Establishment of Pediatric Reference Intervals for Routine Laboratory Tests in Korean Population: A Retrospective Multicenter Analysis.

BACKGROUND: Reference intervals defined for adults or children of other ethnicities cannot be applied in the evaluation of Korean pediatric patients. Pediatric reference intervals are difficult to establish because children are in their growing stage and their physiology changes continuously. We aimed to establish reference intervals for routine laboratory tests for Korean pediatric patients through retrospective multicenter data analysis. METHODS: Preoperative laboratory test results from 1,031 pediatric patients aged 0 month-18 years who underwent minor surgeries in four university hospitals were collected. Age- and sex-specific reference intervals for routine laboratory tests were defined based on the Clinical and Laboratory Standards Institute (CLSI) EP28-A3c guidelines. RESULTS: The pediatric reference intervals determined in this study were different from existing adult reference intervals and pediatric reference intervals for other ethnicities. Most tests required age-specific partitioning, and some of those required sex-specific partitioning for at least one age-partitioned subgroup. Erythrocyte sedimentation rate, monocyte percentage, basophil percentage, activated partial thromboplastin time, glucose, cholesterol, albumin, bilirubin, chloride, and C-reactive protein did not show any difference between age- or sex-partitioned subgroups. CONCLUSIONS: We determined Korean pediatric reference intervals for hematology, coagulation, and chemistry tests by indirect sampling based on medical record data from multiple institutions. These reference intervals would be valuable for clinical evaluations in the Korean pediatric population.

Evolutionary History of Mitochondrial Genomes in Discoba, Including the Extreme Halophile Pleurostomum flabellatum (Heterolobosea).

Data from Discoba (Heterolobosea, Euglenozoa, Tsukubamonadida, and Jakobida) are essential to understand the evolution of mitochondrial genomes (mitogenomes), because this clade includes the most primitive-looking mitogenomes known, as well some extremely divergent genome information systems. Heterolobosea encompasses more than 150 described species, many of them from extreme habitats, but only six heterolobosean mitogenomes have been fully sequenced to date. Here we complete the mitogenome of the heterolobosean Pleurostomum flabellatum, which is extremely halophilic and reportedly also lacks classical mitochondrial cristae, hinting at reduction or loss of respiratory function. The mitogenome of P. flabellatum maps as a 57,829-bp-long circular molecule, including 40 coding sequences (19 tRNA, two rRNA, and 19 orfs). The gene content and gene arrangement are similar to Naegleria gruberi and Naegleria fowleri, the closest relatives with sequenced mitogenomes. The P. flabellatum mitogenome contains genes that encode components of the electron transport chain similar to those of Naegleria mitogenomes. Homology searches against a draft nuclear genome showed that P. flabellatum has two homologs of the highly conserved Mic60 subunit of the MICOS complex, and likely lost Mic19 and Mic10. However, electron microscopy showed no cristae structures. We infer that P. flabellatum, which originates from high salinity (313‰) water where the dissolved oxygen concentration is low, possesses a mitochondrion capable of aerobic respiration, but with reduced development of cristae structure reflecting limited use of this aerobic capacity (e.g., microaerophily).

Toll-like receptor 2 downregulation and cytokine dysregulation predict mortality in patients with Staphylococcus aureus bacteremia.

BACKGROUND: Staphylococcus aureus bacteremia (SAB) presents heterogeneously, owing to the differences in underlying host conditions and immune responses. Although Toll-like receptor 2 (TLR2) is important in recognizing S. aureus, its function during S. aureus infection remains controversial. We aimed to examine the association of TLR2 expression and associated cytokine responses with clinical SAB outcomes. METHODS: Patients from a prospective SAB cohort at two tertiary-care medical centers were enrolled. Blood was sampled at several timepoints (≤5 d, 6-9 d, 10-13 d, 14-19 d, and ≥ 20 d) after SAB onset. TLR2 mRNA levels were determined via real-time PCR and serum tumor necrosis factor [TNF]-α, interleukin [IL]-6, and IL-10 levels were analyzed with multiplex-high-sensitivity electrochemiluminescent ELISA. RESULTS: TLR2 levels varied among 59 SAB patients. On days 2-5, TLR2 levels were significantly higher in SAB survivors than in healthy controls (p = 0.040) and slightly but not significantly higher than non-survivors (p = 0.120), and SAB patients dying within 7 d had lower TLR2 levels than survivors (P = 0.077) although statistically insignificant. IL-6 and IL-10 levels were significantly higher in non-survivors than in survivors on days 2-5 post-bacteremia (P = 0.010 and P = 0.021, respectively), and those dying within 7 d of SAB (n = 3) displayed significantly higher IL-10/TNF-α ratios than the survivors did (P = 0.007). CONCLUSION: TLR2 downregulation and IL-6 and IL-10 concentrations suggestive of immune dysregulation during early bacteremia may be associated with mortality from SAB. TLR2 expression levels and associated cytokine reactions during early-phase SAB may be potential prognostic factors in SAB, although larger studies are warranted.

