Reconstructing the history of helminth prevalence in the UK.

Intestinal helminth parasites (worms) have afflicted humans throughout history and their eggs are readily detected in archaeological deposits including at locations where intestinal parasites are no longer considered endemic (e.g. the UK). Parasites provide valuable archaeological insights into historical health, sanitation, hygiene, dietary and culinary practices, as well as other factors. Differences in the prevalence of helminths over time may help us understand factors that affected the rate of infection of these parasites in past populations. While communal deposits often contain relatively high numbers of parasite eggs, these cannot be used to calculate prevalence rates, which are a key epidemiological measure of infection. The prevalence of intestinal helminths was investigated through time in England, based on analysis of 464 human burials from 17 sites, dating from the Prehistoric to Industrial periods. Eggs from two faecal-oral transmitted nematodes (Ascaris sp. and Trichuris sp.) and the food-derived cestodes (Taenia spp. and Diphyllobothrium latum syn Dibothriocephalus latus) were identified, although only Ascaris was detected at a high frequency. The changing prevalence of nematode infections can be attributed to changes in effective sanitation or other factors that affect these faecal-oral transmitted parasites and the presence of cestode infections reflect dietary and culinary preferences. These results indicate that the impact of helminth infections on past populations varied over time, and that some locations witnessed a dramatic reduction in parasite prevalence during the industrial era (18th-19th century), whereas other locations continued to experience high prevalence levels. The factors underlying these reductions and the variation in prevalence provide a key historical context for modern anthelmintic programs.

Introduction

The eggs of intestinal helminths have been detected in a variety of archaeological contexts including those in Europe and North America (reviewed in [1-6]) indicating that these parasites had a much wider historical distribution than the present day [7-9]. Helminth infections remain a concern in numerous countries, particularly those that are considered less economically developed (LEDC) in tropical and subtropical regions. Indeed, according to the World Health Organization (WHO) helminth infections rate amongst the top neglected tropical diseases [9].

Intestinal helminths are often identified in archaeological excavations because the eggs are environmentally resilient and remain identifiable by morphology for thousands of years (e.g. [10-18]). Helminth eggs can be identified to the genus level by light microscopy [19] and their presence indicates infection of a host with adult parasites. Since many parasites have complex life histories, the detection of eggs can be used to comment upon the human practices that contribute to the propagation of helminth infections. For example, detection of faecal-oral transmitted helminths (e.g. the nematodes Ascaris and Trichuris) indicates a failure of hygiene or sanitation practices to limit parasite transmission. In contrast, detection of eggs from some cestodes (tapeworms) in human derived deposits indicate the acquisition of infections from specific food sources such as freshwater fish (Diphyllobothrium latum, syn. Dibothriocephalus latus) or red meat (Taenia spp.), and in both cases indicate a lack of or insufficient cooking processes that would otherwise prevent parasitic infection.

Helminth eggs have been detected archaeologically in different types of faecal-associated material including latrines and other communal deposits (e.g. waste ditches), and samples associated with single individuals (e.g. the abdominal region of mummified or skeletal remains [11,14,17,20-27]. The detection of parasites in each type of context provides different kinds of information. For example, communal deposits often contain larger concentrations of parasite eggs but do not provide information related to the number of infected individuals. Although the detection of parasite eggs associated with individual remains is technically more challenging since fewer eggs are present, prevalence rates can be determined and assessed relative to other osteologically-derived characteristics including age and sex. To be most informative, individual-based datasets need to be sufficiently large to develop reliable estimates of prevalence.

Most intestinal helminths are no longer endemic in Europe, including the UK, although the timing and circumstances that led to their demise are not well understood. Although it is likely this disappearance was linked to improvements in sanitation during the 19th and early 20th centuries, prior to the development of modern anthelmintic drugs, empirically-based estimates of parasite prevalence would help to support or dispel this theory. Having established that prevalence rates of helminth infections in Medieval Europe were similar to those found in modern endemic regions [26] it was then important to consider how helminth prevalence may have changed over time. This temporal approach may reveal factors that influenced the rates of helminth infection in past populations, in particular those that led to a level insufficient to maintain an endemic status.

To assess helminth prevalence through time in a geographically limited region (historic England), we examined soil samples from the abdominal region of 464 individual skeletal remains for evidence of helminth eggs. The term prevalence usually refers to the proportion of infected individuals at a specific time. Since the samples in this study were from the pelvic (sacral) region of skeletal remains prevalence is reported as the number or percentage of burials positive for helminth eggs. Four helminths: Ascaris, Trichuris, Taenia and D. latum were detected and their prevalence varied across the sample sets. The goal of this study was to establish a foundation for understanding the epidemiology of parasite infections in a historical context. The changes in helminth prevalence over time can be linked with changes in the levels of urbanization, living conditions, sanitation and social practices of people in the UK. A historical perspective considering the factors which may have affected helminth prevalence in the past (without access to modern anthelmintics) has potential to inform efforts to control these infections in modern endemic contexts.

