Determination of genotypic varieties and genotyping of multiple drug-resistant tuberculosis by the RFLP and spoligotyping methods

Abstract

IntroductionAlthough tuberculosis is one of the oldest diseases in history, it is still a serious public health problem. Tuberculosis ranks first (25%) among the causes of preventable mortality worldwide (1). Today there are more than 100 species of mycobacteria other than tuberculosis (MOTT) in the genus Mycobacterium besides the members of the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, and M. africanum). Identification of the mycobacteria produced with various methods is an essential stage in the diagnosis of infection. The rapid and accurate identification of mycobacteria will allow both taking measures against transmission and determining appropriate treatment alternatives. Depending on the increase in immune-suppressed cases and the developments in diagnostic techniques, an increasing number, and more and more species, of MOTT bacteria are isolated as disease agents every passing day (2). Since conventional methods take a long time, molecular methods were put into use as supplementary methods for quick diagnosis (3). Background/aim:The purpose of the present study was to determine the distribution and epidemiological features of mycobacteria with molecular methods.Materials and methods: Fifty-five culture-positive samples were analyzed by polymerase chain reaction-restriction enzyme length polymorphism (PCR-RFLP) at species level, and their molecular typing was performed with spoligotyping. The IS6110 region and the locus of gene coding for Hsp65 were amplified. RFLP profiles were obtained by cutting the Hsp65 region with the Hae III and BstE II (Eco91I) enzymes. Spoligotyping was carried out by commercial kit. The H37Rv strain was used as the control.Results: All samples showed the same cutting pattern with the H37Rv strain. The RFLP profiles of 9 strains identified as "mycobacteria other than tuberculosis" were compatible with the M. tuberculosis complex. Spoligotyping of 55 isolates detected 13 different genetic profiles. The Beijing genotype was not detected. One isolate was described as an orphan strain according to the SpolDB4 database. The most frequently detected family was T1 with 32 strains (64%), followed by 9 isolates (18%) belonging to the LAM7 TUR family. Conclusion:PCR-RFLP is a specific, rapid, and effective method in routine diagnosis of mycobacteria. Spoligotyping is an ideal method in the determination of genotypic varieties of mycobacteria.

Biodiversity of Mycobacterium tuberculosis in Bulgaria Related to Human Migrations or Ecological Adaptation

“…The Turkish and Bulgarian genetic MTBC landscape remains dominated by L4, and more specifically by L4.2. Less than 1% of the genotypes of the L1/EAI, L2/Beijing, and L3/CAS sublineages are found in Turkey [12,[74][75][76][77]. At the same time, Bulgarian Turks are not carriers of the CAS and EAI genotypes, although each year several hundred thousand Turks visit each other from both sides of the border.…”

“…The low incidence of the CAS, EAI, and Beijing sublineages in Turkey leads to the logical supposition that lineages L1-L3 were somehow ecologically limited and did not spread in this region of the Middle East [68,[74][75][76][77]. These discrepancies cannot be fully explained with a specific human genetic background.…”