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

Ibrahim M Sayed; Ahmed R A Hammam; Mohamed Salem Elfaruk; Khalid A Alsaleem; Marwa A Gaber; Amgad A Ezzat; Eman H Salama; Amal A Elkhawaga; Mohamed A El-Mokhtar

doi:10.3390/microorganisms8081231

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

Microorganisms. 2020 Aug 12;8(8):1231. doi: 10.3390/microorganisms8081231.

Authors

Ibrahim M Sayed^{1

2}, Ahmed R A Hammam^{3

4}, Mohamed Salem Elfaruk^{3

5}, Khalid A Alsaleem^{3

6}, Marwa A Gaber⁷, Amgad A Ezzat⁸, Eman H Salama⁹, Amal A Elkhawaga¹, Mohamed A El-Mokhtar¹

Affiliations

¹ Department of Medical Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut 71515, Egypt.
² Department of Pathology, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
³ Dairy and Food Science Department, South Dakota State University, Brookings, SD 57007, USA.
⁴ Dairy Science Department, Faculty of Agriculture, Assiut University, Assiut 71526, Egypt.
⁵ Medical Technology College, Nalut University, Nalut 00218, Libya.
⁶ Department of Food Science and Human Nutrition, College of Agriculture and Veterinary Medicine, Qassim University, Buraydah 51452, Saudi Arabia.
⁷ Department of Medical Biochemistry, Faculty of Medicine, Assiut University, Assiut 71515, Egypt.
⁸ Department of Microbiology and Immunology, Faculty of Medicine, Al-Azhar University, Assiut 71524, Egypt.
⁹ Department of Clinical Pathology, Faculty of Medicine, Sohag University, Sohag 82524, Egypt.

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 LoD_95% 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 LoD_95% 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.

Keywords: HEV; RNA extraction; limit of detection.; milk; milk serum; molecular assay; serology; skim milk; virus recovery.

Grants and funding

2018-07-17-001/Faculty of Medicine, Assiut University