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Evaluation of different strategies for direct detection of B. melitensis in clinical samples from infected fetus

Comunicación oral en Brucellosis 2011-International Research Conference

23 de septiembre de 2011

Perez-Sancho M., Alvarez J., Duran M., Garcia N., Martinez I., Garcia-Seco T., Gonzalez S., Garrido F. y Dominguez L.

Due to specificity limitations of indirect diagnosis tests, direct detection techniques are usually required to confirm Brucella infection. Bacteriological culture is usually the gold-standard technique for brucellosis confirmation, but it has several drawbacks (time consuming, need of BSL3 facilities, limited sensitivity…). Molecular-based direct detection techniques can partially overcome these problems, thus representing a useful complementary alternative for detection of the pathogen. Among them, Loop-Mediated Isothermal Amplification (LAMP) is rapid and specific detection method that can be performed without sophisticated equipment, therefore becoming
a good option for implementation in developing countries. The present study aimed at the evaluation of the usefulness of two direct molecular-techniques (Real time PCR and an in-house designed LAMP) compared to classical bacteriological methods for B melitensis detection on clinical samples from aborted fetus (abomasal content, n=17; liver, n=16; spleen, n=15; lungs, n=15) from infected ewes. Both RT- PCR and LAMP targeted the multiple insertion element IS711. Isolation of B. melitensis was performed according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2010).
Overall sensitivity of RT-PCR and in house-LAMP compared with culture was 51.22% and 53.66% respectively. Both direct molecular techniques showed similar using the culture as gold-standard. However, differences were observed depending on the type of clinical specimens analyzed: the highest sensitivity of direct detection techniques was observed in abomasal content (60-75% for both molecular-based direct detection techniques). Lack of detection of Brucella DNA in culture positive samples could be explained by the existence of inhibitors
of amplification reactions in clinical samples. Still, a number of samples (n=5) were positive in the direct molecular techniques and negative in the microbiological analysis, thus highlighting the usefulness of these tests. In summary, direct detection techniques constitute a feasible complementary technique for rapid Brucella
infection confirmation, as even though a limited sensitivity might be observed (most likely due to sample issues)they yield a rapid result that could be useful at the flock-diagnosis level for diagnostic confirmation. Analysis of multiple samples from aborted fetuses contributed to ensure optimal Brucella detection. Our results show
that LAMP technique shows a similar sensitivity to that of RT-PCR, and therefore can become a diagnostic alternative for Brucella detection/identification, especially in areas where poor laboratory resources exist, due to its easy implementation




Participantes:

Universidad ComplutenseServicio de Zoonosis Emergentes, de Baja Prevalencia y Agresivos Biológicos (NED). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).

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


Enlace a Brucellosis 2011-International Research Conference





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Brucellosis 2011-International Research Conference


21-23 septiembre de 2011
Buenos Aires
Argentina

TÍTULO: Evaluation of different strategies for direct detection of B. melitensis in clinical samples from infected fetus


TIPO: Comunicación oral


AUTORES: Perez-Sancho M., Alvarez J., Duran M., Garcia N., Martinez I., Garcia-Seco T., Gonzalez S., Garrido F. y Dominguez L.


PARTICIPANTES VISAVET


First
Marta Pérez Sancho
2nd
Julio Álvarez Sánchez
4th
Nerea García Benzaquén
5th
Irene Martínez Alares
6th
Teresa García-Seco Romero
7th
Sergio González Domínguez
Last
Lucas Domínguez Rodríguez

FECHA: 23 de septiembre de 2011



CITA ESTA COMUNICACIÓN:

Perez-Sancho M., Alvarez J., Duran M., Garcia N., Martinez I., Garcia-Seco T., Gonzalez S., Garrido F. y Dominguez L. Evaluation of different strategies for direct detection of B. melitensis in clinical samples from infected fetus. Brucellosis 2011-International Research Conference, Asociación Argentina de Microbiología, Buenos Aires, Argentina, 23 de septiembre de 2011. (Comunicación oral)


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