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Peter M Hawkey - One of the best experts on this subject based on the ideXlab platform.
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Utilizing Rapid Multiple-Locus Variable-Number Tandem-Repeat Analysis Typing To Aid Control of Hospital-Acquired Clostridium difficile Infection: a Multicenter Study
Journal of Clinical Microbiology, 2012Co-Authors: Katherine J. Hardy, Susan Manzoor, Claire Marriott, Helen Parsons, Claire Waddington, Savita Gossain, Ala Szczepura, Nigel Stallard, Peter M HawkeyAbstract:The early identification of outbreaks is crucial for the control of Clostridium difficile infection. This study aimed to determine if the number of hospital-acquired C. difficile infections could be reduced by rapidly typing C. difficile strains using multiple-locus variable-number tandem-repeat analysis (MLVA) compared to typing using PCR Ribotyping. A total of 16 hospitals were recruited to the study, and all periods of increased incidence (PIIs) of C. difficile infection were identified. The hospitals were randomized into two study arms, the test and the control, with all isolates typed in the test using MLVA and in the control using PCR Ribotyping. Following a PII, each hospital received a structured questionnaire regarding control measures implemented or stopped prior to or following the typing results. During the study period, there were a total of 1,682 hospital-apportioned C. difficile toxin-positive cases, with 868 in the control and 814 in the test, with modeling demonstrating no differences between the two arms. A total of 245 PIIs occurred, involving 785 patients. There was a significant difference in the mean turnaround time between the Ribotyping and MLVA typing (13.6 and 5.3 days, respectively [P < 0.001]). The discriminatory ability of MLVA was greater than Ribotyping, with 85 outbreaks being confirmed by Ribotyping and 62 by MLVA. In the test arm, 40.6% of respondents strongly agreed that the typing result had aided their management of clusters, as opposed to 9.9% in the control. The study demonstrated the utility of rapidly typing C. difficile strains, demonstrating that it aided the management of clusters, enabling effective targeting of infection control resources.
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extended multilocus variable number tandem repeat analysis of clostridium difficile correlates exactly with Ribotyping and enables identification of hospital transmission
Journal of Clinical Microbiology, 2011Co-Authors: Susan Manzoor, Peter M Hawkey, Hannah E Tanner, Clare Marriott, J S Brazier, Katie Hardy, S PlattAbstract:PCR Ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR Ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR Ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR Ribotyping.
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validation of use of whole cell repetitive extragenic palindromic sequence based pcr rep pcr for typing strains belonging to the acinetobacter calcoaceticus acinetobacter baumannii complex and application of the method to the investigation of a hospi
Journal of Clinical Microbiology, 1996Co-Authors: Andrea M Snelling, C Porter, P Parnell, Peter Gernersmidt, Peter M Hawkey, Andrew R. Bodenham, Timothy J J InglisAbstract:Acinetobacter spp. are being reported with increasing frequency as causes of nosocomial infection. In order to identify reservoirs of infection as quickly as possible, a rapid typing method that can differentiate epidemic strains from environmental and nonepidemic strains is needed. In 1993, a cluster of Acinetobacter baumannii isolates from five patients in the adult intensive therapy unit of our tertiary-care teaching hospital led us to develop and optimize a rapid repetitive extragenic palindromic sequence-based PCR (REP-PCR) typing protocol for members of the Acinetobacter calcoaceticus-A. baumannii complex that uses boiled colonies and consensus primers aimed at repetitive extragenic palindromic sequences. Four of the five patient isolates gave the same REP-PCR typing pattern as isolates of A. baumannii obtained from the temperature probe of a Bennett humidifier; the fifth isolate had a unique profile. Disinfection of the probe with 70% ethanol, as recommended by the manufacturer, proved ineffective, as A. baumannii with the same REP-PCR pattern was isolated from it 10 days after cleaning, necessitating a change in our decontamination procedure. Results obtained with REP-PCR were subsequently confirmed by Ribotyping. To evaluate the discriminatory power (D) of REP-PCR for typing members of the A. calcoaceticus-A. baumannii complex, compared with that of Ribotyping, we have applied both methods to a collection of 85 strains that included representatives of six DNA groups within the complex. Ribotyping using EcoRI digests yielded 53 patterns (D = 0.98), whereas 68 different REP-PCR patterns were observed (D = 0.99). By computer-assisted analysis of gel images, 74 patterns were observed with REP-PCR (D = 1.0). Overall, REP-PCR typing proved to be slightly more discriminatory than Ribotyping. Our results indicate that REP-PCR typing used boiled colonies is a simple, rapid, and effective means of typing members of the A. calcoaceticus-A. baumannii complex.
