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PubMed1984

Blood-free selective medium for isolation of Campylobacter jejuni from feces.

A blood-free selective agar is described which contains charcoal, ferrous sulfate, sodium pyruvate, casein hydrolysates, cefazolin, and sodium deoxycholate (CCD agar). CCD agar was compared with Preston medium for isolation of Campylobacter jejuni from human feces, and isolation rates were similar on both media, but CCD agar was less selective. Temperature studies at 37 and 42 degrees C confirmed that incubation of direct plates at 42 degrees C for 48 h was necessary for maximum isolation of C. jejuni.

Agar↗
PubMed1984

Selective medium for isolation of Clostridium butyricum from human feces.

A selective medium, Clostridium butyricum isolation medium (BIM), is described for the isolation of C. butyricum from human feces. The BIM is a synthetic minimal medium and contains trimethoprim (16 micrograms/ml), D-cycloserine (10 micrograms/ml), and polymyxin B sulfate (20 micrograms/ml) as selective inhibitory agents. Qualitative tests indicated that C. butyricum and other butyric acid-producing clostridia grew on BIM, Clostridium sphenoides and Bacillus cereus produced small colonies, and other clostridia and other obligate anaerobic or facultatively anerobic bacteria were inhibited. Quantitative recovery of C. butyricum from cultures or seeded fecal samples was comparable with BIM and with complex medium, but the quantitative recovery of the other butyric acid-producing clostridia tested (C. beijerinckii, C. acetobutylicum) was lower with BIM than with complex medium. The BIM should aid the rapid isolation of C. butyricum from fecal samples and should be useful for bacteriological investigation of neonatal necrotizing enterocolitis.

Anti-Bacterial Agents↗
PubMed1986

Microbiological and biochemical characterization of spirochetes isolated from the feces of homosexual males.

Spirochetes were isolated from the feces of 11 homosexual males who had diarrhea. The anaerobic organisms were isolated from a selective medium that consisted of Trypticase soy agar supplemented with either 5% horse or human blood, 400 micrograms of spectinomycin per ml, and 5 micrograms of polymyxin B per ml. Nonselective media that permitted good growth of these fastidious organisms were developed, and selected biochemical tests were performed. The tests included carbohydrate utilization, detection of certain enzymes, and determination of volatile fatty-acid end products of metabolism. Two growth patterns were noted on solid media, a haze of growth and production of small colonies. Based on the results of biochemical tests, patterns of preformed enzymes, and volatile fatty-acid production, we believe that the 11 isolates represent a heterogeneous group of spirochetes. The data suggest that the human colon may harbor unique strains of cultivable spirochetes; additional study of the taxonomy of the organisms and assessment of their virulence for humans are needed.

Culture Media↗
PubMed1987

Usefulness of an enzyme-linked immunosorbent assay for detection of Giardia antigen in feces.

The usefulness of a recently developed enzyme-linked immunosorbent assay which detects Giardia lamblia antigen in feces was determined in experimentally infected humans. Giardia antigen was determined in serially collected fecal specimens from humans inoculated with two Giardia isolates, GS/M and Isr. A total of 277 stools from 18 volunteers were tested, 74 from Isr-inoculated volunteers and 203 from GS/M-inoculated volunteers. None of the five Isr-inoculated volunteers became infected, and none of their stools contained Giardia antigen. In contrast, all of the 13 GS/M-inoculated volunteers became infected, and Giardia antigen was present one or more times in the stools of each. Of 203 stools from GS/M-inoculated volunteers, 73 contained Giardia cysts, and 69 of these (94.5%) had detectable antigen. In contrast, 108 of the 203 specimens were positive for Giardia antigen and only 73 had cysts. Most antigen-positive but cyst-negative specimens occurred during treatment, but during patency 71 stools contained antigen and 65 had cysts. The enzyme-linked immunosorbent assay is infections and is easier to perform.

Antigens, Protozoan↗
PubMed1988

Urease-positive thermophilic Campylobacter (Campylobacter laridis variant) isolated from an appendix and from human feces.

Urease-positive thermophilic campylobacters were isolated for the first time from the feces of two adults with diarrheal disease and from the appendix of a child with appendicitis. They were identified as Campylobacter laridis by a hybridization dot blot assay. Urease testing should be included in the tests used for the identification of campylobacters at the species level, even for those strains which are not of gastric origin.

Appendix↗
PubMed1989

Direct isolation of atypical thermophilic Campylobacter species from human feces on selective agar medium.

Campylobacter upsaliensis is the name which has been proposed for a new group of thermophilic campylobacter strains which differ from C. jejuni and C. coli in having a negative or weak catalase reaction. Primary isolation of these strains from human feces has been achieved only by use of filtration techniques. We report here direct isolation of strains corresponding to C. upsaliensis from stools of six children. The strains were isolated on a newly described campylobacter-selective medium. The strains were oxidase positive, hippurate negative, nitrate positive, negative for H2S in triple sugar iron, and susceptible to cephalothin (30-micrograms disk) and nalidixic acid (30-micrograms disk), and they grew at 37 and 43 degrees C, but not at 25 degrees C. The selective medium used was a blood-free, charcoal-based medium consisting of Columbia agar base, activated charcoal, cefoperazone (32 micrograms/ml), vancomycin (20 micrograms/ml), and cycloheximide (100 micrograms/ml). The medium supported the growth of the weakly reacting or catalase-negative strains, with colony counts equivalent to those obtained on antibiotic-free horse blood agar. These strains could not be isolated directly from stool on Skirrow medium, and colony counts confirmed that this medium could not support a low inoculum of these organisms. The clinical significance of these strains is unknown. We conclude that C. upsaliensis can be isolated directly from stool by using a selective medium, without the need for filtration.

Agar↗
PubMed1990

Identification of Campylobacter cinaedi isolated from blood and feces of children and adult females.

Five Campylobacter-like organisms isolated from blood and feces were identified by numerical analysis of gel electrophoretic protein profiles and immunotyping as Campylobacter cinaedi. Two of these strains were isolated from adult females; the remaining three strains were isolated from children, two of whom were girls. C. cinaedi has hitherto been isolated only from rectal swabs and blood of homosexual and bisexual males with gastrointestinal symptoms. The results presented extend our knowledge of the features and the habitat of C. cinaedi.

Adult↗
PubMed1991

Prevalence of virulent Rhodococcus equi in isolates from soil and feces of horses from horse-breeding farms with and without endemic infections.

The prevalence of virulent Rhodococcus equi in isolates from soil and feces of foals on a farm with endemic R. equi infections was significantly higher than that of a farm with no history of the disease. Foals bred on a farm with the endemic disease might be constantly exposed to virulent R. equi in their environment.

Actinomycetales Infections↗
PubMed1992

Detection of Shigella dysenteriae type 1 and Shigella flexneri in feces by immunomagnetic isolation and polymerase chain reaction.

A combination of immunomagnetic separation (IMS) and a polymerase chain reaction (PCR) procedure was used for direct isolation and identification of Shigella dysenteriae type 1 and Shigella flexneri from feces. Immunomagnetic particles were coated with monoclonal antibody MASFB, which is specific for a common epitope of the O polysaccharides of S. dysenteriae type 1 and S. flexneri. Bacteria bound to the beads were boiled in water, and target DNA was amplified with a primer pair specific for a gene coded on the invasion-associated locus (ial) of the large virulence plasmid of all four Shigella spp. and enteroinvasive strains of Escherichia coli. A 320-bp DNA fragment was generated and detected by an alkaline phosphatase-conjugated probe. Nonviable cells were also captured and detected by this technique. The method is simple and fast (7 h) and has a detection limit of ca. 10 Shigella organisms per g in fecal samples. The combined IMS-PCR assay correctly identified all 57 samples carrying S. dysenteriae type 1 and 68 samples carrying S. flexneri from 238 fecal specimens and also permitted detection of 17 samples carrying Shigella spp. from 113 specimens from diarrheal patients in whom shigellae were not detected by conventional culture.

Antibodies, Monoclonal↗
PubMed1992

Detection of genes in feces by booster polymerase chain reaction.

A 321-bp fragment intragenic to the gene ereA carried by Escherichia coli BM2195 was used as a model target to study the conditions under which DNA amplification by booster polymerase chain reaction can be used to detect specific bacterial DNA sequences in fecal specimens. When target E. coli cells were mixed with 41 freshly obtained fecal specimens, the polymerase chain reaction detection limit varied from 4.5 to 7.1 log CFU/g of feces, depending on the individual fecal specimen used to prepare the test sample. These variations were not statistically related to the sex or age of the subject from whom the specimen was obtained. After storage of the samples for 4 weeks at room temperature on swabs or filter papers, no loss in sensitivity was observed.

