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A high-throughput method for the simultaneous detection of drug resistance and genotypic mutations in M.tuberculosis isolates

Abstract number: O234

Bergval I., Schuitema A., Klatser P., Anthony R.

Objective: To make the genetic screening of tuberculosis (TB) isolates suitable for low-income countries as well as endemic countries with high prevalence/incidence of drug-resistant TB. The emerging epidemic of drug-resistant TB calls for rapid diagnosis and early detection of drug resistance. This allows immediate and appropriate treatment of the patient and could thereby reduce the spread of multidrug-resistant (MDR) or extensively drug resistant TB (XDR-TB).

We have recently developed an MTB-specific multiplex assay, based on Multiplex Ligation-dependent Probe Amplification (MLPA). This method allows simultaneous detection of multiple dispersed DR mutations and genotype-specific mutations in the Mycobacterium tuberculosis (MTB) genome and has proved to be highly specific.

Method: The current read-out of MLPA is done by capillary electrophoresis, a method that is expensive and time-consuming and therefore difficult to implement and sustain in low-income countries. Recent developments in biotechnology have raised the opportunity to transfer the assay to a liquid array. Current MLPA-probes were modified to be compatible with such a system.

To cover the increasing prevalence of MDR- and XDR-TB isolates, targets revealing resistance to several important second-line drugs (e.g. fluoroquinolones) were included in the MLPA assay, as well as additional genotypic markers.

Results and Conclusion: The newly included MLPA-probes were very specific for the targeted SNPs, allowing the detection of prevalent second-line drug resistance mutations and a further delineation of MTB complex members. The genotyping abilities of the MLPA assay were additionally increased by the discovery of a new genotypic marker. There will be an explosion of SNP discovery in the next years as data from high throughput sequencing projects become available. Methods allowing informative SNPs to be rapidly detected will then become increasingly valuable.

Furthermore, we feel that the new analysis method for MLPA will specifically be of added value in low-income countries with high endemicity of DR-TB. The liquid array allows multi-parameter testing and could provide a standard platform for several diagnostic and screening tests, which are traditionally performed by several different methods. Therefore, MLPA combined with this detection system can bring molecular typing of MTB clinical isolates closer to the patient than is currently feasible.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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