Blood Culture Broth Research Articles

An Improved In-house MALDI-TOF MS Protocol for Direct Cost-Effective Identification of Pathogens from Blood Cultures.

Background: Bloodstream infection is a major cause of morbidity and mortality in hospitalized patients worldwide. Delays in the identification of microorganisms often leads to a poor prognosis. The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) directly to blood culture (BC) broth can potentially identify bloodstream infections earlier, and facilitate timely management.Methods: We developed an “in-house” (IH) protocol for direct MALDI-TOF MS based identification of organisms in positive BCs. The IH protocol was initially evaluated and improved with spiked BC samples, and its performance was compared with the commercial Sepsityper™ kit using both traditional and modified cut-off values. We then studied in parallel the performance of the IH protocol and the colony MS identifications in positive clinical BC samples using only modified cut-off values. All discrepancies were investigated by “gold standard” of gene sequencing.Results: In 54 spiked BC samples, the IH method showed comparable results with Sepsityper™ after applying modified cut-off values. Specifically, accurate species and genus level identification was achieved in 88.7 and 3.9% of all the clinical monomicrobial BCs (284/301, 94.4%), respectively. The IH protocol exhibited superior performance for Gram negative bacteria than for Gram positive bacteria (92.8 vs. 82.4%). For anaerobes and yeasts, accurate species identification was achieved in 80.0 and 90.0% of the cases, respectively. For polymicrobial cultures (17/301, 5.6%), MALDI-TOF MS correctly identified a single species present in all the polymicrobial BCs under the Standard mode, while using the MIXED method, two species were correctly identified in 52.9% of the samples. Comparisons based on BC bottle type, showed that the BACTEC™ Lytic/10 Anaerobic/F culture vials performed the best.Conclusion: Our study provides a novel and effective sample preparation method for MALDI-TOF MS direct identification of pathogens from positive BC vials, with a lower cost ($1.5 vs. $ 7) albeit a slightly more laborious extracting process (an extra 15 min) compared with Sepsityper™ kit.

Applications of copolymer for rapid identification of bacteria in blood culture broths using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify pathogens in blood culture samples. However, sample pretreatment is needed for direct identification of microbes in blood culture bottles. Conventional protocols are complex and time-consuming. Therefore, in this study, we developed a method for collecting bacteria using polyallylamine–polystyrene copolymer for application in wastewater treatment technology. Using representative bacterial species Escherichia coli and Staphylococcus capitis, we found that polyallylamine–polystyrene can form visible aggregates with bacteria, which can be identified using MALDI-TOF MS. The processing time of our protocol was as short as 15min. Hemoglobin interference in MALDI spectra analysis was significantly decreased in our method compared with the conventional method. In a preliminary experiment, we evaluated the use of our protocol to identify clinical isolates from blood culture bottles. MALDI-TOF MS-based identification of 17 strains from five bacterial species (E. coli, Klebsiella pneumoniae, Enterococcus faecalis, S. aureus, and S. capitis) collected by our protocol was satisfactory. Prospective large-scale studies are needed to further evaluate the clinical application of this novel and simple method of collecting bacteria in blood culture bottles.

Usefulness of matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify pathogens, including polymicrobial samples, directly from blood culture broths.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (TOF-MS) is now widely used to detect pathogens in clinical settings in Japan. Here, we report the ability of TOF-MS to detect bacteria from blood culture (BC) broths, and compare the efficacy of TOF-MS to that of conventional culture methods. Bacteria were correctly detected from 63 monomicrobial samples within 80 minutes; results matched those obtained by conventional BC methods, although the conventional methods took 2–3 days. In addition to the 63 monomicrobial samples, another three polymicrobial samples were tested; notably, the infecting bacteria were not correctly identified in two of these three samples. To better assess the TOF-MS detection of polymicrobial samples, we tested various ratios of mixed broth samples, including combinations of the bacteria that we were unable to detect in clinical samples. Combinations of Enterobacter cloacae and Pseudomonas aeruginosa were correctly detected at a culture ratio of 2:1, but not in the 3:1 mixture. These results suggested that TOF-MS is a strong tool for the rapid and correct detection of pathogens from monomicrobial BC samples, though results need to be carefully checked when handling known or suspected polymicrobial samples.

Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI- TOF MS) for Early Identification of Septic Patients.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful technique for the rapid identification of bacteria from growing colonies in routine cultures. In this study, we evaluated the feasibility of a 5-hour incubation on solid medium after sub-cultivation of positive blood culture broth without any preparation steps in order to speed up the identification of bacteria. In addition to standard laboratory protocols, a Columbia agar plate with 5% sheep blood was inoculated with 1 drop from the blood culture broth. After a 5-hour incubation period, a colony from the culture plate was submitted to MALDI-TOF MS. A total of 1351 positive blood cultures (1299 monomicrobial and 51 polymicrobial) were analyzed. When compared to routine identification procedure results for positive blood cultures, 79.3% of isolates were correctly identified to the species level. When manufacturer-recommended score values were taken into account, MALDITOF MS correctly identified 98.4% of the isolates to the species level with a score of > 2.0, 89.1% with a score between 1.7 and 2.0, and 75.4% with a score of < 1.7. ln our evaluation, a large majority of the S. aureus (91.5%) and Enterobacteriaceae (87.6%) were correctly identified at the species level. A 5-hour incubation period was found to be associated with moderate identification results for CoNS, Enterococcus spp., and nonfermentative gram negative bacilli, with failure being mostly observed with Streptococcus spp., Candida spp., and other gram positive bacteria. We believe that the performance of MALDI-TOF MS identification after short-term culture is directly related to the sufficient growth of microorganisms at 5 hours.

MALDI-TOF MS in Adult Inpatients with Bloodstream Infections: Pre- and Post-intervention Study

BackgroundDelays in diagnosis of bloodstream infections (BSI) can lead to adverse outcomes. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can rapidly identify bacteria directly from blood culture bottles. We describe our experience in patients with BSI before and after implementation of MALDI-TOF MS.MethodsPatients: adult inpatients with BSI. Design: pre-intervention group (August-November 2015); bacterial identification and susceptibility testing performed by Vitek®2. Post-intervention group (August–November 2016); bacterial identification on liquid blood culture broth by MALDI-TOF MS; susceptibility testing performed by Vitek®2. Both groups received baseline antimicrobial stewardship program (ASP) intervention. Outcomes: times to bacterial identification, susceptibilities, effective and optimal antibiotics, length of stay (LOS), 30-day readmission and 30-day all-cause mortality. Statistical analysis: univariable analysis performed; continuous variables analyzed using a two-tailed t-test; discrete variables analyzed using a chi-square test or Fisher exact test.Results267 cases of BSI occurring in 256 patients were analyzed (137 pre-intervention, 130 post-intervention). Time to bacterial identification was significantly shorter in the MALDI-TOF MS group (40 vs. 63 hours, P < 0.001). Times to susceptibilities, effective, and optimal antibiotic therapy did not differ between the two groups. There was no significant difference in LOS or 30-day readmission rates. 30-day mortality was significantly higher in the pre-intervention group (25 vs. 13 percent, P = 0.026). The pre-intervention group had significantly more BSI due to multidrug-resistant (MDR) Gram-negative bacteria and vancomycin-resistant enterococci (VRE).ConclusionMALDI-TOF MS significantly shortened time to bacterial identification in patients with BSI. Differences in times to effective and optimal antibiotic therapy were not observed. This may be due to high rates of early appropriate empiric antibiotic use at our institution and limited real-time MALDI-TOF MS and ASP interventions. Higher mortality in the pre-intervention group may be due to higher prevalence of multidrug-resistant bacteria.Disclosures All authors: No reported disclosures.

Utility of direct susceptibility testing on blood cultures: is it still worthwhile?

