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Direct PCR on tissue samples to detect Mycobacterium tuberculosis complex: an alternative to the bacteriological culture

Investigation article published in Journal of Clinical Microbiology

November 25th, 2020

Bovine tuberculosis (bTB) is an ongoing issue in several countries within the European Union (EU). Microbiological culture is the official confirmation technique for the presence of Mycobacterium tuberculosis complex (MTBC) members in bovine tissues, but several methodological issues, such as moderate sensitivity and long incubation times, require the development of more sensitive and rapid techniques. This study evaluates the analytical and diagnostic performance, comparatively to culture, of a real-time PCR targeting the MTBC-specific IS6110 transposon using a panel of bovine tissue samples sourced from the Spanish bTB eradication campaign. Robustness and repeatability were evaluated in an inter-laboratory trial between EU National Reference Laboratories. The limit of detection with 95% confidence was established at 65 fg/reaction of purified genomic. Diagnostic sensitivity and specificity was, respectively, 96.45% and 93.66%, and the overall agreement (κ) was 0.88. Cross-reactivity was detected against two mycobacterial isolates identified as M. marinum and "M. avium subsp. hominissuis" species, and Whole Genome Sequence analysis of the latter isolate revealed an IS6110-like sequence with 83% identity. An identical IS-like element was found in other Mycobacterium avium Complex species in the NCBI nucleotide and WGS databases. Despite this finding, this methodology is considered a valuable alternative to culture and the strategy of use should be defined depending on the control or eradication programmes




Lorente-Leal V., Liandris E., Pacciarini M., Botelho A., Kenny K., Loyo B., Fernandez R., Bezos J., Dominguez L., de Juan L. and Romero B..




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Direct PCR on tissue samples to detect Mycobacterium tuberculosis complex: an alternative to the bacteriological culture

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Direct PCR on tissue samples to detect Mycobacterium tuberculosis complex: an alternative to the bacteriological culture



Participants:

Universidad ComplutenseServicio de Micobacterias (MYC). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).

Universidad ComplutenseDepartamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM).

Centro Nazionale di Referenza per la Tubercolosi Bovina. Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia Romagna (IZSLER).

Instituto Nacional de Investigação Agrária e Veterinária (INIAV).

Central Veterinary Research Laboratory (CVRL).

Department of Agriculture, Food and the Marine (DAFM).

Junta de Castilla y LeónServicio de Sanidad Animal. Junta de Castilla y León.

Junta de Castilla y LeónConsejería de Agricultura y Ganadería. Junta de Castilla y León.

Ministerio de Agricultura, Pesca y AlimentaciónMinisterio de Agricultura, Pesca y Alimentación (MAPA).







Journal of Clinical Microbiology
FACTOR YEAR Q
5.948 2020

NLMID: 7505564

PMID: 33239374

ISSN: 0095-1137



TITLE: Direct PCR on tissue samples to detect Mycobacterium tuberculosis complex: an alternative to the bacteriological culture


JOURNAL: J. Clin. Microbiol.


NUMERACIÓN: 01404-20


AÑO: 2020


PUBLISHER: American Society for Microbiology


AUTHORS: Lorente-Leal V., Liandris E., Pacciarini M., Botelho A., Kenny K., Loyo B., Fernandez R., Bezos J., Dominguez L., de Juan L. and Romero B..


VISAVET PARTICIPANTS


First
Victor Lorente Leal
8th
Javier Bezos Garrido
9th
Lucas Domínguez Rodríguez
10
Lucía de Juan Ferré
Last
Beatriz Romero Martínez

DOI: https://doi.org/ 10.1128/JCM.01404-20


CITE THIS PUBLICATION:

Lorente-Leal V., Liandris E., Pacciarini M., Botelho A., Kenny K., Loyo B., Fernandez R., Bezos J., Dominguez L., de Juan L. and Romero B. Direct PCR on tissue samples to detect Mycobacterium tuberculosis complex: an alternative to the bacteriological culture. Journal of Clinical Microbiology. 01404-20. 2020. (A). ISSN: 0095-1137. DOI: 10.1128/JCM.01404-20


UNITS: