Enteric Infections in Young Children are Associated with Environmental Enteropathy and Impaired Growth.

OBJECTIVE: To investigate the relationship between faecal contamination in child play spaces, enteric infections, environmental enteropathy (EE) and impaired growth among young children. METHODS: A prospective cohort study was conducted of 203 children 6-30 months of age in rural Bangladesh. Stool samples were analysed by quantitative PCR for Shigella, Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni, Giardia intestinalis and Cryptosporidium spp. Four faecal markers of intestinal inflammation were also measured: alpha-1-antitrypsin, myeloperoxidase, neopterin and calprotectin. Child growth was measured at baseline and 9 months after enrolment. E. coli was measured in soil in child play spaces. RESULTS: Forty-seven percent of study children had three or more enteric pathogens in their stool. Thirty five percent (71/203) of children had Shigella, 30% (61/203) had ETEC, 73% (148/203) had C. jejuni, 79% (160/203) had Giardia intestinalis and none had Cryptosporidium. Children with ETEC had significantly higher calprotectin concentrations (Coefficient: 1.35, 95% Confidence Interval [CI]: 1.005, 1.82). Children with Shigella had a significantly higher odds of being stunted at our 9-month follow-up (OR: 2.01, 95% CI: 1.02, 3.93). Children with Giardia intestinalis had significantly higher E.coli counts in the soil collected from their play spaces (OR: 1.23, 95% CI: 1.02, 1.48). CONCLUSION: Enteric infections were significantly associated with EE and impaired growth in rural Bangladesh. These findings provide further evidence to support the hypothesis that contaminated soil in child play spaces can lead to enteric infections, many of which are likely subclinical, resulting in EE and impaired growth in young children.

Mouthing of Soil Contaminated Objects is Associated with Environmental Enteropathy in Young Children.

OBJECTIVE: To characterise childhood mouthing behaviours and to investigate the association between object-to-mouth and food-to-mouth contacts, diarrhoea prevalence and environmental enteropathy. METHODS: A prospective cohort study was conducted of 216 children ≤30 months of age in rural Bangladesh. Mouthing contacts with soil and food and objects with visible soil were assessed by 5-h structured observation. Stool was analysed for four faecal markers of intestinal inflammation: alpha-1-antitrypsin, myeloperoxidase, neopterin and calprotectin. RESULTS: Overall 82% of children were observed mouthing soil, objects with visible soil, or food with visible soil during the structured observation period. Sixty two percent of children were observed mouthing objects with visible soil, 63% were observed mouthing food with visible soil, and 18% were observed mouthing soil only. Children observed mouthing objects with visible soil had significantly elevated faecal calprotectin concentrations (206.81 µg/g, 95% confidence interval [CI]: 6.27, 407.36). There was also a marginally significant association between Escherichia coli counts in soil from a child's play space and the prevalence rate of diarrhoea (diarrhoea prevalence ratio: 2.03, 95% CI 0.97, 4.25). CONCLUSION: These findings provide further evidence to support the hypothesis that childhood mouthing behaviour in environments with faecal contamination can lead to environmental enteropathy in susceptible paediatric populations. Furthermore, these findings suggest that young children mouthing objects with soil, which occurred more frequently than soil directly (60% vs. 18%), was an important exposure route to faecal pathogens and a risk factor for environmental enteropathy.

The dominance of pandemic serovars of Vibrio parahaemolyticus in expatriates and sporadic cases of diarrhoea in Thailand, and a new emergent serovar (O3 : K46) with pandemic traits.

