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[Bacterial studies on the occurrence of Mycobacterium paratuberculosis in fecal samples of zoo ruminants].

Mycobacterium (M.) paratuberculosis was isolated from fecal samples of 3 (21.4%) from 14 mouflons, of 10 (20.4%) from 49 dwarf goats, of 5 (14.3%) from 35 Cameroon sheep and of 1 (9.1%) from 11 alpine ibex. M. paratuberculosis could not found by cultural method in fecal samples of 22 Pinzgauer goats, of 15 bantengs, of 9 wild goats, of 9 skuddens, of 6 four-horned sheep, of 3 red-head sheep, and of 1 chamois. From all 19 animals with cultural positive fecal samples complement binding antibodies against M. paratuberculosis could not be found in the corresponding serum samples. The results confirm that M. paratuberculosis is more frequently in small zoo ruminants than up to now was suspected. The cultural examination of fecal samples has been proved to be a better method for detecting animal excretors than serological investigations by means of the complement fixation test.

Animals

Comparison of results using electron microscope, immunodiffusion and fluorescent antibody analyses to detect rotavirus in diarrheic fecal samples of calves.

Seventy-nine diarrheic calf fecal samples were examined by electron microscopy, immunodiffusion and the fluorescent antibody technique for the presence of rotavirus (reovirus-like agent). Thirty-eight (48%) of the samples were positive by electron microscopy, 59% by immunodiffusion and 20% positive by fluorescent antibody technique analyses. Another 9% were suspect-positive by fluorescent antibody technique. Chymotrypsin treatment of the fecal samples increased the ease of observing the viral particles by electron microscopy and also intensified the immunodiffusion arcs obtained. Immunodiffusion analyses using specific antisera to the virus would appear to be a practical method of detecting rotavirus in diarrheic fecal samples.

Animals

Metaproteomic Analysis to Assess the Impact of Storage Media on Human Gut Microbiome in Fecal Samples.

The human gut microbiome is a diverse community of microorganisms residing in the gastrointestinal tract. The storage condition of fecal samples may impact the taxonomic and protein compositions of microbiomes in these samples. Here, we performed a mass spectrometry-based metaproteomic study to assess the impact of storage media on human gut microbiome in fecal samples. We evaluated FDA-authorized OMNIgene·GUT (OG), phosphate-buffered saline (PBS), and RNALater (RNAL) buffers and identified 38,185 microbial peptides corresponding to 7348 microbial proteins, which matched 16 phyla, 20 classes, 50 orders, 104 families, 332 genera, and 453 species. We found a high similarity among the fecal microbiomes preserved in OG, PBS, and RNAL in terms of the identification of proteins, taxa, and functional annotations. Both alpha and beta diversity suggested the high similarity among samples stored in the three media. Nonetheless, we also found some notable differences among buffers regarding the abundances of a few taxon groups. A partial human proteome (over 400 proteins) was identified in the fecal samples, with most of these proteins associated with the membrane and extracellular regions. The findings indicate the similarity among microbiomes in the fecal samples stored in OG, PBS, and RNAL regarding proteome profile, taxa, and functional capacity. SUMMARY: This study thoroughly analyzed and compared the metaproteomes of fecal samples preserved at -80°C in PBS, RNALater, and OMNIgene·GUT Dx buffers, offering novel insights into the effectiveness of these buffers in maintaining the stability and composition of the human gut microbiome. We found a high similarity in the identification and quantification of proteins, taxa, and functional annotations across the three buffers, with notable quantitative differences highlighting subtle yet important variations in preservation efficacy. The unique datasets and findings could offer valuable revelations into the impact of fecal sample preservation on translational and clinical analyses of the human gut microbiome.

Humans

The development and standaridization of an ELISA method for the detection of Entamoeba histolytica antigens in fecal samples.

Recognizing the difficluties associated with the microscopic detection and identification of Entamoeba histolytica in fecal samples, an immunochemical method of detection of E. histolytica antigens in fe-es has been developed. For a number of reasons, e.g., the known fragility of E. histolytica trophozoites, it was felt that intestinal amebiasis would be associated with detectable levels of E. histolytica anti-ens in feces from infected patients. A technique for harvesting amebic antigens in fecal samples was developed. An enzyme labeled antibody technique was used to visualize the harvested antigens. The inherent problems of performing any immunochemical assay in fecal samples have been addressed. The enzyme labeled antibody method is simple and is designed to be more sensitive than microscopic stool examination methods. Preliminary results indicate that the method is specific and sensitive.

Animals

Detection of Clostridium perfringens enterotoxin in human fecal samples and anti-enterotoxin in sera.

