disk diffusion testing. The patient was empirically treated with intravenous amoxicillin-clavulanate and oral clarithromycin, which were subsequently changed to ampicillin-sulbactam on the basis of susceptibility testing. However, on the third day of admission, he developed a fever, and a repeat chest X-ray showed progression of consolidation. Repeat cultures were negative, and he was treated with piperacillin-tazobactam. He completed 10 days of antibiotics in total and returned home in a stable condition. This case illustrates that A. baumannii can present with unusual Gram stain findings in clinical specimens. Both laboratory and clinical personnel should be aware that in the appropriate clinical settings, A. baumannii infection should be considered a differential diagnosis despite seemingly incongruent Gram stain findings. (See page 2797 in this issue [doi:10.1128/JCM.02551-13] for photo quiz case presentation.)

In vitro aggregate formation and unusual colony morphology impairing identification of Roseomonas sp. from a septic patient

Introduction: The Gram-stained morphology of organisms from positive blood culture bottles often suggests diagnoses and therapeutic options. In vitro aggregate formation by the organism in this case led to initial confusion in the diagnosis. Case presentation: A 57-year-old woman with a recent stroke returned to the emergency department with acute exacerbation of her neurological deficit. Although afebrile, she had an elevated white blood cell count. Urine analysis revealed 4+ blood, nitrates, leukocyte esterase and bacteria. Blood and urine cultures were collected and intravenous piperacillin/tazobactam treatment was begun. The urine culture grew >100 000 colonies Escherichia coli ml− 1; the antibiotic was changed to ceftriaxone when susceptibilities became available at 48 h. At 61 h, the aerobic blood culture bottle was flagged as positive. A Gram stain revealed spherical structures, which stained predominantly Gram-negative but were Gram-positive in some areas of the smear. Subcultures grew overnight on blood, chocolate and MacConkey agars and on brain–heart infusion (BHI) broth. Growth on MacConkey agar was sparse. A Gram stain of the snow-white, mucoid colonies revealed Gram-negative rods, spherical aggregates and thick-walled tetrads. Subsequent cultures on solid medium grew only Gram-negative rods, but inoculation of isolated colonies from these cultures into BHI broth or negative blood culture bottles caused the thick-walled spheres and tetrads to reappear. Conclusion: MicroScan and matrix-assisted laser desorption/ionization time of flight failed to identify the organism, but 16S rRNA gene sequencing identified it as Roseomonas genomospecies 5 (100 % match). To the best of our knowledge, there are no reports in the literature of aggregate-forming Roseomonas.

Randomized Trial of Rapid Multiplex Polymerase Chain Reaction-Based Blood Culture Identification and Susceptibility Testing.

The value of rapid, panel-based molecular diagnostics for positive blood culture bottles (BCBs) has not been rigorously assessed. We performed a prospective randomized controlled trial evaluating outcomes associated with rapid multiplex PCR (rmPCR) detection of bacteria, fungi, and resistance genes directly from positive BCBs. A total of 617 patients with positive BCBs underwent stratified randomization into 3 arms: standard BCB processing (control, n = 207), rmPCR reported with templated comments (rmPCR, n = 198), or rmPCR reported with templated comments and real-time audit and feedback of antimicrobial orders by an antimicrobial stewardship team (rmPCR/AS, n = 212). The primary outcome was antimicrobial therapy duration. Secondary outcomes were time to antimicrobial de-escalation or escalation, length of stay (LOS), mortality, and cost. Time from BCB Gram stain to microorganism identification was shorter in the intervention group (1.3 hours) vs control (22.3 hours) (P < .001). Compared to the control group, both intervention groups had decreased broad-spectrum piperacillin-tazobactam (control 56 hours, rmPCR 44 hours, rmPCR/AS 45 hours; P = .01) and increased narrow-spectrum β-lactam (control 42 hours, rmPCR 71 hours, rmPCR/AS 85 hours; P = .04) use, and less treatment of contaminants (control 25%, rmPCR 11%, rmPCR/AS 8%; P = .015). Time from Gram stain to appropriate antimicrobial de-escalation or escalation was shortest in the rmPCR/AS group (de-escalation: rmPCR/AS 21 hours, control 34 hours, rmPCR 38 hours, P < .001; escalation: rmPCR/AS 5 hours, control 24 hours, rmPCR 6 hours, P = .04). Groups did not differ in mortality, LOS, or cost. rmPCR reported with templated comments reduced treatment of contaminants and use of broad-spectrum antimicrobials. Addition of antimicrobial stewardship enhanced antimicrobial de-escalation. NCT01898208.

