Reprint of “Assessment of marker proteins identified in whole cell extracts for bacterial speciation using liquid chromatography electrospray ionization tandem mass spectrometry”

Staphylococcal strains (CoNS) were speciated in this study. Digests of proteins released from whole cells were converted to tryptic peptides for analysis. Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS, Orbitrap) was employed for peptide analysis. Data analysis was performed employing the open-source software X!Tandem which uses sequenced genomes to generate a virtual peptide database for comparison to experimental data. The search database was modified to include the genomes of the 11 Staphylococcus species most commonly isolated from man. The number of total peptides matching each protein along with the number of peptides specifically matching to the homologue (or homologues) for strains of the same species were assessed. Any peptides not matching to the species examined were considered conflict peptides. The proteins typically identified with the largest percentage of sequence coverage, number of matched peptides and number of peptides corresponding to only the correct species were elongation factor Tu (EF Tu) and enolase (Enol). Additional proteins with consistently observed peptides as well as peptides matching only homologues from the same species were citrate synthase (CS) and 1-pyrroline-5-carboxylate dehydrogenase (1P5CD).Protein markers, previously identified from gel slices, (aconitate hydratase and oxoglutarate dehydrogenase) were found to provide low confidence scores when employing whole cell digests. The methodological approach described here provides a simple yet elegant way of identification of staphylococci. However, perhaps more importantly the technology should be applicable universally for identification of any bacterial species.     

Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus

Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria.     

Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory

This study evaluates matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) capability for the identification of difficult-to-identify microorganisms. A total of 150 bacterial isolates inconclusively identified with conventional phenotypic tests were further assessed by 16S rRNA sequencing and by MALDI-TOF MS following 2 methods: a) a simplified formic acid-based, on-plate extraction and b) performing a tube-based extraction step. Using the simplified method, 29 isolates could not be identified. For the remaining 121 isolates (80.7%), we obtained a reliable identification by MALDI-TOF: in 103 isolates, the identification by 16S rRNA sequencing and MALDI TOF coincided at the species level (68.7% from the total 150 analyzed isolates and 85.1% from the samples with MALDI-TOF result), and in 18 isolates, the identification by both methods coincided at the genus level (12% from the total and 14.9% from the samples with MALDI-TOF results). No discordant results were observed. The performance of the tube-based extraction step allowed the identification at the species level of 6 of the 29 unidentified isolates by the simplified method. In summary, MALDI-TOF can be used for the rapid identification of many bacterial isolates inconclusively identified by conventional methods.     

Evaluation of MALDI-TOF mass spectrometry and Sepsityper Kit? for the direct identification of organisms from sterile body fluids in a Canadian pediatric hospital

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit – the Sepsityper Kit (Bruker Daltonik, Germany) – and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8%) and 39 of 50 (78%) blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%.MALDI-TOF MS is a promising tool for the direct identification of organisms cultured from sterile sites.     

Sensor-based surgical activity recognition in unconstrained environments

Abstract

Introduction : Automatic surgical activity recognition in the operating room (OR) is mandatory to enable assistive surgical systems to manage the information presented to the surgical team. Therefore the purpose of our study was to develop and evaluate an activity recognition model. Material and methods : The system was conceived as a hierarchical recognition model which separated the recognition task into activity aspects. The concept used radio frequency identification (RFID) for instrument recognition and accelerometers to infer the performed surgical action. Activity recognition was done by combining intermediate results of the aspect recognition. A basic scheme of signal feature generation, clustering and sequence learning was replicated in all recognition subsystems. Hidden Markov models (HMM) were used to generate probability distributions over aspects and activities. Simulated functional endoscopic sinus surgeries (FESS) were used to evaluate the system. Results and discussion: The system was able to detect surgical activities with an accuracy of 95%. Instrument recognition performed best with 99% accuracy. Action recognition showed lower accuracies with 81% due to the high variability of surgical motions. All stages of the recognition scheme were evaluated. The model allows distinguishing several surgical activities in an unconstrained surgical environment. Future improvements could push activity recognition even further.     