Base Sequence↗
PubMed1993

Differentiation of pathogenic Entamoeba histolytica infections from nonpathogenic infections by detection of galactose-inhibitable adherence protein antigen in sera and feces.

We determined whether epitope-specific monoclonal antibodies to the galactose-inhibitable adherence protein (GIAP) of Entamoeba histolytica could be used in an enzyme-linked immunosorbent assay (ELISA) to detect antigen in serum and feces and differentiate between nonpathogenic zymodemes and the potentially invasive pathogenic organisms that require treatment. Overall, 57% of subjects from Cairo, Egypt, with symptomatic intestinal amebiasis and 42% with asymptomatic infection possessed GIAP antigen in their sera, whereas 4% of uninfected controls or subjects with other parasitic infections possessed GIAP antigen in their sera (P < 0.001). In subjects from Durban, South Africa, only 6% of uninfected controls or those with nonpathogenic E. histolytica infection were positive for GIAP in serum, whereas 3 of 4 with asymptomatic pathogenic intestinal infection and 75% with amebic liver abscess were positive for GIAP in serum. Fifteen stool samples from patients with intestinal amebiasis were available for study; all had a positive ELISA result for fecal GIAP antigen. Epitope-specific monoclonal antibodies identified 8 of 15 subjects with fecal antigen from pathogenic strains. Seven of those eight subjects had adherence protein antigen in their sera, whereas none of seven with apparent nonpathogenic E. histolytica infection had adherence protein antigen in their sera. In summary, we were able to detect E. histolytica adherence protein antigen directly in serum and fecal samples by ELISA. The presence of amebic antigen in serum demonstrated 94% specificity for pathogenic E. histolytica infection, and amebic antigen is present during asymptomatic intestinal infection. In conjunction with antibody detection, this method should be very useful in the diagnosis and management of intestinal amebiasis.

Animals↗
PubMed1994

Cryptosporidium antigen detection in human feces by reverse passive hemagglutination assay.

A reverse passive hemagglutination (RPH) assay was developed for Cryptosporidium oocyst antigen with an antioocyst monoclonal antibody (MAb; MAb-C1) coupled to stabilized sheep erythrocytes. RPH was compared with microscopy of auramine-phenol-stained smears of 56 oocyst-positive fecal samples, each of which was tested blindly by RPH with two oocyst-negative samples received on the same day (a total of 112 controls). Thirty-nine additional fecal samples from human immunodeficiency virus type 1 antibody-positive patients with diarrhea (10 of which were positive in auramine-phenol-stained smears) were stored at -20 degrees C before testing. Thirty specimens with a variety of other fecal pathogens (all negative for oocysts) were also tested. Of the 237 samples tested, 69 were positive by one or both methods: 65 by RPH and 66 by microscopy. The kappa coefficient of agreement between the methods was very high at 0.926. The sensitivity of RPH was 93.9%, the specificity was 98.2%, the positive predictive value was 95.4%, and the negative predictive value was 97.7%. Visible oocyst numbers and RPH titers were measured after storage of fecal samples and oocyst concentrates for 8 days at 4 degrees C. Oocyst morphology was generally poor in specimens from the human immunodeficiency virus type 1 antibody-positive group, and it degenerated during the 8-day storage experiments. MAb-C1-reactive antigen eluted from oocysts to give progressively higher reciprocal titers during storage, and it was partially removed from the oocysts by concentration. RPH is a promising technique for the detection of Cryptosporidium antigen in human feces and may be useful when specimens are stored before testing. Studies of the sensitivity of Cryptosporidium immunoassays should take into account the possible release of antigen from oocysts.

Adolescent↗
PubMed1996

Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay.

