Hepatitis C virus (HCV) sequences recovered from serum, peripheral blood mononuclear cells (PBMCs), and various tissues from human immunodeficiency virus type 1 (HIV-1) positive patients were compared by single strand conformational polymorphism (SSCP) and sequencing. In five patients, paired serum and PBMCs samples were analyzed while in two other patients multiple autopsy tissues were studied. Sequences amplified from the NS5 and E2 regions were consistently identical in the same patient; however, three PBMCs samples and three different tissue samples (pancreas and adrenal gland in one patient and lymph node in the other patient) contained 5' untranslated region (5'UTR) sequences that were different from circulating sequences. The presence of 5' UTR sequences differing from circulating sequences correlated with the presence of HCV RNA negative strand, as the latter was detected by a Tth-based strand-specific assay in all but one of these samples. These two independent lines of evidence: viral sequence differences and the presence of RNA negative strand in the same tissues strongly argue for the genuine presence of extrahepatic HCV replication, at least in the setting of HIV-1 infection.

BackgroundNational surveillance of antimicrobial resistance becomes more important for the control of antimicrobial resistance and determination of treatment guidelines. We analyzed Korean Antimicrobial Resistance Monitoring System (KARMS) data collected from 2013 to 2015.MethodsOf the KARMS participants, 16 secondary or tertiary hospitals consecutively reported antimicrobial resistance rates from 2013 to 2015. Data from duplicate isolates and institutions with fewer than 20 isolates were excluded. To determine the long-term trends, previous KARMS data from 2004 to 2012 were also considered.ResultsThe prevalence of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium from 2013 to 2015 was 66–72% and 29–31%, respectively. The resistance rates of Escherichia coli to cefotaxime and cefepime gradually increased to 35% and 31%, respectively, and fluoroquinolone resistance reached 48% in 2015. The resistance rates of Klebsiella pneumoniae to cefotaxime, cefepime, and carbapenem were 38–41%, 33–41%, and <0.1–2%, respectively, from 2013 to 2015. The carbapenem susceptibility rates of E. coli and K. pneumoniae decreased from 100% and 99.3% in 2011 to 99.0% and 97.0% in 2015, respectively. The resistance rate of Pseudomonas aeruginosa to carbapenem increased to 35% and the prevalence of carbapenem-resistant Acinetobacter baumannii increased from 77% in 2013 to 85% in 2015.ConclusionsBetween 2013 and 2015, the resistance rates of E. coli to third- and fourth-generation cephalosporins increased continuously, while carbapenem-susceptibility gradually decreased, particularly in K. pneumoniae. The prevalence of carbapenem-resistant P. aeruginosa and A. baumannii increased significantly; therefore, few treatment options remain for these resistant strains.

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cThe emerging fungal pathogens Candida haemulonii and Candida pseudohaemulonii often show high-level resistance to amphotericin B (AMB). We compared the utilities of five antifungal susceptibility testing methods, i.e., the Etest using Mueller-Hinton agar supplemented with glucose and methylene blue (Etest-MH), the Etest using RPMI agar supplemented with glucose (Etest-RPG), the Vitek-2 yeast susceptibility system, and the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution methods, for the detection of AMB-resistant isolates of C. haemulonii and closely related species. Thirty-eight clinical isolates (8 C. haemulonii, 10 C. pseudohaemulonii, and 20 Candida auris isolates) were analyzed. Of the 18 C. haemulonii and C. pseudohaemulonii isolates, 18, 15, 18, 10, and 9 exhibited AMB MICs of >1 g/ml by the Etest-MH, Etest-RPG, Vitek-2, CLSI, and EUCAST methods, respectively. All 20 C. auris isolates showed AMB MICs of <1 g/ml by all five methods. Of the methods, the Etest-MH generated the broadest distribution of AMB MICs for all 38 isolates and showed the best discrimination between the C. haemulonii and C. pseudohaemulonii isolates (4 to 32 g/ml) and those of C. auris (0.125 to 0.5 g/ml). Taking the Etest-MH as the reference method, the essential agreements (within two dilutions) for the Etest-RPG, Vitek-2, CLSI, and EUCAST methods were 84, 92, 55, and 55%, respectively; the categorical agreements were 92, 92, 79, and 76%, respectively. This study provides the first data on the efficacy of the Etest-MH and its excellent agreement with Vitek-2 for discriminating AMB-resistant from AMB-susceptible isolates of these Candida species.

