Sung Kuk Hong

Clinical significance of the mixing test in laboratory diagnoses of lupus anticoagulant: the fate of the mixing test in integrated lupus anticoagulant test systems.

The mixing test is used to determine the presence of inhibitors in laboratory diagnoses of lupus anticoagulant. Updated international guidelines state that an integrated lupus anticoagulant test system does not require the mixing test; an appraisal of the mixing tests in integrated lupus anticoagulant test systems is, therefore, required. We investigated the clinical relevance of mixing tests by using the best cutoff value of the mixing test through thrombotic risk analysis. A retrospective analysis was performed on 525 specimens with positive screening tests by using two integrated lupus anticoagulant tests: diluted Russells Viper venom (dRVVT) and silica clotting time. The diagnostic performance of two interpretation formulas (percentage correction, Rosner index) was assessed, and the thrombotic risk of a subgroup based on the mixing results was investigated. Finally, the thrombotic risk of lupus anticoagulant positivity based on the integrated lupus anticoagulant test system procedures was assessed for the appraisal of mixing test exclusion in integrated lupus anticoagulant test systems. The best cutoff values of mixing test interpretation methods based on dRVVT were as follows: 60.1% for percentage correction and 15.7 for Rosner index. There was no substantial difference in the thrombotic risk between percentage correction and the Rosner index. The mixing-positive group showed a higher lupus anticoagulant titer and higher thrombotic risk than the mixing-negative group. However, even the mixing-negative group carried a significant risk of thrombosis. Finally, lupus anticoagulant positivity determined by the updated two-step procedure (screening and confirmation tests) showed higher thrombotic risk than that determined by the traditional three-step procedure (screening, mixing, and confirmation tests). Although a positive mixing result can predict a high risk of thrombosis, negative mixing results are also associated with a substantial thrombotic risk. The updated two-step procedure without the mixing test is relevant for lupus anticoagulant detection in the context of thrombotic risk estimation.

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.

Campylobacter hyointestinalis isolated from a human stool specimen.

Dear EditorCampylobacter are curved, gram-negative, non-spore-forming rods that usually grow in a microaerophilic environment. To date, 22 species have been assigned to Campylobacter. Although most Campylobacter species are zoonotic, C. jejuni, C. coli, and C. fetus are well-known human pathogens; C. upsaliensis and C. lari are also considered enteropathogenic bacteria [1]. C. hyointestinalis was first isolated from pigs with proliferative enteritis in 1983 [2] and was initially considered a pathogen of pigs and rodents; however, there have been two previous re-ports of isolation from patients with proctitis and diarrhea [3, 4]. C. hyointestinalis is now thought to be a human pathogen, but reported cases are rare [1, 3, 4]. We report a case of gastroen-teritis possibly induced by C. hyointestinalis, which was identi-fied in stool specimens and confirmed with 16S ribosomal RNA (rRNA) gene sequencing, biochemical testing, and matrix-as-sisted laser desorption/ionization time-of-flight mass spectrome-try (MALDI-TOF MS). The patient was an 88-yr-old male who visited the emergency room because of right-side weakness. He had a history of left middle cerebral artery infarction and rectal cancer and under-went low anterior resection with coloanal anastomosis and post-operative adjuvant radiotherapy five years earlier. He was diag-nosed as having recurrent left middle cerebral artery infarction and admitted for antithrombotic therapy. On day three after ad-mission, he complained of diarrhea, which was unresponsive to antidiarrheal agents. Physical examination showed mild hypoes-thesia of the right extremities unrelated to the diarrhea. Labora-tory investigation showed hemoglobin level of 13.0 g/dL, leuko-cyte count of 5.58×10

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.

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.

Complete genome sequence of the 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.