Earlier targeted therapy for bacteraemia optimizes patient outcomes and reduces broad spectrum antibiotic use. Standardized susceptibility testing results are available at 36-48 h. Direct disc susceptibility testing from blood culture broth reduces time to results but the inoculum is not standardized. No studies have looked at the clinical utility of direct susceptibility results. This retrospective cohort study aimed to assess the correlation between direct and formal testing methods as well as the clinical utility of direct susceptibility results. 160 episodes of bacteraemia with paired direct and formal susceptibility testing were studied. Direct disc testing was performed on blood culture broth. Formal testing was performed on isolates, using automated broth microdilution or Etests. The rate of error was 9.0 % (95 % CI 7.0-11.6 %). In 10 cases (6.3 %, 95 % CI 3.0-11.2 %), inappropriate antibiotics were used due to direct susceptibility results, including two cases with ineffective (as opposed to too broad) antibiotics being used. Antibiotics were changed in 28.1 % of cases once direct susceptibility data was available. There was a decreased time to effective antibiotics in 9.3 % (95 % CI 5.3-15.0 %), and a decreased time to a targeted antibiotics in 14.3 % (95 % CI 9.3-20.8 %) of cases. Despite the error rate, the advantages of earlier times to effective and targeted antibiotics justifies continuing direct testing in bacteraemia episodes with Gram-negative rods. In the Gram-positive group, given the contamination rate, the availability of adjunctive PCR, and the fact that early identification of the isolate could equally influence antibiotic choices, direct susceptibility testing may no longer be warranted.

Recent Progress in the Diagnosis of Pathogenic Candida Species in Blood Culture.

Candidemia has become an emerging invasive fungal disease. Prompt treatment with appropriate antifungal agent is crucial to reduce the mortality of candidemia. The conventional blood culture method, which is considered the gold standard for candidemia diagnosis, has a low sensitivity and is time-consuming to perform. Recently, several novel advanced diagnostic methods that have a higher sensitivity and a shorter turnaround time than the conventional blood culture method have been developed for the early detection of Candida in blood samples or in blood culture broth. Most of these newer methods were developed using various molecular techniques, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, peptide nucleic acid fluorescence in situ hybridization, and a number of DNA-based techniques including in-house and commercial polymerase chain reactions. In this article, we review and summarize the novel molecular methods that have been recently used for the detection and identification of Candida organisms in blood specimens.

Evaluation of direct sensitivity testing as a method for early initiation of treatment in Gram-negative sepsis

Purpose: For antibiotic susceptibility results, conventional culture and sensitivity methods take 48 h after a blood culture are flagged positive by automated systems. Early initiation of targeted antibiotic therapy is essential for effective management of sepsis to reduce morbidity, mortality, cost of treatment and prevent antibiotic resistance. The objective of this study was to evaluate direct sensitivity test (DST) as a potential tool to get reliable antibiotic susceptibility results 24 h earlier. Materials and Methods: Blood cultures that flagged positive from 1st January 2015 to 30th June 2015 by BacT/ALERT were stained by Gram stain. All blood cultures with only one kind of Gram-negative bacteria by Gram stain were simultaneously cultured and sensitivity tests put up directly (DST) from the broth using disk-diffusion method according to the British Society of Antimicrobial Chemotherapy guidelines. DST results available next day were compared with conventional antibiotic susceptibility test (AST). Results of DST (test method) and AST (reference method) were compared for agreements or errors. Results: Of the fifty Gram-negative isolates tested, we observed 91.5% categorical agreement or no error (κ = 0.407, P < 0.001). Conclusions: DST using disk diffusion from positive blood culture broths helps initiate early targeted antibiotic therapy. There is a high concordance between DST and AST.

Enhanced Diagnosis of Pneumococcal Bacteremia Using Antigen- and Molecular-Based Tools on Blood Specimens in Mali and Thailand: A Prospective Surveillance Study.

Prior antibiotic use, contamination, limited blood volume, and processing delays reduce yield of blood cultures for detection of Streptococcus pneumoniae. We performed immunochromatographic testing (ICT) on broth from incubated blood culture bottles and real-time lytA polymerase chain reaction (PCR) on broth and whole blood and compared findings to blood culture in patients with suspected bacteremia. We selected 383 patients in Mali and 586 patients in Thailand based on their blood culture results: 75 and 31 were positive for pneumococcus, 100 and 162 were positive for other pathogens, and 208 and 403 were blood culture negative, respectively. ICT and PCR of blood culture broth were at least 87% sensitive and 97% specific compared with blood culture; whole blood PCR was 75–88% sensitive and 96–100% specific. Pneumococcal yields in children < 5 years of age increased from 2.9% to 10.7% in Mali with > 99% of additional cases detected by whole blood PCR, and from 0.07% to 5.1% in Thailand with two-thirds of additional cases identified by ICT. Compared with blood culture, ICT and lytA PCR on cultured broth were highly sensitive and specific but their ability to improve pneumococcal identification varied by site. Further studies of these tools are needed before widespread implementation.

