Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples

Area of Science:

• Clinical Virology
• Molecular Diagnostics
• Infectious Diseases

Background:

• Adenoviruses (AdV) cause significant disease, especially in immunocompromised patients.
• Quantifying AdV DNA is difficult due to 71 serotypes and lack of a WHO standard, leading to lab variability.
• Accurate AdV quantification is crucial for patient management and treatment efficacy.

Purpose of the Study:

• To compare the performance of a novel laboratory-developed multiplex PCR assay (Octaplex) against two other methods for AdV DNA quantification.
• To evaluate the impact of different PCR strategies and reference materials on AdV detection and quantification accuracy.
• To identify a reliable method for sensitive and specific AdV DNA detection in clinical samples.

Main Methods:

• A laboratory-developed multiplex PCR assay (Octaplex) targeting AdV groups A-G and subgroup E4 was compared to a modified Jothikumar assay and a bisulfite pretreatment assay (Genetic Signatures).
• Assays were tested using low-copy-number clinical samples, high-copy-number cultured strains, and 51 AdV types for endpoint detection.
• Standard curves were generated using two different materials to assess quantitative accuracy.

Main Results:

• The Octaplex assay successfully detected all low-copy-number clinical samples, while other assays missed some.
• The modified Jothikumar assay showed inefficient amplification of high-copy strains, and the Genetic Signatures assay exhibited a positive bias.
• Both Octaplex and modified Jothikumar assays yielded lower AdV levels than a commercial platform for culture fluids, but not patient samples.

Conclusions:

• The Octaplex multiplex PCR assay demonstrates superior sensitivity for detecting low-copy adenovirus DNA in clinical samples.
• Significant variations in AdV detection and quantification exist between different PCR methods and reference materials.
• Standardization of AdV quantification methods and reference materials is essential for reliable clinical diagnostics.