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World Health Organization (WHO) recommended standard ORS solution has sodium (90 mmol/L) and glucose (111 mmol/L) almost in the ratio of 1:1 and a total osmolarity of 311 mmol/L. There are concerns that the sodium or glucose concentration and the overall osmolarity in the formulation is not appropriate. Therefore, the efficacy of standard and reduced-osmolarity ORS solutions in young children with acute diarrhea was evaluated in a recent WHO supervised multicentre trial conducted in India (New Delhi), Brazil, Mexico and Peru. The implications of trial results are discussed. In non-cholera diarrhea, both the standard and reduced osmolarity ORS solutions were effective in achieving clinical rehydration. The stool output was 39% higher in the standard ORS solution group as compared to the reduced-osmolarity ORS solution group. The duration of diarrhea was 22% higher in the standard ORS solution group. The risk of requiring supplementary intravenous infusion was increased in children treated with standard ORS solution [relative risk 1.4 (0.9-2.4)]; this benefit was not observed in Indian patients due to high breast feeding rate. The mean sodium concentration at 24 hours after admission was lower in the reduced osmolarity ORS solution group [135 (134-136) vs 138 (136-139), p < 0.01). The low osmolarity ORS deserves to be evaluated in adult cholera to determine its efficacy and any excess hyponatreamia. Meanwhile, it is reassuring that the WHO formulation was effective and its use was not associated with hypenatremia even in young children. Efforts must continue to be made to promote WHO-ORS while research to improve it further is welcome.

Diarrhoea that begins acutely but lasts longer than two weeks is defined to be persistent. Revised estimates in developing countries including India showed that acute diarrhoea accounts for 35 per cent, dysentery 20 per cent and non-dysenteric persistent diarrhoea (PD) for 45 per cent of total diarrhoeal deaths. PD also often changes marginal malnutrition to more severe forms. Factors that increase the risk of acute diarrhoea becoming persistent have been identified in India and other developing countries. These include antecedent malnutrition, micronutrient deficiency particularly for zinc and vitamin A, transient impairment in cell mediated immunity, infection with entero aggregative Escherichia coli and cryptosporidium, sequential infection with different pathogens and lack of exclusive breast feeding during the initial four months of life particularly use of bovine milk. Several issues regarding the management of persistent diarrhoea in hospitalized children in India have been resolved. Diets providing modest amounts of milk mixed with cereals are well tolerated. In those who fail on such diets providing carbohydrate as a mixture of cereals and glucose or sucrose hasten recovery. The role of antimicrobial agents and individual micronutrients in PD is currently being investigated. A management algorithm appropriate for India and other developing countries has been developed and found to substantially reduce case fatality in hospital settings to about 2-3 per cent. Recent epidemiological and clinical research related to persistent diarrhoea is also reviewed.

Persistent diarrhea (PD) and dysentery (DD) account for most diarrhea-associated deaths among children in developing countries. Zinc deficiency can cause stunting and impaired immune function, both of which are risk factors for these diarrheal illnesses. We investigated the effect of zinc supplementation on the incidence of PD and DD in a community-based, double-blind randomized trial in children 6-35 mo of age. Increase over baseline in plasma zinc concentrations in the supplemented group compared with a control group (3.61 vs. 0.009 mumol.L-1), indicated successful supplementation. The overall reductions in the zinc supplemented group of 21% in the incidence of PD (95% CI -6 to 42%) and 14% in the incidence of dysentery (95% CI -15 to 36%) were not significant. There was a significant interaction of treatment effect with baseline plasma zinc concentration and age for PD and with gender for DD. In the zinc-supplemented group compared with the control group, the incidence of PD was reduced by 73% (P < 0.05; 95% CI 34 to 91%) in children with a baseline zinc < 7.65 mumol.L-1 and by 49% (P < 0.05; 95%CI 24 to 66%) in children > 11 mo of age. Zinc supplementation resulted in a 38% (P < 0.05 95%CI 8 to 59%) reduction in the incidence of DD in boys. There was no effect on PD among children 6-11 mo old or on DD in girls. In conclusion, zinc supplementation had a significant impact on the incidence of persistent diarrhea in children > 1 y old and in children with low plasma zinc, as well as on dysentery in boys. These findings may have important implications for reducing diarrhea-related morbidity and mortality.

Background: In developing countries the duration and severity of diarrheal illnesses are greatest among infants and young children with malnutrition and impaired immune status, both factors that may be associated with zinc deficiency. In children with severe zinc deficiency, diarrhea is common and responds quickly to zinc supplementation., Methods: To evaluate the effects of daily supplementation with 20 mg of elemental zinc on the duration and severity of acute diarrhea, we conducted a double-blind, randomized, controlled trial involving 937 children, 6 to 35 months of age, in New Delhi, India. All the children also received oral rehydration therapy and vitamin supplements., Results: Among the children who received zinc supplementation, there was a 23 percent reduction (95 percent confidence interval, 12 percent to 32 percent) in the risk of continued diarrhea. Estimates of the likelihood of recovery according to the day of zinc supplementation revealed a reduction of 7 percent (95 percent confidence interval, -9 percent to +22 percent) in the risk of continued diarrhea during days 1 through 3 and a reduction of 38 percent (95 percent confidence interval, 27 percent to 48 percent) after day 3. When zinc supplementation was initiated within three days of the onset of diarrhea, there was a 39 percent reduction (95 percent confidence interval, 7 percent to 61 percent) in the proportion of episodes lasting more than seven days. In the zinc-supplementation group there was a decrease of 39 percent (95 percent confidence interval, 6 percent to 70 percent) in the mean number of watery stools per day (P = 0.02) and a decrease of 21 percent (95 percent confidence interval, 10 percent to 31 percent) in the number of days with watery diarrhea. The reductions in the duration and severity of diarrhea were greater in children with stunted growth than in those with normal growth., Conclusion: For infants and young children with acute diarrhea, zinc supplementation results in clinically important reductions in the duration and severity of diarrhea.

Reviewed are all the published clinical trials of glycine-based oral rehydration salts (ORS), L-alanine-based ORS, L-glutamine-based ORS, maltodextrin-based ORS, and rice-based ORS, as well as the results of several recently completed, but unpublished, studies of these formulations that were supported by WHO. All experimental ORS formulations contained the same concentrations of salts as citrate-based WHO-ORS; all trials were randomized comparisons with WHO-ORS, and all except those with rice-based ORS were double-blind studies. The rate of stool loss and, less frequently, the duration of diarrhoea were used as indicators of clinical performance to compare ORS formulations. The following conclusions were reached concerning the efficacy and use of modified ORS formulations. Rice-based ORS (50 g/l) is superior to WHO-ORS for patients with cholera, and for such patients it can be recommended in any situation where its preparation and use are practical. Rice-based (50 g/l) and WHO-ORS solutions are equally effective for treating children with acute non-cholera diarrhoea, when feeding is resumed promptly following initial rehydration, as has been consistently recommended by WHO. Since rice-based ORS is not superior to WHO-ORS for such children, there is no apparent reason to advise a change from glucose to pre-cooked rice in the recommended formulation for WHO-ORS. Maltodextrin-based ORS formulations (50 g/l) and WHO-ORS appear to be equally effective for treating children with acute non-cholera diarrhoea; there is no reason to advise a change from glucose to maltodextrin in the recommended formulation for WHO-ORS. Amino-acid-containing ORS formulations are not recommended for either non-cholera or cholera diarrhoea, since they are more costly and have no clinical advantage over WHO-ORS for children with acute non-cholera diarrhoea or over rice-based ORS for persons with cholera.

Rotavirus strain 116E was isolated from the fecal specimen of a newborn infant from New Delhi who had no symptoms of diarrhea. The strain could not be P typed using the PCR method recently developed in our laboratory so we have sequenced the 4th gene segment that encodes the VP4 protein using viral transcript RNA prepared in vitro. This gene is 2353 base pairs in length and codes for a protein 772 amino acids long which begins with a methionine initiation codon at nucleotides 11 to 13 and ends with a single termination codon at nucleotides 2327 to 2329. Northern blot analysis demonstrates that the VP4 protein of this strain is encoded by genome segment 4. This gene is closely related to the VP4 gene of the bovine B223 strain (P type 11) at both the nucleotide (90.8% identity) and amino acid (92.2% identity) levels, but is unique from those of strains from P types 1 to 10. The close relatedness of the VP4 proteins of strains 116E and B223 is demonstrated by the amino acid composition of the potential trypsin cleavage sites and their flanking sequences, the size of the cleavage fragments, and conservation of most cysteine and proline residues. Comparative amino acid analyses of the variable regions thought to be important in VP4 antigenicity are consistent with the hypothesis that strain 116E may represent the first reported human P type 11 strain.

