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Optimising extraction and RT-qPCR-based detection of hepatitis E virus (HEV) from pork meat and products (HEVDETECT)

Optimising extraction and RT-qPCR-based detection of hepatitis E virus (HEV) from pork meat and products: Appendices

Appendices for the report.

Diweddarwyd ddiwethaf: 31 July 2023
Gweld yr holl ddiweddariadau
Diweddarwyd ddiwethaf: 31 July 2023
Gweld yr holl ddiweddariadau

Appendix A: Full report of market share and consumption data of pork products in the UK.

The aim of this review was to help identify specific matrices which we will use for the next phase of the HEVDETECT project, so that we can optimize a detection method suitable for as wide a range of product types as possible, whilst this reflects the most consumed pork products in the UK. A case control study of 25 cases conducted in England identified pork pies (OR 6.33), and ham and sausages purchased from one particular supermarket chain (OR 10.12) as significantly associated with indigenous infection. (Said et al, 2014).  A subsequent study in England supported this association of ham and sausages from a particular supermarket chain, with the G3-2 phylotype detected in cases not detected in indigenous pigs suggesting infection from pork originating outside the UK (Said et al, 2017).  Recently Health Protection Scotland carried out an enhanced surveillance study for Scottish individuals confirmed positive for HEV.   Among the clinical cases, the consumption of pork (i.e., pork chops, roast pork) at least once a week was reported by 17% of cases, 46% consumed sliced prepacked ham at least once a week, 40% consumed bacon at least once a week and 21% consumed pork sausages at least once a week.   
Overall, 80% reported the consumption of at least one pork product in the nine weeks prior to onset.  Further data will be added to the compilation to determine target products.  

Review of available data  

Three sets of data have been acquired. The first set is from the NDNS (National Diet and Nutrition Survey) covering a rolling program for 8 years from 2008 – 2016 (Bates et al 2014; Bates et al. 2016 and Roberts et al. 2018). This dataset includes consumption data split into 2 age groups of 19-40 years and 40 years plus and are also split by gender. Table 1 details the chronic consumption of pork carcass meat, whilst Table 2 details the acute consumption of pork carcass meat. Table 3 details the chronic consumption of processed pork and Table 4 details the acute consumption of processed pork. Statistical significance was calculated using a z score calculator for 2 populations to assess the difference in male and female consumption in each age grouping for each consumption category (https://www.socscistatistics.com/).  Table 5 shows the number of respondents in the population groups.  

Table 1: Chronic Consumption of pork carcase meat  

Gender Age group Number of consumers (g/person/day) Mean (g/person/day) 97.5th percentile (g/person/day) Maximum (g/kg bw/day) Mean (g/kg bw/day) 97.5th percentile (g/kg bw/day) Maximum
Female 19 to 40 years 132 31 80 127 0.46 1.3 1.9
3Female 40+ years 379 27 68 121 0.39 1.0 1.8
Male 19 to 40 years 105 38 87 106 0.46 1.1 1.6
Male 40+ years 317 37 92 150 0.43 1.1 1.8

Table 2: Acute Consumption of pork carcase meat

Gender Age group Number of consumers (g/person/day) Mean (g/person/day) 97.5th percentile (g/person/day) Maximum (g/kg bw/day) Mean (g/kg bw/day) 97.5th percentile (g/kg bw/day) Maximum
Female 19 to 40 years 132 31 80 127 0.46 1.3 1.9
3Female 40+ years 379 27 68 121 0.39 1.0 1.8
Male 19 to 40 years 105 38 87 106 0.46 1.1 1.6
Male 40+ years 317 37 92 150 0.43 1.1 1.8

Table 3: Chronic Consumption of processed pork

Gender Age group Number of consumers (g/person/day) Mean (g/person/day) 97.5th percentile (g/person/day) Maximum (g/kg bw/day) Mean (g/kg bw/day) 97.5th percentile (g/kg bw/day) Maximum
Female 19 to 40 years 384 22 64 94 0.32 0.9 1.8
3Female 40+ years 781 26 73 105 0.36 1.1 1.8
Male 19 to 40 years 311 36 100 187 0.45 1.4 2.4
Male 40+ years 718 34 97 148 0.40 1.2 2.1

Table 4: Acute Consumption of processed pork  

 

