Development of a high throughput PCR to detect Coxiella burnetii and its application in a diagnostic laboratory over a 7-year period

Abstract

Q fever is a worldwide zoonotic infectious disease due to Coxiella burnetii . The clinical presentation may be acute (pneumonia and/or hepatitis) or chronic (most commonly endocarditis). Diagnosis mainly relies on serology and PCR. We therefore developed a quantitative real-time PCR. We first tested blindly its performance on various clinical samples and then, when thoroughly validated, we applied it during a 7-year period for the diagnosis of both acute and persistent C. burnetii infection. Analytical sensitivity (< 10 copies/PCR) was excellent. When tested blindly on 183 samples, the specificity of the PCR was 100% (142/142) and the sensitivity was 71% (29/41). The sensitivity was 88% (7/8) on valvular samples, 69% (20/29) on blood samples and 50% (2/4) on urine samples. This new quantitative PCR was then successfully applied for the diagnosis of acute Q fever and endovascular infection due to C. burnetii , allowing the diagnosis of Q fever in six patients over a 7-year period. During a local small cluster of cases, the PCR was also applied to blood from 1355 blood donors; all were negative confirming the high specificity of this test. In conclusion, we developed a highly specific method with excellent sensitivity, which may be used on sera for the diagnosis of acute Q fever and on various samples such as sera, valvular samples, aortic specimens, bone and liver, for the diagnosis of persistent C. burnetii infection.

SdrA, an NADP(H)‐regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly

“…HeLa CCL 2 cells were seeded into 24‐well plates at a density of 5 × 10 4 with or without glass coverslips 1 day before infection. C. burnetii strains grown in axenic medium for 7 days were harvested and resuspended in DMEM +5% FCS and the bacteria quantified by qPCR amplifying ompA , as described previously (Jaton et al, ), to determine an multiplicity of infection (MOI) of 50. Cells were infected by C. burnetii diluted in DMEM +5% FCS and incubated for 4 hr.…”

The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii

“…At this time (day 0), as well as 1, 3 and 5 days post infection, cells were lysed in dH 2 O and pelleted by centrifugation. Genomic DNA was extracted from the samples and genome equivalents (GE) were quantified by qPCR specific for the Coxiella ompA gene [47]. Samples were collected for immunofluorescence analysis at day 3 as described above.…”

From Q Fever to Coxiella burnetii Infection: a Paradigm Change

“…They found that multiple combinations of DNA extraction kits and qPCR assays gave equivalent results for Q fever diagnosis. In Switzerland, a qPCR system targeting the ompA gene has been used for 7 years for detection of C. burnetii in clinical samples (361). The sensitivity was 88% for valvular samples, 69% for blood samples, and 50% for urine samples.…”