Application of hybridisation probe-based real-time PCR assay to monitor the Brucella DNA load in patients with brucellosis throughout different stages of the disease

Abstract number: 1733_955

Vrioni G., Priavali E., Pappas G., Gartzonika C., Levidiotou S.

Different methods have been described for diagnosis of human brucellosis. Among them, peripheral blood real-time PCR assay (RTPCR) seems to be a very promising technique not only for the initial diagnosis of the disease, but also for the post-treatment follow-up by the accurate quantification of Brucella DNA. A quantitative RTPCR assay using the LightCycler instrument (Roche Diagnostics, Mannheim, Germany) was employed for the monitoring of bacterial DNA load in patients with brucellosis throughout different stages of the disease.

A total of 130 peripheral blood specimens were examined from 39 patients with acute brucellosis as determined by blood culture, serologic tests, and the patients' clinical picture. A minimum of 3 specimens (one at diagnosis, one at the end of treatment and at least one during the post-therapy follow-up period) were obtained per patient. RTPCR assay was based on direct amplification of a 207-bp DNA sequence of a gene that codes for the synthesis of an immunogenetic 31-kDa protein specific for Brucella genus (BCSP31). The amplification product was detected by using as fluorescence technique hybridisation probes labelled with LightCycler Red 640 (detected in channel F2). Primers and probes were designed by TIB MOLBIOL (Berlin, Germany) and fluorescence curves were analysed with LightCycler software v. 3.5. Following amplification, melting curve analysis was performed to verify the specificity of PCR products. A standard curve, comprising 10-fold dilutions of Brucella BCSP31 DNA from 101 to 107 target equivalents, allowed quantification of unknown samples.

RTPCR assay was 100% sensitive and specific for B. melitensis at the time of diagnosis. Despite the decrease of bacterial DNA load at the completion of treatment, the RTPCR results for 87% of patients remained positive. Six months after therapy, 77% of patients presented with residual bacterial DNA without experiencing relapse. From 21 patients who were monitored for >1 year after therapy, 13 continued to have positive RTPCR results, albeit asymptomatic.

In conclusion, B. melitensis DNA may persist on finalising and after therapy, despite of the appropriate treatment and apparent recovery from infection. The significance of these findings remains to be elucidated in reference with their pathogenic and therapeutic implications.