J S Brazier - One of the best experts on this subject based on the ideXlab platform.
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extended multilocus variable number tandem repeat analysis of clostridium difficile correlates exactly with Ribotyping and enables identification of hospital transmission
Journal of Clinical Microbiology, 2011Co-Authors: Susan Manzoor, Peter M Hawkey, Hannah E Tanner, Clare Marriott, J S Brazier, Katie Hardy, S PlattAbstract:PCR Ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR Ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR Ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR Ribotyping.
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comparison of toxinotyping and pcr Ribotyping of clostridium difficile strains and description of novel toxinotypes
Microbiology, 2001Co-Authors: Maja Rupnik, J S Brazier, Brian I Duerden, Miklavz Grabnar, Simon L J StubbsAbstract:Toxinotyping and PCR Ribotyping are two methods that have been used to type Clostridium difficile isolates. Toxinotyping is based on PCR-RFLP analysis of a 19 kb region encompassing the C. difficile pathogenicity locus. PCR Ribotyping is based on comparison of patterns of PCR products of the 16S–23S rRNA intergenic spacer region. Representative strains (101) from a C. difficile PCR ribotype library and 22 strains from previously described toxinotypes were analysed to compare Ribotyping with toxinotyping. Within this panel of strains all 11 toxinotypes (0–X) described previously and an additional 5 novel toxinotypes (XI–XV) were observed. PCR Ribotyping and toxinotyping correlated well and usually all strains within a given ribotype had similar changes in toxin genes. The new toxinotype XI comprises strains that did not express toxins TcdA or TcdB at detectable levels, but contained part of the tcdA gene. Strains of toxinotype XII exhibit changes only in the 5′ end of the tcdB gene. Toxinotype XIV is composed of strains that have a large insertion at the beginning of the tcdA gene. A total of 25 of the 89 tested PCR ribotypes of C. difficile contained variant strains. It was estimated that they represent 7·7% of the total number of strains in the Anaerobe Reference Unit collection.
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modification of a pcr Ribotyping method for application as a routine typing scheme forclostridium difficile
Anaerobe, 1996Co-Authors: G L Oneill, J S Brazier, F T Ogunsola, Brian I DuerdenAbstract:Abstract A modification of a PCR Ribotyping procedure based on polymorphisms in the 16S-23S intergenic spacer region was evaluated for use as a typing method for Clostridium difficile . This procedure depends on the variation that can occur in the intergenic space between the 16S and 23S rRNA genes of the ribosomal RNA gene complex. The primers used in this study were chosen by examining the sequence of the 16S gene of C. difficile and the 23S gene of C. botulinum . The primers used were: CTG GGG TGA AGT CGT AAC AAG G (positions 1445-1466 in the 16S rRNA gene) and GCG CCC TTT GTA GCT TGA CC (positions 1-20 in the 23S rRNA gene) and the PCR parameters were optimised for this primer pair. To evaluate the discriminatory power of the method, PCR Ribotyping was performed on strains of C. difficile serotyped by Delmee (serogroups A-X and sub-serogroups A2-A10). Each isolate gave multiple DNA bands in PCR Ribotyping and a series of products ranging in size from 260 to 585 bp in length was obtained. All of the 19 different serogroups gave different banding patterns and these patterns were reproducible. This modification of PCR Ribotyping offers several advantages over the original method and appears to hold much promise as a method for typing wild isolates of C. difficile .
