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Article ; Online: Diagnostic yield of broad-range 16s rRNA gene PCR varies by sample type and is improved by the addition of qPCR panels targeting the most common causative organisms.

Harris, Kathryn A / Brown, Julianne R

2023 Volume 71, Issue 12

Abstract: Introduction. ...

Abstract	Introduction.
MeSH term(s)	Humans ; Bacteria/genetics ; DNA, Bacterial/genetics ; DNA, Bacterial/analysis ; Genes, rRNA ; Polymerase Chain Reaction/methods ; Retrospective Studies ; RNA, Ribosomal, 16S/genetics ; Bacterial Infections/diagnosis
Chemical Substances	DNA, Bacterial ; RNA, Ribosomal, 16S
Language	English
Publishing date	2023-02-06
Publishing country	England
Document type	Journal Article
ZDB-ID	218356-0
ISSN	1473-5644 ; 0022-2615
ISSN (online)	1473-5644
ISSN	0022-2615
DOI	10.1099/jmm.0.001633
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Article: Untargeted metagenomics protocol for the diagnosis of infection from CSF and tissue from sterile sites.

Atkinson, Laura / Lee, Jack Cd / Lennon, Alexander / Shah, Divya / Storey, Nathaniel / Morfopoulou, Sofia / Harris, Kathryn A / Breuer, Judy / Brown, Julianne R

Heliyon

2023 Volume 9, Issue 9, Page(s) e19854

Abstract: Metagenomic next-generation sequencing (mNGS) is an untargeted technique capable of detecting all microbial nucleic acid within a sample. This protocol outlines our wet laboratory method for mNGS of cerebrospinal fluid (CSF) specimens and tissues from ... ...

Abstract	Metagenomic next-generation sequencing (mNGS) is an untargeted technique capable of detecting all microbial nucleic acid within a sample. This protocol outlines our wet laboratory method for mNGS of cerebrospinal fluid (CSF) specimens and tissues from sterile sites. We use this method routinely in our clinical service, processing 178 specimens over the past 2.5 years in a laboratory that adheres to ISO:15189 standards. We have successfully used this protocol to diagnose multiple cases of encephalitis and hepatitis.
Language	English
Publishing date	2023-09-09
Publishing country	England
Document type	Journal Article
ZDB-ID	2835763-2
ISSN	2405-8440
ISSN	2405-8440
DOI	10.1016/j.heliyon.2023.e19854
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Article ; Online: Molecular techniques for diagnosing prosthetic joint infections.

Hartley, John C / Harris, Kathryn A

The Journal of antimicrobial chemotherapy

2014 Volume 69 Suppl 1, Page(s) i21–4

Abstract: Prosthetic joint infections (PJI) can be broadly classed into two groups: those where there is a strong clinical suspicion of infection and those with clinical uncertainty, including 'aseptic loosening'. Confirmation of infection and identification of ... ...

Abstract	Prosthetic joint infections (PJI) can be broadly classed into two groups: those where there is a strong clinical suspicion of infection and those with clinical uncertainty, including 'aseptic loosening'. Confirmation of infection and identification of the causative organism along with provision of antibiotic susceptibility data are important stages in the management of PJI. Conventional microbiological culture and susceptibility testing is usually sufficient to provide this. However, it may fail due to prior antimicrobial treatment or the presence of unusual and fastidious organisms. Molecular techniques, in particular specific real-time and broad-range PCR, are available for diagnostic use in suspected PJI. In this review, we describe the techniques available, their current strengths, limitations and future development. Real-time pathogen-specific and broad-range PCR (with single sequence determination) are suitable for use as part of the routine diagnostic algorithm for clinically suspected PJI. Further development of broad-range PCR with high-throughput (next-generation) sequencing is necessary to understand the microbiome of the prosthetic joint further before this technique can be used for routine diagnostics in clinically unsuspected PJI, including aseptic loosening.
MeSH term(s)	High-Throughput Nucleotide Sequencing/methods ; Humans ; Molecular Diagnostic Techniques/methods ; Osteoarthritis/diagnosis ; Polymerase Chain Reaction/methods ; Prosthesis-Related Infections/diagnosis
Language	English
Publishing date	2014-09
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	191709-2
ISSN	1460-2091 ; 0305-7453
ISSN (online)	1460-2091
ISSN	0305-7453
DOI	10.1093/jac/dku249
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Zs.A 1197: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: What is broad-range 16S rDNA PCR?

