Prevalence of ST131 and ST1193 Among Bloodstream Isolates of Escherichia coli not Susceptible to Ciprofloxacin in a Tertiary Care University Hospital in Korea, 2013 - 2014.

We investigated the ST distribution of bloodstream isolates of E. coli that were not susceptible to ciprofloxacin (CIP-NS isolates), collected from 2013 to 2014 in a tertiary care hospital. Fifty-nine CIP-NS isolates were collected. ST131 was the most frequent ST (37.3%) isolate, followed by ST1193 (23.7%). ST131 and ST1193 showed significant differences in ESBL production and lactose fermentation. Further study should be continued on the monitoring of resistant strains.

Fluoroquinolone Resistance Mechanisms by Molecular Epidemiologic Study of Ciprofloxacin-Nonsusceptible Escherichia coli Sequence Types Isolated from Clinical Specimens in a Tertiary Care University Hospital in Korea: Emergence of Clone ST131, 2006-2008.

To investigate the sequence types (STs) and fluoroquinolone resistance related mutations among ciprofloxacin (CIP)-non-susceptible extended-spectrum ß-lactamase (ESBL)-producing E. coli isolated from Korean patients from 2006-2008. The prevalence of fluoroquinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, and parE and plasmid-mediated quinolone resistance (PMQR) genes were also studied. Multilocus sequence typing (MLST) was performed to identify STs. The most common ST was ST131 (33/51, 64.7%). All isolates, except one isolate, showed three mutations at codons 83 (S83L) and 87 (S87N) in gyrA and 80 (S80I) in parC The prevalence of ST131 in our hospital was much higher than reported in other Asian studies during a similar time period. The mutations found in ST131 were concordant with other studies.

Mutations in DNA Gyrase and Topoisomerase IV in Ciprofloxacin-Nonsusceptible Extended-Spectrum -Lactamase-Producing Escherichia coli and Klebsiella pneumoniae.

BACKGROUND: We investigated mutations in the quinolone resistance-determining region (QRDR) of ciprofloxacinnonsusceptible extended-spectrum -lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae by a statistical analysis. METHODS: We collected 97 clinical isolates of ciprofloxacin-nonsusceptible ESBL-producing E. coli (55 strains) and K. pneumoniae (42 strains) from a tertiary-care university hospital in Seoul, Republic of Korea, between 2006 and 2008. The QRDR of the gyrA, gyrB, parC, and parE genes were amplified by PCR and sequenced. RESULTS: Most E. coli isolates (53/55; 96.4%) with a minimum inhibitory concentration of ≥ 64 mg/L against ciprofloxacin had double mutations in gyrA (Ser-83Leu and Asp-87Asn) and at least one mutation in parC (Ser-80 Ile or Glu-84Val), with or without one in parE. Fifty E. coli (90.9%) isolates had a mutation in parE, of which Ile-529Leu (70.9%) was the most frequent. However, we could not find statistically significant variables in increasing ciprofloxacin resistance in E. coli isolates. Thirty-six K. pneumoniae isolates (36/42; 85.7%) had at least one mutation in gyrA, gyrB, or parC, and the mutation in gyrA might have been associated with plasmid-mediated quinolone-resistance (PMQR). Ser-80Ile in parC and aac(6')-Ib-cr in the K. pneumoniae isolates were significantly associated with an increased MIC of ciprofloxacin by ordinal logistic regression analysis. CONCLUSIONS: The Ser-80Ile in parC and aac(6')-Ib-cr in K. pneumoniae are supposed to play an important role in increased ciprofloxacin resistance, but statistically significant variables could not be found in E. coli isolates in the present study.

Comparison of the Xpert MTB/RIF Assay and Real-time PCR for the Detection of Mycobacterium tuberculosis.

