title: A Rapid and Sensitive Diagnostic Screening Assay for Detection of Mycobacteria Including Mycobacterium tuberculosis Directly from Sputum without Extraction.
abstract: We report a novel approach utilising a real-time PCR screening assay targeting a 53 bp tandemly repeated element present at various loci within the Mycobacterium tuberculosis (Mtb) genome. Positive samples were identified within a discriminatory melting curve range of 90-94°C, with results obtained in under one hour directly from decontaminated sputum samples without extraction. A panel of 89 smear-positive sputa were used for analytical validation of the assay with 100% concordance, with sensitivity matching that of culture. Cross reactivity was detected within a narrow range of mycobacteria other than tuberculosis (MOTT) (five sputa, three in silico), with the highest sensitivity within M. avium complex (MAC). A year-long head to head evaluation of the test with the GeneXpert platform was carried out with 104 consecutive samples at the Royal Free Hospital, UK. Receiver operating characteristics (ROC) analysis of the data revealed that the two tests are approximately equivalent in sensitivity, with the area under the curve being 0.85 and 0.80 for the GeneXpert and our assay, respectively, indicating that the test would be a cost effective screen prior to GeneXpert testing.
authors.affiliation: Genomic Services & Development Unit, Public Health England, Microbiology Services Operations, 61 Colindale Avenue, London NW9 5HT, UK.
authors.forename: Lisa Jane
authors.identifier: 0000-0002-6168-4720
authors.initials: LJ
authors.lastname: Cross
authors.affiliation: Genomic Research Unit, Public Health England, Microbiology Services, 61 Colindale Avenue, London NW9 5HT, UK; Centre for Clinical Microbiology, Royal Free Campus, UCL and Department of Medical Microbiology, Royal Free Hampstead NHS Trust, Royal Free Hospital, Pond Street, London NW3 2PF, UK.
authors.forename: Catherine
authors.identifier: 0000-0003-3049-2256
authors.initials: C
authors.lastname: Anscombe
authors.affiliation: Centre for Clinical Microbiology, Royal Free Campus, UCL and Department of Medical Microbiology, Royal Free Hampstead NHS Trust, Royal Free Hospital, Pond Street, London NW3 2PF, UK.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: McHugh
authors.affiliation: Centre for Infectious Disease Epidemiology, Mortimer Market Centre, UCL and TB Section, Public Health England, London WC1E 6JB, UK.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Abubakar
authors.affiliation: Centre for Clinical Microbiology, Royal Free Campus, UCL and Department of Medical Microbiology, Royal Free Hampstead NHS Trust, Royal Free Hospital, Pond Street, London NW3 2PF, UK.
authors.forename: Robert John
authors.initials: RJ
authors.lastname: Shorten
authors.affiliation: Genomic Research Unit, Public Health England, Microbiology Services, 61 Colindale Avenue, London NW9 5HT, UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Thorne
authors.affiliation: Genomic Services & Development Unit, Public Health England, Microbiology Services Operations, 61 Colindale Avenue, London NW9 5HT, UK.
authors.forename: Cath
authors.initials: C
authors.lastname: Arnold