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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 degrees C, room temperature, and 37 degrees 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 approximately 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.

SUBMITTER: Desbois D

PROVIDER: S-EPMC2681854 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Use of dried serum spots for serological and molecular detection of hepatitis a virus.

Desbois Delphine D Roque-Afonso Anne-Marie AM Lebraud Patricia P Dussaix Elisabeth E

Journal of clinical microbiology 20090325 5

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 degrees C, room temperature, and 37 degrees C for 1, 2, and 4 weeks. Sensitivity and specificity of the serological assa ...[more]

PMID: 19321728

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Project description:BackgroundNigeria has one of the highest burdens of pneumococcal disease in the world, but accurate surveillance is lacking. Molecular detection of infectious pathogens in dried blood spots (DBS) is an ideal method for surveillance of infections in resource-limited settings because of its low cost, minimal blood volumes involved, and ease of storage at ambient temperature. Our study aim was to evaluate a Streptococcus pneumoniae real-time polymerase chain reaction (rt-PCR) assay on DBS from febrile Nigerian children on Whatman 903 and FTA filter papers, compared to the gold standard of culture.MethodsBetween September 2011 to May 2015, blood was collected from children 5 years of age or under who presented to six hospital study sites throughout northern and central Nigeria with febrile illness, and inoculated into blood culture bottles or spotted onto Whatman 903 or FTA filter paper. Culture and rt-PCR were performed on all samples.ResultsA total of 537 DBS specimens from 535 children were included in the study, of which 15 were culture-positive for S. pneumoniae. The rt-PCR assay detected S. pneumoniae in 12 DBS specimens (2.2%). One positive rt-PCR result was identified in a culture-negative specimen from a high-risk subject, and two positive rt-PCR results were negative on repeat testing. Six culture-confirmed cases of S. pneumoniae bacteremia were missed. Compared to culture, the overall sensitivities of Whatman 903 and FTA DBS for detection of S. pneumoniae were 57.1% (95% CI 18.4-90.1%) and 62.5% (95% CI 24.5-91.5%), respectively. Nonspecific amplification was noted in an additional 22 DBS (4.1%). Among these, six were positive for a non-S. pneumoniae pathogen on culture.ConclusionsRt-PCR was able to detect S. pneumoniae from clinical DBS specimens, including from a culture-negative specimen. Our findings show promise of this approach as a surveillance diagnostic, but also raise important cautionary questions. Several DBS specimens were detected as S. pneumoniae by rt-PCR despite growth of a non-S. pneumoniae pathogen on culture. A precise definition of what constitutes a positive result is required to avoid falsely over-identifying specimens.

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Use of dried serum spots for serological and molecular detection of hepatitis a virus.

Publications

Use of dried serum spots for serological and molecular detection of hepatitis a virus.

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