The simplicity of enterotoxigenic Escherichia coli (ETEC) stool blot hybridization, where the total bacterial growth of a fecal inoculum is examined directly for the presence of enterotoxin genes, has been marred by reports of unsatisfactory sensitivity and/or specificity. To assess the accuracy of stool blot hybridization and to study the effect of varying proportions of ETEC among fecal E. coli (ETEC/E. coli) on test performance, a detailed 'blind' study of 166 stool specimens from children with diarrhea was performed. Oligonucleotide probes were found to be superior to polynucleotide probes, having a sensitivity of 80%, a specificity of 99%, a positive predictive value of 89% and a negative predictive value of 97%. The sensitivity was found to be 100% when ETEC/E. coli > 2/12, as compared with 20% when ETEC/E. coli < or = 2/12 (p = 0.001), showing that the proportion of ETEC among fecal E. coli is of paramount importance for test sensitivity.

The role of Hep-2 cell adherent Escherichia coli (EAEC) of localized (EAEC-L), diffuse (EAEC-D) and aggregative (EAggEC) phenotype, was investigated in 254 children aged < or = 48 months seeking treatment for non-bloody diarrhea at an outpatient clinic, and in 107 age-matched controls. The stool excretion rates of single isolates from patients/controls for the different phenotypes of Hep-2 cell adherent E. coli were: EAEC-L, 5.9/2.8%, p = 0.33; EAEC-D, 7.5/12.1%, p = 0.22; and EAggEC, 11.8/3.7%, p = 0.03. When patients were categorized by pre-admission diarrheal duration > or = 14/< 14 days, the excretion rates were EAEC-L, 0/7.1%; EAEC-D, 9.5/7.1%, and EAggEC, 21.4/9.9%, the difference approaching significance only for EAggEC (p = 0.06). These findings suggest that EAggEC may be an important cause of diarrhea in children, with a predilection to cause prolonged disease.

The ability of HEp-2 cell adherent Esch. coli of aggregative phenotype (EA-Agg EC) to cause diarrhoea and to colonize the bowel of rabbits was studied. Thirty six rabbits were challenged with one of three EA-Agg EC strains (F23A; H766C and F17A-15, 3 and 3 rabbits respectively) or a control strain (K12-15 rabbits) in reversible ileal-tie in adult rabbit diarrhoea (RITARD) model. The animals were sacrificed 72 h post challenge. Severe diarrhoea occurred in greater number of F23A challenged rabbits than the controls (P < 0.05). Mucosal cultures from proximal and distal small intestine and colon yielded about 1000 times more Esch. coli in the test than control rabbits (P < 0.001 in each case). EA-Agg EC were consistently grown from mucosa in the test rabbits who commonly showed mild to moderate villous stunting and grade + to nuclear fragmentation (karyorrhexis) in the small and large bowel epithelium. The control animals had either normal villi or very mild villous stunting. Results comparable to F23A were obtained with the other two EA-Agg EC strains tested in a smaller number of animals.

Mortality associated with diarrhea was investigated in a longitudinally followed cohort of children under six years of age in rural North India. During the follow-up, 1663 episodes of diarrhea and 23 diarrhea related deaths were recorded in 1467 children followed up for 20 months. The case fatality rate was 0.56% for acute watery diarrhea, 4.27% for dysentery and 11.94% for non-dysenteric persistent diarrhea. Most of the episodes lasted less than a week; 5.2% became persistent (duration > 14 days). The case fatality rate was similar in episodes of one and two weeks' duration (0.64% and 0.8%) and increased to 13.95% for persistent episodes. Of the total 86 persistent episodes, 22.1% were dysenteric; the case fatality rate for such dysenteric persistent episodes was 21.1% and for watery persistent diarrhea 11.4%. Diarrheal attack rates were similar among different nutritional groups, but diarrheal case fatality rates progressively increased with increasing severity of malnutrition, these were 24 times higher in children with severe malnutrition (7.48%) compared to those normally nourished (0.31%). With availability and use of oral rehydration therapy, dysentery and persistent diarrhea emerge as major causes of diarrhea related mortality, with underlying malnutrition as a key associated factor.

The role of feeding breast milk, unmodified bovine milk or adapted infant formula during acute diarrhoea in protecting against or causing persistence of the episodes was investigated in a population-based case control study in an urban area of north India. After adjustment for confounding variables, exclusive breast-feeding was associated with an odds ratio of 0.06 (95% CI 0.002-2.1), a 16.5 times lower odds in favour of developing persistence of an episode. Infants fed unmodified bovine milk in addition to breast milk had an odds of 2.5 times (95% CI 1.0-9.9) in favour of developing persistence of acute diarrhoea (p = 0.04). In infants receiving unmodified bovine milk and no breast milk, this odds ratio was 11.1 (95% CI 1.0-228.8) (p = 0.05). This study indicates that promoting exclusive breastfeeding may reduce the persistence of diarrhoea over and above its effect in decreasing the incidence of acute diarrhoea. In urban areas of the developing countries where working mothers often use milk supplementation beyond the age of three months, our findings suggest that use of adapted spray dried formula may be safer than unmodified bovine milk with respect to the risk of developing persistent diarrhoea.