INTRODUCTION: We compared the Xpert MTB/RIF assay with a real-time PCR assay using samples from culture-positive patients with TB. In addition, drug susceptibility test results were compared to evaluate the usefulness of these methods. MATERIALS AND METHODS: Fifty-two clinical specimens were analyzed by standard smear-microscopy, mycobacterial growth indicator tube (MGIT) culture, solid culture, MGIT drug-susceptibility testing, TB real-time PCR, and the Xpert MTB/RIF assay. RESULTS: Diagnostic sensitivity of AdvanSure TB/NTM real-time PCR was 80.0%. As shown from smear positive and negative specimens, sensitivities were 87.5% and 75.9%, respectively. The diagnostic sensitivity of Xpert MTB/RIF assay was 75.5%, and from smear positive and negative specimens, sensitivities were 93.8% and 65.5%, respectively. There were 10 cases with discrepant results between two methods. 2 cases were found resistant to rifampin, although Xpert MTB/RIF assay was able to detect rifampin resistance in only one specimen. DISCUSSION: Xpert MTB/RIF assay is an easier method to conduct and while its ability to detect rifampin resistance simultaneously is a benefit, its sensitivity from smear negative-culture positive specimens was lower than Advansure TB/NTM real-time PCR. Further investigation to increase the sensitivity and detect other drug resistances by kit-based assays is required for the rapid and accurate diagnosis of tuberculosis.

Prevalence of plasmid-mediated quinolone resistance genes among ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from blood cultures in Korea.

OBJECTIVES: To analyze the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from patients at a tertiary care hospital in Korea. METHODS: A total of 102 nonduplicate isolates of ciprofloxacin-intermediate or ciprofloxacin-resistant E coli (n=80) and K pneumoniae (n=22) from blood cultures were obtained. The qnr (qnrA, qnrB, qnrS), aac(6')-Ib-cr, qepA and oqxAB genes were detected using polymerase chain reaction (PCR) and confirmed using direct sequencing. To determine whether the PMQR-positive plasmid was horizontally transferable, conjugation experiments were performed. RESULTS: Of the 102 isolates, 81 (79.4%) had one or more PMQR genes; these consisted of 59 (73.8%) E coli and 22 (100%) K pneumoniae isolates. The qnr genes were present in 15 isolates (14.7%): qnrB4 was detected in 10.8% and qnrS1 was detected in 3.9%. The aac(6')-Ib-cr, qepA and oqxAB genes were detected in 77.5%, 3.9% and 10.8%, respectively. In conjugation experiments, PMQR genes were successfully transferred from seven (8.6%) isolates. The range of minimum inhibitory concentrations of ciprofloxacin for these seven transconjugants increased to 0.5 mg/L to 1 mg/L, which was 16- to 33-fold that of the recipient E coli J53 bacteria. CONCLUSIONS: PMQR genes were highly prevalent among ciprofloxacin-nonsusceptible E coli and K pneumoniae from blood cultures in the authors' hospital. Therefore, it is necessary to monitor for the spread of PMQR genes of clinical isolates and to ensure careful antibiotic use in a hospital setting.

OBJECTIFS: Analyser la prévalence des déterminants de la résistance à la quinolone à médiation plasmidique (RQMP) en cas d'Escherichia coli et de Klebsiella pneumoniae non susceptibles à la ciprofloxacine, isolés chez des patients d'un hôpital de soins tertiaires de la Corée. MÉTHODOLOGIE: Au total, les chercheurs ont obtenu 102 isolats non dupliqués d'E coli (n=80) et de K pneumoniae (n=22) moyennement résistants ou résistants à la ciprofloxacine dans les hémocultures. Ils ont décelé les gènes qnr (qnrA, qnrB, qnrS), aac(6')-Ib-cr, qepA et oqxAB au moyen de la réaction en chaîne de la polymérase (PCR) et les ont confirmés par séquençage direct. Pour déterminer si les plasmides ayant une RQMP pouvaient opérer un transfert horizontal, les chercheurs ont effectué des expériences de conjugaison. RÉSULTATS: Sur les 102 isolats, 81 (79,4 %) avaient au moins un gène de RQMP. De ce nombre, 59 (73,8 %) étaient des isolats d'E coli et 22 (100 %), de K pneumoniae. Les gènes qnr étaient présents dans 15 isolats (14,7 %), soit 10,8 % de gène qnrB4 et 3,9 % de gène qnrS1. Les gènes aac(6')-Ib-cr, qepA et oqxAB ont été décelés dans 77,5 %, 3,9 % et 10,8 % des isolats, respectivement. Dans les expériences de conjugaison, sept isolats (8,6 %) ont entraîné un transfert des gènes de RQMP. La plage de concentrations inhibitrices minimales de la ciprofloxacine de ces sept produits de transconjugaison est passée de 0,5 mg/L à 1 mg/L, soit 16 fois à 33 fois plus que celles des bactéries d'E coli J53 des receveurs. CONCLUSIONS: Les gènes de RQMP étaient hautement prévalents dans les hémocultures d'E coli et de K pneumoniae non susceptibles à la ciprofloxacine à l'hôpital des auteurs. Par conséquent, il faut surveiller la propagation des gènes de RQMP dans les isolats cliniques et vérifier attentivement l'utilisation des antibiotiques en milieu hospitalier.

