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Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease
Héctor David de Paz1, Pedro Brotons1,2,3, Cristina Esteva1,2
1Department of Molecular Microbiology, Institut de Recerca Pediatrica, Hospital Sant Joan de Déu, Barcelona, Spain.
Abstract:
Current molecular PCR-based techniques used for detecting Streptococcus pneumoniae, the causative pathogen of invasive pneumococcal disease (IPD), are accurate but have a run time of several hours. We aimed to develop and validate a novel real-time loop mediated amplification (LAMP) assay for rapid detection of pneumococcus in normally sterile samples with accuracy comparable to a gold standard real-time PCR. Conserved regions of lytA were used for the design of the LAMP test. Analytical validation included assessment of linearity, limit of detection (LOD), intra-assay and inter-assay precision and analytical specificity, which was evaluated by using reference strain S. pneumoniae R6 and a quality control panel. Clinical performance was assessed on all samples collected from children with suspicion of IPD attended in Hospital Sant Joan de Deu (Barcelona, Spain) during the period April-September 2015. Fresh samples were analyzed after DNA extraction. The following values of analytical parameters were determined: linearity within the range 108-104 copies/mL; limit of detection, 5·103 copies/mL; intra- and inter-assay precision measured by mean coefficient of variance, 3.61 and 6.59%; analytical specificity, 9/9 pathogens similar to S. pneumoniae and 14/14 strains of different S. pneumoniae serotypes correctly identified as negative and positive results, respectively. Diagnostic sensitivity and specificity values were 100.0 and 99.3%. Median time of DNA amplification was 15 min. The new LAMP assay showed to have similar accuracy as PCR while being 5-fold faster and could become a useful diagnostic tool for early diagnosis of IPD.
Insights
A new rapid Loop-Mediated Amplification (LAMP) assay detects Streptococcus pneumoniae, the cause of invasive pneumococcal disease (IPD). This novel method is accurate and significantly faster than traditional PCR, aiding early IPD diagnosis.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Invasive pneumococcal disease (IPD) is caused by Streptococcus pneumoniae.
- Current PCR methods for S. pneumoniae detection are accurate but time-consuming.
- Rapid diagnostic tools are needed for timely IPD management.
Purpose of the Study:
- To develop and validate a novel real-time Loop-Mediated Amplification (LAMP) assay for rapid S. pneumoniae detection.
- To compare the accuracy and speed of the LAMP assay against real-time PCR.
- To assess the clinical performance of the LAMP assay in identifying IPD.
Main Methods:
- Designed a real-time LAMP assay targeting conserved regions of the S. pneumoniae lytA gene.
- Performed analytical validation including linearity, limit of detection (LOD), precision, and specificity.
- Assessed clinical performance on samples from children suspected of IPD.
Main Results:
- The LAMP assay demonstrated high analytical performance: LOD of 5·10^3 copies/mL, good precision (CV < 6.59%), and high specificity.
- Clinical evaluation showed diagnostic sensitivity of 100.0% and specificity of 99.3%.
- Median DNA amplification time was 15 minutes, significantly faster than PCR.
Conclusions:
- The novel real-time LAMP assay provides accurate detection of S. pneumoniae.
- This LAMP assay is substantially faster (5-fold) than real-time PCR.
- The LAMP assay is a promising tool for rapid and early diagnosis of IPD.