Patel, Amani / Harris, Kathryn A / Fitzgerald, Felicity

Archives of disease in childhood. Education and practice edition

2017 Volume 102, Issue 5, Page(s) 261–264

MeSH term(s)	Bacteria/genetics ; Cell Culture Techniques ; Humans ; Polymerase Chain Reaction ; RNA, Bacterial/analysis ; RNA, Ribosomal, 16S/analysis
Chemical Substances	RNA, Bacterial ; RNA, Ribosomal, 16S
Language	English
Publishing date	2017-04-17
Publishing country	England
Document type	Journal Article
ZDB-ID	2148818-6
ISSN	1743-0593 ; 1743-0585
ISSN (online)	1743-0593
ISSN	1743-0585
DOI	10.1136/archdischild-2016-312049
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Article ; Online: Single base mutations in the nucleocapsid gene of SARS-CoV-2 affects amplification efficiency of sequence variants and may lead to assay failure.

Storey, Nathaniel / Brown, Julianne R / Pereira, Rui P A / O'Sullivan, Denise M / Huggett, Jim F / Williams, Rachel / Breuer, Judith / Harris, Kathryn A

Journal of clinical virology plus

2021 Volume 1, Issue 3, Page(s) 100037

Abstract: Reverse transcriptase quantitative PCR (RT-qPCR) is the main diagnostic assay used to detect SARS-CoV-2 RNA in respiratory samples. RT-qPCR is performed by specifically targeting the viral genome using complementary oligonucleotides called primers and ... ...

Abstract	Reverse transcriptase quantitative PCR (RT-qPCR) is the main diagnostic assay used to detect SARS-CoV-2 RNA in respiratory samples. RT-qPCR is performed by specifically targeting the viral genome using complementary oligonucleotides called primers and probes. This approach relies on prior knowledge of the genetic sequence of the target. Viral genetic variants with changes to the primer/probe binding region may reduce the performance of PCR assays and have the potential to cause assay failure. In this work we demonstrate how two single nucleotide variants (SNVs) altered the amplification curve of a diagnostic PCR targeting the Nucleocapsid (N) gene and illustrate how threshold setting can lead to false-negative results even where the variant sequence is amplified. We also describe how
Language	English
Publishing date	2021-08-15
Publishing country	England
Document type	Journal Article
ISSN	2667-0380
ISSN (online)	2667-0380
DOI	10.1016/j.jcvp.2021.100037
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Article ; Online: Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Molecular Typing of Acinetobacter baumannii in Comparison with Orthogonal Methods.

Busby, Eloise J / Doyle, Ronan M / Leboreiro Babe, Clara / Harris, Kathryn A / Mack, Damien / Méndez-Cervantes, Gema / O'Sullivan, Denise M / Pang, Vicky / Sadouki, Zahra / Solanki, Priya / Huggett, Jim F / McHugh, Timothy D / Wey, Emmanuel Q

Microbiology spectrum

2023 Volume 11, Issue 3, Page(s) e0499522

Abstract: Colonization and subsequent health care-associated infection (HCAI) with Acinetobacter baumannii are a concern for vulnerable patient groups within the hospital setting. Outbreaks involving multidrug-resistant strains are associated with increased ... ...

