Background: The application of molecular tools has revealed that infection by Mycobacterium tuberculosis (MTB) is more complex than initially assumed. Genotyping is generally performed on cultures. However, there is no information about bacterial clonal complexity in clinical specimens or whether standard culture procedures can modify this complexity.
Methods: An in vitro assay was performed to determine whether culture can modify the clonal complexity of the MTB population in clinical specimens. Pairs of MTB strains (10 pairs) or stain-positive sputa (4 pairs) were mixed in different volumetric proportions. The DNA extracted from the mixtures before and after culture was genotyped using mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis to detect potential changes in the proportion of the mixed strains.
Results: In 6/10 pairs of MTB strains and 2/4 pairs of sputa, marked changes were observed in clonal composition after culture, even in mixtures of strains differing in their drug-susceptibility patterns. In some cases, only one of the mixed strains was detected after culture.
Conclusions: The initial clonal composition in bacteriologically complex clinical specimens could be underestimated if genotyping analysis is performed after culture. Genotyping strategies aimed at analyzing clinical samples must be optimized to reveal the real dimension of clonal complexity in infection by MTB.
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