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Tang H.-L.,National Chung Hsing University | Tang H.-L.,China Medical University at Taichung | Chiang M.-K.,National Chung Cheng University | Liou W.-J.,Chung Shan Medical University | And 8 more authors.
European Journal of Clinical Microbiology and Infectious Diseases | Year: 2010

Klebsiella pneumoniae-caused liver abscess (KLA) is an emerging infectious disease. However, factors other than K1-specific loci that contribute to the pathogenesis of this disease have not been identified. pLVPK is a 219,385-bp plasmid of K. pneumoniae CG43, an invasive K2 strain associated with KLA. We aimed in this study to evaluate the involvement of pLVPK in K. pneumoniae virulence and its clinical significance in abscess formation. A pLVPK-cured CG43 was isolated and its virulence was examined in a mouse model. The prevalence of pLVPK-derived loci terW, iutA, rmpA, silS, and repA was investigated in 207 clinical isolates by screening with specific primers. Loss of pLVPK abolished the ability of K. pneumoniae to disseminate into extraintestinal sites and, consequently, attenuated abscess formation in mice. Primary K. pneumoniae abscess isolates (n=94) were more likely to be terW +-iutA +-rmpA +-silS + than those related to non-abscess infections (n=113) (62% vs. 27%; p<0.0001). Logistic regression analysis indicated that the presence of the terW-rmpA-iutA-silS loci was a significant risk factor (odds ratio, 4.12; 95% confidence interval, 2.02-8.4; p<0.0001) for abscess formation. pLVPK is a determinant for K. pneumoniae virulence and infection with strains carrying the pLVPK-derived terW-rmpA-iutA-silS loci may predispose patients to abscess formation. © 2010 Springer-Verlag.

Wu S.-L.,A-Life Medical | Wu S.-L.,Center for Research and Diagnostics | Wang S.-C.,National Health Research Institute | Tsou H.-H.,National Health Research Institute | And 10 more authors.
PLoS ONE | Year: 2013

Background and Objectives:Heroin-dependent patients typically contract hepatitis C virus (HCV) at a disproportionately high level due to needle exchange. The liver is the primary target organ of HCV infection and also the main organ responsible for drug metabolism. Methadone maintenance treatment (MMT) is a major treatment regimen for opioid dependence. HCV infection may affect methadone metabolism but this has rarely been studied. In our current study, we aimed to test the hypothesis that HCV may influence the methadone dosage and its plasma metabolite concentrations in a MMT cohort from Taiwan.Methods:A total of 366 MMT patients were recruited. The levels of plasma hepatitis B virus (HBV), HCV, human immunodeficiency virus (HIV) antibodies (Ab), liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) were measured along with the urine morphine concentration and amphetamine screening.Results:Of the 352 subjects in our cohort with HCV test records, 95% were found to be positive for plasma anti-HCV antibody. The liver functional parameters of AST (Wilcoxon Rank-Sum test, P = 0.02) and ALT (Wilcoxon Rank-Sum test, P = 0.04), the plasma methadone concentrations (Wilcoxon Rank-Sum test, P = 0.043) and the R-enantiomer of methadone concentrations (Wilcoxon Rank-Sum test, P = 0.032) were significantly higher in the HCV antibody-positive subjects than in the HCV antibody-negative patients, but not the S-EDDP/methadone dose ratio. The HCV levels correlated with the methadone dose (β̂ = 14.65 and 14.13; P = 0.029 and 0.03) and the S-EDDP/methadone dose ratio (β̂ = -0.41 and -0.40; P = 0.00084 and 0.002) in both univariate and multivariate regression analyses.Conclusions:We conclude that HCV may influence the methadone dose and plasma S-EDDP/methadone dose ratio in MMT patients in this preliminary study. © 2013 Wu et al.

