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High-performance liquid chromatographic determination of xanthohumol in rat plasma, urine, and fecal samples.

Xanthohumol (XN) is the major prenylated flavonoid in hop plants and as such a constituent of beer. Pharmacological studies have shown that XN possesses marked antioxidant and antiproliferative effects. In order to study the resorption and metabolism of this compound, reversed-phase high-performance liquid chromatography is used for the determination of XN in rat plasma, urine, and feces. In session one, rats receive either oral or intravenous (iv) administration (20 mg/kg body weight) of XN. In session two, rats receive oral administration of 50, 100, 200, 400, and 500 mg/kg body weight XN for bioavailability studies at various dose levels. Plasma, urine, and feces are collected at varying time points and assayed for their XN content. Plasma levels of XN fell rapidly within 60 min after iv administration; no XN is detected in plasma after oral administration in either session. XN and its metabolites are excreted mainly in feces within 24 h of administration. The method is a reliable tool for performing studies of XN in different biological material.

Animals↗

Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test.

A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples.

Clostridium perfringens↗

Bifidobacterium, Bacteroides, and Clostridium spp. in fecal samples from breast-fed and bottle-fed infants with and without iron supplement.

Bifidobacterium, Bacteroides, and Clostridium spp. isolated from the feces of 23 neonates during the first 3 months of life were identified. Of the 23 neonates, 10 were breast fed, 6 received an infant formula with iron supplement (5 mg/liter), and 7 received the formula without iron supplement (iron concentration, less than 0.5 mg/liter). The Bifidobacterium spp. most frequently isolated from the three groups of infants were B. longum, B. breve, B. adolescentis, and B. bifidum. The bacteroides spp. most frequently isolated were B. fragilis and B. vulgatus. The most common Clostridium sp. in the three groups of infants was C. perfringens. The type of milk did not select for species of Bifidobacterium, Bacteroides, or Clostridium, except for Clostridium butyricum, which was isolated significantly more often from bottle-fed infants with iron supplement than from the other groups, and Clostridium tertium, which was more often isolated from breast-fed infants. The species of the three anaerobic genera did not persist for a long period of time in the three groups of infants.

Bacteria, Anaerobic↗

Quantitation of Giardia cysts and Cryptosporidium oocysts in fecal samples by direct immunofluorescence assay.

The lack of quick, simple, and sensitive quantitative tests has impeded studies on infection patterns and treatment of Giardia spp. and Cryptosporidium spp. A quantitative direct immunofluorescence assay (FA) using a commercial FA kit was developed and evaluated. Recovery rates of the FA for Cryptosporidium oocysts in calf feces seeded with 1,000, 10,000, 100,000, and 1,000,000 oocysts per g were 14.8, 40.8, 84.2, and 78.2%, respectively. Interassay coefficients of variation were 10.6 to 47.1%. Recovery rates of the FA for Giardia cysts in feces seeded with 1,000, 10,000, and 100,000 cysts per g were 76.4, 96.9, and 89.6%, respectively. Interassay coefficients of variation were 7.4 to 22.1%. By comparison, recovery rates of Giardia cyst by sucrose gradient flotation were only 20.5, 51.2, and 42.9%, respectively. Counts of cysts-per-gram obtained by sucrose gradient flotation with samples from calves, lambs, and ewes were only 49.1 to 54.8% of those obtained by the FA. Zinc sulfate flotation detected only 36.4% of infections when there were < or = 1,000 cysts per g. The quantitative FA offers a useful technique for epidemiological and control studies of these two parasites.

Animals↗

Mitomycin immunoblot colony assay for detection of Shiga-like toxin-producing Escherichia coli in fecal samples: comparison with DNA probes.

We developed a direct screening immunoblot assay for the detection of Shiga-like toxin (SLT)-producing organisms in stool samples. The assay takes advantage of the phage-mediated nature of SLT production in Escherichia coli and the phage-inducing effects of mitomycin. The addition of mitomycin significantly enhanced the amount of toxin available for immunologic detection. By using the mitomycin-enhanced immunoblot assay, SLT-producing E. coli could be distinguished from non-toxin-producing E. coli and normal stool flora in ratios of 1:1,000 to 1:5,000. The immunoblot assay was examined in a field setting and compared with direct DNA probing for SLT-I and SLT-II. The assay was able to detect SLT-producing E. coli with a high level of sensitivity and specificity. Specificity was markedly improved by using a monoclonal antibody which cross-reacts with both SLT-I and SLT-II B subunits in place of the polyclonal antitoxin sera. We conclude that the mitomycin-enhanced immunoblot colony assay is a rapid and reliable alternative to DNA probing for the detection of phage-mediated SLT-producing organisms in stool samples, especially when the production and use of nucleic acid probes are not feasible. In addition, it permits isolation of positive colonies for further study and confirmation.

Animals↗

Detection, typing, and subtyping of enteric adenoviruses 40 and 41 from fecal samples and observation of changing incidences of infections with these types and subtypes.