Utility of Conventional Culture and MALDI-TOF MS for Identification of Microbial Communities in Bronchoalveolar Lavage Fluid in Comparison with the GS Junior Next Generation Sequencing System.

BACKGROUND: Diverse microbiota exist in the lower respiratory tract. Although next generation sequencing (NGS) is the most widely used microbiome analysis technique, it is difficult to implement NGS in clinical microbiology laboratories. Therefore, we evaluated the performance of conventional culture methods together with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying microbiota in bronchoalveolar lavage (BAL) fluid. METHODS: BAL fluid samples (n=27) were obtained from patients undergoing diagnostic bronchoscopy for lung mass evaluation. Bacterial and fungal culture was performed with conventional media used in clinical microbiology laboratories. On an average, 20 isolated colonies were picked from each agar plate and identified by MALDI-TOF MS. Microbiome analysis using 16S rRNA NGS was conducted for comparison. RESULTS: Streptococcus spp. and Neisseria spp. were most frequently cultured from the BAL fluid samples. In two samples, Enterobacteriaceae grew predominantly on MacConkey agar. Actinomyces and Veillonella spp. were commonly identified anaerobes; gut bacteria, such as Lactobacillus, Bifidobacterium, and Clostridium, and fungi were also isolated. NGS revealed more diverse bacterial communities than culture, and Prevotella spp. were mainly identified solely by NGS. Some bacteria, such as Staphylococcus spp., Clostridium spp., and Bifidobacterium spp., were identified solely by culture, indicating that culture may be more sensitive for detecting certain bacteria. CONCLUSIONS: Culture and NGS of BAL fluid samples revealed common bacteria with some different microbial communities. Despite some limitations, culture combined with MALDI-TOF MS might play a complementary role in microbiome analysis using 16S rRNA NGS.

Comparison of a New Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Platform, ASTA MicroIDSys, With Bruker Biotyper for Species Identification.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, with its accuracy and speed, is widely used for bacterial identification. The ASTA MicroIDSys system (ASTA, Korea) was recently developed for species identification. We compared its performance with that of Bruker Biotyper (Bruker Daltonics, Germany). Microbes were recovered from sputum, urine, and pus samples from patients admitted to a tertiary care hospital in Korea from January to April 2016. Matrix solution (α-cyano-4-hydroxycinnamic acid) was used, and the peptide profiles acquired from the Microflex LT (Bruker Daltonics) and Tinkerbell LT (ASTA) were analyzed by using their respective software. From 5,322 isolates, Bruker Biotyper identified 163 species; fifty species from 4,919 isolates were identified more than 10 times, including Klebsiella pneumoniae (n=571), Acinetobacter baumannii (n=436), Pseudomonas aeruginosa (n=358), Escherichia coli (n=372), Staphylococcus aureus (n=511), S. epidermidis (n=444), Enterococcus faecium (n=262), E. faecalis (n=220), and Candida albicans (n=248). Identical results, confidence scores (≥ 2.0 for Bruker Biotyper), and acceptable scores (≥140 for ASTA MicroIDSys) were obtained for 86.1% of isolates. Of 4,267 isolates, 99.2% showed acceptable scores in both systems. Results from the ASTA MicroIDSys showed good agreement with those from the Bruker Biotyper. The ASTA MicroIDSys could reliably identify clinically important microorganisms.

Characterization of microbiome in bronchoalveolar lavage fluid of patients with lung cancer comparing with benign mass like lesions.

