Polymerase chain reaction based method for the detection of BCG retention after intravesical instillation in guinea pig bladders.

Abstract

In intravesical Bacille bilié de Calmette-Guérin (BCG) immunotherapy of superficial bladder cancer, a T cell mediated immunological reaction is associated with the antitumor activity. To gain insight into the approximate number of BCG bacteria retained in the normal, noninjured, urinary bladder after intravesical application responsible for induction of the immune reaction, the utility of a sensitive polymerase chain reaction (PCR) based assay was investigated in a guinea pig model. After one single or six subsequent weekly instillations with 1x10(7) CFU of BCG, the bladders were resected and processed for BCG determination with PCR. The bladders were resected 24 h after instillation, aiming at (semi)quantifying the number of BCG organisms able to resist the natural voiding washout of the bladder. The PCR was based on amplification of a 249 base pair fragment of the insertion element IS6110 and is specific for bacteria belonging to the Mycobacterium tuberculosis complex which includes Mycobacterium bovis BCG. After one single instillation no detectable BCG retention was found. However, after six weekly instillations, BCG bacteria could be demonstrated in 2 out of 5 guinea pig bladders, indicating that the number of adhering BCG organisms was around the detection limit of the assay (600-1,000 BCG bacteria per bladder). The data suggest that after six instillations, the retention of BCG in the guinea pig bladder is enhanced as compared with one single instillation. This finding is suggestive of a role of the inflammatory process that is, besides immune system mediated reactions, associated with intravesical BCG instillations. The nature of the molecules involved in enhanced BCG retention after repeated instillations remains to be investigated.