A Case of Hepatitis A Virus Detection in River Water Flowing into Tidal Flats

In 2019, an outbreak of hepatitis A was reported in South Korea due to the ingestion of salted shellfish contaminated with hepatitis A virus. In a survey of the contamination of salted shellfish, hepatitis A virus (HAV) type IA was detected, and it was confirmed that it originated from clams produced in one of the clam farms located in the western parts of the country. The aim of this study was to determine the cycle of human infection by HAV contaminating


Introduction
Hepatitis A is an infectious liver disease caused by hepatitis A virus. In 2019, an outbreak of hepatitis A was reported in the Republic of Korea due to the ingestion of salted shellfish. Notably, the number of infected individuals dramatically decreased when all individuals were advised to stop ingesting salted shellfish. Hepatitis A is known to spread from one human to another via water and food contaminated with the virus. However, the long incubation period of this virus, which is an average of 4 weeks, makes it difficult to determine the exact route of infection [1]. Different types of food that are directly consumed without heating, such as vegetables (e.g. lettuce, frozen strawberries) and shellfish (e.g. mussels, clams, and raw oysters), have been identified as sources of infection by hepatitis A [2,3].
In particular, bivalve shellfish (e.g. clams), which ingest organic matter from the surrounding water, tend to harbor several pathogens in their digestive tracts and are known to be a major infectious agent causing several foodborne diseases, such as norovirus infection [4,5]

Materials and Methods
Water samples were first collected from a river close to a clam farm for analysis. A contamination survey was conducted on this farm, which is known to be one of the largest producers of clams in this area.

Results
In

Discussion
In this study, the genotype of the HAV detected in the river water flowing into the clam farm under investigation was found to be type IA. This is the most common genotype found in human infection cases worldwide and is the same as that found to be were type IB [10. 11]. Therefore, most of the genotypes detected in the environment are estimated to be type IB. In addition, although no genotyping has been performed, the genotype of the HAV detected in the rivers of Ansan, as well as in various strawberry farms, was also estimated to be type IB [12,13].
However, the presence of type IA in the water is commonly associated with human infection, proving that the HAV detected in the river water flowing into the clam farm under investigation is mostly due to human infection [14,15]. Although the HAV detected in the water and the virus that caused human infection were not analyzed and compared, they are estimated to be significantly related.
Boryeong is a shellfish-rich area, especially with respect to clams, because of its tidal flats. The clams produced in this area are consumed not only as salted seafood and raw material, but also as ready-made food in shops and restaurants. The clam harvest area is located at a site where a river flows through Boryeong, with several streams of unpurified sewage treatment water directly flowing close to the river. In other words, most of this unpurified river water flows directly into the clam farm. Clams, the raw material of salted shellfish, were collected from July to August 2019, the time at which the incidence of hepatitis A is highest in Chungnam and Boryeong (Table 1). However, we performed our tests in November, after the outbreak had sharply declined.
Notably, it is estimated that the level of contamination in the clam farm had increased from July to August when the clams were harvested.
Normally, because the concentration of HAV is generally low, a detection test is performed after a concentration process to detect the virus in water.
This method, which has been recommended by the United States Environmental Protection Agency, is used worldwide and is recommended to detect viruses by concentrating 1,500 L of purified water (e.g. groundwater) and 300 L or more of highly polluted water (e.g. surface water) [16]. However, in practice, there is a limit with regard to concentrating a large amount of water. Therefore, various volumes of water, ranging from 10 to 300 L, were used to concentrate the virus in the water [17][18][19]. In addition, several reports have shown that the virus recovery rate (45~64%) does not largely differ depending on the properties of water, the amount of water to be concentrated, and the method of con- centration [20]. In this study, the virus was concentrated using a Concentrating Pipette Select water concentrator (InnovaPrep) after collecting 1 L of water twice from one sampling point. The virus recovery rate of this concentration equipment is allegedly more than 50%; however, our actual experiment showed a low recovery rate of only 4.5%.
In addition, in this experiment, the amount of virus recovered was expected to be low as suspended matter was removed twice. Hence, although the amount of virus present in the water was small, the possibility of detecting the viral gene was extremely low because the virus has a low recovery rate during the pretreatment process. Furthermore, in the gene amplification process, no HAV was detected in the 40-reaction scenario, but rather it was confirmed in the 45-reaction scenario. Genetic amplification is generally recommended using the 40-reaction scenario because of the RT-PCR threshold in clinical samples [7], whereas the 45-reaction scenario is sometimes recommended to increase the sensitivity of gene detection in environmental samples [11].
Therefore, even though this viral gene was detected in only one of 15 samples, it is very significant that HAV was detected in the river water.

Conclusion
In conclusion, it is difficult to conclude whether the