OBJECTIVES: Disruption in the stability of respiratory microbiota is known to be associated with many chronic respiratory diseases. However, only few studies have examined microbiomes in lung cancer. Therefore, we characterized and compared the microbiomes of patients with lung cancer and those with benign mass-like lesions. MATERIALS AND METHODS: Bronchoalveolar fluid was collected prospectively to evaluate lung masses in patients who had undergone bronchoscopies from May to September 2015. Twenty-eight patients (20 male, 8 female) were enrolled: 20 diagnosed with lung cancer and 8 diagnosed with benign diseases. Samples were analysed by 16S rRNA-based next-generation sequencing. RESULTS: The participants' mean age was 64±11years. Bacterial operational taxonomic units were classified into 26 phyla, 44 classes, 81 orders, 153 families, 288 genera, and 797 species. The relative abundance of two phyla (Firmicutes and TM7) was significantly increased in patients with lung cancer (p=0.037 and 0.035, respectively). Furthermore, two genera (Veillonella and Megasphaera) were relatively more abundant in lung cancer patients (p=0.003 and 0.022, respectively). The area under the curve of a combination of these two genera used to predict lung cancer was 0.888 (sensitivity=95.0%, specificity=75.0% and sensitivity=70.0%, specificity=100.0%; p=0.002). CONCLUSION: The results indicate that differences exist in the bacterial communities of patients with lung cancer and those with benign mass-like lesions. The genera Veillonella and Megasphaera showed the potential to serve as biomarkers to predict lung cancer. Thus, the lung microbiota may change the environment in patients with lung cancer.

Disk Carbapenemase Test for the Rapid Detection of KPC-, NDM-, and Other Metallo-ß-Lactamase-Producing Gram-Negative Bacilli.

BACKGROUND: Rapid detection of carbapenemase-producing gram-negative bacilli (GNB) is required for optimal treatment of infected patients. We developed and assessed a new disk carbapenemase test (DCT). METHODS: Paper disks containing 0.3 mg of imipenem and bromothymol blue indicator were developed, and the performance of the DCT were evaluated by using 742 strains of GNB with or without carbapenemases. RESULTS: The paper disks were simple to prepare, and the dried disks were stable at -20°C and at 4°C. The DCT detected 212 of 215 strains (98.6% sensitivity with 95% confidence interval [CI] 96.0-99.5%) of GNB with known class A (KPC and Sme) and class B (NDM, IMP, VIM, and SIM) carbapenemases within 60 min, but failed to detect GES-5 carbapenemase. The DCT also detected all two Escherichia coli isolates with OXA-48, but failed to detect GNB with OXA-232, and other OXA carbapenemases. The DCT showed 100% specificity (95% CI, 99.2-100%) in the test of 448 imipenem-nonsusceptible, but carbapenemase genes not tested, clinical isolates of GNB. CONCLUSIONS: The DCT is simple and can be easily performed, even in small laboratories, for the rapid detection of GNB with KPC, NDM and the majority of IMP, VIM, and SIM carbapenemases.

Differential expression of leukocyte receptors in disseminated intravascular coagulation: prognostic value of low protein C receptor expression.

BACKGROUND: The protein C receptor (PROCR), toll-like receptor (TLR) and L-selectin leukocyte receptor play roles in systemic inflammatory response including disseminated intravascular coagulation (DIC). Expression of these receptors, which mediate systemic immune or coagulation responses, is tightly regulated by physiologic or pathologic signals. We investigated whether the expressions of 3 leukocyte receptors (PROCR, TLR4, L-selectin) was related to clinical outcomes in patients suggestive of having DIC. METHODS: RNA was extracted from the peripheral blood buffy coats of patients suggestive of having DIC. After reverse transcription, mRNA expression levels of PROCR, TLR4, and L-selectin were measured using Taqman Gene Expression Assays. The 28-day hospital mortality rate was used as a clinical outcome. RESULTS: The expression level of PROCR mRNA in leukocytes was lower in those with overt-DIC as compared to those without overt-DIC, however, this difference was not statistically significant. As for TLR4 and L-selectin mRNA expression, there were no significant differences observed between those with and without overt-DIC. A Kaplan-Meier survival analysis revealed that patients with low PROCR mRNA expression levels showed significantly lower survival rates than those with high expression levels. On multivariate cox regression analysis, low levels of PROCR mRNA expression were an independent prognostic marker. However, expression levels of TLR4 and L-selectin mRNA were not associated with any prognostic value. CONCLUSION: Considering that the PROCR is an important anticoagulant receptor, low PROCR mRNA expression levels associated with a poor prognosis in patients with DIC represents an exhaustion of the natural anticoagulant system, and reflects the final decompensate stage of DIC. The leukocyte PROCR may contribute to a dampening of florid activation of coagulation reactions in vivo.

Large deletion in KCNQ1 identified in a family with Jervell and Lange-Nielsen syndrome.

Long QT syndrome (LQTS) is a genetically heterogeneous disorder associated with sequence variations in more than 10 genes; in some cases, it is caused by large deletions or duplications among the main, known LQTS-associated genes. Here, we describe a 14-month-old Korean boy with congenital hearing loss and prolonged QT interval whose condition was clinically diagnosed as Jervell and Lange-Nielsen syndrome (JLNS), a recessive form of LQTS. Genetic analyses using sequence analysis and multiplex ligation-dependent probe amplification (MLPA) assay revealed a large deletion spanning exons 7-10 as well as a frameshift mutation (c.1893dup; p.Arg632Glnfs*20). To our knowledge, this is the first report of a large deletion in KCNQ1 identified in JLNS patients. This case indicates that a method such as MLPA, which can identify large deletions or duplications needs to be considered in addition to sequence analysis to diagnose JLNS.

