Abstract
Hepatitis E virus (HEV) infection is endemic in developing and developed countries. HEV was reported to be excreted in the milk of ruminants, raising the possibility of transmission of HEV infection through the ingestion of contaminated milk. Therefore, the detection of HEV markers in milk samples becomes pivotal. However, milk includes inhibitory components that affect HEV detection assays. Previously it was reported that dilution of milk matrix improves the performance of HEV molecular assay, however, the dilution of milk samples is not the best strategy especially when the contaminated milk sample has a low HEV load. Therefore, the objective of this study is to compare the effect of extraction procedures on the efficiency of HEV RNA detection in undiluted milk samples. In addition, we assessed the effect of the removal of milk components such as fats and casein on the performance of the molecular and serological assays of HEV. Phosphate buffered saline (PBS) and different milk matrices (such as whole milk, skim milk, and milk serum) were inoculated with different HEV inoculums and subjected to two different extraction procedures. Method A includes manual extraction using spin column-based extraction, while method B includes silica-based automated extraction. Method A was more sensitive than method B in the whole milk and skim milk matrices with a LoD95% of 300 IU/mL, and virus recovery yield of 47%. While the sensitivity and performance of method B were significantly improved using the milk serum matrix, with LoD95% of 96 IU/mL. Interestingly, retesting HEV positive milk samples using the high sensitivity assay based on method B extraction and milk serum matrix increased the HEV RNA detection rate to 2-fold. Additionally, the performance of HEV serological assays such as anti-HEV IgG and HEV Ag in the milk samples was improved after the removal of the fat globules from the milk matrix. In conclusion, HEV RNA assay is affected by the components of milk and the extraction procedure. Removal of inhibitory substances, such as fat and casein from the milk sample increased the performance of HEV molecular and serological assays which will be suitable for the low load HEV milk with no further dilutions.
Highlights
Hepatitis E virus (HEV) is the most causative agent for acute viral hepatitis globally
HEV-7 and HEV-8 were identified in camels [15,16], HEV-7 is zoonotic, chronic HEV infection was documented in a liver transplant patient through the frequent ingestion of the camel meat and milk, raising the possibility of HEV transmission via the contaminated milk [17]
HEV Molecular Assay is affected by the Milk Matrix 3.1
Summary
Hepatitis E virus (HEV) is the most causative agent for acute viral hepatitis globally. It infects about 20 million people resulting in 70000 deaths annually [1]. HEV-3 and HEV-4 infect humans and animals such as pigs, wild boars, deer, rabbits, cows, and goats [6,7]. They are zoonotic, infection is mainly transmitted to humans either by contact with these animals or the ingestion of undercooked meat, sausages, liver, or other products from them [8,9,10]. Other HEV isolates such as HEV-5, HEV-6, and HEV-8 have not been confirmed as human pathogens [3,18]
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