Among 222 Proteus mirabilis clinical isolates collected from 17 hospitals in Korea in 2008, 28 (12.6%) and 8 (3.6%) isolates exhibited extended-spectrum ␤-lactamase (ESBL) and AmpC phenotypes, respectively. The most common type of ESBL gene identified by PCR and sequencing experiments was bla CTX-M-14a (n ‫؍‬ 12). The bla CTX-M-90 (n ‫؍‬ 4), bla CTX-M-15 (n ‫؍‬ 3), bla CTX-M-12 (n ‫؍‬ 3), bla CTX-M-2 (n ‫؍‬ 2), bla CTX-M-14b (n ‫؍‬ 1), bla TEM-52 (n ‫؍‬ 5), and bla SHV-12 (n ‫؍‬ 1) genes were also detected. Eight isolates carried an AmpC ␤-lactamase gene, such as bla CMY-2 (n ‫؍‬ 6) or bla DHA-1 (n ‫؍‬ 2). All bla genes encoding CTX-M-1-and CTX-M-9-type enzymes and all bla CMY-2 genes were preceded by ISEcp1-like elements. The bla CTX-M-2 gene found in two isolates was located on a complex class 1 integron. The bla DHA-1 gene was preceded by a transcriptional regulator gene and was followed by phage shock protein genes. The bla CTX-M genes were located on the chromosome in 21 isolates. A plasmid location for the bla CTX-M gene was found in only four isolates: the bla CTX-M-14a gene was located on ϳ150-kbp IncA/C plasmids in three isolates and on a ϳ50-kbp IncN plasmid in one isolate. The bla TEM-52 gene was located on ϳ50-kbp IncN plasmids in all five isolates. The AmpC ␤-lactamase genes were located on the chromosome in seven of eight isolates; one isolate carried the bla CMY-2 gene on a ϳ150-kbp IncA/C plasmid. Our results show that a chromosomal location of CTX-M ESBL and AmpC ␤-lactamase genes in P. mirabilis is no longer an unusual phenomenon in hospital environments.

We conducted a prospective study to target toxR in the blood of patients with skin and soft tissue infections who were admitted to four tertiary hospitals to assess the clinical usefulness of real-time quantitative PCR (Q-PCR) as a diagnostic technique. We performed conventional PCR (C-PCR), nested PCR (N-PCR), and Q-PCR assays and compared the results to those obtained using the "gold standard" of microbiological culture. The lower detection limit for the Q-PCR assay was 5 ؋ 10 0 copies/l. By use of blood samples of patients with skin and soft tissue infections, the sensitivities of the C-PCR and N-PCR assays against the target toxR gene of V. vulnificus as diagnostic tools were determined to be 45% and 86%, respectively. The C-PCR and N-PCR assays had specificities of 100% and 73%, respectively. When we adopted a crossing-point (cp) cutoff value of <38 cp as a positive result, the Q-PCR assay had 100% sensitivity and specificity. Q-PCR to detect V. vulnificus-specific genes is not only the most sensitive and specific of the techniques but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay using blood is useful for the rapid diagnosis and subsequent treatment of V. vulnificus sepsis.Vibrio vulnificus can cause severe and life-threatening disease in those who eat contaminated seafood or have a wound that is exposed to seawater (2,11,15,24). The disease develops rapidly and mortality is high. Hence, these patients require rapid diagnosis and subsequent treatment. Microbiological culture methods for the identification of causative organisms take several days; they are time-consuming and laborious but have good specificity (13). PCR assays have proven useful for early diagnosis. Conventional PCR (C-PCR) has been used to detect specific target genes in various microorganisms (5, 6, 13). Nested PCR (N-PCR) was developed to improve sensitivity but can give erroneous positive results due to DNA contamination (1). Multiplex PCR has the advantage of detecting several target genes at the same time, but it is time-consuming and laborious like 14). Real-time quantitative PCR (Q-PCR) can detect V. vulnificus-specific genes within 2 h (4, 15); there is no agarose gel-loading step (23), and the assay is not laborious and has high sensitivity and specificity (22).Up to now there has been little comparative evaluation of these three PCR methods, namely, C-PCR, N-PCR, and Q-PCR, for targeting V. vulnificus-specific genes. The toxR gene is known as a gene encoding a transmembrane DNA binding regulatory protein in Vibrio species. The partial sequences of toxR differ among Vibrio species. The difference in toxR sequences among Vibrio species has been used as an effective marker for the identification of Vibrio species (22). To assess the clinical usefulness of Q-PCR as a diagnostic technique, we conducted a prospective study targeting the toxR gene of V. vulnificus in blood samples of patients with skin and soft tissue infections who were admitted to four tertiary-care hospitals. We carr...