First case of Mycobacterium longobardum infection.

Mycobacterium longobardum is a slow-growing, nontuberculous mycobacterium that was first characterized from the M. terrae complex in 2012. We report a case of M. longobardum induced chronic osteomyelitis. A 71-yr-old man presented with inflammation in the left elbow and he underwent a surgery under the suspicion of tuberculous osteomyelitis. The pathologic tissue culture grew M. longobardum which was identified by analysis of the 65-kDa heat shock protein and full-length 16S rRNA genes. The patient was cured with the medication of clarithromycin and ethambutol without further complications. To the best of our knowledge, this is the first report of a M. longobardum infection worldwide.

Prevalence of plasmid-mediated quinolone resistance genes and ciprofloxacin resistance in pediatric bloodstream isolates of Enterobacteriaceae over a 9-year period.

A total of 279 isolates of Escherichia coli (n = 128) and Klebsiella pneumoniae (n = 151) were obtained from blood samples from children at the Seoul National University Children's Hospital, Seoul, Korea from 1999 to 2007. Five plasmid-mediated quinolone resistance (PMQR) genes, qnrA, qnrB, qnrS, qepA, and aac(6')-Ib-cr, and the minimal inhibitory concentration (MIC) values for ciprofloxacin were tested for all the strains. Mutations in both gyrA and parC were analyzed in 57 representative strains. Twenty-seven strains (9.7%) had at least 1 of the 5 PMQR genes: qnrB in 20 isolates, qnrS in 1, aac(6')-Ib-cr in 5, and both qnrB and aac(6')-Ib-cr in 1 isolate. The overall PMQR prevalence rates tended to increase over time (P = 0.001). The non-susceptibility rate to ciprofloxacin was 11.0% (31/279). PMQR-harboring isolates tended to have increased ciprofloxacin MIC values among both quinolone resistance-determining region (QRDR) mutation-present (P = 0.016) and QRDR mutation-absent isolates (P < 0.001). The increasing prevalence of PMQR genes was associated with increase in quinolone use over time (P < 0.001) and increasing frequency of non-susceptibility to ciprofloxacin (P < 0.001).

Changes in molecular epidemiology of community-associated and health care-associated methicillin-resistant Staphylococcus aureus in Korean children.

Widespread emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has changed the epidemiology of S. aureus infections. We examined the molecular types and antibiotic susceptibility of CA-MRSA and health care-associated MRSA (HA-MRSA) among Korean children. MRSA isolates were obtained from patients admitted to university-affiliated tertiary hospitals in Korea, between 2006 and 2010. Molecular studies including multilocus sequence typing, SCCmec typing, and polymerase chain reaction amplification of PVL genes and antibiotic susceptibility tests were performed. SCCmec type IV was most frequently found for both CA-MRSA (80.0%) and HA-MRSA (56.4%). ST72-MRSA-SCCmec type IV and its single-locus variants were the most prevalent MRSA clones in the Korean pediatric population, both in community and in health care settings. The PVL genes were detected in 10% (4/40) of CA-MRSA isolates. Most of the clinical MRSA isolates showed vancomycin MIC ≥1.0 µg/mL. In conclusion, the molecular characteristics of HA-MRSA have been changing and CA-MRSA genotype overtook HA-MRSA genotype in health care settings.

Molecular characterization of methicillin-resistant Staphylococcus aureus obtained from the anterior nares of healthy Korean children attending daycare centers.

