Lyophilization to improve the sensitivity of qPCR for bacterial DNA detection in serum: the Q fever paradigm

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Abstract
Quantitative real-time PCR (qPCR) on serum provides significant added value to the diagnosis of Q fever, mainly at the acute stage of the disease in seronegative patients and in patients with endocarditis. We evaluated the benefits of C. burnetii DNA concentration in serum by lyophilization to improve qPCR sensitivity. The detection limit of qPCR was determined by comparing six 10-fold dilutions of serum (calibrated with 104 bacteria/ml) with and without lyophilization. We also tested, after lyophilization, 73 sera from patients with acute Q fever and 10 sera from patients with endocarditis for which specific qPCR for C. burnetii performed under our usual conditions remained negative. Lyophilization of DNA was found to improve sensitivity of the qPCR; the limit of detection of C. burnetii DNA by qPCR was 100-fold lower in lyophilized sera (1 bacterium/ml) than in non-lyophilized sera (102 bacteria/ml). Among the 73 sera from patients with acute Q fever, 26 (36%) were positive after lyophilization, demonstrating a sensitivity gain of 44% for early negative sera and 30% for positive sera compared to our usual qPCR conditions. Sensitivity was also higher in sera from patients with endocarditis for which 8/10 (80%) were positive after lyophilization. Our results serve as a proof of concept that lyophilization increases the sensitivity of qPCR in serum by concentrating bacterial DNA. This technique may be applied for the earlier diagnosis of other fastidious bacteria or viruses and extended to other clinical samples.

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