04 May 2018
The role of whole genome sequencing in hepatitis A surveillance and outbreak investigation
Hepatitis A (HAV) infections occur worldwide, with approximately 1.5 million clinical cases occurring annually . The rate of infection is likely at least ten times higher, however, as most cases are asymptomatic and young children rarely show signs of disease  . The incidence rate of HAV is an indirect marker of socioeconomic status, with infection rates decreasing with increased access to safe drinking water and improved sanitation measures .
HAV transmission occurs most commonly through the faecal-oral route, either by person-to-person contact or the ingestion of HAV contaminated food or water . Intrinsic to the transmissibility of HAV is that the virus is hardy. The virus can survive passage through the gastrointestinal tract, and virus shedding in stool peaks before symptoms appear, which enhances person-to-person spread and contamination of food by food handlers . While occurring predominantly in developing countries, clusters of HAV infection can also occur in developed countries. In Australia, there have been two recent HAV outbreaks, one associated with semi-dried tomatoes and another with frozen berries  .
Identifying the source of HAV outbreaks can be difficult. The most common method relies on epidemiological investigation, but tracing the source of outbreaks can be problematic due to the long incubation period of the virus (on average one month ), leading to difficulties in cases being able to recall foods consumed and exposure to other risk factors during this period.
One method of improving source identification is sequencing selected regions of the HAV genome to determine the genetic relatedness of isolates. In 2016, the Victorian Infectious Diseases Reference Laboratory (VIDRL) began a project to genotype any HAV RNA-positive samples sent to VIDRL for diagnostic testing by using specific PCR primer sets designed by the Hepatitis A laboratory network HAVNET. These are international consensus primers which are used worldwide, primarily by the reference laboratories working under the HAV network. The use of these specific primers allows the sharing of HAV sequence data for international mapping and source tracing.
Additionally, VIDRL established a local epidemiological and nucleotide sequence database of HAV strains using a software platform, Geneious, which also has a collection of bioinformatic tools for subsequent molecular and epidemiological data analysis. The database was then used to explore and investigate the genetic relationship between current diagnostic samples and previous HAV cases. Two notable hepatitis A clusters were identified in 2017. Five samples received in 2017 showed 100% identity to the index case of the 2015 frozen berries outbreak, strongly suggesting that the patients were infected by the same strain. This information, combined with epidemiological evidence gathered by OzFoodNet epidemiologists, led to the issuing of public health alerts and a voluntary product recall of the frozen berries .
The database was also able to identify local HAV strains linked to an international HAV outbreak. The European Union (EU) reported an HAV outbreak that mostly affected men who have sex with men (MSM). Between June 1 2016 and June 26 2017, 1,500 confirmed HAV cases were reported by 16 EU countries . Phylogenetic analysis revealed that the EU cases could be grouped into three genetically-related clusters of infection, all belonging to HAV genotype IA . As of December 2017, VIDRL had identified 66 Australian cases that had sequences associated with HAV IA clusters from the EU MSM outbreak. A public health alert was issued in November by the DHHS in response to laboratory and epidemiological findings .
These results show that molecular epidemiology plays a critical role in suspected HAV outbreak investigations and should become part of the standard workup. It should not exclude standard and proven epidemiological approaches, but should be seen as complementary. A National Enhanced Hepatitis A pilot surveillance scheme which was instigated by OzFoodNet in 2017 is building on this principle. Running from the 1st of July 2017 to the 30th of June 2019, the pilot involves the collaboration of three public health laboratories: VIDRL, the Microbiological Diagnostic Unit Public Health Laboratory (MDU), and Queensland Health Forensic and Scientific Services (QHFSS). VIDRL and QHFSS receive, sequence, and analyse HAV samples from their respective jurisdictions. MDU then collates and analyses all national HAV sequences obtained from VIDRL and QHFSS, and shares the results with OzFoodNet epidemiologists, providing them with the opportunity to analyse case epidemiological metadata and sequence data simultaneously.
This national approach provides a way of comparing sequences between jurisdictions which has rarely occurred previously, with the aim of early identification of local and national transmission clusters as well as potential source attribution of locally acquired cases due to imported food. Real-time monitoring of local circulating HAV strains can inform public health authorities of a potential outbreak and enable interventions, such as additional investigations, introduction of vaccination, and product recalls, which may limit transmission before the results from traditional epidemiological investigations are available.
Authors: Scott Bowden (VIDRL), Linda Viberg (MDU)