Real-time polymerase chain reaction on filter paper spotted samples: a gateway to molecular diagnosis of invasive bacterial diseases for rural areas in low-income countries

Background: Bacterial culture is the gold standard for the diagnosis of invasive bacterial diseases (IBDs) but molecular methods are more specific and sensitive. Fresh liquid samples (FLSs) show patent [imitations for shipping and storage. We aimed to evaluate the sensitivity and specificity of real-time polymerase chain reaction (PCR) performed on dried sample spots (DSSs) obtained from different biological fluids compared with real-time PCR or culture performed on FLSs. Methods: FLSs positive for Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, Bordetella pertussis and/or Pseudomonas aeruginosa were spotted on filter paper. Real-time PCR was performed on both FLSs and DSSs and results were compared. The stability of the DSS results over time was evaluated. Results: Real-time PCR performed on 114 DSSs showed a specificity of 99.1% and a sensitivity of 91.2% for IBD diagnosis. A positive correlation was found between FLS cycle threshold (Ct) and DSS Ct (r=0.84; r 2 =0.71) with the Pearson statistical test and Bland-Altman analysis showing that 95% of the specimens were within agreeable limits. Although we observed a trend towards signal reduction over time in the DSSs, there was no statistical evidence of an increase in Ct values. Real-time PCR on DSSs was 2.2 times more sensitive than culture. Conclusions: Real-time PCR applied to DSSs may be a useful approach in different situations, such as IBD diagnosis, both for rural areas of low-income countries and family practitioners in various settings.

Automated summary

The study found that real-time PCR performed on dried sample spots showed high specificity and sensitivity for the diagnosis of invasive bacterial diseases, indicating potential value in clinical applications. Additionally, real-time PCR on DSSs was 2.2 times more sensitive than traditional bacterial culture, offering a more convenient diagnostic method for family practitioners in various settings, including rural areas of low-income countries.