Rapid identification of Beijing Mycobacterium tuberculosis strains by high-resolution melting analysis
Abstract number: P2038
Navarro Y., Alonso M., Barletta F., Martinez Lirola M., Gotuzzo E., Bouza E., García de Viedma D.
Background and Objective: Genotypic analysis of Mycobacterium tuberculosis (MTB) has enabled us to define several lineages. Among these, the Beijing family is considered highly virulent and transmissible, has been associated with resistance in certain settings and involved in severe outbreaks, making it one of the most closely monitored lineages. Therefore, rapid identification of cases infected by Beijing MTB strains could be relevant at a clinical and epidemiological level. Our aim in this study was to develop and evaluate a new rapid procedure to identify Beijing MTB isolates using high-resolution melting (HRM) analysis.
Methods: We designed a real-time polymerase chain reaction followed by HRM analysis based on the identification of the single-nucleotide polymorphism A191C in the Rv2629 gene, which is a marker for the Beijing lineage. We analysed thirty-two MTB strains (18 representatives of the Beijing genotype and 14 representatives of the non-Beijing genotype, according to their spoligotypes) and 44 smear-positive clinical respiratory samples. Finally, 84 MTB strains were selected from a prospective cohort of 145 isolates from 5 areas in Peru between December 2008 and March 2009.
Results: HRM analysis efficiently differentiated Beijing and non-Beijing strains from the 32 reference strains. HRM was able to assign a Beijing/non-Beijing genotype in 100% of respiratory specimens with a high bacterial load and in 73.1% of those with a low/intermediate load. An indirect analysis based on the identification of heteroduplexes when Beijing and non-Beijing DNA were combined in the reaction succeeded in assigning a genotype in 50% of the cases where the direct HRM analysis had failed. Our HRM design identified 10.7% of the Peruvian strains as belonging to the Beijing genotype; this proportion reached 20% in the North Lima area.
Conclusion: Our HRM-based method is a rapid, reliable, and sensitive method for the efficient identification of cases infected by high-risk Beijing MTB strains. A Beijing/non-Beijing genotype was succesfully assigned in 100% of the MTB isolates and in 90.9% of the respiratory specimens assayed. Its application to a Peruvian sample of MTB isolates revealed high representativity of Beijing strains, especially in the Lima area.
Y. Navarro was receiving a grant for International Cooperation of the "Fundación para la Investigación Biomédica del Hospital Gregorio Marañón".
This study was partially funded by the FIS (FIS060882; FIS061467; FIS06/90490; 06/90357; PS09/02205).
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
|Back to top|