Sayed Ibrahim M, Hammam Ahmed R A, Elfaruk Mohamed Salem, Alsaleem Khalid A, Gaber Marwa A, Ezzat Amgad A, Salama Eman H, Elkhawaga Amal A, El-Mokhtar Mohamed A
Department of Medical Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut 71515, Egypt.
Department of Pathology, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Microorganisms. 2020 Aug 12;8(8):1231. doi: 10.3390/microorganisms8081231.
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 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 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.
戊型肝炎病毒（HEV）感染在发展中国家和发达国家均为地方性流行。据报道，反刍动物乳汁中会排出HEV，这增加了通过摄入受污染乳汁传播HEV感染的可能性。因此，检测乳汁样本中的HEV标志物至关重要。然而，乳汁中含有影响HEV检测分析的抑制成分。此前有报道称，稀释乳汁基质可提高HEV分子检测的性能，然而，稀释乳汁样本并非最佳策略，尤其是当受污染的乳汁样本中HEV载量较低时。因此，本研究的目的是比较提取程序对未稀释乳汁样本中HEV RNA检测效率的影响。此外，我们评估了去除乳汁成分（如脂肪和酪蛋白）对HEV分子和血清学检测性能的影响。用不同的HEV接种物接种磷酸盐缓冲盐水（PBS）和不同的乳汁基质（如全脂牛奶、脱脂牛奶和乳清），并采用两种不同的提取程序。方法A包括使用基于离心柱的提取方法进行手动提取，而方法B包括基于二氧化硅的自动提取。在全脂牛奶和脱脂牛奶基质中，方法A比方法B更灵敏，检测限为300 IU/mL，病毒回收率为47%。而使用乳清基质时，方法B的灵敏度和性能显著提高，检测限为96 IU/mL。有趣的是，使用基于方法B提取和乳清基质的高灵敏度检测方法对HEV阳性乳汁样本进行重新检测，可使HEV RNA检测率提高2倍。此外，从乳汁基质中去除脂肪球后，乳汁样本中抗HEV IgG和HEV Ag等HEV血清学检测的性能得到改善。总之，HEV RNA检测受乳汁成分和提取程序的影响。从乳汁样本中去除脂肪和酪蛋白等抑制物质可提高HEV分子和血清学检测的性能，适用于无需进一步稀释的低载量HEV乳汁。
