The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

Alison S. Devonshire, Denise M. O'Sullivan, Isobella Honeyborne, Gerwyn Jones, Maria Karczmarczyk, Jernej Pavšič, Alice Gutteridge, Mojca Milavec, Pablo Mendoza, Heinz Schimmel, Fran Van Heuverswyn, Rebecca Gorton, Daniela Maria Cirillo, Emanuele Borroni, Kathryn Harris, Marinus Barnard, Anthenette Heydenrych, Norah Ndusilo, Carole L. Wallis, Keshree Pillay & 11 others Thomas Barry, Kate Reddington, Elvira Richter, Erkan Mozioğlu, Sema Akyürek, Burhanettin Yalçinkaya, Muslum Akgoz, Jana Žel, Carole A. Foy, Timothy D. McHugh, Jim F. Huggett

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Original languageEnglish
Article number366
JournalBMC Infectious Diseases
Volume16
Issue number1
DOIs
Publication statusPublished - Aug 3 2016

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Molecular Pathology
Tuberculosis
Polymerase Chain Reaction
Physiologic Monitoring
Mycobacterium tuberculosis
Calibration
Real-Time Polymerase Chain Reaction
Microscopy
Reference Values
Therapeutics

Keywords

  • Diagnostics
  • Digital PCR
  • Mycobacterium tuberculosis

ASJC Scopus subject areas

  • Infectious Diseases

Cite this

Devonshire, A. S., O'Sullivan, D. M., Honeyborne, I., Jones, G., Karczmarczyk, M., Pavšič, J., ... Huggett, J. F. (2016). The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis. BMC Infectious Diseases, 16(1), [366]. https://doi.org/10.1186/s12879-016-1696-7

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis. / Devonshire, Alison S.; O'Sullivan, Denise M.; Honeyborne, Isobella; Jones, Gerwyn; Karczmarczyk, Maria; Pavšič, Jernej; Gutteridge, Alice; Milavec, Mojca; Mendoza, Pablo; Schimmel, Heinz; Van Heuverswyn, Fran; Gorton, Rebecca; Cirillo, Daniela Maria; Borroni, Emanuele; Harris, Kathryn; Barnard, Marinus; Heydenrych, Anthenette; Ndusilo, Norah; Wallis, Carole L.; Pillay, Keshree; Barry, Thomas; Reddington, Kate; Richter, Elvira; Mozioğlu, Erkan; Akyürek, Sema; Yalçinkaya, Burhanettin; Akgoz, Muslum; Žel, Jana; Foy, Carole A.; McHugh, Timothy D.; Huggett, Jim F.

In: BMC Infectious Diseases, Vol. 16, No. 1, 366, 03.08.2016.

Research output: Contribution to journalArticle

Devonshire, AS, O'Sullivan, DM, Honeyborne, I, Jones, G, Karczmarczyk, M, Pavšič, J, Gutteridge, A, Milavec, M, Mendoza, P, Schimmel, H, Van Heuverswyn, F, Gorton, R, Cirillo, DM, Borroni, E, Harris, K, Barnard, M, Heydenrych, A, Ndusilo, N, Wallis, CL, Pillay, K, Barry, T, Reddington, K, Richter, E, Mozioğlu, E, Akyürek, S, Yalçinkaya, B, Akgoz, M, Žel, J, Foy, CA, McHugh, TD & Huggett, JF 2016, 'The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis', BMC Infectious Diseases, vol. 16, no. 1, 366. https://doi.org/10.1186/s12879-016-1696-7
Devonshire, Alison S. ; O'Sullivan, Denise M. ; Honeyborne, Isobella ; Jones, Gerwyn ; Karczmarczyk, Maria ; Pavšič, Jernej ; Gutteridge, Alice ; Milavec, Mojca ; Mendoza, Pablo ; Schimmel, Heinz ; Van Heuverswyn, Fran ; Gorton, Rebecca ; Cirillo, Daniela Maria ; Borroni, Emanuele ; Harris, Kathryn ; Barnard, Marinus ; Heydenrych, Anthenette ; Ndusilo, Norah ; Wallis, Carole L. ; Pillay, Keshree ; Barry, Thomas ; Reddington, Kate ; Richter, Elvira ; Mozioğlu, Erkan ; Akyürek, Sema ; Yalçinkaya, Burhanettin ; Akgoz, Muslum ; Žel, Jana ; Foy, Carole A. ; McHugh, Timothy D. ; Huggett, Jim F. / The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis. In: BMC Infectious Diseases. 2016 ; Vol. 16, No. 1.
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abstract = "Background: 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. Methods: 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. Results: 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. Conclusions: 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.",
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AU - Devonshire, Alison S.

AU - O'Sullivan, Denise M.

AU - Honeyborne, Isobella

AU - Jones, Gerwyn

AU - Karczmarczyk, Maria

AU - Pavšič, Jernej

AU - Gutteridge, Alice

AU - Milavec, Mojca

AU - Mendoza, Pablo

AU - Schimmel, Heinz

AU - Van Heuverswyn, Fran

AU - Gorton, Rebecca

AU - Cirillo, Daniela Maria

AU - Borroni, Emanuele

AU - Harris, Kathryn

AU - Barnard, Marinus

AU - Heydenrych, Anthenette

AU - Ndusilo, Norah

AU - Wallis, Carole L.

AU - Pillay, Keshree

AU - Barry, Thomas

AU - Reddington, Kate

AU - Richter, Elvira

AU - Mozioğlu, Erkan

AU - Akyürek, Sema

AU - Yalçinkaya, Burhanettin

AU - Akgoz, Muslum

AU - Žel, Jana

AU - Foy, Carole A.

AU - McHugh, Timothy D.

AU - Huggett, Jim F.

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