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.     

Oligonucleotide gene probes may detect enterotoxigenic Escherichia coli directly in fecal smears

Diarrhoeal stools from 86 Indian children were examined for the presence of enterotoxigenic Escherichia coli (ETEC) by DNA hybridization on bacterial colonies as well as directly on fecal smears. Colony hybridization showed that 8 of the samples contained ETEC. Only in three of these specimens were all the colonies tested positive for ETEC. These samples were correctly identified by the stool smear hybridization assay. The remaining 5 specimens produced enterotoxigenic as well as nonenterotoxigenic colonies. These samples were all negative by the stool smear hybridization technique. Methodological improvements of the stool smear assay may provide a substantially simplified method for the otherwise cumbersome identification of ETEC.     

Toxin detection after storage or cultivation of enterotoxigenic with colicinogenic Escherichia coli: a possible mechanism for toxin-negative pools.

Of 100 non-enterotoxigenic Escherichia coli isolated from children with diarrhea in Bangkok, Thailand, 24 were found to produce colicin(s). Of these, 87% were active against one or more enterotoxigenic E. coli isolated from the same population. Storage of nine enterotoxigenic E. coli with known inhibitory colicin-producing E. coli in different proportions caused 51 of 96 pools to become negative in the suckling mouse assay (heat-stable toxin) and 17 of 52 to become negative in the Y-1 adrenal cell assay (heat-labile toxin). Cocultivation of the same strains, without prior storage, caused 12 of 96 pools to become negative for heat-stable toxin and 1 of 52 pools to become negative for heat-labile toxin. Storage or cultivation of E. coli in pools may cause negative results in the suckling mouse and Y-1 adrenal cell assays if any of the isolates in the pool produces colicin(s).     

Identification of enterotoxigenic Escherichia coli isolated from swine with diarrhea in Thailand by colony hybridization, using three enterotoxin gene probes.

The DNA colony hybridization assay was used to identify enterotoxigenic Escherichia coli among E. coli isolated from 803 swine with diarrhea at 10 farms in Thailand. Between 5 September and 8 December 1981, enterotoxigenic E. coli were identified in 40% of 58 litters of piglets under 10 days old and 17% of 29 litters between 10 and 21 days old with diarrhea at farms at four different locations in Thailand. All E. coli that hybridized with one or more of the three enterotoxin gene probes produced heat-labile or heat-stable toxin or both, as determined by testing culture supernatants in the Y1 adrenal and suckling mouse assays. The DNA colony hybridization technique is a specific method of identifying enterotoxigenic E. coli from swine and can be used to further characterize these enteric pathogens.     

Evaluation of performance and potential clinical impact of ProSpecT Shiga toxin Escherichia coli microplate assay for detection of Shiga Toxin-producing E. coli in stool samples

Shiga toxin-producing Escherichia coli bacteria (STEC) are emerging pathogens capable of producing sporadic and epidemic diarrhea, hemorrhagic colitis, and potentially life-threatening hemolytic-uremic syndrome. Although the presence of E. coli O157 can be readily detected in stool by sorbitol-MacConkey agar culture (SMAC), STEC non-O157 serotypes cannot. In contrast to culture, testing for the presence of Shiga toxins 1 and 2 in stool detects both O157 and non-O157 STEC serotypes capable of causing disease. Over two consecutive summers, we evaluated the performance of the ProSpecT Shiga toxin E. coli Microplate assay (Alexon-Trend, Ramsey, Minn.), an enzyme immunoassay for the detection of Shiga toxins 1 and 2, on all stools submitted for culture of enteric pathogens, and the potential clinical impact of Shiga toxin detection. Twenty-nine stool specimens were STEC positive by ProSpecT assay. Twenty-seven of 29 STEC-positive isolates were confirmed by SMAC and serotyping or by a second enzyme immunoassay and PCR (positive predictive value, 93%). Thirteen of 27 confirmed Shiga toxin-producing strains were serotype O157. The remaining 14 strains represented 8 other serotypes. The ProSpecT assay was 100% sensitive and specific for detection of E. coli O157 in stool (7 of 7) compared to SMAC. In addition, the ProSpecT assay detected twice as many STEC as SMAC. Fifty-two percent of confirmed STEC-positive stools were nonbloody. Thus, in our population, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. Eleven (41%) STEC-positive patients were hospitalized, and eight (30%) developed severe disease (two developed hemolytic-uremic syndrome, and six developed hemorrhagic colitis). Prior to detection of STEC infection, seven (26%) and eight patients (30%) underwent unnecessary diagnostic procedures or received potentially deleterious empirical treatment, respectively. We propose that establishing a specific diagnosis of STEC may have prevented these potentially harmful interventions. We conclude that the ProSpecT assay is sensitive and specific for the detection of Shiga toxins 1 and 2 in stool and has potentially significant clinical impact for the individual patient and public health. Shiga toxin assays should be considered for routine use in settings where prevalence of STEC disease warrants testing.     

Alkaline phosphatase-conjugated oligonucleotide probes for enterotoxigenic Escherichia coli in travelers to South America and West Africa.

Several studies have demonstrated the usefulness of 32P-labeled recombinant DNA probes for identifying enterotoxigenic Escherichia coli (ETEC). The use of radioisotopes and X-ray development, however, severely handicaps the utility of DNA probes in most clinical laboratories. In this study, enzyme-labeled oligonucleotide probes for ETEC LT (heat-labile toxin) and ST (heat-stable toxin) genes were compared with the standard Y1 adrenal cell and suckling mouse assays for their ability to identify ETEC in a population of American adults experiencing acute episodes of diarrhea in South America and West Africa. The LT probe hybridized with 12% (64 of 529) of the E. coli colonies tested, whereas 11% (57 of 529) were positive by Y1 adrenal cell assay. DNA from 9% (47 of 529) of the E. coli colonies tested hybridized with the ST probe, whereas only 5% (28 of 529) produced ST as measured by the suckling mouse bioassay. For the patient samples tested, correlation between probe and bioassay for LT was 97%, or three discrepancies in 111 patients tested. Overall concordance of the ST probe and bioassay was 95%, or five discrepancies in 111 patients. Enzyme-labeled oligonucleotide probes represent a major advance in the diagnosis of ETEC-associated diarrheal disease and may be used in laboratories with minimal equipment.     

EnterotoxigenicEscherichia coli (ETEC) isolated in the Tel-Aviv (Israel) area

The prevalence of enterotoxigenicE. coli (ETEC) as a pathogenic agent of diarrhoea in the Tel-Aviv (Israel) area was determined, and the isolatedE. coli strains characterized. During three periods (summer 1977, summer 1978, and summer 1979), a total of 335 specimens were tested for the presence ofE. coli producing LT and ST toxin. Most of the specimens were from sporadic ambulatory diarrhoea cases (children and adults) attending a number of health care clinics in Tel-Aviv. Two to five colonies were tested from each sample. ETEC was detected in 69 cases (20%): LT/ST strains were isolated from 9 cases (2.7%); LT from 7 cases (2.1%); and ST from 53 cases (15.2%). ETEC was isolated in all age groups.In 19 specimens, 2 or more of 4 colonies tested were enterotoxigenic and were identical according to biotype, antibiotic sensitivity, and serogroup. These findings suggest that enterotoxigenic strains predominated in the bacterial population of the stool specimen. Part of the isolated ETEC strains belonged to serotypes already known as enterotoxigenic in different geographic areas of the world. The most frequently encountered were serogroups O8 (9 cases) represented by at least three serotypes, among them O8:K40:H9, and serotype O6:K15:H16 (5 cases); a number of serotypes were represented only by two cases or by single cases. Among 16 LT-producing stains (LT/ST and LT-only), 13 belonged to 3 serogroups, while ST-only strains represented a large spectrum of serotypes, some of which are now known as enterotoxigenic. Several serotypes common in other geographical locations were not detected.     

Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR.

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.     

Heat-labile enterotoxin production in isolates from a shipboard outbreak of human diarrheal illness.

As reported elsewhere, an enterotoxigenic strain of Escherichia coli serotype O25:K98:NM was epidemiologically incriminated as the etiological agent in a shipboard outbreak of diarrheal illness. This enterotoxigenic E. coli strain and possibly other enteric isolates were found to produce heat-labile toxin and not heat-stable toxin. Since previous genetic analyses of enterotoxigenic E. coli strains producing heat-labile and heat-stable toxins have shown a plasmid location for both toxin determinants and since in this outbreak more than one bacterial strain appeared to produce only heat-labile toxin, the possibility of an extrachromosomal heat-labile toxin determinant was investigated. Results of endonuclease cleavage and hybridization experiments, as well as apparent heat-labile toxin phenotypic instability, strongly suggest a plasmid mediation of toxin production. Additionally, the stability of this heat-labile toxin production was evaluated after several traditional methods of bacterial cell preservation.     

Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes.

A polymerase chain reaction (PCR) technique for the identification of Shiga-like toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.     

The use of phage-sensitivity patterns for tracing heat labile toxin-positive (LT+) Escherichia coli

The heat-labile toxin (LT) positive Escherichia coli colonies from 785 stool specimens obtained during a cholera vaccine trial were examined for their phage-sensitivity pattern to 31 E. coli phages. These specimens originated from 105 index cases and their contacts. Isolates with common phage-sensitivity patterns were grouped and were studied further both serologically and for their plasmid profile. The largest group (42 isolates) belonged to serogroup O78 and the second largest group (19 isolates) belonged to serogroup O6. There were 23 index cases which had E. coli with the same phage-sensitivity pattern as some of their contact strains. The identity of isolates from 16 index cases with strains from their respective contacts could be verified serologically. For the remaining seven index cases and their contacts, the isolates did not agglutinate with available antisera. However, subsequent studies demonstrated that, when the phage-sensitivity pattern among the strains matched, the plasmid profiles of these strains also matched. This further indicates the ability of phage-sensitivity patterns to serve as markers in tracing strains.     

Phylogenetic comparisons reveal multiple acquisitions of the toxin genes by enterotoxigenic Escherichia coli strains of different evolutionary lineages

Escherichia coli is a diverse bacterial species which is widely distributed in the environment but also exists as a commensal and pathogen of different host species. Human intestinal pathogenic E. coli causes over 160 million cases of diarrhea and an estimated 1 million deaths per year. The majority of deaths are attributable to one pathovar of E. coli, namely, enterotoxigenic E. coli. The pathogenesis of enterotoxigenic E. coli is dependent on the production of a colonization factor to promote adhesion to the intestinal epithelium and the elaboration of heat-labile or heat-stable toxins which induce a secretory diarrhea. Despite the high morbidity and mortality associated with enterotoxigenic E. coli infection, little is known of the genetic background of this global pathogen. Here we demonstrate by multilocus sequence typing that enterotoxigenic E. coli isolates are present in all phylogenetic lineages of E. coli, indicating that acquisition of the toxin genes may be sufficient to generate an enterotoxigenic E. coli strain. In addition, screening of diarrheal isolates for the presence of additional genes previously associated with the virulence of enterotoxigenic E. coli revealed that they were not abundant. These observations have significant implications for disease epidemiology and for the design of effective vaccines.     

Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli.

An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.     

Detection of enterotoxigenic Escherichia coli by colony hybridization with biotinylated enterotoxin probes.

By using biotinylated enterotoxin DNA probes, a method to detect enterotoxigenic Escherichia coli by colony hybridization was developed. The treatment of colonies on nitrocellulose membrane filters with proteinase K and Triton X-100 was essential for obtaining the specific hybridization. A total of 200 E. coli strains isolated from travelers with diarrhea were tested for colony hybridization by using a probe encoding heat-labile toxin (LT) type h. All strains (86 of 86) that produced LT, but none of the non-LT producers, hybridized with 32P-labeled and biotinylated LT type h probes. A total of 36 strains chosen randomly from the 200 isolates were tested for colony hybridization by using heat-stable enterotoxin (ST) probes. All but two strains that hybridized with the 32P-labeled ST type Ia probe also hybridized with the biotinylated ST type Ia probe. All strains that hybridized with the 32P-labeled ST type Ib probe also hybridized with the biotinylated ST type Ib probe. Thus, almost all E. coli strains tested were judged to be the same by colony hybridization with biotinylated or 32P-labeled enterotoxin probes. These results demonstrate that the biotinylated enterotoxin probes are useful in the diagnosis of enterotoxigenic E. coli strains by colony hybridization.     

Rapid detection by a coagglutination test of heat-labile enterotoxin in cell lysates from blood agar-grown Escherichia coli.

Colonies of Escherichia coli from blood agar plates were suspended and lysed in saline containing polymyxin B and a detergent (Triton X-100). The lysates were assayed for heat-labile enterotoxin (LT) by a coagglutination test (coa-test). The coa-test reagent consisted of Formalin-treated and heat-killed cells of Staphylococcus aureus, strain Cowan 1, sensitized with a high-titer rabbit anti-LT serum. Purified LT was detected in the coa-test at the nanogram level (2 to 5 ng), whereas larger amounts of cholera toxin (50 ng) were required to give a positive test. The coa-test was compared with the CHO cell test for detection of LT among E. coli strains isolated from human and animal stool cultures. The results of the coa-test and the CHO cell test correlated with 63 of 67 strains of human origin. Six of nine animal strains, defined as LT positive by the CHO cell test, gave positive results in the coa-test. We conclude that the coa-test is probably accurate and sensitive enough to be used in routine diagnosis of LT-producing E. coli strains isolated from human stool cultures. No special laboratory equipment is required, which makes the coa-test suited for diagnosis of enterotoxigenic E. coli in small hospital laboratories and in developing countries.     

Detection of enterotoxigenic Escherichia coli in water by filter hybridization with three enterotoxin gene probes.

The DNA hybridization assay for genes encoding for Escherichia coli enterotoxins was used to examine water specimens in Thailand. In a reconstruction experiment, the DNA hybridization assay was 10(4) times more sensitive than testing random E. coli in the Y-1 adrenal and suckling mouse assays in identifying enterotoxigenic E. coli (ETEC) in water. Drinking and bathing water collected from 2 of 10 different homes of individuals with ETEC-associated diarrhea and 6% (5 of 78) and 11% (11 of 78) of drinking and bathing water samples collected from homes of individuals with diarrhea without ETEC infections, as well as 6% (5 of 77) and 8% (6 of 77) of drinking and bathing water collected from homes in which no inhabitants had diarrhea, were homologous with the DNA probes. Ten E. coli from each of the 31 water specimens which contained bacteria which were homologous with the DNA probes were tested in the Y-1 adrenal and suckling mouse assay. In only 2 of these 31 specimens could ETEC be identified with the standard assays. The DNA hybridization assay is a much more sensitive means of detecting organisms carrying genes coding for enterotoxin production than testing 10 individual colonies in the Y-1 adrenal and suckling mouse assays. This novel application of recombinant DNA technology provides a sensitive method of detecting organisms carrying genes coding for enterotoxin, and this method will be useful in defining the epidemiology of ETEC.     

Molecular typing of Clostridioides difficile from frozen stool samples to investigate cross-transmissions: A proof of concept

PurposeToxigenic Clostridioides difficile is responsible for up to one third of post antibiotic diarrhea and for more than 95% of pseudomembranous colitis. Nowadays, diagnosis relies on the documentation of the presence of the toxin in stools by specific antigenic or PCR tests. Stool cultures have been mostly abandoned, leading to the absence of isolates for further epidemiological analyses.MethodsAliquots of stool samples, frozen for up to two years, were thawed and inoculated onto commercial C. difficile media. Eighteen stools were recovered from patients hospitalized in the pediatric ward where at that time a chain of transmission was suspected. Eleven stools were recovered from patients hospitalized in a medical ward over a three months period with no suspected transmission event. Up to 16 characteristic colonies were isolates per culture. PCR of toxins genes and molecular typing by Double Locus Sequence Typing (DLST) were performed on these colonies. Whole genome multi locus sequence typing (wgMLST) was performed on selected isolates.ResultsAmong the 29 stool specimens, no growth was observed for four stools and only one colony grew for one stool. Except the latter, all 16 colonies of the 24 stools showed identical toxin genes profiles than the original stool. However, variant DLST genotypes was observed within 20% of investigated stools. The majority of variants were single locus variant due to an IN/DEL of the repeat in one of the two DLST locus.Despite this variation, results of molecular typing overrule the putative transmission chain in the pediatric ward and revealed undetected chains of transmission in the medical ward. These results were confirmed with wgMLST.ConclusionsThe developed protocol allows prospective and retrospective molecular and genomic epidemiological investigation of C. difficile infections for infection control purpose.     

Fecal leukocytes in children infected with diarrheagenic Escherichia coli

The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response.     

Rapid stool-based diagnosis of Clostridium difficile infection by real-time PCR in a children's hospital

Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdA and tcdB. Stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively cultured on cycloserine-cefoxitin-fructose agar following alcohol shock. Six testing modalities were evaluated, including stool EIA, culture EIA, and real-time PCR (tcdA and tcdB) of cultured isolates and stool samples. Real-time PCR detection was performed with tcdA and tcdB gene-specific primers and hydrolysis probes using the LightCycler platforms (Roche Diagnostics, Indianapolis, IN). A total of 157 samples from 96 pediatric patients were analyzed. The sensitivities of stool real-time PCR and stool EIA were 95% and 35%, respectively, with a specificity of 100% for both methods. The lower limit of detection of the stool real-time PCR was 30 CFU/ml of stool sample per reaction for tcdA and tcdB. This study highlights the poor performance of stool toxin EIAs in pediatric settings. Direct detection of C. difficile toxin genes in stool samples by real-time PCR showed sensitivity superior to that of stool and culture EIAs and performance comparable to that of real-time PCR assay of cultured isolates. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for children that should facilitate appropriate patient management and halt the practice of serial testing by EIA.     

Development and evaluation of immunochromatographic assay for simple and rapid detection of Campylobacter jejuni and Campylobacter coli in human stool specimens

An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA, they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 x 10(4) to 8.2 x 10(5) CFU/ml of cell suspension, and those for the C. coli strains ranged from 1.4 x 10(5) to 4.6 x 10(6) CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens, suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent, and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens.