Use of real-time quantitative polymerase chain reaction to monitor the evolution of Brucella melitensis DNA load during therapy and post-therapy follow-up in patients with brucellosis.

BACKGROUND

We performed quantitative real-time polymerase chain reaction (Q-PCR) to monitor the evolution of Brucella melitensis DNA load from initial diagnosis through post-therapy follow-up in patients with brucellosis.

METHODS

On the basis of real-time fluorometric quantification of PCR products, we used the ultra-rapid LightCycler system (Roche Diagnostics). We collected 180 peripheral blood samples from 18 patients with brucellosis. Analysis of bacterial DNA loads was performed for 2 groups: 11 patients who did not experience relapse and 7 patients who experienced relapse in the follow-up phase.

RESULTS

Q-PCR was 100% specific for B. melitensis and showed an analytical sensitivity of 15 fg. Sensitivity of Q-PCR for both initial infections and relapses was 100%. There were no statistically significant differences between groups with respect to bacterial DNA load from initial diagnosis to the end of post-treatment follow-up (P > .05). Evolution of the bacterial DNA load throughout the treatment phase was similar among patients who relapsed and did not relapse. Despite positive response to treatment and a sharp decrease in bacterial DNA load after initiating therapy, the results of Q-PCR on finalizing treatment for 50% of the patients (7 from the relapse group and 2 from the nonrelapse group) were low-level positive. At the conclusion of follow-up, almost 40% of the patients (4 from the relapse group and 3 from the nonrelapse group), most of them asymptomatic, still maintained low bacterial DNA loads.

CONCLUSIONS

Using Q-PCR techniques, we consistently detected B. melitensis DNA in the blood samples of patients with brucellosis throughout treatment and follow-up, despite apparent recovery from infection. These findings may have diagnostic, pathogenic, and therapeutic implications.