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.

Paleoparasitological analysis of the extinct Myotragus balearicus Bate 1909 (Artiodactyla, Caprinae) from Mallorca (Balearic Islands, Western Mediterranean).

Myotragus balearicus (Artiodactyla, Caprinae) is an extinct caprine endemic of the Eastern Balearic Islands or Gymnesics (i.e., Mallorca, Menorca and surrounding islets, Western Mediterranean Sea). In spite of its small size, c. 50cm height at the shoulder, it was the largest mammal inhabiting these islands until the human arrival, and it had peculiar short legs and frontal vision. It disappeared between 2830 and 2210calBCE. The coprolites here studied were recovered from Cova Estreta, in Pollença, Mallorca. The samples were subjected to microscopic examination and enzyme-linked immunosorbent assays (ELISA) for E. histolytica/E. dispar, Giardia intestinalis and Cryptosporidium parvum. This study provides new paleoparasitological data from an extinct animal species of the Holocene period. The microscopy revealed one sample containing uninucleated-cyst of Entamoeba sp., whereas ELISA detected nine positive samples for Cryptosporidium sp. The finding of these protozoans can help in the discussion of its extinction cause and demonstrates the antiquity and the evolutionary history of host-parasite relationships between protozoa and caprines since the Messinian.

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.

Potencialidades da Amazônia para a paleoparasitologia / The potentialities of the Amazon for paleoparasitology

Uma falácia científica muitas vezes leva à falta de interesse em estudar determinadas regiões geográficas no que diz respeito a áreas específicas do conhecimento científico. Este é o caso da Paleoparasitologia na região amazônica, que pretendemos desmitificar com esta revisão. Não é incomum encontrarmos afirmações de que na Amazônia seriam raras as possibilidades de recuperação de vestígios orgânicos preservados, o que limitaria o estudo da Paleoparasitologia. Isso pelo fato de a região haver sido habitada no passado por populações pequenas, o que resultaria em pouco material para o estudo, ou em razão dos baixos índices de preservação do material. Porém, isso não é verdade, uma vez que a região apresenta grande potencial de estudo, sobretudo de vestígios ósseos, mas também do solo arqueológico. A pesquisa deste material traria ganho inestimável para a área e poderia elucidar questões acerca da origem de infecções, se no Velho ou Novo Mundo, do caminho percorrido por elas, das datações e explicações para sua entrada nas Américas. A maioria das infecções que poderiam ser pesquisadas continua presente na região e acomete a população local constituindo, em alguns casos, importantes problemas na área da saúde coletiva. É preciso alertar os pesquisadores e moradores da região que têm conhecimento da existência deste material arqueológico sobre o fato de que, com as técnicas atuais, o menor fragmento ou quantidade do material possibilita seu estudo.

Leishmania tarentolae molecular signatures in a 300 hundred-years-old human Brazilian mummy.

BACKGROUND: L. tarentolae, the lizard-infecting species of Old World geckos, has been classified as non-pathogenic to man. While it has been demonstrated that L. tarentolae is capable of infecting human phagocytic cells and to differentiate into amastigote-like forms, there is no clear evidence for its efficient replication within macrophages. Here we provide first evidence for L. tarentolae ancient DNA sequences from bone marrow and intestines of a 300yo adult male. METHODS: We identified molecular signatures of Leishmania tarentolae, the lizard-infecting species of Old World geckos, in hard and soft tissue biopsies from a Brazilian mummy (A74) uncovered in Itacambira (Brazil) and dating to the Colonial Period (end of 18th/beginning of the 19th century). RESULTS: Our results imply that efficient replication of the parasite occurred within human macrophage and to lead to a systemic spread and visceralization in this individual. The ancient sequences show a 100% similarity with those of isolated L. tarentolae parasites grown on artificial nutrient media and a 99% similarity with two modern sequences isolated from reptiles. CONCLUSIONS: De facto, our findings re-open the debate about the potential survival of ancient L. tarentolae strain within human macrophage and its ability to spread systemically. They also raise ecological issues since it is unknown whether this parasite circulates in the reptilian reservoir in modern day Brazil or not. Investigations on fossil fauna and arthropods are needed to shed light on the interactions between saurian Leishmania and lizards in Brazil's remote and recent past.

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.

PREHISTORICAL Pediculus humanus capitis INFESTATION: QUANTITATIVE DATA AND LOW VACUUM SCANNING MICROSCOPY / Infestação pré-histórica por Pediculus humanus capitis: análise quantitativa e por microscopia de varredura de baixo vácuo

A pre-Columbian Peruvian scalp was examined decades ago by a researcher from the Oswaldo Cruz Foundation. Professor Olympio da Fonseca Filho described nits and adult lice attached to hair shafts and commented about the origin of head lice infestations on mankind. This same scalp was sent to our laboratory and is the subject of the present paper. Analysis showed a massive infestation with nine eggs/cm2 and an impressive number of very well preserved adult lice. The infestation age was roughly estimated as nine months before death based on the distance of nits from the hair root and the medium rate of hair growth. A small traditional textile was associated with the scalp, possibly part of the funerary belongings. Other morphological aspects visualized by low-vacuum scanning electron microscopy are also presented here for adults and nits.

