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PubMed1996

Detection of Cryptosporidium parvum DNA in human feces by nested PCR.

Cryptosporidium parvum is a coccidian protozoan that causes diarrhea in humans, often chronic and severe in patients with AIDS. Conventionally, diagnosis is made by concentration of stools followed by acid-fast staining (AF) or immunofluorescent staining. The threshold of detection in human stool specimens by these methods may require the presence of 50,000 (immunofluorescent staining) to 500,000 (AF) oocysts per g of stool. In this study, a nested PCR assay was developed to detect C. parvum DNA directly from stool specimens. After extraction of DNA from formalinized stool, a 400-bp fragment of C. parvum DNA was amplified with two 26-mer outer primers. The amplicon from this reaction was amplified with a second primer pair. With these nested primers, a 194-bp DNA fragment was amplified and confirmed as C. parvum DNA by internal probing with an enzyme-linked chemiluminescence system. This PCR-based test allowed the detection of 500 oocysts per g of stool or 100 ng of C. parvum DNA. Studies indicate that the primers utilized are specific for the DNA of C. parvum. DNA sequences were also detected in stool specimens from 4 of 28 patients previously reported negative by AF. In summary, a rapid, sensitive, and specific assay for the detection of C. parvum directly from stool specimens has been developed. This test has the potential for detecting asymptomatic infection, monitoring the response to therapy, and detecting the organism in environmental sources.

AIDS-Related Opportunistic Infections↗
PubMed1996

Isolation of Campylobacter concisus from feces of children with and without diarrhea.

A prospective study compared fecal isolation rates of Campylobacter concisus for children with diarrhea and without diarrhea by a filter technique in which media were incubated for 4 days in a microaerobic atmosphere. No statistically significant difference in isolation rates was found (13.2% in patients with diarrhea and 9% in controls). Moreover, 35 of 37 children attending the same day care center harbored different C. concisus strains, as was demonstrated by arbitrary primer PCR DNA fingerprinting. These data suggest a lack of a pathogenic role for C. concisus in enteritis.

Campylobacter↗
PubMed1999

Typing and characterization of mechanisms of resistance of Shigella spp. isolated from feces of children under 5 years of age from Ifakara, Tanzania.

Eighty-six strains of Shigella spp. were isolated during the dry season from stool samples of children under 5 years of age in Ifakara, Tanzania. The epidemiological relationship as well as the antimicrobial susceptibility and mechanisms of resistance to ampicillin, chloramphenicol, and co-trimoxazole were investigated. Four different epidemiological tools, pulsed-field gel electrophoresis (PFGE), repetitive extragenic palindromic (REP)-PCR, plasmid analysis, and antibiogram, were compared for typing Shigella strains. Seventy-eight (90%) strains were Shigella flexneri and were distributed into four groups, by either PFGE or REP-PCR, with 51, 17, 7, and 3 strains. The four strains of Shigella dysenteriae belonged to the same group, and the four strains of Shigella sonnei were distributed in two groups with three and one strain each. Plasmid analysis showed a high level of heterogeneity among strains belonging to the same PFGE group, while the antibiogram was less discriminative. REP-PCR provided an alternative, rapid, powerful genotyping method for Shigella spp. Overall, antimicrobial susceptibility testing showed a high level of resistance to ampicillin (81.8%), chloramphenicol (72.7%), tetracycline (96.9%), and co-trimoxazole (87.9%). Ampicillin resistance was related to an integron-borne OXA-1-type beta-lactamase in 85.1% of the cases and to a TEM-1-type beta-lactamase in the remaining 14.8%. Resistance to co-trimoxazole was due to the presence of a dhfr Ia gene in all groups except one of S. flexneri, where a dhfr VII gene was found within an integron. Chloramphenicol resistance was associated in every case with positive chloramphenicol acetyltransferase activity. All strains were susceptible to nalidixic acid, ciprofloxacin, ceftazidime, cefotaxime, and cefoxitin. Therefore, these antimicrobial agents may be good alternatives for the treatment of diarrhea caused by Shigella in Tanzania.

Bacterial Typing Techniques↗
PubMed2001

Magnetic immuno-PCR assay with inhibitor removal for direct detection of Helicobacter pylori in human feces.

A PCR protocol was developed to detect Helicobacter pylori in human stool specimens. This protocol was based on the association of a magnetic immuno-PCR assay with a technique to remove inhibitors (agarose-embedded DNA preparation). Of the 47 H. pylori-positive and 57 H. pylori-negative patients included in this study, 38 were positive and 66 were negative by this new protocol. The sensitivity, specificity, and predictive values for a positive or a negative result were 80.9% (95% confidence interval [CI], 66.3 to 90.4), 100% (95% CI, 92.1 to 100), 100% (95% CI, 88.6 to 100), and 86.4% (95% CI, 75.2 to 93.2), respectively.

Adolescent↗
PubMed2001

Evaluation of methods for detection of toxins in specimens of feces submitted for diagnosis of Clostridium difficile-associated diarrhea.

Clostridium difficile is the principal pathogen associated with hospital-acquired acute diarrheal disease. We have evaluated the performances of six approaches for diagnosis of C. difficile-associated diarrhea (CDAD). Consecutive stool specimens (n = 200) from 133 patients were examined by cytotoxin assay, by culture of C. difficile on cycloserine-cefoxitin-fructose agar, and by toxin detection using four rapid immunoassay systems (Oxoid Toxin A test, ImmunoCard Toxin A test, TechLab Tox A/B II test, and Premier Toxins A&B test). A diagnosis of CDAD was established for 35 (27%) patients (representing 29% of specimens). The adjusted sensitivity and specificity of the methods were, respectively, 98 and 99% for the cytotoxin assay, 54 and 99% for ImmunoCard, 50 and 98% for Oxoid, 79 and 98% for TechLab, 80 and 98% for Premier, and 57 and 100% for culture. The TechLab and Premier assays are acceptable tests for diagnosis of CDAD but are not equivalent to the cytotoxin assay.

