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ABSTRACTThe prospect that rotavirus diarrhea in children may soon be prevented by vaccines has placed a new priority on understanding the diversity of rotavirus strains and the mechanism by which these strains evolve over time. We have characterized a total of 465 rotavirus strains collected in North India from 2000 to 2007 for G and P types by reverse transcription-PCR and sequencing. The novel G12 rotavirus strains recently detected in other countries were first detected in India in 2001 and have emerged as the predominant strains in Delhi, India, during 2005 to 2007. While the VP7 sequence was highly homologous among G12 strains isolated in Delhi, suggesting recent emergence from a common ancestor, the strains had a diverse constellation of other gene segments, demonstrating substantial reassortment. For the entire period, the common rotavirus G types G1 (26%), G2 (25%), and G9 (14%) comprised 65% of the strains, and common P types, P[4] (19%), P[6] (22%), and P[8] (35%), comprised 76% of the total P types. Of note, we detected a high percentage of unusual (17%) strains and fecal specimens with mixed (12% G and 15% P) rotavirus infections having a variety of genomic constellations. For the first time, we identified two novel rotavirus strains with unusual G/P combinations, G2P[11] and G3P[11], in patients with diarrhea. The study highlights the great diversity among rotaviruses isolated from Indian children, the opportunity for genetic reassortment between strains, and the emergence of a novel G12 strain in our country. Due to the demonstrated effect of antigenic diversity on rotavirus vaccines, it will be important to continue careful monitoring of these strains as rotavirus vaccine programs are implemented in India.

ABSTRACTRotavirus genotype G12 strains were detected for the first time among newborns with asymptomatic rotavirus infection (74% of 39 rotavirus strains isolated from the infected infants were genotype G12) in the nursery of the All India Institute of Medical Sciences during a period from 2005 to 2006. Sequence analysis of the VP7 genes from these neonatal strains indicated a high level of homology to other G12 strains reported worldwide, suggesting the recent emergence of these strains in humans. Such nosocomial infections of newborns represent a potential source of introduction of novel rotavirus serotypes into the community.

The effect of seasonal changes on oil content of a new clone of Jamrosa 'RL-931' (Cymbopogon nardus var. confertiflorus × C. jwarancusa) was investigated during 1998-99. The pre-monsoon and onset of the monsoon period (May-July) was characterised by a higher oil content, whereas the post-monsoon and winter period (August-December) showed a comparatively lower oil content. The quality of the oil is best in May-July. High temperature and low humidity favours the accumulation of geraniol, geranyl acetate, and a low citral content in the oil. Maximum oil content was recorded when both maximum and minimum temperatures were high.

ABSTRACTWhile only four globally important rotavirus G serotypes (1 to 4) have been documented, many studies suggest that serotype G9 viruses may be widely distributed and more important than previously recognized. We have evaluated 10 serotype G9 rotavirus-neutralizing monoclonal antibodies (MAbs) directed to VP7, which bound by direct enzyme immunoassay (EIA) to P1A[8], G9 rotaviruses F45, WI61, and AU32, for their ability to recognize the New Delhi G9 rotavirus 116E. Only one MAb (MAb F45:1) bound to P[11], G9 virus 116E to a high titer by EIA. This MAb was incorporated into an indirect EIA for G serotyping, which was validated with prototype cultivable human rotaviruses of G types 1 to 4 and 9. The EIA was compared with genotyping by reverse transcriptase PCR (RT-PCR) under code for the determination of the G types of rotaviruses obtained from neonates in New Delhi, India. The sensitivities of RT-PCR and EIA (after two additional freeze-thaw cycles) for the typing of G9 rotaviruses were 91 and 86%, respectively, for 24 culture-adapted rotavirus strains. The untypeable culture-adapted rotavirus samples also were unreactive with VP7 group antigen-reactive MAb 60. After two additional freeze-thaw cycles, only 26 of 42 (62%) of stools containing rotavirus typed as G9 by RT-PCR were positive for G9 rotavirus by EIA. Stools containing rotavirus untypeable by EIA contained significantly less MAb 60-reactive VP7 antigen (P= 0.0001) than the stools containing typeable rotavirus. Thus, RT-PCR genotyping was the more sensitive method for determination of G9 type, but a serotype was readily determined in rotavirus samples containing MAb 60-reactive VP7 antigen by an EIA that incorporates MAb F45:1.

Although rotavirus is the most common cause of diarrhea in children older than 3 months of age, neonatal infections, which are asymptomatic, have rarely been surveyed and have been identified in only a few discrete nosocomial outbreaks. After such a nosocomial outbreak of rotavirus infection among newborns at a hospital in Delhi, we screened infants born at five other nurseries in the immediate area to assess the prevalence of neonatal infections and to determine whether the unique neonatal rotavirus strain, 116E, previously identified in Delhi, was present in other settings. Infection was documented in 43 to 78 of hospitalized infants between 4 and 6 days of life born at five of the six hospitals. Infection with strains related to 116E were the most common, but other unusual strains and no strains common in the community were detected. In addition a shift in genotype was observed among specimens collected from two of these hospitals during a 2-year period. Our observation that neonatal rotavirus infections are more common than recognized previously would encourage the administration of rotavirus vaccines during the newborn period and suggests that the low efficacy of vaccines observed during trials in developing countries may be caused by early natural exposure of infants before immunization. The extraordinary predisposition of neonates for unusual rotavirus strains not commonly found in the community should encourage others to screen neonates for this infection, characterize the strains more fully and attempt to understand at a molecular level the unique relationship between the infecting strain type and the age of the host.

