Evaluation of a Two-Step Approach for Large-Scale, Prospective Genotyping of Mycobacterium tuberculosis Isolates in the United States

Abstract

ABSTRACT Genotyping of Mycobacterium tuberculosis isolates is useful in tuberculosis control for confirming suspected transmission links, identifying unsuspected transmission, and detecting or confirming possible false-positive cultures. The value is greatly increased by reducing the turnaround time from positive culture to genotyping result and by increasing the proportion of cases for which results are available. Although IS 6110 fingerprinting provides the highest discrimination, amplification-based methods allow rapid, high-throughput processing and yield digital results that can be readily analyzed and thus are better suited for large-scale genotyping. M. tuberculosis isolates ( n = 259) representing 99% of culture-positive cases of tuberculosis diagnosed in Wisconsin in the years 2000 to 2003 were genotyped by using spoligotyping, mycobacterial interspersed repetitive unit (MIRU) typing, and IS 6110 fingerprinting. Spoligotyping clustered 64.1% of the isolates, MIRU typing clustered 46.7% of the isolates, and IS 6110 fingerprinting clustered 29.7% of the isolates. The combination of spoligotyping and MIRU typing yielded 184 unique isolates and 26 clusters containing 75 isolates (29.0%). The addition of IS 6110 fingerprinting reduced the number of clustered isolates to 30 (11.6%) if an exact pattern match was required or to 44 (17.0%) if the definition of a matching IS 6110 fingerprint was expanded to include patterns that differed by the addition of a single band. Regardless of the genotyping method chosen, the addition of a second or third method decreased clustering. Our results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS 6110 fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.