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1. A culture-independent nucleic acid diagnostics method for use in the detection and quantification of Burkholderia cepacia complex contamination in aqueous finished pharmaceutical products.

Author

Duong, Huong, Minogue, Elizabeth, Fullbrook, Shannon, Barry, Thomas, and Reddington, Kate

Subjects
BURKHOLDERIA cepacia, NUCLEIC acids, ANTIMICROBIAL preservatives, QUALITY control, PHARMACEUTICAL industry
Abstract

The Burkholderia cepacia complex (Bcc) is the number one bacterial complex associated with contaminated Finished Pharmaceutical Products (FPPs). This has resulted in multiple healthcare related infection morbidity and mortality events in conjunction with significant FPP recalls globally. Current microbiological quality control of FPPs before release for distribution depends on lengthy, laborious, non-specific, traditional culture-dependent methods which lack sensitivity. Here, we present the development of a culture-independent Bcc Nucleic Acid Diagnostic (NAD) method for detecting Bcc contaminants associated with Over-The-Counter aqueous FPPs. The culture-independent Bcc NAD method was validated to be specific for detecting Bcc at different contamination levels from spiked aqueous FPPs. The accuracy in Bcc quantitative measurements was achieved by the high degree of Bcc recovery from aqueous FPPs. The low variation observed between several repeated Bcc quantitative measurements further demonstrated the precision of Bcc quantification in FPPs. The robustness of the culture-independent Bcc NAD method was determined when its accuracy and precision were not significantly affected during testing of numerous aqueous FPP types with different ingredient matrices, antimicrobial preservative components and routes of administration. The culture-independent Bcc NAD method showed an ability to detect Bcc in spiked aqueous FPPs at a concentration of 20 Bcc CFU/mL. The rapid (≤ 4 hours from sample in to result out), robust, culture-independent Bcc NAD method presented provides rigorous test specificity, accuracy, precision, and sensitivity. This method, validated with equivalence to ISO standard ISO/TS 12869:2019, can be a valuable diagnostic tool in supporting microbiological quality control procedures to aid the pharmaceutical industry in preventing Bcc contamination of aqueous FPPs for consumer safety. [ABSTRACT FROM AUTHOR]

Published
2024
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9. Erratum:Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function (GigaScience DOI: 10.1093/gigascience/giaa053)

Author

Reddington, Kate, Eccles, David, O'Grady, Justin, Drown, Devin M., Hansen, Lars Hestbjerg, Nielsen, Tue Kjærgaard, Ducluzeau, Anne Lise, Leggett, Richard M., Heavens, Darren, Peel, Ned, Snutch, Terrance P., Bayega, Anthony, Oikonomopoulos, Spyridon, Ragoussis, Jiannis, Barry, Thomas, van der Helm, Eric, Jolic, Dino, Richardson, Hollian, Jansen, Hans, Tyson, John R., Jain, Miten, Brown, Bonnie L., Reddington, Kate, Eccles, David, O'Grady, Justin, Drown, Devin M., Hansen, Lars Hestbjerg, Nielsen, Tue Kjærgaard, Ducluzeau, Anne Lise, Leggett, Richard M., Heavens, Darren, Peel, Ned, Snutch, Terrance P., Bayega, Anthony, Oikonomopoulos, Spyridon, Ragoussis, Jiannis, Barry, Thomas, van der Helm, Eric, Jolic, Dino, Richardson, Hollian, Jansen, Hans, Tyson, John R., Jain, Miten, and Brown, Bonnie L.

Abstract

In the original publication of this article, the author Jiannis Ragoussis was not part of the author byline. His name, affiliation, and funding information have been added to this article. The authors regret this error.

