Use of Dried Serum Spots for Serological and Molecular Detection of Hepatitis A Virus

• Published in: Journal of Clinical Microbiology 2009-05, Vol.47 (5), p.1536-1542
• Main Authors: Desbois, Delphine, Roque-Afonso, Anne-Marie, Lebraud, Patricia, Dussaix, Elisabeth
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Desiccation; Fundamental and applied biological sciences. Psychology; Hepatitis A - diagnosis; Hepatitis A Antibodies - blood; Hepatitis A virus; Hepatitis A virus - isolation & purification; Humans; Microbiology; Miscellaneous; Molecular Sequence Data; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral - genetics; Sensitivity and Specificity; Sequence Analysis, DNA; Sequence Homology; Serum - virology; Specimen Handling - methods; United Kingdom; Virology
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.02191-08

We assessed the feasibility of using dried serum spots (DSS) for the serological and molecular diagnosis of hepatitis A virus (HAV) infection. Sixty-eight sera spotted onto filter papers (Whatman International Ltd., United Kingdom) were used for detection of total anti-HAV antibodies, and 64 sera were used for detection of immunoglobulin M antibody to HAV. DSS were stored at 4°C, room temperature, and 37°C for 1, 2, and 4 weeks. Sensitivity and specificity of the serological assays were 100% regardless of temperature and storage duration. To assess the stability of HAV RNA, we performed qualitative and quantitative reverse transcription-PCRs (RT-PCRs) with human plasma spiked with serial dilutions of cultured HAV spotted on Flinders Technology Associates filter paper cards (Whatman International Ltd.). Filter papers were stored at room temperature and processed for RT-PCR assays. No reduction of viral load was observed after 5, 15, and 30 days of storage. The ~10-fold reduction of sensitivity from DSS was attributable to a smaller sample input in DSS samples. This method was further evaluated using 35 frozen sera. HAV RNA amplification showed 100% specificity and 92.3% sensitivity, and sequence analysis from DSS and sera provided identical results. HAV RNA can be accurately recovered from DSS for molecular epidemiology purposes, and we confirm the reliability of blotted samples in the serological diagnosis of HAV infection. The DSS method facilitates storage and shipment of samples from routine laboratories to reference centers for further investigations and large epidemiological studies.