Use of an automated PCR assay, the GenomEra S. pneumoniae, for rapid detection of Streptococcus pneumoniae in blood cultures

Background: Streptococcus pneumoniae is recognized as a major cause of pneumonia, meningitis, and bacteremia. Since the mortality rate for pneumococcal bacteremia remains high, the reliable detection of the bacterium in blood samples is important. In this study, the performance of a new automated PCR assay, the GenomEra™ S. pneumoniae, for direct detection of S. pneumoniae in blood cultures was investigated. Methods: In total, 200 samples were analyzed, including 90 previously identified culture collection isolates and 110 blood culture specimens. The species identification was confirmed with routine diagnostic methods including MALDI-TOF or 16S rDNA sequencing. Results: From culture collection, the GenomEra S. pneumoniae assay correctly identified all 37 S. pneumoniae isolates consisting of 18 different serotypes, while all 53 non-S. pneumoniae isolates yielded negative test results. Of 110 blood culture specimens, 46 grew S. pneumoniae and all were positive by the GenomEra assay direct from bottle. The detection sensitivity and specificity of the GenomEra assay for direct analysis of S. pneumoniae in signal positive blood culture bottles was 100%, respectively. With a straightforward sample preparation protocol of blood cultures the results were available within 55 min, thus being significantly quicker than by the routinely used identification methods (18–48 h). The two-step, time-resolved fluorometric measurement mode employed by the GenomEra CDX™ instrument showed no interference from blood or charcoal. Conclusion: The GenomEra S. pneumoniae assay is a tool that performs well for the rapid and reliable detection of S. pneumoniae in blood cultures.

Identificación rápida de microorganismos de frascos de hemocultivos por espectrometría de masas. Comparación de 2 procedimientos diagnósticos

Rapid identification of microorganisms is critical in hospitalized infected patients. Blood culture is currently the gold standard for detecting and identifying microorganisms causing bacteremia or sepsis. The introduction of mass spectrometry by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) in microbiology laboratories, especially in microorganisms growing in blood culture bottles, provides rapid identification.This study evaluates the performance of the Maldi Sepsityper Biotyper procedure (hereinafter, MS) compared to that of an in-home method (hereinafter, HF). Eight hundred and forty (840) positive blood culture bottles were processed using the HF procedure, 542 of which were also processed using MS. The organisms were identified in 670 (79.76%) and 391 (72.14%) bottles respectively (p = 0,0013). This study demonstrates the effectiveness of both procedures for identifying microorganisms directly from positive blood culture bottles. However, the HF procedure proved to be more effective than MS, especially in the presence of Gram positive organisms.

Development of a real-time polymerase chain reaction method for the identification of Candida species

Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 μl of extracted DNA, 2 μl of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 μl of MgCl(2) (5 mmol), 2 μl of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 μl of each primer (0.01 nmol/μl) and 1 μl of each probe (0.1 μmol/μl) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95°C for 10 mins and 50 cycles of denaturation at 95°C for 10 secs, annealing at 62°C for 10 secs and polymerisation at 72°C for 20 secs. A melting curve was created by cooling the producs at 50°C for 30 secs and then heating to 80°C at a rate of 0.1°C/sec measuring of the fluorescence simultaneously. For the quantitation of fungal DNA according to the standard curve, serial dilutions of C.albicans ATCC 10231 DNA from 3 x 10(5) to 3 x 10(2) ng/μl were used. All of the strains were also identified by conventional methods and sequence analysis in order to compare the results obtained by Rt-PCR. In our study, all patient and standard samples could be amplified, identified and quantitated by this developed Rt-PCR method. A total of 50 strains, of them 26 were C.parapsilosis, 15 were C.glabrata, 6 were C.albicans, and 3 were C.tropicalis have been detected and identified among patient samples. The results were completely concordant with the sequencing and conventional methods, so the sensitivity and specificity of this method were estimated as 100 percent. In conclusion, it was novel Rt-PCR developed and evaluated in this study is considered as a rapid, accurate, reproducible, sensitive and specific method for the detection, identification and quantitation of commonly observed Candida spp. strains.

MALDI-TOF MS for the diagnosis of infectious diseases.

First introduced into clinical microbiology laboratories in Europe, MALDI-TOF MS is being rapidly embraced by laboratories around the globe. Although it has multiple applications, its widespread adoption in clinical microbiology relates to its use as an inexpensive, easy, fast, and accurate method for identification of grown bacteria and fungi based on automated analysis of the mass distribution of bacterial proteins. This review provides a historical perspective on this new technology. Modern applications in the clinical microbiology laboratory are reviewed with a focus on the most recent publications in the field. Identification of aerobic and anaerobic bacteria, mycobacteria, and fungi are discussed, as are applications for testing urine and positive blood culture bottles. The strengths and limitations of MALDI-TOF MS applications in clinical microbiology are also addressed. MALDI-TOF MS is a tool for rapid, accurate, and cost-effective identification of cultured bacteria and fungi in clinical microbiology. The technology is automated, high throughput, and applicable to a broad range of common as well as esoteric bacteria and fungi. MALDI-TOF MS is an incontrovertibly beneficial technology for the clinical microbiology laboratory.

Direct identification of bacteria from positive BD-Bactec blood culture bottles on the Vitek MS

Direct identification of bacteria from positive BD-Bactec blood culture bottles on the Vitek MS

Rapid identification of pathogens in positive blood culture of patients with sepsis: review and meta-analysis of the performance of the sepsityper kit.

Sepsis is one of the leading causes of deaths, and rapid identification (ID) of blood stream infection is mandatory to perform adequate antibiotic therapy. The advent of MALDI-TOF Mass Spectrometry for the rapid ID of pathogens was a major breakthrough in microbiology. Recently, this method was combined with extraction methods for pathogens directly from positive blood cultures. This review summarizes the results obtained so far with the commercial Sepsityper sample preparation kit, which is now approved for in vitro diagnostic use. Summarizing data from 21 reports, the Sepsityper kit allowed a reliable ID on the species level of 80% of 3320 positive blood culture bottles. Gram negative bacteria resulted consistently in higher ID rates (90%) compared to Gram positive bacteria (76%) or yeast (66%). No relevant misidentifications on the genus level were reported at a log(score)cut-off of 1.6. The Sepsityper kit is a simple and reproducible method which extends the MALDI-TOF technology to positive blood culture specimens and shortens the time to result by several hours or even days. In combination with antibiotic stewardship programs, this rapid ID allows a much faster optimization of antibiotic therapy in patients with sepsis compared to conventional workflows.

A new approach to determine the susceptibility of bacteria to antibiotics directly from positive blood culture bottles in two hours