BackgroundSeveral factors contribute to differences in Streptococcus pneumoniae serotype distribution. We investigated the serotype distribution and antimicrobial resistance of S. pneumoniae isolated between 2014 and 2016 in Korea.MethodsWe collected a total of 1,855 S. pneumoniae isolates from 44 hospitals between May 2014 and May 2016, and analyzed the serotypes by sequential multiplex PCR. We investigated the distribution of each serotype by patient age, source of the clinical specimen, and antimicrobial resistance pattern.ResultsThe most common serotypes were 11A (10.1%), followed by 19A (8.8%), 3 (8.5%), 34 (8.1%), 23A (7.3%), and 35B (6.2%). The major invasive serotypes were 3 (12.6%), 19A (7.8%), 34 (7.8%), 10A (6.8%), and 11A (6.8%). Serotypes 10A, 15B, 19A, and 12F were more common in patients ≤5 years old, while serotype 3 was more common in patients ≥65 years old compared with the other age groups. The coverage rates of pneumococcal conjugate vaccine (PCV)7, PCV10, PCV13, and pneumococcal polysaccharide vaccine 23 were 11.8%, 12.12%, 33.3%, and 53.6%, respectively. Of the 1,855 isolates, 857 (46.2%) were multi-drug resistant (MDR), with serotypes 11A and 19A predominant among the MDR strains. The resistance rates against penicillin, cefotaxime, and levofloxacin were 22.8%, 12.5%, and 9.4%, respectively.ConclusionsThere were significant changes in the major S. pneumoniae serotypes in the community. Non-PCV13 serotypes increased in patients ≤5 years old following the introduction of national immunization programs with the 10- and 13-polyvalent vaccines.

BackgroundAcinetobacter baumannii infections are difficult to treat owing to the emergence of various antibiotic resistant isolates. Because treatment options are limited for multidrug-resistant (MDR) A. baumannii infection, the discovery of new therapies, including combination therapy, is required. We evaluated the synergistic activity of colistin, doripenem, and tigecycline combinations against extensively drug-resistant (XDR) A. baumannii and MDR A. baumannii.MethodsTime-kill assays were performed for 41 XDR and 28 MDR clinical isolates of A. baumannii by using colistin, doripenem, and tigecycline combinations. Concentrations representative of clinically achievable levels (colistin 2 µg/mL, doripenem 8 µg/mL) and achievable tissue levels (tigecycline 2 µg/mL) for each antibiotic were used in this study.ResultsThe colistin-doripenem combination displayed the highest rate of synergy (53.6%) and bactericidal activity (75.4%) in 69 clinical isolates of A. baumannii. Among them, thedoripenem-tigecycline combination showed the lowest rate of synergy (14.5%) and bacteri-cidal activity (24.6%). The doripenem-tigecycline combination showed a higher antagonistic interaction (5.8%) compared with the colistin-tigecycline (1.4%) combination. No antagonism was observed for the colistin-doripenem combination.ConclusionsThe colistin-doripenem combination is supported in vitro by the high rate of synergy and bactericidal activity and lack of antagonistic reaction in XDR and MDR A. baumannii. It seems to be necessary to perform synergy tests to determine the appropri-ate combination therapy considering the antagonistic reaction found in several isolates against the doripenem-tigecycline and colistin-tigecycline combinations. These findings should be further examined in clinical studies.