The role of IL-1beta in reduced IL-7 production by stromal and epithelial cells: a model for impaired T-cell numbers in the gut during HIV-1 infection.

OBJECTIVES: Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines produced during inflammatory conditions, may impact on IL-7 production. DESIGN: We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST. RESULTS: We found that IFN-gamma enhanced the expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant down-regulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL-1alpha,IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs and of important chemo-attractants for T cells. The microarray results were verified by additional methods. CONCLUSIONS: Our results are discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.

Identification of newly recognized serotype 1c as the most prevalent Shigella flexneri serotype in northern rural Vietnam.

We investigated the identity of 37 Shigella flexneri strains that had previously been isolated from northern rural Vietnam (Son Tay Province) and described as untypable. Twenty-four isolates reacted with MASF 1c, a monoclonal antibody specific for S. flexneri serotype 1c. A further ten untypable isolates were found to be rough mutants (no longer expressing O-antigen) that were derived from serotype 1c strains. Pulsed-field gel electrophoresis demonstrated that these strains consisted of many different clones, indicating serotype 1c was well established in this region in the late 1990s. Serotype 1c was the most prevalent S. flexneri serotype isolated in the Son Tay Province, accounting for about 40% of S. flexneri isolates. Subsequent isolation of S. flexneri serotype 1c in this region and elsewhere in Vietnam confirmed that serotype 1c is of genuine importance in Vietnam.

Practice of using human excreta as fertilizer and implications for health in Nghean Province, Vietnam.

The ancient practice of applying latrine wastes to agricultural land has maintained soil fertility in Vietnam for several centuries but may be associated with health risks if the wastes are inadequately treated before usage. This study aimed at investigating the perceptions and handling practices using latrine wastes as fertilizers in a community in central Vietnam. Information was collected through structured questionnaire interviews administered to 75 farming households, focus group discussions, and key informant interviews. The majority (64%) of households had a single vault latrine, a possession that was associated with low income (chi2= 12.45; p < 0.05). Most households (85%) used latrine waste in agriculture that was composted before usage (98%). Households often mixed the composted excreta with kitchen ashes and powdered lime likely to increase pH and pathogen die-off. About 28% of households that were applying latrine waste as fertilizer composted three to six months, and only 11 (18%) households composted human excreta for more than the recommended six months. Households with double vault latrines were 7.8 (chi2= 9.4; p<0.05) times more likely to compost human excreta more than six months as compared with households having single vault latrine. Most farmers distributed the latrine wastes with bare hands (66%) because of convenience during application. Respondents with a high educational level used protective gloves more often when distributing latrine wastes in the fields compared to respondents with a low educational level (chi2 = 7.6; p<0.05). If any negative health impacts of latrine waste use in agriculture are to be reduced, then it is suggested that sustainable interventions should take into consideration farmers current excreta-use practices.

Evaluation of community-based serologic screening for identification of chronic Salmonella typhi carriers in Vietnam.

OBJECTIVES: To determine the utility of screening anti-Vi antibodies to detect chronic Salmonella Typhi carriers in an endemic community. METHODS: We conducted a community-based serologic survey for anti-Vi antibodies to identify chronic Salmonella Typhi carriers in a typhoid endemic region in Vietnam. RESULTS: We tested sera from 3209 (67.2%) of 4772 eligible adults. The median age was 37 years (range 20-92), 57.3% were female, 4.6% reported a history of typhoid fever and 0.3% reported typhoid vaccination. Anti-Vi antibody titers tested in Vietnam were < 1:40 in 2759 (86.0%), 1:40 in 194 (6.0%), 1:80 in 168 (5.2%), 1:160 in 57 (1.8%), and > or = 1:320 in 31 (1.0%). On re-testing in the USA, an additional 19 sera with titers > or = 1:160 were identified. We collected 589 rectal swabs from 103 (96.3%) of 107 persons with Vi antibody titers > or = 1:160 and 183 swabs from 33 persons with antibody titers < 1:80. No Salmonella Typhi was isolated. CONCLUSIONS: Community-based serologic screening is a feasible, but impractical method for identifying chronic Salmonella Typhi carriers. Background levels of anti-Vi antibody titers in this endemic area may be high despite a low prevalence of chronic carriers.

Characterization of antimicrobial resistance, plasmids, and gene cassettes in Shigella spp. from patients in vietnam.

The objective of the study was to investigate antimicrobial resistance, plasmids and class 1 integrons in 150 Shigella strains isolated from patients with diarrhea in Vietnam. Most isolates were resistant to the majority of antimicrobial agents used for treatment in the isolation areas and 90% were resistant to three or more antibiotics. A total of 20 strains yielded class 1 integrons, which harbored oxa1, dfrA, orfF, and aadA gene cassettes. The most common gene cassette, aadA2, was always located closest to the 3' conserved segment of the integrons and oxa1 and dfrA closest to the 5' end. Plasmid profiles of the 20 class 1 integron-positive strains all contained more than one plasmid, and 14 different profiles were found. No correlation was found between species, antibiograms, plasmid profiles, or presence of class 1 integrons. Conjugation resulted in 25 transconjugants, which all were resistant to four or more antimicrobial agents and all harbored at least one plasmid (>60 kb). Class 1 integrons were detected in 64% of the transconjugants. Phenotypic resistance pattern and plasmid profiles of the transconjugants seemed independent of the presence of an integron. Class 1 integrons seemed of less importance in phenotypic antibiograms and in transfer of resistance genes than conjugative plasmids.

[Cyclospora cayetanensis presence in aquatic surroundings in Hanoi (Vietnam). Environmental study (well water, lakes and rivers)]. / Cyclospora cayetanensis et sa présence en milieu hydrique à Hanoï (Vietnam). Etude dans l'environnement (eaux de forage, lacs et rivières).

Cyclospora cayetanensis is a pathogenic agent originating from the intertropical zone. It causes diarrhoeal diseases in local populations as well as in travellers visiting these zones. In the first part of this work, an epidemiological study on drinking water supply (reservoirs and consumers' taps) was conducted in Hanoi over 12 months; samples were daily collected and have revealed the presence of Cyclospora cayetanensis oocysts during the whole year in tanks and only during monsoon season. Molecular methods were used for species identification. In the second part, we tried to investigate different water sources in Hanoi city in order to detect Cyclospora cayetanensis environmental contamination: groundwaters, surface waters collected in lakes and rivers and also waters from treatment plants. Our results show that none Cyclospora cayetanensis oocyst was found in the groundwaters and in the desinfected finished waters after treatment. In contrast, in rivers and lakes samples, the level of positivity reached about 63.6% with significant differences between the districts regarding the rates of oocysts recovery: only 24% positive specimens in Hoan Kiem district, whilst respectively 80.4%, 78.3% and 65% positive samples in Hai Ba Trung, Dong Da and Ba Dinh districts. The results of this study seem to confirm that environmental water is contaminated by Cyclospora cayetanensis oocysts and points out the importance of water as a significant source of human transmission. It is quite obvious that observation could be probably extended to the other endemic areas.

Investigations into the safety and immunogenicity of a killed oral cholera vaccine developed in Viet Nam.

OBJECTIVE: To evaluate a killed oral cholera vaccine produced in Viet Nam, and to compare the Vietnamese vaccine with one that is licensed internationally. METHOD: Two-dose regimens of a locally produced, bivalent, anti-O1, anti-O139 killed oral whole-cell cholera vaccine (biv-WC) and of a commercially available, monovalent (anti-O1) oral recombinant B subunit-killed whole-cell cholera vaccine (rBS-WC) were compared in two trials in Viet Nam. In the first trial, 144 adults were randomized to biv-WC with or without buffer, rBS-WC with buffer, or placebo without buffer. In the second, 103 children aged 1-12 years were randomized to biv-WC without buffer, rBS-WC with buffer, or placebo without buffer. FINDINGS: No regimen was associated with significant side-effects. In adults, ca 60% of recipients of either vaccine exhibited at least fourfold serum anti-O1 vibriocidal antibody responses and ca 40% of recipients of biv-WC demonstrated anti-O139 vibriocidal responses. Both anti-O1 (ca 90% in each vaccine groupand anti-O139 (68% in the biv-WC group) vibriocidal responses occurred more frequently in children. The responses to biv-WC were unaffected by the receipt of buffer. CONCLUSION: It was concluded that biv-WC was safe and immunogenic, that it could be administered without buffer, and that it could elicit robust immune responses even in children, for whom the risk of endemic cholera is highest.

Prospective study of the incidence of diarrhoea and prevalence of bacterial pathogens in a cohort of Vietnamese children along the Red River.

