New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

Probert, William S.; Hacker, Jill K.

doi:10.1128/jcm.00500-19

Cited by 5 publications

(5 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These efforts will expand nanoPCR's utility in remote or resource-limited settings 46,47 . NanoPCR could also be applied for the prompt diagnosis of other infections, such as acquired immunodeficiency syndrome, tuberculosis and hepatitis [48][49][50] .…”

Section: Resultsmentioning

confidence: 99%

Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device

et al. 2020

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 99%

Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device

et al. 2020

View full text Add to dashboard Cite

“…This study and others have also designed assays using FRET-based probes such as TaqMan or molecular beacon to target the VP1 capsid region or the 5 -UTR. Consistent with the design of our assay, targeting the well-conserved 5 -UTR is a common approach for molecular detection of HAV because the virus is genetically diverse [1,24]. The development of a consensus assay that can detect multiple HAV genotypes is cost-effective and allows for rapid diagnosis of infections, and a first important step will be determining an internationally agreed-upon set of primers and probes, likely targeting the 5 -UTR [25].…”

Section: Discussionmentioning

confidence: 91%

“…Although the assay was able to accurately identify the six different HAV sub-types from patient samples, single-plex assays can increase the turnaround time and ease of application in a clinical setting. By contrast, the California Department of Public Health developed an improved multiplex RT-PCR assay for sub-genomic identification of HAV genotypes IA, IB and IIIA, and the sensitivity and specificity were greater than 97% [23,24]. This study and others have also designed assays using FRET-based probes such as TaqMan or molecular beacon to target the VP1 capsid region or the 5 -UTR.…”

Section: Discussionmentioning

confidence: 99%

Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens

Kozak

Rutherford

Richard-Greenblatt

et al. 2022

Viruses

View full text Add to dashboard Cite

Hepatitis A virus (HAV) is an emerging public health concern and there is an urgent need for ways to rapidly identify cases so that outbreaks can be managed effectively. Conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10–30% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis, which is key to prevent the spread of infections. In this study, we developed a real-time PCR (RT-PCR) assay to detect HAV RNA for the identification of acute HAV infection. Primers were designed to target the conserved 5′-untranslated region (5′-UTR) of HAV, and the assay was optimized on both the Qiagen Rotor-Gene and the BD MAX. We successfully detected HAV from patient serum and stool samples with moderate differences in sensitivity and specificity depending on the platform used. Our results highlight the clinical utility of using a molecular assay to detect HAV from various specimen types that can be implemented in hospitals to assist with diagnostics, treatment and prevention.

show abstract

“…Regardless, such recovery at this Cτ is remarkable given the difficulty of detecting subgenotype IIIA. Recently, a reverse transcription-PCR (rRT-PCR) HAV subgenotyping assay found the LOD for IIIA to be 10 RNA copies per reaction, equivalent to 34.2 and 33.9 Cτ for singleplex and triplex reactions, respectively ( 56 ). Whereas a subgenotype-specific optimized reverse transcription quantitative real-time PCR (RT-qPCR) assay found the LOD for IIIA to be 5,000 copies per reaction, equivalent to Cτ near 41.3 ( 57 ).…”

Section: Discussionmentioning

confidence: 99%

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

Zufan,

Mercoulia,

Kwong

et al. 2024

Microbiol Spectr

View full text Add to dashboard Cite

Hepatitis A virus (HAV) infections are an increasing public health concern in low-endemicity regions due to outbreaks from food-borne infections and sustained transmission among vulnerable groups, including persons experiencing homelessness, those who inject drugs, and men who have sex with men, which is further compounded by aging, unvaccinated populations. DNA sequence characterization of HAV for source tracking is performed by comparing small subgenomic regions of the virus. While this approach has been successful when robust epidemiological data are available, poor genetic resolution can lead to the conflation of outbreaks with sporadic cases. HAV outbreak investigations would greatly benefit from the additional phylogenetic resolution obtained by whole virus genome sequence comparisons. However, HAV genomic approaches can be difficult because of challenges in isolating the virus, low sensitivity of direct metagenomic sequencing in complex sample matrices like various foods, such as fruits, vegetables, and molluscs, and difficulty designing highly multiplexed PCR primers across diverse HAV genotypes. Here, we introduce a proof-of-concept pan-HAV oligonucleotide hybrid capture enrichment assay from serum and frozen berry specimens that yields complete and near-complete HAV genomes from as few as four input HAV genome copies. We used this method to recover HAV genomes from human serum specimens with high Cτ values (34.7–42.7), with high assay performance for all six human HAV subgenotypes, both contemporary and historical. Our approach provides a highly sensitive and streamlined workflow for HAV whole-genome sequencing from diverse sample types that can be the basis for harmonized and high-resolution molecular epidemiology during HAV outbreak surveillance. IMPORTANCE This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making them suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations as well as our understanding of the global diversity and transmission dynamics of HAV.

show abstract

New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

Cited by 5 publications

References 32 publications

Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device

Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device

Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks

Contact Info

Product

Resources

About