Quantification of Streptococcus pneumoniae in the Human Nasopharynx: Assay Validation Studies for the FDA Öffentlichkeit
Watson, David (2016)
Published
Abstract
Introduction: Diagnosing a case of pneumococcal pneumonia can be very difficult due to the drawbacks of the current gold-standard methods based on bacterial culture. Recently several new qPCR procedures have been developed to identify genes specific to the pneumococcus, making accurate identification easier. We set out to validate the new lytA assay for use in future clinical studies.
Methods: The lytA assay was conducted as described in this report, with careful attention to quality control measures and strict adherence to predetermined acceptance criteria. In order to establish the performance characteristics of the lytA assay, a series of experiments were conducted. These experiments included a linearity study, a limit-of-detection study, a replication experiment, an interference study and a comparison of methods study. These experiments were designed to determine the assay's reportable range, analytical sensitivity, precision, analytical specificity, and accuracy, respectively.
Results: In the linearity study linear regression provided an R2 of 0.9941, indicating a strong linear relationship between assigned values of standard concentrations and values measured using the assay. In the limit-of-detection study the overall LOD for the assay was 4.28 copies/reaction. In the replication experiment the assay performed very reliably with low standard deviations in run and across runs. In the interference study, all replicates of the 29 organisms tested were not detected by the lytA qPCR assay, while TIGR4 positive controls were detected. Finally, in the comparison of methods study the lytA assay found all of the 40 S. pneumoniae-spiked specimens to be positive, matching the gold standard methods.
Discussion and Conclusion: The experiments conducted in this study display the efficacy of the lytA assay and allow us to establish thorough performance specifications for this laboratory-developed assay. The lytA assay demonstrates excellent linearity across the entire linear range. The assay also demonstrates a high level of precision and repeatability, with acceptable levels of random error. Further, the sensitivity and specificity of the assay are both very high, so the test identifies pneumococcus in extremely low concentrations, while at the same time not detecting similar organisms. Finally, we find that the lytA assay is in 100% agreement with S. pneumoniae gold-standard reference identification methods.
Table of Contents
Introduction. 1
Abbreviation Listing. 3
Literature Review. 4
Epidemiology of pneumonia. 4
Physiopathology of pneumonia. 5
Pneumococcal pneumonia. 5
Vaccination with pneumococcal vaccines. 6
Community acquired bacterial pneumonia (CABP) and Clinical Diagnosis. 7
Laboratory diagnostics for the identification of pneumococcal pneumonia. 9
Cultures and identification of S. pneumoniae. 9
Polymerase chain reaction. 12
Methods. 13
The lytA Assay. 13
DNA extraction from nasopharyngeal samples. 13
DNA extraction of S. pneumoniae reference strain TIGR4. 14
Preparation of standards for quantitative polymerase chain reaction. 15
Quantitative PCR (qPCR). 16
Assay Quality Assurance. 16
Quality controls. 16
Acceptance criteria. 17
Recording of Data/Observations. 18
Experiments. 18
Reportable Range. 19
Analytical Sensitivity. 19
Precision. 20
Analytical Specificity. 20
Accuracy. 21
Results. 22
Reportable Range (Linearity). 22
Analytical Sensitivity (Limit of Detection). 23
Precision. 23
Analytical Specificity. 24
Accuracy. 25
Discussion. 25
Conclusions and Recommendations. 27
Appendices. 31
Figures. 31
Figure 1. Linearity of the lytA qPCR Assay. 31
Tables. 32
Table 1. lytA qPCR Specificity Panel Organisms. 32
Table 2 lytA qPCR Accuracy Panel Organisms: S. pneumoniae Strains. 33
Table 3. TIGR4 DNA Standards and Conversions. 34
Table 4 lytA qPCR Sensitivity Results. 34
Table 5. Precision Testing in the lytA qPCR Assay by S. pneumoniae Strain. 35
Table 6. Summary of Precision Testing in the lytA qPCR Assay. 35
Table 7. Accuracy Panel lytA qPCR results. 36
Works Cited. 38
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