Há décadas um escalpo peruano, datado do período pré-colombiano, foi examinado por um pesquisador da Fundação Oswaldo Cruz. O Professor Olympio da Fonseca Filho descreveu lêndeas e adultos fixos a fios de cabelos e fez comentários sobre a origem da infecção por piolhos na espécie humana. Este mesmo escalpo foi enviado ao nosso laboratório e é objeto deste artigo. Sua análise mostrou maciça infestação, com nove lêndeas/cm2 em impressionante número de adultos muito bem preservados. O tempo de infestação foi estimado em cerca de nove meses antes da morte, baseado na maior distância entre lêndeas e o couro cabeludo, levando em consideração taxa média de crescimento capilar de 1 cm por mês. Um pequeno pedaço de tecido tradicional peruano foi encontrado associado ao escalpo, provavelmente pertencente ao conjunto de peças usado no ritual funerário. Aqui, apresentamos alguns aspectos morfológicos de adultos e lêndeas vizualizados por microscopia eletrônica de varredura de baixo vácuo.

Paleoparasitology: the origin of human parasites / Paleoparasitologia: a origem dos parasitas humanos

Parasitism is composed by three subsystems: the parasite, the host, and the environment. There are no organisms that cannot be parasitized. The relationship between a parasite and its host species most of the time do not result in damage or disease to the host. However, in a parasitic disease the presence of a given parasite is always necessary, at least in a given moment of the infection. Some parasite species that infect humans were inherited from pre-hominids, and were shared with other phylogenetically close host species, but other parasite species were acquired from the environment as humans evolved. Human migration spread inherited parasites throughout the globe. To recover and trace the origin and evolution of infectious diseases, paleoparasitology was created. Paleoparasitology is the study of parasites in ancient material, which provided new information on the evolution, paleoepidemiology, ecology and phylogenetics of infectious diseases.

O parasitismo é composto por três subsistemas: o parasito, o hospedeiro e meio ambiente. Não existe organismo que não possa ser parasitado. A relação entre um parasito e suas espécies de hospedeiro não resulta, na maior parte das vezes, em dano ou doença ao hospedeiro. Entretanto, numa doença parasitária, a presença de um determinado parasito é sempre necessária, pelo menos em um determinado momento da infecção. Algumas espécies de parasitos que infectam humanos foram herdados dos pré-hominídeos, e foram compartilhados com outras espécies de hospedeiro filogeneticamente próximas, mas outras espécies foram adquiridas pelo meio ambiente à medida que os humanos se desenvolveram. As migrações humanas espalharam os parasitos adquiridos por todo o mundo. Para recuperar e traçar a origem e evolução das doenças infecciosas foi criada a paleoparasitologia. Paleoparasitologia é o estudo dos parasitos em materiais antigos, que trouxe novas informações na evolução, paleoepidemiologia, ecologia e filogenética das doenças infecciosas.

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.

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.

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.

A hipótese clássica sobre a origem da doença de Chagas propõe que tenha surgido entre as populações pré-históricas dos Andes quando começaram a domesticar animais, mudaram para hábitos sedentários e adotaram a agricultura. Estas mudanças em seus hábitos de vida aconteceram há aproximadamente 6.000 anos. Entretanto, os dados da paleoparasitologia, baseados na biologia molecular, mostraram que a infecção por Trypanosoma cruzi e a doença de Chagas eram comuns tanto em populações pré-históricas da América do Sul e América do Norte muito antes deste período. De acordo com os dados paleoparasitológicos, a doença de Chagas pode ser tão antiga quanto a presença humana no continente americano. O estudo sobre a origem e dispersão da infecção por Trypanosoma cruzi entre populações humanas pré-históricas pode auxiliar na compreensão de questões clínicas e epidemiológicas sobre a doença de Chagas que ainda permanecem sem resposta.

Fundamentos da paleoparasitologia

Iniciativa inédita no mundo, o livro compila o conhecimento disponível sobre o assunto e apresenta o estado da arte em paleoparasitologia – termo cunhado há cerca de 30 anos por um brasileiro, o pesquisador da Fiocruz Luiz Fernando Ferreira, pioneiro dessa ciência voltada para o estudo dos parasitos no passado. Multidisciplinar por essência, a paleoparasitologia reúne contribuições de cientistas sociais, biólogos, historiadores, arqueólogos, farmacêuticos, médicos e tantos outros profissionais, sejam da área biomédica ou das humanidades. Com aplicações variadas, como nos estudos evolutivos ou das migrações, seus resultados, muitas vezes, dependem da associação entre os achados de laboratório e os vestígios culturais. A obra está dividida em quatro partes - Os Parasitos, Hospedeiros Humanos e o Ambiente; Vestígios de Parasitos Preservados em Diversos Materiais, Técnicas de Microscopia e Diagnóstico Molecular; O Encontro de Parasitos em Material Antigo: uma visão paleogeográfica; e Estudos Especiais e Perspectivas. Assinado por autores de vários países, como Argentina, Estados Unidos, Alemanha e França, o livro tem capítulos dedicados às descobertas da paleoparasitologia em todos os continentes

Fundamentos da paleoparasitologia / Fundamentals of paleoparasitology

A obra está dividida em quatro partes - Os Parasitos, Hospedeiros Humanos e o Ambiente; Vestígios de Parasitos Preservados em Diversos Materiais, Técnicas de Microscopia e Diagnóstico Molecular; O Encontro de Parasitos em Material Antigo: uma visão paleogeográfica; e Estudos Especiais e Perspectivas. Assinado por autores de vários países, como Argentina, Estados Unidos, Alemanha e França, o livro tem capítulos dedicados às descobertas da paleoparasitologia em todos os continentes.