Enhancement of the Molecular and Serological Assessment of Hepatitis E Virus in Milk Samples

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.

Clinical Outcomes and Prevalence of Hepatitis E Virus (HEV) Among Non-A-C Hepatitis Patients in Egypt

“…35,50 Recently we reported HEV-3 in the cow and goat milk in Assiut rural communities. 28,51,52 Interestingly, this patient (HEV-3 infected) was living in the same village where HEV-3 identified in the cow milk and the diagnosis was done within the same period. The patient reported that he often consumed unpasteurized cow milk and its products.…”

Hepatitis E virus (HEV) open reading frame 2: Role in pathogenesis and diagnosis in HEV infections

“…The HEV Ag assay might be helpful in screening new compounds due to its low cost and rapid performance compared with the molecular approaches. Interestingly, HEV ORF2 Ag was detected in cow and goat milk samples, indicating that this assay can be used for human safety studies as a food contamination marker 20,21,115 …”

“…Interestingly, HEV ORF2 Ag was detected in cow and goat milk samples, indicating that this assay can be used for human safety studies as a food contamination marker. 20,21,115…”

Milk-Borne Diseases

“…Hepatitis E virus (HEV) is the leading global cause of acute viral hepatitis. It infects approximately 20 million people annually, resulting in 70,000 deaths [10,11]. However, milk contains several substances, such as fats, casein, whey proteins, and lactose, that interfere with the extraction of viral nucleic acid and the recovery of enteric viruses [12].…”