Multidrug Resistance in Mycobacterium Tuberculosis - Essay Example

INVESTIGATIONS ON THE MOLECULAR ASPECTS OF MULTIDRUG RESISTANT TUBERCULOSIS (MDR-TB) BY ALOZIEUWA CYRIL IKECHUKWU Despite advances in different fields of Medicine and drastic improvements in diagnostic technological know-how, the diagnosis and management of tuberculosis remain elusive. The prominent reason for this is emergence of multi-drug resistant strains of Mycobacterium tuberculosis throughout the globe. In an attempt to answer questions as to why particularly this is a problem, researches revealed that despite newer drugs are developed that are effective against tuberculosis, stringent adherence to therapy with baseline drugs offered in a multi-drug regimen is still the best line of therapy, and non-adherence to the regimen with incomplete therapeutic course leads to changes in the Mycobacterium tuberculosis in the genetic and molecular levels are responsible for this. Thus, the world is threatened with the risk of multi-drug resistant tuberculosis in the form of a pandemic that thrusts on the financial state of the State. Apart from that, emergence of immune deficiency viral disease has also predisposed to rapid spread of multi-drug resistant tuberculosis. Present knowledge on the molecular basis of development of multi-drug resistance may be utilized to develop a diagnostic protocol applying improved genetic analysis to detect the point mutations in the multi-drug resistant strains of Mycobacterium tuberculosis, so the clinicians may diagnose and treat this disease better. This research proposal aims to analyze the scientific basis of such a protocol, not only to add to the existing knowledge but also to devise a method that at least theoretically has far-reaching implications in the control of the disease that could be a global threat. Background: Recent years have witnessed a phenomenal upsurge in number of reported cases of multi-drug resistant Mycobacterium tuberculosis (MDR-TB) infection. Multi-drug resistant tuberculosis is defined as bacterial isolate strains that are resistant to both isoniazid and rifampin with or without resistance to other anti-tuberculosis drugs. Since there is a gradual increase in the number of cases throughout the world including the developed countries, World Health Organization (WHO) has termed this as a global emergency. The rise of MDR-TB showing resistance to conventional treatments is a serious threat to control of tuberculosis disease load throughout the world in high-prevalence countries and low prevalence countries as well. According to estimates of WHO, 50 million people world-wide are infected with MDR-TB. In the year 2000, the number of new tuberculosis cases was 8.7 million, of which 3.1% was MDR-TB yielding a count of 273,000 (World Health Organization, 2005). Drug Resistance: Strains of Mycobacterium tuberculosis resistant to individual drugs arise by spontaneous point mutations in the mycobacterial genome that occurs at low but predictable rates (Dye, C., Williams, B.G., Espinal, M.A., and Raviglione, M.C., 2002). This is just one of the many strategies these living organisms employ to resist the antimicrobial agents, and this is applicable to the whole typical and atypical mycobacterial family of organisms (World Health Organization/International Union against Tuberculosis and Lung Disease, 1994). Isoniazid and rifampin are cornerstones of antitubercular therapy (van Rie, A., Gie, R.P., Enarson, D., and Beyers, N., 2000). The development of multidrug resistance poses a great threat to the individual in the sense that resistance to either isoniazid and rifampin may be managed with other first-line drugs, MDR-TB demands treatment with second-line drugs that have limited sterilizing capacity against Mycobacterium tuberculosis, and these drugs are less effective and more toxic. This has particularly created a situation of pandemic of antibiotic resistance that threatens the globe (Pablos-Mendez, A., Lazlo, A., and Bustreo, F., 1997). Mechanism: To solve the problem and to find out an effective solution against this problem, it is important to know the mechanism of development of resistance. The mycobacterial cell wall is surrounded by a specialized, highly hydrophobic cell wall. This can modify itself in a given situation to disallow entry of drugs inside the bacteria to be able to kill it (Snider, D.E. and Castro, D.K., 1998). This is in a nutshell resistance, and in a resistant strain of bacteria, active drug efflux system and degrading or inactivating enzymes that are controlled by specific genetic modification have been noted. From the molecular perspective, spontaneous chromosomally borne mutations occurring in Mycobacterium tuberculosis at a predictable rate are thought to confer resistance against anti-tubercular drugs. These genes encode either the target of the drug or enzymes that are involved in drug activation. Resistance is conferred by point mutations, deletions, or insertions specific for all drugs, primary, secondary, or newer drugs (Mitchison, D.A, 1984). Molecular Basis: Mutations at the catalase-peroxidase gene, KatSer G315Thr gene for