International Journal of Paleopathology 31 (2020) 34–37

Contents lists available at ScienceDirect

International Journal of Paleopathology

journal homepage: www.elsevier.com/locate/ijpp

First report in pre-Columbian mummies from Bolivia of Enterobius

vermicularis infection and capillariid eggs: A contribution to
Paleoparasitology studies
Guido Valverde a, Viterman Ali a, b, Pamela Durán a, b, Luis Castedo c, José Luis Paz c,
Eddy Martínez a, b, *
a
Instituto de Investigación en Salud y Desarrollo (IINSAD), Calle Claudio Sanjinés s/n, Complejo Hospitalario de Miraflores, La Paz, Bolivia
b
Cátedra de Parasitología, Facultad de Medicina, Universidad Mayor de San Andrés (UMSA), Av. Saavedra 2246, La Paz, Bolivia
c
Museo Nacional de Arqueología (MUNARQ), Unidad de Arqueología y Museos (UDAM), Viceministerio de Interculturalidad, Ministerio de Culturas y Turismo, Estado
Plurinacional de Bolivia, Calle Tiahuanacu No. 93, Esq. Federico Suazo, La Paz, Bolivia

A R T I C L E I N F O A B S T R A C T

Keywords: Objective: This study was designed to search for ancient parasites in abdominal content and coprolites from
Paleoparasitology Bolivian mummies.
Andean mummies Materials: Twelve mummified individuals from the Andean highlands, housed at the National Museum of
Pinworm
Archaeology (MUNARQ) in La Paz, Bolivia.
Capillaria
Paleoparasites
Methods: Microscopic analysis of rehydrated samples (coprolites and abdominal content), following Lutz’s
spontaneous sedimentation technique.
Results: Eggs of Enterobius vermicularis were identified in coprolites from one mummy, and capillariid eggs in the
organic abdominal content from another individual.
Conclusions: This is the first evidence of ancient intestinal parasites in Bolivian mummies.
Significance: This pioneering study focused on the search of ancient intestinal parasites in human remains of the
Bolivian Andes and contributes to greater knowledge of paleoparasitology in South America.
Limitations: All mummies in the MUNARQ belonged to the Andean Bolivian highlands (post-Tiwanaku era or Late
Intermediate Period), although the exact provenance of the material and the associated contexts are not well
recorded.
Suggestions for further research: Considering the great number of well-known archaeological sites and other un­
explored sites in Bolivia, in addition to large collections in museums, further paleopathological and paleopar­
asitological molecular studies in mummies and skeletons are called for.

1. Introduction
Since the findings of Trichuris trichiura and Ascaris lumbricoides eggs
in two bodies from Prussia, dated 600 BCE and 500 CE (Szidat, 1944),
nematodes were the most frequently identified parasites in archaeo­
logical remains. In South America, Trichuris trichura eggs identified in a
Chilean Inca mummy of a child of 8–9 years old constitute the first ev­
idence of a nematode in this region (Pizzi and Schenone, 1954). Sub­
sequently, there were many publications reporting nematodes: Ascaris
lumbricoides in Argentina, Peru and Brazil (Fugassa et al., 2008;
Gonçalves et al., 2003); T. trichura in Argentina, Brazil, Chile and Peru
(Fugassa et al., 2006; Gonçalves et al., 2003; Pizzi and Schenone, 1954);
Ancylostomids in Brazil, Chile and Peru (Allison et al., 1974; Ferreira
et al., 1980; Gonçalves et al., 2003); and Enterobius vermicularis in
Argentina, Chile and Peru (Gonçalves et al., 2003; Iñiguez et al., 2003;
Zimmerman and Morilla, 1983). Other studies performed on parasites in
archaeological and paleontological remains explained aspects of their
origin, migration, and geographic distribution (Araujo et al., 2008;
Araújo et al., 2011), as well as the living conditions and behavior of
ancient groups (Novo and Ferreira, 2016).

