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DISSARM
TUBERCULOSIS
Framework programme: 5
Project number:
QLK2-CT-2000-00765
EC contribution: € 899 695
Duration: 36 months
Type: RS
Starting date: 1 September 2000
Website: http://www.nmrc.ie/projects/dissarm/
Graphic element Development of Integrable Sensors for Screening of Antibiotic Resistance in Mycobacterium
Keywords: Tuberculosis; multi-drug resistance; rifampicin; sensor array; molecular biology methods

Summary:

The overall aim of this project is the development of multisequence DNA PCR-ELISA and sensor arrays for the identification of multidrug resistant (MDR) strains of Mycobacterium tuberculosis.  The methodologies developed will allow assessment from clinical samples within hours, in comparison to the laborious and time-consuming methods presently in use.  The use of a 3 x 3 final configuration sensor array allows for the development of a disposable, rapid, simple and one-step measurement system.

Problem:

Before the discovery of specific antibiotics for the treatment of tuberculosis (TB), there was no cure.  The mortality rate of those with pulmonary disease (disease of the lungs) was about 50%. The introduction of anti-TB drugs in the 1950s and the development of the various drug regimens meant that by the 1980s there was a 98% chance of cure. However, treatment had to be continued with good quality drugs for as long as six months to ensure cure. The difficulties in ensuring this occurs, especially in resource-poor countries, have resulted in an increasing incidence of tubercle bacteria resistant to the most effective drug.   In the last decade, TB has re-emerged as one of the leading causes of death (nearly 3 million die annually). The estimated 8.8 million new cases every year correspond to 52 000 deaths per week or more than 7 000 each day, which translates into more than 1 000 new cases every hour of every day.  These death rates, however, only partially depict the global TB threat; more than 80% of TB patients are in the economically productive age of 15 to 49 years. A decline in socio-economic standards and, to a lesser extent AIDS, are contributing to the disease's resurgence in industrialised countries.  In most developing countries, although the disease has always been endemic, its severity has increased, probably because of extensive social restructuring due to rapid industrialisation, conflicts and to the global HIV pandemic. A major public health problem worldwide, TB is now a global emergency.  In 1993, the World Health Organisation initiated a Global Programme on Drug Resistance in 1994 1,2 and in 1998 announced improved treatment regimens3. It seems, therefore, reasonable that a detection device for mutations responsible for MDRTB is chosen in this project as a demonstration of a molecular diagnostics tool that can be applied in all types of DNA diagnostics, using the same generic technology.  Thus, with a relatively simple, disposable array, the concept will be demonstrated and a useful diagnostic tool will be developed.

Rifampicin (RIF), first introduced in 1972 as an anti-tubercular drug, is extremely effective against M. tuberculosis.  Because of its high bactericidal action, rifampicin, along with isoniazide, forms the backbone of short-course chemotherapy.  Resistance to rifampicin is increasing because of widespread application and results in selection of mutants resistant to other components of short-course chemotherapy.  In this context, resistance to rifampicin can be assumed to be a surrogate marker for MDRTB.  With the combination of the new molecular biology methods and the advances in the fields of sensors and microsystems, rapid, inexpensive and point-of-care genetic testing becomes a possibility.  In this project, the aim is to tackle some of the problems of detecting antibiotic resistance of M. tuberculosis, using rifampicin as a surrogate marker for MDRTB, allowing for a more effective treatment of the disease.

 

Aim:

This project focuses on the resistance of M. tuberculosis to rifampicin.  Resistance to this front line anti-mycobacterial agent is conferred by mutations in the rpoB gene, encoding the b subunit of RNA polymerase. The work will centre on a subset of the mutations, which confer rifampicin resistance to M. tuberculosis and will establish a set of protocols for release, amplification and detection of PCR product.

 

This project does not aim to compete with the high information density, research-focused optical arrays, but to provide a useful, cheap, disposable, easy-to-use alternative to existing diagnostic tests.  The identification of a relatively small number of targets, required for routine diagnostics such as pathogen detection and genotyping, is being addressed.