We prospectively studied diarrhoea incidence among 1655 children < 5 years of age in northern Vietnam for 1 year using primarily passive surveillance. Standard culture methods were used to detect bacterial pathogens. Overall 2160 cases occurred (13 cases/child per year). Peak rates of diarrhoea occurred in children < 12 months old. Rates ranged from 3.3 cases/child per year in children < 1 year old, to 0.7 cases/child per year in 4-year-olds. Campylobacter, shigella and enterotoxigenic Escherichia coli were most commonly isolated. Rates detected by active surveillance were about twice those detected passively. S. flexneri was the most common shigella serogroup (65 %). S. flexneri serotypes 6, 4, 1 and Y were most common, but 40% were untypable using commercial antisera. The data illustrate important regional differences in pathogen prevalence and shigella serotype distribution. Shigella vaccine development strategies, commonly targeting S. flexneri 2a, S. sonnei and S. dysenteriae 1, will have little impact on diarrhoea rates in Vietnam.

Antimicrobial resistance of Shigella spp isolated from diarrheal patients between 1989 and 1998 in Vietnam.

Shigellosis is an important cause of infectious diarrhea in Vietnam, caused mainly by Shigella flexneri. This study provides for the first time in the international literature, data on the development of antimicrobial resistance in Shigella between 1988 to 1998, including data reported to the National Program for Surveillance of Antimicrobial Resistance (NPSAR). Our studies show that about 80% of the Shigella strains tested were resistant to ampicillin, chloramphenicol, oxytetracycline, trimethoprim and sulfonamides. This combination of drugs was also the most common antibiogram among multiple-resistant S. flexneri (57%). Resistance to tetracyclines, sulfonamides and, in particular trimethoprim (p<0.001), increased during the study period. Our findings indicate that tetracyclines and co-trimoxazole (a combination of a sulfonamide and trimethoprim), which are recommended and commonly used drugs for the treatment of shigellosis in Vietnam, may have limited therapeutic effect. In contrast to neighboring countries, low percentages of resistance were found to nalidixic acid and norfloxacin (3-5%) and no resistance was found to ciprofloxacin, indicating that nalidixic acid with its low cost and safety in children could be recommended for the treatment of shigellosis. The NPSAR provides a useful picture of the levels and development of antimicrobial resistance in Vietnam and should receive continued support for further improvement by increasing the number of provinces covered, the numbers of isolates tested from rural areas, and the communication of results to medical practitioners and others prescribing and/or selling antimicrobials.

Cholera in Vietnam: changes in genotypes and emergence of class I integrons containing aminoglycoside resistance gene cassettes in vibrio cholerae O1 strains isolated from 1979 to 1996.

The number of cholera cases and the mortality rates reported from different regions of Vietnam varied considerably in the period from 1979 to 1996, with between 2,500 and 6,000 cases reported annually from 1992 to 1995. Annual mortality rates ranged from 2.0 to 9.6% from 1979 to 1983 to less than 1.8% after 1983. Major cholera outbreaks were reported from the High Plateau region for the first time in 1994 and 1995; this is an area with limited access to health services and safe drinking-water supplies. All cases were associated with Vibrio cholerae O1. Using ribotyping, cholera toxin (CT) genotyping, and characterization of antibiotic susceptibility patterns and antibiotic resistance genes by PCR, we show that strains isolated after 1990 were clearly different from strains isolated before 1991. In contrast to strains isolated before 1991, 94% of 104 strains isolated after 1990 showed an identical ribotype R1, were resistant to sulfamethoxazole and streptomycin, and showed a different CT genotype. Furthermore, PCR analysis revealed that sulfamethoxazole-resistant strains harbored class I integrons containing a gene cassette ant(3")-1a encoding resistance to streptomycin and spectinomycin. This is, to our knowledge, the first report of class I integrons in V. cholerae. The development of cholera and the changes in the phenotypic and genotypic properties of V. cholerae O1 shown in the present study highlight the importance of monitoring V. cholerae O1 in Vietnam as in other parts of the world. In particular, the emergence of the new ribotype R1 strain containing class I integrons should be further studied.

The use of an IpaC-specific ELISA to identify enteroinvasive Escherichia coli strains of unusual serogroups.

Twenty five Escherichia coli isolates expressing O antigens different from the conventionally recognized enteroinvasive E. coli were tested in the Sereny test, with an invasion plasmid-specific DNA probe, and by an enzyme-linked immunosorbent assay recognizing the secreted IpaC antigen. These results indicate that the IpaC enzyme-linked immunosorbent assay is a sensitive method to recognize enteroinvasive E. coli, irrespective of their serogroups.

Immune responses in Vietnamese children after a single dose of the auxotrophic, live Shigella flexneri Y vaccine strain SFL124.

The live, auxotrophic Shigella flexneri vaccine strain SFL124 was given in a single dose of 10(7), 10(8) or 10(9) colony forming units (cfu), respectively, to each of three groups of 10 Vietnamese children aged 9-14 years. The vaccine was well tolerated by all the children without any severe side effects such as diarrhoea or fever being observed. Mild symptoms were reported by five children. Only five children were found by culture to excrete SFL124 but, by PCR, 28 of 30 children were found to excrete the vaccine strain for up to 5 days (mean 2.8 days) with insignificant differences among the groups. Local mucosal immune responses and antibody secreting cell (ASC) responses to S. flexneri lipopolysaccharide (LPS) and invasion plasmid-coded antigens (Ipa) were elicited in the children in a dose-dependent manner. Doses of 10(9) cfu induced most prominent responses, followed by those of 10(8) and 10(7) cfu. The sIgA responses were the highest whereas the ASC were modest. High titres of serum antibodies to Shigella LPS and Ipa were found in all the children before ingestion of the vaccine which elicited increases in serum antibody titres in only a few of them. The immune response patterns seen indicate a booster rather than a primary response and may be a consequence of the endemic nature of shigellosis in Vietnam.

Immune response against lipopolysaccharide and invasion plasmid-coded antigens of shigellae in Vietnamese and Swedish dysenteric patients.

The peripheral immune responses of adult Vietnamese patients infected with Shigella dysenteriae 1 and Shigella flexneri 1b and 2a and those of S. flexneri-infected Swedish patients were studied against various lipopolysaccharide and invasion plasmid-coded antigens (Ipa-s) and compared with the titers for the corresponding local healthy populations. Both Vietnamese and Swedish patients reacted with significant (P, < 0.05) titer increases in the immunoglobulin A and G classes against the homologous lipopolysaccharide antigen. However, significant titer responses against the Ipa-s were seen among only the Swedish patients. We surmise that the weak-to-moderate responses against the Ipa-s in Vietnamese patients are due to the fact that the high level of titers induced by previous infections in the local population could not be considerably increased further by a recent infection.

Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in adult Vietnamese volunteers.

The live, auxotrophic dependent Shigella flexneri Y vaccine strain SFL124 with a deleted aroD gene was tested in 30 healthy adult male Vietnamese volunteers. A single dose of 2 x 10(9) live bacteria was given orally to 15 volunteers, whereas 15 received three doses every other day. None of the volunteers reacted with fever or diarrhoea and SFL124 was excreted by all for a mean of 2.8 (single dose) and 2.6 (three doses) days. A total of 27 of 30 (90%) and 26 of 30 (87%) responded with significantly (0.001 < p < 0.01) increased antibody-secreting cell (ASC) numbers against Shigella flexneri Y lipopolysaccharide (LPS) and invasion plasmid-coded antigens (Ipa). A faecal IgA antibody response to LPS and Ipa was seen in 20 of the 30 (67%) volunteers against both antigens. Serum antibody responses were seen in 23 of 30 (77%) against the LPS and in 17 of the 30 against Ipa. The three-dose schedule elicited only somewhat stronger immune responses than the single-dose schedule. A booster dose of 2 x 10(9) live bacteria was given to half of the volunteers in each group after 6 months, the other half received the same dose after 12 months. Following the booster at 6 or 12 months (i) the excretion of SFL124 was significantly shorter (p < 0.05) than after primary vaccination; (ii) the anti-S. flexneri LPS and anti-Ipa faecal sIgA titres were significantly higher (p < 0.05 to p < 0.01) than after primary vaccination; (iii) the anti-LPS and anti-Ipa ASC responses were significantly lower (p < 0.05) and of shorter duration than after primary vaccination, and (iv) the serum anti-LPS and anti-Ipa responses were significantly elevated (p < 0.05) and similar to those seen after primary vaccination. The results indicate that SFL124 is a safe, live vaccine strain with a negligible reactogenicity in adults living in a Shigella endemic area. SFL124 induces specific immune responses against LPS and Ipa with a mucosal memory lasting for at least 1 year.

