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Indian Journal of Medical Sciences, Vol. 59, No. 6, June, 2005, pp. 253-258 Original Article Investigation of genetic heterogeneity in Mycobacterium tuberculosis isolates from tuberculosis patients using DNA fingerprinting Khosravi Azar D., Barazandeh B. Department of Microbiology, School of Medicine and Infectious and Tropical Diseases Research Center, Ahvaz Jundi Shapur University of Medical Sciences, Ahvaz ABSTRACT 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. Keywords: Fingerprinting, Mycobacterium tuberculosis, restriction enzyme INTRODUCTION Tuberculosis (TB) is still a major health hazard in both developed and
developing countries. According to the latest WHO data (2004), one third
of the world′s population is currently infected by Mycobacterium tuberculosis (MTB), and approximately 10 million people become sick with TB each year.[1],[2] MATERIALS AND METHODS It was a prospective cross-sectional study conducted in TB reference center, PHLS, Ahvaz, Iran, over a period of 9 months (March-November 2002). A total of 180 isolates derived from all new-diagnosed TB patients referred to the center, were analyzed. Despite that the sampling was a part of patients′ diagnosis protocol, informed consent was taken in the presence of the research assistants. Besides, permission was obtained from human ethics committee at the university and the relevant authorities during the approval of the proposal. The patients were 133 men and 47 women and their age ranged from 20 to 60 years with a mean of 34.6. The isolates were identified as MTB by acid fast staining of direct smears prepared from sputum samples, culture in Lowenstein Jensen (LJ) medium and subsequently tested biochemically, using the niacin accumulation test, the nitrate reduction test, and heat-labile catalase test.[13]Chromosomal DNA was extracted from growth harvested from the surface of LJ medium by the simple boiling method. In short, few colonies were removed and suspended in 500 ml of sterile double distilled water in a microfuge tube and was boiled for 10 min. After centrifugation at 12 000 g for 3 min in a microcentrifuge, 10 ml of supernatant was used for the PCR.[12] An IS 6110 -based PCR approach was performed with a single primer, 5¢-GAGTCTCCGGACTCACCGG-3¢, targeted at the inverted repeat sequence of the IS 6110 insertion.[14] Reaction volumes were 25 ml and contained 1 ´ PCR buffer, 1.5 mmol MgCl2, 0.2 mmol of each deoxynucleotide triphosphate, 25 p mol of primer, 1 unit of Taq polymerase and 5 ml of DNA template primer. The reaction conditions were as follows. An initial denaturation at 95°C for 120 s; 1 cycle of 95°C for 20 s, 45°C for 360 s and 72°C for 120 s; 30 cycles of 95°C for 20 s, 62°C for 30 s and 72°C for 180 s; and a final extension at 72°C for 10 min.[14] The PCR products were loaded on an 1.5% (w/vol.) agarose gel with 0.5 mg/ml of ethidium bromide and were analyzed by gel electrophoresis. HaeIII restriction enzyme was used in this study and the experiment was carried out as described by other investigators.[15] In brief, 10 ml of PCR product was added to 6 ml of sterile distilled water, 2 ml of restriction enzyme HaeIII (Cinnagen Co., Tehran, Iran), and 2 ml of corresponding buffer. The mixture was incubated for 1-2 h at 37°C in a water bath. The results were analyzed on a 2% (w/vol.) agarose gel with 0.5 mg/ml of ethidium bromide. Gels were photographed and the digestion bands were measured. The base pair size of each DNA fragment was determined by comparing the migration distance of the bands with the molecular markers visually. Statistical analysis SPSS software was used for data analysis. The data was analyzed using SPSS software, chi-square test and Fishers′ exact test was applied wherever applicable.RESULTS Based on the REA data, four clusters were identified. One cluster (I) contained 71.1% of the strains (128 isolates) with two fragments of 72 and 118 base pair (bp). Cluster II contained three fragments of 72, 118, and 194 bp (11 isolates); cluster III contained three fragments of 118, 194, and 234 bp (eight isolates); and cluster IV contained four fragments of 72, 118, 194, and 234 bp (33 isolates) [Figure - 1].Clustering was observed more in males than females [Table - 1]. We did not find any significant correlation between diversity of the REA patterns and sex of the patients. Similarly neither correlation was seen between patients with different age groups and REA patterns nor any relevance was seen between the patterns and patients′ occupation according to data extracted from questionnaires. In order to investigate whether direct smear results could be related to heterogeneity in fingerprint patterns, patients were divided into four groups of 3.9% of negative; 44.4% of 1+; 33.3% of 2+ and 18.4% of 3+, according to their direct smear results. No significant correlation was seen between the diversity of REA patterns and the smear results or degree of smear positivity. Based on a survey which was undertaken during the study, probable correlation was seen between patients′ residential area and living conditions to diversity of patterns. According to [Table - 2], the results indicated that most of the identical REA patterns were found in patients who were lived in certain overcrowded suburb with low quality sanitation (data from questionnaire), which the correlation was statistically significant ( P = 0.049, d.f. = 2). DISCUSSION Mycobacterial identification to the species level is not only of academic interest but also is important because by use of fingerprinting techniques, that provides a great deal of useful information on the epidemiology and pathogenesis of the organisms, suggesting potential intervention strategies including successful treatment of patients.[15]In present study, we investigated whether the PCR-based REA can be apply as a suitable fingerprinting technique for studying genetic variation among MTB isolates in the region of study , which is one of the endemic foci of TB in Iran. Based on the results, the prevalence of IS 6110- REA patterns with two fragments in the area of survey were high which suggests a high genetic similarity among the isolates. We were not able to find any evidence for transmission or possible laboratory contamination events under these circumstances. All the strains under investigation underwent duel REA testing and the patterns remained constant in both tests. It is unclear whether grouping of IS 6110 -REA is of significance but it seems that a secondary typing method is required in such a large-scale surveillance study. The possible relevance between diversity of the REA patterns and residential area (three categories of Ahvaz suburb, central, and province) of the patients were investigated. Despite that collected data suggested possible relationship and infection from the same source in each category; we still need to have a precise interpretation for such data by using a more discriminatory method, as.[16],[17] In conclusion, 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. ACKNOWLEDGEMENTS We wish to thank Dr Yad Yad, the coordinator Physician of TB in the Khuzestan province for his collaboration. We also acknowledge the TB reference laboratory staff, PHLS, Ahvaz, Iran, for providing the subjects and their assistance.REFERENCES
Copyright 2005 - Indian Journal of Medical Sciences The following images related to this document are available:Photo images[ms05038t1.jpg] [ms05038f1.jpg] [ms05038t2.jpg] |
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