In order to make a rapid and definite diagnosis of Salmonella enteritis in children, an enrichment broth culture-multiplex PCR combination assay was devised to identify Salmonella serovars directly from fecal samples. Two pairs of oligonucleotide primers were prepared according to the sequences of the chromosomal invA and plasmid spvC genes. PCR with these two primers would produce either one amplicon (from the invA gene) or two amplicons (from the invA and spvC genes), depending on whether or not the Salmonella bacteria contained a virulence plasmid. The fecal sample was diluted 10- to 20-fold into gram-negative enrichment broth and incubated to eliminate inhibitory compounds and also to allow selective enrichment of the bacteria. One or two amplicons were obtained, the expected result if Salmonella bacteria were present. The detection limit of this PCR was about 200 bacteria per reaction mixture. The primers were specific, as no amplification products were obtained with 18 species and 22 isolates of non-Salmonella bacteria tested which could be present in the feces or cause contamination. In contrast, when 23 commonly seen Salmonella serovars (38 isolates) were tested, all were shown to carry the invA gene and seven concomitantly harbored the spvC gene of the virulence plasmid. This assay was applied to the diagnosis of Salmonella enteritis in 57 children who were suffering from mucoid and/or bloody diarrhea. Of the 57 children, 38 were PCR positive and 22 were culture positive. There were two culture-positive samples that were not detected by PCR. Thus, this PCR assay showed an efficiency of 95% (38 of 40), which is much higher than the 60% (24 of 40) by culture alone. Not only is this method more sensitive, rapid, and efficient but it will cause only an incremental increase in the cost of stool processing, since enrichment cultivation of fecal samples from diarrheal patients using gram-negative enrichment broth is a routine practice for identification in many diagnostic microbiology laboratories. This PCR method, therefore, has clinical application.

Bacterial Proteins↗
PubMed1997

An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.

Animals↗
PubMed1999

Isolation of Providencia heimbachae from human feces.

Providencia heimbachae was first described in 1986. It has been isolated from penguin feces and an aborted bovine fetus. To date, there has been no reported isolation of this organism from human specimens. We now report the isolation of P. heimbachae from the stool of a 23-year-old woman with idiopathic diarrhea. The identity of the human strain was determined biochemically and by DNA relatedness to the type strain of P. heimbachae.

Adult↗
PubMed2001

Detection of salmonellae in chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis.

This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonella genus-specific 281-bp PCR product was detected when Salmonella strains but not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml(-1), respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml(-1), respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella could not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harbor Salmonella by both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detect Salmonella with the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased birds, the total time for PCR and detection is reduced to 2 h since the 18-h PE is not required. These results indicate that TTB enrichment, bacterial lysis, and genus-specific capillary PCR combined with capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identify Salmonella-infected flocks.

Animals↗
PubMed2002

Detection of Tropheryma whipplei DNA in feces by PCR using a target capture method.

Whipple's disease is a rare multisystemic bacterial infection with variable clinical manifestations. For decades, the laboratory diagnosis was based on the demonstration of periodic acid Schiff-positive inclusions in macrophages of gastrointestinal biopsies. PCR has improved the diagnosis of Whipple's disease due to its increased sensitivity compared to histopathological analysis. To avoid invasive procedures for taking specimens, we have investigated the possibility of detecting Tropheryma whipplei DNA in feces rather than in biopsies or gastric aspirate of patients with and without Whipple's disease. Total bacterial DNA was isolated from stool specimens using Qiagen columns followed by a T. whipplei-specific hybridization step with a biotinylated capture probe and streptavidin-coated magnetic particles. The captured DNA was then amplified using the same seminested PCR targeting the 16S rRNA gene of the organism that had been applied to other specimens without capturing. For five of eight patients with Whipple's disease, duodenal biopsies and stool samples were PCR positive, whereas for the three other patients, both specimens were PCR negative. Of 84 patients without Whipple's disease, 75 tested negative in the duodenal biopsy and in the stool sample. For four, both specimens were positive. Five patients tested positive in the stool sample but not in the biopsy. However, for three of these five patients, the gastric aspirate had been PCR positive, indicating that the stool PCR result was true rather than false positive. Compared to PCR from duodenal biopsies, stool PCR has a sensitivity of 100% and a specificity of 97.3%. Additionally, 15 PCR-positive and 22 PCR-negative stool samples were extracted using the Invisorb Spin Stool DNA kit. The simplified stool extraction showed 93.3% sensitivity and 95.5% specificity compared to the target capture method. We conclude that PCR with stool specimens with either extraction method is a sensitive and specific diagnostic tool for the detection of T. whipplei DNA and one not requiring invasive sampling procedures.

Actinobacteria↗