Free light chain levels in pediatric patients with inflammatory conditions.

BACKGROUND: Because free light chain assays measure polyclonal as well as monoclonal free light chain components, some previous studies focused on the potential utility of the free light chain assay for detecting chronic immune stimulation, which occurs in autoimmune diseases and allergies. However, most of these trials have been limited to adult patients. In this study, we evaluated the paired serum and urinary free light chain levels of pediatric patients with various inflammatory conditions to investigate the clinical significance of free light chain measurement in pediatrics. METHODS: The study included 227 paired serum and urine specimens from 134 pediatric patients at our hospital between January and February of 2012. Serum and urinary FLC levels were measured using a Freelight Kit (The Binding Site, Ltd., Birmingham, UK). RESULTS: The serum lambda and urine kappa and lambda components were significantly increased only in the renal impairment group, not in the mild inflammatory group. FLC ratios were not significantly different among these groups. CONCLUSIONS: In serum, only the L components were significantly increased. This result may indicate the presence of a dimeric L structure, in contrast with monomeric K. FLC levels might also be influenced by renal conditions other than mild inflammation. Therefore, as shown in previous studies of adult patients, renal reference ranges might be needed to interpret FLC results, especially for dimeric L components.

Evaluation of a chromogenic culture medium for the detection of Clostridium difficile.

PURPOSE: Clostridium difficile (C. difficile) is an important cause of nosocomial diarrhea. Diagnostic methods for detection of C. difficile infection (CDI) are shifting to molecular techniques, which are faster and more sensitive than conventional methods. Although recent advances in these methods have been made in terms of their cost-benefit, ease of use, and turnaround time, anaerobic culture remains an important method for detection of CDI. MATERIALS AND METHODS: In efforts to evaluate a novel chromogenic medium for the detection of C. difficile (chromID CD agar), 289 fecal specimens were analyzed using two other culture media of blood agar and cycloserine-cefoxitin-fructose-egg yolk agar while enzyme immunosorbent assay and polymerase chain reaction-based assay were used for toxin detection. RESULTS: ChromID showed the highest detection rate among the three culture media. Both positive rate and sensitivity were higher from chromID than other culture media. ChromID was better at detecting toxin producing C. difficile at 24 h and showed the highest detection rate at both 24 h and 48 h. CONCLUSION: Simultaneous use of toxin assay and anaerobic culture has been considered as the most accurate and sensitive diagnostic approach of CDI. Utilization of a more rapid and sensitive chromogenic medium will aid in the dianogsis of CDI.

Comparison of two molecular methods for detecting toxigenic Clostridium difficile.

BACKGROUND: Clostridium difficile is one of the most common causes of nosocomial diarrhea, and diagnostic methods for detecting C. difficile infection have shifted from conventional to more recent molecular techniques. This study aimed to compare the performance of two molecular assays (Meridian Illumigene™ and AdvanSure CD real-time PCR) in detecting C. difficile using a toxigenic culture as a reference standard. MATERIALS AND METHODS: This study was conducted at Kyung Hee University Hospital, a tertiary university teaching hospital in Seoul, Korea, from July 2010 to February 2011. The study used 203 fresh diarrheal stools. All fecal specimens were immediately tested by culture and the VIDAS C. difficile toxin A & B assay using an automated VIDAS immunoanalyzer. The remainder was stored at -70°C until required for AdvanSure CD real-time polymerase chain reaction and Illumigene™. The alcohol shock procedure was then performed. Aliquots were inoculated directly on C. difficile-selective agar and blood agar and then incubated in an anaerobic jar for 48 h at 35°C. The Rapid ID 32 A test was used for specifying colonies on plates. The AdvanSure CD real-time PCR was used to detect the tcdA and tcdB gene, and PCR Illumigene™ kits were used to detect the tcdA gene of the pathogenicity locus (PaLoc) harboring toxigenic C. difficile. RESULTS: Of 203 clinical samples, 197 showed identical results between the two molecular assays, with a concordance rate of 97.0%. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: Illumigene: 92.3, 99.4, 96.0, and 98.9, respectively; AdvanSure CD real-time PCR: 84.6, 98.3, 88.0, and 97.8, respectively. CONCLUSIONS: Both molecular assays demonstrated good sensitivity and specificity. Additionally, both molecular assays showed comparable results to those of a toxigenic culture, albeit with a slight decrease in test sensitivity and specificity.

Factors influencing discordant results of the QuantiFERON-TB Gold In-tube test in patients with active TB.

OBJECTIVES: Indeterminate or negative results from the QuantiFERON-TB Gold In-tube test (QFT-GIT) for TB-confirmed patients indicate the lower sensitivity of this method. The aim of this study was to determine the factors associated with indeterminate and negative QFT-GIT results in active TB patients. METHODS: We analyzed retrospectively the laboratory and clinical data of patients diagnosed with TB between December 2009 and April 2012 at a tertiary university hospital in Seoul, Korea. RESULTS: Among 1301 patients who underwent QFT-GIT, TB-PCR and TB-culture, 168 (12.9%), those with positive TB-PCR or TB-culture were diagnosed with TB. Thirty-nine (23.2%) had indeterminate or negative results by QFT-GIT assay, which did not correlate with positive results of TB-PCR or TB-culture. These patients were older, had lower lymphocyte, total protein and albumin levels, and showed significantly higher CRP levels than the positive group. Multivariate logistic regression analysis showed that the probability of indeterminate and negative QFT-GIT results increased as CRP (odd ratio, 1.069; 95% CI, 1.013-1.127; P = 0.014) or age (1.030, 1.005-1.056, 0.02) increases. CONCLUSIONS: When levels of markers of inflammation, such as CRP, are high or the patient is older, QFT-GIT results should be interpreted carefully and correlated with additional tests for TB.

Investigation of mutation distribution in DNA gyrase and topoisomerase IV genes in ciprofloxacin-non-susceptible Enterobacteriaceae isolated from blood cultures in a tertiary care university hospital in South Korea, 2005-2010.

This study investigated the distribution of mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes and compared the distribution of these mutations with the distribution of plasmid-mediated quinolone resistance (PMQR) genes and extended-spectrum ß-lactamase (ESBL) production in 101 ciprofloxacin-non-susceptible Enterobacteriaceae from blood culture isolates (80 Escherichia coli and 21 Klebsiella pneumoniae) isolated in Kyung Hee University Hospital, a tertiary care university hospital in Seoul, South Korea. Among the 101 isolates, 80 (79.2%) contained PMQR genes and 28 (27.7%) produced ESBL. Mutations in the gyrA and parC genes were observed more frequently than in the gyrB and parE genes as well as more frequently in E. coli than in K. pneumoniae isolates, even in the same ciprofloxacin minimum inhibitory concentration (MIC) range of the two species. In E. coli isolates, the distribution of the codon 529 mutation (Ile→Leu) in parE was increased with an increase in the ciprofloxacin MIC. An increase in high-level resistance to quinolones may occur with double mutations compared with a single mutation in gyrA as well as with additional mutations in parC. However, this finding could not be applied to ciprofloxacin-resistant K. pneumoniae. A higher level of quinolone resistance may be correlated with an additional mutation in parE, especially Ile529→Leu.

Clinical significance of abnormal serum free light chain ratio: diagnostic confusion or underlying monoclonality?

BACKGROUND: The spectrum of laboratory tests used to detect monoclonal components (M-components) include serum and urine protein electrophoresis (PEP), immunofixation electrophoresis, and immunonephelometric methods such as free light chain assay (sFLC). METHODS: In this study, we retrospectively analyzed 78 patients who were to be tested with FLC without previous evidence of MG in order to investigate the clinical meaning of K/L screening. Abnormal K/L in sFLC was found in 25 samples from 21 patients (21/78, 26.92%). RESULTS: Among them, serum electrophoresis was requested for 16 patients where 5 were diagnosed as either normal or polyclonal gammopathy, 5 as plasma cell myeloma, 5 as monoclonal gammopathy of undetermined significance and I as amyloidosis. In total, 11 patients were revealed to have MG related diseases (11/25, 44.0 %). CONCLUSIONS: The clinical function of sFLC as a MG screening tool turned out to be effective based on the result where 16 out of 21 patients who were subject to further study led to diagnoses of MG related diseases in 11 patients. To provide an accurate evaluation for the performance of sFLC as a screening tool for MG, further studies should include additional confirmation of PEP results for patient groups that showed normal K/L ratios.