Materials and methods

Sample material

Samples were obtained from 17 sites (Fig 1 and Table 1) which were grouped into five time periods. The time periods are: Prehistoric (Bronze Age and earlier), Roman Britain (1st c BCE - 5th c CE), Anglo-Saxon/Early Medieval (5th- early 11th c CE), High/Late Medieval (11th-16th c CE) and Industrial Era (18-19th c CE). The sites include Taplow (Prehistoric, 1 sample), Evesham Vale Crematorium (Prehistoric, 1 sample), Durrington Larkhill (Prehistoric, 6 samples), Datchet (Prehistoric, 4 samples), Bulford (Prehistoric, 3 samples), York All Saints in the Marsh (Roman, 1 sample; High/Late Medieval, 35 samples), Churchill South Strategic Support Mains (Roman, 1 sample), Bleadon Wentwood Drive (Roman, 9 samples), Bletchingley North Park Quarry (Roman, 3 samples), Canterbury Peugeot Garage (Roman, 80 samples), Worcester Cathedral (Anglo-Saxon/Early Medieval, 65 samples), Ipswich Stoke Quay (Anglo-Saxon/Early Medieval, 14 samples; High/Late Medieval, 64 samples), Christchurch Priory (High/Late Medieval, 8 samples), Southampton Chapel Riverside (High/Late Medieval, 52 samples), Oxford Radcliffe Infirmary (Industrial, 56 samples), London St James (Industrial, 30 samples), and Birmingham Park Street (Industrial, 30 samples). Two of the sites, Ipswich Stoke Quay and York All Saints in the Marsh, had skeletal remains spanning two different time periods and samples from different time periods were grouped separately resulting in 19 skeletal groups for analysis. Sediment samples were collected from the pelvic region (immediately ventral to the sacrum) of the skeleton during excavation, except York All Saints in the Marsh where samples were taken from sediment adhering to the pelvis/sacrum of excavated skeletons during the cleaning process. Some of the data from York, Ipswich and Worcester has been presented in the context of prevalence rates in Medieval Europe [26]. Although “control” (e.g. sediment from the skull) were not analysed for all graves this was performed for a subset of samples; in all cases these were negative for parasite eggs. The remaining unprocessed material is archived at the Department of Zoology, University of Oxford and/or by the site archaeologist organisations.

Fig 1

Temporal and spatial distribution of the samples.

The pie chart shows the number of samples, the proportion of the sample set and the time period for each of the 17 sites of the study. The groups of samples were collected from sites depicted on the map: Taplow (Maidenhead, A), Vale Crematorium (Evesham, B), Larkhill (Durrington, C), Datchet (Slough, D), Bulford (Salisbury, E), All Saints in the Marsh (York, Roman Fi and High-Late Medieval Fii), Southern Strategic Support Main (Churchill, G), Wentwood Drive (Bleadon, H), North Park Quarry (Bletchingley, I), Peugeot Garage (Canterbury, J), Worcester Cathedral (Worcester, K), Stoke Quay (Ipswich, Anglo-Saxon Li and High-Late Medieval, Lii), Christchurch Priory (Christchurch, M), Chapel Riverside (Southampton, N), Radcliffe Infirmary (Oxford, O), St James (London, P), and Park Street (Birmingham, Q). The map was modified from the NASA SEDAC centre resource: https://sedac.ciesin.columbia.edu/maps/gallery/search?contains=United%20Kingdom.

Table 1

Key information on the sampled sites and the numbers positive for helminth eggs.

							Number of helminth positive samples
Map ID1	Site location	Total2	♀n3	♂n3	Period	Dating4	Ascaris	Trichuris	Taenia	D. latum
ABCDE	TaplowEveshamDurringtonDatchetBulford	11643	10100	00100	PrehistoricPrehistoricPrehistoricPrehistoricPrehistoric	PrehistoricPrehistoricLN-BA5LN-BA5Bronze Age	00001	00000	00001	00000
FiGHIJ	YorkChurchillBleadonBletchingleyCanterbury	119380	000015	000028	RomanRomanRomanRomanRoman	c49-45 BCERomanRomanRoman2nd–4th c CE	116028	00000	01003	00001
KLi	WorcesterIpswich	6514	147	264	Anglo-SaxonEarly MedievalAnglo-SaxonEarly Medieval	680-10669th-10th c CE	65	11	02	01
FiiLiiMN	YorkIpswichChristchurchSouthampton	3564852	030019	028815	High/Late MedievalHigh/Late MedievalHigh/Late MedievalHigh/Late Medieval	11th-16th c CE11th-15th c CE11th-16th c CE13th-16th c CE	1315020	3400	0401	0400
OPQ	OxfordLondonBirmingham	563030	0010	0011	IndustrialIndustrialIndustrial	1770-1855 CE1788-mid 19thc1810-1873 CE	080	001	010	020

1Map Identifier used on Fig 1.

2Total numbers examined.

3 Number of individuals with osteological definition of sex.

4Provided by the site archaeologists.

5LN-BA: late Neolithic to Early Bronze Age.