Gender Age group Number of consumers (g/person/day) Mean (g/person/day) 97.5th percentile (g/person/day) Maximum (g/kg bw/day) Mean (g/kg bw/day) 97.5th percentile (g/kg bw/day) Maximum
Female 19 to 40 years 384 71 170 310 1.0 2.5 3.9
3Female 40+ years 781 80 188 374 1.1 2.8 5.7
Male 19 to 40 years 311 103 260 402 1.3 3.5 5.5
Male 40+ years 718 102 254 432 1.2 3.2 5.7

Table 5. Number of respondents in the population group  

Gender Age group Number of respondents in Population group
Female 19 - 40 years 927
Female 40+ years 1866
Male 19 - 40 years 601
Male 40+ years 1394

Key:   

The number of people in the age group 19-40 years who took part in the NDNS survey = 927 female and 601 male.  
The number of people in the age group 40 plus years who took part in the NDNS survey = 1866 female and 1394 male.  
The number of consumers relates to the number of people in the population group who ate the food e.g. 397 people consumed pork carcass meat out of 1866.  
“Chronic consumption” relates to food consumed over a long period of time continuously or intermittently i.e. daily average  
“Acute consumption” relates to food consumed over a short time i.e. maximum day.  
g/kg bw/day = g per kg bodyweight per day consumed  
97.5th percentile = 97.5 percentage of the sample  
The pork carcass meat consists generally of the following: 

  • Pork belly rashers  
  • Pork loin chops  
  • Pork leg joints  
  • Pork fat  
  • Pork belly  
  • Pork blood  
  • Pork shoulder  
  • Pork crackling  
  • Pork spare rib  
  • Pork hand or spring joint  
  • Pork loin joint  
  • Pork chump chops  
  • Pork leg steaks  
  • Diced / minced pork  
  • Pork tongue  
  • Kidneys  
  • Livers  

The processed pork consists generally of the following:  

  • Chorizo  
  • Canned ham / pork  
  • Pork luncheon meat  
  • Black pudding  
  • Frankfurter
  • Pork sausages  
  • Pork and beef sausages  
  • Saveloy  
  • Economy sausages  
  • Skinless sausages  
  • Premium sausages  
  • Pepperami  
  • Smoked pork sausages  
  • Sausage cakes  
  • Pork burgers  
  • Smoked / unsmoked / streaky bacon rashers  
  • Gammon / bacon joint  
  • Bacon fat  

As a very general conclusion from the data in Tables 1-4 above, the daily average (Chronic consumption) and the maximum per day (Acute consumption) shows that males in both age categories consume more pork carcass meat (chronic consumption p=0.03; acute consumption p<0.0001) and processed pork meat in the 19-40 age group (chronic consumption p=0.0056; acute consumption p<0.0001) than females. For processed meat, in the 40+ age group, chronic consumption appears to be equivalent between males and females (p>0.5; NS) but still significant in the acute consumption of processed meat (p=0.018).    

It can be assumed that the majority of the above cuts of meat, both carcass and processed is intended to be cooked before consumption, however there are certain products which may not have had any heat treatment, e.g. Chorizo, pepperami, etc., and are intended to be eaten without any cooking step. It is not possible to derive from the data how much of each of the listed products is consumed.  

The second dataset is derived from “Family Food datasets: countries and regions (CR) (updated with revised 2016/17 data and new 2017/18 data): Household purchases”, DEFRA, 2020, and shows the Purchased quantities of household food & drink by Government Office Region and Country. Table 6 shows the averages of pork products purchased per person per week (grams) and per category and is the latest figures for 2017/2018 with the data in Figure 1. in funnel chart format.  