Quinolone-resistant Shigella flexneri isolated in a patient who travelled to India.

We report a recent case in which ciprofloxacin-resistant Shigella flexneri was isolated from a 23-yr-old female patient with a history of travel to India. Prior to her admission to our internal medicine department, she experienced symptoms of high fever and generalized weakness from continuous watery diarrhea that developed midway during the trip. S. flexneri was isolated from the stool culture. Despite initial treatment with ciprofloxacin, the stool cultures continued to show S. flexneri growth. In the susceptibility test for antibiotics of the quinolone family, the isolate showed resistance to ciprofloxacin (minimum inhibitory concentration [MIC], 8 µg/mL), norfloxacin (MIC, 32 µg/mL), ofloxacin (MIC, 8 µg/mL), nalidixic acid (MIC, 256 µg/mL), and intermediate resistance to levofloxacin (MIC, 4 µg/mL). In molecular studies for quinolone resistance related genes, plasmid borne-quinolone resistance genes such as qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA, and oqxAB were not detected. Two mutations were observed in gyrA (248C→T, 259G→A) and 1 mutation in parC (239G→T). The molecular characteristics of the isolated S. flexneri showed that the isolate was more similar to the strains isolated from the dysentery outbreak in India than those isolated from Korea.

Prevalence of the plasmid-mediated quinolone resistance genes, aac(6')-Ib-cr, qepA, and oqxAB in clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae in Korea.

In this study, we sought to determine the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes aac(6')-Ib-cr, qepA, and oqxAB in extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in South Korea. In total, 104 isolates (63 E. coli and 41 K. pneumoniae) were collected. We found that 23 of the 63 (36.5%) E. coli and nine of the 41 (22.0%) K. pneumoniae isolates were positive for aac(6')-Ib-cr. No isolate was positive for qepA, while transferable oqxAB was detected only in 10 (24.4%) K. pneumoniae isolates. Among the 32 aac(6')-Ib-cr-positive isolates, 30 (93.8%) were positive for both aac(6')-Ib-cr and bla(CTX-M) (CTX-M-15, -14, and -57). Our results suggest that PMQR determinants are highly prevalent in ESBL-producing E. coli and K. pneumoniae isolates in Korea.

Characteristics of outpatients with pandemic H1N1/09 influenza in a tertiary care university hospital in Korea.

The pandemic H1N1/09 emerged rapidly in Korea. Here, we describe the clinical characteristics of outpatients in Seoul, Korea who were infected in the 2009 H1N1 pandemic. We reviewed the cases of outpatients with pandemic H1N1/09 who visited a tertiary care teaching hospital between September 1 and December 31, 2009. Infection with pandemic H1N1/09 was confirmed by molecular tests. Of a total of 7,182 tests, 3,020 (42.0%) were positive. Compared with 473 cases of influenza- like illness (ILI), the 586 confirmed cases of pandemic H1N1/09 differed in age [odds ratio (OR) 0.975] and fulfilling at least one of the following factors: age < 5 or ≥ 65 years, history of contact with other pandemic H1N1/09-infected individuals (OR 0.611), fever ≥ 37.8°C (OR 3.567), cough (OR 2.290), and myalgia (OR 1.559). The sensitivity of the best criteria, "fever (≥ 37.8°C) plus cough" (41.03%) in this study was lower than that of the Korea Centers for Disease Control and Prevention (KCDC) criteria (47.95%), whereas the positive likelihood ratio (3.55) and positive predictive value (81.6) of this criteria was higher than those of the KCDC criteria (2.98 and 78.7, respectively). The clinical characteristics of pandemic H1N1/09 are, in many regards, indistinguishable from those of ILI. Moreover, the accuracy and predictability of criteria which include only symptoms or signs were not sufficient to diagnose pandemic H1N1/09 infection. Therefore, use of a combination of symptoms with confirmatory laboratory testing is necessary for accurate diagnosis of pandemic H1N1/09.