Processing and microscopy

In our standard approach 5-7g of the pelvic/sacral soil sample was rehydrated in 20 ml ultrapure water with gentle agitation (Titer-Tek plate shaker, setting 3/10; Titertek-Berthold, Pforzheim, Germany) over-night to disaggregate. The samples were then filtered (disposable nylon sieves, 1030 μm and 500 μm; Plastok Associates Ltd, Birkenhead, UK) and centrifuged at 400g for 5min (Heraeus Multifuge X3R). The supernatant was discarded and the pellet re-suspended in 20ml MilliQ-water and a 1ml aliquot removed for microscopy. Two subsamples (total volume 50μl) were examined for parasite eggs (Nikon Eclipse E400 with Nikon 10x/0.25 Ph1 DL and 40x/0.65 Ph2 DL objectives and QImaging MP5.0 RTV camera). Any putative eggs were recorded and images were assessed against reference images from archaeological and/or clinical contexts. Although numbers were recorded (detection limit ~80 eggs/gram of original sample) we only report the samples as positive or negative as many taphonomic factors and the size of collected sample may influence enumeration.

Statistics

Wilson Score intervals (95% confidence level [28] and Fisher’s Exact Test were calculated using R within RStudio [29,30].

Results

Estimation of helminth prevalence rates in historic England

Accurate determination of the prevalence of infection is an important epidemiological characteristic. Here, we present primary data on a large dataset of single grave samples derived from different historic periods and locations within England (Fig 1 and Table 1). Overall, 134 of the 464 samples contained helminth eggs and of the 19 skeletal groups in this study, 12 showed evidence of helminth infections (Table 1). The nematodes Ascaris and Trichuris were detected in 12 groups, whilst the cestodes Taenia spp. and D. latum were detected in six groups (example photomicrographs shown in Fig 2). Six of the seven groups with no evidence of helminths had low numbers of skeletal samples (n≤8) with the exception of the Oxford Radcliffe Infirmary Industrial period site (n = 56). Groups with less than three samples were excluded from site-specific statistical analysis to avoid small sample size effects.

Fig 2

Prevalence rates of helminths through time.

Prevalence of Ascaris, Trichuris, Taenia and Diphyllobothrium latum for four different time periods (Roman [n = 94], Anglo-Saxon [n = 79], High/Late Medieval [n = 159], and Industrial era [n = 116]). The confidence intervals are calculated using the Wilson Score interval (95% confidence level). Example photomicrographs of parasite eggs from left to right: Ascaris, Trichuris, Taenia and Diphyllobothrium latum Scale bar: 20 μm.

The overall prevalence of soil-transmitted nematodes (in particular Ascaris) was greater (24.4%, range 0–38.3%) than the prevalence of cestodes (4.1%, range 0–12.5%). This pattern was also evident when samples were grouped according to four historic intervals between the Roman and Industrial periods (Fig 2, Table 1). We have reported the detection of parasites in the Prehistoric period (Table 1) but these sample sets represented very few individuals (n = 15 from five sites, two of which only contained one sample) therefore it was inappropriate to report them as site or period prevalence estimates. Of the nematodes Ascaris was more prevalent (22.8%, range 0–38.3%) than Trichuris (2.2%, range 0–7.1%) and of the cestodes, Taenia was more prevalent (2.6%, range 0–14.3%) than D. latum (1.7%, range 0–7.1%). Ascaris was detected in 9 sites, and although Trichuris is transmitted by a similar faecal-oral process it was only detected in 4 sites. Of these one site contained a single Trichuris positive sample and no Ascaris positive individuals (Birmingham, Industrial period, Table 1). D. latum was found in 3 sites (in Ipswich during both Anglo-Saxon and High/Late Medieval time periods) and all of these sites also contained Taenia eggs (Table 1).

Since Ascaris was the most frequent parasite in all time periods, it represented the most robust dataset to explore changes in prevalence over time (Fig 3 and Table 1). In this analysis, the prevalence rates were generated by grouping all individuals analysed in sites associated with particular time periods and not subdivided according to location. The prevalence rates for Ascaris were highest in the Roman (36/94, 38.3%) and High/Late Medieval periods (50/159, 31.5%). These rates were significantly greater than those detected in the Anglo-Saxon (11/79, 13.9%; Fisher Exact p-values 0.0003 and 0.004, respectively) and the Industrial periods (8/116, 6.9%; Fisher Exact p-values both <0.0001). Although not depicted in the figure due to the relatively low numbers of graves examined, the prevalence for Ascaris in the Prehistoric period (1/15, 6.7%), was lower than for the Roman period (Fisher Exact p-value 0.030; Fig 3 and Table 1).

Fig 3

Variation of Ascaris prevalence rates in different sites.

The prevalence of Ascaris in skeletal individuals from fifteen sites (group size n>1) across the five time periods is displayed with a 95% confidence interval (Wilson Confidence Interval). Sites with very few samples (<10) provide a less reliable estimate for the prevalence and are thus displayed in grey. The sites included are Durrington, DU, Datchet, DA, and Bulford, BU (Late Neolithic/Early Bronze Age); Bleadon, BD, Bletchingley, BY, and Canterbury, CB (Roman); Worcester, WO, and Ipswich (Anglo-Saxon/Early Medieval); Ipswich, IP, York, YO, Christchurch, CC, and Southampton, SH (High/Late Medieval); Oxford, OX, London, LO, and Birmingham, BH (Industrial). The dating range for the sites is visualized by the horizontal bar chart below, with a continuous time axis from the Anglo-Saxon (Early Medieval) to the Industrial periods. The dates for the earliest samples are not displayed as they are labelled Late Neolithic/Early Bronze Age (DU/DA) or Bronze Age (BU).

Considerable variation in prevalence rates was observed for infection with Ascaris between sites within each of the time periods (Fig 3). Sites with a high number of samples were considered more likely to be representative of prevalence rates in the living population. Groups with n<10 (depicted in grey in Fig 3) showed both the highest (Bleadon, 6/9, 66.7%) and lowest prevalence rates (Durrington 0/3, Datchet 0/4, Bletchingley 0/3 and Christchurch 0/8). However, of the sites with low numbers of individuals, only Christchurch was significantly different in prevalence rate to one of the other sites from the same High/Late Medieval period (Southampton, Fisher Exact p-value 0.043).

The only sites with n>10 where Ascaris was not detected were dated to the Industrial period (Oxford, n = 56 and Birmingham, n = 30), and there was a significant difference between these and the third site from the same period (London, 8/30, 26.7%, Fisher Exact p-values <0.005 for both sites). The prevalence rates at the two Anglo-Saxon sites were also significantly different (Fisher Exact p-value 0.021), and the site with the larger sample size (Worcester, n = 65) had the lower prevalence rate. When comparing sites with group sizes n>10, it was apparent that there was no significant difference between the Roman and the Medieval sites. These prevalence rates are aligned with the sites with the highest prevalence rates from the Anglo-Saxon and Industrial periods but the average prevalence rates in these periods were significantly lower than that of the Roman and Medieval periods.

Estimated osteological age was available for 265 skeletal individuals. Age estimation was based on standard osteological markers [31]. Infections with soil-transmitted helminths (STH) are often more prevalent in children of school age (e.g. [32-34]), To explore this potential effect whilst maintaining sufficient group sizes for statistical analysis the data was partitioned into two groups: according to reported skeletal age: adults (>13 years old) and juveniles (<13 years old). Overall, there was a greater number of adults (n = 215) than juveniles (n = 50) in the overall dataset. To avoid any bias introduced by low sample numbers for a particular location or previously noted changes in prevalence rates over time the samples were analysed within each time period (Fig 4A). A higher prevalence of Ascaris was detected in juveniles (compared with adults) from the High/Late Medieval period (n = 124, 12/22 infected juveniles vs. 25/102 infected adults, Fisher-Exact p = 0.009), but not for the Roman (n = 73, 2/8 infected juveniles vs. 26/65 infected adults, Fisher-Exact p = 0.70) or Anglo-Saxon (n = 68, 4/20 infected juveniles vs. 7/48 infected adults, Fisher-Exact p = 0.72) periods. The two cestodes Taenia and D. latum were detected at low prevalence rates however, cestode eggs were only detected in older individuals (>13 years old).

Fig 4

Age and Sex-associated effects on prevalence rates for Ascaris.

Samples with associated osteological age were grouped into juveniles (0–12 years old, n = 50) and adults (>13 years old, n = 207) and according to period (A). The prevalence in juveniles was significantly higher in the High/Late Medieval period (Fisher Exact two-tailed p-value 0.02). Individuals where osteological sex was identified (adult individuals only, n = 194 with nfemale = 85 and nmale = 109) were grouped by period (B). No significant differences were identified.

Osteological derived identification of sex was available for 218 individuals with a slight bias towards males (n = 121) compared to females (n = 97). None of the individuals with sex information from the Prehistoric (2 females, 1 male) or Industrial periods (10 females, 11 males) were infected by Ascaris. There were no significant differences in infection rates between male and female individuals within any time period (Roman: 7/15 females vs. 9/28 males, Fisher-Exact p = 0.51; Anglo-Saxon: 3/21 females vs. 3/30 males, Fisher-Exact p = 0.68; High/Late Medieval: 14/49 females vs. 10/51 males, Fisher-Exact p = 0.35) (Fig 4B).

The prevalence rates of Trichuris in the seven sites analysed here ranged from 0% to 8.6%. The highest prevalence rate detected in this study is lower than the rate recently reported from Medieval European sites [26], where the prevalence of Trichuris was 28.6%. The total number of individuals in this study from Early to Late Medieval sites in which Trichuris was identified (9/208) was significantly lower (Fisher-Exact p = 0.0022) than the numbers reported in non-English sites (Pohansko, Brno, Ellwangen and Rottenburg) that date to the same time period (41/406). This difference, however, was due to the inclusion of the Czech site of Pohansko, which contained a high proportion of juveniles which are more likely to contain eggs of Trichuris [26]. When the English and European [26] data sets were segregated into juveniles and adults there were no significant differences in the Medieval prevalence rates of Trichuris. An alternative way of comparing Trichuris between sites whilst correcting for local factors that might affect prevalence for all faecal-oral transmitted nematodes would be to consider each species as a fraction of total STH detected. The prevalence of Trichuris as a fraction of total STH prevalence was not different between any of the Medieval English sites and those in continental Europe (Pohansko, Brno, Ellwangen and Rottenburg; primary data from [26]). It is noteworthy that very few Trichuris positive individuals were detected in English sites outside of the Medieval era with no Trichuris eggs detected in the Roman period or earlier (109 individuals) and only a single individual in the Industrial period (116 individuals).