An enterotoxigenic Escherichia coli (ETEC) strain producing a previously undescribed putative colonization factor was isolated from a child with diarrhea in India. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial heat extracts revealed a polypeptide band of 20.8 kDa when the bacteria were grown at 37 degrees C which was absent after growth at 22 degrees C. A specific rabbit antiserum raised against the purified 20.8-kDa protein bound specifically to the fimbriae, as shown by immunoelectron microscopy, and inhibited bacterial adhesion to tissue-cultured Caco-2 cells. Transformation with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator for several ETEC fimbriae, induced hyperexpression of the 20.8-kDa fimbrial subunit and a substantial increase in the proportion of bacterial cells that were fimbriated. The N-terminal amino acid sequence of the polypeptide showed 65 and 60% identity to the PCFO20 and 987P fimbriae of human and porcine ETEC, respectively. We propose the term CS20 for this new putative colonization factor of human ETEC.

Enteroaggregative Escherichia coli (EAggEC) is a distinct category of diarrheal pathogen implicated as the cause of persistent diarrhea. The pathogen exhibits a characteristic "stacked-brick" pattern of aggregation when incubated with HEp-2 cells. The outer membrane protein (OMP) profile of a prototype EAggEC strain (F03) reflected the presence of one major 30-kDa protein. The OMP is expressed in the presence of the 60-MDa plasmid that the strain harbors. Antibodies were raised against the OMP by injecting the protein into a rabbit. The manifestation of an adherence phenotype on HEp-2 cells was observed for F03 and other strains that express OMP in the presence and absence of anti-OMP serum. Clumps of bacteria forming an aggregative pattern were observed in the HEp-2 cell assay in the absence of OMP antibodies, whereas a few bacteria attached to the cells in the presence of OMP antibodies. Mannose-resistant hemagglutination of human erythrocytes observed in the presence of EAggEC strains was inhibited in the presence of anti-OMP serum. Sequence analysis of a peptide generated by protease digestion of OMP exhibited 90% homology to a peptide of flagellin protein encoded by the hag gene of Serratia marcescens. Immunolabeling of the outer membrane by colloidal gold confirmed the protein to be an OMP. Our results suggest that the OMP of EAggEC have common antigenic properties. Antibodies raised against the protein can prevent adherence in vitro and could potentially interrupt the natural disease.

Enteropathogenic Escherichia coli (EPEC) strains designated on the basis of their serotypes are epidemiologically associated with diarrhea. They adhere to the intestinal mucosa, producing the characteristic attaching and effacing (AE) lesion in an in vitro organ culture system. EPEC manifest localized adherence (LA) in the HEp-2 cell assay, and this is commonly used for clinical diagnosis. Recently, the fluorescence actin staining (FAS) test was proposed for the identification of E. coli causing the AE lesion. We therefore compared the FAS test with the HEp-2 cell assay and the EPEC adherence factor (EAF) probe assay for the detection of EPEC strains. Among 240 stool samples from children with diarrhea examined, 176 yielded E. coli and 14 of these strains showed the LA pattern in the HEp-2 cell assay; 11 of these were positive by both the EAF and the FAS tests. By using the HEp-2 cell assay as the "gold standard," the FAS test gave a sensitivity of 78.5% and a specificity of 100%. The three localized adherent FAS-negative strains tested subsequently were positive by the enteroaggregative E. coli DNA Probe and failed to produce the AE lesions characteristic of EPEC. When these strains were not considered, the sensitivity of the FAS test for detecting isolates that manifest LA was 100%. Against the EAF probe, the sensitivity and specificity of the FAS test were 91.6 and 100%, respectively. The FAS test avoids infrequent but nevertheless important phenotypic misclassifications in the HEp-2 cell assay, and it may therefore serve as a confirmatory test for EPEC.

