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Purpose/Objective(s) Radiation-induced sarcoma (RIS) is a rare secondary malignancy resulting from ionizing radiation-related treatment after a long latency period. Despite the unfavorable clinical outcomes, the genomic footprints of ionizing radiation in RIS development remain largely unknown. Hence, this study aimed to characterize RIS genomes and the genomic alterations in them. Materials/Methods We performed whole-genome sequencing (WGS) of 11 cases of RIS matched with normal genomes to identify somatic alterations potentially associated with RIS development. Results The mutation abundance of RIS genomes including one hypermutated genome was variable. Cancer-related genes might show different types of genomic alterations. For instance, NF1, NF2, NOTCH1, NOTCH2, PIK3CA, RB1, and TP53 showed singleton somatic mutations; MYC, CDKN2A, RB1, and NF1 showed recurrent copy number alterations; and NF2, ARID1B, and RAD51B showed recurrent structural variations (SVs). The effects of non-homologous end joining on short insertions-deletions and SVs were substantial in RIS genomes, compared with in spontaneous osteosarcoma genomes, representing the genomic hallmark of RIS genomes. In addition, frequent chromothripsis and predisposing germline variants in DNA damage-repair pathways were identified. Conclusion Taken together, WGS-scale characterization of RIS genomes may pave the way for advanced diagnostic and therapeutic strategies for RIS.

Objectives To compare a microarray-based identification and resistance determination system (blood culture gram-negative [BC-GN]; Nanosphere, Northbrook, IL) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct blood cultures (BCs). Methods BC-GN and MALDI-TOF MS assay results from direct BCs were compared with conventional test results after pure culture. Results Among 124 BCs, 130 gram-negative rods (GNRs), including six cultures with mixed GNRs (117 bacteria were covered by the BC-GN panel), were detected. The BC-GN test presented 116/117 (99.1%) concordance for the identification of targeted GNRs. Among the six polymicrobial BCs, 10 targeted GNRs were correctly identified. Among the 100 BCs tested by MALDI-TOF MS, 88/106 (86.7%) GNRs were correctly identified, and 18 GNRs were not identified. Among the six polymicrobial samples, seven of 12 GNRs (58.3%) were correctly identified. Conclusions The BC-GN assay exhibited superior performance compared with MALDI-TOF MS for the identification of targeted GNRs in direct BCs, particularly in polymicrobial samples.

Cord blood units (CBUs) for transplantation should be free of communicable disease and must contain a specific amount of total nucleated cells and CD34+ cells. Although posttransplantation cytomegalovirus (CMV) infections are from latent infection in patients, ensuring CMV-free CBUs by performing CMV-specific IgM and nucleic acid amplification testing (NAT) is one of the mandatory procedures for the safety of CBUs. However, the exclusion policies (based on these test results) vary among nations and institutions. We tested 28,000 processed CBUs between May 2006 and June 2014. The cord blood leukocytes from CMV IgM-positive samples were then subjected to NAT. The total nucleated cell and CD34+ cell counts were measured for each CBU, and the results were compared to the CMV IgM and IgG results. The seroprevalence of CMV among pregnant women was 98.1% (18,459/18,818) for IgG and 1.7% (441/25,293) for IgM. The concentration and the total number of CD34+ cells were significantly higher in CBUs from IgM-negative mothers compared to those from IgM-positive mothers (72.4/μl vs. 57.2/μl, respectively, p < 0.0001; 1.45 × 106/unit vs. 1.15 × 106/unit, respectively, p < 0.0001). Among CBUs with positive CMV IgM in their mothers' plasma or cord blood plasma, only 0.58% of the samples (3/517) had a positive NAT. The number of excluded CBUs from inventory due to positive CMV IgM in the cord blood was 54 of 18,326 (0.3%). For inventory purposes, it is appropriate to remove CBUs with positive cord blood CMV IgM findings irrespective of the NAT status as well as positive maternal CMV IgM in South Korea.

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 (bioMerieux, 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.

Persistent human papillomavirus type 16 (HPV16) is the major risk factor for cervical cancer. HPV16 intratypic variants differ in their geographical distribution and oncogenic potential. This study aimed to analyze the distribution of HPV16 variants and their association with cervical lesion histopathology in Korean women. In total, 133 HPV16-positive cervical samples from women admitted to Seoul National University Boramae Hospital were analyzed by sequencing E6, E7, and L1 genes and the long control region (LCR), and the variant distribution according to cervical lesion grade was determined. Isolates were grouped into a phylogenetic lineage, and A1-3, A4, C, and D sublineages were detected in 54.1, 37.8, 0.7, and 7.4% of samples, respectively. The most commonly observed LCR variations were 7521G>A (91.5%), 7730A>C (59.6%), and 7842G>A (59.6%). Furthermore, A4 or D sublineage-positive women had a higher risk for cervical cancer than women who were positive for A1-3. Among HPV phylogenetic clusters, A1-3 was the predominant sublineage, and within A1-3, the 350G polymorphism was highly frequent. These results differed from those of previous studies in Korea and other Asian countries. The findings suggest that cervical neoplasia incidence in HPV16-infected patients could be affected by the distribution of HPV16 variants in the population.

Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.

Background The largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection outside Middle East Asia in 2015 has necessitated the rapid expansion of laboratories that conduct MERS-CoV molecular testing in Korea, together with external quality assessment (EQA) to evaluate the assays used. Methods The EQA program consisted of two phases; self-validation and blind assessment. For the first EQA phase, in vitro transcribed upstream region of the envelope gene (upE) and the open reading frame (ORF)1a RNAs were used at a concentration of 1,000 copies/µL. The test panel for the second EQA phase consisted of RNA extracts from three samples, which were obtained from two MERS-CoV positive patients and one MERS-CoV negative patient. Results The first EQA phase results for 46 participants showed a linear relationship between the threshold cycle (CT) values of RNA materials and the logarithmic concentrations for both upE and ORF1a gene targets (R2=0.73 and 0.75, respectively). The mean CT value for each concentration was different depending on which commercial kit was used for the assay. Among the three commonly used kits, PowerChek MERS Real-Time PCR kit (KogeneBiotech, Korea) showed the lowest CT values at all concentrations of upE and most concentrations of ORF1a. The second EQA phase results for 47 participants were 100% correct for all tested samples. Conclusions This EQA survey demonstrates that the MERS-CoV molecular testing performed in Korea during the 2015 outbreak is of robust capability. However, careful establishment and validation of a cut-off value are recommended to ensure good analytical sensitivity.

