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

Abstract

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 limitations 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; r2=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.

Evaluation of Dried Blood and Cerebrospinal Fluid Filter Paper Spots for Storing and Transporting Clinical Material for the Molecular Diagnosis of Invasive Meningococcal Disease

“…Our dried blood spot assay using the crude DNA extraction method showed a high sensitivity (89%) and specificity (99%) for the detection of Nm in dried blood spots. These results are comparable to the sensitivity and specificity of 90.3% and 99%, respectively reported by De Vitis and colleagues for their dried spot assay which utilised the MagCore automated NucleicAcid Extractor with MagCore Nucleic Acid Tissue Extraction Kit [ 22 ]. Crude DNA extraction from blood and other clinical specimens therefore could be a viable alternative to traditional extraction kits and make dried spot assays for the diagnosis of bacterial meningitis more accessible in resource-limited settings.…”

“…Of the 213 dried CSF specimens, they tested for Nm, S. pneumoniae and Haemophilus influenzae , the specificities were 100% and the sensitivities ranged from 96 to 100% across all three pathogens. Likewise, De Vitis and colleagues found that among 44 CSF specimens for which a bacterial infection was confirmed by qPCR of the liquid specimen, 41 (93.2%) were positive by testing on dried filter paper [ 22 ]. Our study provides further evidence that compared to real-time PCR on liquid specimens, dried filter spot assays are highly specific and sensitive for the detection of Nm in CSF.…”

“…Among febrile children in Nigeria, dried blood spot assays had sensitivities ranging from 57% to 63% for the detection of S. pneumoniae compared to culture and qPCR on liquid specimens [ 14 , 23 ]. Fukasawa and colleagues reported sensitives of 73%, 88% and 100% for the detection of Nm, S. pneumoniae and H. influenzae , respectively, in serum dried spots using FTA TM cards [ 22 ]. False negatives in DBS testing may result from the presence of inhibitors in blood such as immunoglobulin, haemoglobin, and metal ions [ 24 , 25 ].…”