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Background Despite vaccination programs, Streptococcus pneumoniae remains among the main microorganisms involved in bacterial pneumonia, notably in terms of severity. The prognosis of pneumococcal infections is conditioned in part by the precocity of the diagnosis. The aim of this study was to evaluate the impact of a Rapid Diagnostic Test (RDT) targeting cell wall polysaccharide of Streptococcus pneumoniae and performed directly in respiratory samples, on the strategy of diagnosis of respiratory pneumococcal infections in children. Results Upper-respiratory tract samples from 196 children consulting at hospital for respiratory infection were tested for detecting S. pneumoniae using a newly-designed RDT (PneumoResp, Biospeedia), a semi-quantitative culture and two PCR assays. If positive on fluidized undiluted specimen, the RDT was repeated on 1:100-diluted sample. The RDT was found highly specific when tested on non-S. pneumoniae strains. By comparison to culture and PCR assays, the RDT on undiluted secretions exhibited a sensitivity (Se) and negative predictive value (NPV) of more than 98%. By comparison to criteria of S. pneumoniae pneumonia combining typical symptoms, X-ray image, and culture ≥107 CFU/ml, the Se and NPV of RDT on diluted specimens were 100% in both cases. Conclusions In case of negative result, the excellent NPV of RDT on undiluted secretions allows excluding S. pneumoniae pneumonia. In case of positive result, the excellent sensitivity of RDT on diluted secretions for the diagnosis of S. pneumoniae pneumonia allows proposing a suitable antimicrobial treatment at day 0.

International audience; Meningococcal meningitis remains a life-threatening disease worldwide, with high prevalence in the sub-Saharan meningitis belt. A rapid diagnosis is crucial for implementing adapted antimicrobial treatment. We describe the performances of a new immunochromatographic test (MeningoSpeed, BioSpeedia, France) for detecting and grouping Neisseria meningitidis. Cerebrospinal fluids (CSFs) were collected from 5 African countries and France. For the rapid diagnostic test (RDT), the CSF sample was deposited on each of the 3 cassettes for a total volume of 90 μl. The results of the RDT were compared to those of a reference multiplex PCR assay detecting the major serogroups of N. meningitidis on 560 CSF specimens. Five specimens were found uninterpretable by RDT (0.9%). The results of interpretable specimens were as follows: 305 positive and 212 negative samples by both techniques, 14 positive by PCR only, and 24 positive by RDT only (sensitivity, specificity, and positive and negative predictive values of 92.7%, 93.8%, 95.6%, and 89.8%, respectively, with an accuracy of 93.2% and a kappa test of 0.89; P < 0.05). From 319 samples positive by PCR for serogroups A, C, W, X, or Y, the grouping results were concordant for 299 specimens (sensitivity of 93.0%, 74.4%, 98.1%, 100%, and 83.3% for serogroups A, C, W, X, and Y, respectively). The MeningoSpeed RDT exhibited excellent performances for the rapid detection of N. meningitidis antigens. It can be stored at room temperature, requires a minimal amount of CSF, is performed in 15 minutes or less, and is easy to use at bedside.

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.

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.

We studied isotype profiles of anti-HIV antibodies (Ab) in HIV-1-infected African patients with high viral loads and major B cell dysfunction. We focussed on IgG1, IgG3, and IgA as these classes and subclasses tend to support neutralizing functions against HIV. Total IgG1, IgG3 and IgA were detected in the plasma of both HIV-1-infected and HIV-negative African individuals, but there was significantly more IgG3, a rare subclass, in HIV-1-infected patients (P

To study the progression of HIV-1 infection and coreceptor usages in Central African Republic, clinical data, plasma viral load, and coreceptor usage of sequential HIV-1 isolates were analyzed in a seroincident prospective cohort (PRIMOCA). Twenty-three HIV-1 infected individuals from the Central African Armed Forces were followed from 1995 to 2000. Viruses were isolated from 17 patients at various time points after seroconversion and their coreceptor usage was examined using GHOST cells expressing CD4 and one of the HIV-1 chemokine coreceptors CCR5, CXCR4, BOB/GPR15, and Bonzo/STRL33/CXCR6. Eleven patients died from AIDS. Eight of them died between 2 and 5 years after seroconversion, after a brief symptomatic stage. Patients who rapidly progressed to AIDS and death displayed the highest viral loads after seroconversion. All isolates obtained soon after seroconversion used CCR5, albeit, in some cases, CXCR4, BOB, or Bonzo were also used. Most isolates remained R5 (59 out of 61 isolates), although viruses using CXCR4 appeared in some cases of progression to AIDS. In several cases, a broad tropism was observed during the course of infection, with a frequent usage of BOB and Bonzo in addition to CCR5. Rapid progression to disease and short survival time among Central African HIV-1 patients appear more frequent than those reported in industrialized countries. Viral coreceptor used was mainly CCR5, but, interestingly, a large part of isolates also used BOB and Bonzo. However, there was no strict correlation between the clinical outcome and extended viral tropism.

