Detection of rotavirus in stool specimens with monoclonal and polyclonal antibody-based assay systems.

Accurate diagnosis of rotavirus is important in both clinical and research situations. A total of 100 stool specimens from children with diarrhea were tested for rotavirus by electron microscopy. These specimens were then coded and tested for rotavirus by four procedures: a monoclonal antibody-based enzyme immunoassay (EIA) (Pathfinder; Kallestad Laboratories, Inc., Austin, Tex.), two polyclonal antibody-based EIAs (Rotazyme II; Abbott Laboratories, North Chicago, Ill.; and an EIA performed with reagents from the National Institutes of Health, Bethesda, Md. [NIH reagent EIA]), and a latex agglutination (LA) assay (Rotalex; Medical Technology Corp., Somerset, N.J.). The sensitivity of the monoclonal antibody EIA (95%) was superior to those of the polyclonal antibody EIAs (73% for Rotazyme II and 57% for the NIH reagent EIA) and the LA assay (61%). The specificity of the LA assay (98%) was slightly better than those of the other systems (88 to 96%). The positive and negative predictive values of the monoclonal antibody EIA (93 and 96%, respectively) were better than those of Rotazyme II (82 and 80%, respectively), the LA assay (96 and 76%, respectively), and the NIH reagent EIA (93 and 74%, respectively). The visual readings of the monoclonal antibody EIA correlated better with the spectrophotometric optical density readings than did the visual readings of the polyclonal antibody EIAs; however, the agreement of both with electron microscopy results was poor when 1+ or plus-minus readings were observed. The monoclonal antibody EIA is more sensitive and predictive than other rotavirus detection systems and second only to the LA assay in specificity in detecting rotavirus in stool specimens.     

Detection of adenoviruses in stool specimens by nucleic acid spot hybridization

Nucleic acid hybridization was used for the detection of adenovirus DNA in stool specimens, and the results were compared with those obtained by a radioimmunoassay (RIA) for adenovirus hexon antigen. DNA from 40 specimens, 18 of which were positive by RIA, were spotted onto nitrocellulose filters and analyzed by hybridization using radioactively labeled adenovirus-2 DNA or a cloned DNA fragment from enteric adenovirus-41 as probes. With the adenovirus-2 DNA probe, 15 of the 18 RIA-positive specimens were also positive in the hybridization assay, and one of the RIA negative specimens was also scored as positive. The cloned adenovirus-41 fragment gave a positive signal with five specimens, all of which were also detected with the adenovirus-2 DNA probe. The results show that hybridization is an alternative method for detection of adenovirus in stool specimens. The sensitivity of the assay is comparable to that of the RIA.     

Detection of adenovirus types 40 and 41 in stool specimens by immune electron microscopy

An immune electron microscope (IEM) test was developed that allowed the direct detection of adenovirus type 40 (ad 40) or ad 41 in stools specimens. The polyclonal rabbit antisera used differentiated ad 40 and 41 from other ad serotypes but not from each other. The method was evaluated in a 13 month prospective study of stools from children with gastroenteritis. Seventy-two specimens found to contain ad by conventional electron microscope screening were retested by IEM. Results were typically obtained within 2 hr and showed that 55 (76%) viruses typed as ad 40/41. No ads were recovered from conventional virus isolation attempts on these specimens. Additionally, 39 of these 55 viruses were tested by restriction endonuclease analysis (REA) after growth in 293 cells, and results showed that all produced digest patterns typical of ad 40 (seven cases) or ad 41 (32 cases). Twenty-four percent (17/72) of viruses could not be typed by IEM; 9/17 (53%) yielded ads [ad 1 (1), ad 2 (4), ad 5 (1), ad 6 (1), ad 7 (2)] in routine culture, whereas REA identified the other eight as ad 2 (6), ad 1 (1), and ad 41 (1). The concordance between IEM and the reference methods was therefore 100% specificity and 97.5% sensitivity. The method described allows the clinically useful diagnosis of ad 40/41 infection to be rapidly made and will be a particularly useful technique in laboratories screening faeces by electron microscopy.     

Detection of enterotoxigenic Escherichia coli colonization factor antigen I in stool specimens by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was employed to detect and quantitate the fimbrial colonization factor antigen (CFA/I) of enterotoxigenic Escherichia coli in stool specimens obtained from adult cases of diarrhea in which CFA/I-positive E. coli was the known causative agent. The inhibition method, or blocking technique, was used. In this method, a standardized dilution of human anti-CFA/I serum was preincubated with dilutions of stool extract before transfer to CFA/I-coated microtiter plate wells, and then ELISA was performed with alkaline phosphatase-conjugated anti-human immunoglobulin. CFA/I purified from E. coli strain H-10407 (O78:H11) was used. Acute-phase diarrheal stool specimens were found to contain approximately 3.0 mg of antigen (mean value) per g stool, whereas control (CFA/I-negative) specimens contained insignificant amounts (less than 0.03 mg/g) of antigen. Also, CFA/I was detected in culture fluids of CFA/I positive enterotoxigenic E. coli belonging to a variety of serotypes and was undetectable in similar preparations from P-strains (spontaneous CFA/I-negative derivatives) of the same test cultures. Equivalent results were obtained in ELISA tests by using bacterial cells taken from isolated colonies grown on CFA agar. These results indicate that the ELISA technique will be useful for the diagnosis of diarrhea caused by CFA/I-positive enterotoxigenic E. coli.     

Concomitance of cytotoxigenic and non-cytotoxigenic Clostridium difficile in stool specimens.

Six patients with antibiotic-associated diarrhea and one patient with diarrhea unrelated to antibiotic use yielded both cytotoxigenic and non-cytotoxigenic isolates of Clostridium difficile from the same stool specimens. In addition, these isolates were shown to be pathogenic and nonpathogenic, respectively, in the hamster model of antibiotic-associated colitis. These data imply that more than one toxin type of C. difficile may be harbored simultaneously. If toxin testing is used to identify C. difficile, more than one colony must be tested.     

Fluorescent-antibody test for detection of Clostridium difficile in stool specimens.

We evaluated a direct fluorescent-antibody test to detect Clostridium difficile, the most frequent cause of antibiotic-associated colitis. C. difficile organisms were injected into the ear veins of New Zealand White rabbits to induce antibodies, and the globulin fractions of their sera were conjugated to fluorescein isothiocyanate. The resulting conjugate strongly stained all 40 isolates of C. difficile tested. It also stained isolates of C. sordellii, C. bifermentans, C. chauvoei, and C. sporogenes, but not 20 other clostridial isolates or 10 isolates from other species. Results of testing fecal smears with the direct fluorescent-antibody method were compared with results of testing stools for C. difficile toxin and of culturing for C. difficile on a selective medium. A total of 158 fecal specimens from patients with antibiotic-associated diarrhea were tested. In these patients, the fluorescent-antibody test agreed with culture and toxin testing in 93% of the specimens. However, in normal adults, 62% of the fecal specimens from which C. difficile could not be cultured were positive by the fluorescent-antibody test. Absorption of the conjugate with C. sordellii led to a loss of reactivity to other clostridia as well as to 18 of 20 isolates of C. difficile.     

Specific detection of toxigenic strains of Clostridium difficile in stool specimens.

Clostridium difficile is the infectious agent responsible for antibiotic-associated colitis. We report the use of the polymerase chain reaction technique to identify toxigenic strains of C. difficile in human stool specimens. A set of primers based on the nucleotide sequence of the toxin B gene, which amplified a 399-bp fragment from isolates producing toxin B, was designed. We examined 28 known toxigenic strains, which were all positive by this assay. DNAs from the nontoxigenic strains examined and from strains of Clostridium sordellii and C. bifermentans were not amplified with these primers. The sensitivity of this assay allowed us to identify as little as 10% toxigenic C. difficile cells in the presence of 90% nontoxigenic cells and to detect the toxin B gene in 1 pg of DNA from a toxigenic strain. DNAs extracted from 18 clinical stool specimens that were positive for toxin B by the tissue culture cytotoxicity assay were also positive by this assay. In addition, we detected toxin B sequences in DNA from 2 of 18 stool specimens that were negative for toxin B by the cytotoxicity assay. These two stool specimens were from patients who had a clinical pattern of colitis that was compatible with C. difficile causation. This rapid, sensitive assay will be useful for specific identification of toxigenic C. difficile and for revealing cases that are undetected by analysis of fecal samples for toxin B alone.     

Rapid dimethyl sulfoxide-modified acid-fast stain of Cryptosporidium oocysts in stool specimens.

A rapid dimethyl sulfoxide modification of an acid-fast technique was applied to direct fecal smears to monitor cryptosporidiosis in nonhuman primates. Brilliantly stained pink oocysts against a pale green background demonstrated well-preserved internal morphology and facilitated rapid, simple, noninvasive diagnosis without fluorescent or phase-contrast microscopy.     

Identification of Clostridium difficile in stool specimens by culture-enhanced gas-liquid chromatography.

We have developed a sensitive and specific method for the identification of Clostridium difficile in stool specimens based on the detection of metabolic breakdown products of the organism by gas-liquid chromatography after incubation of stool samples in a selective broth medium containing cefoxitin. Use of this approach to test samples from two different populations of patients at separate medical centers showed this method to be superior to plate cultures or cytotoxin testing alone for both populations. The combined results from the two patient populations showed that 225 of 226 confirmed isolates were identified correctly, resulting in a sensitivity of 99.6% and a specificity of 99.0%. This method eliminates the delay caused by subculturing for tests requiring a pure isolate. The culture phase amplifies even low numbers of C. difficile in fecal samples (due to low in vivo concentrations or delayed transport) and thus increases sensitivity. Other advantages include the ability to detect C. difficile in the mixed flora of the stool and the ability of most clinical laboratories to use this procedure. Given the complexities of the detection of C. difficile toxins and the increasing importance of this organism as a nosocomial agent, culture-based methods remain the preferred approach to screening and routine workup for cases of diarrhea.     

In vitro growth of some fastidious adenoviruses from stool specimens.

Sixty-seven stool specimens from 51 children, positive for adenoviruses by electron microscopy and negative for growth in human-embryo kidney cells, were tested for growth in Chang conjunctiva cells. Twenty-eight specimens caused a cytopathic effect over more than one passage in these cultures, and several adenovirus strains grew better at 33 degree C than at 37 degree C. Most of the culture-positive specimens also induced the development of adenovirus antigens in KB cells detectable by a group-specific indirect immunofluorescence test. Twenty-four of the 25 fastidious strains tested were antigenically related and were distinct from the established serotypes commonly isolated from stools.     

Identification of microsporidia in stool specimens by using PCR and restriction endonucleases.

We report the development of a PCR-based assay for the detection of microsporidia in clinical specimens. A single primer pair complementary to conserved sequences of the small-subunit rRNA enabled amplification of DNA from the four major microsporidian pathogens of humans: Encephalitozoon cuniculi, Encephalitozoon hellem, Enterocytozoon bieneusi, and Septata intestinalis. The extraction method allowed PCR amplification of E. bieneusi and S. intestinalis DNA from sodium hypochlorite-treated stool specimens. Differentiation of the microsporidian gastrointestinal pathogens E. bieneusi and S. intestinalis could be accomplished by restriction endonuclease digestion of PCR products using PstI and HaeIII.     

Improved stool concentration procedure for detection of Cryptosporidium oocysts in fecal specimens.

Epidemiologic and laboratory data suggest that coprodiagnostic methods may fail to detect Cryptosporidium oocysts in stool specimens of infected patients. To improve the efficacy of stool concentration procedures, we modified different steps of the Formalin-ethyl acetate (FEA) stool concentration technique and evaluated these modifications by examining stool samples seeded with known numbers of Cryptosporidium oocysts. Because these modifications failed to improve oocyst detection, we developed a new stool concentration technique that includes FEA sedimentation followed by layering and flotation over hypertonic sodium chloride solution to separate parasites from stool debris. Compared with the standard FEA procedure, this technique improved Cryptosporidium oocyst detection. The sensitivities of the two concentration techniques were similar for diarrheal (watery) stool specimens (100% of watery stool specimens seeded with 5,000 oocysts per g of stool were identified as positive by the new technique, compared with 90% of stools processed by the standard FEA technique). However, the most significant improvement in diagnosis occurred with formed stool specimens that were not fatty; 70 to 90% of formed stool specimens seeded with 5,000 oocysts were identified as positive by the new technique, compared with 0% of specimens processed by the standard FEA technique. One hundred percent of formed specimens seeded with 10,000 oocysts were correctly diagnosed by using the new technique, while 0 to 60% of specimens processed by the standard FEA technique were found positive. Similarly, only 50 to 90% of stool specimens seeded with 50,000 oocysts were identified as positive by using the standard FEA technique, compared with a 100% positive rate by the new technique. The new stool concentration procedure provides enhanced detection of Cryptosporidium oocysts in all stool samples.     

Hafnia alvei in stool specimens from patients with diarrhea and healthy controls.

We found an epidemiological association of Hafnia alvei with diarrhea, because the organism was isolated from 12 of 77 (16%) adult Finnish tourists to Morocco who developed diarrhea and from 0 of 321 tourists without diarrhea (P < 0.001). From another group of 112 adult Finnish diarrheal patients, only 2 (2%) yielded H. alvei. In contrast to some Bangladeshi strains of H. alvei, the Finnish strains were negative for the attachment-effacement lesion by an in vitro fluorescent acting staining test and also did not show homology to the Escherichia coli attachment-effacement gene (eaeA) by PCR. These results suggest that a mechanism or mechanisms other than the attachment-effacement lesion may also be involved in the association of H. alvei with diarrhea.