At an intensive care unit, four neonates died consecutively within 80 minutes. Citrobacter freundii was isolated from blood samples of the 4 patients. It was also cultured from the leftover SMOFlipid that had been infused intravenously into the patients. In this in vitro study, we evaluated the bacterial growth kinetics and change in size of fat globules in SMOFlipid contaminated with C. freundii. Following the growth of bacteria, pH of SMOFlipid decreased to < 6, and the number of fat globules larger than 5 μm increased. Pulmonary fat embolism is proposed as a possible cause of the sudden deaths as well as fulminant sepsis.

There have been few clinical studies on the association between the vancomycin 24-h area under the concentration-time curve (AUC24) to minimum inhibitory concentration (MIC) ratio and vancomycin treatment outcomes in methicillin-resistant Staphylococcus aureus (MRSA) infections. To examine this association and to establish a suitable cut-off value for AUC24/MIC, a multicentre prospective observational study was conducted in patients with MRSA bacteraemia. Data were collected on all patients aged ≥18 years with MRSA bacteraemia treated with vancomycin for ≥72 h without dialysis. The MIC was determined by broth microdilution (BMD) and Etest. Treatment failure was defined as (i) 30-day mortality, (ii) persistent bacteraemia (≥7 days) and (iii) recurrence (≤30 days after completion of therapy). AUC24 was estimated by a Bayesian approach based on individual vancomycin concentrations. The AUC24/MIC cut-off value for differentiating treatment success and failure was calculated by Classification and Regression Tree (CART) analysis. In total, 117 patients were enrolled, among which vancomycin treatment failure occurred in 38 (32.5%). In univariate analysis, high vancomycin MIC and low trough levels were unrelated to treatment outcomes. In the CART analysis, low vancomycin AUC24/MIC [392.7 (BMD) and397.2 (Etest)] was associated with treatment failure. In multivariate analysis, low AUC24/MIC was a risk factor for treatment failure [adjusted odds ratio (aOR)=3.50, 95% confidence interval (CI) 1.39-8.82 by BMD; aOR=5.61, 95% CI 2.07-15.24 by Etest]. AUC24/MIC is associated with vancomycin treatment outcomes in MRSA bacteraemia, and seeking individualised AUC24/MIC ratios above target (400) may improve treatment outcomes.

Little is known about the incidence of and risk factors for glucose intolerance after distal pancreatectomy. This study investigated the clinicopathologic factors associated with the development of glucose intolerance or overt diabetes mellitus (DM) after distal pancreatectomy, and the correlation between resected pancreas volume and endocrine function impairment.After excluding patients with preoperative DM, 101 patients who underwent distal pancreatectomy with a minimum of 1-year postoperative follow-up were enrolled in this prospective cohort. Patients were assessed preoperatively and 1 week and 12 months postoperatively by oral glucose tolerance tests and by measures of HbA1c and pancreatic volume.Mean patient age was 54.1 years, mean body mass index (BMI) was 23.3 kg/m(2), and the male-to-female ratio was 1:1.8. Of the 101 patients, 21 (20.8 %) had pancreatic ductal adenocarcinoma. The median percent resected pancreas volume was 28.0 % (range 5.0-71.3 %). One year after distal pancreatectomy, 51 patients (50.5 %) had glucose intolerance, including 26 with impaired fasting glucose and 25 with DM. ROC curve analysis showed that a resected pancreas volume of 25 % showed maximum diagnostic value for development of glucose intolerance. Univariate analysis showed that female sex (58.5 vs. 36.1 %, P = 0.031), BMI (24.1 vs. 22.5 kg/m(2), P = 0.010), larger resected volume (36.5 vs. 28.0 %, P = 0.026), and lower remnant volume relative to BMI (1.7 × 10(-3) vs. 2.1 × 10(-3) m(5)/kg, P = 0.021) were risk factors for postoperative endocrine function impairment. Multivariate analysis revealed that female sex (odds ratio [OR] 5.818, P = 0.003), higher BMI (OR 10.556, P = 0.006), and resected pancreatic volume (OR 3.192, P = 0.035) were independent risk factors for endocrine impairment.About 50 % of patients without preoperative DM developed impaired glucose tolerance or overt DM following distal pancreatectomy. Female sex, higher BMI, and resection of pancreatic volume25 % were risk factors for endocrine function impairment, indicating the need for preoperative evaluation and careful perioperative glucose monitoring in these patients.

Middle East respiratory syndrome coronavirus continues to circulate in the Middle East. During a recent outbreak in Korea, changes in MERS coronavirus viral load were determined during the course of illness in 17 patients.

For the timely treatment of patients with infections in bloodstream and cerebrospinal fluid, a rapid antimicrobial susceptibility test (AST) is urgently needed. Here, we describe a direct and rapid antimicrobial susceptibility testing (dRAST) system, which can determine the antimicrobial susceptibility of bacteria from a positive blood culture bottle (PBCB) in six hours. The positive blood culture sample is directly mixed with agarose and inoculated into a micropatterned plastic microchip with lyophilized antibiotic agents. Using microscopic detection of bacterial colony formation in agarose, the total time to result from a PBCB for dRAST was only six hours for a wide range of bacterial concentrations in PBCBs. The results from the dRAST system were consistent with the results from a standard AST, broth microdilution test. In tests of clinical isolates (n = 206) composed of 16 Gram-negative species and seven Gram-positive species, the dRAST system was accurate compared to the standard broth microdilution test, with rates of 91.11% (2613/2868) categorical agreement, 6.69% (192/2868) minor error, 2.72% (50/1837) major error and 1.45% (13/896) very major error. Thus, the dRAST system can be used to rapidly identify appropriate antimicrobial agents for the treatment of blood stream infection (BSI) and antibiotic-resistant strain infections.