Children with acute diarrhea and moderate dehydration between 3 months and 5 years of age were randomly assigned to receive treatment with standard WHO oral rehydration solution (ORS) (n = 33) and two other solutions in which the 20 g/L glucose was substituted by 50 g/L of pop rice (n = 31) and 60 g/L of mung bean (lentil) powder (n = 29). Satisfactory oral rehydration, as assessed clinically and by changes in PCV and total serum solids (TSS), was achieved in 90.9 with WHO ORS, 96.8 with pop rice, and 96.6 in the mung bean ORS treated group (p > 0.05). The purging rates (ml/kg/h) until recovery were 2.49 ± 1.5 (pop rice); 2.91 ± 2.0 (WHO), and 3.41 ± 1.7 in the mung bean group (p > 0.05). The percentage of patients recovering from diarrhea within the 72 h study period was 58.0 (pop rice), 48.4 (WHO), and 44.8 for mung bean group (p > 0.05). Though differences in stool volumes and duration in the three groups were not statistically different, there was a trend toward improvement in efficacy with the pop rice ORS in several parameters: greater weight gain, higher percentage decline in TSS, higher urine output despite lower ORS intake, and lower purging rates. The intake of semisolids in the 24–72 h study period was also higher in the pop rice group as compared to the other two groups (p < 0.05). The number of breast feeds and intake of artificial milk was however similar in all groups (p > 0.05).

Sixty children < 2 years of age suffering from mild acute gastroenteritis with < 5 dehydration were randomly assigned to two different isocaloric feeding regimens, viz., a locally prepared milk-free formulation (group A) of rice, lentil, sugar, and coconut oil and a spray dried commercial cow's milk formula (group B). There were two treatment failures in group A and one in group B. The postintervention duration of diarrhea (days) in group A (11.0 ± 10.0) was higher than in group B (7.6 ± 10.8), but these differences were not significant (p > 0.05). The energy intake (kcal/kg/24 h) on postintervention day 4 was 78.7 ± 31.7 in group A and 101.3 ± 41.1 in group B (p > 0.05). The corresponding values for day 7 were 74.2 ± 29.1 and 110.0 ± 41.1, respectively (p < 0.05). The mean weight gain (g/kg/24 h) between admission and the day of recovery in group A (2.0 ± 4.2) was significantly lower (p < 0.05) than in group B (5.8 ± 7.8). Similar trends in weight gain were observed at days 4 and 7. These findings suggest that a cow's milk-based formula is well tolerated by majority of the infants with mild acute gastroenteritis after initial rehydration with ORS. The infants who were fed the milk-free cereal-based diet showed significantly less energy intake and gained weight less rapidly than those who were fed the cow's milk-based formula.

Enteroaggregative Escherichia coli (EAggEC) is a distinct category of diarrheal pathogen implicated as the cause of persistent diarrhea. The pathogen exhibits a characteristic "stacked-brick" pattern of aggregation when incubated with HEp-2 cells. The outer membrane protein (OMP) profile of a prototype EAggEC strain (F03) reflected the presence of one major 30-kDa protein. The OMP is expressed in the presence of the 60-MDa plasmid that the strain harbors. Antibodies were raised against the OMP by injecting the protein into a rabbit. The manifestation of an adherence phenotype on HEp-2 cells was observed for F03 and other strains that express OMP in the presence and absence of anti-OMP serum. Clumps of bacteria forming an aggregative pattern were observed in the HEp-2 cell assay in the absence of OMP antibodies, whereas a few bacteria attached to the cells in the presence of OMP antibodies. Mannose-resistant hemagglutination of human erythrocytes observed in the presence of EAggEC strains was inhibited in the presence of anti-OMP serum. Sequence analysis of a peptide generated by protease digestion of OMP exhibited 90% homology to a peptide of flagellin protein encoded by the hag gene of Serratia marcescens. Immunolabeling of the outer membrane by colloidal gold confirmed the protein to be an OMP. Our results suggest that the OMP of EAggEC have common antigenic properties. Antibodies raised against the protein can prevent adherence in vitro and could potentially interrupt the natural disease.

Rotavirus (RV) strains infecting newborns often have unique neutralization antigens (P serotypes) on their outer capsids that are distinct from those found on RV strains that cause diarrhea in older children. We examined the hypothesis that unusual RV strains preferentially infect newborns because the newborns lack maternal neutralizing antibodies to these strains. To test this hypothesis, sera and saliva samples collected from neonates infected with 116E-like (P[11]G9) strains in the maternity ward of the All India Institute of Medical Sciences (AIIMS) hospital in New Delhi were tested for neutralizing antibodies against common RV strains and those infecting newborns and these titers were compared with those of newborns who did not become infected (controls). The infected neonates had significantly lower levels of cord blood neutralizing antibodies to 116E than the controls, suggesting that immunity to neonatal RV infection is acquired transplacentally through maternal antibodies. Further, this study confirmed the immunogenicity of the AIIMS neonatal strain 116E, a vaccine candidate, in its ability to evoke a potent RV-specific immunoglobulin A and neutralizing antibody response in serum and saliva among the infected babies. Our findings have important implications for the development of an effective RV vaccine. In India, where G9 strains are common in the community, the use of 116E as a vaccine, together with the rhesus tetravalent vaccine, may provide a broader protection against all the circulating RV serotypes, including serotype G9, which is not represented in the current rhesus RV tetravalent vaccine (G1-G4).

