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Characterization of drug-resistant Mycobacterium tuberculosis strains isolated in Nepal

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Title: Characterization of drug-resistant Mycobacterium tuberculosis strains isolated in Nepal
Other Titles: ネパールにおいて分離された薬剤耐性結核菌株の特徴
Authors: Poudel, Ajay Browse this author
Issue Date: 28-Jun-2013
Abstract: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, which currently presents an immense global health challenge. It ranks as the second leading cause of death from an infectious disease worldwide. An estimated one third of the world’s population is infected with the tubercle bacilli; with 95% of TB cases occur in developing countries. The World Health Organisation (WHO) estimates that there were 9 million new cases of TB and 1.4 million TB deaths globally in 2011. TB normally affects the lungs (pulmonary TB) and can affect other sites as well (extrapulmonary TB). The disease is spread by the air when people who are sick with pulmonary TB spout bacteria during coughing, sneezing and speaking. In general, infection by the pathogen does not necessarily result in the development of clinical symptoms and relatively small proportion (5-10%) of these individuals will progress to active disease each year. The remaining proportion (90-95%) of infected individuals will initially be asymptomatic and undergo latent infection, from which reactivation may occur when immune system of the patient becomes weakened.If patients are not treated properly, TB can be fatal. New cases of drug-susceptible TB are treated with a 6-month regimen of four first-line drugs: isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and pyrazinamide (PZA). RIF is a broad spectrum rifamycin derivative that interferes the synthesis of mRNA by binding to the ß subunit of RNA polymerase (RpoB) in bacterial cells. INH is a prodrug that requires activation by M. tuberculosis catalase-peroxidase (KatG), to generate a range of reactive oxygen species and reactive organic radicals, which then attack multiple targets in the tubercle bacillus. The primary target of the inhibition is the cell wall mycolic acid synthesis pathway (Figure 1). EMB inhibits the formation of mycobacterial membrane. The mechanism of action of PZA is poorly understood. The only known mechanisms are it disrupts membrane energetics and inhibits membrane transport function in M. tuberculosis.Fluoroquinolones (FQs) are considered to be important second-line drugs recommended for the treatment of multidrug-resistant TB (MDR-TB). They act by inhibiting DNA supercoiling, thus preventing replication and cell division. They block a type II topoisomerase (called DNA gyrase) of M. tuberculosis, a heterotetramer consisting of two A and B subunits coded by the gyrA and gyrB genes. Among second-line anti-TB drugs, aminoglycosides (kanamycin [KAN] and amikacin [AMK]) and cyclic peptides (capreomycin [CAP] and viomycin) inhibit protein synthesis by inhibiting the normal function of ribosomes (Figure 1).The emergence and spread of drug-resistant strains of M. tuberculosis throughout the world possess a serious threat to TB control. Resistance to anti-TB drugs arises due to a variety of reasons, such as the failure to detect resistance to TB drugs, interrupting treatment, omitting one or more drugs from the recommended prescription and suboptimal dosage. Thus, M. tuberculosis can become resistant to multiple drugs in the period of a few months. Essentially, drug resistance arises in areas with improper TB control programmes. A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals.MDR-TB, which is caused by bacteria that are resistant to least two first-line drugs including INH and RIF and, extensively drug resistant TB (XDR-TB), which is caused by bacteria that are resistant to INH and RIF as well as any FQs and any of the second-line anti-TB injectable drugs (AMK, KAN or CAP). WHO estimates that from 220,000 to 400,000 of MDR-TB cases occur among TB cases notified in the world in 2011. About 60% of these drug-resistant TB cases occur in Brazil, China, India, the Russian Federation and South Africa (that are referred as BRICS countries) (Figure 2). By the end of 2010, 68 countries had reported at least one case of XDR-TB (Figure 3).Genotyping of M. tuberculosis plays an increasing role for understanding the epidemiology and biology of TB. Genotyping techniques are important for molecular epidemiological investigations of TB such as defining chains of ongoing transmission and differentiating patient relapse from exogenous re-infection, and defining the evolutionary background of clinical isolates. Over the past years, several methods have been developed to discriminate M. tuberculosis strains: insertion sequence 6110 restriction fragment length polymorphism, spacer oligonucleotide typing (spoligotyping), variable-number tandem repeat (VNTR), single-nucleotide polymorphisms and large sequence polymorphisms.Nepal is a landlocked country in South East Asia, bounded to the north by China and to the south by India, sharing an open border with India. In Nepal, TB is a major public health problem. The incidence of all forms of TB was estimated to be 173/ 100,000 population while the incidence of new smear-positive cases was at 77 per 100,000 in 2008. The four surveillances conducted between 1996 and 2007 have indicated the fluctuating prevalence of MDR-TB among new cases of between 1.1% and 3.7% (1.1% in 1996, 3.7% in 1999, 1.4% in 2001 and 2.9% in 2007). The latest estimate of MDR-TB is 2.9% and 11.7% among new and recurrent cases, respectively.Rapid determination of the antimicrobial susceptibility pattern in clinical isolates of M. tuberculosis is important for the early administration of appropriate therapeutic agents for the prevention of additional resistance development. In this context, the molecular characterization of drug resistance by identifying mutations in associated genes will be applicable for developing a potential rapid molecular drug susceptibility test as an alternative to conventional methods.The present thesis consists of two chapters; in chapter I, I have investigated the type and frequency of drug resistance-conferring mutations that occurred among M. tuberculosis clinical isolates that were phenotypically MDR by DNA sequencing. I have also compared the frequency of different mutations with those in isolates circulating in the surrounding countries. In chapter II, I have described drug resistance-associated mutations in XDR isolates and analyzed the genetic background of these isolates by using a molecular approach.
Conffering University: 北海道大学
Degree Report Number: 甲第11043号
Degree Level: 博士
Degree Discipline: 獣医学
Examination Committee Members: (主査) 教授 鈴木 定彦, 教授 杉本 千尋, 教授 東 秀明, 准教授 伊藤 公人
Degree Affiliation: 獣医学研究科(獣医学専攻)
Type: theses (doctoral)
Appears in Collections:学位論文 (Theses) > 博士 (獣医学)
課程博士 (Doctorate by way of Advanced Course) > 獣医学研究科(Graduate School of Veterinary Medicine)

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