Abstract	Colonization and subsequent health care-associated infection (HCAI) with Acinetobacter baumannii are a concern for vulnerable patient groups within the hospital setting. Outbreaks involving multidrug-resistant strains are associated with increased patient morbidity and mortality and poorer overall outcomes. Reliable molecular typing methods can help to trace transmission routes and manage outbreaks. In addition to methods deployed by reference laboratories, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) may assist by making initial in-house judgments on strain relatedness. However, limited studies on method reproducibility exist for this application. We applied MALDI-TOF MS typing to A. baumannii isolates associated with a nosocomial outbreak and evaluated different methods for data analysis. In addition, we compared MALDI-TOF MS with whole-genome sequencing (WGS) and Fourier transform infrared spectroscopy (FTIR) as orthogonal methods to further explore their resolution for bacterial strain typing. A related subgroup of isolates consistently clustered separately from the main outbreak group by all investigated methods. This finding, combined with epidemiological data from the outbreak, indicates that these methods identified a separate transmission event unrelated to the main outbreak. However, the MALDI-TOF MS upstream approach introduced measurement variability impacting method reproducibility and limiting its reliability as a standalone typing method. Availability of in-house typing methods with well-characterized sources of measurement uncertainty could assist with rapid and dependable confirmation (or denial) of suspected transmission events. This work highlights some of the steps to be improved before such tools can be fully integrated into routine diagnostic service workflows for strain typing.
MeSH term(s)	Humans ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods ; Acinetobacter baumannii/genetics ; Reproducibility of Results ; Bacterial Typing Techniques/methods ; Pandemics ; COVID-19/epidemiology ; Molecular Typing ; Cross Infection/epidemiology ; Cross Infection/microbiology ; Anti-Infective Agents
Chemical Substances	Anti-Infective Agents
Language	English
Publishing date	2023-05-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2807133-5
ISSN	2165-0497 ; 2165-0497
ISSN (online)	2165-0497
ISSN	2165-0497
DOI	10.1128/spectrum.04995-22
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Article ; Online: Outcome according to subspecies following lung transplantation in cystic fibrosis pediatric patients infected with Mycobacterium abscessus.

Kavaliunaite, Ema / Harris, Kathryn A / Aurora, Paul / Dixon, Garth / Shingadia, Delane / Muthialu, Nagarajan / Spencer, Helen

Transplant infectious disease : an official journal of the Transplantation Society

2020 Volume 22, Issue 3, Page(s) e13274

Abstract: Background: Mycobacterium abscessus infection has been associated with variable outcomes following lung transplantation. M abscessus comprises three subspecies (M abscessus subsp abscessus, M abscessus subsp massiliense, and M abscessus subsp bolletii). ...

Abstract	Background: Mycobacterium abscessus infection has been associated with variable outcomes following lung transplantation. M abscessus comprises three subspecies (M abscessus subsp abscessus, M abscessus subsp massiliense, and M abscessus subsp bolletii). We investigated whether lung transplantation outcome in cystic fibrosis (CF) patients in a single center was related to the M abscessus subspecies and genetic cluster. Methods: CF patients with chronic M abscessus infection transplanted at Great Ormond Street Hospital between 2004 and 2017 were retrospectively examined. All M abscessus isolates were identified to subspecies level by polymerase chain reaction and sequencing. Genetic cluster was determined by variable number tandem repeat profiling and whole-genome sequencing (WGS), and sequence type inferred from WGS. Results: Thirteen patients with chronic M abscessus infection underwent heart/lung or lung transplantation. Subspecies identification showed n = 1 with M abscessus bolletii, n = 5 with M abscessus massiliense, and n = 7 with M abscessus abscessus infection. Eight (62%) patients (one with M abscessus massiliense and seven with M abscessus abscessus) died post-lung transplant. The patient with M abscessus bolletii and three patients with M abscessus massiliense did well post-transplant. One patient with M abscessus massiliense is receiving ongoing treatment. Conclusions: Dramatically worse outcomes are observed in patients infected with M abscessus subspecies abscessus, the majority of whom were infected with ST-1 and ST-26 strains. Patients infected with other M abcsessus strains can have acceptable outcomes.
MeSH term(s)	Adolescent ; Child ; Cystic Fibrosis/complications ; Cystic Fibrosis/microbiology ; Cystic Fibrosis/surgery ; DNA, Bacterial/genetics ; Female ; Humans ; Lung Transplantation/adverse effects ; Male ; Microbial Sensitivity Tests ; Mycobacterium Infections, Nontuberculous/microbiology ; Mycobacterium Infections, Nontuberculous/physiopathology ; Mycobacterium abscessus/classification ; Mycobacterium abscessus/pathogenicity ; Outcome and Process Assessment, Health Care ; Phylogeny ; Retrospective Studies ; Sequence Analysis, DNA ; Whole Genome Sequencing
Chemical Substances	DNA, Bacterial
Language	English
Publishing date	2020-03-18
Publishing country	Denmark
Document type	Journal Article
ZDB-ID	1476094-0
ISSN	1399-3062 ; 1398-2273
ISSN (online)	1399-3062
ISSN	1398-2273
DOI	10.1111/tid.13274
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Article ; Online: Mycobacterium abscessus infection in cystic fibrosis: molecular typing and clinical outcomes.