Vibrio parahaemolyticus is a major cause of gastroenteritis worldwide. A total of 95 V. parahaemolyticus isolates belonging to 23 different serovars were identified in a case-control study of expatriates and Thai adults from 2001 to 2002 in Thailand. Fifty-two per cent of isolates (49/95) were resistant to ampicillin and sulfisoxazole, but all isolates were susceptible to ciprofloxacin and trimethoprim-sulfamethoxazole, two antibiotics commonly used to treat traveller's diarrhoea. All isolates were positive for the species-specific toxR gene, and 91 and 5 were positive for the thermostable direct haemolysin (tdh) gene and the tdh-related (trh) gene, respectively. Sixty-five isolates were assigned to the pandemic group of V. parahaemolyticus by a group-specific PCR and the presence of the orf8 gene. The pandemic isolates belonged to three recognized serovars (O3 : K6, O1 : K25, O1 : KUT) and a new serovar, O3 : K46. This new serovar harboured pandemic traits. PFGE analysis revealed that all pandemic isolates including serovar O3 : K46 were closely related and clearly distinct from the non-pandemic isolates. In summary, three well-known serovars of pandemic V. parahaemolyticus isolates were identified as a major cause of diarrhoea in Thailand and a new V. parahaemolyticus isolate, serovar O3 : K46, with pandemic traits was detected.

Evaluation of three rapid diagnostic tests for cholera: does the skill level of the technician matter?

OBJECTIVE: To evaluate SMART, Medicos Dip Stick and an Institut Pasteur (IP) cholera dipstick tests for accuracy and ease of use. METHOD: Every 50th patient presenting with diarrhoea at ICDDR,B between 1 April 2003 and 30 November 2003 was enrolled. The rapid diagnostic tests were performed by field and laboratory technicians, and sensitivity (Se), specificity (Sp), positive (PPV) and negative (NPV) predictive values calculated. RESULTS: We isolated Vibrio cholerae O1 from 116 (38%) of 304 patients. The Se, Sp, PPV and NPV of the SMART test were 58%, 95%, 84% and 84% for field technicians, and 83%, 88%, 83% and 88% for laboratory technicians. The Se, Sp, PPV and NPV of the IP dipstick test were 93%, 67%, 63% and 94% for field technicians, and 94%, 76%, 70% and 95% for laboratory technicians. The Se, Sp, PPV and NPV of the Medicos test were 84%, 79%, 71% and 90% for field technicians, and 88%, 80%, 72% and 92% for laboratory technicians. A high proportion of indeterminates (30%) hampered the performance of the SMART test. The IP dipstick had the highest Se, irrespective of technician skill level. CONCLUSION: The IP dipstick is the most appropriate rapid diagnostic assay for the detection of V. cholerae O1 in locations where the skill level of personnel may be low, such as remote areas or refugee camp settings. High cost may limit the utility of any diagnostic test in the developing world.

Genomic relatedness of the new Matlab variants of Vibrio cholerae O1 to the classical and El Tor biotypes as determined by pulsed-field gel electrophoresis.

The genomes of the recently described Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes were compared with those of El Tor and classical biotypes by the use of pulsed-field gel electrophoresis. Dendrograms constructed using the unweighted-pair group method using average linkages generated from NotI restriction patterns of whole-chromosomal DNA grouped these strains into two major clusters that were found to be similar but not identical to those of either of the biotypes. Strains that clustered with the classical biotype appear to have been derived from the classical strains, which are thought to be extinct.

Pandemic strains of O3:K6 Vibrio parahaemolyticus in the aquatic environment of Bangladesh.

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahaemolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic beta-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.

Adaptive and inflammatory immune responses in patients infected with strains of Vibrio parahaemolyticus.

In patients with diarrhea caused by Vibrio parahaemolyticus, antibody-secreting cell responses to thermostable direct hemolysin (TDH), lipopolysaccharide (LPS), and whole-cell bacteria were seen. TDH- and LPS-specific responses were seen in serum samples, and immunoglobulin A antibody responses were observed in stool. Levels of C-reactive protein and nitric oxide metabolites increased in the systemic circulation at the onset of illness. Tumor necrosis factor-alpha and lactoferrin levels were high during the acute stage in mucosal secretions and in plasma, whereas interleukin-1beta levels were high only in mucosal secretions. Duodenal and rectal biopsy specimens obtained at the onset of illness showed an acute inflammatory response. The lamina propria showed edema, congestion of blood vessels, and hemorrhage, with an increase in levels of polymorphonuclear neutrophils and macrophages. Strains belonging to different serotypes exhibited varying resistance to killing by serum; the O8:K21 strain was most sensitive. Infection with V. parahaemolyticus results in B cell responses and an acute inflammatory response that is self-limiting.