Investigation of genetic heterogeneity in Mycobacterium tuberculosis isolates from tuberculosis patients using DNA fingerprinting

BACKGROUND: DNA fingerprinting of Mycobacterium tuberculosis (MTB) based on IS6110 has been shown to be a powerful epidemiologic tool. Restriction enzyme analysis (REA) is a fingerprinting technique, which is used for differentiation and investigation of genetic diversity among mycobacterial species.
AIMS: To investigating the genetic heterogeneity in MTB isolates in Ahvaz, Iran.
SETTINGS AND DESIGN: It was a cross-sectional study conducted in Ahvaz, Iran.
METHODS AND MATERIAL: One hundred and eighty clinical isolates of MTB were collected from TB reference unit, PHLS, Ahvaz, Iran. The PCR-REA employed uses a simple DNA extraction followed by a PCR step involving a single primer based on the insertion sequence IS6110. Restriction enzyme analysis was performed on the amplification products using HaeIII enzyme.
STATISTICAL ANALYSIS: Data was analyzed using SPSS software and chi-square test/Fishers′ exact test was applied wherever applicable. RESULTS: The isolates were divided into four clusters based on their REA patterns. Cluster I contained 71.1% of strains with two fragments of 72 and 118. Cluster II with three fragments of 72, 118, and 194; cluster III with three fragments of 118, 194, and 234; and cluster IV with four fragments of 72, 118, 194, and 234 base pairs. As many as 73.8% of the identical fingerprint patterns were seen in male patients. Accounting the men as the major population in the study, there was no significant difference between REA patterns and sex; similarly, with age, patients′ occupation and degree of smear positivity. However, we found significant correlation between REA patterns and patients′ origin. As many as 61.6% of identical patterns were found in the patients who were lived in the same suburb.
CONCLUSIONS: By PCR-based REA typing, the isolates studied were grouped into four clusters each containing between two and four fragments. However, in order to ascertain the level of heterogeneity of MTB isolates in their sample, further testing with a more discriminatory method is needed.