The impact of protein extraction protocols on the performance of currently available MALDI-TOF mass spectrometry for identification of mycobacterial clinical isolates cultured in liquid media.

BACKGROUND: Protein extraction step is particularly important for identification of mycobacterial isolates by MALDI-TOF mass spectrometry (MS) because of its thick and solid cell wall. This study compared the performance of MALDI-TOF MS for identification of mycobacterial clinical isolates cultured in liquid media between heating-based protocol and non-heating protocol. METHODS: Clinical mycobacterial isolates cultured in liquid media were prospectively analyzed. Reference identification was real-time PCR and restriction fragment length polymorphism. The specimens prepared by heating protocol and non-heating protocol were tested using MALDI Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMérieux, Marcy l'Etoile, France), respectively. RESULTS: Among the 206 clinical specimens prepared by heating method, identification rates were 90.3% and 60.7% in MALDI Biotyper and Vitek MS, respectively. Identification accuracy of MALDI Biotyper and Vitek MS was 100% for the isolates of Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus and Mycobacterium fortuitum. Among the 121 clinical specimens prepared by non-heating method, identification rate for MALDI Biotyper and Vitek MS were 61.2% and 69.4%, respectively. Identification accuracy of MALDI Biotyper/Vitek MS were 92.9%/94.1% for MTBC, 92.9%/100% for M. avium, 90%/100% for M. intracellulare, 100%/100% for M. abscessus and 100%/100% for M. fortuitum. CONCLUSIONS: The performance of MALDI-TOF MS for identification of mycobacterial clinical isolates is affected by protein extraction protocol. For best performance, protein extraction protocol should be chosen considering the MALDI-TOF MS system. In the present study, heating protocol with MALDI Biotyper system showed reliable identification results for mycobacterial clinical isolates.

Evaluation of three phenotypic identification systems for clinical isolates of Raoultella ornithinolytica.

Raoultella spp. have recently been separated from the genus Klebsiella based on their molecular characteristics. It was discovered that Raoultella ornithinolytica can be misidentified as Klebsiella oxytoca by commonly used phenotypic identification systems. Therefore, this study evaluated the ability of three phenotypic systems to identify R. ornithinolytica compared with the genotypic methods sequence-specific primer PCR (SSP-PCR), 16S rRNA gene sequence analysis using the MicroSeq 500 system16S rDNA bacterial identification system or comparison with GenBank sequences using blast. The phenotypic systems examined in this study were the VITEK 2 GN ID card, the MicroScan Neg Combo 32 panel and API 20E. The SSP-PCR panel was able to distinguish the R. ornithinolytica reference strain from other Raoultella spp. and K. oxytoca. Of the 27 isolates identified as R. ornithinolytica by SSP-PCR, VITEK 2 identified all of them as R. ornithinolytica. MicroScan and API identified 25 isolates (92.6%) and 24 isolates (88.9%) as K. oxytoca, respectively. These isolates were ornithine decarboxylase (ODC) negative in all three phenotypic systems. MicroSeq 500 identified 24 isolates (88.9%) as R. ornithinolytica, whereas GenBank identification was heterogeneous. Of the 68 isolates identified as K. oxytoca by SSP-PCR, 66 isolates (97.1%) were identified as K. oxytoca by VITEK 2, MicroScan and API. MicroScan and API require additional biochemical tests to differentiate between ODC-negative R. ornithinolytica and K. oxytoca.

Comparison of the MicroScan, VITEK 2, and Crystal GP with 16S rRNA sequencing and MicroSeq 500 v2.0 analysis for coagulase-negative Staphylococci.

BACKGROUND: Three phenotypic identification systems (MicroScan, VITEK 2, and Crystal GP) were evaluated for their accuracy to identify coagulase-negative staphylococci (CNS). A total of 120 clinical isolates confirmed to be CNS via 16S rRNA sequencing and analysis with the MicroSeq 500 v2.0 database were assessed. RESULTS: The MicroScan, VITEK 2, and Crystal GP systems correctly identified 82.5%, 87.5%, and 67.5% of the isolates, respectively. Misidentification was the main problem in MicroScan (10.8%) and Crystal GP (23.3%) systems, whereas the main problem of VITEK 2 was low-level discrimination (7.5%). CONCLUSION: None of the 3 phenotypic systems tested could accurately and reliably identify CNS at the species level. Further verifications such as biochemical testing or 16S rRNA sequencing together with analysis using a comparable database might be helpful in this regard.

[Validity and reliability of a clinical performance examination using standardized patients].

PURPOSE: The purpose of this study was to test the validity of a modified clinical performance examination (CPX) for preclinical students in nursing. METHOD: 70 nursing students in their second semester of the junior year at C University participated in CPX. Scenarios and checklists were developed by our research team from September to October 2005. Six stations were organized. Evaluation included physical examination of a patient with lung cancer, education on usage of a metered dosage inhaler, and lobectomy postoperative care. Students were randomly assigned to a station. RESULT: There was a difference in the CPX scores according to stations. The agreement of scoring between trained faculty members and SPs was more than moderate (r=.647). The correlation between the CPX score and the average grade in the previous semester and between the CPX score and the average grade of a paper and pen test of the pulmonary system of adults was low (r=.276; r=.048). CONCLUSION: Traditional CPX is generally recommended, however, modified CPX is appropriate for preclinical students in the current Korean Nursing school setting if there are additional scoring systems to balance the testing level at each station.

Evaluation of sodium lactate as a replacement for conventional chemical preservatives in comminuted sausages inoculated with Listeria monocytogenes.

Sodium lactate (SL) as a potential replacer for potassium sorbate (PS) or sodium benzoate (SB) in comminuted sausages was evaluated. Sausages manufactured with 3.3% SL were compared with a control and 0.05 or 0.1% of PS and SB with regard to its influence on changes of chemical composition, physico-chemical and textural properties, and the growth of inoculated Listeria monocytogenes (LM) stored at 4 °C for up to 8 weeks. The sausages contained 62-64% moisture, 15-17% fat and 12-14% protein with pH range of 6.10-6.15 and water activity (a(w)) range of 0.936-0.941. Sausages containing 3.3% SL alone had lower (P<0.05) thiobarbituric acid reacting substances (TBARS) values than the control and those of PS (0.05-0.1%). Lightness values of sausages varied (P<0.05) among preservatives and storage times, while yellowness values tended to increase with storage time. Textural attributes (springiness and hardness) were reduced after 2 and 6 weeks storage, respectively. Sodium lactate at an incorporation level of 3.3% to sausage formulation had an antilisterial effect similar to those of 0.05-1.0% of PS or SB and delayed the lag phase for the growth of Listeria monocytogenes at least 2 weeks, compared with the control.