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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., Competing Interests: The authors declare that there are no conflicts of interest., (© 2019 The Authors.)

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.

Background: We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs., Methodology/principal Findings: The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10(6) CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%-98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%-88.6%) and 100 %, respectively., Conclusion: This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.

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.

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-1beta, but not of MIP-1alpha 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.

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-gp160 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.

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 < 0.05). Anti-HIV-gp160 specific antibodies were detected in sera from nearly all HIV-1-infected individuals tested, but not in HIV- individuals: 10/10, 9/10 and 8/10 individuals displayed specific IgG1, IgG3 and IgA, respectively. In the corresponding PBMC cultures carried out in the presence of IL-10 and IL-2, there was specific IgG1 and IgA in 5/10, and 3/10, respectively, but no IgG3 was detected. When HAART-treated European HIV-infected PBMC cultures were tested using the same protocol, specific IgG3 was detected in 4/10 cultures, and was unaffected by the addition of soluble CD40L molecules. The present study thus shows that, despite lymph node disorganization in HIV-infected drug-naïve Africans, these individuals retain the ability to produce HIV-specific IgG1, IgG3 and IgA. However, the specific mechanisms controlling the selective production of IgG3, probably the most potent subclass and a potential target of immuno-intervention, warrants further investigation.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.