Drug resistance arises in Mycobacterium tuberculosis (MTB) generally
by mutation of chromosomal genes, but despite combined drug therapy, the spread
of multidrug-resistant (MDR) strains is increasing alarmingly. The LONG-DRUG
project is a longitudinal study, carried out in an area with high MDR-TB
prevalence, which aims to document the generation of multidrug resistant MTB
strains through the use of novel molecular techniques. The combined use of a
variety of techniques on longitudinally obtained samples and primary cultures
will enable clinically significant knowledge to be generated on the evolution of
multidrug resistance in Mycobacterium.
MTB positive clinical samples will be collected in the central TB Hospital of
Abkhazia (former Soviet Union), an area of increased MDR-TB prevalence, by
Médecins Sans Frontières (MSF). An already ongoing medical aid programme of MSF
is a guarantee for longitudinal sample collection. After sample shipment and
routine microbiological examination, the minimal inhibitory concentration to
primary and secondary anti-tuberculosis drugs will be determined. The
determination of the resistance phenotype will target the qualitative molecular
analysis to resistance-related genes. Real time PCR and RFLP-PCR will be used to
screen for heterogeneous MTB populations. Quantitative real time PCR using
molecular beacons on sequenced single nucleotide polymorphisms will finally
elucidate prevalence of single resistant clones in bacterial populations. The
genetic relationship of all significant strains will be analysed by IS-RFLP and
spoligotyping. Centralised data management and correlation of clinical,
bacteriological and molecular data should enable elucidation of the contribution
of resistant subpopulations to the development of MDR-TB and thus the clinical
significance of drug-resistant TB clones in mixed populations.
High prevalence of drug resistance in TB in various geographical settings
suggests that MDR-TB may become a more significant problem in the future, even
for industrialised countries. The fact that no significant novel first line
anti-TB drugs have been developed over the last 40 years does contribute to the
significance of the problem.
The first aim of the LONG-DRUG consortium is the generation of a
collection of longitudinally collected samples from patients with MDR-TB.
Diverse molecular tools will be generated and validated for an in-depth
characterisation of these samples. The primary objectives are;
1) the molecular characterisation of multiple drug resistance development in
MTB populations over time
2) the clarification of the epidemiological relationship of the strains and
sub-clones identified in the study population
3) the elucidation of the generation of resistant sub-populations of MTB are
of clinical relevance.
Generate knowledge on (i) the resistance development in a bacterial
population infecting the human host over time, (ii) the fate of the different
resistant subpopulations generated during this process, and (iii) the clinical
relevance of these microbial populations.
Over 300 samples have been collected to which the minimal inhibitory
concentration MIC for the classical first line anti TB drugs is being
determined. About 200 of these are resistant to at least one drug, and the
determination of the MIC for second line anti TB drugs is in progress. The
collection of samples already contains an extensive follow-up from over 15
Novel molecular tools will be prepared and validated during the project and
will be available to diagnostic laboratories promptly after publication. It is
possible that the careful analysis of drug resistance development in MTB will
develop novel recommendations for use of molecular technologies in drug
resistance detection. The partners also envisage that the molecular tools being
developed, exploiting the SNPs generated by the selective pressure of drugs, may
enable the consortium to develop more general considerations on the
characterisation of the progression and outcome of human pulmonary TB.
Marco R. Oggioni
LAMMB, Dept. Biol Mol.
Università di Siena
LAMMB, Policlinico Le Scotte 1S
Tel: +39 0577 233101
Fax: +39 0577 233334