Wu F.-T.,Center for Research and Diagnostics | Wu F.-T.,National Yang Ming University | Banyai K.,Veterinary Medical Research Institute | Huang J.C.,National Yang Ming University | And 10 more authors.
Clinical Microbiology and Infection | Year: 2011

Genotype P[25] rotaviruses are rare and to date have been reported to occur only in a few countries of mainland Asia. Here we report the molecular characterization of a novel human rotavirus genotype combination, G3P[25], detected in a 17-month-old child hospitalized due to severe gastroenteritis during 2009 in central Taiwan. Sequencing and phylogenetic analysis of the VP4 gene demonstrated a distinct origin from other strains bearing the P[25] VP4 gene, whereas the VP7, VP6 and NSP4 gene phylogenies identified common origins with cognate genes of other, presumed human-porcine reassortment Taiwanese strains. These results suggest that interactions between human and animal strains appear to contribute to the generation of genetic and antigenic diversity of rotavirus strains, with potential public health importance in Taiwan. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Wei H.L.,Center for Research and Diagnostics | Wei H.L.,National Chung Hsing University | Kao C.,Center for Research and Diagnostics | Wei S.H.,Third Branch Office | And 2 more authors.
BMC Microbiology | Year: 2011

Background: Polymerase chain reaction (PCR) ribotyping is one of the globally accepted techniques for defining epidemic clones of Clostridium difficile and tracing virulence-related strains. However, the ambiguous data generated by this technique makes it difficult to compare data attained from different laboratories; therefore, a portable technique that could supersede or supplement PCR ribotyping should be developed. The current study attempted to use a new multilocus variable-number tandem-repeat analysis (MLVA) panel to detect PCR-ribotype groups. In addition, various MLVA panels using different numbers of variable-number tandem-repeat (VNTR) loci were evaluated for their power to discriminate C. difficile clinical isolates. Results: At first, 40 VNTR loci from the C. difficile genome were used to screen for the most suitable MLVA panel. MLVA and PCR ribotyping were implemented to identify 142 C. difficile isolates. Groupings of serial MLVA panels with different allelic diversity were compared with 47 PCR-ribotype groups. A MLVA panel using ten VNTR loci with limited allelic diversity (0.54-0.83), designated MLVA10, generated groups highly congruent (98%) with the PCR-ribotype groups. For comparison of discriminatory power, a MLVA panel using only four highly variable VNTR loci (allelic diversity: 0.94-0.96), designated MLVA4, was found to be the simplest MLVA panel that retained high discriminatory power. The MLVA10 and MLVA4 were combined and used to detect genetically closely related C. difficile strains. Conclusions: For the epidemiological investigations of C. difficile, we recommend that MLVA10 be used in coordination with the PCR-ribotype groups to detect epidemic clones, and that the MLVA4 could be used to detect outbreak strains. MLVA10 and MLVA4 could be combined in four multiplex PCR reactions to save time and obtain distinguishable data. © 2011 Wei et al; licensee BioMed Central Ltd.

Wang S.-C.,National Health Research Institute | Ho I.-K.,National Health Research Institute | Wu S.-L.,A-Life Medical | Wu S.-L.,Center for Research and Diagnostics | And 4 more authors.
Biomedical Chromatography | Year: 2010

A liquid chromatography-photodiode array (LC-PDA) method using a chiral analytical column was developed to determine the plasma levels of enantiomers of methadone and its chiral metabolite, 2-ethylidene-1,5-dimethyl-3,3- diphenylpyrrolidine (EDDP), without the standard compounds of R-form or S-form enantiomers. This method was established by the characteristics of recombinant cytochrome P-450 (CYP) isozymes, where CYP2C19 prefers to metabolize R-methadone and CYP2B6 prefers to metabolize S-methadone. We incubated the racemic methadone standard with either enzyme for 24 h. We identifi ed the retention times of R- and S-methadone to be around 10.72 and 14.46 min, respectively. Furthermore, we determined the retention times of R- and S-EDDP to be approximately 6.76 and 7.72 min, respectively. No interferences were shown through the retention times of morphine, buprenorphine and diazepam. With the high recovery rate of a solid-phase extraction procedure, this method was applied in analyzing plasma concentrations of seven methadone maintenance patients where R- and S-methadone and R- and S-EDDP were 233.4 ± 154.9 and 185.9 ± 136.3 ng/mL and 84.4 ± 99.4 and 37.6 ± 22.9 ng/mL, respectively. These data suggest that the present method can be applied for routine assay for plasma methadone and EDDP concentrations for patients under treatment. Copyright © 2009 John Wiley & Sons, Ltd.