心脏手术相关医疗纠纷的法医学分析

目的分析心脏手术相关医疗纠纷的临床及法医学特点,并就发生原因进行剖析及提出相应防范措施。方法对2002年1月-2011年12月四川华西法医学鉴定中心受理的四川省各级医疗机构发生的17例与心脏手术相关的医疗纠纷法医学鉴定资料进行回顾性分析。结果17例心脏手术相关医疗纠纷中,12例进行了尸体解剖死因鉴定,死亡原因有心脏传导系统出血,术后感染,低心排量综合症、肺动脉高压、失血性休克致死等。其余5例加上尸体解剖2例在内共7例进行了医疗过错鉴定,存在的医疗过错包括术前检查不完善,告知不充分,手术操作不细致,术后观察、处理不足,医疗记录不完整等。结论心脏手术相关医疗纠纷与术后并发症关系密切,医护人员应重视对心脏术后并发症的防治。尸体解剖对解决心脏术后死亡引起的医疗纠纷具有重要意义。     

RNA恒温扩增实时检测技术快速鉴定鸟分枝杆菌

目的建立RNA恒温扩增技术快速鉴定鸟分枝杆菌(RIARD-MA)的方法,评估鉴定分枝杆菌临床分离株的应用效果。方法以鸟分枝杆菌(MA)的16S rRNA的特异序列为检测靶标,设计RNA探针和带有T7启动子的逆转录扩增引物,42℃恒温扩增实时检测,对21种分枝杆菌标准株、4株非分枝杆菌和259株分枝杆菌临床分离株进行鉴别检测;同时以PCR测序结果为参考对照。结果 RIARD-MA可以在2 h内完成检测,能准确鉴定出21种分枝杆菌标准株及4株非分枝杆菌检测中的MA,检测灵敏度达103CFU/mL;检测分枝杆菌临床分离株中MA的敏感性、特异性均可达100%。结论 RIARD-MA鉴定MA具有较高的敏感性、特异性,检测快速,有望作为一种新的MA临床分离株鉴定方法。     

肝癌早期鉴定标志物研究进展

肝癌全球发病率不断增高,多数病人确诊时已不能进行有效的治疗。因此寻找能够早期鉴定肝癌的标志物成为降低肝癌致死率的重要举措之一。肝硬化病人传统的定期超声波检测以及血清AFP含量检测灵敏度和准确率有限。目前科学家正致力于寻找一系列高效的生物标志物检测早期肝癌,取得了一定的成效,为以后肝癌的早期鉴定提供了一种科学的方法。     

医院内假丝酵母菌感染的菌种构成和耐药性分析

目的 分析医院内感染假丝酵母菌菌种构成和耐药性。 方法 对临床标本分离的酵母样真菌,用VITEK-Ⅱ(生物梅里埃公司)生化鉴定仪、API20c生化鉴定试纸条和念珠菌显色琼脂以及聚合酶链反应(polymerase chain reaction, PCR)方法进行酵母样真菌菌种的鉴定,应用ATB Fungus3 进行药敏试验。 结果 96株酵母样真菌,可分为6个种,包括白色念珠菌40株 (41.7%)、热带念珠菌36株 (37.5%)、光滑念珠菌13株 (13.54%)、近平滑念珠菌5株 (5.21%)、克柔念珠菌1株 (1.04%)、挪威念珠菌 1株(1.04%)。各种假丝酵母菌对5种抗真菌药呈现不同的敏感性,对两性霉素B和5-氟胞嘧啶的敏感率为100%,而对氟康唑、伊曲康唑、伏立康唑则表现出一定的耐药性。 结论 基因间隔转录区分子生物学分析结合传统培养和生化方法,可有效提高假丝酵母菌鉴定的准确性。本研究结果提示医院内非白假丝酵母菌感染有增多趋势。重症监护病房(intensive care unit, ICU)是重要的假丝酵母菌来源科室。60岁以上的老龄患者是医院内真菌感染的高危人群。体外药敏试验提示部分假丝酵母菌出现了唑类药物(氟康唑和伊曲康唑)的耐药性。