* Corresponding author at: Instituto de Investigación en Salud y Desarrollo (IINSAD), Calle Claudio Sanjinés s/n, Complejo Hospitalario de Miraflores, La Paz,
Bolivia.
E-mail addresses: eddy.martinez.a@gmail.com, eddy.martinez@umsalud.edu.bo (E. Martínez).

https://doi.org/10.1016/j.ijpp.2020.08.002
Received 14 February 2020; Received in revised form 21 August 2020; Accepted 22 August 2020
Available online 12 September 2020
1879-9817/© 2020 Elsevier Inc. All rights reserved.
G. Valverde et al. International Journal of Paleopathology 31 (2020) 34–37

Parasites belonging to the Capillaria genus were identified in 2.2. Analytic methods
archaeological remains of different mammalian species (domestic and
wild) and in human hosts, with about 40 articles published in the All samples were processed in the laboratory of the Unidad de Par­
Americas, the great majority from Argentina and some from Brazil asitología, Medicina Tropical y Medio Ambiente (UPAMETROP) in the
(Araújo et al., 2011; Borba et al., 2019). Nevertheless, the exact role that Instituto de Investigación en Salud y Desarrollo (IINSAD), and in the
humans play as hosts in parasites’ biological cycle remains controversial Parasitology Research Laboratory, both within the Faculty of Medicine
(Beltrame et al., 2018). of the Universidad Mayor de San Andrés in La Paz, Bolivia.
With the exception of only one study on Trypanosoma cruzi in Approximately two grams of abdominal content or coprolites were
mummies (Orellana, 2008), paleoparasitological research has been maintained for rehydration during three days (up to five days for copro­
neglected in Bolivia. Archeologists and other Bolivian researchers have lites) in 0.5 % trisodium phosphate aqueous solution (Na3PO4), following
not focused on paleopathology/paleparasitology, despite the existence Callen and Cameron (1960). After this time, a suspension was obtained
of many mummified bodies and skeletons associated with Andean cul­ and filtered through triple-folded gauze in conical glass jars, where
tures in museums. Thus, our study represents the first effort to recover samples were kept for sedimentation for 24 h following Lutz’s sponta­
intestinal parasites from pre-Columbian human remains in Bolivia, neous sedimentation technique to improve performance (Camacho et al.,
generating new evidence on the health and other conditions of ancient 2018, 2013; Lutz, 1919). The supernatant was decanted and few drops of
human populations. the sediment were placed between a glass microscope slide and a coverslip
for observation under light microscopy using 10x and 40x magnification
2. Materials and methods objectives (Olympus CX33, Japan), attached to a camera (Olympus LC30)
provided with Cell Sens® Software. A minimum of 20 slides for each
2.1. Material studied sample were examined, and parasites were identified based on morpho­
logical characteristics (without quantification).
The National Museum of Archeology (Museo Nacional de Arqueo­
logía, MUNARQ) in La Paz stores the most important anthropological 2.3. Ethics statement
collection of mummified bodies of Bolivia. Unfortunately, the prove­
nance of most of the individuals at the museum is unknown, as this All necessary permissions to collect samples from the National
archaeological material was donated from private collections or Museum of Archaeology were granted by the Bolivian Ministry of Cul­
confiscated from traffickers, without historical records and documen­ tures and Tourism (AUTORIZACION MDCyT – UDAM No. 070/2018).
tation. Faced with the absence of paleoparasitological information in
Bolivia, after requesting the MUNARQ’s permission, we were granted 3. Results
access to 12 mummies (see Table 1). Individuals were classified as pre-
Columbian mummies belonging to the post-Tiwanaku era or the Late Parasites were identified in two of the 12 samples studied: i) a
Intermediate Period (1150–1450 AD), based on mummification type in coprolite from the mummy MUNARQ-10327 (Fig. 1A, 1B) contained
the Andean region according to Ponce and Linares (1966) and their typical eggs of Enterobius vermicularis (pinworm) (Fig. 1C); the exact
preservation features (i.e. natural mummification). We collected the origin of this mummy is unknown; ii) Capillariid eggs were found in the
samples for analysis (3 coprolites and 9 organic content material from sample of organic abdominal content from the mummy MUNARQ - 4296
abdominal cavities) following standard protocols (Sianto and Luísa, (Figure 2A, 2B, 2C); the origin of this mummy was attributed by Ponce
2016). and Linares (1966) to the Carangas Province in the Department of
Oruro.
The E. vermicularis eggs measured 56.7 ± 1.9 μm (length) by 27.1 ±
1.1 μm (width), and showed typical characteristics: transparent and
Table 1
Details of the archaeological material studied from the anthropological collec­
elongated with one of their faces flattened and the other convex, and a
tion (mummies) at the National Museum of Archaeology, La Paz, Bolivia. thin eggshell (Fig. 1C). The average size of the capillariid eggs was 60.9
± 1.2 μm (length) by 32.8 ± 1.9 μm (width), with an elliptical shape and
# Museum ID Sex Approx. Type of sample
Age
polar plugs, flattened, and with the outermost membrane crossed by
minuscule channels that give it a characteristic striated appearance
1 MUNARQ-10 * M Organic content abdominal
>50
(Figure 2C).
region
2 MUNARQ- M 25− 30 Coprolite
10327 4. Discussion
3. MUNARQ-4296 M 25− 30 Organic content abdominal
region This is the first report of intestinal paleoparasites identified in sam­
4 MUNARQ-4324 ND 30− 35 Coprolite
5 MUNARQ-4319 M 25− 30 Coprolite
ples from Bolivian mummies. Several authors have reported parasites
6 MUNARQ-4297 ND 25− 30 Organic content abdominal infecting American prehistoric populations (Gonçalves et al., 2003),
region suggesting that nematodes might have pre-Columbian antiquity (Araújo
7 MUNARQ-4283 F 25− 30 Organic content abdominal et al., 2011; Novo and Ferreira, 2016), and that they might have been
region
introduced across the Bering Land Bridge or alternative prehistoric
8 MUNARQ-4321 M 25− 30 Organic content abdominal
region coastal or transoceanic migratory routes accompanying their human
9 MUNARQ- F >60 Organic content abdominal hosts (Araujo et al., 2008). E. vermicularis eggs were found in human
15335 region coprolites dated at 7837 BCE from caves in western Utah, USA (Fry and
10 MUNARQ- M 25− 30 Organic content abdominal Moore, 1969), being considered the oldest human parasite (Iñiguez
15334 region
11 MUNARQ-4288 ND ND Organic content abdominal
et al., 2003). Similarly, we found this parasite in a coprolite obtained
region directly from the abdominal cavity, thus making it impossible that the
12 MUNARQ- ND ND Organic content abdominal sample should be contaminated.
15415 region Considering that E. vermicularis is transmitted among humans by the
*With cranial deformation. ND: No data. All individuals had gone through a oral route or by inhalation, other findings of pinworms in archaeological
natural mummification process and are related to the post-Tiwanaku era or Late remains from Argentina, Mexico, Chile, Peru and North America (Gon­
Intermediate Period (1150–1450 AD). çalves et al., 2003; Hugot et al., 1999; Reinhard et al., 2016) suggest

35
G. Valverde et al.
Fig. 1. Mummies positive for parasites. (1A) MUNARQ-10327; (1B) Coprolite from mummy 1A; (1C) Enterobius vermicularis egg found in coprolite; (2A) MUNARQ-
4296; (2B) Abdominal content of mummy 2A; (2C) Capillariid egg found in abdominal content.
close contact among people in the past. According to some authors,
airborne transmission emerged when humans changed their
hunter-gatherer behavior to more sedentary habits (Hugot et al., 1999),
and with the development of agriculture to farming lifestyles (Reinhard
et al., 2016), a well-known situation in ancient Andean populations.
Capillariids include around 300 species belonging to different
genera, with similar morphology among their eggs, and it is not possible
to define the genus by microscopy, making molecular tools necessary
(Borba et al., 2019). Similar to findings by Fugassa et al. (2008), the
present study found Capillariid eggs in an organic abdominal content
sample associated with the pelvic region of an adult male individual.
Capillaria eggs have been recorded from several human coprolites from
Neolithic sites of the French Alps, without confirmation if the people
were definitive, intermediary, paratenic or transitory hosts (Bouchet,
1997). Thus, the presence of these parasites in human remains may be
due to contamination of the samples or an incidental presence, or related
to dietary habits or practices (Borba et al., 2019). An example of the
latter is liver ingestion from infected animals (Roberts and Janovy,
36
International Journal of Paleopathology 31 (2020) 34–37
2009), recently supported with the identification of Brazilian indigenous
peoples eliminating Capillaria sp. eggs in feces due to consumption of
raw rodent liver, but without infection (Camargo et al., 2010; Coimbra
and Mello, 1981), a condition known as "false parasitism". Some pale­
oparasitological studies developed in Patagonia, Argentina showed
capillariid eggs in human coprolites (Fugassa and Guichón, 2005),
camelids and other mammals (Taglioretti et al., 2017). Human infection
by capillariids is also a possibility, considering some reports arguing that
the parasitic infection is due to fish consumption (El-Dib et al., 2015;
Wang et al., 2013).
Our pioneering study contributes to greater knowledge of paleo­
parasitology in South America, especially considering the scant infor­
mation on paleoparasites in Bolivia compared to neighboring countries.
It is therefore essential to promote initiatives which encourage paleo­
parasitology research using mummified remains (Araújo et al., 2000).
The inclusion of organic material and sediments from archaeological
sites will provide new insights into ancient parasitic infections and will
begin to address contemporary public health problems.
G. Valverde et al.
Considering the existence of anthropological material (mummies and
skeletons) in museums, and several well-known but inadequately or
insufficiently explored archaeological sites, we hope that the dissemina­
tion of our findings will contribute to promoting similar studies in Bolivia.
Authors’ contributions
EM conceived and coordinated the study. GV, EM set up collabora­
tion with the MUNARQ and designed the study protocol. LC, JLP granted
access to the analyzed materials and related information. GV, EM, VA,
PD, LC performed sampling of mummies. GV, VA, EM, PD performed the
paleoparasitological analyses. EM, GV, PD, VA wrote the manuscript. All
authors reviewed, discussed the contents and accepted the final version.
Financial support
This study was partially supported by the Facultad de Medicina,
Universidad Mayor de San Andrés, La Paz, Bolivia.
Declaration of Competing Interest
We declare that we have no conflict of interest.
Acknowledgments
We thank the Bolivian staff of the Museo Nacional de Arqueología -
MUNARQ in La Paz, Bolivia for allowing access to the mummy collec­
tion. We are grateful to Susanna Rance for linguistic revision and helpful
suggestions.
References
Allison, M.J., Pezzia, A., Hasegawa, I., Gerszten, E., 1974. A case of hookworm
infestation in a precolumbian American. Am. J. Phys. Anthr. 41, 103–106.
Araújo, A., Reinhard, K.J., Ferreira, L.F., 2000. The role of Mummy studies in
Paleoparasitology. Chungará (Arica).
Araujo, A., Reinhard, K.J., Ferreira, L.F., Gardner, S.L., 2008. Parasites as probes for
prehistoric human migrations? Trends Parasitol. 24, 112–115. https://doi.org/
10.1016/j.pt.2007.11.007.
Araújo, A., Reinhard, K., Leles, D., Sianto, L., Iñiguez, A., Fugassa, M., Arriaza, B.,
Orellana, N., Ferreira, L.F., 2011. Paleoepidemiology of intestinal parasites and lice
in pre-Columbian South America. Chungara (Arica) 43, 303–313. https://doi.org/
10.4067/S0717-73562011000200011.
Beltrame, M.O., Bellusci, A., Fernández, F.J., Sardella, N.H., 2018. Carnivores as zoonotic
parasite reservoirs in ancient times: the case of the Epullán Chica archaeological
cave (Late Holocene, northwestern Patagonia, Argentina). Archaeol. Anthropol. Sci.
10, 795–804. https://doi.org/10.1007/s12520-016-0399-8.
Borba, V.H., Machado-Silva, J.R., Le Bailly, M., Iñiguez, A.M., 2019. Worldwide
paleodistribution of capillariid parasites: paleoparasitology, current status of
phylogeny and taxonomic perspectives. PLoS One 14, 1–19. https://doi.org/
10.1371/journal.pone.0216150.
Bouchet, F., 1997. Intestinal capillariasis in neolithic inhabitants of Chalain (Jura,
France). Lancet 349, 256. https://doi.org/10.1016/S0140-6736(05)64868-4.
Callen, E., Cameron, T., 1960. A prehistoric diet revealed in coprolites. New Sci. 8,
35–40.
Camacho, M., Pessanha, T., Leles, D., Dutra, J.M.F., Silva, R., de Souza, S.M., Araujo, A.,
2013. Lutz’s spontaneous sedimentation technique and the paleoparasitological
analysis of sambaqui (shell mound) sediments. Mem. Inst. Oswaldo Cruz 108,
155–159. https://doi.org/10.1590/0074-0276108022013005.
Camacho, M., Araújo, A., Morrow, J., Buikstra, J., Reinhard, K., 2018. Recovering
parasites from mummies and coprolites: an epidemiological approach. Parasit.
Vectors 11, 1–17. https://doi.org/10.1186/s13071-018-2729-4.
37
International Journal of Paleopathology 31 (2020) 34–37
Camargo, L.M.A., De Souza Almeida Aranha Camargo, J., De Souza Vera, L.J., Di Tarique
Crispim Barreto, P., Tourinho, E.K., De Souza, M.M., 2010. Capillariaisis (Trichurida,
Trichinellidae, Capillaria hepatica) in the Brazilian Amazon: low pathogenicity, low
infectivity and a novel mode of transmission. Parasit. Vectors 3, 11. https://doi.org/
10.1186/1756-3305-3-11.
Coimbra, C.E., Mello, D.A., 1981. Enteroparasitas e Capillaria sp. entre o grupo Suruí,
Parque Indigena Aripuanã, Rondônia. Mem. Inst. Oswaldo Cruz 76, 299–302.
https://doi.org/10.1590/s0074-02761981000300008.
El-Dib, N.A., El-Badry, A.A., Ta-Tang, T.H., Rubio, J.M., 2015. Molecular detection of
Capillaria philippinensis: an emerging zoonosis in Egypt. Exp. Parasitol. 154,
127–133. https://doi.org/10.1016/j.exppara.2015.04.011.
Ferreira, L.F., De Araújo, A.J.G., Confalonieri, U.E.C., 1980. The finding of eggs and
larvae of parasitic helminths in archaeological material from Unai, Minas Gerais.
Brazil. Trans. R. Soc. Trop. Med. Hyg. 74, 798–800. https://doi.org/10.1016/0035-
9203(80)90205-9.
Fry, G.F., Moore, J.G., 1969. Enterobius vermicularis: 10,000-Year-old human infection.
Science 166, 1620. https://doi.org/10.1126/science.166.3913.1620.
Fugassa, M., Guichón, R., 2005. Análisis paleoparasitológico de coprolitos hallados en
sitios arqueológicos de Patagonia Austral: definiciones y perspectivas. Magallania
(Punta Arenas) 33, 13–19. https://doi.org/10.4067/s0718-22442005000200002.
Fugassa, M.H., Araújo, A., Guichón, R.A., 2006. Quantitative paleoparasitology applied
to archaeological sediments. Mem. Inst. Oswaldo Cruz 101, 29–33. https://doi.org/
10.1590/S0074-02762006001000006.
Fugassa, M.H., Sardella, N.H., Guichón, R.A., Denegri, G.M., Araújo, A., 2008.
Paleoparasitological analysis applied to museum-curated sacra from Meridional
Patagonian collections. J. Archaeol. Sci. 35, 1408–1411. https://doi.org/10.1016/j.
jas.2007.10.006.
Gonçalves, M.L.C., Araújo, A., Ferreira, L.F., 2003. Human intestinal parasites in the past:
new findings and a review. Mem. Inst. Oswaldo Cruz 98, 103–118. https://doi.org/
10.1590/S0074-02762003000900016.
Hugot, J.P., Reinhard, K.J., Gardner, S.L., Morand, S., 1999. Human enterobiasis in
evolution: origin, specificity and transmission. Parasite 6, 201–208. https://doi.org/
10.1051/parasite/1999063201.
Iñiguez, A.M., Reinhard, K.J., Araújo, A., Ferreira, L.F., Vicente, A.C.P., 2003. Enterobius
vermicularis: ancient DNA from North and South American human coprolites. Mem.
Inst. Oswaldo Cruz 98, 67–69. https://doi.org/10.1590/S0074-
02762003000900013.
Lutz, A., 1919. Schistosomum Mansoni and Schistosomatosis observed in Brazil. Mem.
Inst. Oswaldo Cruz 11, 121–155.
Novo, S.P.C., Ferreira, L.F., 2016. The paleoparasitology in Brazil and findings in human
remains from South America: a review. Korean J. Parasitol. 54, 573–583. https://
doi.org/10.3347/kjp.2016.54.5.573.
Orellana, N., 2008. Paleogenética de populações pré-colombianas da Bolívia: análises do
mtDNA humano, e infecções por Trypanosoma cruzi e vírus linfotrópico das células T
humanas (HTLV) (Tese Mestrado). IOC/Fiocruz.
Pizzi, T., Schenone, H., 1954. Hallazgo de huevos de Trichuris trichiura en contenido
intestinal de un cuerpo arqueológico incaico. Boletín Chil. Parasitol. 9, 73–75.
Ponce, C., Linares, E., 1966. Comentario Antropológico acerca de la determinación
paleoserológica de grupos sanguíneos en momias prehispanicas del Altiplano
Boliviano. Acad. Nac. Ciencias Boliv. Publicación 72.
Reinhard, K.J., Araújo, A., Morrow, J.J., 2016. Temporal and spatial distribution of
Enterobius vermicularis (Nematoda: Oxyuridae) in the prehistoric Americas. Korean
J. Parasitol. 54, 591–603. https://doi.org/10.3347/kjp.2016.54.5.591.
Roberts, L.S., Janovy, J.J., 2009. Foundations of parasitology, 8va ed. The McGraw-Hill,
New York. https://doi.org/10.1016/0169-4758(90)90219-t.
Sianto, L., Luísa, A., 2016. Sampling guidelines for paleoparasitological and paleodietary
studies. Cad. do GEEvH 5, 43–50.
Szidat, L., 1944. Über die Erhaltungsfähigkeit Von Helmintheneiern in vor-und
Frühgeschichtlichen Moorleinchen. Zeitschrift für Parasitenkd. 13, 265–574.
Taglioretti, V., Fugassa, M.H., Rindel, D., Sardella, N.H., 2017. New parasitological
findings for pre-Hispanic camelids. Parasitology 144, 1763–1768. https://doi.org/
10.1017/S0031182017000932.
Wang, Z., Lin, X., Wang, Y., Cui, J., 2013. The emerging but neglected hepatic
capillariasis in China. Asian Pac. J. Trop. Biomed. 3, 146–147. https://doi.org/
10.1016/S2221-1691(13)60039-8.
Zimmerman, M.R., Morilla, R.E., 1983. Enterobiasis in precolumbian America.
Paleopathol. Newsl. 42, 8.