We recently reported that the culture-adapted neonatal rotavirus strain 116E represented the first P type 11 human rotavirus, based on the close relationship of its VP4 protein to that of the bovine serotype G₁₀P₁₁ strain B223. In this study, we demonstrated by sequence analysis and cross-neutralization studies that the VP7 protein of 116E is closely related to those of the human serotype G9 strains. F45 and WI61, but distinct from B223 and other rotaviruses. Low-level cross-neutralization was also observed between strains 116E and B223, probably because of the antigenic similarity of their VP4 proteins. We have demonstrated by RNA-RNA hybridization that strain 116E is a reassortant between strains from the Wa and bovine (KK3-like) genogroups, deriving at least seven genes from the former and at least one gene from the latter. Together with the recent identification of serotype G₁₀P₁₁ newborn rotavirus strains in Bangalore, India (M. Das et al., Virology, 194, 374-379, 1993), these results are consistent with the hypothesis that reassortment may be an important mechanism for generation of rotavirus strains of newborns. Copyright 1993, 1999 Academic Press

A collection of 44 enteroaggregative Escherichia coli (EAggEC) strains isolated from infants with diarrhea in India and the United Kingdom were examined for their ability to adhere in vitro to human intestinal mucosa and by electron microscopy for production of putative adherence factors. None of the strains adhered to human duodenal mucosa, and six strains tested did not adhere to ileal mucosa; all 44 strains, however, adhered to human colonic mucosa in localized aggregates. Electron microscopy of infected colonic mucosa indicated fimbrially mediated adhesion of the EAggEC strains. Four morphologically distinct kinds of fimbriae, including a new morphological type of E. coli fimbriae consisting of bundles of fine filaments, were identified among the EAggEC strains; this new type of fimbria was observed in 43 of the 44 EAggEC strains. Forty-three of the 44 EAggEC strains were positive with a DNA probe developed to identify EAggEC, and most of the strains belonged to serotypes unrelated to the other major classes of diarrheic E. coli. These results suggest that EAggEC may be a large-bowel pathogen and colonize the colon by a fimbrially mediated adhesion mechanism.

A polynucleotide probe comprising the gene encoding a major structural subunit protein of coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) was developed. Eighty-nine ETEC isolates were examined in parallel with the probe in a colony hybridization assay and in a recently developed polyclonal-antibody-based inhibition enzyme-linked immunosorbent assay (ELISA). The two assays showed a high level of concordance in the detection of CS6-positive ETEC (kappa = 0.84, P < 0.00001). Thus, 36 of the 89 ETEC isolates were identified as CS6-positive by both assays. Six strains that were negative for other colonization factor antigens were positive with the CS6 probe but negative in the ELISA, suggesting lack of surface CS6 expression in these strains. One strain was probe negative but positive in the ELISA, while the remaining 46 strains were negative in both assays. The phenotypic and genotypic assays will prove useful in vaccine-oriented studies of ETEC disease.

The present study describes a colony hybridization setup for identification of enterotoxigenic Escherichia coli obviating the need for advanced equipment and radioactive isotopes. With a modest laboratory arrangement, polynucleotide gene probes were produced in large quantities. The probes were labeled with digoxigenin and, after hybridization, detected with an antidigoxigenin alkaline phosphatase conjugate. With an established isotope-based oligonucleotide hybridization assay as reference, a blinded study on a large battery of enterotoxigenic and nonenterotoxigenic bacteria revealed a satisfactory sensitivity and specificity of the nonradioactive assay.

The duodenal juice and fecal aerobic microflora was investigated in 54 patients with persistent diarrhea age ≤2 years. The duodenal aspirates yielded increased aerobic bacteria >105organismsml in 28 51.9 of the patients. Established enteric pathogens were isolated from the duodenal aspirates of 12 22.2 of the 54 patients, viz., enteroadherent Escherichia coliEAEC 5, enterotoxigenic E. coliETEC 3, enteropathogenic E. coliEPEC 1, nontyphoidal Salmonella1, and Giardia lamblia2. The total aerobic bacterial count was >105in all 12 patients positive for specific pathogens apart from one case in whom E. colishowing diffuse adherence to HEp2 cells were identified. Among the remaining 42 specific pathogennegative patients, 19 45.2 also had >105aerobic organismsml in the small bowel. Eight strains of Klebsiellafrom four of the patients were negative for enterotoxin production in a rabbit ileal loop assay and for adherence to HEp2 cells. In contrast, 2854 51.9 of the same patients had known enteric pathogens in their stools, viz., nontyphoidal Salmonella8 or 14.8, Shigella2 or 3.7, Campylobacter1 or 1.9, ETEC 4 or 7.4, EPEC 2 or 3.7, EAEC 7 or 13.0, and G. lambliaand Entameba histolytica3 or 5.6. Further search for potential virulence factors among aerobic bacteria colonizing the upper small intestine in persistent diarrhea is warranted.

