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.

Feline heartworm (Dirofilaria immitis) infection: first case report of serological diagnosis in Brazil, confirmed by molecular assay.

The clinical importance of heartworm infection in cats has indeed increased in recent years. Dirofilaria immitis infection has been reported worldwide in cats and continues to be regularly diagnosed in endemic areas. The diagnosis can be overlooked easily, especially in Brazil, where there is not a specific feline immunodiagnostic test, forcing the veterinarians to use a test made for the canine host. In 2015, a 10-year-old female neutered cat was diagnosed with D. immitis using an antigen serological test, based on imunocromatography and designed for dogs. The modified Knott test was negative. As the disease progressed, the cat showed clinical signals of respiratory distress, such as dyspnoea and polypnea in addition to prostration and emaciation, and died a few weeks after the diagnosis. During necropsy, one adult nematode was found in the pulmonary artery. D. immitis infection was confirmed by molecular amplification, performed in the worm fragment. This is the first report of serological diagnosis of feline dirofilariasis in Brazil. A chemoprophylaxis routine in cats should be done, as is done in dogs from endemic areas.

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.

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.

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.

Evaluation of the bioremoval of Cr(VI) and TOC in biofilters under continuous operation using response surface methodology.

In the present study, the bioremoval of Cr(VI) and the removal of total organic carbon (TOC) were achieved with a system composed by an anaerobic filter and a submerged biofilter with intermittent aeration using a mixed culture of microorganisms originating from contaminated sludge. In the aforementioned biofilters, the concentrations of chromium, carbon, and nitrogen were optimized according to response surface methodology. The initial concentration of Cr(VI) was 137.35 mg l(-1), and a bioremoval of 85.23% was attained. The optimal conditions for the removal of TOC were 4 to 8 g l(-1) of sodium acetate, >0.8 g l(-1) of ammonium chloride and 60 to 100 mg l(-1) of Cr(VI). The results revealed that ammonium chloride had the strongest effect on the TOC removal, and 120 mg l(-1) of Cr(VI) could be removed after 156 h of operation. Moreover, 100% of the Cr(VI) and the total chromium content of the aerobic reactor output were removed, and TOC removals of 80 and 87% were attained after operating the anaerobic and aerobic reactors for 130 and 142 h, respectively. The concentrations of cells in both reactors remained nearly constant over time. The residence time distribution was obtained to evaluate the flow through the bioreactors.

Back to basics: Could simple experiments resolve important parasitology enigmas? The rarity of Ascaris eggs compared with Trichuris eggs in archeology and other contexts.

Most parasitologists face a conundrum throughout their careers: 'which parasite eggs are more resistant to environmental stress, Ascaris spp. or Trichuris spp.?' In this analysis, our experimental and statistical analyses demonstrated that Trichuris sp. is more resistant than Ascaris sp. We highlight that desiccation exerts a major effect on the conservation of Ascaris eggs, and this may result in an underestimation of Ascaris eggs in paleoparasitological records. This observation can be extrapolated to more modern scenarios, for example, parasitological research in animal feces from semiarid environments, where whipworms are more common than roundworms. Similarly, this could be a plausible explanation for the higher frequency and abundance of whipworms than roundworms, when other hypotheses are unsupported.

PARASITES OF THE BRAZILIAN ROCK CAVY, KERODON RUPESTRIS: REVEALING THEIR HISTORY IN THE BRAZILIAN SEMIARID REGION.

Using microscopy and/or immunodiagnosis, the authors analyzed 284 fecal samples from the Brazilian rock cavy, Kerodon rupestris, that were collected between 1984 and 2015 in Serra da Capivara National Park for the presence of helminths and protozoa. Fourteen morphospecies of helminth eggs of the following taxa were found: Trematoda, Nematoda, Strongylidae, Lagochilascaris sp., Strongylida, Trichuris (2 species), Oxyuridae (3 species), Ancylostomatidae (2 species), and Ascarididae (2 species), along with 3 protozoan taxa: Coccidia, Cryptosporidium sp., and Balantidium sp. During the last 30 yr, the population of K. rupestris has increased in the region as a consequence of the creation and management of the National Park, and data from this study show a concurrent increase in the diversity of intestinal parasites in this host, including new reports. Some of these species have zoonotic potential, which suggests that K. rupestris may be in contact with domestic farm animals and/or human feces. These results show the importance of integrating different diagnostic approaches for the identification of protozoa in the region and indicate that further methods need to be employed to increase recovery. This work highlights the usefulness of parasite studies in assessing the health of ecosystems, especially in protected areas, which should be considered by park managers and health agencies.