The objective of this study is to improve the viability after freeze-drying and during storage of delicate or recalcitrant strains safeguarded at biological resource centers. To achieve this objective, a joint experimental strategy was established among the different involved partner collections of the EMbaRC project ( www.embarc.eu ). Five bacterial strains considered as recalcitrant to freeze-drying were subjected to a standardized freeze-drying protocol and to seven agreed protocol variants. Viability of these strains was determined before and after freeze-drying (within 1 week, after 6 and 12 months, and after accelerated storage) for each of the protocols. Furthermore, strains were exchanged between partners to perform experiments with different freeze-dryer-dependent parameters. Of all tested variables, choice of the lyoprotectant had the biggest impact on viability after freeze-drying and during storage. For nearly all tested strains, skim milk as lyoprotectant resulted in lowest viability after freeze-drying and storage. On the other hand, best freeze-drying and storage conditions were strain and device dependent. For Aeromonas salmonicida CECT 894(T), best survival was obtained when horse serum supplemented with trehalose was used as lyoprotectant, while Aliivibrio fischeri LMG 4414(T) should be freeze-dried in skim milk supplemented with marine broth in a 1:1 ratio. Freeze-drying Campylobacter fetus CIP 53.96(T) using skim milk supplemented with trehalose as lyoprotectant resulted in best recovery. Xanthomonas fragariae DSM 3587(T) expressed high viability after freeze-drying and storage for all tested lyoprotectants and could not be considered as recalcitrant. In contrary, Flavobacterium columnare LMG 10406(T) did not survive the freeze-drying process under all tested conditions.

Considerant le role important du gene eaeA dans la physiopathologie des diarrhees dues a des souches de E. coli enteropathogenes (ECEP) et la specificite de la sonde derivee de ce gene, nous avons developpe un test PCR pour faciliter la detection du gene eaeA dans les souches de ECEP par les laboratoires d'analyses medicales.

Strains 1517T and 61DT were characterized by phenotypic and molecular taxonomic methods. These Gram-positive lactic acid bacteria were homo-fermentative, facultatively anaerobic short rods. They were phylogenetically related to the genus Lactobacillus according to 16S rRNA gene sequence analysis, with 99 % similarity between strain 1517T and the type strain of Lactobacillus gigeriorum , and 98.6, 98.5 and 98.4 % between strain 61DT and Lactobacillus gasseri , Lactobacillus taiwanensis and Lactobacillus johnsonii , respectively. Multilocus sequence analysis and metabolic analysis of both strains showed variation between the two strains and their close relatives, with variation in the position of the pheS and rpoA genes. The DNA–DNA relatedness of 43.5 % between strain 1517T and L. gigeriorum , and 38.6, 29.9 and 39.7 % between strain 61DT and L. johnsonii , L. taiwanensis and L. gasseri , respectively, confirmed their status as novel species. Based on phenotypic and genotypic characteristics, two novel species of Lactobacillus are proposed: Lactobacillus pasteurii sp. nov., with 1517T ( = CRBIP 24.76T = DSM 23907T) as the type strain, and Lactobacillus hominis sp. nov., with 61DT ( = CRBIP 24.179T = DSM 23910T) as the type strain.

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.

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.

The role of enterotoxigenic Escherichia coli (ETEC) in childhood diarhoea in New Caledonia was demonstrated in previous epidemiological works. This study was under-taken 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-, 02: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 posesess 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. Only 7 of 24 ETEC expressed resistance to ampicillin, trimethoprimsulphamethoxazole or tetracycline. Data indicate that several ETEC isolates would be refractory to current vaccine candidates, and that for vaccine to be effective in our region, other antigens must be included.

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.

Background Environmentally driven immune activation was suggested to contribute to high rates of HIV-1 infection in Africa. We report here a study of immune activation markers and susceptibility to HIV-1 infection in vitro of forty-five highly exposed uninfected partners (EUs) of HIV-1 infected individuals in Central African Republic, in comparison with forty-four low-risk blood donors (UCs). Results Analysis of T lymphocyte subsets and activation markers in whole blood showed that the absolute values and the percentage of HLA-DR+CD4 T cells and of CCR5+CD4 T cells were lower in the EUs than in the UCs (p = 0.0001). Mutations in the CCR5 coding region were not found in either group. Susceptibility to in vitro infection of unstimulated peripheral blood mononuclear cells, prior of PHA activation, was decreased in EUs compared to UCs, either using a CXCR4-tropic or a CCR5-tropic HIV-1 strain (p = 0.02 and p = 0.05, respectively). Levels of MIP-1β, but not of MIP-1α or RANTES, in the supernatants of PHA-activated PBMC, were higher in the EUs than in the UCs (p = 0.007). Conclusion We found low levels of CD4 T cell activation and reduced PBMC susceptibility to HIV-1 infection in Central African EUs, indicating that both may contribute to the resistance to HIV-1 infection.