Eleven (13.8) children (4–14 months; mean of 7.8 ± 3.6 months) presenting with protracted diarrhea (duration >21 days) and weight loss had associated infection with Salmonella typhimurium. All had documented weight loss of 16–25 and progressive clinical deterioration. On admission, they had high purging rates (>4 ml/kg/h), hyponatremia (5/11), mucosal injury, and malabsorption as measured by 1 h blood D-xylose, fecal α,-antitrypsin, and oral fat tolerance test. Diarrhea was secretory in 8 of 11 and fecal sodium was high (54–142 mEq/L; mean of 102 ± 27 mEq/L). The organism showed multiple drug resistance. All patients received antibiotics (amikacin and nalidixic acid/norfloxacin) for 10–14 days, which was followed by rapid improvement in clinical status and absorption studies. The two youngest patients died. Due to ethical reasons, an untreated control group was not included. Use of appropriate antibiotics may benefit children with S. typhimurium-associated severe protracted diarrhea and rapid progressive weight loss.

A gene probe derived from the colonization factor antigen I (CFA/I) operon cross-hybridized at very low stringency to plasmid DNA from coli surface antigen 17 (CS17)-producing enterotoxigenic Escherichia coli (ETEC) and from the ETEC strain F595C, which was negative for previously described CFAs, CSs, and putative colonization factors (PCFs). A 16-kDa protein was identified in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat extracts prepared after growth of strain F595C at 37 degrees C on CFA agar containing bile salts. Transmission electron microscopy revealed bile salt- and temperature-dependent expression of fimbriae with a diameter of 7 nm. After transformation with a recombinant plasmid harboring the cfaR gene, which encodes a positive regulator of several CFAs, PCFs, and CSs, the 16-kDa protein was hyperexpressed. Polyclonal antibodies raised against this protein bound to the fimbriae and inhibited the adhesion of F595C bacteria to tissue-cultured Caco-2 cells. Nucleotide sequence determination of the gene encoding the 16-kDa fimbrial subunit revealed a high degree of amino acid sequence homology to the CFA/I, CS1, CS2, CS4, CS14, and CS17 polypeptides. The term CS19 is proposed for the new fimbria.

Eleven (13.8%) children (4–14 months; mean of 7.8 ± 3.6 months) presenting with protracted diarrhea (duration >21 days) and weight loss had associated infection with Salmonella typhimurium. All had documented weight loss of 16–25% and progressive clinical deterioration. On admission, they had high purging rates (>4 ml/kg/h), hyponatremia (5/11), mucosal injury, and malabsorption as measured by 1 h blood D-xylose, fecal a,-antitrypsin, and oral fat tolerance test. Diarrhea was secretory in 8 of 11 and fecal sodium was high (54–142 mEq/L; mean of 102 ± 27 mEq/L). The organism showed multiple drug resistance. All patients received antibiotics (amikacin and nalidixic acid/norfloxacin) for 10–14 days, which was followed by rapid improvement in clinical status and absorption studies. The two youngest patients died. Due to ethical reasons, an untreated control group was not included. Use of appropriate antibiotics may benefit children with S. typhimurium-associated severe protracted diarrhea and rapid progressive weight loss.

Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E. coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon. Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA. Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes. The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression. A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy. The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.

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 =70% of NCHS, diarrheal duration =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< 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. Our findings are consistent with improved efficacy of WHO-ORS following alanine fortification but the clinical benefits achieved in these mildly dehydrated cases fall short of what may be clinically meaningful and of relevance to public health

Sixty children < 2 years of age suffering from mild acute gastroenteritis with < 5% dehydration were randomly assigned to two different isocaloric feeding regimens, viz., a locally prepared milk-free formulation (group A) of rice, lentil, sugar, and coconut oil and a spray dried commercial cow's milk formula (group B). There were two treatment failures in group A and one in group B. The postintervention duration of diarrhea (days) in group A (11.0 ± 10.0) was higher than in group B (7.6 ± 10.8), but these differences were not significant (p > 0.05). The energy intake (kcal/kg/24 h) on postintervention day 4 was 78.7 ± 31.7 in group A and 101.3 ± 41.1 in group B (p > 0.05). The corresponding values for day 7 were 74.2 ± 29.1 and 110.0 ± 41.1, respectively (p < 0.05). The mean weight gain (g/kg/24 h) between admission and the day of recovery in group A (2.0 ± 4.2) was significantly lower (p < 0.05) than in group B (5.8 ± 7.8). Similar trends in weight gain were observed at days 4 and 7. These findings suggest that a cow's milk-based formula is well tolerated by majority of the infants with mild acute gastroenteritis after initial rehydration with ORS. The infants who were fed the milk-free cereal-based diet showed significantly less energy intake and gained weight less rapidly than those who were fed the cow's milk-based formula.