Table 6. Averages purchased per person per week (grams)

Product England Wales Scotland Northern Ireland
Pigs liver 1 0 0 1
Pork chops 6 9 10 11
All other pork 11 12 6 9
Pork fillets and steaks 14 14 12 9
Pork joints 17 18 5 7
Bacon and ham joints uncooked 16 29 7 39
Bacon and ham rashers uncooked 40 48 31 59
Bacon and ham cooked 39 61 46 62
Pork sausage uncooked 56 69 54 72
Totals 200 251 171 271

Table 7 – Regional data for England – average consumption of pork products (g) 2017/2018

Product North East North West Yorkshire & Humber East Midlands West Midlands East London South East South West Totals
Pork joints 18 19 26 26 7 19 14 12 17 158
Pork chops 7 6 5 7 9 10 4 7 4 59
Pork fillets and steaks 24 9 17 12 13 17 10 16 13 131
All other pork 5 4 12 9 8 11 18 12 9 88
Pigs liver 2 1 1 0 2 1 0 0 0 7
Bacon and ham joints uncooked 17 21 16 10 18 18 12 18 16 146
Bacon and ham rashers uncooked 50 48 39 45 54 48 19 37 38 378
Bacon and ham cooked 56 34 40 40 39 36 26 35 57 363
Pork sausages uncooked 56 57 44 78 60 63 42 51 65 516
Totals 235 199 200 227 210 223 145 188 219 1846
HEV Regional Data (16--17) 77 197 185 160 255 228 258 300 353 2013

Figure 1. Funnel chart detailing the weight (g) of pork products by category purchased in the UK during 2017/2018.  

Funnel chart detailing the weight (g) of pork products by category purchased in the UK during 2017/2018.  

This data shows that uncooked pork sausages; cooked bacon and ham and uncooked bacon and ham rashers account for the majority of weekly purchases across all countries in the UK, followed by uncooked ham and bacon joints. It is not entirely clear from this dataset which of the above relates to “processed” pork as detailed in the first dataset, however the “all other pork” category may well include processed products such as chorizo and pepperami. It is not apparent from the data if uncooked / dried / fermented pork products including salamis are included in the “uncooked” categories in the top 4 listed above. Data for pies or pâtés were unavailable in this dataset. Table 7 illustrates the breakdown of consumption per region in England and also provides the number of cases which have been laboratory confirmed in that region for the period 16-17 (figure 3; Oeser et al 2019).  The highest incidence of cases is in the South East and South West, which appears to coincide with the largest consumption of pork sausages. Further detailed statistical regression analysis is required to identify if any particular product is related in this data set. It has previously been shown that the increased autochthonous incidence was first recognised in the S. West (Dalton et.al 2006).

Figure 3: Estimated log relative risk for all hepatitis E virus (HEV) genotype 3 (G3) infections (A), HEV G3 group 1 infections (B), and HEV G3 group 2 infections (C). Tolerance contours are superimposed as solid lines at the 95% confidence level. Solid lines indicate areas of significantly higher risk. Dashed lines indicate areas of significantly lower risk.  Data reproduced from J Infect Dis, Volume 220, Issue 5, 1 September 2019, Pages 802–810, https://doi.org/10.1093/infdis/jiz207   
 Table 8 shows the 3-year average of pork products purchased per person per week (grams) and per category and Figure 2 is in Funnel chart format.   

Bat chart of estimated log relative risk for all hepatitis E virus (HEV) genotype 3 (G3) infections (A), HEV G3 group 1 infections (B), and HEV G3 group 2 infections

 Table 8. 3-year average of pork products purchased per person per week (grams) in the UK  

Product England Wales Scotland Northern Ireland
Pigs liver 1 0 0 1
Pork chops 6 9 10 13
All other pork 11 12 6 9
Pork fillets and steaks 14 14 12 9
Pork joints 17 18 5 7
Bacon and ham joints uncooked 16 20 7 39
Bacon and ham rashers uncooked 40 48 31 59
Bacon and ham cooked 39 61 46 62
Pork sausages uncooked 56 69 54 72
Totals 200 251 171

271

Funnel 2. Funnel chart detailing the 3-year average of weight (g) of pork products by category purchased in the UK  

Funnel chart detailing the 3-year average of weight (g) of pork products by category purchased in the UK  

Both the one, and three year averages show the same pattern of consumption, with uncooked pork sausages being the largest by weight of product purchased in the UK, followed by cooked bacon and ham and uncooked bacon and ham rashers being the third.  
  
The third dataset has been obtained from Kantar UK – a data, insights and consulting company and details the purchase of pork products by UK retailers during the year 2015. The graphs below show the retailers purchase of chilled processed pork; cooked chilled pork products and fresh cuts as a yearly total in ‘000s of units sold. Again, this is not strictly consumption data, but for the purposes of this review we are assuming purchases relate to amounts consumed to give a general conclusion as to which product categories are consumed most across the UK.  

2015 Chilled processed pork yearly total ('000s of units) 2015 Cooked chilled meat yearly total ('000s of units) 2015 Fresh Cuts Yearly total ('000s of units)

This dataset does not break down the products by type in the same way as the first two datasets, and neither does it break down the data between locally produced and imported products but it does give an overall picture of the sheer volumes of pork products sold over the course of a year by different retailers. A summary of the data for the product categories across all regions of the UK and across all retailers, whether Prepacked or loose, is shown in Table 8, with the funnel chart below.  

Table 8. Number of units of each category purchased by UK retailers in 2015  

Product category Number if units purchased
Chilled processed 261,392,000  
Fresh cuts 231,383,000  
Cooked chilled meat 164,536,000  
Fresh joint 123,474,000  
Frozen processed 57,611,400  
Chilled ingredient 26,343,800  
Fresh cooking 12,984,000  
Frozen cuts 11,459,900  
Frozen ingredient 1,794,030  
Frozen joint 1,443,440  
Frozen cooking 17,441  
Funnel chart showing Product Category/Number of units purchased (all retailers/UK regions 2015)

Conclusions  

In reviewing the data from the 3 datasets available, there are no solid conclusions we can come to with regard to specific product types consumed across the UK e.g., premium vs. economy pork sausages, salami, chorizo, hams etc. The general conclusion from these data shows that pork sausages are one of the highest consumed products across the UK and this is certainly one matrix which will be included in the next phase of the project. Many of the pork products listed in the datasets are intended to be cooked, including of course pork sausages, however we know already that pork sausages have had HEV RNA detected in them (Berto. et al 2012) and that inadequate cooking of such products may present a risk of HEV infection. Perhaps of more concern are the “uncooked” categories which are intended to be eaten uncooked or RTE. These may include chorizo, salami, fermented sausage products etc. We will continue to analyse the data from Public Health England as it becomes available and may include products which are linked to high case rates in the development of the method.

References

Bates, B.; Lennox, A.; Prentice, A.; Bates, C.; Page, P.; Nicholson, S.; Swan, G. (2014) National Diet and Nutrition Survey Results from Years 1, 2, 3 and 4 (combined) of the Rolling Programme (2008/2009 – 2011/2012) (PDF)

2. Bates, B.; Cox, L.; Nicholson, S.; Page, P.; Prentice, A.; Steer, T.; Swan, G. (2016) National Diet and Nutrition Survey Results from Years 5 and 6 (combined) of the Rolling Programme (2012/2013 – 2013/2014) (PDF)

Berto A, Martelli F, Grierson S, Banks M. Hepatitis E virus in pork food chain, United Kingdom, 2009-2010. Emerg. Infect Dis 2012; 18:1358–60.  

Dalton, H. R.; Thurairajah, P. H.; Fellows, H. J.; Hussaini, H. S.; Mitchell, J.; Bendall, R.; Banks, M.; Ijaz, S.; Teo, C.-G.; Levine, D. F (2007). Autochthonous hepatitis E in southwest England. Journal of Viral Hepatitis. Vol. 14 Issue 5, p304-309   

Family Food datasets: countries and regions (CR) (updated with revised 2016/17 data and new 2017/18 data): Household purchases (PDF)

Kantar dataset kindly provided by FSS.  

Oeser,C; Vaughan, A; Said, B; Ijaz, S; Tedder, R; Haywood, B; Warburton, F; Charlett, A; Elson, R and Morgan, D. (2019). Epidemiology of Hepatitis E in England and Wales: A 10-Year Retrospective Surveillance Study, 2008–2017. The Journal of Infectious Diseases, pp 802-10.  

Roberts, C.; Steer, T.; Maplethorpe, N.; Cox, L.; Meadows, S.; Page, P.; Nicholson, S.; Swan, G. (2018) National Diet and Nutrition Survey Results from Years 7 and 8 (combined) of the Rolling Programme (2014/2015 – 2015/2016) (PDF)

Said, B., et al. (2014). Hepatitis E virus in England and Wales: indigenous infection is associated with the consumption of processed pork products. Epidemiol.Infect. 142:1467-1475  

Said, B., et al. (2017).  Pork products associated with human infection caused by an emerging phylotype of hepatitis E virus in England and Wales Epidemiol.Infect. 145:2417-2423 

Appendix B: HEV recovery from pork sausage (high and low grade) and smooth pâté using four extraction and three different detection methods

Protocol Source Elution buffer 1 step RT-qPCR Mean CT and SD 2 step RT-qPCR Mean CT and SD Ceeram Mean CT and SD 1 step RT-qPCR % recovery 2 step RT-qPCR % recovery Ceeram % recovery
GCU Low grade Pork sausage 1 RLT lysis 35.184/0.338 38.438/0.118 34.871/0.382 64.16 27.72 5.49
GCU Low grade Pork sausage 2 RLT lysis 35.154/0.224 39.402/0.000 35.243/0.485 65.05 12.67 4.23
GCU High grade Pork sausage 1 RLT lysis 34.333/0.510 37.995/0.772 34.461/1.264 124.5 43.54 8.85
GCU High grade Pork sausage 2 RLT lysis 32.715/0.049 35.691/0.005 31.968/0.041 407.5 256.33 44.74
GCU Smooth Brussels Pâté 1 RLT lysis 0/0 36.867/0.484 33.738/0.651 0.000 102.63 13
GCU Smooth Brussels Pâté 2 RLT lysis 0/0 35.622/0.025 32.777/0.039 0.000 271.1 24.79
Di Barolo et al (2015) Low grade Pork sausage 1 RLT lysis 38.547/0 34.423/0.096 34.948/0.118 9.82 122.39 8.94
Di Barolo et al (2015) Low grade Pork sausage 2 RLT lysis 33.452/0.067 35.521/0.199 31.945/0.012 355.75 59.62 64.59
Di Barolo et al (2015) High grade Pork sausage 1 RLT lysis 36.713/0.220 32.260/0.005 31.135/0.042 35.97 508.27 110.37
Di Barolo et al (2015) High grade Pork sausage 2 RLT lysis 37.411/0.199 34.933/0.000 33.669/0.246 21.97 87.41 20.82
Di Barolo et al (2015) Smooth Brussels Pâté 1 RLT lysis 0.000/0.000 32.849/0.097 32.709/0.174 0.000 345.11 39.13
Di Barolo et al (2015) Smooth Brussels Pâté 2 RLT lysis 0.000/0.000 34.709/0.044 32.033/0.074 0.000 101.3 60.98
Martin Latil et al (2015) Low grade Pork sausage 1 Distilled water 0/0 37.440/0.181 33.293/0.031 0 28.58 144.25
Martin Latil et al (2015) Low grade Pork sausage 2 Distilled water 0/0 38.638/0.706 33.739/0.277 0 21.97 73.98
Martin Latil et al (2015) High grade Pork sausage 1 Distilled water 0/0 37.465/0.222 33.622/0.069 0 23.42 142.39
Martin Latil et al (2015) High grade Pork sausage 2 Distilled water 0/0 38.137/0.075 33.410/0.230 0 26.76 95.34
Martin Latil et al (2015) Smooth Brussels Pâté 1 Distilled water 0/0 38.263/0.492 33.976/0.354 0 19.11 90.33
Martin Latil et al (2015) Smooth Brussels Pâté 2 Distilled water 0/0 38.142/0.117 33.680/0.059 0 22.6 95.14
CBRI Low grade Pork sausage 1 Trizol 36.867/0.415 34.791/0.501 33.764/0.582 7.34 50.33 19.78
CBRI Low grade Pork sausage 2 Trizol 37.072/0.257 34.563/0.400 33.537/0.246 6.17 59.32 22.27
CBRI High grade Pork sausage 1 Trizol 35.745/1.598 36.738/0.751 33.769/0.179 23.82 15.29 19.1
CBRI High grade Pork sausage 2 Trizol 36.931/0.043 35.107/0.269 34.069/0.381 6.8 38.32 15.9
CBRI Smooth Brussels Pâté 1 Trizol 38.939/0.000 0.000/0.000 36.874/0.590 1.45 0 2.62
CBRI Smooth Brussels Pâté 2 Trizol 38.098/0.875 36.345/0.621 35.778/0.296 3.09 15.29 5.2