Three-dimensional visualization of cerebral blood vessels and neural changes in thick ischemic rat brain slices using tissue clearing.

Blood vessels are three-dimensional (3D) in structure and precisely connected. Conventional histological methods are unsuitable for their analysis because of the destruction of functionally important topological 3D vascular structures. Tissue optical clearing techniques enable extensive volume imaging and data analysis without destroying tissue. This study therefore applied a tissue clearing technique to acquire high-resolution 3D images of rat brain vasculature using light-sheet and confocal microscopies. Rats underwent middle cerebral artery occlusion for 45 min followed by 24 h reperfusion with lectin injected directly into the heart for vascular staining. For acquiring 3D images of rat brain vasculature, 3-mm-thick brain slices were reconstructed using tissue clearing and light-sheet microscopy. Subsequently, after 3D rendering, the fitting of blood vessels to a filament model was used for analysis. The results revealed a significant reduction in vessel diameter and density in the ischemic region compared to those in contralesional non-ischemic regions. Immunostaining of 0.5-mm-thick brain slices revealed considerable neuronal loss and increased astrocyte fluorescence intensity in the ipsilateral region. Thus, these methods can provide more accurate data by broadening the scope of the analyzed regions of interest for examining the 3D cerebrovascular system and neuronal changes occurring in various brain disorders.

Same-Day Identification and Antimicrobial Susceptibility Testing of Bacteria in Positive Blood Culture Broths Using Short-Term Incubation on Solid Medium with the MicroFlex LT, Vitek-MS, and Vitek2 Systems.

BACKGROUND: Early and appropriate antibiotic treatment improves the clinical outcome of patients with septicemia; therefore, reducing the turn-around time for identification (ID) and antimicrobial susceptibility test (AST) results is essential. We established a method for rapid ID and AST using short-term incubation of positive blood culture broth samples on solid media, and evaluated its performance relative to that of the conventional method using two rapid ID systems and a rapid AST method. METHODS: A total of 254 mono-microbial samples were included. Positive blood culture samples were incubated on blood agar plates for six hours and identified by the MicroFlex LT (Bruker Daltonics) and Vitek-MS (bioMeriéux) systems, followed by AST using the Vitek2 System (bioMeriéux). RESULTS: The correct species-level ID rates were 82.3% (209/254) and 78.3% (199/254) for the MicroFlex LT and Vitek-MS platforms, respectively. For the 1,174 microorganism/antimicrobial agent combinations tested, the rapid AST method showed total concordance of 97.8% (1,148/1,174) with the conventional method, with a very major error rate of 0.5%, major error rate of 0.7%, and minor error rate of 1.0%. CONCLUSIONS: Routine implementation of this short-term incubation method could provide ID results on the day of blood culture-positivity detection and one day earlier than the conventional AST method. This simple method will be very useful for rapid ID and AST of bacteria from positive blood culture bottles in routine clinical practice.

Implementation of biological variation-based analytical performance specifications in the laboratory: Stringent evaluation of Improvacutor blood collection tubes.

Recently, because the quality of laboratory analyses has increased along with the need for quality improvement, several external quality control bodies have adapted performance specifications using the Desirable Biological Variation Database, termed "Ricos goals"; these criteria are more stringent than those presented in CLIA 88. In this study, we aimed to validate newly introduced serum separator tubes, Improvacutor, for routine clinical chemistry testing in accordance with Ricos goals and CLIA 88. Blood samples were collected from 100 volunteers into three types of serum vacuum tubes: Greiner Vacuette, Becton Dickinson (BD) Vacutainer, and Improve Improvacutor. The samples were subjected to 16 routine chemistry tests using a TBA-200fr NEO chemistry autoanalyzer. In the comparison analysis, all 16 test results were acceptable according to CLIA 88. However, in the comparison of Improve and BD tubes, creatinine showed 4.31% (+0.08 µmol/L) bias. This slightly exceeded the Desirable Specification for Inaccuracy Ricos limit of ±3.96%, but still satisfied the CLIS88 limit of ±26.52 µmol/L. The remaining 15 analytes performed acceptably according to the Desirable Specifications of Ricos. The correlation coefficient of 12 analytes was greater than 0.95 in Passing-Bablok regression analysis among the three tubes, but was lower for four analytes: calcium, sodium, potassium, and chloride. In the stability assay, only potassium tested in the Greiner tube revealed a larger positive bias (2.18%) than the Ricos Desirable Specification for Inaccuracy based on biologic variation (1.8%). The BD tube also showed a positive bias of 1.74%, whereas the new Improve tube showed the smallest positive bias of 1.17% in potassium level after 72 h storage. Thus, the results of this study demonstrate that recently introduced analytical performance specifications based on components of biological variation (Rico's goal) could be extended to criterion for performance evaluation and applied.

MALDI-TOF-MS Fingerprinting Provides Evidence of Urosepsis caused by Aerococcus urinae.

Urosepsis due to Aerococcus urinae is rare in clinical settings with only a few of reported cases worldwide by 16S rRNA sequencing. Here we report a case of sepsis caused by A. urinae in a 86 year-old male with complicated urinary tract infection which was confirmed through peptide mass fingerprinting of matrix-assisted laser desorption ionization time of flight mass spectrometry.

Analysis of the Vaginal Microbiome by Next-Generation Sequencing and Evaluation of its Performance as a Clinical Diagnostic Tool in Vaginitis.

BACKGROUND: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis. METHODS: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis. RESULTS: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%). CONCLUSIONS: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.

