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...

BackgroundHepatitis A (HAV) and E (HEV) viruses are responsible for enterically transmitted hepatitis. Tunisia is reported to be of intermediate endemicity for HAV and of low seroprevalence for HEV; however, data from rural areas of South Tunisia are lacking.MethodsSera from 216 asymptomatic pregnant women and from 92 patients with acute hepatitis were collected between October 2014 and November 2015. Total and IgM anti-HAV immunoglobulins and anti-HEV IgG and IgM were investigated. Anti-HAV IgM-positive samples were subjected to RT-PCR targeting the VP1/2A region and sequenced. HEV IgM positive samples and all samples from acute hepatitis patients were assessed for HEV RNA.ResultsAmong pregnant women (mean age 32+/-8), HAV seroprevalence was 98.6%, none presented anti-HAV IgM; HEV seroprevalence was 5.1% and three presented weakly reactive anti-HEV IgM without detectable RNA. Among acute hepatitis patients (mean age 18.5 +/- 14), HEV seroprevalence was 19,5%, none presented anti-HEV IgM, nor HEV RNA. HAV seroprevalence exceeded 90% by age 5 and acute HAV infection was detected in 20 patients (21,7%), younger than patients with other hepatitis causes (9,8 years vs. 20,4 years, p = 0,004); 65% were male. Most acute HAV infections were observed in a coastal area where HAV infections represented 52% of hepatitis etiology. Phylogenetic analysis identified genotype IA strains, clustering close to previously published Tunisian sequences.ConclusionThe present study confirmed a low HEV endemicity and evidenced a still high level of HAV circulation in Southern Tunisia, suggesting distinct dissemination patterns for these viruses.

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