Performance of Matrix-Assisted Laser Desorption Ionization Time-of-Fight Mass Spectrometry for Rapid Discrimination of Methicillin-Resistant Staphylococcus aureus (MRSA): First Report of a Relation Between Protein Peaks and MRSA spa Type

Dear Editor, Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely applied for the rapid identification of bacterial species [1]. However, an antimicrobial susceptibility test (AST) should be performed by using conventional methods following MALDI-TOF MS, and this delay represents a significant hurdle for the rapid adjustment of administered antimicrobial treatments. To overcome this limitation, many methods to improve MALDI-TOF MS for the production of rapid AST results have been proposed [2–4]. For example, carbapenemase-producing Enterobacteriaceae can be detected by analyzing the MALDI-TOF MS peaks of carbapenems and their metabolites [2, 3]. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multidrug-resistant organism in hospital and community settings. To date, few studies have attempted to differentiate MRSA and methicillin-susceptible S. aureus (MSSA) using MALDI-TOF MS. Despite the subsequent identification of many MRSA-specific peaks, no indisputable evidence has been obtained for reliable markers for MRSA and MSSA discrimination [5–7]. Furthermore, no study has evaluated Korean S. aureus isolates to date. Therefore, we aimed to identify MRSA isolates in Korea using MALDI-TOF MS peak analysis, and evaluated the feasibility of employing this method in clinical microbiology laboratories. This study was approved by institutional review board of Yonsei university health system (Approval number 4-2017-0456) and informed consent was waived due to the retrospective manner of this study. We collected S. aureus isolated from the anterior nares of patients with atopic dermatitis from 2012 to 2013. Of 81 isolates, 47 were MSSA and 34 were MRSA as confirmed by the cefoxitin disc diffusion test according to the CLSI document M100-S25 [8]. All isolates were processed for protein extraction by using Microflex LT (Bruker Daltonics GmbH, Bremen, Germany) according to the manufacturer protocol. In Phase 1, peptide profiles were analyzed by using the QuickClassifier algorithm of ClinProTools 3.0 software (Bruker Daltonics GmbH). This model showed good sensitivity and specificity (Ta-

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.

Two non-otic cases of POM-1 metallo-β-lactamase-producing Pseudomonas otitidis infection: Necrotizing fasciitis and pan-peritonitis

Pseudomonas otitidis was first described in 2006 causing otic infections [1]. P. otitidis has phenotypic similarity to Pseudomonas aeruginosa and the rarity of its detection may partly be due to misidentification of P. otitidis as P. aeruginosa. One of the important characteristics of P. otitidis is its inherent possession of the metallob-lactamase gene blaPOM-1 [2]. Although blaPOM-1 is intrinsic to this species, only 10% and 35% of isolates were intermediate or resistant to imipenem and meropenem, respectively. Interestingly, the carbapenem-non-susceptible P. otitidis isolates were susceptible to piperacillin and ceftazidime and intermediate to aztreonam [2], potentially due to the higher catalytic efficiency of POM-1 against carbapenems than against piperacillin and antipseudomonal cephalosporins [3]. The aim of this study was to determine the presence of misidentified P. otitidis among phenotypically identified P. aeruginosa isolates and to compare the relative prevalence of P. otitidis and P. aeruginosa among consecutive isolates identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). All specimens were obtained from patients in a university hospital in Seoul, South Korea. The VITEK2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France), MALDI-TOF/MS (MALDI Biotyper; Bruker Daltonics, Bremen, Germany), PCR for detection of the blaPOM-1 gene [2] and 16S rRNA gene sequence analysis were used to identify the Pseudomonas isolate to species level. The EzTaxon database (http://www.ezbiocloud.net/eztaxon) was used to compare the 16S rRNA gene sequence. Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined with the AST N212 card (bioMérieux) or by Etest (bioMérieux) and were interpreted according to Clinical and Laboratory Standards Institute (CLSI) recommendations for non-Enterobacteriaceae [4], except for tigecycline susceptibility for which the US Food and Drug Administration (FDA) breakpoint was used [5]. Among the phenotypically identified P. aeruginosa isolates, a total of 201 isolates that were non-susceptible to carbapenems but were susceptible to other b-lactams (12 and 189 from ear and other sites, respectively) were collected between January 2012 and December 2013. Among them, one isolate (Isolate 1) was identified as P. otitidis by the MALDI-TOF/MS system. Another P. otitidis isolate (Isolate 2) from routine clinical workup in 2014 was identified by the MALDI-TOF/MS system. The blaPOM-1 gene was detected in both isolates and species identification by the MALDI-TOF/MS system was confirmed by 16S rRNA gene sequencing. Compared with the sequence of P. otitidis (GenBank AY953147), Isolates 1 and 2 were