Mycobacterium arupense, a slow-growing nontuberculous mycobacterium (NTM), was first isolated from clinical samples in 2006 [1]. It is a member of the M. terrae complex, which includes M. arupense, M. engbaekii, M. heraklionense, M. hiberniae, M. kumamotonense, M. longobardum, M. nonchromogenicum, M. senuense, and M. terrae [1, 2, 3, 4, 5]. Although the M. terrae complex is generally considered nonpathogenic [6], infections caused by the M. terrae complex have been reported [7, 8, 9]. The pathogenicity of M. arupense is debatable, but a few cases of M. arupense infection involving bacteremia, osteomyelitis, pulmonary infection, and tenosynovitis have been reported [10, 11, 12, 13, 14]. We report a case of M. arupense tenosynovitis in a patient with a history of puncture injury to the finger caused by a crab. A 56-yr-old woman presenting with symptoms of redness, pain, and swelling in the second and fifth fingers of the right hand was admitted to our hospital. One year previously, the patient had suffered from a puncture injury to the right second finger caused by a crab; the injured finger showed swelling and redness 2 months later. She received antibiotics and corticosteroid injections at a local hospital, but the infected finger did not improve and the fifth finger of her right hand also began to show symptoms of infection. The patient had a history of being treated for hypertension and had undergone trans-sphenoidal surgery 4 yr ago for a non-functioning pituitary adenoma. After the surgery, she was treated with prednisolone and levothyroxine, but prednisolone was stopped on the day after admission. On admission, her body temperature was 36.0℃, blood pressure was 117/79 mm Hg, pulse was 56/min, and respiratory rate was 20 breaths/min. Laboratory tests confirmed that the patient's electrolytes, liver function, hormones, and complete blood counts were normal, with the exception of an elevated erythrocyte sedimentation rate (43 mm/hr) and decreased thyroid-stimulating hormone level (0.12 µIU/mL). Magnetic resonance imaging of the right hand revealed synovial hypertrophy with enhancement along the flexor tendons, which was especially evident along the second and fifth flexor tendons. Under suspicion of tuberculous tenosynovitis, her right hand was incised and drained. Histological examination of the resected tissue showed chronic granulomatous inflammation, but no organisms were detected on Gram and Ziehl-Neelsen stainings. Molecular analysis of the resected tissue for M. tuberculosis and NTM by using the AdvanSure TB/NTM real-time PCR kit (LG Life Science, Seoul, Korea) showed a negative result, and direct cultures of the tissue sample for bacteria and fungi were sterile. After the surgical site had healed, the patient was discharged on cephradine. The biopsied tissue was cultured for mycobacteria, and mycobacterial growth was observed on Lowenstein-Jensen medium 27 days later. The colonies were rough and unpigmented in the dark and after light exposure. The mycobacterium was identified as NTM by using the Seeplex MTB/NTM ACE Detection Kit (Seegene Inc., Seoul, Korea). For NTM identification, 16S rRNA and 65-kDa heat shock protein genes (hsp65) of this isolate were sequenced. The primers and PCR setting used to amplify the target regions were as previously described [15, 16, 17]. Sequences were analyzed by using an ABI PRISM 3730 series DNA Analyzer (Applied Biosystems, Foster City, CA, USA). For the first 500 bp of the 16S rRNA gene sequence, the isolate showed a 100% identity match with GenBank sequences {"type":"entrez-nucleotide","attrs":{"text":"NR_043588","term_id":"343198755"}}NR_043588 (M. arupense), a 99.6% identity match with {"type":"entrez-nucleotide","attrs":{"text":"GU084182","term_id":"343796636"}}GU084182 (M. heraklionense), and a 99.1% identity match with {"type":"entrez-nucleotide","attrs":{"text":"DQ058406","term_id":"66932807"}}DQ058406 (M. nonchromogenicum). The 415 bp of hsp65 showed a 100% identity match with {"type":"entrez-nucleotide","attrs":{"text":"AB239922","term_id":"110796715"}}AB239922 (M. arupense) and a 96.9% identity match with {"type":"entrez-nucleotide","attrs":{"text":"JX154109","term_id":"394987442"}}JX154109 (M. engbaekii). The phylogenetic tree of the hsp65 showed that this isolate was most closely related to {"type":"entrez-nucleotide","attrs":{"text":"AB239922","term_id":"110796715"}}AB239922 (M. arupense) and was classified as M. arupense (Fig. 1). Fig. 1 Phylogenetic tree based on 65-kDa heat shock protein gene sequences constructed by using the neighbor-joining method. Nucleotide sequences were used to align the reference panels provided by the National Center for Biotechnology Information Reference ... After the identification of M. arupense, the patient was prescribed clarithromycin, ethambutol, and rifampin empirically. We used Clinical and Laboratory Standards Institute breakpoints for M. kansasii [18] to interpret the results of the drug susceptibility test (DST) performed by using the broth microdilution method, which indicated that the isolate was susceptible to clarithromycin, ethambutol, and linezolid, but resistant to amikacin, ciprofloxacin, moxifloxacin, rifampin, and trimethoprim-sulfamethoxazole (Table 1). Therapy was maintained and the patient was cured without further complications. Table 1 Antimicrobial susceptibility testing results for Mycobacterium arupense M. arupense was first described in 2006 by Cloud et al. [1] as a non-pigmented organism, negative for niacin accumulation and positive for 68℃ catalase and hydrolysis of Tween 80. Although the clinical significance of M. arupense is controversial, infections caused by this organism are occasionally reported [10, 11, 12, 13, 14]. To date, only 6 patients, besides the present case, have been infected with M. arupense, although 1 pulmonary isolate from an immunocompetent patient was classified as colonization according to the American Thoracic Society criteria [6]. The cases include tenosynovitis of the hand (n=2), osteomyelitis of the wrist (n=1), pulmonary infection in an immunocompetent patient (n=1), pulmonary infection in an HIV-positive patient (n=1), and bacteremia in an HIV-positive patient (n=1). In all 4 extrapulmonary M. arupense infections in the immunocompetent patients, there was a predisposing factor such as corticosteroid injection or injury. Accurate identification is important for the treatment of M. terrae complex infection, because a substantial number of patients infected with these organisms show persistent disease owing to misdiagnosis [7]. Using mycolic acid analysis by high-performance liquid chromatography, it is difficult to distinguish M. arupense from M. nonchromogenicum because of their similar mycolic acid patterns [1]. Thus, sequencing of the 16S rRNA gene, 16S-23S internal transcribed spacer region 1 region, and especially the hsp65, is useful for identifying M. arupense [1]. In our case, the hsp65 showed better discriminatory power than 16S rRNA gene. The present strain was susceptible to clarithromycin, ethambutol, and linezolid. Among the 8 isolates studied by Cloud et al. [1], all were susceptible to clarithromycin, ethambutol, and rifabutin, and 1 isolate was resistant to clarithromycin. However, the DST results for amikacin and trimethoprim-sulfamethoxazole were variable, and all isolates were resistant to ciprofloxacin, linezolid, moxifloxacin, rifampicin, and streptomycin. Although the optimal therapy for M. arupense is not well established in the literature, a multiple antibiotic regimen including clarithromycin and ethambutol is a useful initial approach to manage these patients. In conclusion, this is the first case of M. arupense tenosynovitis in Korea. Our findings indicate that M. arupense can be an opportunistic pathogen in patients with a history of corticosteroid injection or injury.