Between April and December 1993, we determined P and G genotypes of group A rotavirus strains obtained from children admitted to diarrhea treatment centers in five Indian cities. From a total of 63 rotavirus-positive specimens, we identified 10 different strains with five different G genotypes and four distinct P types by using reverse transcription-PCR. The common worldwide strains G1P8, G2P4, G3P8, and G4P8 were underrepresented among Indian children (33%), whereas strains of P type 6 (G1P6, G2P6, G3P6, G4P6, and G9P6), which primarily infect asymptomatic newborns but are rare in children with diarrhea were common in India (43%). Of these, G9P6, a strain not previously reported to be found in children with diarrhea, was the most prevalent (22%). Eleven percent of the strains were nontypeable, and another 11% of the specimens had mixed infections. Using digoxigenin-labeled, genotype-specific hybridization probes, we confirmed all G9 strains and mixed infections tested and identified three nontypeable strains (one G9 and two P8). The epidemiological significance of G9 rotavirus strains, if confirmed in other settings, may have important implications for vaccine development.

We developed an accurate nonradioactive colony hybridization assay (NCHA) using a digoxigenin-labeled polynucleotide probe and an antidigoxigenin alkaline phosphatase conjugate for the identification of enterotoxigenic Escherichia coli (ETEC) harboring genes for colonization factor antigen I (CFA/I), coli surface antigen 4 (CS4), or putative colonization factor O166 (PCFO166). In this 2-day assay, visual registration of color intensity could be used to distinguish between CFA/I-positive strains and strains with the genetic potential to express CS4 or PCFO166. A rapid NCHA was developed by which the results could be read visually 7 h and 45 min after inoculation of the bacteria. In the rapid NCHA, densitometry verified the visual discrimination between four groups of E. coli; ETEC with the CFA/I gene, ETEC with the CS4 gene, ETEC with the PCFO166 gene, and E. coli strains that lack such genes. As a confirmatory test, plasmids from ETEC with the CFA/I, CS4, or PCFO166 gene were differentiated by their characteristic restriction fragment patterns in nonradioactive Southern blot hybridization.

Five genetically distinct human rotavirus (HRV) gene 4 groups have been described on the basis of comparative nucleotide sequencing and the predicted amino acid sequences, and at least four of them represent distinct VP4 antigenic types. To identify each gene 4 type and investigate its distribution in HRV isolates from patients with diarrhea, we developed a polymerase chain reaction (PCR) typing method using sequence information available for four genetically distinct gene 4 types. Rotavirus double-stranded RNAs (dsRNAs) isolated from stool samples were first reverse transcribed and amplified by PCR by using two oligonucleotide primers that correspond to regions that are highly conserved among all known HRV gene 4 types. The 876-bp dsDNA products were then reamplified by PCR in the presence of a cocktail containing one conserved plus-sense primer and four type-specific minus-sense primers (selected from the hypervariable region of gene 4), resulting in products of 345, 483, 267, and 391 bp corresponding to gene 4 types 1, 2, 3, and 4, respectively. This method reliably identified the gene 4 types of 16 well-characterized HRV isolates. Our results were independently confirmed for all 16 strains by reverse transcription and PCR amplification of HRV dsRNA in the presence of alternate type-specific primer pairs. For direct gene 4 typing of HRV in stool samples, we developed a method to extract rotavirus dsRNA from stool specimens by using glass powder. Our results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.

The duodenal juice and fecal aerobic microflora was investigated in 54 patients with persistent diarrhea (age ≤2 years). The duodenal aspirates yielded increased aerobic bacteria (>105organisms/ml) in 28 (51.9%) of the patients. Established enteric pathogens were isolated from the duodenal aspirates of 12 (22.2%) of the 54 patients, viz., enteroadherent Escherichia coli(EAEC) (5), enterotoxigenic E. coli(ETEC) (3), enteropathogenic E. coli(EPEC) (1), nontyphoidal Salmonella(1), and Giardia lamblia(2). The total aerobic bacterial count was >105in all 12 patients positive for specific pathogens apart from one case in whom E. colishowing diffuse adherence to HEp‐2 cells were identified. Among the remaining 42 specific pathogen‐negative patients, 19 (45.2%) also had >105aerobic organisms/ml in the small bowel. Eight strains of Klebsiellafrom four of the patients were negative for enterotoxin production in a rabbit ileal loop assay and for adherence to HEp‐2 cells. In contrast, 28/54 (51.9%) of the same patients had known enteric pathogens in their stools, viz., nontyphoidal Salmonella(8 or 14.8%), Shigella(2 or 3.7%), Campylobacter(1 or 1.9%), ETEC (4 or 7.4%), EPEC (2 or 3.7%), EAEC (7 or 13.0%), and G. lambliaand Entameba histolytica(3 or 5.6%). Further search for potential virulence factors among aerobic bacteria colonizing the upper small intestine in persistent diarrhea is warranted.

We compared a new colony hybridization assay with an established enzyme-linked immunosorbent assay for detection of enterotoxigenic Escherichia coli (ETEC) expressing colonization factor antigen I (CFA/I). The tests were applied to 135 human ETEC strains. Of these isolates, 30 had previously been characterized for CFAs. A strain harboring the plasmid vector of the polynucleotide gene probe, nine non-ETEC strains from healthy infants, and eight ETEC strains of animal origin were included for further evaluation of probe specificity. The two assays showed a high level of concordance in the specific detection of ETEC strains expressing CFA/I. A total of 24 strains tested positive in the CFA/I hybridization assay, while 23 of those strains were positive in the CFA/I enzyme-linked immunosorbent assay. The single discrepant result could be explained by the loss of a regulatory gene. The strain harboring the plasmid vector of the probe, the non-ETEC E. coli strains, and the ETEC strains of animal origin were all negative in the CFA/I probe assay.

Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with 32P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays.

A gene probe derived from the colonization factor antigen I (CFA/I) operon cross-hybridized at very low stringency to plasmid DNA from coli surface antigen 17 (CS17)-producing enterotoxigenic Escherichia coli (ETEC) and from the ETEC strain F595C, which was negative for previously described CFAs, CSs, and putative colonization factors (PCFs). A 16-kDa protein was identified in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat extracts prepared after growth of strain F595C at 37 degrees C on CFA agar containing bile salts. Transmission electron microscopy revealed bile salt- and temperature-dependent expression of fimbriae with a diameter of 7 nm. After transformation with a recombinant plasmid harboring the cfaR gene, which encodes a positive regulator of several CFAs, PCFs, and CSs, the 16-kDa protein was hyperexpressed. Polyclonal antibodies raised against this protein bound to the fimbriae and inhibited the adhesion of F595C bacteria to tissue-cultured Caco-2 cells. Nucleotide sequence determination of the gene encoding the 16-kDa fimbrial subunit revealed a high degree of amino acid sequence homology to the CFA/I, CS1, CS2, CS4, CS14, and CS17 polypeptides. The term CS19 is proposed for the new fimbria.

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 ≥70 of NCHS, diarrheal duration ≤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< 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. Our findings are consistent with improved efficacy of WHO-ORS following alanine fortification but the clinical benefits achieved in these mildly dehydrated cases fall short of what may be clinically meaningful and of relevance to public health

Serum anti-gliadin antibody (AGA) titres were estimated by diffusion in a gel enzyme-linked immunosorbent assay in children with coeliac disease (n = 11), protracted diarrhoea of non-coeliac causes (n = 110), acute gastroenteritis (n = 20), protein energy malnutrition (n = 20), and asymptomatic, well-nourished children (n = 66). The mean IgG and IgA AGA titres were significantly higher (p < 0.001) in children with coeliac disease than in any other groups. There was no significant difference (p ± 0.01) in AGA titres in relation to age, nutritional status, or severity of villous injury. In patients with coeliac disease AGA titres showed a good correlation with disease activity. The specificity and sensitivity of the assay are discussed.

Children with acute diarrhea and moderate dehydration between 3 months and 5 years of age were randomly assigned to receive treatment with standard WHO oral rehydration solution (ORS) (n = 33) and two other solutions in which the 20 g/L glucose was substituted by 50 g/L of pop rice (n = 31) and 60 g/L of mung bean (lentil) powder (n = 29). Satisfactory oral rehydration, as assessed clinically and by changes in PCV and total serum solids (TSS), was achieved in 90.9% with WHO ORS, 96.8% with pop rice, and 96.6% in the mung bean ORS treated group (p > 0.05). The purging rates (ml/kg/h) until recovery were 2.49 ± 1.5 (pop rice); 2.91 ± 2.0 (WHO), and 3.41 ± 1.7 in the mung bean group (p > 0.05). The percentage of patients recovering from diarrhea within the 72 h study period was 58.0 (pop rice), 48.4 (WHO), and 44.8 for mung bean group (p > 0.05). Though differences in stool volumes and duration in the three groups were not statistically different, there was a trend toward improvement in efficacy with the pop rice ORS in several parameters: greater weight gain, higher percentage decline in TSS, higher urine output despite lower ORS intake, and lower purging rates. The intake of semisolids in the 24–72 h study period was also higher in the pop rice group as compared to the other two groups (p < 0.05). The number of breast feeds and intake of artificial milk was however similar in all groups (p > 0.05).

An enterotoxigenic Escherichia coli (ETEC) strain producing a previously undescribed putative colonization factor was isolated from a child with diarrhea in India. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial heat extracts revealed a polypeptide band of 20.8 kDa when the bacteria were grown at 37 degrees C which was absent after growth at 22 degrees C. A specific rabbit antiserum raised against the purified 20.8-kDa protein bound specifically to the fimbriae, as shown by immunoelectron microscopy, and inhibited bacterial adhesion to tissue-cultured Caco-2 cells. Transformation with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator for several ETEC fimbriae, induced hyperexpression of the 20.8-kDa fimbrial subunit and a substantial increase in the proportion of bacterial cells that were fimbriated. The N-terminal amino acid sequence of the polypeptide showed 65 and 60% identity to the PCFO20 and 987P fimbriae of human and porcine ETEC, respectively. We propose the term CS20 for this new putative colonization factor of human ETEC.