Bacterial overgrowth in the upper small intestine is postulated to cause persistent diarrhea. We compared the fecal and upper intestinal flora in 82 patients with persistent diarrhea aged < or = 36 months and weight-for-length < or = 90% of National Center for Health Statistics standards (NCHS) and 39 non-diarrheal children with age < or = 36 months, nutritionally matched with the patients and residing in the same environment. In the age groups < or = 12 months and > 12 months the duodenal fluid bacterial counts > or = 10(5)/ml were found with similar frequency in patients and controls for aerobic (p = 0.33; p = 0.1) and anaerobic (p = 1.0; p = 1.0) bacteria. However, the duodenal isolation rates of any aerobic bacteria (p < 0.05) without regard to counts and Enterobacteriaceae (p = 0.06) were higher in patients than in controls. Colonization with Enterobacteriaceae was directly correlated with increased stool weight over a 7-day observation period (p < 0.05; p < 0.01). One or more pathogens were isolated in the feces of 58% of the patients and 43.8% of the controls. Enteroadherent E. coli of the localized [EAEC-L] (p < 0.01) and aggregative [EAggEC] (p = 0.22) phenotypes were isolated more commonly from the feces of patients. The duodenal fluid detection rates for Klebsiella were significantly greater (p < 0.01) in patients, while Giardia lamblia was detected more commonly in the duodenal fluid of controls (p < 0.01). The presence of specific pathogens in the feces did not seem to be related to the extent of small bowel colonization.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study describes a nonisotopic DNA-DNA hybridization assay for the detection of enterotoxigenic Escherichia coli (ETEC) in which the probes were labelled with the hapten molecule digoxigenin and after hybridization, DNA hybrids were detected by antidigoxigenin alkaline phosphatase conjugate. A blinded study carried out on a battery of enterotoxigenic and nonenterotoxigenic Esch. coli by dig-probe hybridization assay were compared with the results of a radiolabelled toxin gene probe hybridization assay (performed earlier). The three digoxigenin labelled probes gave a 100 per cent specificity and sensitivities of 95.45, 100 and 100 per cent for LT, STh and STp respectively. These results were comparable to those with the radioactive probes.

We examined the ability of a colonization factor antigen I (CFA/I) polynucleotide probe to identify coli-surface antigen 4 producing (CS4+) strains of enterotoxigenic Escherichia coli (ETEC). At low stringency (LS) the probe hybridized to colony lysates of strains previously shown to produce CS4 or CFA/I fimbriae. Only DNA from CFA/I+ strains maintained a stable probe-target hybrid under high stringency (HS) conditions. On examination of several clones from three previous CS4 producers, identified as positive in LS and negative in HS colony hybridization, spontaneous loss of nucleotide sequences homologous to a gene encoding a positive CFA/I regulator, CfaD, was found to be associated with lacking expression of CS4. Our findings indicate that, on stored or subcultured isolates of ETEC, identification of CS4 strains may benefit from applying gene probe technology.

The association between persistent diarrhoea and 'recent morbidity' defined as that occurring within the two-month period immediately preceding the onset of persistent diarrhoea was investigated in a population-based case-control study in rural North India. In two separate matched case-control analyses children with persistent diarrhoea (cases) were compared to population controls (five controls matched to each case) and acute diarrhoeal controls (three controls matched to each case). After correcting for possible confounding variables, comparing children with persistent diarrhoea and matched population controls, presence of a recent diarrhoeal illness was significantly associated with persistent diarrhoea with an odds ratio (OR) 2.6 (95%) confidence interval (CI): 1.1-7.1; p less than 0.05); during infancy this OR was 5.2 (95% CI: 1.0-31.9; p less than 0.01). Comparing children with persistent diarrhoea to matched acute diarrhoeal controls, presence of recent diarrhoeal illness was associated with an OR of 5.1 (95% CI: 1.3-20.3) in favour of the episode becoming persistent; in infants this OR was 10.4 (95% CI: 1.1-132.4; p less than 0.001).