OBJECTIVES: This study was performed to investigate the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolated from the anterior nares of Korean children attending daycare centers. METHODS: During September and October 2008, a survey of nasal carriage of MRSA was conducted among healthy children who were attending daycare centers in Seoul, Korea. Nasal swab samples were cultured to isolate S. aureus, and antimicrobial susceptibility was assessed using a disk diffusion test. All MRSA isolates were archived for subsequent molecular tests, including multilocus sequence typing, Panton-Valentine leukocidin (PVL) genes polymerase chain reaction (PCR), and staphylococcal cassette chromosome mec (SCCmec) typing. RESULTS: Among 428 preschool-aged children enrolled, 9.3% (40/428) were colonized with MRSA. Among the 40 MRSA isolates, antibiotic susceptibilities to clindamycin and erythromycin were 97.5% (39/40) and 45% (18/40), respectively. All of the 21 strains susceptible to clindamycin and resistant to erythromycin had MLS(B)-inducible phenotypes. Sequence type (ST) 72-SCCmec type IV was the predominant clone (n=23; 57.5%), followed by ST72-SCCmec type II (n=6; 15%), ST1765-SCCmec type IV (n=4; 10%), ST1765-SCCmec type II (n=2; 5%), and ST1-SCCmec type IV (n=2; 5%). No clone was positive for PVL genes. CONCLUSIONS: ST72 strains, which were previously found in hospital-associated MRSA, are now widely distributed in healthy Korean children. In addition, the prevalence of inducible resistance of clindamycin should be considered when selecting empirical antibiotics for community-associated MRSA infections in Korea.

Role of human coronavirus NL63 in hospitalized children with croup.

BACKGROUND: Human coronavirus NL63 (HCoV-NL63) has recently been implicated as a common cause of croup in children. This study was performed to evaluate viruses associated with croup in children, with an emphasis on HCoV-NL63. METHODS: Nasopharyngeal aspirates were prospectively collected from 182 children hospitalized with croup at Seoul National University Bundang Hospital from January 2005 to June 2009. Multiplex reverse-transcriptase polymerase chain reaction was conducted for detection of 11 respiratory viruses, and medical records were reviewed. RESULTS: Viruses were identified in 147 (80.8%) of the 182 croup patients. The 3 most commonly detected viruses were parainfluenza virus type 1 (PIV1) in 44 (24.2%) patients, HCoV-NL63 in 30 (16.5%) patients, and influenza A virus in 25 (13.7%) patients. Other detected viruses were rhinovirus in 22 (12.1%) patients, PIV type 3 and respiratory syncytial virus in 15 (8.2%) patients, human bocavirus in 8 (4.4%) patients, and several others in a few patients. Coinfections with > or = 2 viruses were found in 20 (11%) patients. HCoV-NL63 was identified primarily in winter, which coincides with the peak occurrence of croup. Patients with HCoV-NL63 infection were younger than those who were positive for PIV1 (median age 13 months versus 21 months, P = 0.006) and had shorter fever duration than influenza A virus infection (median 1.5 days versus 4 days, P = 0.000). CONCLUSIONS: PIV1 and HCoV-NL63 were most commonly associated with patients who were hospitalized with croup. HCoV-NL63 is prevalent in winter and is associated with younger age and with shorter fever duration.

The association of newly identified respiratory viruses with lower respiratory tract infections in Korean children, 2000-2005.

BACKGROUND: This study was performed to evaluate the associations of newly recognized viruses, namely, human metapneumovirus (hMPV), human coronavirus (HCoV)-NL63, and human bocavirus (HBoV) with lower respiratory tract infections (LRTIs) in previously healthy children. METHODS: To determine the prevalences of 11 viruses--respiratory syncytial virus (RSV), adenovirus, rhinovirus, parainfluenza viruses (PIVs) 1 and 3, influenza viruses A and B, hMPV, HCoV, HCoV-NL63, and HBoV--among infants or children with LRTIs, in association with their epidemiologic characteristics, we performed multiplex reverse-transcriptase polymerase chain reaction on nasopharyngeal aspirates obtained from 515 children < or =5 years old with LRTIs during the period 2000-2005. RESULTS: Viruses were identified in 312 (60.6%) of the 515 patients. RSV was detected in 122 (23.7%), HBoV in 58 (11.3%), adenovirus in 35 (6.8%), PIV-3 in 32 (6.2%), rhinovirus in 30 (5.8%), hMPV in 24 (4.7%), influenza A in 24 (4.7%), PIV-1 in 9 (1.7%), influenza B in 9 (1.7%), and HCoV-NL63 in 8 (1.6%). Coinfections with > or =2 viruses were observed in 36 patients (11.5%). Twenty-two patients (37.9%) infected with HBoV had a coinfection. Bronchiolitis was frequently diagnosed in patients who tested positive for RSV, PIV-3, or rhinovirus, whereas influenza A, PIV-1, and HCoV-NL63 were commonly found in patients with croup. The age distributions of patients with viral infections differed; notably, RSV was responsible for 77% of LRTIs that occurred in infants < or =3 months old. The number of hMPV infections peaked between February and April, whereas the number of HCoV-NL63 infections peaked between April and May. CONCLUSIONS: This study describes the features of LRTIs associated with newly identified viruses in children, compared with those associated with known viruses. Additional investigations are required to define the role of HBoV in LRTI.