Background Nucleic acid amplification tests have allowed simultaneous detection of multiple respiratory viruses. Methods We compared the results of a liquid bead array xTAG Respiratory Virus Panel (RVP; (Luminex Corporation, Toronto, Canada) and a solid microarray Verigene Respiratory Virus Plus (RV+; Nanosphere, Northbrook, IL) for the detection of influenza A virus (INF A), influenza B virus (INF B), and respiratory syncytial virus (RSV) in 170 respiratory specimens from hospitalized patients. Results Overall, xTAG RVP demonstrated sensitivities and specificities of 97.6 and 100% for INF A, 100 and 99.4% for INF B, and 100 and 100% for RSV, while the Verigene RV+ test sensitivities and specificities were 95.1 and 98.5%, 100.0 and 99.4%, and 97.1 and 100%, respectively. There were no significant differences in the area under the curves between the two assays for each virus (P = 0.364 for INF A, P = 1.000 for INF B, P = 0.317 for RSV). Comparing the results of two assays, discordant results were present mostly due to subtype assignments and identification of coinfections. The detection of viruses was not significantly different (P = 1.000) and the virus/subtype assignment showed good agreement with kappa coefficients of 0.908. Conclusion The xTAG RVP and Verigene RV+ showed high sensitivities and specificities, and good overall agreement in detection and identification of INF and RSV. These assays can be used in clinical settings for a reliable detection of respiratory viruses found commonly in hospitalized patients.

Direct fluorescent antibody (DFA) staining of induced sputum is frequently used to diagnose Pneumocystis pneumonia (PCP) in patients infected with human immunodeficiency virus, although induction can provoke nausea and bronchospasm. Since the diagnostic value of expectorated sputum examined with DFA stain has not been well evaluated, we reviewed the medical records of HIV-infected patients who were clinically diagnosed as having PCP between 1999 and 2011. Over this 13-year period, we found 76 patients whose records included the results of DFA staining of expectorated sputum and noted that 42 (55.3%) were positive. Polymerase chain reaction to detect Pneumocystis in the sputum of 65 of the patients resulted in the finding of 43 (66.2%) who were positive. Our findings suggest that DFA staining of expectorated sputum could be a useful initial diagnostic method in HIV-infected patients with PCP.

Dear Editor Flavobacterium species are nonfastidious, oxidase-positive, glucose nonfermenting, gram-negative rods. Few Flavobacterium species have been extensively studied [1]. Flavobacterium ceti was first isolated from the lung and liver of two beaked whales, as described in 2007 by Vela et al. [2]. Flavobacterium species are mainly found in aquatic animals and are rarely isolated from human clinical specimens. We present a case in which F. ceti was identified in the blood of a patient admitted to Seoul Metropolitan Government-Seoul National University Boramae Medical Center, Seoul, Korea. A 43-yr-old man with alcoholic liver cirrhosis presented with mild fever and abdominal distension. Laboratory investigation revealed mild leukocytosis with neutrophilia (10.34×109/L; neutrophils, 83%) and hepatic dysfunction. Ascitic fluid showed leukocyte counts >1×109 cells/L (neutrophils, 62%), whereas the serum ascites albumin gradient was >1.1 g/dL, total protein was 0.8 g/dL, glucose was 107 mg/dL, and lactate dehydrogenase was 97 U/L. Blood culture was drawn from two separate sites on the day of admission, but ascites cultures were not performed. Intravenous antibiotics were administered from the day of admission: one day of cefotaxime followed by ceftriaxone for the next 20 days. Follow-up blood cultures on day 4, 11, and 14 were all negative. Ascitic cultures performed after 14 days of antibiotics revealed no organisms. Signs of inflammation were gradually resolved, and the patient was discharged 25 days after admission. After two days of incubation, bacterial growth was detected in aerobic culture bottles of two separate blood cultures. The next day, circular, orange-colored, non-hemolytic colonies were observed on the blood agar plates (Fig. 1A), and gram stain smear revealed gram-negative rods (Fig. 1B). No colonies were observed on MacConkey agar plates. Vitek2 GN system (bioMerieux, Marcy l'Etoile, France) identified the isolated organism as Brevundimonas diminuta/vesicularis with 95% probability. The contraindicating typical biopattern was phosphatase negative, enzyme activity that is almost always observed in Brevundimonas [3]. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; BrukerDaltonik GmbH, Bremen, Germany) revealed the most matched pattern as Acinetobacter lwoffii, but showed no reliability with a score of 1.191 (Biotyper Version 3.1, Bremen, Germany). To confirm the identification, 16S rRNA sequence was analyzed by using polymerase chain reaction with 27F and 1492R primers and sequencing with 518F and 801R primers in order to span all bacteria [4]. Sequencing reactions were performed by using a DNA Engine Tetrad 2 Peltier Thermal Cycler (BioRad, Hercules, CA, USA) with a ABI BigDye(R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and were analyzed by using electrophoresis in an ABI 3730xl DNA Analyzer (Applied Biosystems) with ChromasPro 1.7.6 (Technelysium Pty Ltd, South Brisbane, Australia). The 1,367 bp of the 16S rRNA gene sequence shared 99% identity with the GenBank sequence {"type":"entrez-nucleotide","attrs":{"text":"NR_042540","term_id":"343202254","term_text":"NR_042540"}}NR_042540 (F. ceti strain 454-2). Fig. 1 Colony morphology and microscopic characteristics of Flavobacterium ceti. (A) Orange-colored, non-hemolytic colonies were observed after two days of aerobic culture on a blood agar plate. (B) Gram-negative rods from positive aerobic blood culture smear ... Flavobacterium species are well-known pathogens of bacterial cold-water disease, with some causing opportunistic infections. There have been some reports of human infections due to pathogens that were previously known as Flavobacterium but are currently classified under other genera such as Chryseobacterium [5] and Myroides [6]. Thus, known human infections from current Flavobacterium are limited, with only a few reports of different species [7, 8]. Mosayebi et al. [9] reported a Flavobacterium sepsis outbreak due to contaminated distilled water in a neonatal intensive care unit. To our knowledge, our report discusses the first case of F. ceti infection in human. Physical examination and ascites lab findings were consistent with spontaneous bacterial peritonitis. F. ceti was considered the causative pathogen because it was isolated from two separate blood cultures collected on the day of admission and the cause of bacteremia was presumed to be peritonitis although ascitic culture was not performed. Clinical manifestations were relatively mild, with good outcomes. Identification of gram-negative non-fermenting rods is quite challenging, and commercial systems often provide unreliable results. Molecular sequencing is one of the most accurate ways characterizing these bacteria to the species level. In this case, conventional methods and MALDI-TOF MS were insufficient in identifying Flavobacterium. The MALDI database contained a limited number of Flavobacterium species, and F. ceti was not included. 16S rRNA sequence analysis successfully confirmed the isolate as F. ceti. Here, we report the first probable human case of F. ceti detected in the blood of a patient with liver cirrhosis. Flavobacterium may cause an opportunistic infection in humans, although its significance remains unclear owing to the limited number of human infections reported. Molecular sequencing is currently the most reliable method for identifying this unusual pathogen.