isoniazid, rpo526; 531 for rifampin; rpsL43 for streptomycin; and embB306 for ethambutol are implicated to cause resistance. MDR develops by sequential acquisition of mutations of different loci as a result of inadequate and inappropriate therapy. No single phenotypic locus has been found that may be involved in MDR. While certain mutations are widely present indicating polymorphism, certain mutations can be seen rarely, indicating diversity and unpredictability (Riska, E., Jacobs, W.R., and Alland, D., 2000). This is hence postulated that sometimes, to generate a single drug resistance, it needs mutations in multiple genes. MDR strains can be the result of cumulative mutations, to avoid resistance, three to four drugs are combined routinely to treat patients with tuberculosis (Cole, S.T. and Telenti, A., 1995). Predicated Strategy: To avoid this threat and to gain control over the bacteria, as mentioned earlier, it is important to know the molecular basis resistance to the frequently used antitubercular drugs. Using this knowledge, more specific and sensitive laboratory tests can be developed to diagnose the specific resistant strain, and an appropriate therapeutic strategy can be developed to control the disease better (Espinal, M.A., Laszlo, A., and Simonsen, L., et al., 2001). Rationale: Genetically, the multi-drug resistance is a result of spontaneous point mutations in genes that encode either the drug targets or enzymes that activate the drugs exerting bactericidal effects. All these point mutations, deletions, or alterations are directed to almost all the drugs commonly used for therapy, be it first-line or second-line drugs. To better understand the infection by a resistant strain, it would be pertinent to look into the phenotype mutation, and investigations, such as, P-glycoprotein, efflux pump protein, and structure-activity relationship of the drugs involved (Albert, H., Heydenrych, A., Mole, R., Trollip, A., and Blumberg, L., 2001). Aims: Treatment of MDR-TB includes second-line drugs, which are more expensive and less effective with more serious and damaging side effects. As a result, the cost of management of the disease goes up by prolonged hospitalization since these patients will need close monitoring and supervision in the inpatient setting. The management is problematic in that it would need a firm laboratory diagnosis that needs tedious procedures to carry out the investigations (World Health Organization, Global Tuberculosis Programme, 1997). Laboratory workload will increase astronomically, and more time will be consumed for early detection of multidrug resistance. Turn-around time for receipt of specimen to reporting of results is long at present. MDR cases are very difficult to result in cure, and most often to abolish an MDR focus, it will need surgical removal of the affected organ. The most worrying aspect of MDR-TB is that newly infected patients with drug resistant strains die much quicker than those with an uncomplicated infection (Foulds, J. and O'Brien, R., 1998). Outcome Expected: This research is directed to an outcome where discovery/manufacture of new effective drugs would to reduce the period of treatment with fewer side effects. This needs new and improved diagnostic tools and methods to assist in the detection, prevention, and treatment of tuberculosis with improved speed, sensitivity, specificity, and reliability of diagnosis based on molecular biologic technologies (World Health Organization, 1998). The Polymerase Chain Reaction method of investigation that is based on DNA amplification in its present format is not as sensitive or as specific as it was originally thought would be. Better knowledge of the TB genome in the perspective of mechanism of drug resistance and overcoming it would be the final goal of such advances in research (Roth, A., Schaberg, T., and Mauch, H., (1997). Objective: The objective of this proposed research is to identify the presence of MDR types and other genotypes of Mycobacterium tuberculosis and to develop an investigative protocol that would isolate MDR-TB strains better. This would enable the investigator to study the genotypic polymorphism of these strains and would enable grouping them (genotyping) in order to establish a database to identify and detect outbreaks originated by the same strains or types using molecular typing. This would automatically involve unraveling the genome and study the mutations, deletions, insertions, etc., and characterization of the mutations responsible for the resistance phenotype; thus, new resistance mechanism can be characterized. Looking a little far, libraries of insert ional mutants using transposons can be constructed that will allow the selection of those mutants with decreased pathogenicity. From these mutants, genes that have been inactivated by the transposons can be identified. Inactivation of these genes may generate a new vaccine candidate. Specific Objectives: The molecular characterization of multidrug resistance development in MDR TB population and its elucidation are of clinical relevance. Using Fluorescent Amplified Fragment Length Polymorphism (FAFLP) analysis