[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.

Increase of HIV-1 subtype A in Central African Republic.

The concomitant presence of five distinct HIV-1 subtypes and of unclassified HIV-1 was reported in Bangui, Central African Republic (C.A.R.) between 1990 and 1991. This previous study was conducted in individuals belonging to the C.A.R. Armed Forces (FACA) Cohort and in patients from the University Hospital of Baugui. To follow the HIV-1 subtype distribution in Bangui over time, we conducted a cross-sectional surveillance of HIV-1 subtypes between 1987 and 1997 in three groups of individuals in Bangui: 47 men belonging to the FACA Cohort, 38 patients from the CNHUB hospital, and 51 individuals consulting the sexually transmitted diseases (STD) clinic. One hundred and ten HIV-1 were subtyped by heteroduplex mobility assay (HMA) and/or sequencing of env regions encompassing the V3 domain. By comparing the HIV-1 distribution in two time periods (1987-1991 and 1991-1996) in the FACA cohort, we observed a significant increase of subtype A from 43.7% to 83.9%. This subtype distribution does not seem specific to the FACA cohort, in that subtype A accounted for 46.7% of the HIV-1 infections in CNHUB patients in the first time period studied and for 69.6% in the second time period. In STD patients, subtype A infections were predominant in 1995 (72.7%) and 1997 (89.7%). Subtype E viruses could be identified in the second time period, but represented only between 6.5% and 21.8% of the infections in the three groups of individuals studied. Other subtypes (B, C, H) and non-classified HIV-1 in C2-V3 were detected with only a 3.2% to 9.1% frequency for each in the second time period. Phylogenetic analysis excluded infection by a single source for the individuals included in the study. Our data demonstrate an increase in the proportion of HIV-1 subtype A infections in Bangui that raises the question of a preferential transmissibility of specific HIV-1 variants.

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.

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.

Comparison of two GM1-erythrocyte assays to detect heat-labile Escherichia coli enterotoxin in stool specimens.

Two erythrocyte immunoassay techniques to detect the presence of Escherichia coli heat-labile enterotoxin (LTh) in stool supernatants and cell-free culture supernatants were compared. In the competitive assay, GM1 ganglioside was coated onto V-shaped-well microdilution plates and enterotoxin was coupled to sheep erythrocytes. As little as 0.8 ng of LTh per ml was detected by this method, which was based on the competition between the LTh of the test sample and the sensitized erythrocytes. The second assay made use of chimera antibody prepared by coupling polyclonal anti-LTh antibody to a monoclonal antibody specific for sheep erythrocytes. In this case, LTh, which was specifically bound to a GM1 ganglioside-coated plate, was detected by successively adding the chimera antibody and sheep erythrocytes. The limit of detection of the chimera antibody erythrocyte immunoassay was 0.2 ng/ml. Stool samples were collected from 167 infants hospitalized for diarrhea in the hospital of Noumea, New Caledonia. False-negative reactions due to proteases present in the stool samples were avoided by the addition of phenylmethylsulfonyl fluoride.

[Direct detection in diarrheal stools of Escherichia coli adhesion factors CFA/I, CFA/II and E8775 by the india ink immune reaction]. / Dépistage direct dans les selles diarrhéiques des Escherichia coli porteurs des adhésines CFA/I, CFA/II et E8775 par la réaction immuno/encre de Chine.

An India ink immune reaction was used for the direct detection, in diarrhoeal stools, of Escherichia coli possessing the CFA/I, CFA/II and E8775 fimbriae. With this method, a presumptive diagnosis of enterotoxigenic E. coli (ETEC) can rapidly be made. Staining required the bivalency of rabbit anti-fimbriae IgG; the F(ab')2 fragment was necessary. The reaction was impossible with purified Fab fragment. A comparative study of this technique and detection by culture of ETEC strains showed good correlation when the India ink immune reaction was performed at the beginning of infection.

[Specific detection of the thermolabile enterotoxin of Escherichia coli in human feces by competitive immunoenzyme tests in the presence of protease inhibitors]. / Détection spécifique de l'entérotoxine thermolabile de Escherichia coli dans les selles humaines par épreuves immunoenzymatiques compétitives en présence d'inhibiteur des protéases.

Two simple two-step competitive enzyme-linked immunoassays for human E. coli heat-labile enterotoxin (LTh) employing microtitration plates coated with rabbit anti-LTh antibody (ELISA) or GM1 ganglioside (GM1-ELISA) are described. LTh of the test sample competed with the same toxin coupled with horse-radish peroxidase. ELISA and GM1-ELISA were able to detect, respectively, as low as 5 ng and 6.5 ng TLh/ml and up to 9 and 11 micrograms TLh/ml. Both techniques were applied to the study of 167 infant diarrhoeas; ETEC producing LTh were identified in 17 diarrhoeal stools. When the faeces were diluted in phosphate buffer, only 17.6% (3 stools) and 29% (5 stools) of LTh-positive faeces were identified in ELISA and GM1-ELISA. When the stools were diluted with phenylmethylsulphonyl fluoride (PMSF), a synthetic protease inhibitor, 82% (14 stools) and 88% (15 stools) of LTh-positive stool supernatants were detected. Aprotinin, another protease inhibitor, was without effect and foetal calf serum, horse serum and bovine serum albumin enabled detection of only a low percentage of LTh-positive stools.

Antibody chimera technique applied to the detection of Escherichia coli heat-labile enterotoxin.

Covalently prepared chimera antibodies were tested in a ganglioside GM1 erythro-immunoassay (CERIA) for E. coli heat-labile enterotoxin (LT) detection. The antibody specific for LT was conjugated with a polyclonal antibody specific for sheep erythrocytes. The assay is based on the specific binding of LT to polystyrene-adsorbed GM1 and subsequent erythro-adsorption via chimera antibody by which the bound toxin is visualized. Enterotoxin titers determined with this CERIA method were similar to those obtained with the Vero cell assay and with ELISA. 5 ng of cholera toxin/ml may be detected with the assay. The CERIA, as described, may be used either qualitatively or quantitatively and is well suited for routine laboratory diagnosis of LT in a culture supernatant of E. coli.

[Assay of thermolabile enterotoxins of Escherichia coli and Vibrio cholerae by inhibition of erythroadsorption on the GM1 ganglioside]. / Dosage de l'entérotoxine thermolabile de Escherichia coli et de Vibro cholerae par inhibition de l'érythro-adsorption sur le ganglioside GM1.

A GM1 erythroassay (GERYDO) for heat-labile Escherichia coli enterotoxin (LT) and cholera toxin (CT) is described. This assay was developed for use in poorly equipped laboratories in developing countries. It uses GM1-coated polystyrene plates and is based on the competition between the toxin to be assayed and CT covalently bound to sheep red blood cells. GERYDO can detect 0.9 or 0.5 ng of CT per ml depending on the method of sensitization of erythrocytes. Good quantitative and qualitative correlation with the enzyme-linked immunosorbent assay and the Vero cell test was observed.

Assay for heat-labile Escherichia coli enterotoxin using sandwich erythroimmunoassay.

We investigated the possibility of using a sheep erythrocyte-antibody conjugate as reagent in a sandwich erythroimmunoassay (SERIA) procedure to detect and titrate Escherichia coli heat-labile enterotoxin (LT) with the naked eye. In this assay, which is based on the immunological similarity between Vibrio cholerae toxin (CT) and LT, rabbit anti-CT IgG was used as immunosorbent, and sheep erythrocytes, sensitized with the rabbit anti-CT antibodies, were used as indicator. The sensitivity of the test was demonstrated by a comparative study using an enzyme-linked immunosorbent assay (ELISA). The results obtained by SERIA with 130 samples correlated well with those of a Vero cell assay and a GM1-ELISA. The test is easy, relatively cheap and as sensitive as other standard techniques; it is particularly suited for field laboratories, especially in tropical countries, and a large number of strains may be examined daily.

GM1 erythroimmunoassay for detection and titration of Escherichia coli heat-labile enterotoxin.

A GM1 ganglioside erythroimmunoassay for the detection of heat-labile Escherichia coli enterotoxin (LT) was developed for use in poorly equipped laboratories in developing countries. This assay is based on the immunological similarity between Vibrio cholerae toxin and LT and uses cholera toxin antiserum and sheep anti-rabbit immunoglobulin covalently coupled to sheep erythrocytes as conjugate. This assay has the following advantages over other currently available techniques: the reagents it uses are stable, in particular, tanned and sensitized sheep erythrocytes; GM1 ganglioside is commercially available; erythro-adsorption can be read with the naked eye; the test can be completed in 1 day; and as little as 4 ng of V. cholerae toxin or LT per ml can be detected accurately. The GM1 ganglioside erythroimmunoassay showed good quantitative and qualitative correlation with the Vero cell assay and the conventional GM1 enzyme-linked immunosorbent assay. The GM1 ganglioside erythroimmunoassay was somewhat less sensitive than the GM1 enzyme-linked immunosorbent assay but more sensitive than the Vero cell assay. Results obtained for 12 LT-positive and 138 LT-negative E. coli strains correlated with results obtained with GM1 enzyme-linked immunosorbent and Vero cell assays.

[Application of the vapor test for the detection and immunologic determination of Escherichia coli heat labile enterotoxin]. / Application du test à la vapeur à la détection et au dosage immunologique de l'entérotoxine thermolabile de Escherichia coli.

The principle of this thin-layer immunoassay (vapour condensation technique or TVAP) is based on the ability of antibodies to absorb firmly to polystyrene surfaces and to retain their reactivity. A condensation pattern consisting of large confluent water drops is noticable when an antibody-antigen reaction takes place. We used this technique to detect and assay the Escherichia coli heat-labile enterotoxin (ETEC LT+) and compared the results of 53 strains (40 positives and 13 negatives) with single radial immune haemolysis, Gm1-ELISA and Vero cell culture tests. With the reagents used, this reaction was specific for a toxin dilution up to 1/14. As little as 0.025 micrograms/ml of cholera toxin could be detected. The TVAP-test is simple, rapid and cost-effective. It is thus quite suitable for use in diarrhoeal endemic areas.