This randomized, double-blind trial determined whether adding 90 mmol/L of alanine with a reduction in glucose to 90 mmol/L (alanine ORS) improves the efficacy of the standard oral rehydration solution (WHO-ORS). One hundred twenty-nine males aged 3-48 months with weight for length greater than or equal to 70% of NCHS, diarrheal duration less than or equal to 96 h, and clinical signs of mild to moderate dehydration were randomly allocated to either treatment group. During 0-6 h of treatment, ORS was offered at 120 ml/kg for rehydration without food or water. Beyond 6 h, ORS was offered as a volume-to-volume replacement for stool losses and a mixed diet of uniform composition was offered in amounts standardized for body weight. The most frequently isolated pathogens in alanine ORS and WHO-ORS groups were rotavirus (42 and 48%, respectively) and enterotoxigenic Escherichia coli (15 and 12%, respectively). In the 0-6 h period when food was withheld, median urine output in ml/kg (8;5, p less than 0.05) and percentage decrease in total serum solids (9:7%, p = 0.06) was significantly greater in alanine ORS than in WHO-ORS; median ORS intake and stool output were marginally lower in the alanine group but the differences were statistically not significant. Between 0 h and recovery, although the median values for duration of diarrhea (56.5 and 65.0 h), ORS consumption (260 and 323 ml/kg), and stool output (188.4 and 216.3 g/kg) were lower in the alanine ORS group, these differences with the WHO-ORS group were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

The reliability of saliva as an indicator of rotavirus infection was assessed among 15 infants (3-12 months) with rotaviral and 15 with nonrotaviral diarrhea. Paired salivary samples collected during acute and convalescent phases were tested for rotavirus-specific IgA and IgM by an ELISA. The sensitivity of IgA or IgM alone to predict infection was 53.3% and 46.6%, respectively; used in conjunction, the sensitivity rose to 80%. It seems that infants with rotaviral diarrhea mount mucosal antibody responses as reflected in their saliva; possibly salivary antibodies could be used to evaluate vaccine "take" in rotavirus vaccine trials.

Esch. coli strains manifesting localised (17), diffuse (8) or aggregative (17) phenotypes of adherence to HEp-2 cells were tested for their ability to adhere to human enterocytes isolated from duodenal biopsies of adult volunteers to obtain further evidence of their enteropathogenecity. Esch. coli strains H10407+; CFAI+ and LT+ STp+ STh+, F 294 B; a localised adherent strain positive with entero-adherent factor probe reported previously to attach to small intestinal enterocytes and F 582 C; LT- STp+ STh+ were the positive controls: H10407P (CFAI- mutant of H10407+) and K12 served as negative control strains. Adherence of variable degree was seen with 35.3 per cent of enteroaggregative Esch. coli (EAggEc) and with 58.8 per cent of enteroadherent Esch. coli localised (EAEC-L); EAEC-diffuse (EAEC-D) did not adhere to the human enterocytes. The possibility that EAgg EC and diffuse phenotypes may adhere better to lower small intestine or the large intestine, needs to be investigated.

The associated factors in 80 children (less than 2 yrs) with protracted diarrhea (greater than 21 days duration) and weight loss were: secondary carbohydrate intolerance (36): enteric pathogens (non typhoidal salmonella (11), enteropathogenic E. coli 'EPEC' (6), giardia (4), and shigella (3); cow's milk protein intolerance (3), gluten intolerance (3); miscellaneous (5); and undiagnosed enteropathy (9). Three of the EPEC showed localised pattern of adherence in vitro with HEP-2 cells. Most patients with salmonella and EPEC had severe secretory diarrhea with large fecal sodium losses. All 6 patients who died had secretory diarrhea and very high fecal sodium. All but 4 patients could be effectively managed with a chicken puree-glucose-coconut oil based diet.

Brush border lactase, sucrase and glucoamylase activities were assessed in jejunal mucosal biopsy specimens from 34 children (median age 11 months; range 1.5-38) having protracted diarrhoea with failure to thrive and 8 well nourished children with normal jejunal mucosal histology (median age 10.2 months; range 2-37). All enzymes showed progressive decrease in activity which was directly in relation to increasing degree of mucosal injury (P less than 0.002). Lactase was significantly reduced even in patients with protracted diarrhoea and normal mucosa (P less than 0.05). Glucoamylase and sucrase were significantly reduced only in the presence of mucosal injury (P less than 0.01). Our data suggest that most children with protracted diarrhoea may not tolerate lactose containing feeds and may need lactose-free diets preferably based on starch. A small number of children with protracted diarrhoea, who have severe mucosal injury may not be able to handle even starch and may require diets based on short chain glucose polymers. The findings of this study, need to be corroborated with well-controlled metabolic balance studies.