of Mycobacterium tuberculosis to develop high-resolution fingerprints of Mycobacterium tuberculosis and other pathogenic isolates as well as predictive modeling of single base substitutions associated with molecular pathogenesis, drug resistance, and virulence. Study Plan: Study Population: All study participants will give informed consent. Patients are to answer a brief questionnaire and undergo physical examination. Medical record is to be reviewed. A tuberculin skin test (TST) is to be performed. Sample population is to be mixed male and female to avoid bias. Methodology: Total sample size of 1150 patients of equal sex distribution will be chosen randomly who were previously diagnosed as sputum positive tuberculosis will be chosen. A questionnaire will be supplied to the patient, where duration of the disease, contact history, history of drug therapy will be documented. A TST will be performed to support the diagnosis. The sputum will be cultured in each patient and the isolated strain of Mycobaterium tuberculosis will be analyzed for in vitro sensitivity profile to indicate resistance. If a drug-resistant strain is encountered, FAFLP analysis of the bacteria will be done. The isolated strain will be subjected to virulence analysis. The collected data will be statistically analyzed. The patients will be accessed from the Department of Chest of 7 different hospital from the area, and the ancillary data will be accessed from the medical records section of these hospitals. Contribution: This proposed study will definitely better elucidate the gap in the knowledge about multi-drug resistant Mycobacterium tuberculosis in the sense that genetic characteristics of these strains will be documented, and the inciting factors for these changes will be known so that prophylactic measures can be designed and appropriate pharmacotherapeutic strategies can be planned to help reduce the burden of MDR-TB. Apart from that, it might possibly throw some light on the easiest and cheapest method of diagnosing the MDR-TB. Conclusion: This proposed study, this researcher believes, will be of help to devise laboratory tests and knowledge-guided pharmacotherapeutic strategies to control the MDR-TB. This might also throw some light on the clinical strategies so that an uncomplicated infective strain of Mycobacterium tuberculosis does not turn out to be a strain of MDR-TB. References Albert, H., Heydenrych, A., Mole, R., Trollip, A., and Blumberg, L., (2001). Evaluation of FASTPlaqueTB-RIFTM, a rapid, manual test for the determination of rifampicin resistance from Mycobacterium tuberculosis cultures. International Journal of Tuberculosis and Lung Disease; 5: 906-911. Cole, S.T. and Telenti, A., (1995). Drug Resistance In Mycobacterium Tuberculosis. European Respiratory Journal; 8: Suppl. 20, pp. 701s-713s. Dye, C., Williams, B.G., Espinal, M.A., and Raviglione, M.C., (2002). Erasing The World's Slow Stain: Strategies To Beat Drug Resistant Tuberculosis. Science. Espinal, M.A., Laszlo, A., and Simonsen, L., et al., (2001). Global Trends In Resistance To Antituberculosis Drugs. New England Journal of Medicine; 344: pp. 1294-1303. Foulds, J. and O'Brien, R., (1998). New tools for the diagnosis of tuberculosis: the perspective of developing countries. International Journal of Tuberculosis and Lung Disease; 2: pp. 778-783. Mitchison, D.A, (1984). Drug Resistance In Mycobacteria. British Medical Bulletin; 40: pp. 84-90. Pablos-Mendez, A., Lazlo, A., and Bustreo, F.,(1997). Antituberculosis drug resistance in the world. Publication No. WHO/GTP/97.229. Geneva, World Health Organization. Global Tuberculosis Programme, 1997. Riska, E., Jacobs, W.R., and Alland, D., (2000). Molecular Determinants Of Drug Resistance In Tuberculosis. International Journal of Tuberculosis and Lung Diseases; 42: S4-S10. Roth, A., Schaberg, T., and Mauch, H., (1997). Molecular Diagnosis Of Tuberculosis: Current Clinical Validity And Future Perspectives. European Respiratory Journal; 10: pp. 1877-1891. Snider, D.E. and Castro, D.K., (1998). The Global Threat Of Drug Resistant Tuberculosis. New England Journal of Medicine; 338: pp.1689-1690. van Rie, A., Gie, R.P., Enarson, D., and Beyers, N., (2000). Classification Of Drug-Resistant Tuberculosis In An Epidemic Area. Lancet; 356: pp. 22-25. World Health Organization (2005). Tuberculosis: the global burden;global TB fact sheet 2005. Available at: http://www.who.int/tb/publications/tb_global_facts_sep05_en.pdf. Accessed August, 4, 2007. World Health Organization, (1998). Laboratory Services in Tuberculosis Control 1998. Publication No. WHO/TB/98.258. Geneva, World Health Organization, 1998. World Health Organization/International Union against Tuberculosis and Lung Disease, (1994). Antituberculosis drug resistance in the world. Report No. 2. Prevalence and trends (2000). Publication No. WHO/CDS/TB/2000.278. Geneva, Communicable Diseases, World Health Organization, 1994. World Health Organization, Global Tuberculosis Programme, 1997. Treatment of tuberculosis: guidelines for national programmes. 2nd Edn. Publication No. WHO/TB/97.220. 1997. Geneva, World Health Organization, 1997. Read More