To the Editor, Cedecea, a genus of the Enterobacteriaceae family, was named in 1981 from the initials of U.S. Centers for Disease Control and Prevention [1]. To date, a total five species have been identified; currently, three of these have been named (C. davisae, C. lapagei, and C. neteri), whereas two remain unnamed [1, 2]. Infections caused by the Cedecea species are infrequent [2, 3, 4, 5, 6]. Here, we report a case of C. lapagei pneumonia in a patient with chronic obstructive pulmonary disease (COPD). A 76-yr-old man, with a history of COPD, was admitted with dyspnea. While being treated with empirical antibiotics, he was admitted to the intensive care unit owing to respiratory failure and was provided mechanical ventilator care. After 5-day ventilator care, he was extubated and transferred to a general ward. After 1-week supportive care without antibiotics, he developed an increase in sputum and difficulty in breathing. Physical examination revealed decreased breath sounds and increased wheezing in both lung fields. Chest radiography revealed an infiltration of the left lower lobe. His temperature was 36.8℃; blood pressure, 110/63 mmHg; pulse, 115/min; respiratory rate, 20 breaths/min; and O2 saturation, 94% without O2 supply. His hematological tests showed the following findings: Hb, 11.5 g/dL; leukocyte count, 10.0×109/L (neutrophils 72.1%); platelet count, 461×109/L; C-reactive protein (CRP) level, 5.55 mg/dL. A sputum culture and two sets of blood cultures from separate peripheral veins were performed. The blood cultures tested negative, but many gram-negative rods and some neutrophils containing intracellular organisms were observed on the smear preparation of the sputum sample (Fig. 1A). White colored, non-hemolytic colonies predominantly grew on a blood agar plate (Fig. 1B), and gram-negative coccobacilli were observed on gram stain smear preparations (Fig. 1C). The isolate was colorless on a MacConkey agar plate and catalase-positive and oxidase-negative. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker Daltonik GmbH, Bremen, Germany) and the Vitek2 GN system (bioMerieux, Marcy l'Etoile, France), the organism was identified as C. lapagei, with a score of 2.322 in the Bruker system and with a 99.9% probability in the Vitek2 system. Fig. 1 Colonial and microscopic morphology of Cedecea lapagei. (A) Neutrophil containing intracellular gram-negative coccobacilli from smear preparation of the sputum sample (Gram stain, ×1,000). (B) White colored and non-hemolytic colonies on blood ... To confirm the identity of the isolate, 16S rRNA gene sequencing was conducted. However, the 1,402 bp 16S rRNA gene sequence of the isolate shared a 99.6% identity with {"type":"entrez-nucleotide","attrs":{"text":"AB086230","term_id":"21355062","term_text":"AB086230"}}AB086230 (C. neteri) in the eZtaxon database (http://www.ezbiocloud.net). To solve the discrepancy, additional tests were performed: biochemical tests as well as sequencing of the dnaJ gene, which was reported to be useful to identify Enterobacteriacea [7]. The biochemical tests were compatible with the characteristics of C. lapagei; orinithine decarboxylase and acid production from sucrose, D-sorbitol, raffinose, D-xylose, and melibiose were negative [8]. The 719 bp dnaJ gene sequence of the isolate shared a 99.3% identity with {"type":"entrez-nucleotide","attrs":{"text":"AB272620","term_id":"157059916","term_text":"AB272620"}}AB272620 (C. lapagei), and a 91.1% identity with {"type":"entrez-nucleotide","attrs":{"text":"AB272621","term_id":"157059918","term_text":"AB272621"}}AB272621 (C. neteri) in the GenBank database. The phylogenetic trees showed that C. lapagei has 16S rRNA gene intra-species heterogeneity and this isolate was most close to C. lapagei, based on the dnaJ gene (Fig. 2). Fig. 2 Neighbor-joining phylogenetic trees based on partial 16S rRNA (1,300 bp) and dnaJ (719 bp) gene sequences of rare Enterobacteriaceae strains. (A) Phylogenetic tree of 16S rRNA gene showed that Cedecea lapagei has 16S rRNA gene intra-species heterogeneity. ... Antimicrobial susceptibility was tested by using an AST-GN27 card (bioMerieux). Using the CLSI breakpoints for Enterobacteriaceae for the interpretation [9], the isolate was determined susceptible to piperacillin, cefotaxime, ceftazidime, cefepime, imipenem, amikacin, ciprofloxacin, tetracycline, and trimethoprim-sulfamethoxazole, but resistant to amoxicillin-clavulanic acid and cefoxitin. After determining the presence of the pathogen, the patient was treated with cefpodoxime for one week. The pathogen was not observed in follow-up respiratory cultures, and the patient was discharged. Clinical significance and disease spectrum of Cedecea have not yet been clearly elucidated; however, in elderly patients (age >60 yr) who are immunocompromised or have multiple comorbidities, Cedecea has been reported to cause bacteremia and pneumonia [2]. Accurate identification at species level is important in understanding Cedecea infections. Identification of Cedecea at the species level is possible by using conventional biochemical tests [8]; so its identification using molecular characteristics is not well established. Therefore, thus far, the number of sequences on GenBank is small, and whole genome analyses have not been established. This case study showed that sequencing the dnaJ gene could be useful in the molecular identification of Cedecea, as it has 16S rRNA gene intra-species heterogeneity. Although C. lapagei was isolated only in a sputum sample from the patient, we concluded that the patient had C. lapagei pneumonia on the basis of his respiratory symptoms, elevated CRP levels, and the phagocytosis by neutrophils. Therefore, we report the first case of C. lapagei pneumonia in Korea. The present findings indicate that good care must be taken in its molecular identification, as it has intra-species heterogeneity due to the 16S rRNA gene.

