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.
Experimental Research Unit, University General Hospital, Albacete, Spain.
Elena
E
Navarro
Juan Carlos
JC
Segura
María Jesús
MJ
Castaño
Javier
J
Solera
D000328:Adult; D000368:Aged; D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D017347:Brucella melitensis / Q000235:genetics* / Q000302:isolation & purification*; D002006:Brucellosis / Q000188:drug therapy* / Q000382:microbiology*; D004269:DNA, Bacterial / Q000097:blood*; D004334:Drug Administration Schedule; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012008:Recurrence; D012680:Sensitivity and Specificity; D013997:Time Factors
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial