Hepatitis A virus investigation to establish epidemiological and molecular database for source tracing during foodborne outbreak

Hepatitis A virus (HAV) is one of the potential hazards to public health. Although it is relatively harmless, it may cause high expenses for medical treatment and loss of productivity. Further concern is the ability of HAV in causing outbreaks which can affect large population. It is because HAV has various routes of transmission. The most common route of HAV transmission is by faecal-oral route. However, it can be transmitted through close person to person contact or contact with inanimate objects. The other transmissions are among men having sex with men (MSM), injecting drug use and via contaminated food and water which is less common. Although it is less common, foodborne transmission can spread the infection to a wider area. Foodborne transmission can lead into a global problem because many food products are exported around the world. The best way to prevent an outbreak is rapid detection of the infection source. The most common method for identifying detect the source is doing epidemiology and trace-back investigation. However, it is difficult to use this method for food borne transmission because HAV has a long incubation period. Therefore, by the time the symptoms occur, the patients might not remember what they have eaten. Furthermore, the contaminated food might have been thrown away, so it would be impossible to isolate the virus from the suspected food. One of the effective tools to rapidly confirm the investigation is doing sequencing characterization. It is done by sequence the HAV and compares it with the available sequences from the previous cases. The current research project genotyped HAV RNA by using protocols and primer sets designed by HAVNET. It collected and genotyped previous HAV-positive samples in VIDRL sample bank, and the results were used to establish epidemiological and molecular database of the HAV strains. The database was used to investigate the relationship between the current diagnostic samples and the previous HAV infection cases. It was expected to identify the source of the hepatitis A infection. The project was divided into part A and part B; part A retrospectively genotyped the HAV RNA positive samples collected from VIDRL sample bank, and part B prospectively genotyped current diagnostic samples and compared the result against the database. One hundred and forty four samples were collected during part A with 130 samples were nested RT-PCR positive. These positive samples were sequenced, and the results showed 71 samples genotype IA, 37 IB and 22 IIIA. They were grouped into 24 clusters by the Geneious R7. The two largest clusters were the 2009 semi-dried tomato outbreak and the 2015 frozen berries outbreak, which consisted of 19 and 17 sequences respectively. Meanwhile, the other 22 clusters only have two to three sequences and consisted of cases which were geographically related, among family members and close contact and patients with travel history to HAV endemic countries. In part B, there were 195 samples with 188 were positive in the nested RT-PCR assay. The comparison between the sequences and the database identified five samples received in 2017 showed 100% identity to the index case of the 2015 frozen berries outbreak. These findings led to a health alert issued by the DHHS along with a product recall of the frozen berries. It was suspected that the frozen berries came from the same plant and area and in the same time frame as the berries associated with the 2015 frozen berries outbreak. Another notable finding was the identification of HAV outbreaks related to HAV outbreaks associated to MSM group in Europe. A total of 66 samples were identified among Australian which consisted of three different clusters. Genetic analysis found that cases in Cluster 1 were related but not identical with the European case. Most cases had two nucleotide differences with the index case from Europe. Meanwhile, cases in Cluster 2 and 3 were identical with the Europe index case of each cluster. These findings also led to a health alert issued by the DHHS. Besides those two major clusters, there were several smaller clusters identified during part B. These clusters consisted of families and their close contact, and among patients with travel history to HAV endemic countries. The project showed that molecular investigation has a critical role in investigating the source of HAV infection. It should be used to support the epidemiological approaches, especially during HAV outbreak investigation