Discussion

In the past, many intestinal helminths were geographically more widely distributed and endemic in Europe and North America (e.g. [1-4,26,35-40]), but they are now largely restricted to tropical and sub-tropical regions [9]. Investigating the epidemiology of helminth parasites in historical contexts helps us understand the living conditions, diet, health and hygiene practices, as well as providing insights into the biology and control of infectious disease in the past (e.g.1-6.41–43). The reductions in helminth infections in Europe occurred prior to the development of modern anthelminthic drugs and identifying factors that influenced helminth epidemiology in historic populations may provide information relevant to modern control regimens.

Most archaeological studies of helminths report eggs within communal deposits, such as latrines, pit fills or waste deposits. The density of parasite eggs in communal deposits serves as an indicator of the overall amount of infection in a locality, although the number of contributing individuals cannot be determined. Analyses based on single individuals, such as mummified bodies, burial-derived sediments or coprolites, are less common. Unfortunately, most individual-based studies involve very few individuals and therefore reliable prevalence rates cannot be estimated. In the most extensive study of historic prevalence, the numbers of infected individuals in Medieval Europe was shown to be similar to modern day endemic regions [26], indicating that later reductions in local transmission were not facilitated by low initial prevalence rates. The current study builds upon these findings by determining helminth prevalence at sites within England from Prehistoric to Industrial periods. It is important to note that the prevalence rates based upon detection of eggs in pelvic/sacral soil may represent an underestimate due to taphonomic processes or sample processing (reviewed in [39-44]). The current study employed a simple water-based rehydration/filtration/microscopy approach and identified the eggs of four helminths: Ascaris, Trichuris, Taenia and Diphyllobothrium latum. All of these parasites have been reported in archaeological sites within the UK, but previous data sets were unsuitable for calculating prevalence either by being based upon communal deposits or low numbers of individuals. While burial-derived skeletal remains may not represent a totally unbiased sample of the living population (e.g. age/sex based mortality bias, local customs or taphonomic processes) these factors can be taken into account. Since school age children are more often affected by STH in modern endemic areas [32-34] in some analyses we considered adults and juveniles separately. A higher rate of infection with Ascaris was detected in juveniles compared with adults from the High/Late Medieval period but not in samples other periods. In contrast, eggs of food-derived cestodes (Taenia spp. and D. latum) were only detected in adults.

Ascaris was the most common infection detected throughout the whole dataset with prevalence rates greater than 20% in Roman (Bleadon, Canterbury), Anglo-Saxon (Ipswich), High/Late Medieval (Ipswich, York and Southampton) and Industrial period (London) sites. These rates are similar to those reported from late 4th century Florence (27.7%) [25], Medieval Rottenburg (22%), Ellwangen (22.1%), Pohansko (35.1%) and Brno (42.9%) [26]. Ascaris was also detected in one of three late Neolithic/early Bronze Age sites (Bulford), but due to the small sample sizes the prevalence rates cannot be accurately estimated. Ascaris was detected in all Roman and Medieval period sites except for Bletchingley and Christchurch, where no parasite eggs were detected albeit in a small number of individuals (Table 1). Ascaris eggs were previously reported in pre-Roman English sites including Somerset [45], York [46] and Carlisle [47], as well as in a number of sites on mainland Europe (e.g. [1,10,17,38]).

Trichuris eggs were readily detected in Anglo-Saxon (early Medieval) and High/Late Medieval sites but were not detected in any pre-Roman (n = 15) or Roman period dated samples (n = 94) despite 37 being positive for Ascaris eggs. The lack of Trichuris in Roman and earlier samples was surprising given that Trichuris and Ascaris share a common faecal-oral pattern of infection. Although we did not detect any Trichuris eggs in pre-Roman sites, these parasites have previously been reported from excavations in the UK [18,45,48] and continental Europe (e.g. [1,10,38]). Interestingly, Trichuris has been regularly detected in Roman sites across continental Europe (summarised in [49,50]) and has been reported in the UK [41,47,51-57]. It is noteworthy that in UK sites which cover multiple time periods, Trichuris is more often associated with medieval deposits than those dated to earlier periods [46,51,58,59]. Hence, it is clear that Trichuris was present in the UK during Roman and pre-Roman periods, but may have been less common than Ascaris.

During the Medieval period both Trichuris and Ascaris were prevalent in English communities being found at similar levels to those in continental Europe [26]. The High/Late Medieval period represented a peak in prevalence rates in England with overall lower numbers detected in samples dated from the Anglo-Saxon/Early Medieval period (albeit with variation between sites). The eggs of both parasites are readily detected in communal medieval deposits from the UK and often found in high numbers [14,46,47,51,58-68]. Unfortunately, the data was often not quantitative or based on single samples, which makes it difficult to compare between studies. Nevertheless, it is clear that both Ascaris and Trichuris were common infections during the Medieval period. Various factors could have been associated with increased STH rates including the magnitude or patterns of trade, increased urbanisation, issues with waste disposal, sanitation or hygiene, and the use of night soil as a fertiliser [69-71]. However, with current data sets it is difficult to identify factors that adequately explain the emergence of Trichuris as a more common infection.

The pattern of STH prevalence rates observed in the Industrial period sites (Oxford, Birmingham and London) was different to the Medieval period. No eggs were detected in samples from Oxford (n = 56) and only one individual from Birmingham (n = 30) had Trichuris. In contrast, a high prevalence of Ascaris was evident in the samples from London although no Trichuris eggs were detected. There are very few reports of parasite analyses in the UK covering the Industrial period. Jones and Phipps [72] reported no helminth eggs in 12 individuals from the Spitalfields area of London whereas Anastasiou et al, [73] reported Trichuris (2 of 5 coprolites) and Ascaris (1 of 5). Akeret et al. [68] studied communal/pit fill samples from York (Low Petergate) and reported a single Trichuris egg in one of the Industrial period samples compared with larger numbers of Ascaris and Trichuris eggs in multiple Medieval period samples. The overall pattern of STH infections during the Industrial period suggests that prevalence rates were highly variable. The high prevalence of Ascaris in London clearly indicates efficient STH transmission, as might be expected from a high-density urban population with poor sanitation. The absence of helminths in the Oxford Radcliffe Infirmary burial ground samples was unexpected, but the association with a hospital that had a largely rural catchment [74] and a well-based water supply [75] may be important. Birmingham was even more surprising since the sampled site represented a densely populated urban population. However, it is noteworthy that Birmingham was also much less affected by the cholera outbreaks of the mid 1800s compared with many other towns and cities. The low cholera rates were attributed to a greater reliance on spring and well water rather than other, more easily contaminated surface water supplies [History of the Family. 2019 ">76-78] which would also affect water-based sources of STH infections. Some of the low infection rates during this period may also reflect changes in the use of night soil [70].

The prevalence of STH was variable between sites in all time periods examined which probably reflects local differences in the conditions that promote transmission (e.g. faecal contamination of food and water) or maturation/survival of eggs. Interestingly, there were significant differences in prevalence rates during different historic periods. The highest rates of STH were seen in samples from Roman and High/Late Medieval sites, with a slight decrease in the overall rate during the Anglo-Saxon/Early Medieval period. These high rates may be due to population density alongside poor separation of contaminated faecal material from the local environment, food or water sources. The slight reduction during the Anglo-Saxon/Early Medieval period may have been due to the decentralisation of people and a less urbanised society [79] reducing faecal contamination of the environment. However, the levels of STH in Anglo-Saxon sites were variable and more sites will be required to provide a clearer picture of this period. The lack of STH in two of three sites representing the Industrial period is noteworthy and may represent the beginnings of wider scale reductions in STH infection levels that led to the modern non-endemic state.

The rates of many faecal-oral infections would have been reduced by the large-scale municipal improvements to sewage and water treatment in the 1860s and the declining use of night soil to fertilise crops. The cholera outbreaks between 1831–1866, famously linked to contaminated drinking water by John Snow [80] were a key driver in sanitary reform (alongside endemic Typhoid) involving infrastructural improvement, legislation and promotion of municipal responsibility [76,81,82]. However, many interventions were piecemeal with some areas benefiting earlier than others [82]. Our data identifying very low STH levels in some locations during the Industrial period suggests that some areas may have been reaping benefits from improvements in sanitation and water supplies prior to the Victorian sanitary revolution. It is possible that our small sample (3 locations) is not representative of the Industrial period as a whole. However, given the numbers of individuals analysed and the fact that all Medieval and Roman sites with more than 10 individuals were positive suggests that this is an accurate observation. Since the enteric bacterial pathogens remained problematic during the Industrial period it is worth considering how different pathogens might be affected by variations in sanitary improvement. Helminth eggs are large compared with bacterial pathogens, which means that settlement processes (e.g. sedimentation tank or pit), used for many centuries in different guises [83], may differentially retain parasite eggs. However, the solid matter from these features needed to be periodically removed and since helminth eggs remain viable for months (e.g. [84,85]) the disposal of this material may have facilitated STH transmission. Improved settlement techniques, (e.g. holding reservoirs) and the development of sand filtration in the early 1800s [81] would have reduced contamination of water with the relatively large STH eggs. Similarly, the locations of sewage outlets and potable water intakes might differentially affect pathogens depending on sedimentation rates, water conditions and environmental resilience. Linking STH rates with local water and waste management practices during the Industrial and post-Industrial periods will be an interesting avenue for future studies.

Even though the great sanitary reforms of the Victorian period clearly impacted STH prevalence there are reports of a problem persisting into more modern times [86] Evidence for the continued, spatially focussed, problem includes STH reported in Cornish tin mine-associated populations but not similar mining communities in Staffordshire or Shropshire [87]. Significant STH infection rates (12.5%) were also reported in patients at Guys Hospital, London in 1905 [88]. Outbreaks of STH infections were reported in various mental health institutions across England in the late 1960s but there was no evidence for significant transmission of STH in the general population at this time [89].

Many factors may affect overall STH prevalence including age-structure of the sampled population ([26,32,90] and this paper) as well as a variety of site-specific factors. Therefore we considered the contribution of Trichuris or Ascaris to the overall STH burden and confirmed the observation that the level of Trichuris (in relation to total STH) was unexpectedly low before and after the Medieval period. In the Medieval period, Trichuris contributed to the STH burden at a higher level, comparable to that in Medieval samples from continental Europe. A similar approach was used to show that the parasite assemblage in North America was dominated by Trichuris in the 18th century, but changed during the early to mid-19th century when Ascaris became dominant [91]. STH eggs are readily detected in archaeological contexts across a wide temporal range (e.g. [10,17,18,21,38,92,93]) which argues against differential preservation as an explanation for the lack of Trichuris in some time periods. The changes in the proportions of the two dominant STH parasites suggest that there are factors which differentially influence transmission and these may relate to embryonation, survival or persistence of viable eggs [84,85,94].

The detection of cestode eggs (Taenia spp. and D. latum), albeit at lower rates than seen with Ascaris provides information on diet and culinary practices in English sites through the ages. Both parasites are transmitted to humans via uncooked or undercooked meat (or their products) with Taenia acquired from pork or beef and D. latum acquired from freshwater fish. Cestode eggs were identified in samples that spanned a wide range of time from the Bronze Age (Bulford) to the Industrial period (London). Interestingly in four of the six sites where Taenia was detected, D. latum was also detected suggesting local culinary preferences. To complete the life cycle the eggs from human faeces would also need to access appropriate intermediate hosts. Hence, the presence of D. latum eggs in humans indicates faecal contamination of freshwater sources of fish. In contrast, Taenia sp. utilise pigs or cattle as intermediate hosts necessitating exposure of livestock to human faecal material perhaps through the use of night-soil applied to pasture or animal-feed crops. Interestingly, and in contrast to STH, the cestode eggs were only detected in adults. This pattern has been reported previously [26] and may be expected from a long-lived infection acquired by occasional consumption of contaminated food.

In summary, the prevalence of helminth infections is poorly understood in past populations, despite being a key factor in the epidemiology of these infections. The findings presented indicate that the Medieval period was a high point of STH prevalence in the UK. Interestingly, outside of the Medieval period there was much less Trichuris present than expected which may relate to as yet undefined environmental or anthropological factors. The Industrial period saw some sites with very low levels of STH infections, but this reduction was patchy and high Ascaris prevalence persisted in London. Defining factors that affected the past prevalence of helminths help us understand historical patterns of disease and could be relevant to modern control programmes.

23 Dec 2021

Dear Prof Smith,

Thank you very much for submitting your manuscript "Reconstructing the history of helminth prevalence in the UK" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Subash Babu

Associate Editor

PLOS Neglected Tropical Diseases

Maria Periago

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: This is a well written and interesting manuscript and I have only few comments.

Introduction

First line: in which country/countries?

Page 6: I think the paragraph on eradication should be deleted as it is a bit out of focus. Moreover, it is elimination (as a public health problem) and not eradication

Results

Page 9 last paragraph (first paragraph of the results): this paragraph should be deleted as it summarized M&M and introduction.

Page 14 last paragraph and page 15: the comparison of the Czech site and the last paragraph should be deleted or moved as this already part of the discussion

Discussion

The discussion is a bit long, hence I would suggest condensing it a bit.

Are there any study limitations?

It might be worth mentioning that hookworms rapidly hatch and therefore cannot be diagnosed unless samples are frozen or conserved, hence there might have been hookworm infections as well.

Last sentence: Understanding the factors that may have affected the past prevalence of STH infections may provide key information for modern control efforts, particularly where anthelminthic programmes have struggled to provide a long-lasting solution.

I would delete this sentence: we know what is needed to control these infections but implementation is often challenging

Reviewer #2: Material & method

The sampling process could be more precise. Information should be added on where exactly samples from the pelvis were taken, i.e., above or below the pelvis.

The last sentence page 8 is written in a different police size.

In the processing and microscopy section: why authors decided to use a rehydration method different to all what is readable in the bibliography dealing with ancient parasites? The rehydration is performed with ultrapure water. No screening steps. “Two rounds of microscopy” which isn’t clear according to me. Does it mean two slides?

The rehydration techniques used in the different groups working and developing paleoparasitology or archaeoparasitology, even if not exactly the same, consist in more or less 3 steps:

The rehydration, generally in TSP, which help in disaggregation of the sample and the parasite egg suspension.

The screening step, discarding the bigger elements which present no interest and can be problematic during microscopic observation.

The sedimentation.

The choice of the authors has to be explained.

Reviewer #3: 1. The objectives of the study were clearly articulated, well-written and easy to understand.

2. The study was designed in detail.

3. The population was clearly described and appropriate.

4. The sample size was sufficient to support the conclusions.

5. I did not find any concerns regarding ethical or regulatory requirements not being met.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: Results:

The first lines of this paragraph were already written in the previous section. This should be shortened or deleted.

Page 11, the use of the acronym STH isn’t explained before. Probably Soil Transmitted Helminths. But the entire terminology has to be used one time with the acronym to be clear and correct.

Page 13: authors decide to divide individuals into two age-groups, but here again, this choice isn’t explained. Because of the collection, this appears to be motivated by the low number of individuals for some age class, having consequences on the statistic treatment. But this has to be state.

As a consequence, page 14, it is mentioned that cestods (Taenia and Dibothriocephalus) were only detected in older individuals, older than 13 yo.

Page 15, line 7, Trichuris has to be italicized.

Reviewer #3: 1. The results presented were clear, precise and well-described.

2. The figures (tables, images) were clear. Legends were detailed and self-explanatory.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: Discussion:

My previous comment on the bibliography also concerned this part. This paper proposition concerns European archaeological sites and the authors cite 6 American papers on the 8 citations appearing in the first sentence…

Some affirmation in the discussion appears problematic according to me. Of course, they are directly linked to the results, and the previous results from Flammer et al 2020 (cited 16 times in the all text), but no discussion are made on the samples themselves neither the extraction method and observation.

I mean, working on skeleton remains never allowed to observe the maximum of parasite remains under microscopy. Because of taphonomical processes, including postmortem body preparation (exposition, washing, intestine removal…), and the intense microbiological activity, eggs disappear.

The extraction method, which isn’t conventional (very different from the one used in the other labs), also is a problem. The microscopical observation, if only two slide per sample were observed also is a problem.

This led to conclusion which are not true, and in opposition with previous observation from cesspit/latrine contexts for example.

Page 18, author states that previous data in England were unsuitable for calculating prevalence. Why? It is not possible to consider the number of positive samples from a site on which latrine are analyzed?

Same page, line 8, Ascaris has to be italicized.

A sentence page 20, lines 13-14, justify an observation of the results from the Industrial period, on the low rate of Trichuris, referring to Akeret et al 2006. It refers to samples prepared using palynological techniques, including acids and bases for preparation, known as destroying parasite eggs, except Ascaris which are more resistant.

Reviewer #3: The authors have done an excellent job of combining history/archeology with parasitic epidemiology. The entire paper was a delight to read. However I have 2 points of concern :- 1. I believe the references listed in the paper may not be the correct way to present. 2. Please add line numbers to the manuscript, so that it's easier to review.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Bibliography

The entire bibliography has to be check as many ref do not appear in the text :

Cox, 2002

Loreille et al 2001

Pullan & Brooker 2012

Soe et al 2015

Torok et al 2016.

Wrigley 1985.

The date in the ref Lynch et al 1985 has to be check as different in the text and the bibliography section.

Finally, some ref in the text are not cited in the bibliography:

Ledger et al 2020

Senecal et al 2020

The photo of the Trichuris egg isn't in a good quality. This has to be modified.

Reviewer #3: 1. I believe the references listed in the paper may not be the correct way to present.

2. Please add line numbers to the manuscript, so that it's easier to review.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: General comments:

The article proposition deals with the analysis the variation of the percentage of positive samples (rate) after microscopic analysis of intestinal parasites in samples taken from human skeleton from archaeological sites in England.

This paper is well written and understandable.

However, many complements has to be integrate before being acceptable for publication.

Below the authors could find some comments and modification proposition for the text.

Abstract:

In the all text, the author should use the new accepted name for the fish tapeworm which is no more Diphyllobothrium latum, but as stated page 4, Dibothriocephalus latus.

Introduction:

From the beginning of the text, and later in the discussion, I am perplexed on the fact that large part of the European bibliography on ancient parasites is omitted. Bouchet, in France, published articles on the subject from 1989 to 2016, most part on European contexts, and is only cited one time. Herrmann, in Germany, published articles on cites dated to the medieval period in the late 1980’s. In a general comment, almost all the French work is forgotten. Dommelier, Le Bailly, Maicher, Dufour. All publishing in international journals.

Of course, English researchers as Jones, Pike, and Mitchell are well cited.

In the first introduction sentence, please add European references. Bouchet et al 2003 should be added.

The statement of the authors (page 5) writing that “prevalence rates can be determined” based on the egg recovery on skeleton isn’t true, when considering the real definition of the “prevalence” in epidemiology. When sampling on skeletons, the sample do not only contain ancient fecal matter, but fecal matter mixed with an unknown amount of earth/soil, which block the calculation of the prevalence.

The only case I know from archaeology on which such calculation was pertinent is in Racz et al 2015 (JAS).

The sentence and the following work are valid only if the authors explain their definition of the “prevalence”. This explanation arrives too late page 6 and should be mentioned earlier.

Reviewer #3: (No Response)

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

4 Feb 2022

Submitted filename: Response to reviewers comments final.docx

Click here for additional data file.

7 Mar 2022

Dear Prof Smith,

We are pleased to inform you that your manuscript 'Reconstructing the history of helminth prevalence in the UK' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Subash Babu

Associate Editor

PLOS Neglected Tropical Diseases

Maria Periago

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: All comments were considered and received were discussed in the author answer or added in the new version.

Reviewer #3: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: All comments were considered and received were discussed in the author answer or added in the new version.

Reviewer #3: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: All comments were considered and received were discussed in the author answer or added in the new version.

Reviewer #3: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have addressed all comments

Reviewer #2: All comments were considered and received were discussed in the author answer or added in the new version.

Reviewer #3: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

28 Mar 2022

Dear Prof Smith,

We are delighted to inform you that your manuscript, "Reconstructing the history of helminth prevalence in the UK," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases