IS6110 restriction fragment length polymorphism typing of Mycobacterium tuberculosis isolates from East Azerbaijan Province of Iran

Abstract

To investigate the genetic variation among Mycobacterium tuberculosis isolates in the East Azerbaijan Province of Iran and to evaluate the level of and risk factors for recent transmission of tuberculosis (TB),Key words: tuberculosis -restriction fragment length polymorphism -IS6110 -Iran DNA fingerprinting of Mycobacterium tuberculosis is a valuable tool to study tuberculosis (TB) epidemiology. Many other studies on this methodology have been done in various parts of the world including developed and developing countries (Pineda-Garcia et al. 1997, Gutierrez et al. 1998, Dahle et al. 2001, Diaz et al. 2001, Bhanu et al 2002. The method of fingerprinting based on IS6110 repetitive element has become standard (Hermans et al. 1990, Van Embden et al. 1993) and its stability (Niemann et al. 2000, Warren et al. 2002 and discriminatory power have been proven (Kremer et al. 1999).The usefulness of DNA fingerprinting has already been defined in source tracing (Kiers et al. 1997) and understanding of TB transmission in the general population (Alland et al. 1994). Fingerprinting results from various parts of the world demonstrate that in areas with low incidence of TB, the majority of cases are due to reactivation of previous infection whereas in high incidence areas reinfection is responsible for the majority of TB cases (Alland et al. 1994, Dahle et al. 2001) but there are some exceptions (Bauer et al. 1998, Narayanan et al. 2002.The East Azerbaijan Province is located in the North West of Iran, in neighbor-hood of Nakhichevan state of Republic of Azerbaijan (Fig. 1A). The estimated population of the Province is 3,500,000 of which about two-fifth are inhabitants of Tabriz, the capital city of the Province. The estimated rate of TB in Iran in 2002 was 29 in 100,000 and notification rate is 17 in 100,000 (WHO 2004). However, TB incidence is not homogeneous in different parts of this country. In East Azerbaijan Province the estimated rate of TB in 2002 was low; this can be due both to low case finding or low prevalence of TB in this part of the country.The aims of this study were to determine the genetic diversity of M. tuberculosis population in East Azerbaijan Province, and to detect the manner of transmission of the disease in this area. The species identification of the isolates was based on polymerase chain reaction (PCR) method and standard microbiological tests. The susceptibilities of the isolates to isoniazid (INH), rifampin (RF), streptomycin (SM), and ethambutol (ETB) were determined by the proportional method. MATERIALS AND METHODS Patient population and bacterial isolatesRestriction fragment length polymorphism analysis -RFLP analysis was performed as described previously (Van Soolingen et al. 1994). Briefly, extracted mycobacterial DNA was digested with PvuII, subjected to electrophoresis, and hybridized with a 245-bp PCR-amplified probe directed against the right arm of IS6110. After hybridization, the insertion sequences were visualized with a colorimetric system, the DIG DNA labeling and detection kit (Roc...

Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis

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DNA Fingerprinting of Resistant Mycobacterium tuberculosis Isolates in Iran by IS6110-RFLP Method

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The role of Azeri patients in transmission of tuberculosis to East Azerbaijan, Iran

“…Extraction of deoxyribonucleic acid (DNA) from MTB isolates and fingerprinting was performed as described by van Soolingen et al 12 A 245 bp fragment amplified by polymerase chain reaction (PCR) on purified chromosomal mycobacterium bovis Bacillus Calmette-Guérin (BCG) DNA by using used from these oligonucleotides INS-1 (5΄-CGT GAG GGC ATC GAG GTG GC) and INS-2 (5΄-GCG TAG GCG TCG GTG ACA AA) TIB Molbiol, Germany, 13 and then this fragments purified and after labeling with digoxigenin, used as IS6110 probe. The extracted mycobacterial DNA was digested with PvuII enzyme (Cinnagen, Iran) and restriction fragments were separated in 0.8% agarose gel electrophoresis at 20 v for 18 h. The fragments were transferred from gel to positively charged nylon membranes then hybridized with IS6110 probe and insertion sequences were visualized with a colorimetric system.…”