Monoclonal antibody (MAb) preparations specific for the enteric adenoviruses of subgenus F (AdF) were generated and evaluated as typing reagents in virus neutralization tests and enzyme-linked immunosorbent assays (ELISAs). A panel of 11 genome types of adenovirus 40 (Ad40), 24 genome types of Ad41, and 47 adenovirus prototype strains was used to determine the specificities of the MAbs in the two assays. In this way two MAbs, MAb 40-1 (anti-Ad40) and MAb 41-1 (anti-Ad41) were selected. These two MAbs showed strict type specificity in both assays. A third MAb reacted in an ELISA with all 47 human adenovirus types. With two other MAbs, three antigenic subtypes of Ad41 could be distinguished by their reactivities in virus neutralization tests and ELISAs. On the basis of the five selected MAbs, a sensitive ELISA system was developed for the direct detection and simultaneous typing and subtyping of Ad40 and Ad41 present in stool specimens. The five MAbs were also used to study the epidemiology of infections with Ad40 and Ad41 in The Netherlands in the period 1981 through 1989. It was shown that there were no significant fluctuations in the annual incidence of the cluster of enteric adenoviruses as a whole. This cluster should therefore be considered to belong to the "endemic" rather than the "epidemic" adenoviruses. The relative incidence of Ad40 infections compared with that of Ad41 infections changed considerably during the period studied; the proportion of Ad41 infections rose from about 30% in 1981 to about 95% in 1986, after which it stabilized at 90 to 95%. The proportion of one of the subtypes of Ad41 (Ad41 subtype M3) increased from about 40 to 80% in the same period.

Adenovirus Infections, Human↗

Detection of Helicobacter pylori DNA in fecal samples from infected individuals.

Stool, gastric biopsy, and serum samples were collected from 22 subjects. DNA from stool was extracted, amplified, and hybridized with primers specific for the 16S rRNA gene of Helicobacter pylori. DNA from gastric biopsy specimens was analyzed similarly for comparison. Universal primers were used to confirm successful extraction of DNA from samples. Histologic, serologic, and DNA analyses were scored in a blinded fashion. Universal primer amplification verified successful DNA extraction from all stool and gastric tissue specimens. The gastric tissue DNA assay was positive for H. pylori in 11 of the 22 subjects, correlating completely with histologic and serologic results. Stool DNA was positive for H. pylori by our molecular assay in 8 of these 11 H. pylori-positive subjects. All subjects who were negative by histologic, serologic, and gastric tissue DNA analyses were also negative by stool DNA analysis. Compared to histology, serology, and gastric tissue DNA analyses, the sensitivity of our stool DNA assay was 73%, with a specificity of 100%.

DNA, Bacterial↗

Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp. in human fecal samples as estimated by a reevaluation of isolation methods for Campylobacters.

The aims of this study were to investigate the prevalence of campylobacteria including Campylobacter jejuni subsp. jejuni (C. jejuni) and Campylobacter coli in human clinical samples and in samples from healthy individuals and to reevaluate the efficacies of conventional selective methods for isolation of Campylobacter spp. Two charcoal-based selective media, modified charcoal cefoperazone deoxycholate agar (mCCDA) and cefoperazone-amphotericin-teicoplanin (CAT) agar, were compared with Skirrow's blood-based medium and with a filter method (filter) applied to a yeast-enriched blood agar. A total of 1,376 specimens were tested on all four media, and the percentages of thermophilic Campylobacter-positive specimens isolated on Skirrow's medium, filters, CAT agar, and mCCDA were 82, 83, 85, and 95%, respectively. When additional samples were processed with the three selective media, mCCDA recovered significantly more thermophilic Campylobacter spp. than Skirrow's medium (P = 0.0034). No significant difference between Skirrow's medium and CAT agar was observed in this study. Another six taxa were identified, namely, Campylobacter concisus, Campylobacter curvus-like bacteria, Arcobacter butzleri, Arcobacter cryaerophilus, Helicobacter cinaedi, and Sutterella wadsworthensis. Most of these strains were isolated after 5 to 6 days of incubation by use of the filter technique. This paper provides evidence for the existence of S. wadsworthensis in human feces from clinical cases of gastrointestinal disorders and in feces from a healthy individual. Furthermore, C. concisus was isolated from a large number of diarrheal cases, particularly those at the extremes of age, but was additionally isolated from the feces of healthy people. Further investigations to establish the role of C. concisus and S. wadsworthensis in enteric disease is needed. We conclude that a range of campylobacteria may cause infections in Denmark.

Adolescent↗

Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples.

A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica- or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.

Animals↗

Antibiotic resistance in Staphylococcus isolates obtained from fecal samples of healthy children.

Thirty-nine Staphylococcus isolates with different mechanisms of resistance were recovered from the feces of 50 healthy children and tested for their susceptibilities to 17 antibiotics. The percentages of resistance of the staphylococci to some antibiotics were as follows: penicillin, 87%; erythromycin, 64%; tobramycin, 36%; tetracycline, 20.5%; kanamycin, 15%; and gentamicin, 13%. The mecA gene was detected in nine coagulase-negative staphylococci.

Aminoglycosides↗

Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR.

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.

Animals↗