The rapid identification and antibiotic susceptibility test of bacteria causing bloodstream infections are given a very high priority by clinical laboratories. In an effort to reduce the time required for performing antibiotic susceptibility test (AST), we have developed a new method to be applied from positive blood culture bottles. The design of method was performed using blood culture bottles prepared artificially with five strains which have a known susceptibility. An aliquot of the blood culture was subcultured in the presence of specific antibiotics and bacterial counts were monitored using the Sysmex UF-1000i flow cytometer at different times up to 180min. Receiver operating curve (ROC) analysis allowed us to find out the cut-off point for differentiating between sensitive and resistant strains to the tested antibiotic. This procedure was then validated against standard commercial methods on a total of 100 positive blood culture bottles from patients. First, bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) directly from positive blood culture bottles as we have previously reported. Secondly, antibiotic susceptibility test was performed in the same way that was carried out in artificially prepared blood culture bottles. Our results indicate that antibiotic susceptibility test can be determined as early as 120min since a blood culture bottle is flagged as positive. The essential agreement between our susceptibility test and commercial methods (E-test, MicroScan and Vitek) was 99%. In summary, we conclude that reliable results on bacterial identification and antibiotic susceptibility test performed directly from positive blood culture bottles can be obtained within 3h.

Rapid detection of enterobacteriaceae producing extended spectrum beta-lactamases directly from positive blood cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Bacteria that produce extended-spectrum β-lactamases (ESBLs) are an increasing healthcare problem and their rapid detection is a challenge that must be overcome in order to optimize antimicrobial treatment and patient care. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been used to determine resistance to β-lactams, including carbapenems in Enterobacteriaceae, but the methodology has not been fully validated as it remains time-consuming. We aimed to assess whether MALDI-TOF can be used to detect ESBL-producing Enterobacteriaceae from positive blood culture bottles in clinical practice. In the assay, 141 blood cultures were tested, 13 of them were real bacteraemias and 128 corresponded to blood culture bottles seeded with bacterial clinical isolates. Bacteraemias were analysed by MALDI-TOF after a positive growth result and the 128 remaining blood cultures 24 h after the bacterial seeding. β-lactamase activity was determined through the profile of peaks associated with the antibiotics cefotaxime and ceftazidime and its hydrolyzed forms. Clavulanic acid was added to rule out the presence of non-ESBL mechanisms. Overall data show a 99% (103 out of 104) sensitivity in detecting ESBL in positive blood cultures. Data were obtained in 90 min (maximum 150 min). The proposed methodology has a great impact on the early detection of ESBL-producing Enterobacteriaceae from positive blood cultures, being a rapid and efficient method and allowing early administration of an appropriate antibiotic therapy.

Update pneumococcal infections

Pneumococci remain the most important cause for community-acquired pneumonia. The colonization of the nasopharynx is prerequisite for their transmission and the colonization of toddlers are an important reservoir also for infections in the elderly. Since pneumococcal conjugate vaccines reduce the colonization rate of children its widespread use has also resulted in a reduction of invasive pneumococcal disease in adults. Advanced age and comorbidity are the most important risk factors for pneumococcal pneumonia and the mortality in this risk group is still very high. The strongest risk factor for invasive pneumococcal infections, however, is HIV/AIDS and solid or hematologic malignancies. A more rapid identification could aid de-escalation of empiric antibiotic therapy. The pneumococcal urinary antigen test, however lacks sensitivity for this purpose. In contrast, the direct identification of pathogens from positive blood culture bottles by MALDI-TOF is a very encouraging development in diagnostic microbiology. In several cohort studies patients treated with a combination of a betalactam plus macrolide had a lower mortality than patients treated with betalactam monotherapy. If these observations reflect a truly beneficial effect of combination therapy or rather confounding of the studies remains unclear and evidence from interventional studies is needed.

Use of MALDI-TOF MS technique for rapid identification of bacteria from positive blood cultures

We evaluated the feasibility of same-day routine aerobic bacterial identification using the following procedures: Picking colonies from 4 and 6 h incubated subculture from positive blood culture bottle and analyzing them by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The matched identification rate of this procedure at the species level was 80.6% (141/175) for the 4-h cultures compared with overnight cultures and 90.9% (159/175) for the 6-h cultures. Thus, our technique provides an easy and rapid method for identification of aerobic bacteria in routine clinical microbiology laboratories.

Rapid identification of microorganisms from positive blood cultures by MALDI-TOF mass spectrometry subsequent to very short-term incubation on solid medium

Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure.