[Point-of-care tests for the rapid diagnosis of shigellosis]. / Tests de diagnostic rapide des shigelloses.

Worldwide, it is estimated that 140 million people suffer from shigellosis annually. The traditional identification of Shigella spp. by culture lacks sensitivity. Rapid diagnosis of shigellosis is important because it allows to engage appropriate antimicrobial treatment that shortens the duration and severity of the illness and reduces microbial carriage, thus the spread of infection in the community. Onestep immunochromatographic dipstick tests have been successfully developed at Institut Pasteur for Shigella spp., Shigella flexneri 2a, Shigella sonnei, and Shigella dysenteriae 1. The present work describes the evaluation of these four rapid diagnostic tests (RDT) that addressed the issue of rapid diagnosis of Shigella diarrhea and dysentery testing from bacterial cultures, stools, and rectal swabs which is usually how the specimen is often collected or received from the field or from remote settings. The evaluations have been performed in Chile, Democratic Republic of Congo, Senegal, Djibouti, Vietnam, India, and France, in dispensaries, in emergency room, on the field, in public health laboratories, and by the French Army. The dipstick method used requires minimal technical skill, and the test can be read between 5 and 15 minutes. Stool cultures and the immunochromatographic test showed concordant results in the comparative studies when RDT for S. sonnei was tested in Chile, Vietnam, India, and France; specificity (Sp) was 96% and sensitivity (Se) was 100%. When RDT for S. flexneri 2a was tested in Vietnam, Se was 91.5% and Sp was 99.2%. In Chile, Se was 83.3% and Sp was 100%. When RDT for S. dysenteriae 1 was tested in India, Vietnam, Senegal, and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the Sp was 98.7% and the Se was 91.7%. In Chile, the initial finding for a simple RDT to diagnose Shigella spp. demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys. Additionally, the dipsticks can be stored at room temperature in a humidity-proof plastic bag, making them easily transportable. Considering the potential impact these RDT have for the clinical management of the disease and for epidemiological studies, industrialization of these tests is in progress.

Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr(+) adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr(-) adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr(-) adhesin) was found to predominate in afa-positive isolates from sepsis patients (75%); it was frequent in afa-positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr(+) strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa-positive strains.

An epidemic of bloody diarrhea: Escherichia coli O157 emerging in Cameroon?

Between November 1997 and April 20, 1998, bloody diarrhea sickened 298 persons in Cameroon. Laboratory investigation of the epidemic (case-fatality rate, 16.4%) documented amoebiasis in one of three patients and three types of pathogens: multidrug-resistant Shigella dysenteriae type 1, S. boydii, and enterohemorrhagic Escherichia coli. We report the first isolation of E. coli O157:H7 in Cameroon and the second series of cases in the Central African region.

Etiologies of acute, persistent, and dysenteric diarrheas in adults in Bangui, Central African Republic, in relation to human immunodeficiency virus serostatus.

A study of the etiologies of diarrhea in adults in relation to their human immunodeficiency virus (HIV) serostatus and number of CD4+ cells was carried out in the Central African Republic. In cases and controls, multi-parasitism was observed. Salmonella spp. were identified mainly during acute diarrhea, with 50% of the S. enteritidis isolated during the study being responsible for septicemia and/or urinary tract infection in immunodeficient patients. Enteroaggregative Escherichia coli (EAggEC) were the most frequently identified agent in HIV+ patients with persistent diarrhea; 42.8% of the patients with EAggEC as sole pathogens had bloody diarrhea, and these strains were negative for the presence of a virulence plasmid. Coccidia were found in those with acute and persistent diarrhea. Blood was observed in 53.3% of infections involving coccidia as the sole pathogen. Microsporidium spp. and Blastocystis hominis were found only in HIV+ patients with persistent diarrhea. Shigella spp., Campylobacter spp., and Entamoeba histolytica were found in HIV+ and HIV- dysenteric patients; bacteria resembling spirochetes that could not be cultivated were identified only in HIV+ cases with dysentery. Shiga-like toxin-producing E. coli O157:H- was isolated from two cases with hemolytic-uremic syndrome. Fungi were identified as the sole pathogen in 6.4% of the HIV+ patients with persistent diarrhea. Most of enteropathogenic bacteria identified were resistant to ampicillin and trimethoprim-sulfamethoxazole, remained susceptible to ampicillin plus clavulanic acid, and were susceptible to amikacin, gentamicin, and ciprofloxacin.

Enterohaemorrhagic Escherichia coli in Central African Republic.

PIP: A dysentery outbreak in the Central African Republic village of Zemio was diagnosed as "Shigella flexneri" by the Pasteur Institute in Bangui (IPB) in February 1996; 2 months later there was an outbreak of hemorrhagic colitis. 108 patients presented with bloody diarrhea; cramping abdominal pain, fever, nausea, and vomiting were uncommon. The illness lasted between 5 days and 3 weeks (average, 8 days). Antibiotics were ineffective. Four patients died and several developed hemolytic-uremic syndrome. Stool cultures done at IPB tested negative. PCR was used to detect enterohemorrhagic Shiga-like toxin (SLT) 1 and 2, the invasivity gene ipaH, and the attaching and effacing gene eaeA. DNA fragments of 130 and 494 nucleotides corresponding to amplified SLT1 and eaeA were found in 80% of the specimens tested. No amplification was obtained for SLT2 or for ipaH in specimens collected during the second epidemic. These results suggest the presence of enterohemorrhagic Escherichia coli and the absence of Shigella. The number of reported cases of acute bloody diarrhea in infants and adults in Bangui has increased since 1996. E. coli O157:H7 was isolated from two fatal adult cases. Smoked zebu meat was suspected in several hospital cases (bloody diarrhea, hemolytic anemia, and renal insufficiency) in which non-fermenting sorbitol E. coli O157:H7 was not isolated. In two cases of acute diarrhea, other serotypes of E. coli were indicated by retrospective PCR on stools which were positive for SLT1 and for eaeA and negative for invasivity. A study was conducted in Bangui on 290 cases (33 with bloody diarrhea) and 140 controls. Patients were not paired because of civil unrest in the city. The questionnaire included demographic and socioeconomic characteristics, environmental factors, and habitual food consumption. The major contributing factor was consumption of locally made meat pies (kanda), which were made with smoked zebu meat. Kanda is stored at ambient temperature, often for days, before it is sold in markets or along roads. Before 1996, E. coli was not reported as a cause of bloody diarrhea in the Central African Republic.^ieng

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy.

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.

Prevalence of enteropathogenic, enteroaggregative, and diffusely adherent Escherichia coli among isolates from children with diarrhea in new Caledonia.

The clinical significance of HEp-2-adherent Escherichia coli in children with diarrhea in New Caledonia has been examined by testing isolates from stools of ill children and matched controls in a HEp-2 cell binding assay and by hybridizing the same clones with DNA probes identifying the enteropathogenic (EPEC), enteroaggregative (EAggEC), and diffusely adherent (DAEC) E. coli. From the 100 patient-control pairs, 35 HEp-2-adherent strains were isolated; 24 were identified as the only pathogen in stools of ill children, and 11 were from controls. EPEC strains were significantly associated with diarrheal disease (P < .008) in children in the first 2 years of life. For the DAEC strains, the difference in rate of isolation between patients and controls was significant only when the presence of afa/daa sequences in the strains was considered (P = .03, Fisher's exact test). The afa/daa-positive DAEC isolates were characterized from children 2-6 years old. EAggEC strains were isolated equally in patients and controls.

Escherichia coli heat-stable enterotoxin (STa)-biotin enzyme-linked immunosorbent assay (STa-biotin ELISA).

We have previously shown that an Escherichia coli heat-stable enterotoxin (STa)-biotin conjugate binds to polystyrene microtitre plates coated with avidin (Germani et al., 1992). In the present study the STa-biotin ELISA, based on inhibition of binding of anti-STa antibodies to avidin-bound STa-biotin conjugates, was compared with the conventional suckling mouse assay for the identification of STa from Biken agar extracts and from culture supernatants, using 150 E. coli isolates (50 STa-positive and 100 ST-negative). Pieces of Biken agar were a good source of toxin, 142 of 150 strains gave consistent results by both tests: 100 were negative and 42 were positive; seven of the remaining eight E. coli gave questionable but positive results in the STa-biotin ELISA and were positive by the suckling mouse test; the last E. coli gave negative result by both tests. The STa-biotin ELISA was 85.7% sensitive and 100% specific; the negative predictive value was 0.935 and the positive predictive value was 1. All the 150 strains tested for STa production from standard liquid cultures gave consistent results by both techniques. The STa-biotin ELISA detected 20 pg of partly purified STa compared to 15 pg in the suckling mouse assay.

Two-year study of endemic enteric pathogens associated with acute diarrhea in New Caledonia.

A longitudinal study of diarrheal disease among patients of all ages with acute diarrhea was carried out in New Caledonia from January 1990 to December 1991. Stool samples from 2,088 diarrheal patients were examined for parasites, rotavirus, and bacterial pathogens. Potential sources of contamination (drinking water, seawater and bovine and porcine feces) were investigated. One or more enteric pathogens were identified in 41.8 and 40.6% of the persons with diarrhea, in 1990 and 1991, respectively. Salmonella spp., Shigella spp., HEp-2 cell adherent Escherichia coli (diffuse adherent and enteroaggregative), enteropathogenic E. coli (EPEC) (EPEC adherence factor-positive strains belonging to classical serotypes), localized adherent E. coli (non-EPEC), and enterotoxigenic E. coli were the frequently identified enteropathogenic bacteria. Other major enteropathogens were Entamoeba histolytica and Giardia lamblia. Campylobacter jejuni, Clostridium difficile, Clostridium perfringens, Yersinia enterocolitica, and rotavirus were isolated from only a few patients. No Vibrio spp., Aeromonas spp., Plesiomonas spp., Shiga-like-toxin-producing E. coli, enterohemorrhagic E. coli, or enteroinvasive E. coli were identified. Shiga-like toxin I-producing E. coli were present in adult bovines and calves, and heat-stable enterotoxin II-producing enterotoxigenic E. coli were found in pigs.

Easy-to-perform modified Elek test to identify Shiga-like toxin-producing diarrhoeogenic Escherichia coli.

We determined whether Shiga-like toxin I (SLT-I) -producing diarrhoeogenic Escherichia coli could be detected by a modified Elek tests. The test (SLT Elek test) is based on the principle of the Elek test and the Ouchterlony double-gel diffusion. The development of the SLT Elek test was preceded by a preliminary study; the purpose of the later was to establish whether a simplified purification procedure of SLT-I (involving bacterial sonic extract, "Affi-Gel Blue" chromatography and anion- and cation-exchange liquid chromatography) could be employed in the preparation of rabbit antisera to SLT-I. SLT-I-specific antisera were obtained after adsorption of sera with bacterial sonic extract from non-toxigenic E. coli. A total of 135 strains of E. colo were tested by the SLT Elek test (100 SLT-I-negative and 35 SLT-I-positive). The results of the SLT Elek test and the Vero cell test correlated well: 30 strains gave positive results and 100 strains gave negative results in both tests. Only 5 strains gave discrepant results: they were weakly positive in the Vero cell assay, whereas 3 gave borderline reactions and 2 were negative in the SLT Elek test. Positive predictive value was 1, negative predictive value was 0.98; the SLT Elek test was 91% sensitive and 100% specific.

Molecular characterization of enterotoxigenic Escherichia coli (ETEC) isolated in New Caledonia (value of potential protective antigens in oral vaccine candidates).

The role of enterotoxigenic Escherichia coli (ETEC) in childhood diarrhoea in New Caledonia was demonstrated in previous epidemiological works. This study was undertaken in order to characterize these strains and to determine whether bacterial components of current vaccine candidates (toxin, colonization factor antigens, O:H antigens) would be useful in our region. A total of 24 ETEC strains were studied: 5 strains produced heat-labile enterotoxin, 17 strains produced heat-stable enterotoxin (9 STp and 8 STh), and 2 strains produced both toxins (1 LT/STp/STh and 1 LT/STh). E. coli strains were screened for the presence of genes encoding for enterotoxins (DNA dot blot and Southern hybridization assays); results obtained with probes were closely correlated and were in agreement with biological assays. No two ETEC strains possessed similar plasmid profiles, and DNA sequences encoding for enterotoxins were located on plasmids ranging from 58 to 75 MDa. The O:H (O1:H-,O2:H7, O6:H16, O25:H-, O27:H7, O28ab:H9, O52:H10, O64:H5, O70:H-, O78:H12, O88:H25, O99:H6, O101:H-, O126:H12, O166:H30) serotypes are presented (all the strains were typable, but some ETEC serotypes were unusual). By using antisera against colonization factor antigens (CFA) I and II, results showed that 9 of the 24 ETEC strains expressed CFA (2 CFA/II and 7 CFA/I). These strains possessed high bacterial surface hydrophobicity. Fifteen ETEC did not possess CFA; among these, 11 did not exhibit high hydrophobicity or show haemagglutination activity. Four of the 15 CFA-negative strains exhibited high hydrophobicity (two O64:H45, one O70:H- and one O88:H25) but no haemagglutination in the presence or absence of mannose.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of diarrheogenic Escherichia coli in children less than ten years old with and without diarrhea in New Caledonia using seven acetylaminofluorene-labeled DNA probes.

We report the use of seven acetylaminofluorene (AAF)-labeled DNA probes in evaluating the incidence of various Escherichia coli pathotypes in New Caledonia among 448 children with acute diarrhea (1,278 E. coli pathotypes studied) and 88 controls (264 E. coli pathotypes studied) in 1990. Diarrheogenic E. coli were detected using cloned gene probes for heat-labile and heat-stable enterotoxins, Shiga-like cytotoxins (SLTI and SLTII), the cell invasion phenotype (INV), and enteropathogenic-adherence factor (EAF). Isolates were also studied using bioassays and radioactive DNA probes as reference methods. Enterotoxigenic E. coli (ETEC) were isolated from only 5.36% of the patients; E. coli with localized adherence (LA) to HEp-2 cells was much more common in patients (14.4%) than in controls (3.4%; chi 2 = 7.54, P < 0.01), but most of the E. coli with an LA pattern were members of traditional enteropathogenic E. coli (EPEC) serogroups (chi 2 = 92.95, P < 0.001). Non-enteropathogenic E. coli with an LA pattern were weakly associated with diarrheal disease (8.9%). Escherichia coli with a diffuse or an aggregative pattern did not show a significant association with infantile diarrhea. Eight EPEC serogroups were identified and the frequency of positivity for the LA pattern was 70.5%; the EAF was significantly associated with the 0119:K9 serogroup. No enteroinvasive or SLT-producing E. coli were identified. An evaluation of the AAF probes in comparison with 32P-labeled probes and conventional bioassays was made during this epidemiologic survey. The positive and negative predictive values of the ETEC probes were 0.91 and 1, respectively (overall agreement = 99.8%).(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of enterotoxigenic Escherichia coli in faecal specimens by acetylaminofluorene-labelled DNA probes.

The present study describes acetylaminofluorene(AAF)-modified DNA probes for the identification of heat-labile (LT) and heat-stable (porcine STp and human STh) toxins from enterotoxigenic Escherichia coli (ETEC). AAF probes were compared with established biotinylated probes and bioassays. Ultracentrifugation was not necessary in the preparation of AAF-labelled probes, and the procedures, i.e. chemical modification of probes, hybridization and immunodetection steps, were optimized to detect ETEC by colony hybridization or direct dot blot techniques. The method combines chemical labelling (covalent attachment of AAF group to guanosine is achieved by treatment of DNA with N-acetoxy-2-acetylaminofluorene) and detection of the hybridized target DNA by means of anti-AAF monoclonal antibodies and alkaline phosphatase-labelled second antibodies.

Escherichia coli heat-stable enterotoxin (STa)-biotin conjugates for the titration of STa antisera by an enzyme-linked immunosorbent assay.

The development of a new approach to the diagnosis of infectious diarrhoea, caused by Escherichia coli heat-stable enterotoxin (ST), was preceded by a preliminary study. The purpose of the latter was to establish whether three preparations of ST produced by a human isolate of enterotoxigenic E. coli (STa), obtained at different steps of the purification procedure (involving Amberlite XAD2 resin chromatography (P3), a gel filtration chromatography on a Biogel P4 (P2) or a disc-gel electrophoresis (P1)), could be employed to titrate antisera to STa using an ST-biotin enzyme-linked immunosorbent assay (ELISA). The solid-phase STa was obtained by first coupling the toxin to biotinyl-N-hydroxysuccinimide and then binding this conjugate to avidin adsorbed to flat-bottomed polystyrene microtitre plates. Using these reagents, the assay conditions were examined. Checkerboard tests determined optimal biotin-P3, P2 or P1 toxin conjugate concentrations to be used as the immunosorbent for P3, P2 and P1 antiserum titration. The immunosorbent prepared with STa purified only on Amberlite XAD2 resin was unable to differentiate significantly between P3, P2 or P1 antisera. Immunosorbent prepared with P2 or P1 detected widely differing titres between the three antisera and gave more sensitive results. Only small but questionable differences were observed between P2 and P1 toxin preparations.

Competitive erythroimmunoassay for detecting Clostridium perfringens type A enterotoxin in stool specimens.

A competitive erythroimmunoassay (ERIA) is described for Clostridium perfringens enterotoxin (CPE) detection in stools. This technique uses sheep red blood cells sensitized by CPE and an anti-CPE-antibody-coated plate in which the results are read by eye. ERIA is simple, rapid, economic and more sensitive (2 ng/ml) than the enzyme-linked immunosorbent assay used for evaluation. ERIA is suitable for CPE detection in stool samples protected with phenylmethylsulphonylfluoride.

[Characterization of the enterotoxigenic strains of Escherichia coli isolated from children with diarrhea in Popular Democratic Republic Lao]. / Caractérisation des souches de Escherichia coli entérotoxinogènes isolées d'enfants diarrhéiques en République Démocratique Populaire Lao.

Enterotoxigenic Escherichia coli (ETEC) of infant origin from the Popular Democratic Republic Lao were characterized with respect to there 0 serogroups, biotypes, anti-bioresistances, fimbrial antigens and types of enterotoxins produced. Enterotoxin production was determined by the suckling mice assay, competitive GM1-erythroassay, and cell cultures (CHOK1 and Y1). The presence of genes encoding for the enterotoxins was determined by colony hybridization by using radioactive DNA probes. Profile plasmids from ETEC strains were studied. The plasmids encoding for heat-labile enterotoxin were studied with an acetyl-aminofluorene modified probe.