One third of the world population is infected with Tuberculosis and at risk to develop the disease. The WHO estimates that there are more than nine million new TB cases and 1,5 million deaths from TB each year. Multi-drug resistant (MDR) strains are a growing problem, with 500 000 new cases every year, 10% of these being extremely drug resistant (XDR). Many laboratories and international consortia are searching for effective new drugs and vaccines, and clinical trials are testing new therapeutic regimens to shorten standardized treatment. However, despite considerable efforts for the discovery of new drugs, only ten new antibiotic candidates or repurposed drugs have been subjected to preclinical trials.
Perhaps the greatest progress has been in the introduction of rapid diagnostic tools based on molecular biology. The incorporation of new technologies has revolutionized the detection of microbial pathogens and antibiotic resistance. The growing availability of high capacity sequencing has opened new doors for understanding host-pathogen interactions and immunological responses by identifying critical determinants in both the bacterial and the human genomes.
The training course will review all techniques currently used for the rapid diagnosis of tuberculosis and detection of antibiotic resistance, and provide a background on the genetic basis of the tests.
Genomics markers are used to study tuberculosis transmission and also the phylogeny and evolution of M. tuberculosis complex bacteria. The first techniques were based on the detection of polymorphisms in repeated DNA sequences, but newer, more precise tools are based on single nucleotide polymorphisms. These tools are useful for molecular epidemiology but need to be practiced and evaluated to determine if they are appropriate for local and regional mycobacterial laboratories.
The course will include lectures by international experts to provide up-to-date information on all of these new developments in the study and control of Tuberculosis. Lectures will cover general aspects of Tuberculosis including the immune response, diagnosis, drug susceptibility testing, treatment of MDR-TB, evolution of the M. tuberculosis complex bacteria and molecular epidemiology. In the practical sessions the participants will work with the latest PCR-based diagnostic and drug susceptibility tests commercially available. They will also identify resistance using DNA sequencing of antibiotic resistance determinants and learn how genomic analysis can be used for complete drug sensitivity determination. All of the topics discussed are posted on the WEB site www.moleculartb.org.
The tuberculosis course at the Institut Pasteur in Paris promotes laboratory up-grading, intercontinental exchanges and the discussion of future projects amongst the participants. It is an exceptional opportunity for the participants to acquire new knowledge and to exchange experiences and critically analyze the utility of the techniques in the context of their own countries. The course will stress the importance of implementing the right tools in the right settings for optimal efficacy.
Microscopic examination is a fast and cheap method to detect acid-fast bacilli (AFB) in stained clinical specimens smears. Both living and dead (viable and non-viable) bacilli will stain and be counted. A positive direct smear is suggestive of contagious Tuberculosis.
Gel electrophoresis is a method for separation and analysis of amplified DNA fragments, based on their size and charge. The protocol describes how to prepare agarose gel, load samples and migrate by applying an electric field.
Sequencing of specific genes allows to detection of mutations known to be associated to antibiotic resistance and the prediction of their resistance level Target genes – or part of them – are here amplified and sequenced. Results are compared to wild type sequence of each target gene. Mutations are compared to mutations known to be associated with antibiotic resistance.
Spoligotyping is a molecular fingerprinting method based on the "Direct Repeat" (DR) region, which is uniquely present in Mycobacterium tuberculosis com¬plex bacteria. The DR region in M. bovis BCG consists of directly repeated sequences of 36 base pairs, which are interspersed by non-repetitive DNA spacers. By spoligotyping one can detect the presence or absence of spacers of known sequence to compare M. tuberculosis com¬plex strains.
Multilocus Variable Number Tandem Repeat Genotyping of Mycobacterium tuberculosis. Here is presented the discriminatory subset of 15 loci with the highest evolutionary rates proposed as the MIRU standard for routine epidemiological discrimination of M. tuberculosis isolates, using simpler electrophoresis with agarose gels.
GenoType® is a Line Probe Assay for identification of the M. tuberculosis complex from pulmonary clinical specimens or cultivated samples and its resistance to Rifampicin and/or Isoniazid (GenoType® MTBDRplus kit) and Fluoroquinolones and Aminoglycosides/Cyclic Peptides (GenoType® MTBDRsl ver 2.0 kit). The whole procedure includes DNA extraction from clinical or cultivated samples, a multiplex amplification with biotinilated primers and reverse hybridization.
The Xpert MTB/RIF test for use with the Cepheid GeneXpert® System is a semi-quantitative nested real-time PCR in vitro diagnostic test for detection of M. tuberculosis complex DNA and detection of rifampin- resistance associated mutations of the rpoB gene from clinical samples.