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 language | English |
|---|---|
| Article number | 366 |
| Journal | BMC Infectious Diseases |
| Volume | 16 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Aug 3 2016 |
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Keywords
- Diagnostics
- Digital PCR
- Mycobacterium tuberculosis
ASJC Scopus subject areas
- Infectious Diseases
Cite this
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 journal › Article
}
TY - JOUR
T1 - The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis
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.
PY - 2016/8/3
Y1 - 2016/8/3
N2 - 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.
AB - 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.
KW - Diagnostics
KW - Digital PCR
KW - Mycobacterium tuberculosis
UR - http://www.scopus.com/inward/record.url?scp=84988420095&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84988420095&partnerID=8YFLogxK
U2 - 10.1186/s12879-016-1696-7
DO - 10.1186/s12879-016-1696-7
M3 - Article
VL - 16
JO - BMC Infectious Diseases
JF - BMC Infectious Diseases
SN - 1471-2334
IS - 1
M1 - 366
ER -