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Mycobacteriology and Aerobic Actinomycetes

Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads

Fernando Alcaide, Rocío Trastoy, Raquel Moure, Mónica González-Bardanca, Antón Ambroa, María López, Inés Bleriot, Lucia Blasco, Laura Fernandez-García, Marta Tato, German Bou, María Tomás
and Mycobacterial and GEMARA SEIMC/REIPI Bacterial Clinical Adaptation Study Group
Geoffrey A. Land, Editor
Fernando Alcaide
Department of Microbiology, Hospital Universitari de Bellvitge–IDIBELL, Hospitalet de Llobregat, SpainDepartment of Pathology and Experimental Therapy, Universitat de Barcelona, Hospitalet de Llobregat, SpainTuberculosis Investigation Unit of Barcelona (FUITB), Barcelona, Spain
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Rocío Trastoy
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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Raquel Moure
Department of Microbiology, Hospital Universitari de Bellvitge–IDIBELL, Hospitalet de Llobregat, Spain
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Mónica González-Bardanca
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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Antón Ambroa
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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María López
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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Inés Bleriot
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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Lucia Blasco
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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Laura Fernandez-García
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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Marta Tato
Microbiology Department-Research Institute Biomedical Ramón and Cajal (IRYCIS), Hospital Ramón and Cajal, Madrid, Spain
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German Bou
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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María Tomás
Microbiology Department-Biomedical Research Institute A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain
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Geoffrey A. Land
Carter BloodCare & Baylor University Medical Center
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DOI: 10.1128/JCM.00733-19
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ABSTRACT

Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the Mycobacterium tuberculosis complex (MTBC) have emerged in recent years. Real-time PCR amplification simultaneously combines the amplification and detection of candidate sequences by using fluorescent probes (mainly TaqMan or Molecular Beacons) in automated systems. The multiplex real-time PCR-short assay is performed using locked nucleic acid (LNA) probes (length, 8 to 9 nucleotides) in combination with CodUNG to detect multiple pathogens in clinical samples. In this study, we evaluated the performance of this novel multiplex assay for detecting the MTBC in comparison with that of the conventional culture-based method. The multiplex real-time PCR-shortTUB assay targets two genes, whiB3 (redox-responsive transcriptional regulator) and pstS1 (phosphate-specific transporter), yielding limits of detection (LOD) of 10 copies and 100 copies, respectively, and amplification efficiencies of 92% and 99.7%, respectively. A total of 94 extrapulmonary samples and pulmonary samples with low mycobacterial loads (all smear negative; 75 MTBC culture positive) were analyzed using the test, yielding an overall sensitivity of 88% and a specificity of 95%. For pleural fluid and tissues/biopsy specimens, the sensitivity was 83% and 85%, respectively. In summary, this technique could be implemented in routine clinical microbiology testing to reduce the overall turnaround time for MTBC detection and may therefore be a useful tool for the diagnosis of extrapulmonary tuberculosis and diagnosis using pulmonary samples with low mycobacterial loads.

FOOTNOTES

    • Received 7 May 2019.
    • Returned for modification 28 May 2019.
    • Accepted 9 June 2019.
    • Accepted manuscript posted online 12 June 2019.
  • Supplemental material for this article may be found at https://doi.org/10.1128/JCM.00733-19.

  • Copyright © 2019 American Society for Microbiology.

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Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads
Fernando Alcaide, Rocío Trastoy, Raquel Moure, Mónica González-Bardanca, Antón Ambroa, María López, Inés Bleriot, Lucia Blasco, Laura Fernandez-García, Marta Tato, German Bou, María Tomás Mycobacterial and GEMARA SEIMC/REIPI Bacterial Clinical Adaptation Study Group
Journal of Clinical Microbiology Jul 2019, 57 (8) e00733-19; DOI: 10.1128/JCM.00733-19

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Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads
Fernando Alcaide, Rocío Trastoy, Raquel Moure, Mónica González-Bardanca, Antón Ambroa, María López, Inés Bleriot, Lucia Blasco, Laura Fernandez-García, Marta Tato, German Bou, María Tomás Mycobacterial and GEMARA SEIMC/REIPI Bacterial Clinical Adaptation Study Group
Journal of Clinical Microbiology Jul 2019, 57 (8) e00733-19; DOI: 10.1128/JCM.00733-19
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KEYWORDS

LNATm technology
mycobacterium
real-time PCR
low mycobacterial load
short assay

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