MALDI-TOF identification of Gram-negative bacteria directly from blood culture bottles containing charcoal: Sepsityper® kits versus centrifugation-filtration method.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has dramatically altered the way microbiology laboratories identify clinical isolates. Direct blood culture (BC) detection may be hampered, however, by the presence of charcoal in BC bottles currently in clinical use. This study evaluates an in-house process for extraction and MALDI-TOF identification of Gram-negative bacteria directly from BC bottles containing charcoal. Three hundred BC aliquots were extracted by a centrifugation-filtration method developed in our research laboratory with the first 96 samples processed in parallel using Sepsityper® kits. Controls were colonies from solid media with standard phenotypic and MALDI-TOF identification. The identification of Gram-negative bacteria was successful more often via the in-house method compared to Sepsityper® kits (94.7% versus 78.1%, P≤0.0001). Our in-house centrifugation-filtration method was further validated for isolation and identification of Gram-negative bacteria (95%; n=300) directly from BC bottles containing charcoal.

Riederer K ，Cruz K ，Shemes S ，Szpunar S ，Fishbain JT ... -  《-》

An improved in-house lysis-filtration protocol for bacterial identification from positive blood culture bottles with high identification rates by MALDI-TOF MS.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is now a well-established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of 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 developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-μm membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min. Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores >1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test.

Tsuchida S ，Murata S ，Miyabe A ，Satoh M ，Takiwaki M ，Matsushita K ，Nomura F ... -  《-》

An in-house centrifugation and membrane filtration technique for identifying microorganisms from positive blood culture bottles with high identification rates using matrix-assisted laser desorption ionization-Time-of-flight mass spectrometry: A preliminar

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most promising technologies for the identification of microbial pathogens directly from positive blood culture bottles. As blood culture bottle medium contains various nonbacterial proteins, including those derived from blood cells, pretreatment to effectively remove host cells is key for successful proteome-based identification of microorganisms. Although the Sepsityper® kit is the most widely used pretreatment protocol, its performance is not satisfactory, particularly for gram-positive isolates. We developed a new in-house protocol, the centrifugation and membrane filtration technique (CMFT), in which vacuum-filtration is coupled with differential centrifugation. We prospectively evaluated the performance of this novel method compared with that of the Sepsityper®. For gram-negative bacterial isolates, the species-level identification rates obtained with the CMFT and the Sepsityper® were comparable (98.8% vs 92.9%). By contrast, for gram-positive isolates, the performance of the CMFT was significantly better than that of the Sepsityper® (P < 0.05). Using our new protocol, 81 (95.3%) isolates were identified with a score >2.0, and 85 (100%) isolates were identified with a score >1.7, versus 46 (54.1%) and 69 (81.2%), respectively, for the Sepsityper®. These results are preliminary, but considering that this novel protocol provides notably high species-level identification rates for gram-positive isolates, it deserves assessment in a larger-scale study with a variety of platforms for MS-based identification of microorganisms.

Tsuchida S ，Murata S ，Miyabe A ，Satoh M ，Takiwaki M ，Matsushita K ，Nomura F ... -  《-》

Bacterial rapid identification with matrix assisted laser desorption/ionization time-of-flight mass spectrometry: development of an 'in-house method' and comparison with Bruker Sepsityper(®) kit.

Frédéric Ric S ，Antoine M ，Bodson A ，Lissoir B ... -  《ACTA CLINICA BELGICA》

Parallel Evaluation of the MALDI Sepsityper and Verigene BC-GN Assays for Rapid Identification of Gram-Negative Bacilli from Positive Blood Cultures.

Rapid identification of microorganisms from positive blood cultures has improved clinical management and antimicrobial stewardship. The advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has reduced the time to identification of cultured isolates and is now often the definitive method used in the clinical microbiology laboratory. The commercial in vitro diagnostic MALDI Sepsityper (Sepsityper) kit has the potential for standardization and clinical routine use for the rapid identification of a broad range of bacteria from positive blood cultures. In this study, we performed a parallel evaluation of the Sepsityper (Bruker Daltonics, Billerica, MA) and the Verigene BC-GN (BC-GN) assays (Nanosphere, Inc., Northfield, IL) for the identification of Gram-negative bacilli. A total of 210 Bactec bottles demonstrating Gram-negative bacilli were prospectively enrolled for this study. Among these, 200 monomicrobial cultures were included in the comparative analysis. For monomicrobial cultures, the BC-GN detected 85% (170/200) compared to that detected by routine culture while the Sepsityper detected 94% (188/200) and 91% (181/200) to the genus and species levels, respectively. Comparable positive percentage agreement and negative percentage agreement were observed between the Sepsityper (96.5% and 98.8%, respectively) and the BC-GN (99.4% and 99.8%, respectively) when only (n = 170, 85%) organisms targeted by the latter test were included in the analysis. In conclusion, the two methods evaluated in this study showed excellent performance characteristics for the identification of Gram-negative bacilli commonly isolated from blood cultures. The Sepsityper showed a broader identification range capability that may further improve clinical management and antimicrobial stewardship in patients with less frequent Gram-negative bacilli bloodstream infections.

Arroyo MA ，Denys GA 《-》