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Diagnosis of Bartonella endocarditis by a real-time nested PCR assay using serum
Zaher Zeaiter1, Pierre-Edouard Fournier, Gilbert Greub
1Unité des Rickettsies, IFR 48, CNRS UMR 6020, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 5, France.
Insights
LightCycler PCR (LCN-PCR) offers a sensitive and specific method for diagnosing Bartonella endocarditis, outperforming traditional blood cultures and other PCR techniques. This assay is particularly useful for early detection using serum samples, even in patients on antibiotics.
Area of Science:
- Infectious Diseases
- Molecular Diagnostics
- Bacteriology
Background:
- Bartonella endocarditis is a severe infection often characterized by negative blood cultures.
- Accurate and timely diagnosis is crucial for effective patient management.
- Existing diagnostic methods, including blood cultures and some PCR assays, have limitations in sensitivity and specificity.
Purpose of the Study:
- To evaluate the diagnostic performance of three PCR assays for Bartonella endocarditis using early-stage serum samples.
- To compare the sensitivity and specificity of LightCycler PCR (LCN-PCR), one-step nested PCR (OSN-PCR), and two-step nested PCR (TSN-PCR).
- To assess the utility of LCN-PCR as a diagnostic tool for Bartonella endocarditis.
Main Methods:
- Serum samples from 43 patients diagnosed with Bartonella endocarditis were tested.
- Three PCR assays were employed: TSN-PCR, OSN-PCR, and LCN-PCR, using primers targeting the ribC gene.
- Performance was compared against prolonged blood culturing and other diagnostic findings.
Main Results:
- LCN-PCR demonstrated 100% specificity and 58.1% sensitivity, significantly outperforming OSN-PCR (18.6%) and blood culturing (7.1%).
- TSN-PCR results were uninterpretable due to contamination.
- LCN-PCR efficacy was unaffected by antibiotic treatment but was impacted by prolonged serum storage at -20°C.
Conclusions:
- LCN-PCR is a highly specific and more sensitive molecular diagnostic method for Bartonella endocarditis compared to traditional techniques.
- This assay is effective for diagnosing suspected cases, especially when only serum is available.
- LCN-PCR can identify causative Bartonella species and aids in diagnosing patients with serological evidence alone.
Abstract:
Bartonella endocarditis is a severe disease for which blood cultures frequently remain negative. We tested three PCR assays by using specimens of serum sampled early during the disease from 43 patients diagnosed in our laboratory as having Bartonella endocarditis on the basis of serological, culture, and/or valvular molecular detection. We tested a two-step nested PCR (TSN-PCR), a one-step nested PCR (OSN-PCR) with a regular thermal cycler, and a one-step nested PCR with the LightCycler (LCN-PCR). These assays were performed with primers derived from the riboflavin synthase-encoding gene ribC, never before amplified in our laboratory. Due to contamination of negative controls, the results of the TSN-PCR were not interpretable, and this technique was no longer considered. The LCN-PCR had a specificity of 100% and a sensitivity of 58.1%, higher than those of the OSN-PCR (18.6%; P < 0.01) and prolonged blood culturing (7.1%; P < 0.01). The LCN-PCR results correlated strictly with those of other direct diagnostic tests, when available, and identified the causative species for six patients previously diagnosed on the basis of serological analysis only. The efficacy of the LCN-PCR was not influenced by antibiotics (P = 0.96) but was altered by prolonged storage of serum specimens at -20 degrees C (P = 0.04). Overall, the LCN-PCR is specific and more sensitive than traditional methods (i.e., culturing and/or PCR with EDTA-treated blood). It can easily be applied to the diagnosis of patients with suspected Bartonella endocarditis, especially when only serum is available.