Isolation of Mycobacterium species from respiratory specimens for the diagnosis of tuberculosis using an automated sample preparation system based on magnetic particles

Abstract number: 1133_151

Lysén J., Høidal Berthelsen H.K., Espelund M., Refseth U.H.

Objectives:  

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, is today the single greatest cause of mortality due to an infectious agents with 2 million people dying every year and 8 million new cases. The rising incidence of multi-resistance Mycobacterium tuberculosis (MDR-TB) represent an additional problem to public health, with higher death-rates and the requirement for much more extensive antibiotic treatment. Routinely, mycobacterium infections are diagnosed by microscopic examination for the presence of acid-fast bacilli (AFB) and by conventional culture techniques. Whereas AFB testing lack sensitivity, cultivation may take up to 2–4 weeks to determine the presence of mycobacterium due to the slow growth. Compared to culture, sensitivity using various nucleic acid amplification techniques (NAAT) available is much lower. This may be due to either insufficient sample preparation from respiratory samples or incomplete lysis of the mycobacterium cells. Thus, the requirement for both a rapid and sensitive diagnostic system for TB is still largely unmet.

Method:  

Here we present a new automated system for sample preparation of mycobacterium DNA from respiratory samples utilizing magnetic particles. In this system (chlamCAP, Genpoint, Norway), mycobacterium cells are initially adsorbed to uniquely coated paramagnetic particles and magnetically separated from the respiratory sample. A rapid lysis at RT releases DNA, which is then adsorbed onto the same magnetic particles. After washing, purified DNA is transferred micro-wells for NAAT analysis. Mycobacterium bovis (BCG) of the MTC group was spiked in growth medium and isolation was performed using the chlamCAP system on a Tecan Miniprep 75 customized to this particular application. Following isolation of mycobacterium DNA, the samples were analyzed by real-time PCR using primers specific for species belonging to MTC .

Results:  

The sensitivity obtained was <103 CFU/ml (positive PCR signal) as determined by plating. In addition, results obtained from heat-inactivated sputum samples containing 50% sputasol from patients suspected to be infected with M. tuberculosis, were in agreement with data from cultivation, demonstrating the robustness of the chlamCAP system.

Conclusion:  

Based on these results, we believe that the automated chlamCAP system in the future can promote the implementation of NAAT based methods in mycobacterium diagnostics, complementing today's enrichment systems.