AroD deletion attenuates Shigella flexneri strain 2457T and makes it a safe and efficacious oral vaccine in monkeys.

The aromatic-dependent live Shigella flexneri 2a vaccine strain SFL1070, with a deleted aroD gene, had a much reduced intracellular growth in HeLa cells compared with its parent strain S. flexneri 2457T. S. flexneri SFL1070 gave no adverse effects in eight Macaca fascicularis monkeys orally vaccinated with four doses of 1 x 10(11) live bacteria within a 5-week period, whereas S. flexneri 2457T caused dysentery in all eight non-vaccinated monkeys. Thus the aromatic dependency rendered S. flexneri SFL1070 significantly attenuated (p = 0.00008). Significant intestinal S. flexneri lipopolysaccharide (LPS)-specific sIgA responses were seen in seven of eight vaccinated monkeys (p < 0.01) after four doses with SFL1070. However, serum IgG or IgA responses to various S. flexneri LPS antigens and the invasion plasmid antigens (Ipa-s) were seen in only four of eight vaccinated monkeys. The serum IgG titre increases against S. flexneri Y and 2a LPS reached significant levels (p < or = 0.05). All but one of the vaccinated monkeys were protected against oral challenge with 1 x 10(10) or 1 x 10(11) live S. flexneri 2457T given 2 weeks after the last vaccination. The protection was highly significant (p = 0.0007) as all non-vaccinated monkeys challenged with equal doses of strain 2457T developed dysentery. Three of them succumbed. Challenge infection of vaccinated monkeys elicited serum IgA and IgG responses to the homologous S. flexneri 2a LPS in three monkeys each (0.005 < or = p < or = 0.025). Serum IgA and IgG responses to the Ipa-s were seen in five and four monkeys each (0.01 < p < or = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies against invasion plasmid coded antigens of shigellae in human colostrum and milk.

Colostral and milk samples of Swedish, Vietnamese and Costa Rican mothers living under various socioeconomic conditions were tested for the presence of shigella invasion plasmid coded antigen (Ipa) specific antibodies. IgA antibodies of this specificity were found in significantly higher titres in samples of Vietnamese (600 +/- 338) than in samples of Swedish or high income Costa Rican mothers (190 +/- 224 and 290 +/- 241, respectively; p < 0.05). Specific IgA titres in the low income group of Costa Rican mothers (470 +/- 338) did not differ significantly from the values obtained in Vietnam. While no Ipa specific IgM could be detected in any of the samples tested, specific IgG was found in 90% of the Vietnamese colostrum. These data indicate that antibodies which could be responsible for the dysentery-preventing effect of breast feeding are indeed present in human colostrum and milk in areas where shigellosis occurs with relatively high incidence.

Shigellosis in Vietnam: seroepidemiologic studies with use of lipopolysaccharide antigens in enzyme immunoassays.

The performance of enzyme immunoassays (EIAs) with use of O-antigen-containing lipopolysaccharides (LPSs) extracted with phenol-water from Shigella dysenteriae type 1, Shigella flexneri serotypes 1a-5b, and Shigella sonnei for determination of the serum antibody responses after onset of bacillary dysentery is reviewed. For the purpose of several studies, serum samples from a total of 175 Vietnamese and 47 Swedish patients, for whom Shigella species had been isolated from fecal specimens, were obtained at various intervals until less than or equal to 1 year after the onset of infection. Titers of antibodies in serum samples from infected patients were compared with those in serum samples from healthy control subjects; the combined control population of all studies comprised 426 Vietnamese and 154 Swedes. The sensitivity of the EIAs ranged from 78% to 100% for patients whose fecal culture was positive for Shigella. For diagnosis of S. flexneri, a species-specific but no serotype-specific assay based on LPS antigens is possible. Among Vietnamese patients the EIA with use of S. flexneri was sensitive and diagnostic only for children less than 3 years of age, most likely because healthy older Vietnamese children and adults have high titers of antibody to the O-antigens of S. flexneri. Among Swedish patients the same EIA was diagnostic for adults as well as children. Increased titers of IgA in the early phase and of IgG in the convalescent phase, as determined by EIA, were the best indicators of infection due to Shigella species.