Lin J.-H.,Center for Research and Diagnostics | Lin J.-H.,National Taiwan Ocean University | Chiu S.-C.,Center for Research and Diagnostics | Chiu S.-C.,National Yang Ming University | And 9 more authors.
PLoS ONE | Year: 2011

Background: Many studies concentrate on variation in the hemagglutinin glycoprotein (HA) because of its significance in host immune response, the evolution of this virus is even more complex when other genome segments are considered. Recently, it was found that cytotoxic T lymphocytes (CTL) play an important role in immunity against influenza and most CTL epitopes of human influenza viruses were remarkably conserved. The NP gene has evolved independently in human and avian hosts after 1918 flu pandemic and it has been assigned a putative role as a determinant of host range. Methods and Findings: Phylodynamic patterns of the genes encoding nucleoprotein (NP) of influenza A viruses isolated from 1979-2009 were analyzed by applying the Bayesian Markov Chain Monte Carlo framework to better understand the evolutionary mechanisms of these Taiwanese isolates. Phylogenetic analysis of the NP gene showed that all available H3 worldwide isolates collected so far were genetically similar and divided into two major clades after the year 2004. We compared the deduced amino acid sequences of the NP sequences from human, avian and swine hosts to investigate the emergence of potential adaptive mutations. Overall, selective pressure on the NP gene of human influenza A viruses appeared to be dominated by purifying selection with a mean d N/d S ratio of 0.105. Site-selection analysis of 488 codons, however, also revealed 3 positively selected sites in addition to 139 negatively selected ones. Conclusions: The demographic history inferred by Bayesian skyline plot showed that the effective number of infections underwent a period of smooth and steady growth from 1998 to 2001, followed by a more recent rise in the rate of spread. Further understanding the correlates of interspecies transmission of influenza A virus genes from other host reservoirs to the human population may help to elucidate the mechanisms of variability among influenza A virus. © 2011 Lin et al.

Chiou C.-S.,Center for Research and Diagnostics | Chiou C.-S.,Chung Shan Medical University | Hung C.-S.,Center for Research and Diagnostics | Torpdahl M.,Statens Serum Institute | And 5 more authors.
International Journal of Food Microbiology | Year: 2010

We identified 16 variable number tandem repeat (VNTR) loci for Salmonella enterica serovar Typhimurium. These VNTRs were evaluated with panels of 183 diverse isolates, 203 closely related isolates and 54 isolates from seven outbreaks. The evaluations revealed that five of the 16 VNTRs had diversity values greater than 0.5, and three (STTR5, STTR6 and STTR10) were hypervariable. The results obtained from the outbreak isolates suggested that the 16 VNTRs were considerably stable in isolates recovered during a normal outbreak time course. Multilocus VNTR analysis (MLVA) based on four most variable VNTRs (MLVA4), exhibited a better resolving power over pulsed-field gel electrophoresis (PFGE) in discriminating among isolates, in particular among the closely-related isolates. An MLVA5, which is based on five VNTRs and has been widely used in many European laboratories, displayed a level of discrimination close to MLVA4. The phylogenetic tree established using the MLVA16 profiles presented four distinct clusters, which were associated with four different phage types. Therefore, MLVA based on four or five highly variable VNTRs is sufficiently powerful to supplement or replace PFGE for outbreak investigation and surveillance of S. Typhimurium infections, and MLVA data based on 16 VNTRs can be useful in establishing clonal structures among isolates. © 2010 Elsevier B.V.

