AUTHOR=Kolia-Diafouka Pratt , Godreuil Sylvain , Bourdin Arnaud , Carrère-Kremer Severine , Kremer Laurent , Van de Perre Philippe , Tuaillon Edouard 

TITLE=Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.02224

DOI=10.3389/fmicb.2018.02224

ISSN=1664-302X

ABSTRACT=<p><bold>Background:</bold> When available, nucleic acid tests (NATs) offer powerful tools to strengthen the potential of tuberculosis (TB) diagnosis assays. The sensitivity of molecular assays is critical for detection of <italic>Mycobacterium tuberculosis</italic> (MTB) in paucibacillary sputum.</p><p><bold>Materials and Methods:</bold> The impact of targeting repetitive <italic>IS6110</italic> sequences on the PCR sensitivity was evaluated across <italic>mycobacterium</italic> strains and reference material. Six lysis-extraction protocols were compared. Next, 92 clinical sputum specimens including 62 culture-positive samples were tested and the results were compared to sputum-smear microscopy, culture, and Xpert MTB/RIF test. Finally, the capacity to detect low MTB DNA concentrations was assessed in 40 samples containing &lt;1.5 × 10<sup>2</sup> copies/ml <italic>ex vivo</italic> or after dilution.</p><p><bold>Results:</bold> The lower limit of detection (LOD) using the <italic>IS6110</italic> PCR was 107 genome copies/ml (95% CI: 83–130) using MTB H37Rv as a reference strain, versus 741 genome copies/ml (95% CI: 575–1094) using the <italic>senX3</italic> PCR. The proportion of recovered MTB DNA after lysis and extraction ranged from 35 to 82%. The Chelex<sup>®</sup> method appeared as a more efficient protocol among the six different protocols tested. The sensitivity and specificity in clinical sputum samples were 95.1% (95% CI: 90.7–99.6) and 100% (95% CI: 96.2–100.8), respectively. Among 40 samples with low MTB DNA concentration, 75% tested positive for <italic>IS6110</italic> PCR, versus 55% using the Xpert MTB/RIF assay (<italic>p</italic> = 0.03).</p><p><bold>Conclusion:</bold> Laboratory assays based on an efficient MTB lysis and DNA extraction protocols combined with amplification of <italic>IS6110</italic> repeat sequences appear as a sensitive diagnostic method to detect MTB DNA in sputum with low bacterial load.</p>