Abstract

One third of the world's population is currently infected with tuberculosis (TB), a consuming airborne disease whose main causative agent is Mycobacterium tuberculosis. The majority of these patients are found in the world's poorest areas. Treatment of TB is a lengthy and demanding process utilizing a cocktail of powerful drugs; however, multidrug resistant TB (MDR-TB) strains, defined by resistance to both isoniazid (INH) and rifampicin (RIF), are now emerging worldwide and threatening disease control efforts. The major problem facing efforts to combat MDR-TB spread is its early detection. Conventional fairly affordable methods for drug resistance detection are based on solid culture and are highly time consuming (3-6 weeks in addition to initial pathogen culturing). On the other hand, the more rapid liquid culture-based automated systems are costly to set up and maintain while the very rapid molecular assays (hours to few days) are simply too complex and unaffordable and non-sustainable in limited resource settings. The objective of this work was to evaluate the performance of two liquid culture-based colorimetric assays for detection of drug resistance; nitrate reducate assay (NRA) and colorimetric redox indicator (CRI) methods for detection of MDR-TB. The assays were tested on mycobacterial isolates from Egyptian patients and their performance was compared with microscopic observation drug susceptibility assay (MODS) and the commercial automated culture system MGIT 960. Concordance was 96.7% for CRI and 93.3%, at almost one-tenth of the MGIT cost, and close to that of MODS without the need for an inverted microscope. The NRA format used in this study is more convenient and higher in throughput than the initially developed format. Additionally, DNA was extracted from the mycobacterial isolates and16S rDNA was amplified and sequenced to gain insight on the molecular diversity of Egyptian strains. Moreover, the molecular basis of strain resistance was investigated by DNA sequencing of the genes most commonly containing resistance conferring mutations. Analysis of the 16S rDNA sequencing results confirmed the identity of the samples as mycobacterium tuberculosis and suggested possible presence of two different strains. On the other hand, the analysis of the resistance related genes found common resistance conferring mutations in the MDR samples.

Department

Biotechnology Program

Degree Name

MS in Biotechnology

Graduation Date

2-1-2013

Submission Date

January 2013

First Advisor

Azzazy, Hassan M. E.

Committee Member 1

Mohamed, Moustafa Abdel Fadeel

Committee Member 2

Zada, Suher

Extent

106 p.

Document Type

Master's Thesis

Library of Congress Subject Heading 1

Tuberculosis -- Egypt.

Library of Congress Subject Heading 2

Tuberculosis -- Chemotherapy.

Rights

The author retains all rights with regard to copyright. The author certifies that written permission from the owner(s) of third-party copyrighted matter included in the thesis, dissertation, paper, or record of study has been obtained. The author further certifies that IRB approval has been obtained for this thesis, or that IRB approval is not necessary for this thesis. Insofar as this thesis, dissertation, paper, or record of study is an educational record as defined in the Family Educational Rights and Privacy Act (FERPA) (20 USC 1232g), the author has granted consent to disclosure of it to anyone who requests a copy.

Institutional Review Board (IRB) Approval

Approval has been obtained for this item

Comments

I would like to express my immense gratitude to my advisor and mentor, Dr. Hassan Azzazy. I thank him not only for his support, knowledge opportunities, encouragement, and science he taught me, but most of all for teaching me how to think big and take on challenges effectively. I am privileged to be able to call myself his student, and for all that he taught me, I am forever in his debt. I would like to acknowledge the support and funding the Youssef Jameel Science and Technology Research Center (YJ-STRC) has provided me during the course of my enrollment in the Masters Program. I would like to thank Dr. Moustafa Abdel Fadeel and members of MRU-3 who have welcomed me into their facilities and spared no effort help me out, and provided me with the needed samples for this work. I have learnt a great deal from my time there and met people whom I will always value. I thank Dr. Momtaz Wasfy, and his TB laboratory members Mr. Bassem Abdel Rahman and Mr. Mohamed Abdel Maksoud. They have been more than helpful and have always been available to help me out despite their busy schedule.

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