Mutations frequently occur in oral poliovirus vaccine (OPV) strains upon replication in the human intestine. These strains occasionally revert to being neurovirulent. The more prolonged the excretion of OPV, the higher the risk of reversion. OPV strains can be secreted for several months in humans presenting humoral immune system deficiencies. The duration of excretion of OPV strains or other enteroviruses in individuals infected with the human immunodeficiency virus (HIV) is unknown. We investigated whether HIV infection, which is very prevalent in the Central African Republic, causes prolonged excretion of enteroviruses and, in particular, of OPV strains in adults. We studied 28 HIV-infected adults living with children who were immunized with OPV during national immunization days (NIDs). Blood samples were collected to confirm HIV status and to evaluate immunodeficiency before the NIDs. Stool samples for enterovirus isolation were also collected before the NIDs, between the two rounds of immunization and 2, 4 and 6 months after the second round of immunization. No poliovirus was isolated from any stool sample. Eight enteroviruses were isolated from eight adults (maximum one strain per patient). Enteroviruses were not more frequently isolated from severely immunodeficient patients. Thus, HIV-infected adults do not appear to be at high risk of infection with OPV strains and the excretion of enteroviruses (and thus of polioviruses) does not seem to be prolonged in HIV-infected adults.

Unseparated peripheral blood mononuclear cells (PBMCs) obtained from drug-naïve African individuals living in a context of multi-infections and presenting with high viral load (VL), were cultured in vitro and tested for their ability to produce antibodies (Abs) reacting with HIV-1 antigens. Within these PBMCs, circulating B cells were differentiated in vitro and produced IgG Abs against not only ENV, but also GAG and POL proteins. Under similar experimental conditions, HAART treated patients produced Abs to ENV proteins only. The in vitro antibody production by drug-naïve individuals' PBMCs depended on exogenous cytokines (IL-2 and IL-10) but neither on the re-stimulation of reactive cells in cultures by purified HIV-1-gp 160 antigen nor on the re-engagement of CD40 surface molecules. Further, it was not abrogated by the addition of various monoclonal Abs (mAbs) to co-stimulatory molecules. This suggests that the in vitro antibody production by drug-naïve individuals' PBMCs resulted from the maturation of already envelope and core antigen-primed, differentiated B cells, presumably pre-plasma cells, which are not known to circulate at homeostasy. As in vitro produced Abs retained the capacity of binding antigen and forming complexes, this study provides pre-clinical support for functional humoral responses despite major HIV- and other tropical pathogen-induced B cell perturbations.

To better understand the pathophysiology of B cell populations—the precursors of antibody secreting cells—during chronic human immunodeficiency virus (HIV) infection, we examined the phenotype of circulating B cells in newly diagnosed Africans. We found that all African individuals displayed low levels of naive B cells and of memory-type CD27+B cells, and high levels of differentiated B cells. On the other hand, HIV-infected African patients had a population of germinal center B cells (i.e. CD20+, sIgM-, sIgD+, CD77+, CD138±), which are generally restricted to lymph nodes and do not circulate unless the lymph node architecture is altered. The first observations could be linked to the tropical environment whereas the presence of germinal center B cells may be attributable to chronic exposure to HIV as it is not observed in HIV-negative African controls and HAART treated HIV-infected Europeans. It may impact the management of HIV infection in countries with limited access to HIV drugs and urges consideration for implementation of therapeutic vaccines.

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.

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection., PLoS Pathogens, 8 (7), ISSN:1553-7374, ISSN:1553-7366

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.

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)

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.

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.

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 quaitative correlation with the enzyme-linked immunosorbent assay and the Vero cell test was observed.

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 μg 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 protcase inhibitor, 32 % (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.

An India ink immune reaction was used for the direct detection, in diarrhoel 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.

Summary Fifty-five strains of Escherichia coli isolated from 51 faeces of Melanesian children with acute diarrhoea in New Caledonia were studied; three diarrhoeas were bloody. For each strain, haemagglutination type, adhesion to rabbit enterocytes, serotype, production of heat-labile (LT) or heat-stable (ST) toxins and identification of colonization factor antigens CFA/I or CFA/II were determined. We identified 48 strains able to attach to rabbit enterocytes; 27 produced enterotoxins (21 strains LT+ and 6 ST+) and 19 had CFA (13 CFA/I and 6 CFA/II). Five serotypes were identified: O6, O78, O80, O114 and O127:B8. One strain, O127:B8, which was able to attach to enterocytes, had CFA/I and produced LT toxin.

Summary An ELISA method on microtitration plates to detect and assay Escherichia coli heat-labile enterotoxin (LT) is described. This technique is rapid and simple to perform in any laboratory. It allows detection of the presence of LT with the naked eye within 10 h in a 12-h E. coli culture supernatant. The reaction is based on immunological cross-reaction between LT and the Vibrio cholerae toxin (CT). In place of traditional microtitration plates coated with ganglioside Gm1, we propose, a new polystyrene support coated with purified anti-CT antibodies. This coated support has been conditioned in a kit to be used in laboratories in bush dispensaries of endemic areas. It was tested with 40 enterotoxigenic (LT + ) strains isolated from stools of diarrhoeal children and with 14 LT- strains. All supernatants LT + and LT-were found positive and negative, respectively, with the ELISA method and with the new polystyrene support. Field tests (in Wallis, Futuna et Vanuatu) with the new kit and standard method were satisfactory.

Resume Au cours de la mise au point d'une methode immunoenzymatique de depistage des Escherichia coli porteurs d'adhesines CFA/I ou CFA/II dans des selles diarrheiques, la methode de fixation des anticorps sur le polystyrene par covalence a ete comparee a l'adsorption passive. La fixation covalente des anticorps par covalence permet de conserver les plaques 9 semaines au lieu de 6 par simple adsorption. La sensibilite pour les deux techniques est de 10 5 bacteries/ml pour l'antigene CFA/I et 10 6 pour le CFA/II.

Resume Une technique immunoenzymatique ELISA en micromethode de depistage direct dans les selles des Escherichia coli porteurs des adhesines CFA/I ou CFA/II (Colonization Factor Antigens) est decrite. La methode est comparee a la seroagglutination des subcultures des E. coli. La sensibilite de l'epreuve est de 105 E. coli/ml pour le CFA/I et de 106 pour le CFA/II. La seroagglutination est positive tout au long de la maladie et l'ELISA est positive pendant une periode plus courte.

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.

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.

One hundred and fifty enterotoxigenic strains of Escherichia coli (ETEC) isolated from infants with acute diarrhoeas in New Caledonia, Vanuatu and Wallis and Futuna were examined for serotypes. Twenty serotypes were identified, 5% of E. coli (7 strains) were rough and 8% (12 strains) were untypable. The serotypes 06, 025, and 078 are enterotoxigenic at 100%. The serotypes 027 and 073 classically enterotoxigenic did not produce any enterotoxin. This study opens up a possibility that the predominant enterotoxigenic serotypes in that region of the South Pacific are probably different from the serotypes proposed in the pool of sera to identify ETEC.

An ELISA method on microtitration plates to detect and assay Escherichia coli heat-labile enterotoxin (LT) is described. This technique is rapid and simple to perform in any laboratory. It allows detection of the presence of LT with the naked eye within 10 h in a 12-h E. coli culture supernatant. The reaction is based on immunological cross-reaction between LT and the Vibrio cholerae toxin (CT). In place of traditional microtitration plates coated with ganglioside Gm1, we propose a new polystyrene support coated with purified anti-CT antibodies. This coated support has been conditioned in a kit to be used in laboratories in bush dispensaries of endemic areas. It was tested with 40 enterotoxigenic (LT+) strains isolated from stools of diarrhoeal children and with 14 LT- strains. All supernatants LT+ and LT- were found positive and negative, respectively, with the ELISA method and with the new polystyrene support. Field tests (in Wallis, Futuna et Vanuatu) with the new kit and standard method were satisfactory.

A strain of enteropathogenic Escherichia coli 0126:B16 has been isolated in fifteen children and one adult during a severe outbreak. One infant is dead. The strain produced heat-stable enterotoxin, attach to rabbit enterocytes but did not have colonization factor antigen CFA/I or CFA/II. Its hemagglutination type was the same that the E. coli H10407, CFA/I+. It presented a resistance at eight antibiotics and, with the loss of enterotoxigenicity, there was a loss of resistance at ampicillin and of the capacity to attach to enterocytes.

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.

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.

The focus of the EU regulations on the Nagoya Protocol on Access and Benefit-Sharing leaves the control of access to genetic resources up to each member state. France has chosen to control access and is going to put in place regulations for it. All the materials received should have specific documentation regarding the accession of genetic resources, where there is a National Authority to issue them. The European commission will maintain a list of biological collections with registered status proposed by each country. The member states are responsible for considering inclusion and verification of these collections. In recent years, the Collection of Institut Pasteur (CIP) staff has expressed concern over how to interact with the implementation of the Nagoya Protocol in the collection but also at the national level with the aim that the CIP will be a registered collection. The advantage of accessing resources from a registered collection is that users of genetic resources will be considered as having exercised ‘due diligence’ if they source their genetic resources from these collections. This could facilitate the process for scientists when applying for research funding. The CIP organized the accession of new deposits and the distribution of micro-organisms in connection with it.