The detection of high-risk (HR) HPV in cervical cancer screening is important for early diagnosis of cervical cancer or pre-cancerous lesions. We evaluated the analytical and clinical performances of 3 HR HPV assays in Gynecology patients. A total of 991 specimens were included in this study: 787 specimens for use with a Hybrid Capture 2 (HC2) and 204 specimens for a HPV DNA microarray (DNA Chip). All specimens were tested using an Abbott RealTime High Risk HPV assay (Real-time HR), PGMY PCR, and sequence analysis. Clinical sensitivities for severe abnormal cytology (severe than high-grade squamous intraepithelial lesion) were 81.8% for Real-time HR, 77.3% for HC2, and 66.7% for DNA Chip, and clinical sensitivities for severe abnormal histology (cervical intraepithelial neoplasia grade 2+) were 91.7% for HC2, 87.5% for Real-time HR, and 73.3% for DNA Chip. As compared to results of the sequence analysis, HC2, Real-time HR, and DNA Chip showed concordance rates of 94.3% (115/122), 90.0% (117/130), and 61.5% (16/26), respectively. The HC2 assay and Real-time HR assay showed comparable results to each other in both clinical and analytical performances, while the DNA Chip assay showed poor clinical and analytical performances. The Real-time HR assay can be a good alternative option for HR HPV testing with advantages of allowing full automation and simultaneous genotyping of HR types 16 and 18.

Nontuberculous mycobacteria (NTM) are classified into 2 categories: slow-growing mycobacteria (SGM) and rapid-growing mycobacteria (RGM), based on interval to colony formation by subculture on solid media. However, little is known about the growth rate of NTM in liquid broth media. We evaluated the differences in time to growth detection (TGD) of RGM and SGM in liquid broth media according to acid-fast stain. Among the 696 NTM isolates, 201 were RGM and 495 were SGM. In acid-fast bacilli (AFB)-negative specimens, the mean TGD was 133 h for RGM and 269 h for SGM (P < 0.001). In AFB-positive specimens, the mean TGD was 112 ± 37 h for RGM and 155 ± 125 h for SGM (P = 0.063). In the AFB-negative group, a cut-off value of 6 days was most effective for distinguishing SGM from RGM; however, in the AFB-positive group, an appropriate cut-off value was hard to define with TGD only.

During the 2015 Middle East respiratory syndrome coronavirus outbreak in South Korea, we sequenced full viral genomes of strains isolated from 4 patients early and late during infection. Patients represented at least 4 generations of transmission. We found no evidence of changes in the evolutionary rate and no reason to suspect adaptive changes in viral proteins.

Human papillomavirus (HPV) infection is an important etiologic factor in cervical carcinogenesis. Various HPV DNA detection methods have been evaluated for clinicopathological level. For the specimens with normal cytological finding, discrepancies among the detection methods were frequently found and adequate interpretation can be difficult. 6,322 clinical specimens were submitted and evaluated for real-time PCR and Hybrid Capture 2 (HC2). 573 positive or “Not Detected but Amplified” (NDBA) specimens by real-time PCR were additionally tested using genetic analyzer. For the reliability of real-time PCR, 325 retests were performed. Optimal cut-off cycle threshold (CT) value was evaluated also. 78.7% of submitted specimens showed normal or nonspecific cytological finding. The distributions of HPV types by real-time PCR were not different between positive and NDBA cases. For positive cases by fragment analysis, concordance rates with real-time PCR and HC2 were 94.2% and 84.2%. In NDBA cases, fragment analysis and real-time PCR showed identical results in 77.0% and HC2 revealed 27.6% of concordance with fragment analysis. Optimal cut-off CT value was different for HPV types. NDBA results in real-time PCR should be regarded as equivocal, not negative. The adjustment of cut-off CT value for HPV types will be helpful for the appropriate result interpretation.

Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targetsmecA,femAspecific forS. aureus,femAspecific forS. epidermidis,16S rRNAfor universal bacteria, and16S rRNAspecific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistantS. aureus(MRSA), methicillin-susceptibleS. aureus(MSSA), methicillin-resistantS. epidermidis(MRSE), methicillin-susceptibleS. epidermidis(MSSE), methicillin-resistant non-S. epidermidisCoNS (MRCoNS), and methicillin-susceptible non-S. epidermidisCoNS (MSCoNS) (κ=0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings.

A highly significant increase in anti-Vesivirus (family Caliciviridae) antibody prevalence, along the axis from healthy blood donors; donors with elevated transaminase; patients with clinical hepatitis; and patients with post-transfusion/dialysis hepatitis, has been reported in human sera from the USA and Europe. Asian samples have now been tested retrospectively using serology and enzyme immunoassay (EIA) with a Vesivirus partial-capsid antigen expressed as a fusion protein. Anti-vesiviral antibodies were measured by optical densities (OD650) and compared in patients separated by age, gender and Groups A–F as follows: Control Group A, an Experimental Group B, which was divided further into Group C, patients with elevated enzymes (alanine transaminase (ALT), aspartate transaminase (AST), and γ-glutamyl transpeptidase (γ-GT); Group D, patients receiving transfused blood; Group E, patients with high enzyme levels after transfusion; and Group F, hepatitis B and C positive patients. Using multivariate logistic regression analyses, a significantly greater proportion of patients receiving transfusion(s), were positive for anti-Vesivirus antibody compared with non-transfused patients (P = 0.008; OR: 3.86, 95% CI: 1.43–10.43). Also, anti-Vesivirus antibody was significantly associated with elevated biochemical liver function tests: ALT ≥120 IU or AST ≥ 120 IU (P = 0.017; OR: 4.23, 95% CI: 1.30–13.80). In the blood transfusion group, anti-Vesivirus antibody was significantly correlated with high enzyme levels (ALT, P = 0.018; AST, P = 0.010; γ-GT, P = 0.020). These data provide serologic evidence of vesiviral transfusion–transmission-associated disease, which could include infection of any organ system where cytopathology resulted in high levels of either ALT or AST. J. Med. Virol. 84:1943–1952, 2012. © 2012 Wiley Periodicals, Inc.

Robinsoniella peoriensis has recently been identified as a Gram-positive, spore-forming, anaerobic rod originally recovered from swine manure storage pits. To date, 6 cases of R. peoriensis infection have been reported, including 2 cases of bacteremia, 1 of abdominal fluid collection, and 3 of wound infection. In the present study, we report a 76-yr-old man with R. peoriensis bacteremia who developed aspiration pneumonia. Gram staining of a purified colony revealed Gram-positive, rod-shaped bacteria. Biochemical identification using API 20 A (bioMerieux, France) indicated presence of Clostridium spp. We performed both 500-bp and full-gene sequencing of 16S rRNA of the isolate. The sequence was analyzed with MicroSeq ID 16S rRNA Library v2.0 (Applied Biosystems, USA), GenBank Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/genbank), and EzTaxon database v2.1 (http://www.eztaxon.org). The 500-bp 16S rRNA sequence of the blood culture isolate showed 99.16-99.79% similarity with R. peoriensis and the full-gene 16S rRNA sequence showed 98.87-99.50% similarity with R. peoriensis. The organism was confirmed as R. peoriensis by using all of the mentioned databases except for MicroSeq, which did not include the RNA sequence of this bacterium. This case suggests that identification of R. peoriensis might be challenging in clinical laboratories with no access to molecular methods, as certain commercial identification systems may not identify, or may misidentify, this organism. To the best of our knowledge, this is the first report of the isolation of R. peoriensis in Korea.

In Korea, the incidence of vancomycin-resistant enterococci (VRE) infection has recently been increasing, with a clinical isolation rate of up to 25%. We evaluated the clinical usefulness of real-time PCR for the detection of the vanA gene over conventional culture in patients confirmed as being VRE-colonized. During the evaluation stage, 100 consecutive clinical specimens were analyzed for the presence of the vanA gene via real-time PCR, for which 4 specimen preparations were used. During the application stage, 1,115 specimens from 82 patients were monitored for 20 months by vanA real-time PCR. For isolates prepared via incubation in broth for 24 h, the PCR results were concordant with those of the conventional method. The median value of the time spent in isolation and the number of test repeats from detection to release from isolation was 71 days and 5.5, respectively. The optimal negative test number for the release from isolation was ≥3. In conclusion, the real-time PCR method is more sensitive than, and is expected to replace, the conventional VRE surveillance method.

A total of 10 948 clinical isolates was collected throughout the Asia-Pacific region as part of the Tigecycline Evaluation Surveillance Trial (TEST) during 2004–2010, consisting of 7549 Gram-negative and 3399 Gram-positive pathogens. Susceptibility trends for all species demonstrated several significant species-dependent susceptibility changes to multiple antibiotics. The most notable was minocycline, for which significant decreases in susceptibility (P < 0.001) were observed for six of the ten species studied. In contrast, statistically significant susceptibility changes for tigecycline were observed in only two of the ten species, namely Klebsiella pneumoniae and Serratia marcescens. Seven years following the introduction of tigecycline into clinical use, this agent remains highly active against a wide range of pathogens from the Asia-Pacific region.

Objective.To evaluate the risk factors for surgical site infection (SSI) after gastric surgery in patients in Korea.Design.A nationwide prospective multicenter study.Setting.Twenty university-affiliated hospitals in Korea.Methods.The Korean Nosocomial Infections Surveillance System (KONIS), a Web-based system, was developed. Patients in 20 Korean hospitals from 2007 to 2009 were prospectively monitored for SSI for up to 30 days after gastric surgery. Demographic data, hospital characteristics, and potential perioperative risk factors were collected and analyzed, using multivariate logistic regression models.Results.Of the 4,238 case patients monitored, 64.9% (2,752) were male, and mean age (±SD) was 58.8 (±12.3) years. The SSI rates were 2.92, 6.45, and 10.87 per 100 operations for the National Nosocomial Infections Surveillance system risk index categories of 0, 1, and 2 or 3, respectively. The majority (69.4%) of the SSIs observed were organ or space SSIs. The most frequently isolated microorganisms were Staphylococcus aureus and Klebsiella pneumoniae. Male sex (odds ratio [OR], 1.67 [95% confidence interval (CI), 1.09–2.58]), increased operation time (1.20 [1.07–1.34] per 1-hour increase), reoperation (7.27 [3.68–14.38]), combined multiple procedures (1.79 [1.13–2.83]), prophylactic administration of the first antibiotic dose after skin incision (3.00 [1.09–8.23]), and prolonged duration (≥7 days) of surgical antibiotic prophylaxis (SAP; 2.70 [1.26–5.64]) were independently associated with increased risk of SSI.Conclusions.Male sex, inappropriate SAP, and operation-related variables are independent risk factors for SSI after gastric surgery.

