title: The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.
abstract: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring.
  
  
  
  To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories.
  
  
  
  dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude.
  
  
  
  TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.
authors.affiliation: Molecular and Cell Biology, LGC, Queens Road, Teddington, TW11 0LY, UK.
authors.forename: Alison S
authors.initials: AS
authors.lastname: Devonshire
authors.affiliation: Molecular and Cell Biology, LGC, Queens Road, Teddington, TW11 0LY, UK.
authors.forename: Denise M
authors.initials: DM
authors.lastname: O'Sullivan
authors.affiliation: Centre for Clinical Microbiology, Department of Infection, University College London, Royal Free Campus, London, UK.
authors.forename: Isobella
authors.initials: I
authors.lastname: Honeyborne
authors.affiliation: Molecular and Cell Biology, LGC, Queens Road, Teddington, TW11 0LY, UK.
authors.forename: Gerwyn
authors.initials: G
authors.lastname: Jones
authors.affiliation: European Commission, Joint Research Centre (JRC), Institute for Reference Materials and Measurements (IRMM), Geel, Belgium.
authors.forename: Maria
authors.initials: M
authors.lastname: Karczmarczyk
authors.affiliation: National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
authors.forename: Jernej
authors.initials: J
authors.lastname: Pavšič
authors.affiliation: Molecular and Cell Biology, LGC, Queens Road, Teddington, TW11 0LY, UK.
authors.forename: Alice
authors.initials: A
authors.lastname: Gutteridge
authors.affiliation: National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Milavec
authors.affiliation: Vircell S.L. Molecular Diagnostic Department, The Technology Park of Health Sciences, Granada, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Mendoza
authors.affiliation: European Commission, Joint Research Centre (JRC), Institute for Reference Materials and Measurements (IRMM), Geel, Belgium.
authors.forename: Heinz
authors.initials: H
authors.lastname: Schimmel
authors.affiliation: European Commission, Joint Research Centre (JRC), Institute for Reference Materials and Measurements (IRMM), Geel, Belgium.
authors.forename: Fran
authors.initials: F
authors.lastname: Van Heuverswyn
authors.affiliation: Centre for Clinical Microbiology, Department of Infection, University College London, Royal Free Campus, London, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Gorton
authors.affiliation: TB Supranational Reference Laboratory, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Daniela Maria
authors.initials: DM
authors.lastname: Cirillo
authors.affiliation: TB Supranational Reference Laboratory, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Borroni
authors.affiliation: Microbiology, Virology and Infection Control, Great Ormond Street Hospital NHS Foundation Trust, London, UK.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Harris
authors.affiliation: Centre for Clinical Tuberculosis Research, TASK Applied Science, Cape Town, South Africa.|SA MRC Centre for TB Research, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences - Stellenbosch University, Francie van Zijl Drive, Tygerberg 7505, PO Box 241, Cape Town, 8000, South Africa.
authors.forename: Marinus
authors.initials: M
authors.lastname: Barnard
authors.affiliation: Centre for Clinical Tuberculosis Research, TASK Applied Science, Cape Town, South Africa.|SA MRC Centre for TB Research, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences - Stellenbosch University, Francie van Zijl Drive, Tygerberg 7505, PO Box 241, Cape Town, 8000, South Africa.
authors.forename: Anthenette
authors.initials: A
authors.lastname: Heydenrych
authors.affiliation: KCRI, Moshi, Tanzania.
authors.forename: Norah
authors.initials: N
authors.lastname: Ndusilo
authors.affiliation: BARC-SA and Mycobacteriology Department, Lancet Laboratories, Johannesburg, South Africa.
authors.forename: Carole L
authors.initials: CL
authors.lastname: Wallis
authors.affiliation: BARC-SA and Mycobacteriology Department, Lancet Laboratories, Johannesburg, South Africa.
authors.forename: Keshree
authors.initials: K
authors.lastname: Pillay
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Barry
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Kate
authors.initials: K
authors.lastname: Reddington
authors.affiliation: Forschungszentrum Borstel, National Reference Centre for Mycobacteria, Borstel, D-23845, Germany.
authors.forename: Elvira
authors.initials: E
authors.lastname: Richter
authors.affiliation: UME, Ulusal Metroloji Enstitüsü, Gebze, Kocaeli, Turkey.
authors.forename: Erkan
authors.initials: E
authors.lastname: Mozioğlu
authors.affiliation: UME, Ulusal Metroloji Enstitüsü, Gebze, Kocaeli, Turkey.
authors.forename: Sema
authors.initials: S
authors.lastname: Akyürek
authors.affiliation: UME, Ulusal Metroloji Enstitüsü, Gebze, Kocaeli, Turkey.
authors.forename: Burhanettin
authors.initials: B
authors.lastname: Yalçınkaya
authors.affiliation: UME, Ulusal Metroloji Enstitüsü, Gebze, Kocaeli, Turkey.
authors.forename: Muslum
authors.initials: M
authors.lastname: Akgoz
authors.affiliation: National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
authors.forename: Jana
authors.initials: J
authors.lastname: Žel
authors.affiliation: Molecular and Cell Biology, LGC, Queens Road, Teddington, TW11 0LY, UK.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Foy
authors.affiliation: Centre for Clinical Microbiology, Department of Infection, University College London, Royal Free Campus, London, UK.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: McHugh
authors.affiliation: Molecular and Cell Biology, LGC, Queens Road, Teddington, TW11 0LY, UK. jim.huggett@lgcgroup.com.|Centre for Clinical Microbiology, Department of Infection, University College London, Royal Free Campus, London, UK. jim.huggett@lgcgroup.com.|School of Biosciences & Medicine, Faculty of Health & Medical Sciences, University of Surrey, Guildford, UK. jim.huggett@lgcgroup.com.
authors.forename: Jim F
authors.initials: JF
authors.lastname: Huggett
mesh_terms: D000328:Adult; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D008297:Male; D008853:Microscopy; D025202:Molecular Diagnostic Techniques; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D057089:Pathology, Molecular; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*
chemical_list: D004269:DNA, Bacterial
keywords: Diagnostics; Digital PCR; Mycobacterium tuberculosis