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

Abstract

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 transcriptionPCRs (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 ϳ10-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.Hepatitis A virus (HAV) is a major cause of viral hepatitis worldwide. Infection is usually asymptomatic or anicteric in children younger than 6 years of age but can lead to fulminant hepatitis, particularly in adults older than 50 years of age. With the improvement in hygiene conditions in developed countries, there has been a striking reduction of HAV endemicity over the past few decades (13,25). This decrease in natural immunization allows potentially massive outbreaks involving adults with a consequent number of severe clinical forms and an important socioeconomic impact.The diagnosis of acute hepatitis A is based on the detection of the immunoglobulin M (IgM) antibody to HAV (HAVM). Determination of the total anti-HAV antibodies (HAVT) or anti-HAV IgG allows the determination of the HAV immune status. Investigation of outbreaks relies on epidemiological and serological studies, but only molecular investigation is able to link apparently sporadic cases or apparently distinct outbreaks (6,24). This methodology requires costly devices, such as thermal cyclers and sequencers, and expertise in nucleotide sequence analysis that are available in only few reference centers. Many studies have demonstrated the good performances of blotted blood, serum, or plasma samples in serological and molecular diagnosis of different viral infections, including infection with cytomegalovirus (23), human immunodeficiency virus (HIV) (3, 18, 19), he...

An Overview of the Clinical Use of Filter Paper in the Diagnosis of Tropical Diseases

“…NAATs of dried serum spots perform very well for HAV (92.3% and 100%) and HCV (100% and 100%) sensitivity and specificity, respectively, versus liquid serum 111,113. Both hepatitis viruses showed a 10-fold fall in viral load after storage for 4 weeks on paper at room temperature 111,113…”

The use of dried blood spots for assessing antibody response to hepatitis A virus after natural infection and vaccination

“…Disadvantages include requirements for development and validation of assays as well as small volume of samples collected [Parker and Cubit, 1999]. Since its first implementation for the diagnosis of phenylketonuria in newborns in the 1960s [McDade et al, 2007], DBS has been used for the diagnosis of a number of metabolic disorders and for the analysis of a wide range of biomarkers [McDade et al, 2007], including antibodies raised against several viruses, such as the human immunodeficiency virus [Major et al, 1991; Castro et al, 2008], rubella virus [Punnarugsa and Mungmee, 1991; Neto et al, 1995; Helfand et al, 2001, 2007; Karapanagiotidis et al, 2005], measles virus [Helfand et al, 2001; Ridell et al, 2002], HTLV [Parker et al, 1995], Epstein‐Barr virus [Fachiroh et al, 2008], and hepatitis viruses [Villa et al, 1981; Gil et al, 1997; Tappin et al, 1998; De Almeida et al, 1999; McCarron et al, 1999; Desbois et al, 2009]. However, the use of DBS for the detection of antibodies to hepatitis A virus (anti‐HAV) has been limited [Gil et al, 1997; De Almeida et al, 1999; Desbois et al, 2009], and there are no studies which describe the use of DBS for assessing the humoral response after hepatitis A vaccination.…”

Laser Cutting Eliminates Nucleic Acid Cross-Contamination in Dried-Blood-Spot Processing

“…ried blood spots (DBS) are easily collected, stored, and transported and are suitable for many diagnostic tests (2,3,5,7,8,12). However, cross-contamination of molecular DBS assays is a potential problem since the sample-containing region is usually physically touched by manual or automated punching devices.…”