Background/Aims: Early diagnosis and appropriate antimicrobial choice are crucial when managing pneumonia patients, and quantitative culture of bronchoalveolar lavage (BAL) fluid is considered a useful method for identifying pneumonia pathogens. We evaluated the quantitative yield of BAL fluid bacterial cultures in patients being treated with antimicrobials and attempted to identify factors predictive of positive BAL cultures. Methods: Patients over 18 years old and whose BAL fluid was subjected to quantitative culture to identify the organism causative of pneumonia between January 1, 2005, and December 31, 2009, were included. We reviewed the results of BAL fluid bacterial cultures and the clinical records, laboratory tests, and radiographic findings of the patients. Results: BAL was performed on 340 patients with pneumonia. A positive BAL culture, defined as isolation of more than 104 colony forming units/mL bacteria, was documented in 18 (5.29%) patients. Of these, 9 bacteria isolated from 10 patients were classified as probable pathogens. The most frequently isolated bacteria were methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. No independent predictive factor for positive BAL cultures was identified. Conclusions: The yield of quantitative BAL fluid bacterial culture in patients already on antimicrobials was low. Clinicians should be cautious when performing a BAL culture in patients with pneumonia who are already on antimicrobials.

4, National Medical Center, Seoul, Korea Background: Multiple-locus variable-number tandem-repeat fi ngerprinting (MLVF) is based on multiplex PCR, utilizing variable number tandem repeat. Our goal was to compare the performance of MLVF in distinguishing clinical Staphylococcus aureus isolates with that of pulsed-fi eld gel electrophoresis (PFGE), which has traditionally been the gold standard. Methods: Sixty-three clinically signifi cant S. aureus isolates were tested using both PFGE and MLVF. Multiplex PCR for MLVF was performed using PCR primers for clfA, clfB, sdrCDE, sspA, and spa. PFGE was performed with genomic DNA fragments generated by SmaI endonuclease digestion. Banding patterns of MLVF or PFGE were analyzed using InfoQuestFP software. Results: The hands-on time of our modifi ed method was about 3 h, on average, for each of 18 isolates. PFGE (80% cutoff) or MLVF (75% cutoff) separated all of the 63 isolates into 13 and 12 types, respectively. Three types generated by PFGE were identical to those generated by MLVF. PFGE and MLVF yielded similar Simpson’s diversity indices, indicating similar discriminatory power. The overall concordance between PFGE and MLVF was low, as represented by adjusted Rand indices (0.266-0.278). PFGE predicted MLVF type better than MLVF predicted PFGE type, as refl ected by Wallace coeffi cients (PFGE cutoff 80% vs. MLVF cutoff 75%, 0.389 vs. 0.233). Analysis of the relationship between a pair of isolates showed 91.0% concordance between the PFGE (80% cutoff) and MLVF (75% cutoff). Conclusions: Our simple, low-cost, modifi ed MLVF protocol can effectively discriminate between S. aureus clinical isolates. MLVF can replace PFGE for the hospital infection control of S. aureus.

We attempted to determine the benefits of three-channel multiplex real-time PCR and melting curve analysis not only in detecting and distinguishing between nontuberculous mycobacteria (NTM) and the Mycobacterium tuberculosis complex but also in identifying NTM to the species level.

Background: Optimal blood culture performance is critical for successful diagnosis and treatment of sepsis. To understand the sta- tus of blood culture, we investigated several aspects of the procedure at 9 university hospitals. Methods: The process of ordering blood culture sets and sampling volume for adults and children was investigated from January 2010 to April 2010, while the positive rate of detection and growth of skin contaminants were compared in 2009. Microbial growth in aerobic and anaerobic bottles was investigated prospectively. Results: A majority of the hospitals used 2 sets of bottles for adults and 1 bottle for children. The average blood volume in each set was 7.7 mL for adults and 2.1 mL for children. The positive rate of microorganisms was 8.0%, and the isolation rate of the normal flora of the skin was 2.1%. Bacterial growth rates in aerobic and anaerobic bottles only were 31.8% and 24.5% respectively. Conclusions: Ordering blood culture sets and sampling volumes did not comply with CLSI guidelines. However, the rate of positive cultures and skin contamination rates were acceptable. Anaerobic bottles are useful in enhancing the yield of microorganisms.

Propionibacterium acnes is a gram-positive anaerobic bacillus and a normal inhabitant of the skin. Although it is often considered a contaminant of blood cultures, it can occasionally cause serious infections, including postoperative central nervous system infections. Here, we report the case of a 70-yr-old man who developed a large cerebral abscess caused by P. acnes 13 months after neurosurgery. Immediate gram staining of the pus from his brain revealed the presence of gram-positive coccobacilli. However, colony growth was observed only after 5 days of culture. Therefore, we performed 16S rRNA gene sequencing of the pus specimen. The isolate was identified as P. acnes. The colonies developed 9 days after the initial culture. The API Rapid ID 32A test (bioMerieux, France) was performed using a colony, but an unacceptable profile was obtained. Then, the pus was transferred into the enrichment broths of the BACTEC FX (Becton Dickinson, USA) and BacT/Alert 3D (bioMerieux, Organon Teknika, USA) systems, but only the BACTEC FX system could detect growth after 5 days. We performed 16S rRNA gene sequencing and API Rapid 32A profiling with a colony recovered from Brucella agar, which was inoculated with the microbial growth in the enrichment broth from the BACTEC FX system. The organism was identified as P. acnes by both methods. This case suggests that 16S rRNA gene sequencing may be a useful alternative for identifying slowly growing P. acnes from specimens that do not show growth after 5 days of culture.