Adult↗
PubMed2002

Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler.

We have developed a rapid (1-h) real-time fluorescence-based PCR assay with the Smart Cycler thermal cycler (Cepheid, Sunnyvale, Calif.) for the detection of Shiga toxin-producing Escherichia coli (STEC), as well as other Shiga toxin-producing bacteria. Based on multiple-sequence alignments, we have designed two pairs of PCR primers that efficiently amplify all variants of the Shiga toxin genes stx(1) and stx(2), respectively. These primer pairs were combined for use in a multiplex assay. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon. Assays performed with purified genomic DNA from a variety of STEC strains (n = 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (n = 20) were also tested, and no amplification was observed. The PCR results correlated perfectly with the phenotypic characterization of toxin production in both STEC and non-STEC strains, thereby confirming the specificity of the assay. The assay was validated by testing 38 fecal samples obtained from 27 patients. Of these samples, 26 were PCR positive for stx(1) and/or stx(2). Compared with the culture results, both the sensitivity and the negative predictive value were 100%. The specificity was 92%, and the positive predictive value was 96%. Moreover, this assay detected STEC from a sample in which the STEC concentration was at the limit of detection of the conventional culture methods and from a sample in which STEC was not detected by the conventional culture methods. This real-time PCR assay is simple, rapid, sensitive, and specific and allows detection of all Shiga toxin-producing bacteria directly from fecal samples, irrespective of their serotypes.

DNA, Bacterial↗
PubMed1978

Bacteriocin typing of Clostridium perfringens in human feces.

Three hundred and ninety-nine isolates of Clostridium perfringens from enriched stool specimens of 51 individuals (about eight colonies per person) were typed by bacteriocins. Forty-nine percent of these persons carried more than one bacteriocin type in their stool, and some had three or four different stains as determined by bacteriocin typing. Weekly stool specimens obtained from seven positive volunteers over a period of 5 weeks were screened for C. perfringens, and several colonies from each person were typed. This survey demonstrated that the number of types fluctuated with time, several types could be carried simultaneously, and the isolation of the organism was variable. Nine new bacteriocin types of C. perfringens were isolated in this study.

Bacteriocins↗
PubMed1979

ICMSF methods studies. XII. Comparative study for the enumeration of Clostridium perfringens in feces.

As the second phase of an international comparative study for the enumeration of Clostridium perfringens, four methods were compared for "total" and spore counts of C. perfringens in fecal specimens: the SFP (Shahidi-Ferguson perfringens) agar (A), TSC (tryptose-sulfite-cycloserine) agar (B), SC (sulfite-cycloserine) agar (C), and neomycin blood agar (D) methods. In both the total and spore count procedures, the confirmed C. perfringens counts in method D were lower than in methods A, B, and C. Little differences among methods were found in the percentages of presumptive colonies confirmed as C. perfringens. The nonspecific counts in methods A and D were generally greater than in B and C, but nonspecific microorganisms did not interfere in the enumeration of C. perfringens spores by any of the four methods. In overall performance, methods B and C were superior to A and D. The mean C. perfringens spore count was only 0.17 log lower than the mean total count. Spore counts alone are, therefore, adequate in investigations of C. perfringens outbreaks.

Bacteriological Techniques↗
PubMed1978

Purification of hepatitis A virus from human feces.

Hepatitis A virus was purified fecal specimens obtained from 2 patients with naturally acquired hepatitis A. The purification procedure involved differential centrifugation, organic solvent extraction, agarose gel filtration, ion-exchange chromatography, and isopycnic ultracentrifugation in cesium chloride. Using immune electron microscopy and discontinuous SDS-PAGE, this procedure was found to be effective in removing extraneous material from hepatitis A virus. There was significant recovery of virus as judged by immune electron microscopy and solid-phase radioimmunoassay. Using this protocol, it has been possible to obtain virus preparations of sufficient purity and high enough titer to enable biochemical studies to proceed.

Antigen-Antibody Reactions↗
PubMed1981

Origins of neutral sterols in human feces studied by stable isotope labeling (D and 13C). Existence of an external secretion of cholesterol.

An experimental procedure using stable isotope-labeled cholesterol (13C and D) was carried out on 15 healthy subjects to distinguish the different origins of neutral fecal sterols in man: nonabsorption of dietary cholesterol, fecal excretion by transfer of plasmatic cholesterol and external secretion of cholesterol biosynthetized in digestive tract and directly eliminated. For a mean daily mass of 652 mg of fecal cholesterol, unabsorbed dietary cholesterol is 20% (133 mg), excreted cholesterol 67% (434 mg) and cholesterol from external secretion 13% (85 mg). A short treatment (4 days) with cholestyramine or different bile acids was then administered to each subject to study the possible variations in their fecal elimination of cholesterol. The more evident effect was the large stimulation of external secretion of cholesterol (234 mg/day) observed after chenodeoxycholic acid feeding (1 g/day). This treatment tends also to decrease dietary cholesterol absorption and to enhance excretion of cholesterol.

Adult↗