 

Expected results:

  1. Rapid DNA release, < 20 minutes, from clinical samples with no use of enzymes, organic solvents of purification columns.
  2. Sensitivity of suitable level to be able to detect M. tuberculosis from smear positive clinical samples and culture.
  3. PCR amplification specific for M. tuberculosis.
  4. Hybridisation detection of wild type sequence and the four most prevalent mutations causing rifampicin resistance.
  5. Simultaneous detection of wild type and all mutations in a multisequence sensor array with hand-held instrument for signal readout. PCR-ELISA as a gold standard of mutation detection and for validation of sensor arrays.

 

Potential applications:

The system under development is based on a generic technology with application in a number of different diagnostic fields.  The project has chosen a model system determination of multidrug resistant strains of M. tuberculosis.  Methodologies and findings will be published in peer-reviewed journals. Technologies will be tested under laboratory conditions with a view to establishing sensitivity, specificity, usability and rapidity. A marketing plan will assess further commercial routes.  The results will be used by a number of different groups, including molecular biology, and medical microbiology diagnostic and research laboratories.  The results will be used by laboratories specialising in diagnosis of infectious diseases. The percentage of Rifampicin resistant TB cases is considerably increasing, especially in Eastern Europe; this situation will demand new techniques and protocols to detect these cases. Furthermore, it is important to note that these techniques could also be applied to some other infectious diseases such as AIDS.  The consortium believes that it will also see emerging markets in cancer diagnostics and detection of genes and mutation in genes arising from the human genome project.



1 Snider D E, Jr., La Montagne J R, The neglected global tuberculosis problem: a report of the 1992 World Congress on Tuberculosis, Journal of Infectious Diseases, 1994, 169, 1189-1196.

2 Farmer P, Bayona J, Becerra M, The dilemma of MDR-TB in the global era, International Journal of Tuberculosis & Lung Disease, 1998, 2, 869-876.

3 Iseman M D, MDR-TB and the developing world--a problem no longer to be ignored: the WHO announces 'DOTS Plus' strategy,  International Journal of Tuberculosis & Lung Disease, 1998, 2, 867.

Coordinator:

Helen Berney
National Microelectronics
Research Centre
Prospect Row
Cork
Ireland
Tel: +353 21 490 4010
Fax: +353 21 427 0271
E-mail: hberney@nmrc.ie
Website: http://www.nmrc.ie

Partners:

Principal
Scientific
Participants
Official Address Other Information
2Ioannis KatakisUniversitat Rovira i Virgili
Carretera de Salou S/N
ES-43006 Tarragona
Spain
Tel: +34 977 559655
Fax: +34 977 559667
E-mail: ikatakis@etse.urv.es
Web-site: http://www.etse.urv.es/
3Ken ForbesDepartment of Medical Microbiology
Polwarth Building
Foresterhill,
UK-AB25 2ZD Aberdeen
United Kingdom
Tel: +44 1224 663123 Ext. 54953
Fax: +44 1224 685604
E-mail: k.forbes@abdn.ac.uk
Website: http://www.abdn.ac.uk/
4Christine DoigLothian University Hospitals, NHS Trust
Scottish Mycobacteria, Reference Laboratory
Greenbank Drive
UK-EH10 5SB Edinburgh
United Kingdom
Tel: +44 131 536 6357
Fax: +44 131 536 6152
5Hedda SteingrimsdottirMicrozone Ltd
4 Heath Square, Boltro Road
UK-RH16 1BL Haywards Heath
United Kingdom
Tel: +44 1444 452679
Fax: +44 1444 441534
E-mail: h.steingrims@microzone.co.uk
Website: http://www.microzone.co.uk/
6Lucia GarciaPharmagen S. A.
Alcarria, 7. Poligono Industrial, De Coslada
ES-28820 Coslada Madrid
Spain
Tel: +34 91 674 8990, +34 91 444 4500 (Zeltia S.A)
Fax: +34 91 674 8991
E-mail: lgarcia@pharmagen.es
Website: http://www.pharmagen.es
7Martin WielandTRACE Biotech AG
Mittelweg 7
DE-38106 Braunschweig
Germany
Tel: +49 531 261 3327 / 3349
Fax: +49 531 261 3338
E-mail: mw@trace-ag.de
Website: http://www.trace-ag.de

 
 
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