Published
2020

10. Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function

Author

Reddington, Kate, Eccles, David, O'Grady, Justin, Drown, Devin M., Hansen, Lars Hestbjerg, Nielsen, Tue Kjærgaard, Ducluzeau, Anne Lise, Leggett, Richard M., Heavens, Darren, Peel, Ned, Snutch, Terrance P., Bayega, Anthony, Oikonomopoulos, Spyridon, Ragoussis, Jiannis, Barry, Thomas, van der Helm, Eric, Jolic, Dino, Richardson, Hollian, Jansen, Hans, Tyson, John R., Jain, Miten, Brown, Bonnie L., Reddington, Kate, Eccles, David, O'Grady, Justin, Drown, Devin M., Hansen, Lars Hestbjerg, Nielsen, Tue Kjærgaard, Ducluzeau, Anne Lise, Leggett, Richard M., Heavens, Darren, Peel, Ned, Snutch, Terrance P., Bayega, Anthony, Oikonomopoulos, Spyridon, Ragoussis, Jiannis, Barry, Thomas, van der Helm, Eric, Jolic, Dino, Richardson, Hollian, Jansen, Hans, Tyson, John R., Jain, Miten, and Brown, Bonnie L.

Abstract

Background: Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad "aquascape"scale, and few if any have applied the newest nanopore technology. Results: We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5-1.0 ⋅ 106 long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers. Conclusions: Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.

Published
2020

11. Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function:[incl. Erratum]

Author

Reddington, Kate, Eccles, David, O'Grady, Justin, Drown, Devin M., Hansen, Lars Hestbjerg, Nielsen, Tue Kjærgaard, Ducluzeau, Anne-Lise, Leggett, Richard M., Heavens, Darren, Peel, Ned, Snutch, Terrance P., Bayega, Anthony, Oikonomopoulos, Spyridon, Ragoussis, Jiannis, Barry, Thomas, van der Helm, Eric, Jolic, Dino, Richardson, Hollian, Jansen, Hans, Tyson, John R., Jain, Miten, Brown, Bonnie L., Reddington, Kate, Eccles, David, O'Grady, Justin, Drown, Devin M., Hansen, Lars Hestbjerg, Nielsen, Tue Kjærgaard, Ducluzeau, Anne-Lise, Leggett, Richard M., Heavens, Darren, Peel, Ned, Snutch, Terrance P., Bayega, Anthony, Oikonomopoulos, Spyridon, Ragoussis, Jiannis, Barry, Thomas, van der Helm, Eric, Jolic, Dino, Richardson, Hollian, Jansen, Hans, Tyson, John R., Jain, Miten, and Brown, Bonnie L.

Abstract

Background: Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad "aquascape" scale, and few if any have applied the newest nanopore technology. Results: We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5-1.0 . 10(6) long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers. Conclusions: Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.

Published
2020

13. Corrigendum to: Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function

Author

Reddington, Kate, primary, Eccles, David, primary, O'Grady, Justin, primary, Drown, Devin M, primary, Hansen, Lars Hestbjerg, primary, Nielsen, Tue Kjærgaard, primary, Ducluzeau, Anne-Lise, primary, Leggett, Richard M, primary, Heavens, Darren, primary, Peel, Ned, primary, Snutch, Terrance P, primary, Bayega, Anthony, primary, Oikonomopoulos, Spyridon, primary, Ragoussis, Jiannis, primary, Barry, Thomas, primary, van der Helm, Eric, primary, Jolic, Dino, primary, Richardson, Hollian, primary, Jansen, Hans, primary, Tyson, John R, primary, Jain, Miten, primary, and Brown, Bonnie L, primary

Published
2020
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14. Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function

Author

Reddington, Kate, primary, Eccles, David, primary, O'Grady, Justin, primary, Drown, Devin M, primary, Hansen, Lars Hestbjerg, primary, Nielsen, Tue Kjærgaard, primary, Ducluzeau, Anne-Lise, primary, Leggett, Richard M, primary, Heavens, Darren, primary, Peel, Ned, primary, Snutch, Terrance P, primary, Bayega, Anthony, primary, Oikonomopoulos, Spyridon, primary, Ragoussis, Jiannis, primary, Barry, Thomas, primary, van der Helm, Eric, primary, Jolic, Dino, primary, Richardson, Hollian, primary, Jansen, Hans, primary, Tyson, John R, primary, Jain, Miten, primary, and Brown, Bonnie L, primary