Katie Hardy - One of the best experts on this subject based on the ideXlab platform.
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extended multilocus variable number tandem repeat analysis of clostridium difficile correlates exactly with Ribotyping and enables identification of hospital transmission
Journal of Clinical Microbiology, 2011Co-Authors: Susan Manzoor, Peter M Hawkey, Hannah E Tanner, Clare Marriott, J S Brazier, Katie Hardy, S PlattAbstract:PCR Ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR Ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR Ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR Ribotyping.
Susan Manzoor - One of the best experts on this subject based on the ideXlab platform.
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Utilizing Rapid Multiple-Locus Variable-Number Tandem-Repeat Analysis Typing To Aid Control of Hospital-Acquired Clostridium difficile Infection: a Multicenter Study
Journal of Clinical Microbiology, 2012Co-Authors: Katherine J. Hardy, Susan Manzoor, Claire Marriott, Helen Parsons, Claire Waddington, Savita Gossain, Ala Szczepura, Nigel Stallard, Peter M HawkeyAbstract:The early identification of outbreaks is crucial for the control of Clostridium difficile infection. This study aimed to determine if the number of hospital-acquired C. difficile infections could be reduced by rapidly typing C. difficile strains using multiple-locus variable-number tandem-repeat analysis (MLVA) compared to typing using PCR Ribotyping. A total of 16 hospitals were recruited to the study, and all periods of increased incidence (PIIs) of C. difficile infection were identified. The hospitals were randomized into two study arms, the test and the control, with all isolates typed in the test using MLVA and in the control using PCR Ribotyping. Following a PII, each hospital received a structured questionnaire regarding control measures implemented or stopped prior to or following the typing results. During the study period, there were a total of 1,682 hospital-apportioned C. difficile toxin-positive cases, with 868 in the control and 814 in the test, with modeling demonstrating no differences between the two arms. A total of 245 PIIs occurred, involving 785 patients. There was a significant difference in the mean turnaround time between the Ribotyping and MLVA typing (13.6 and 5.3 days, respectively [P < 0.001]). The discriminatory ability of MLVA was greater than Ribotyping, with 85 outbreaks being confirmed by Ribotyping and 62 by MLVA. In the test arm, 40.6% of respondents strongly agreed that the typing result had aided their management of clusters, as opposed to 9.9% in the control. The study demonstrated the utility of rapidly typing C. difficile strains, demonstrating that it aided the management of clusters, enabling effective targeting of infection control resources.
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extended multilocus variable number tandem repeat analysis of clostridium difficile correlates exactly with Ribotyping and enables identification of hospital transmission
Journal of Clinical Microbiology, 2011Co-Authors: Susan Manzoor, Peter M Hawkey, Hannah E Tanner, Clare Marriott, J S Brazier, Katie Hardy, S PlattAbstract:PCR Ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR Ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR Ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR Ribotyping.
Hailu Kinde - One of the best experts on this subject based on the ideXlab platform.
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use of Ribotyping for characterization of salmonella serotypes
Journal of Clinical Microbiology, 1993Co-Authors: E Esteban, Kurt P Snipes, D Hird, Rickie W Kasten, Hailu KindeAbstract:Abstract Forty-five isolates of Salmonella serotype reading, 20 isolates of Salmonella serotype senftenberg, and 56 isolates of Salmonella serotype typhimurium from domestic and wild animals were characterized genotypically to differentiate within serotypes for epidemiologic studies. The genotypic method of characterization used was Ribotyping, a method for highlighting highly conserved rRNA genes and associated sequences. Isolates were obtained from diverse geographic sources (farms located in Fresno, Sonoma, Stanislaus, and Yolo counties) as well as different hosts (avian, equine, bovine, murine, and environmental) during a period of 8 months. Within a given serotype, ribotying was able to establish subclassifications (ribotypes) that grouped isolates by a common source regardless of host or geographic origin. There were four distinct ribosomal banding patterns observed for Salmonella serotype reading, six were observed for Salmonella serotype senftenberg, and two were observed for Salmonella serotype typhimurium.
