Usefulness of Spoligotyping To Discriminate IS 6110 Low-Copy-Number Mycobacterium tuberculosis Complex Strains Cultured in Denmark

Abstract

Mycobacterium tuberculosis complex strains cultured in Denmark have been analyzed by IS6110 restriction fragment length polymorphism (RFLP) on a routine basis from 1992 and onwards. Due to the influx of immigrants with tuberculosis, the number of strains harboring only one to five copies of IS6110 has increased steadily. Since the discriminatory power of IS6110 fingerprinting for such strains is poor, we have performed additional genotyping of all low-copy-number strains by the recently described PCR-based method known as spoligotyping. A total of 311 clinical strains were typed: 14 Mycobacterium bovisBCG, 48 M. bovis, and 249 M. tuberculosisstrains. Spoligotyping correctly differentiated M. bovisand M. bovis BCG from M. tuberculosis strains, but it did not differentiate M. bovis from M. bovis BCG. All M. bovis BCG strains exhibited identical spoligotype patterns. The discriminatory power of spoligotyping of low-copy-number M. tuberculosis strains was higher than that of IS6110 fingerprinting. Based on RFLP typing solely, 83% of the low-copy-number M. tuberculosis strains were found to form part of a cluster, and 75% were found to form a cluster on the basis of spoligotyping. When the two techniques were combined, the amount of clustering decreased to 55%. The combination of these two techniques might be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of the IS6110 element.

Molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements

“…Unfortunately, a proportion of M. tuberculosis isolates contain no, or only a few, copies of IS 6110 , and this proportion differs significantly by geographical area [99, 108–110, 114–116]. Strain typing based on a low copy number of IS 6110 is not sufficiently discriminatory [46, 99, 109, 117–124], so it has been decided to apply an additional genetic typing method when M. tuberculosis isolates contain fewer than five copies of IS 6110. There are several genetic markers that can be used in addition to IS 6110 RFLP typing.…”

“…Some PCR‐based methods, such as the mixed‐linker PCR [141, 148], reveal a level of discrimination and reproducibility almost comparable with IS 6110 RFLP typing [18]. However, because of its simplicity, spoligotyping is currently a frequently used PCR typing method for M. tuberculosis complex [120, 122, 152–154]. Spoligotyping is also used as an additional typing method for strains with fewer than five copies of IS 6110 [120, 122, 154].…”

“…However, because of its simplicity, spoligotyping is currently a frequently used PCR typing method for M. tuberculosis complex [120, 122, 152–154]. Spoligotyping is also used as an additional typing method for strains with fewer than five copies of IS 6110 [120, 122, 154]. This method is based on the visualization of the spacer DNA sequences in between the 36‐bp direct repeats (DRs) in the genomic DR region of M. tuberculosis complex strains [125].…”

“…A clear example of this phenomenon is that all ‘Beijing’ genotype strains invariably only reacted with the last nine spacers in the panel of 43 [133]. Furthermore, most M. bovis strains, but not all [55], lack spacers 39–43 [118–120, 124, 125]. Most M. bovis and all M. bovis BCG strains lack, in addition, spacers 3, 9 and 16 [120, 125, 154].…”

“…Furthermore, most M. bovis strains, but not all [55], lack spacers 39–43 [118–120, 124, 125]. Most M. bovis and all M. bovis BCG strains lack, in addition, spacers 3, 9 and 16 [120, 125, 154]. M. microti strains can be distinguished in ‘vole’ and ‘lama’ types by their characteristic spoligo patterns [41, 49].…”