The role of enteric-type adenoviruses and rotaviruses in mild and severe acute gastroenteritis was investigated among children younger than 5 years of age seeking treatment at an urban hospital (UH) and at a rural health center (RHC) in India. There were 330 children at the UH and 340 at the RHC; 319 and 315 age matched nondiarrheal children served as controls for the respective groups.

R-1 anti-reticulin antibodies (ARA) were present in sera of 10 of the 12 children who subsequently fulfilled the ESPGAN diagnostic criteria for celiac disease (CD), hut was not found in any children in the age-matched control groups, viz., severely malnourished (n = 20). acute gastroenteritis (n = 23), normally nourished asymptomatic (n = 38), and 42 children with protracted diarrhoea and failure to thrive due to nonceliac causes. ARA was a highly specific (100) and sensitive (83) assay for the early diagnosis of CD. A positive ARA assay with an initial subtotal villous atrophy was seen to always suggest CD, and these together would provide a useful basis for instituting gluten-free diets in suspected cases of CD rather than histological findings alone, which may be often present in children in the third world with protracted diarrhoea and associated severe malnutrition.

R-1 anti-reticulin antibodies (ARA) were present in sera of 10 of the 12 children who subsequently fulfilled the ESPGAN diagnostic criteria for celiac disease (CD), hut was not found in any children in the age-matched control groups, viz., severely malnourished (n = 20). acute gastroenteritis (n = 23), normally nourished asymptomatic (n = 38), and 42 children with protracted diarrhoea and failure to thrive due to nonceliac causes. ARA was a highly specific (100%) and sensitive (83%) assay for the early diagnosis of CD. A positive ARA assay with an initial subtotal villous atrophy was seen to always suggest CD, and these together would provide a useful basis for instituting gluten-free diets in suspected cases of CD rather than histological findings alone, which may be often present in children in the third world with protracted diarrhoea and associated severe malnutrition.

DENOUMENT AND DISCUSSION ROTHMUND-THOMSON SYNDROME (POIKELODERMA CONGENITAL) MANIFESTATIONS This rare disorder is characterized by distinctive cutaneous abnormalities. Maculopapular, erythematous lesions with patches of hypopigmentation usually over the cheeks first appear at the age of 3 to 6 months. Subsequently, they are observed over the rest of the face, dorsum of hands and gluteal regions, forearms, and legs. Photosensitivity may be present. In advanced stages, telangiectases, brownish hyperpigmentation, and reticular, depigmented patches of skin atrophy are seen. Keratoses may develop on exposed skin after several years and squamous cell carcinoma may occur in the keratoses or neighboring skin.Bilateral cataracts are present in approximately 50% to 75% of the patients. They usually appear by age 7 years and may be anterior, subcapsular, perinuclear, or posterior stellate.Other features of the syndrome include premature senile appearance, bird-like facies, diminished scalp hair, hypogonadism, short stature, small hands and feet, and hypoplastic thumbms. Mental

Because the prevalence of chlamydial infection in pregnant Indian women, as well as its associations with low birth weight and prematurity, remains unclear, a preliminary study was conducted to ascertain the prevalence of infection with Chlamydia trachomatisin 94 pregnant women in midgestation attending an antenatal clinic. An additional prevalence study focused on 172 women who presented after 26 weeks’ gestation in spontaneous labor. None had multiple pregnancies or received erythromycin antenatally. Placenta previa and stillbirths were other grounds for exclusion. This group included mainly housewives less than 30 years old who had at least a high school education, were married, and did not smoke or drink.

Candidate rotavirus vaccines have been prepared with reassortant strains specifically to protect against the 4 major rotavirus G serotypes (Gl-4). Many studies using P (VP4) genotyping methods have indicated that, worldwide, rotavirus strains of the 4 common G serotypes are each associated with 1 P genotype: Gt, G3, and G4 are associated with P[8], and G2 is associated with P[4]. In contrast, G and P genotyping of rotavirus in specimens from India revealed that a high percentage of the childhood diarrhea strains belong to genotype P[6], and the most common strain had an unusual G serotype, G9. Similarly, in all regions surveyed in Brazil, apparent reassortants of genotype P[8], G5 were found in children with gastroenteritis. These studies indicate that while rotavirus strains have limited diversity in many settings, reassortment between common and uncommon serotypes or animal strains can arise in some settings and, thus, lead to unusual diversity.

A cohort of 452 rural children was followed longitudinally for 13 mo to ascertain the role of HEp-2 cell adherent Escherichia coli and other pathogens in causing acute (≤14 d) and persistent (>14 d) diarrhea. Aeromonas, Campylobacter jejuni, E. coli manifesting localized adherence to HEp-2 cells and enterotoxigenic E. coli were significantly associated with acute diarrhea. E. coli strains that exhibit aggregative adherence, so-called enteroaggregative E. coli, a newly-described category of diarrheagenic E. coli distinct from enterotoxigenic, enteroinvasive, enterohemorrhagic, and enteropathogenic E. coli, were found significantly more often in patients with persistent diarrhea (29.5%) than with acute diarrhea (12.8%) (P = .0052) or controls (9.9%) (P = .0006).