By using counterimmunoelectrophoresis (CIEP), Clostridium perfringens enterotoxin was successfully demonstrated in fecal samples collected within 1 day of attack from sick individuals involved in a bacteriologically and epidemiologically proven outbreak of C. perfringens food poisoning. In contrast, enterotoxin was not demonstrable in fecal samples of apparently healthy individuals both at high- and low-risk exposure to the organism and enterotoxin or in fecal samples collected 4 to 5 days after a food poisoning outbreak. A 100% prevalence of C. perfringens anti-enterotoxin in sera of human volunteers at high- as well as low-risk exposure to the organism and enterotoxin was recorded with CIEP.

Allied Health Personnel

Antibiotic resistance of Escherichia coli in fecal samples of healthy people in two different areas in an industrialized country.

Fecal samples of 310 healthy persons, from two populations from different areas in the Netherlands, were examined for the presence of Escherichia coli resistant to ampicillin, tetracycline, sulfamethoxazole, trimethoprim and nitrofurantoin. High prevalences of resistance were found in both populations, ranging from 28% for trimethoprim to 89% for ampicillin. The percentages of the fecal samples with a dominantly resistant E. coli flora (> 50% resistance) were distinctly lower, ranging from 1% for nitrofurantoin to 21% for tetracycline. No significant differences in the level of resistance were observed between these two comparable populations in two different areas. The susceptibilities to 11 antimicrobial agents of 456 at random isolated E. coli were determined. The percentages of resistance varied widely: from 80% for chloramphenicol to 9% for nitrofurantoin. Only 19% of the isolates were susceptible to all antibiotics tested and 14% were resistant to more than four of the agents tested. Great differences in resistance rates between the two populations examined were seen for chloramphenicol (80% to 41%) and trimethoprim (16% to 36%). The results of this study underscore the presence of a human reservoir of antibiotic resistant microorganisms.

Adolescent

Assessment of mean oocyst count in groups of kids: litter, individual randomized and non-randomized fecal samplings.

A trial was conducted in six pens of kids visited five times monthly to compare different regimes of fecal sampling in order to assess average coccidial infection at a group level. In addition to individual fecal samplings of all the kids in the pen, giving the actual mean oocyst count, three sampling methods were performed: (i) individual sampling of three, five, seven, ten, 15, 20 or more animals according to the numbers of animals in the pen; (ii) sampling of feces in the litter; (iii) individual sampling of six kids, three with the poorest and three with the highest daily weight gain. Results indicated that drawing a random sample of ten kids was necessary to obtain a good estimation, with an accuracy of 25% in more than 90% of pens. Sampling of feces in the litter and individual sampling of six kids selected on daily weight gain considerations gave accurate estimation of the actual mean oocyst counts except in the case of high levels of infection. Further investigations would be needed to assess the diagnostic value of such sampling techniques whatever the coccidial infection level.

Age Factors

[Isolation of Cryptosporidium sp. oocysts from human fecal samples].

Experiments using two sequential discontinuous sucrose gradients, performed with 12%, 18%, 21% and 24% solutions, for the isolation of Cryptosporidium sp. oocysts from human fecal samples were undertaken. The sucrose gradients were centrifuged at 4000 rpm during 20 min. at 10 degrees C or room temperature. After that, 3 bands were observed. Oocysts were recovered mainly from the second band (75.5% after the first gradient, and 44.4% in the next). The two sequential discontinuous sucrose gradients would permit an efficient isolation of Cryptosporidium sp. oocysts from human fecal samples.

Animals

The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples.

Direct polymerase chain reaction (PCR)-based detection with fecal specimens is hampered by inhibitory compounds, such as bilirubin and bile salts. These fecal compounds showed significant inhibition of PCR at low concentrations (10 to 50 micrograms/ml). For direct PCR analysis, fecal samples must be diluted 500-fold to overcome inhibition. Therefore, the magnetic immuno PCR assay (MIPA), which combines immunomagnetic separation by using specific monoclonal antibodies and PCR, was used to directly detect salmonellae in feces from humans. Immunomagnetically extracted stool samples needed to be diluted only 10-fold when 1 microgram of T4 gene 32 protein was added to the PCR. The MIPA sensitivity obtained was 10(5) CFU/ml of feces. A panel of monoclonal antibodies specific for Salmonella serogroups A to E was used to extract salmonellae from clinical samples. MIPA detection of salmonellae occurred with 11 out of 14 stool samples stored at 4 degrees C for 2 months. MIPA detection of salmonellae in stool samples is a promising, fast method for detection and identification.

Antibodies, Monoclonal

A simple method for cloning Giardia duodenalis from cultures and fecal samples.

Using a novel method for cloning Giardia duodenalis from cultures and fecal samples, 47 clones from 7 isolates were established in vitro. Average colony-forming efficiency in established cultures was 43.2% compared to 11.2% when cloning directly from excystation. The highest success rate of cloning was found with the Portland (P1, ATCC No. 30888) isolate, with a colony-forming efficiency of 92.7%. Cloned and parent populations were compared over a range of 13 enzymes using starch gel electrophoresis. No genetic difference was found between any of the clones and the parent isolates.

Animals

Separation and properties of enterovirus and reovirus recovered from a fecal sample of calf with diarrhea.

In April, 1971, a disease with pyrexia and diarrhea as main symptoms broke out collectively among calves. Fecal samples were collected from calves involved and inoculated into bovine kidney (BK) cell cultures. As a result, the diarrheal feces of one calf were suspected to contain two agents simultaneously. One agent (C-121 E strain) was isolated from the primary infected BK cell culture fluid by terminal dilution passages. It had been predominant in replication and shown a cytopathic effect which gave rise to a granular appearance in the early stage of culture. The other agent (C-121 R strain) was isolated from the primary infected BK cell culture fluid by neutralizing the C-121 E strain contained in this fluid with antiserum against this strain. It caused cytoplasmic inclusion bodies to form. On the basis of their physico-chemical properties, the C-121 E strain was identified as bovine enterovirus and the C-121 R strain as reovirus. Serological tests indicated that some of the affected calves had been infected not only with the two strains isolated, but also with bovine viral diarrhea virus, bovine adenovirus type 7, and bovine parvovirus.

Animals

Gas-liquid chromatographic analysis of volatile short chain fatty acids in fecal samples as pentafluorobenzyl esters.

A protocol was developed for the analysis of volatile short chain fatty acids in microsamples of feces, short chain fatty acid (SCFA) extraction was from fecal samples using ethanol incorporating n-hexanoic acid as an internal standard. The SCFAs were converted to pentafluorobenzyl esters with alpha-2,3,4,5,6-pentafluorotoluene and analyzed on a gas-liquid chromatograph equipped with an electron capture detector. One hundred milligrams of sample was routinely used but analysis could be carried out on 20 mg of sample.

Animals

Identification of aviadenovirus and dependoparvovirus in an Adélie penguin fecal sample from Cape Royds (Ross Island, Antarctica).

Adenoviruses (Adenoviridae) and their associated replication-deficient dependoparvoviruses (Parvoviridae) are recognized as significant components of the avian virome. While well-studied in domestic birds, these viruses remain underexplored in wild Antarctic birds. Information is limited on viruses circulating in penguins, particularly outside of the Antarctic Peninsula Region. Here we leverage a fecal sample collected for diet analyses to identify an aviadenovirus (37,731 nt) and two distinct dependoparvoviruses. The Ad&#xe9;lie penguin (Pygoscelis adeliae) feces-derived adenovirus exhibits a genome organization consistent with members of the genus Aviadenovirus but shares only ~64% amino acid identity in the DNA polymerase protein with its closest known relative and thus represents a new species of aviadenovirus identified in penguins. Additionally, we identified two new dependoparvoviruses, one in length of 4,869 nt and a second of 4,162 nt. These two dependoparvoviruses are diverse, sharing <61% Rep amino acid identity with those of other dependoparvoviruses. Penguin dependoparvovirus 1 represents a new species. The co-detection of these viruses in a single seasonal sample could suggest a potential helper-satellite relationship in Ad&#xe9;lie penguins. This study provides the first evidence of aviadenoviruses in Ad&#xe9;lie penguin feces. These findings highlight the importance of surveillance to understand pathogen dynamics in isolated Antarctic populations.

Animals

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.

Animals

[Evaluation of the immunoenzyme test (Elisa) in detecting Clostiridium difficile toxin A in fecal samples].

Currently, the method of choice in diagnosis of Clostridium difficile-associated intestinal diseases is the detection of toxin B in fecal specimens. This method is long (72 h) and can be realized in laboratories which have tissue culture facilities. Commercial agglutination test have been evaluated but they lack in specificity. An immunoenzymatic test has been recently commercialized for detection of toxin A. We have compared the results of this assay on 275 fecal specimens from patients suspected of having Clostridium difficile-associated intestinal diseases with the results obtained with the cytotoxicity test and the culture. Of the 275 fecal specimens, 58 were positive in cytotoxicity and 53 in Elisa. The overall sensitivity and specificity of the Elisa compared with cytotoxicity were 89.5% and 99.0% respectively. The immunoenzymatic test detecting Clostridium difficile toxin A is an easy test to perform in 2 h 15; it displays a good correlation with detection of toxin B and can be very useful in daily laboratory diagnosis.

Bacterial Toxins