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.

Preliminary Evaluation of the Research-Use-Only (RUO) iCubate iC-GPC Assay for Identification of Select Gram-Positive Bacteria and Their Resistance Determinants in Blood Culture Broths

The iC-GPC assay (iCubate, Huntsville, AL) provides a molecular option for the rapid, on-demand analysis of positive blood cultures. A preliminary evaluation of the iC-GPC assay using 203 clinical or seeded specimens demonstrated a sensitivity of 93.8% to 100% and a specificity of 98.0% to 100% for the identification of five Gram-positive bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) and three associated genetic resistance determinants (mecA, vanA, and vanB) in positive blood culture broths.

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.)

A method for early detection of antibiotic resistance in positive blood cultures: Experience from an oncology centre in eastern India

Purpose: For antibiotic susceptibility results, conventional culture and sensitivity methods takes 48 hours after a blood culture is flagged positive by automated systems. Early initiation of targeted antibiotic therapy is essential for effective management of sepsis to reduce morbidity, mortality, cost of treatment and prevent antibiotic resistance. Objective of this study was to evaluate Direct Sensitivity Test (DST) as a potential tool to get reliable antibiotic susceptibility results 24 hours earlier. Materials and Methods: Blood cultures flagged positive between May 2011 to December 2012 by BacT/ALERT were Gram stained. All uni-microbial gram-negative blood cultures were simultaneously cultured and processed for DST from broth using disk diffusion method using British Society of Antimicrobial Chemotherapy (BSAC) guidelines. DST results available next day were compared with conventional antibiotic susceptibility test (AST) performed by Vitek-2 on isolated colonies. Results of DST (test method) and AST (reference method) were compared for agreements or errors. Results: Of the 840 antibiotic gram-negative organism combinations tested, Categorical and essential agreements were 83.7% and 96.2% respectively. Minor, major and very major errors were 12.5%, 3.33% and 0.47%, respectively. Conclusions: DST using disk diffusion from positive blood culture broths helps to initiate early targeted antibiotic therapy. There is high concordance between DST and AST.

Direct identification of Gram-positive bacteria and resistance determinants from blood cultures using a microarray-based nucleic acid assay: in-depth analysis of microarray data for undetermined results.

The Verigene Gram-Positive Blood Culture (BC-GP) nucleic acid assay (Nanosphere, Inc., Northbrook, IL, USA) is a newly developed microarray-based test with which 12 Gram-positive bacterial genes and three resistance determinants can be detected using blood culture broths. We evaluated the performance of this assay and investigated the signal characteristics of the microarray images. At the evaluation stage, we tested 80 blood cultures that were positive for various bacteria (68 bacteria covered and 12 not covered by the BC-GP panel) collected from the blood of 36 patients and 44 spiked samples. In instances where the automated system failed and errors were called, we manually inspected microarray images, measured the signal intensities of target spots, and reclassified the results. With the manual analysis of the microarray images of 14 samples for which error calls were reported, we could obtain correct identification results for 12 samples without the need for retesting, because strong signals in the target spots were clearly discriminable from background noise. With our interpretation strategy, we could obtain 97.1% sensitivity and 100% specificity for bacterial identification by using the BC-GP assay. The two unidentified bacteria were viridans group streptococci, which produced weaker target signals. During the application stage, among 25 consecutive samples positive for Gram-positive bacteria, we identified two specimens with error calls as Streptococcus spp. by using manual analysis. With help of the manual review of the microarray images, the BC-GP assay could successfully identify species and resistance markers for many clinically important Gram-positive bacteria.

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.

Performance of Two Resin-Containing Blood Culture Media in Detection of Bloodstream Infections and in Direct Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) Broth Assays for Isolate Identification: Clinical Comparison of the BacT/Alert Plus and Bactec Plus Systems

We compared the clinical performances of the BacT/Alert Plus (bioMérieux) and Bactec Plus (Becton Dickinson) aerobic and anaerobic blood culture (BC) media with adsorbent polymeric beads. Patients ≥ 16 years old with suspected bloodstream infections (BSIs) were enrolled in intensive care units and infectious disease wards. A single 40-ml blood sample was collected from each and used to inoculate (10 ml/bottle) one set of BacT/Alert Plus cultures and one set of Bactec Plus cultures, each set consisting of one aerobic and one anaerobic bottle. Cultures were incubated ≤ 5 days in the BacT/Alert 3D and Bactec FX instruments, respectively. A total of 128 unique BSI episodes were identified based on the recovery of clinically significant growth in 212 aerobic cultures (106 BacT/Alert and 106 Bactec) and 151 anaerobic cultures (82 BacT/Alert and 69 Bactec). The BacT/Alert aerobic medium had higher recovery rates for Gram-positive cocci (P = 0.024), whereas the Bactec aerobic medium was superior for recovery of Gram-negative bacilli (P = 0.006). BacT/Alert anaerobic medium recovery rates exceeded those of the Bactec anaerobic medium for total organisms (P = 0.003), Gram-positive cocci (P = 0.013), and Escherichia coli (P = 0.030). In terms of capacity for diagnosing the 128 septic episodes, the BacT/Alert and Bactec sets were comparable, although the former sets diagnosed more BSIs caused by Gram-positive cocci (P = 0.008). They also allowed earlier identification of coagulase-negative staphylococcal growth (mean, 2.8 h; P = 0.003) and growth in samples from patients not on antimicrobial therapy that yielded positive results (mean, 1.3 h; P < 0.001). Similarly high percentages of microorganisms in BacT/Alert and Bactec cultures (93.8% and 93.3%, respectively) were identified by direct matrix-assisted laser desorption ionization-time of flight mass spectrometry assay of BC broths. The BacT/Alert Plus media line appears to be a reliable, timesaving tool for routine detection of BSIs in the population we studied, although further studies are needed to evaluate their performance in other settings.

Rapid identification of gram negative bacteria from blood culture broth using MALDI-TOF mass spectrometry.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

In-house Loop Amplification (LAMP) method for the diagnosis of Salmonella Paratyphi A in low-resource settings

IntroductionSalmonella Paratyphi A (S. Paratyphi A) causes paratyphoid fever, a disease endemic in developing countries. It is transmitted via the faecal-oral route, mainly through contaminated food and water. The prevalence or incidence of the disease is increasing especially in China, India and Asia. Current diagnosis of S. Paratyphi A is via culture and molecular methods such as PCR. However, these methods do not offer a rapid, simple, and cost-effective detection of S. Paratyphi A especially for use in resource-limited settings. ObjectiveThe study was aimed at developing an in-house LAMP method as a rapid, sensitive, specific and cost-effective detection of S. Paratyphi A. MethodsAn In-house LAMP method was developed and optimized for the detection of S. Paratyphi A using primers that were designed based on intergenic region of SSPA1723a and SSPA1723 gene of S. Paratyphi A. The primers' specificity was tested on 60 bacteria strains consisting of 25 S. Paratyphi A, 20 other Salmonella serovars, and 15 non-Salmonella bacteria which were obtained from Institute for Research in Molecular Medicine (INFORMM) culture bank. Detection limit of LAMP was determined using 10-fold serial dilutions of S. Paratyphi A strain DNA and compared with culture and PCR results. The assay was further evaluated on 60 BACTEC blood culture broths suspected of S. Paratyphi A. The results were compared with those obtained by PCR assay and culture method. Results & DiscussionIn-house LAMP method was successfully established and optimized. The sensitivity of the assay was determined at 200 CFU. This LAMP assay gave positive result to all the 25 S. Paratyphi A isolates and negative to 20 other Salmonella and 15 non-Salmonella bacteria. This assay detected 8 samples as positive and 52 samples as negative for S. Paratyphi A. These results were similar to those results obtained by PCR assay and culture methods. ConclusionAn in-house LAMP method was established in this study and could potentially be used as a rapid, sensitive, specific and cost-effective detection method for S. Paratyphi A especially in low-resource settings. However this LAMP method is only recommended for screening purposes and need to be further confirmed with gold standard method which is culture method and PCR.

Same Day Identification and Full Panel Antimicrobial Susceptibility Testing of Bacteria from Positive Blood Culture Bottles Made Possible by a Combined Lysis-Filtration Method with MALDI-TOF VITEK Mass Spectrometry and the VITEK2 System

Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001). Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship.