Are immunoenzymatic tests for intestinal protozoans reliable when used on archaeological material?

Intestinal protozoans found in ancient human samples have been studied primarily by microscopy and immunodiagnostic assays. However, such methods are not suitable for the detection of zoonotic genotypes. The objectives of the present study were to utilize immunoenzimatic assays for coproantigen detection of Cryptosporidium sp., Giardia duodenalis, and Entamoeba histolytica/Entamoeba dispar in sixty ancient human and animal samples collected from 14 archaeological sites in South America, and to carry out a critical analysis of G. duodenalis according to results obtained from three diagnostic methodologies: microscopy, immunodiagnostic tests (immunoenzymatic and immunofluorescence), and molecular biology (PCR and sequencing). More than half (31/60) of the samples analyzed using immunoenzymatic tests were positive for at least one of the intestinal protozoans, with 46.6% (28/60) corresponding to G. duodenalis, 26.6% (16/60) to Cryptosporidium sp., and 5% (3/60) to E. histolytica/E. dispar. Cryptosporidium sp. and G. duodenalis coinfection was observed in 15% (9/60) of the samples, whereas all three protozoans were found in 5% (3/60) of samples. In the Northeast Region of Brazil, by immunoenzymatic tests there is evidence that G. duodenlais and Cryptosporidium sp. have infected humans and rodents for at least 7150 years. However, for G. duodenalis, the results from the three diagnostic tests were discordant. Specifically, despite the efficiency of the molecular biology assay in the experimental models, G. duodenalis DNA could not be amplified from the ancient samples. These results raise the following question: Are all ancient samples positive for coproantigen of G. duodenalis by immunoenzymatic tests truly positive? This scenario highlights the importance of further studies to evaluate the sensitivity and specificity of the immunoenzymatic method in the archaeological context.

Recovery of Toxoplasma gondii DNA in experimentally mummified skin and bones: Prospects for paleoparasitological studies to unveil the origin of toxoplasmosis.

Paleoparasitology studies parasite infections by finding the parasites' remains in preserved organic remains such as natural or artificial mummy tissues, skeletons, teeth, and coprolites, among others. However, some currently important infections like toxoplasmosis have not been studied by paleoparasitology. The reasons include this parasite's complex life cycle, the resulting difficulties in locating this protozoan in the intermediate host tissues, and the limitation of coprolite studies to felines, the protozoan's definitive host. The current study thus aimed to produce an experimental model for molecular diagnosis of toxoplasmosis, prioritizing its study in bones and skin, the most abundant materials in archeological collections and sites. The study demonstrated the feasibility of recovering Toxoplasma gondii DNA from desiccated material, including bones and skin, in experimental models both with circulating tachyzoites (RH strain), characteristic of acute infection, and with cysts (ME49 cystogenic strain), characteristic of chronic infection. At present, most individuals with T. gondii infection are in the chronic phase, and the same was probably true in the past. The current study thus expands the odds of finding the parasite in archeological material, enhanced by the nature of the material in which the diagnosis was made. Finding the parasite may help answer questions that are widely debated in the literature on this protozoan's origin (Old World versus New World). In addition, when conditions do not allow ideal storage of samples for molecular tests, the methodology creates the possibility of testing oven-dried samples transported at room temperature.

Prehistoric Pathoecology as Represented by Parasites of a Mummy from the Peruaçu Valley, Brazil.

Paleopathologists have begun exploring the pathoecology of parasitic diseases in relation to diet and environment. We are summarizing the parasitological findings from a mummy in the site of Lapa do Boquete, a Brazilian cave in the state of Minas Gerais. These findings in context of the archaeology of the site provided insights into the pathoecology of disease transmission in cave and rockshelter environments. We are presenting a description of the site followed by the evidence of hookworm, intestinal fluke, and Trypanosoma infection with resulting Chagas disease in the mummy discovered in the cave. These findings are used to reconstruct the transmission ecology of the site.

Temporal and Spatial Distribution of Enterobius vermicularis (Nematoda: Oxyuridae) in the Prehistoric Americas.

Investigations of Enterobius sp. infection in prehistory have produced a body of data that can be used to evaluate the geographic distribution of infection through time in the Americas. Regional variations in prevalence are evident. In North America, 119 pinworm positive samples were found in 1,112 samples from 28 sites with a prevalence of 10.7%. Almost all of the positive samples came from agricultural sites. From Brazil, 0 pinworm positive samples were found in 325 samples from 7 sites. For the Andes region, 22 pinworm positive samples were found in 411 samples from 26 sites for a prevalence of 5.3%. Detailed analyses of these data defined several trends. First, preagricultural sites less frequently show evidence of infection compared to agricultural populations. This is especially clear in the data from North America, but is also evident in the data from South America. Second, there is an apparent relationship between the commonality of pinworms in coprolites and the manner of constructing villages. These analyses show that ancient parasitism has substantial value in documenting the range of human behaviors that influence parasitic infections.

Trichuris trichiura in a post-Colonial Brazilian mummy.

Trichuris trichiura is a soil-transmitted helminth which is prevalent in warm, moist, tropical and subtropical regions of the world with poor sanitation. Heavy whipworm can result either in Trichuris dysenteric syndrome - especially in children - or in a chronic colitis. In heavy infections, worms can spread proximally and may cause ileitis. Here we provide first microscopic evidence for a T. trichiura adult worm embedded in the rectum of a post-Colonial Brazilian adult mummy. During Colonial and post-Colonial times, many European chroniclers described a parasitic disease named Maculo whose symptomatology coincides with heavy helminthiasis. Based on our findings and on comparison of ancient textual evidence with modern description of heavy whipworm, we feel confident in considering that the two syndromes are expressions of the same pathological condition.

A new ascarid species in cynodont coprolite dated of 240 million years.

Cynodonts represent the transition from reptiles to mammals. They are classified as synapsids, or tetrapod animals with mammalian characteristics. We present here the finding of helminth eggs in a coprolite identified as of cynodont origin dated of nearly 240 million years. Microscopy revealed the presence of very well preserved intestinal parasite eggs. Up to now we identified an ascarid egg by morphological characteristics. Based on a previous description of the new genus Ascarites Poinar Jr and Boucot 2006 in coprolites of iguanodons from Belgium, we propose a new species, Ascarites rufferi n.sp. in cynodonts, a host that inhabited the Southern Region of Brazil in the Triassic period.

Paleoparasitological studies on mummies of the Joseon Dynasty, Korea.

Paleoparasitology is the application of conventional or molecular investigative techniques to archeological samples in order to reveal parasitic infection patterns among past populations. Although pioneering studies already have reported key paleoparasitological findings around the world, the same sorts of studies had not, until very recently, been conducted in sufficient numbers in Korea. Mummified remains of individuals dating to the Korean Joseon Dynasty actually have proved very meaningful to concerned researchers, owing particularly to their superb preservation status, which makes them ideal subjects for paleoparasitological studies. Over the past several years, our study series on Korean mummies has yielded very pertinent data on parasitic infection patterns prevailing among certain Joseon Dynasty populations. In this short review, we summarized the findings and achievements of our recent paleoparasitological examinations of Joseon mummies and discussed about the prospects for future research in this vein.

Studies on protozoa in ancient remains--a review.

Paleoparasitological research has made important contributions to the understanding of parasite evolution and ecology. Although parasitic protozoa exhibit a worldwide distribution, recovering these organisms from an archaeological context is still exceptional and relies on the availability and distribution of evidence, the ecology of infectious diseases and adequate detection techniques. Here, we present a review of the findings related to protozoa in ancient remains, with an emphasis on their geographical distribution in the past and the methodologies used for their retrieval. The development of more sensitive detection methods has increased the number of identified parasitic species, promising interesting insights from research in the future.

Lutz's spontaneous sedimentation technique and the paleoparasitological analysis of sambaqui (shell mound) sediments.

Parasite findings in sambaquis (shell mounds) are scarce. Although the 121 shell mound samples were previously analysed in our laboratory, we only recently obtained the first positive results. In the sambaqui of Guapi, Rio de Janeiro, Brazil, paleoparasitological analysis was performed on sediment samples collected from various archaeological layers, including the superficial layer as a control. Eggs of Acanthocephala, Ascaridoidea and Heterakoidea were found in the archaeological layers. We applied various techniques and concluded that Lutz's spontaneous sedimentation technique is effective for concentrating parasite eggs in sambaqui soil for microscopic analysis.

Syphacia sp. (Nematoda: Oxyuridae) in coprolites of Kerodon rupestris Wied, 1820 (Rodentia: Caviidae) from 5,300 years BP in northeastern Brazil.

We present the results of paleoparasitological analyses in coprolites of Kerodon rupestris, rodent endemic to rocky areas of Brazil's semiarid region. The coprolites were collected from excavations at the archaeological site of Toca dos Coqueiros, in the National Park of Serra da Capivara, southeastern of state of Piauí. Syphacia sp. (Nematoda: Oxyuridae) eggs were identified in coprolites dated at 5,300 ± 50 years before present. This is the first record of the genus Syphacia in rodent coprolites in the Americas.

Trichuris sp. from 1,040 +/- 50-year-old Cervidae coprolites from the archaeological site Furna do Estrago, Pernambuco, Brazil.

We present results of the paleoparasitological analysis of Cervidae coprolites that were recovered from the archaeological site Furna do Estrago, Pernambuco, Brazil. Trichuris sp. eggs were recovered from the coprolite samples dated 1,040 ± 50 years before present. This is the first record of Trichuris sp. in semiarid Cervidae, unexpectedly recorded in archaeological material.

Chagas disease in prehistory.

The classical hypothesis proposes that Chagas disease has been originated in the Andean region among prehistoric people when they started domesticating animals, changing to sedentary habits, and adopting agriculture. These changes in their way of life happened nearly 6,000 years ago. However, paleoparasitological data based on molecular tools showed that Trypanosoma cruzi infection and Chagas disease were commonly found both in South and North American prehistoric populations long before that time, suggesting that Chagas disease may be as old as the human presence in the American continent. The study of the origin and dispersion of Trypanosoma cruzi infection among prehistoric human populations may help in the comprehension of the clinical and epidemiological questions on Chagas disease that still remain unanswered.

Possible influence of the ENSO phenomenon on the pathoecology of diphyllobothriasis and anisakiasis in ancient Chinchorro populations.

Current clinical data show a clear relationship between the zoonosis rates of Diphyllobothrium pacificum and Anisakis caused by the El Niño Southern Oscillations (ENSO) phenomenon along the Chilean coast. These parasites are endemic to the region and have a specific habitat distribution. D. pacificum prefers the warmer waters in the northern coast, while Anisakis prefers the colder waters of Southern Chile. The ENSO phenomenon causes a drastic inversion in the seawater temperatures in this region, modifying both the cool nutrient-rich seawater and the local ecology. This causes a latitudinal shift in marine parasite distribution and prevalence, as well as drastic environmental changes. The abundance of human mummies and archaeological coastal sites in the Atacama Desert provides an excellent model to test the ENSO impact on antiquity. We review the clinical and archaeological literature debating to what extent these parasites affected the health of the Chinchorros, the earliest settlers of this region. We hypothesise the Chinchorro and their descendants were affected by this natural and cyclical ENSO phenomenon and should therefore present fluctuating rates of D. pacificum and Anisakis infestations.

Molecular diagnosis of ascariasis from human feces and description of a new Ascaris sp. genotype in Brazil.

It is estimated that 120 million people are infected by Ascaris lumbricoides in Latin America, but few genomic studies have been conducted. We tested protocols for DNA extraction to obtain an Ascaris sp. molecular diagnosis from human feces, with the emphasis on PCR specificity and sensitivity. Ascaris sp. was detected in 100% of positive fecal samples using physico-chemical DNA extraction and the cytb gene and ITS1 as molecular targets. The method was sufficiently sensitive to detect Ascaris sp. from one isolated egg or four eggs in a fecal sample. Regarding the PCR specificity, there was no cross-reactivity when applied to Trichuris trichiura-positive fecal samples or in Ascaris sp. samples also positive to T. trichiura or Enterobius vermicularis. The ITS1 sequence analysis revealed two genotypes among the sample: the G1 genotype, the most prevalent in humans, and a new genotype, G6, described for the first time in Brazilian samples.

Paleoparasitology of Chagas disease--a review.

One hundred years since the discovery of Chagas disease associated with Trypanosoma cruzi infection, growing attention has focused on understanding the evolution in parasite-human host interaction. This interest has featured studies and results from paleoparasitology, not only the description of lesions in mummified bodies, but also the recovery of genetic material from the parasite and the possibility of analyzing such material over time. The present study reviews the evidence of Chagas disease in organic remains excavated from archeological sites and discusses two findings in greater detail, both with lesions suggestive of chagasic megacolon and confirmed by molecular biology techniques. One of these sites is located in the United States, on the border between Texas and Mexico and the other in state of Minas Gerais, in the Brazilian cerrado (savannah). Dated prior to contact with Europeans, these results confirm that Chagas disease affected prehistoric human groups in other regions outside the Andean altiplanos and other transmission areas on the Pacific Coast, previously considered the origin of T. cruzi infection in the human host.

Parasites as probes for prehistoric human migrations?

Host-specific parasites of humans are used to track ancient migrations. Based on archaeoparasitology, it is clear that humans entered the New World at least twice in ancient times. The archaeoparasitology of some intestinal parasites in the New World points to migration routes other than the Bering Land Bridge. Helminths have been found in mummies and coprolites in North and South America. Hookworms (Necator and Ancylostoma), whipworms (Trichuris trichiura) and other helminths require specific conditions for life-cycle completion. They could not survive in the cold climate of the northern region of the Americas. Therefore, humans would have lost some intestinal parasites while crossing Beringia. Evidence is provided here from published data of pre-Columbian sites for the peopling of the Americas through trans-oceanic or costal migrations.

Chinese liver flukes in latrine sediments from Wong Nim's property, San Bernardino, California: archaeoparasitology of the Caltrans District Headquarters.

Parasitological analysis of 5 sediment samples from San Bernardino, California latrine deposits spanning the time period from about 1880 to the 1930s are presented. Two sediment samples are from a latrine used by European-Americans. Three sediment samples are from latrines used by Chinese-Americans on the property of Wong Nim, an important member of the Chinese community. Two of the Chinese latrines were positive for human parasites. The human parasites encountered include the human whipworm (Trichuris trichiura), the giant intestinal roundworm (Ascaris lubricoides, c.f.), and the Chinese liver fluke (Clonorchis sinensis). Evidence of the liver fluke is especially important. This parasite cannot complete its life cycle outside of its endemic range in Asia because suitable intermediate hosts are not present in the American continents. Its presence signals that at least some of the Chinese-Americans who used the latrines were immigrants who were infected in Asia and then sustained infections while in the Americas.

A tick from a prehistoric Arizona coprolite.

Ticks have never been reported in archaeological analyses. Here, we present the discovery of a tick from a coprolite excavated from Antelope Cave in extreme northwest Arizona. Dietary analysis indicates that the coprolite has a human origin. This archaeological occupation is associated with the Ancestral Pueblo culture (Anasazi). This discovery supports previous hypotheses that ticks were a potential source of disease and that ectoparasites were eaten by ancient people.

Recovering parasites from mummies and coprolites: an epidemiological approach.

In the field of archaeological parasitology, researchers have long documented the distribution of parasites in archaeological time and space through the analysis of coprolites and human remains. This area of research defined the origin and migration of parasites through presence/absence studies. By the end of the 20th century, the field of pathoecology had emerged as researchers developed an interest in the ancient ecology of parasite transmission. Supporting studies were conducted to establish the relationships between parasites and humans, including cultural, subsistence, and ecological reconstructions. Parasite prevalence data were collected to infer the impact of parasitism on human health. In the last few decades, a paleoepidemiological approach has emerged with a focus on applying statistical techniques for quantification. The application of egg per gram (EPG) quantification methods provide data about parasites' prevalence in ancient populations and also identify the pathological potential that parasitism presented in different time periods and geographic places. Herein, we compare the methods used in several laboratories for reporting parasite prevalence and EPG quantification. We present newer quantification methods to explore patterns of parasite overdispersion among ancient people. These new methods will be able to produce more realistic measures of parasite infections among people of the past. These measures allow researchers to compare epidemiological patterns in both ancient and modern populations.