The use of digital pcr to improve the application of quantitative molecular diagnostic methods for tuberculosis
| Autor: | Erkan Mozioğlu, Kathryn A. Harris, Rebecca Gorton, Kate Reddington, Pablo Mendoza, Emanuele Borroni, Thomas Barry, Mojca Milavec, Anthenette Heydenrych, Daniela Maria Cirillo, Elvira Richter, Norah Ndusilo, Isobella Honeyborne, Marinus Barnard, Alison S. Devonshire, Jana Žel, Carole A. Foy, Burhanettin Yalcinkaya, Sema Akyürek, Maria Karczmarczyk, Carole L. Wallis, Jernej Pavšič, Fran Van Heuverswyn, Alice Gutteridge, Heinz Schimmel, Denise M. O'Sullivan, Keshree Pillay, Muslum Akgoz, Jim F. Huggett, Gerwyn M. Jones, Timothy D. McHugh |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Adult DNA Bacterial Male Tuberculosis digital pcr assessing treatment response Molecular Diagnostic Method polymerase-chain-reaction Computational biology bacterial load assay Real-Time Polymerase Chain Reaction Sensitivity and Specificity bacillary load Mycobacterium tuberculosis 03 medical and health sciences medicine diagnostics Humans Digital polymerase chain reaction guidelines minimum information Pathology Molecular Tuberculosis Pulmonary mycobacterium tuberculosis Microscopy xpert mtb/rif assay amplification assays biology business.industry mycobacterial load biology.organism_classification medicine.disease Patient management Therapeutic monitoring 030104 developmental biology Infectious Diseases Molecular Diagnostic Techniques human cytomegalovirus Immunology Female business Tb treatment Quality assurance pulmonary tuberculosis Research Article |
| Zdroj: | BMC Infectious Diseases |
| Popis: | 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. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1696-7) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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