Koh X.P.,University of Malaya | Chiou C.S.,Center for Research and Diagnostics | Ajam N.,University of Malaya | Watanabe H.,Japan National Institute of Infectious Diseases | And 2 more authors.
BMC Infectious Diseases | Year: 2012

Background: Shigellosis is a major public health concern worldwide, especially in developing countries. It is an acute intestinal infection caused by bacteria of the genus Shigella, with a minimum infective dose as low as 10-100 bacterial cells. Increasing prevalence of Shigella sonnei as the etiologic agent of shigellosis in Malaysia has been reported. As there is limited information on the genetic background of S. sonnei in Malaysia, this study aimed to characterize Malaysian S. sonnei and to evaluate the prospect of using multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for subtyping of local S. sonnei.Methods: Forty non-repeat clinical strains of S. sonnei isolated during the years 1997-2000, and 2007-2009 were studied. The strains were isolated from stools of patients in different hospitals from different regions in Malaysia. These epidemiologically unrelated strains were characterized using biotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and MLVA.Results: The two biotypes identified in this study were biotype a (n = 29, 73%) and biotype g (n = 11, 27%). All the 40 strains were sensitive to kanamycin, ceftriaxone and ciprofloxacin. Highest resistance rate was observed for streptomycin (67.5%), followed by tetracycline (40%) and trimethoprim-sulfamethoxazole (37.5%). All the S. sonnei biotype g strains had a core resistance type of streptomycin - trimethoprim-sulfamethoxazole - tetracycline whereas the 29 biotype a strains were subtyped into eight resistotypes. All the strains were equally distinguishable by PFGE and MLVA. Overall, PFGE analysis indicated that S. sonnei biotype a strains were genetically more diverse than biotype g strains. Cluster analysis by MLVA was better in grouping the strains according to biotypes, was reflective of the epidemiological information and was equally discriminative as PFGE.Conclusions: The S. sonnei strains circulating in Malaysia throughout the period studied were derived from different clones given their heterogeneous nature. MLVA based on seven selected VNTR loci was rapid, reproducible and highly discriminative and therefore may complement PFGE for routine subtyping of S. sonnei. © 2012 Koh et al.; licensee BioMed Central Ltd.

Tien Y.-Y.,Tamkang University | Ushijima H.,Aino University | Mizuguchi M.,University of Tokyo | Liang S.-Y.,Center for Research and Diagnostics | And 2 more authors.
Journal of Medical Microbiology | Year: 2012

We evaluated 11 variable number tandem repeat (VNTR) markers for the epidemiological investigation of Salmonella enterica serovar Typhi (S. Typhi) infection and compared the results to those obtained by PFGE. PFGE, using one or two restriction enzymes (XbaI and BlnI), was insufficient to differentiate between some isolates that were epidemiologically unlinked. Multilocus variable-number tandem repeat analysis (MLVA)-8, based on analysis of the eight most variable VNTRs, displayed a high level of discrimination when distinguishing between epidemiologically unlinked isolates that could not be discerned by PFGE with two enzymes. An MLVA-8 typing scheme could be implemented as a routine subtyping tool for the epidemiological investigation of S. Typhi infections. Because seven of the 11 VNTRs are highly variable, the VNTR markers may only be useful in determining genetic relationships among very closely related isolates in short-term epidemiological studies and not for discerning S. Typhi clones. © 2012 SGM.

Tien Y.-Y.,Tamkang University | Wang Y.-W.,Center for Research and Diagnostics | Tung S.K.,Center for Research and Diagnostics | Liang S.-Y.,Center for Research and Diagnostics | And 2 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2011

Salmonella enterica serotype Paratyphi A is a highly clonal organism; pulsed-field gel electrophoresis (PFGE) is insufficient in discriminating isolates. A multilocus variable-number tandem repeat analysis (MLVA) was developed, and its usefulness in discriminating isolates was compared. PFGE analysis with XbaI and BlnI discriminated 55 isolates into 14 types, with a discriminatory index (DI) of 0.741 (confidence interval [CI], 0.635-0.847). MLVA divided the isolates into 23 types, with a DI of 0.937 (CI, 0.909-0.964), which was significantly higher than that for PFGE. Clustering analysis of PFGE and MLVA patterns indicated that S. Paratyphi A isolates recovered from 2000 to 2010 in South and Southeast Asia were highly clonal. Although MLVA is not sufficiently powerful in discriminating epidemiologically unrelated isolates, it can complement PFGE for epidemiologic investigation of S. Paratyphi A infections. © 2011 Elsevier Inc.

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