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. 2016 Aug 3:16:366.
doi: 10.1186/s12879-016-1696-7.

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

Alison S Devonshire  1 , Denise M O'Sullivan  1 , Isobella Honeyborne  2 , Gerwyn Jones  1 , Maria Karczmarczyk  3 , Jernej Pavšič  4 , Alice Gutteridge  1 , Mojca Milavec  4 , Pablo Mendoza  5 , Heinz Schimmel  3 , Fran Van Heuverswyn  3 , Rebecca Gorton  2 , Daniela Maria Cirillo  6 , Emanuele Borroni  6 , Kathryn Harris  7 , Marinus Barnard  8   9 , Anthenette Heydenrych  8   9 , Norah Ndusilo  10 , Carole L Wallis  11 , Keshree Pillay  11 , Thomas Barry  12 , Kate Reddington  12 , Elvira Richter  13 , Erkan Mozioğlu  14 , Sema Akyürek  14 , Burhanettin Yalçınkaya  14 , Muslum Akgoz  14 , Jana Žel  4 , Carole A Foy  1 , Timothy D McHugh  2 , Jim F Huggett  15   16   17
Affiliations

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

Alison S Devonshire et al. BMC Infect Dis. .

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.

Keywords: Diagnostics; Digital PCR; Mycobacterium tuberculosis.

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Figures

Fig. 1
Fig. 1
Homogeneity assessment of ten randomly selected tubes of (a) TB Control Plasmid (b) H37Rv gDNA, (c) BCG/ASM and (d) Total MTB control. * solid line depicts mean value and 95 % CI measured using colony forming units. ** solid line depicts value and range provided by the product sheet
Fig. 2
Fig. 2
Assessment of the reproducibility of dPCR measuring (a) TB Control Plasmid and (b) H37Rv gDNA with four laboratories and three different dPCR instruments or (c) BCG/ASM three laboratories and two different dPCR instruments
Fig. 3
Fig. 3
Fold difference between BCG/ASM and Total MTB control when measured using dPCR and compared to eight clinical testing laboratories applying qPCR or Xpert MTB/RIF
See this image and copyright information in PMC

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