Molecular Characterization of Mycobacteria Causing Pulmonary Tuberculosis in Gondar, North-West Ethiopia

Background: In human infections, molecular epidemiological studies have suggested that certain Mycobacterium tuberculosis types can be especially prone to drug resistance acquisition or to global dissemination. Some related types of Mycobacterium tuberculosis also appear to be strongly associated with specific geographic locations. Other Mycobacterium tuberculosis strains, such as the Beijing family are reported more pathogenic. Therefore, this study determined the dominant Mycobacterium tuberculosis spoligotype causing pulmonary tuberculosis in Gondar

Objectives: the aims of this study was to provide an initial and base-line data on the genetic biodiversity of Mycobacterium tuberculosis strains causing pulmonary tuberculosis in Gondar.

Methods: Sputum samples were collected from 116 smear positive tuberculosis patients and decontaminated with N-acetyl-L-cysteine (NALC)/sodium hydroxide. DNA was purified at an elution volume of 100 µl using the QIAamp DNA Mini cube. DNA samples were subjected to 16S rDNA/RD750 real time PCR, RD9 multiplex PCR and spoligotype. For 16S rDNA/ RD750 assay, PCRs were carried out in 25 µl volumes containing 20 µl of PCR Master Mix and DNA standard was diluted to 10⁷, 10⁶, 10⁵, 10⁴, 10³ and 10² M. tuberculosis genomic DNA genomes/µl. For RD9 PCR assay, eighteen µl of the PCR mix were aliquot into a PCR tubes and 2 µl of the DNA template added to the respective tubes and PCR run with the corresponding PCR program in the thermo cycler. The preparations were subjected to gel electrophoresis and image taken using a 12 bit imaging camera. Spoligotype was run by adding 5 µl of DNA and 20µl of the PCR mix followed by Hybridization in water bath at 42^oc and 60^oC.The autorad was developed by subjecting films to infrared light and striped using 1% SDS at 80^oc. The orientation of the autorad was read first using controls and the SIT number of each Mycobacterium tuberculosis complex determined from the SITVIT database.

Results: The 16S rDNA assay result showed that 22 samples were positive for Mycobacterium tuberculosis complex and the RD750 real-time PCR assay showed that tubercle bacilli in 5 sputum samples were with intact RD750. Ninety-four of the one hundred sixteen samples were RD9 intact and the other 22 RD9 deleted. Seventy-two of the 94 Mycobacterium DNA extracts with RD9 intact showed Mycobacterium tuberculosis with different Shared International type numbers (SITs) of which, Mycobacterium tuberculosis SIT 25, Central Asian Strain variant 1 (CAS-1 variant), accounted for 30.6% (n=22). In this study, 15 new Mycobacterium tuberculosis spoligotypes all with intact RD9 (MTb SIT NEW) were also observed. 

Conclusion: This study showed diversity of Mycobacterium tuberculosis spoligotypes causing pulmonary tuberculosis in Gondar. The new Mycobacterium tuberculosis spoligotypes observed in the study area was different from the recently reported Woldiya lineage that suggests the existence of new Mycobacterium tuberculosis strains circulating in the locality. Therefore, we suggest that large scale community based studies are required to determine the prevalence of this new Mycobacterium tuberculosis spoligotypes around Gondar and the neighboring areas. The disease causing ability or virulence of these new Mycobacterium tuberculosis spoligotypes need to be determined by future works.