REVIEW OF PARASITES FOUND IN EXTINCT ANIMALS: WHAT CAN BE REVEALED.

Parasitism is inherent to life and observed in all species. Extinct animals have been studied to understand what they looked like, where and how they lived, what they fed on, and the reasons they became extinct. Paleoparasitology helps to clarify these questions based on the study of the parasites and microorganisms that infected those animals, using as a source material coprolites, fossils in rock, tissue, bone, mummy, and amber, analyses of ancient DNA, immunodiagnosis, and microscopy.

Paleoparasitologic, paleogenetic and paleobotanic analysis of XVIII century coprolites from the church La Concepción in Santa Cruz de Tenerife, Canary Islands, Spain.

We present the results of a paleoparasitologic, paleogenetic and paleobotanic analysis of coprolites recovered during the excavation of the church La Concepción in Santa Cruz de Tenerife. Coprolites (n = 4) were rehydrated and a multidisciplinary analysis was conducted. The paleobotanic analysis showed numerous silicates, seeds and fruits of the family Moraceae. In the paleoparasitologic study, Ascaris sp. eggs (n = 344) were identified. The paleogenetic results confirmed the Ascaris sp. infection as well as the European origin of human remains. These findings contribute to our knowledge of ancient helminthes infections and are the first paleoparasitological record of Ascaris sp. infection in Spain.

Chewing Lice of Fregata magnificens with First Record of Fregatiella aurifasciata (Phthiraptera: Amblycera) in Brazil.

The genus Fregata includes 5 species, with 3 recorded in Brazil, with Fregata magnificens being the most abundant. However, its ectoparasitic fauna is still little known. This study aimed to evaluate the incidence of ectoparasites of F. magnificens residing along the coast of Rio de Janeiro and São Paulo collected by 2 animal rehabilitation centers. Samples were collected from 5 frigatebirds of the Instituto Argonauta in São Paulo and 10 frigatebirds of the Centro de Recuperação de Animais Selvagens (CRAS) in Rio de Janeiro. Species of lice were identified using both morphological and molecular methods. Scanning electron microscopy was also used for identification. Colpocephalum spineum, Fregatiella aurifasciata, and Pectinopygus fregatiphagus were identified. All 3 louse species have previously been recorded from this host outside Brazil, but only P. fregatiphagus has been recorded from Brazil. This paper reports the first occurrence of F. aurifasciata and C. spineum in Brazil. It is also the first record of P. fregatiphagus in the state of Rio de Janeiro.

Detection of Dirofilaria immitis using microscopic, serological and molecular techniques among dogs in Cabo Frio, RJ, Brazil.

Heartworm disease is a health problem for dogs and cats, especially in tropical and subtropical coastal regions of the world. Some studies have compared the efficacy of the diagnostic techniques used to detect this parasitosis. Therefore, the aim of this study was to compare parasitological optical microscopy (POM), serological and molecular techniques for diagnosing canine heartworm infection. Samples were collected between July 2015 and April 2016 from 103 dogs in Cabo Frio, RJ, Brazil. The wet fresh blood, thick smears, thin smears and modified Knott's test were used to detect microfilariae. ELISA (Snap™ 4Dx ® IDEXX) was used to detect antigens and the polymerase chain reaction (PCR) was used to detect DNA and enable sequencing for species differentiation and confirmation. 19.4% of samples were positive according to microscopy. Through PCR, 15.5% of the total were positive. Using ELISA, the positivity rate was 29.1%. Occult heartworm infection was detected in 11.6% of the samples. ELISA sensitivity was shown to be higher than PCR or microscopy (P = 0.001). Sequencing of samples confirmed the presence of Dirofilaria immitis and Acanthocheilonema reconditum . ELISA was more effective for serological diagnosis canine heartworm and should be used in clinical and epidemiological studies.

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.

Increasing the Efficiency of Spontaneous Sedimentation for the Diagnosis of Intestinal Protozoa.

The aim of this study was to evaluate the association between a longer sedimentation time, the reading of a larger number of slides, and the collection of multiple samples on the efficiency of the spontaneous sedimentation technique. Twenty-two patients with a previous parasitological exam positive for intestinal protozoa were recruited to collect new fecal samples (3 samples per patient) before the beginning of antiparasitic treatment. All collected fecal samples were used for spontaneous sedimentation and centrifuge-flotation techniques. Of these, all 22 patients were positive based on spontaneous sedimentation, and 59.1% (13/22) based on centrifuge flotation. The number of samples and the number of slides analyzed by spontaneous sedimentation influenced the number of positive cases. The modifications applied to the spontaneous sedimentation technique increased its performance in protozoa diagnosis.

Pentatrichomonas hominis infection in two domestic cats with chronic diarrhea.

CASE SERIES SUMMARY: Chronic diarrhea is a common and recurring problem in feline medicine. Intestinal trichomonads have been reported as causative agents of diarrhea in cats. While Pentatrichomonas hominis is considered commensal, Tritrichomonas foetus has been found to cause feline large bowel diarrhea in cats. In our report, two young cats infected with the feline leukemia virus (FeLV) and presenting with chronic diarrhea were identified as having trichomonads in their feces, based on direct examination and fecal culture. Molecular assays (PCR and DNA sequencing) revealed that the parasite involved was P hominis, not T foetus, as was suspected. The animals had already been subjected to ineffective therapy with metronidazole, and after the use of ronidazole, their feces became dry and formed. RELEVANCE AND NOVEL INFORMATION: This case report describes P hominis infection as a possible cause of chronic diarrhea in two young cats also infected by FeLV. The parasite was probably resistant to metronidazole, the drug of choice in the literature, and sensitive to ronidazole. Although considered commensal, P hominis infection should be evaluated as a differential diagnosis in cats with chronic diarrhea, especially those that are immunocompromised. Moreover, a combination of techniques such as direct examination and/or fecal culture and PCR is essential for an accurate diagnosis of P hominis infection.

Methodological innovations for the study of irreplaceable samples reveal giardiasis in extinct animals (Nothrotherium maquinense and Palaeolama maior).

The use of diagnostic methods that prevent irreplaceable samples (from museum collections, archaeological and paleontological samples) of being consumed or that increase their yield is relevant. For museum collections, archaeological and paleontological samples it is essential to conserve samples, subsamples or portions for future research. We are addressing methods for conservation of irreplaceable samples that could be fully consumed. Innovations in methodologies that are used in studies of Paleoparasitology and Paleomicrobiology will contribute to the preservation of collections. Therefore, to the development of archaeology and paleontology in the future, we evaluated whether the discarded material of the immunochromatography test could be used for molecular diagnosis and vice versa. We used a genotyped experimental coprolite positive for Giardia duodenalis. The diagnosis was positive for giardiasis in both cases. This methodology can be corroborated with the coprolite of a Paleolama maior (extinct llama) previously diagnosed for G. duodenalis with an immunoenzymatic test. The residue of the pre-digestion step of the DNA extraction before adding Proteinase K was confirmed positive with the immunochromatographic test. Also, the DNA extraction residue from a coprolite of Nothrotherium maquinense (ground sloth) was tested positive with immunochromatographic test for G. duodenalis. These are the oldest findings for G. duodenalis confirming that this intestinal parasite occurred among Northeastern Brazilian Megafauna animals from the late Pleistocene period, correlated to human occupation. The relevance of these results will allow the study by different methodological approaches from a small amount of material, reusing discarded materials.

Study of infectious diseases in archaeological bone material - A dataset.

Bones of human and ground sloth remains were analyzed for presence of Trypanosoma cruzi by conventional PCR using primers TC, TC1 and TC2. Sequence results amplified a fragment with the same product size as the primers (300 and 350pb). Amplified PCR product was sequenced and analyzed on GenBank, using Blast. Although these sequences did not match with these parasites they showed high amplification with species of bacteria. This article presents the methodology used and the alignment of the sequences. The display of this dataset will allow further analysis of our results and discussion presented in the manuscript "Finding the unexpected: a critical view on molecular diagnosis of infectious diseases in archaeological samples" (Pucu et al. 2017) [1].