Harris, Kathryn A / Kenna, Dervla T D

Journal of medical microbiology

2014 Volume 63, Issue Pt 10, Page(s) 1241–1246

Abstract: Mycobacterium abscessus is a significant pathogen in the cystic fibrosis patient population. PCR amplification and sequencing can provide accurate subspecies identification, and can predict macrolide susceptibility, which is becoming increasingly ... ...

Abstract	Mycobacterium abscessus is a significant pathogen in the cystic fibrosis patient population. PCR amplification and sequencing can provide accurate subspecies identification, and can predict macrolide susceptibility, which is becoming increasingly important for patient management. Molecular techniques for further typing of isolates provide tools for the ongoing investigations into the clinical impact of particular M. abscessus strains. Whole-genome sequencing is likely to be the only technique that provides sufficient resolution for investigating transmission events between patients.
MeSH term(s)	Cystic Fibrosis/complications ; Humans ; Molecular Typing ; Mycobacterium/classification ; Mycobacterium/genetics ; Mycobacterium/isolation & purification ; Mycobacterium Infections, Nontuberculous/microbiology ; Mycobacterium Infections, Nontuberculous/transmission ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Treatment Outcome
Language	English
Publishing date	2014-08-08
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	218356-0
ISSN	1473-5644 ; 0022-2615
ISSN (online)	1473-5644
ISSN	0022-2615
DOI	10.1099/jmm.0.077164-0
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Article ; Online: Cross-transmission Is Not the Source of New Mycobacterium abscessus Infections in a Multicenter Cohort of Cystic Fibrosis Patients.

Doyle, Ronan M / Rubio, Marc / Dixon, Garth / Hartley, John / Klein, Nigel / Coll, Pere / Harris, Kathryn A

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

2019 Volume 70, Issue 9, Page(s) 1855–1864

Abstract: Background: Mycobacterium abscessus is an extensively drug-resistant pathogen that causes pulmonary disease, particularly in cystic fibrosis (CF) patients. Identifying direct patient-to-patient transmission of M. abscessus is critically important in ... ...

Abstract	Background: Mycobacterium abscessus is an extensively drug-resistant pathogen that causes pulmonary disease, particularly in cystic fibrosis (CF) patients. Identifying direct patient-to-patient transmission of M. abscessus is critically important in directing an infection control policy for the management of risk in CF patients. A variety of clinical labs have used molecular epidemiology to investigate transmission. However, there is still conflicting evidence as to how M. abscessus is acquired and whether cross-transmission occurs. Recently, labs have applied whole-genome sequencing (WGS) to investigate this further and, in this study, we investigated whether WGS can reliably identify cross-transmission in M. abscessus. Methods: We retrospectively sequenced the whole genomes of 145 M. abscessus isolates from 62 patients, seen at 4 hospitals in 2 countries over 16 years. Results: We have shown that a comparison of a fixed number of core single nucleotide variants alone cannot be used to infer cross-transmission in M. abscessus but does provide enough information to replace multiple existing molecular assays. We detected 1 episode of possible direct patient-to-patient transmission in a sibling pair. We found that patients acquired unique M. abscessus strains even after spending considerable time on the same wards with other M. abscessus-positive patients. Conclusions: This novel analysis has demonstrated that the majority of patients in this study have not acquired M. abscessus through direct patient-to-patient transmission or a common reservoir. Tracking transmission using WGS will only realize its full potential with proper environmental screening, as well as patient sampling.
MeSH term(s)	Cohort Studies ; Cystic Fibrosis/complications ; Humans ; Mycobacterium Infections, Nontuberculous/epidemiology ; Mycobacterium abscessus/genetics ; Retrospective Studies
Language	English
Publishing date	2019-06-17
Publishing country	United States
Document type	Journal Article ; Multicenter Study ; Research Support, Non-U.S. Gov't
ZDB-ID	1099781-7
ISSN	1537-6591 ; 1058-4838
ISSN (online)	1537-6591
ISSN	1058-4838
DOI	10.1093/cid/ciz526
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Article ; Online: Children With Cystic Fibrosis Are Infected With Multiple Subpopulations of Mycobacterium abscessus With Different Antimicrobial Resistance Profiles.

Shaw, Liam P / Doyle, Ronan M / Kavaliunaite, Ema / Spencer, Helen / Balloux, Francois / Dixon, Garth / Harris, Kathryn A

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

2019 Volume 69, Issue 10, Page(s) 1678–1686

Abstract: Background: Children with cystic fibrosis (CF) can develop life-threatening infections of Mycobacterium abscessus. These present a significant clinical challenge, particularly when the strains involved are resistant to antibiotics. Recent evidence of ... ...

Abstract	Background: Children with cystic fibrosis (CF) can develop life-threatening infections of Mycobacterium abscessus. These present a significant clinical challenge, particularly when the strains involved are resistant to antibiotics. Recent evidence of within-patient subclones of M. abscessus in adults with CF suggests the possibility that within-patient diversity may be relevant for the treatment of pediatric CF patients. Methods: We performed whole-genome sequencing (WGS) on 32 isolates of M. abscessus that were taken from multiple body sites of 2 patients with CF who were undergoing treatment at Great Ormond Street Hospital, United Kingdom, in 2015. Results: We found evidence of extensive diversity within patients over time. A clustering analysis of single nucleotide variants revealed that each patient harbored multiple subpopulations, which were differentially abundant between sputum, lung samples, chest wounds, and pleural fluid. The sputum isolates did not reflect the overall within-patient diversity and did not allow for the detection of subclones with mutations previously associated with macrolide resistance (rrl 2058/2059). Some variants were present at intermediate frequencies before the lung transplants. The time of the transplants coincided with extensive variation, suggesting that this event is particularly disruptive for the microbial community, but the transplants did not clear the M. abscessus infections and both patients died as a result of these infections. Conclusions: Isolates of M. abscessus from sputum do not always reflect the entire diversity present within the patient, which can include subclones with differing antimicrobial resistance profiles. An awareness of this phenotypic variability, with the sampling of multiple body sites in conjunction with WGS, may be necessary to ensure the best treatment for this vulnerable patient group.
MeSH term(s)	Anti-Bacterial Agents/pharmacology ; Anti-Bacterial Agents/therapeutic use ; Child ; Cystic Fibrosis/complications ; Cystic Fibrosis/microbiology ; Drug Resistance, Multiple, Bacterial ; Female ; Genetic Variation ; Humans ; Longitudinal Studies ; Lung/microbiology ; Lung Transplantation/adverse effects ; Macrolides/pharmacology ; Macrolides/therapeutic use ; Male ; Microbial Sensitivity Tests ; Mycobacterium Infections, Nontuberculous/microbiology ; Mycobacterium abscessus/drug effects ; Mycobacterium abscessus/genetics ; Phenotype ; Polymorphism, Single Nucleotide ; Sputum/microbiology ; United Kingdom ; Whole Genome Sequencing
Chemical Substances	Anti-Bacterial Agents ; Macrolides
Language	English
Publishing date	2019-01-28
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1099781-7
ISSN	1537-6591 ; 1058-4838
ISSN (online)	1537-6591
ISSN	1058-4838
DOI	10.1093/cid/ciz069
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