Published
2020
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15. ONT Library Preparation v1

Author

Reddington, Kate, primary, Eccles, David, additional, O’Grady, Justin, additional, M., Devin, additional, Hestbjerg, Lars, additional, Kjærgaard, Tue, additional, Ducluzeau, Anne-Lise, additional, M., Richard, additional, Heavens, Darren, additional, Peel, Ned, additional, P., Terrance, additional, Bayega, Anthony, additional, Oikonomopoulos, Spyros, additional, Ragoussis, Jiannis, additional, Barry, Thomas, additional, van, Eric, additional, Jolic, Dino, additional, Richardson, Hollian, additional, Jansen, Hans, additional, R., John, additional, Jain, Miten, additional, and L., Bonnie, additional

Published
2018
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16. Collection and purification of river water samples v1

Author

Reddington, Kate, primary, Eccles, David, additional, O’Grady, Justin, additional, M., Devin, additional, Hestbjerg, Lars, additional, Kjærgaard, Tue, additional, Ducluzeau, Anne-Lise, additional, M., Richard, additional, Heavens, Darren, additional, Peel, Ned, additional, P., Terrance, additional, Bayega, Anthony, additional, Oikonomopoulos, Spyros, additional, Ragoussis, Jiannis, additional, Barry, Thomas, additional, van, Eric, additional, Jolic, Dino, additional, Richardson, Hollian, additional, Jansen, Hans, additional, R., John, additional, Jain, Miten, additional, and L., Bonnie, additional

Published
2018
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17. DNA extraction and purification for MARC phase 3 global river water sequencing v1

Author

Reddington, Kate, primary, Eccles, David, additional, O’Grady, Justin, additional, M., Devin, additional, Hestbjerg, Lars, additional, Kjærgaard, Tue, additional, Ducluzeau, Anne-Lise, additional, M., Richard, additional, Heavens, Darren, additional, Peel, Ned, additional, P., Terrance, additional, Bayega, Anthony, additional, Oikonomopoulos, Spyros, additional, Ragoussis, Jiannis, additional, Barry, Thomas, additional, van, Eric, additional, Jolic, Dino, additional, Richardson, Hollian, additional, Jansen, Hans, additional, R., John, additional, Jain, Miten, additional, and L., Bonnie, additional

Published
2018
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18. Additional file 2: of The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

Author

Devonshire, Alison, O’Sullivan, Denise, Isobella Honeyborne, Gerwyn Jones, Karczmarczyk, Maria, Pavšič, Jernej, Gutteridge, Alice, Milavec, Mojca, Mendoza, Pablo, Schimmel, Heinz, Heuverswyn, Fran Van, Gorton, Rebecca, Cirillo, Daniela, Borroni, Emanuele, Harris, Kathryn, Barnard, Marinus, Anthenette Heydenrych, Ndusilo, Norah, Wallis, Carole, Keshree Pillay, Barry, Thomas, Reddington, Kate, Richter, Elvira, Mozioğlu, Erkan, Akyürek, Sema, Yalçınkaya, Burhanettin, Akgoz, Muslum, Žel, Jana, Foy, Carole, McHugh, Timothy, and Huggett, Jim

Abstract

Protocol for gDNA extraction and dPCR quantification of materials. (PDF 394 kb)

Published
2016
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19. Additional file 1: of The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

Author

Devonshire, Alison, O’Sullivan, Denise, Isobella Honeyborne, Gerwyn Jones, Karczmarczyk, Maria, Pavšič, Jernej, Gutteridge, Alice, Milavec, Mojca, Mendoza, Pablo, Schimmel, Heinz, Heuverswyn, Fran Van, Gorton, Rebecca, Cirillo, Daniela, Borroni, Emanuele, Harris, Kathryn, Barnard, Marinus, Anthenette Heydenrych, Ndusilo, Norah, Wallis, Carole, Keshree Pillay, Barry, Thomas, Reddington, Kate, Richter, Elvira, Mozioğlu, Erkan, Akyürek, Sema, Yalçınkaya, Burhanettin, Akgoz, Muslum, Žel, Jana, Foy, Carole, McHugh, Timothy, and Huggett, Jim

Abstract

Method for restriction digestion of the TB Control Plasmid, dPCR analysis using the QuantStudio 3D platform, dPCR primers and thermal cycling conditions information, dPCR partition volume and partition number and mean λ values. Results from stability testing of the materials and analysis of BCG/ASM and Total MTB Control materials by Xpert MTB/RIF end-user laboratories including instrument and assay information, extraction and qPCR methods used by qPCR end user laboratories and their reported Cq values. (DOCX 114 kb)

Published
2016
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20. The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

Author

Devonshire, Alison S., primary, O’Sullivan, Denise M., additional, Honeyborne, Isobella, additional, Jones, Gerwyn, additional, Karczmarczyk, Maria, additional, Pavšič, Jernej, additional, Gutteridge, Alice, additional, Milavec, Mojca, additional, Mendoza, Pablo, additional, Schimmel, Heinz, additional, Van Heuverswyn, Fran, additional, Gorton, Rebecca, additional, Cirillo, Daniela Maria, additional, Borroni, Emanuele, additional, Harris, Kathryn, additional, Barnard, Marinus, additional, Heydenrych, Anthenette, additional, Ndusilo, Norah, additional, Wallis, Carole L., additional, Pillay, Keshree, additional, Barry, Thomas, additional, Reddington, Kate, additional, Richter, Elvira, additional, Mozioğlu, Erkan, additional, Akyürek, Sema, additional, Yalçınkaya, Burhanettin, additional, Akgoz, Muslum, additional, Žel, Jana, additional, Foy, Carole A., additional, McHugh, Timothy D., additional, and Huggett, Jim F., additional

Published
2016
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22. Design, development and validation of a series of multiplex real-time PCR diagnostics assays for the rapid and accurate detection and differentiation of the Mycobacterium tuberculosis complex

Author

Reddington, Kate and Barry, Thomas

Subjects
Mycobacterium tuberculosis complex, Molecular diagnostics, Tuberculosis, Microbiology, Real-time PCR
Abstract

Tuberculosis (TB) remains a major health concern both in developed and developing countries due to the high rates of morbidity and mortality associated with disease. In humans, TB is caused by members of the Mycobacterium tuberculosis complex (MTC) namely M. tuberculosis, M. canettii, M. africanum, M. bovis, M. caprae, M. microti, M. pinnipedii and the attenuated M. bovis BCG vaccine strain. This group of microorganisms are ~99% similar on a nucleotide sequence level and have a wide range of natural hosts. While M. tuberculosis is responsible for the majority of cases of human TB, accurate identification of other members of the MTC causing infection is not routinely performed. As a result, the global frequency and distribution of each member of the MTC remains largely unknown with some studies suggesting that TB caused by members of the MTC other than M. tuberculosis are in fact underreported. Therefore, the capability to accurately identify each member of the MTC causing human TB infection would be desirable. This would enable unambiguous TB epidemiological studies and monitoring of human to human and/or zoonotic TB transmission. Equally, differentiation of the MTC is clinically important for treatment management decisions due to the inherent natural resistance of some members of the complex to the first line anti-TB drug pyrazinamide (PZA). Currently there is only one commercially available diagnostics assay for differentiation of the MTC, the Genotype MTBC kit. This diagnostics kit is limited by its inability to accurately identify M. tuberculosis, M. canettii, M. africanum and M. pinnipedii. There are a number of molecular based assays for MTC differentiation described in the literature which are also limited by an inability to differentiate all members of the MTC and most require post amplification processing increasing method complexity, analysis time and potential contamination. The overall aim of this study was to design, develop, optimise and validate a robust, internally controlled, multiplex real-time PCR based method for the rapid and accurate identification of all members of the MTC. This was achieved using a sequential experimental design consisting of three main studies. In the first study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. In the second study, a multiplex real-time PCR assay was developed for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. In the third study two additional biomarkers were incorporated into the previous two multiplex assays specific for the two clades of M. africanum. By incorporation of these two additional targets, it was possible to devise a diagnostic algorithm to differentiate all 8 members of the MTC. The specificity of this method was optimised and validated against a panel of 119 MTC isolates which had been previously characterised using methods such as spoligotyping, mycobacterial interspersed repetitive units - variable number tandem repeats (MIRU-VNTR), IS6110-based typing methods, RD analysis, biochemical testing in addition to morphological examination. Specificity was also demonstrated against 44 Non Tuberculosis Mycobacteria (NTM) and 17 other bacterial species. Analytical sensitivities of less than 100 genome equivalents were determined for each diagnostics assay developed in multiplex format. Further evaluation of SeekTB was performed by blindly testing 125 Mycobacteria Growth Indicator Tube (MGIT) positive cultures. The results of SeekTB were compared to those obtained using the commercially available GenoType MTBC and TBc ID tests. SeekTB and GenoType MTBC test results were 100% concordant identifying 84 isolates as M. tuberculosis and 41 as non MTC. Nine discordant results were observed between the molecular methods and the TBc ID culture confirmation test, however, nucleotide sequencing of the discordant isolates confirmed the results obtained with SeekTB and GenoType MTBC tests In summary, SeekTB, the diagnostic method developed in this study, is the first description of an internally controlled multiplex real-time PCR based diagnostics method for the accurate identification of all eight members of the MTC. This method, designed for use on cultured patient samples, is specific, sensitive and rapid with a turnaround time to results of approximately 1.5 to 3.5 h, depending on which, if any, member of the MTC is present.

Published
2012

24. Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections

Author

Reddington, Kate, primary, Schwenk, Stefan, additional, Tuite, Nina, additional, Platt, Gareth, additional, Davar, Danesh, additional, Coughlan, Helena, additional, Personne, Yoann, additional, Gant, Vanya, additional, Enne, Virve I., additional, Zumla, Alimuddin, additional, and Barry, Thomas, additional

Published
2015
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27. Evaluation of the effectiveness and cost-effectiveness of Families for Health V2 for the treatment of childhood obesity: study protocol for a randomized controlled trial

Author

Robertson, Wendy, primary, Stewart-Brown, Sarah, additional, Stallard, Nigel, additional, Petrou, Stavros, additional, Griffiths, Frances, additional, Thorogood, Margaret, additional, Simkiss, Douglas, additional, Lang, Rebecca, additional, Reddington, Kate, additional, Poole, Fran, additional, Rye, Gloria, additional, Khan, Kamran A, additional, Hamborg, Thomas, additional, and Kirby, Joanna, additional

Published
2013
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33. Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpBReal-Time PCR Assay for Specific Detection of Haemophilus influenzaeIsolates Associated with Respiratory Tract Infections

Author

Reddington, Kate, Schwenk, Stefan, Tuite, Nina, Platt, Gareth, Davar, Danesh, Coughlan, Helena, Personne, Yoann, Gant, Vanya, Enne, Virve I., Zumla, Alimuddin, and Barry, Thomas

Abstract

ABSTRACTHaemophilus influenzaeis a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzaediagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzaein RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpBgene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilusspp. Subsequently, 44 Haemophilusclinical isolates were collected, and 36 isolates were identified as H. influenzaeusing a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and a fucKdiagnostic assay. Using the novel smpBdiagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n= 98) were blindly tested with the gold standard and smpBdiagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpBassay when used directly on respiratory specimens.

Published
2015
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34. SeekTB, a Two-Stage Multiplex Real-Time-PCR-Based Method for Differentiation of the Mycobacterium tuberculosisComplex

Author

Reddington, Kate, Zumla, Alimuddin, Bates, Matthew, van Soolingen, Dick, Niemann, Stefan, Barry, Thomas, and O'Grady, Justin

Abstract

ABSTRACTTuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosiscomplex (MTC). The accurate identification of the MTC member causing human infection is important because the treatment of TB caused by some MTC members requires an alteration of the standard drug regimen, it can inform whether transmission is human to human or zoonotic, and it enables accurate epidemiology studies that help improve TB control. In this study, an internally controlled two-stage multiplex real-time PCR-based method, SeekTB, was developed for the accurate identification of all members of the MTC. The method was tested against a panel of well-characterized bacterial strains (n= 180) and determined to be 100% specific for members of the MTC. Additionally, 125 Mycobacteria Growth Indicator Tube (MGIT)-positive cultures were blindly tested by using SeekTB, and the results were compared to those of the GenoType MTBC and TBc ID tests. The SeekTB and GenoType MTBC results were 100% concordant, identifying 84 of these isolates as M. tuberculosisisolates and 41 as non-MTC isolates. Nine discordant results between the molecular methods and the TBc ID culture confirmation test were observed; however, nucleotide sequencing confirmed the results obtained with GenoType MTBC and SeekTB. SeekTB is the first-described internally controlled multiplex real-time PCR diagnostic method for the accurate identification of all eight members of the MTC. This method, designed for use on cultured patient samples, is specific, sensitive, and rapid, with a turnaround time to results of approximately 1.5 to 3.5 h, depending on which, if any, member of the MTC is present.

Published
2012
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35. Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosisComplex Strains

Author

Reddington, Kate, O'Grady, Justin, Dorai-Raj, Siobhan, Maher, Majella, van Soolingen, Dick, and Barry, Thomas

Abstract

ABSTRACTTuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosiscomplex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosisand M. canettii. The lepAgene was targeted for the detection of members of the MTC, the wbbl1gene was used for the differentiation of M. tuberculosisand M. canettiifrom the remainder of the complex, and a unique region of the M. canettiigenome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.

Published
2011
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36. SeekTB, a Two-Stage Multiplex Real-Time-PCR-Based Method for Differentiation of the Mycobacterium tuberculosis Complex

Author

Reddington, Kate, Zumla, Alimuddin, Bates, Matthew, van Soolingen, Dick, Niemann, Stefan, Barry, Thomas, O'Grady, Justin, Reddington, Kate, Zumla, Alimuddin, Bates, Matthew, van Soolingen, Dick, Niemann, Stefan, Barry, Thomas, and O'Grady, Justin

Abstract

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). The accurate identification of the MTC member causing human infection is important because the treatment of TB caused by some MTC members requires an alteration of the standard drug regimen, it can inform whether transmission is human to human or zoonotic, and it enables accurate epidemiology studies that help improve TB control. In this study, an internally controlled two-stage multiplex real-time PCR-based method, SeekTB, was developed for the accurate identification of all members of the MTC. The method was tested against a panel of well-characterized bacterial strains (n = 180) and determined to be 100% specific for members of the MTC. Additionally, 125 Mycobacteria Growth Indicator Tube (MGIT)-positive cultures were blindly tested by using SeekTB, and the results were compared to those of the GenoType MTBC and TBc ID tests. The SeekTB and GenoType MTBC results were 100% concordant, identifying 84 of these isolates as M. tuberculosis isolates and 41 as non-MTC isolates. Nine discordant results between the molecular methods and the TBc ID culture confirmation test were observed; however, nucleotide sequencing confirmed the results obtained with GenoType MTBC and SeekTB. SeekTB is the first-described internally controlled multiplex real-time PCR diagnostic method for the accurate identification of all eight members of the MTC. This method, designed for use on cultured patient samples, is specific, sensitive, and rapid, with a turnaround time to results of approximately 1.5 to 3.5 h, depending on which, if any, member of the MTC is present.

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