Establishing quality control ranges for antimicrobial susceptibility testing of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus: a cornerstone to develop reference strains for Korean clinical microbiology laboratories.

Quality control (QC) processes are being performed in the majority of clinical microbiology laboratories to ensure the performance of microbial identification and antimicrobial susceptibility testing by using ATCC strains. To obtain these ATCC strains, some inconveniences are encountered concerning the purchase cost of the strains and the shipping time required. This study was focused on constructing a database of reference strains for QC processes using domestic bacterial strains, concentrating primarily on antimicrobial susceptibility testing. Three strains (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) that showed legible results in preliminary testing were selected. The minimal inhibitory concentrations (MICs) and zone diameters (ZDs) of eight antimicrobials for each strain were determined according to the CLSI M23. All resulting MIC and ZD ranges included at least 95% of the data. The ZD QC ranges obtained by using the CLSI method were less than 12 mm, and the MIC QC ranges extended no more than five dilutions. This study is a preliminary attempt to construct a bank of Korean QC strains. With further studies, a positive outcome toward cost and time reduction can be anticipated.

Setting the Gray Zone of Near-Cutoff Values in a Real-Time PCR Test for the Detection of Mycobacterium tuberculosis in an Intermediate Burden Country.

BACKGROUND: Recently, molecular detection of Mycobacterium tuberculosis in a respiratory specimens is accepted as one of the standard procedures for diagnosis of tuberculosis in Korea. When detecting tuberculosis using a real-time polymerase chain reaction (PCR), results showed the repeated near-cutoff values in a specimen make it difficult for the laboratory to give definitive reports as positive or negative. METHODS: We retrospectively evaluated clinical state of ninety-eight patients who were not currently taking antituberculosis medications and had near-cutoff values of respiratory specimens using a real-time PCR test. RESULTS: Sixty-eight percent of the patients had clinical tuberculosis. In subgroup analysis, patients less than the age of 50, 94.3% had tuberculosis while only 55.6% of the patients with the age of equal and over 50 had tuberculosis (p < 0.001). CONCLUSIONS: Setting a gray zone for real-time PCR result and give additional tailored information onto the interpretative report would be suggested for the clinical practice in an intermediate burden of tuberculosis country.

In vitro activity of tedizolid against gram-positive bacteria in patients with skin and skin structure infections and hospital-acquired pneumonia: a Korean multicenter study.

We compared the activities of tedizolid to those of linezolid and other commonly used antimicrobial agents against gram-positive cocci recovered from patients with skin and skin structure infections (SSSIs) and hospital-acquired pneumonia (HAP) in Korean hospitals. Gram-positive isolates were collected from 356 patients with SSSIs and 144 patients with HAP at eight hospitals in Korea from 2011 to 2014. SSSIs included impetigo, cellulitis, erysipelas, furuncles, abscesses, and infected burns. Antimicrobial susceptibility was tested by using the CLSI agar dilution method. All of the gram-positive isolates were inhibited by ≤1 µg/mL tedizolid. The minimum inhibitory concentration [MIC]90 of tedizolid was 0.5 µg/mL for methicillin-resistant Staphylococcus aureus, which was 4-fold lower than that of linezolid. Tedizolid may become a useful option for the treatment of SSSIs and HAP caused by gram-positive bacteria.

Insufficient Discriminatory Power of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Dendrograms to Determine the Clonality of Multi-Drug-Resistant Acinetobacter baumannii Isolates from an Intensive Care Unit.

While pulsed-field gel electrophoresis (PFGE) is recognized as the gold standard method for clonality analysis, MALDI-TOF MS has recently been spotlighted as an alternative tool for species identification. Herein, we compared the dendrograms of multi-drug-resistant (MDR) Acinetobacter baumannii isolates by using MALDI-TOF MS with those by using PFGE. We used direct colony and protein extraction methods for MALDI-TOF MS dendrograms. The isolates with identical PFGE patterns were grouped into different branches in MALDI-TOF MS dendrograms. Among the isolates that were classified as very close isolates in MALDI-TOF MS dendrogram, PFGE band patterns visually showed complete differences. We numeralized similarity among isolates by measuring distance levels. The Spearman rank correlation coefficient values were 0.449 and 0.297 between MALDI-TOF MS dendrogram using direct colony and protein extraction method versus PFGE, respectively. This study is the first paper focusing solely on the dendrogram function of MALDI-TOF MS compared with PFGE. Although MALDI-TOF MS is a promising tool to identify species in a rapid manner, our results showed that MALDI-TOF MS dendrograms could not substitute PFGE for MDR Acinetobacter baumannii clonality analysis.

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.

Complete Genome Sequence of the Siphoviral Bacteriophage YMC/09/04/R1988 MRSA BP: A lytic phage from a methicillin-resistant Staphylococcus aureus isolate.

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing cause of serious infection, both in the community and hospital settings. Despite sophisticated strategies and efforts, the antibiotic options for treating MRSA infection have been narrowed due to the limited number of newly developed antimicrobials. Herein, we analyze the completely sequenced genome of a novel virulent phage YMC/09/04/R1988 MRSA BP as a potential alternative anti-MRSA agent, which lysed clinical isolates from a patient admitted to the hospital due to hip disarticulation. The phage contains a linear double-stranded DNA genome of 44,459 bp in length, with 33.37% GC content, 62 predicted open reading frames (ORFs), and annotated functions of only 23 ORFs that are associated with structural assembly, host lysis, DNA replication, and modification. It showed a broad host range (17 of 30 strains) against MRSA strains in clinical isolates. This article is protected by copyright. All rights reserved.