When is a randomised controlled trial health equity relevant? Development and validation of a conceptual framework.

BACKGROUND: Randomised controlled trials can provide evidence relevant to assessing the equity impact of an intervention, but such information is often poorly reported. We describe a conceptual framework to identify health equity-relevant randomised trials with the aim of improving the design and reporting of such trials. METHODS: An interdisciplinary and international research team engaged in an iterative consensus building process to develop and refine the conceptual framework via face-to-face meetings, teleconferences and email correspondence, including findings from a validation exercise whereby two independent reviewers used the emerging framework to classify a sample of randomised trials. RESULTS: A randomised trial can usefully be classified as 'health equity relevant' if it assesses the effects of an intervention on the health or its determinants of either individuals or a population who experience ill health due to disadvantage defined across one or more social determinants of health. Health equity-relevant randomised trials can either exclusively focus on a single population or collect data potentially useful for assessing differential effects of the intervention across multiple populations experiencing different levels or types of social disadvantage. Trials that are not classified as 'health equity relevant' may nevertheless provide information that is indirectly relevant to assessing equity impact, including information about individual level variation unrelated to social disadvantage and potentially useful in secondary modelling studies. CONCLUSION: The conceptual framework may be used to design and report randomised trials. The framework could also be used for other study designs to contribute to the evidence base for improved health equity.

Salivary IgA from the sublingual compartment as a novel noninvasive proxy for intestinal immune induction.

Whole-saliva IgA appears like an attractive noninvasive readout for intestinal immune induction after enteric infection or vaccination, but has failed to show consistent correlation with established invasive markers and IgA in feces or intestinal lavage. For reference, we measured antibodies in samples from 30 healthy volunteers who were orally infected with wild-type enterotoxigenic Escherichia coli. The response against these bacteria in serum, lavage, and lymphocyte supernatants (antibody-in-lymphocyte-supernatant, ALS) was compared with that in targeted parotid and sublingual/submandibular secretions. Strong correlation occurred between IgA antibody levels against the challenge bacteria in sublingual/submandibular secretions and in lavage (r=0.69, P<0.0001) and ALS (r=0.70, P<0.0001). In sublingual/submandibular secretions, 93% responded with more than a twofold increase in IgA antibodies against the challenge strain, whereas the corresponding response in parotid secretions was only 67% (P=0.039). With >twofold ALS as a reference, the sensitivity of a >twofold response for IgA in sublingual/submandibular secretion was 96%, whereas it was only 67% in the parotid fluid. To exclude that flow rate variations influenced the results, we used albumin as a marker. Our data suggested that IgA in sublingual/submandibular secretions, rather than whole saliva with its variable content of parotid fluid, is a preferential noninvasive proxy for intestinal immune induction.

Prevalence of iron deficiency and anemia among healthy women of reproductive age in Bhaktapur, Nepal.

OBJECTIVE: To determine the prevalence of anemia and iron status as assessed by biochemical markers and to explore the associations between markers of iron status and iron intake. STUDY AREA AND POPULATION: Five hundred healthy women of reproductive age from the Bhaktapur district of Nepal were included in the study. METHODS: A cluster sampling procedure was applied for this cross-sectional study. Women without any ongoing infection aged 13-35 years were selected randomly from the population. We measured the plasma concentration of hemoglobin (Hb), ferritin and transferrin receptors. Dietary information was obtained by a food frequency questionnaire and two 24-h dietary recalls. RESULTS: The prevalence of anemia (Hb concentration <12 g/dl) was 12% (n=58). The prevalence of depleted iron stores (plasma ferritin <15 microg/l) was 20% (n=98) whereas the prevalence of iron deficiency anemia (anemia, depleted iron stores with elevated transferrin receptor i.e. >1.54 mg/l) was 6% (n=30). Seven percent (n=35) of women were having iron-deficient erythropoiesis (depleted iron stores and elevated transferrin receptor but normal Hb). Out of the 58 anemic women, 41 (71%) and 31 (53%) were also having elevated plasma transferrin receptor and depleted iron stores, respectively. Fifty-four percent of the women ate less than the recommended average intake of iron. The main foods contributing to dietary iron were rice, wheat flour and green and dry vegetables. CONCLUSIONS: The prevalence of anemia in our study was substantially lower than the national figure for non-pregnant women. Only about half of the women with anemia were also having depleted iron stores, suggesting that other causes of anemia may be prevalent in this population. SPONSORSHIP: Norwegian Universities Committee for Development, Research and Education (NUFU).

Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections.

Among 167 rotavirus specimens collected from young children in a suburban area of Bissau, Guinea-Bissau, from 1996 to 1998, most identifiable strains belonged to the uncommon P[6], G2 type and approximately 50% remained incompletely typed. In the present study, 76 such strains were further characterized. Due to interprimer interaction during the standard multiplex PCR approach, modifications of this procedure were implemented. The modified analyses revealed a high frequency of G2, G8, and G9 genotypes, often combined with P[4] and/or P[6]. The Guinean G8 and G9 strains were 97 and 98%, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus strains. Detection of such strains among the previously incompletely typed strains indicates a potential underestimation of mixed infections, if only a standard multiplex PCR procedure is followed. Furthermore cross-priming of the G3 primer with the G8 primer binding site and silent mutations at the P[4] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality underscores the need for extensive strain surveillance as a basis to develop appropriate rotavirus vaccine candidates.

The immune response to pneumococcal polysaccharide vaccine in zinc-depleted mice.

Zinc depletion affects several facets of the immune system and the resistance to infections. We assessed the effect of zinc deprivation on the immune response to the pneumococcal polysaccharide antigens in the commercially available Pneumovax pneumococcal vaccine. Young female BALB/c mice were fed diets with 2.7, 5.8 or 25 micro g of elemental zinc per mg diet. After six weeks of pair feeding, there were significant differences in the mean body weights between the feeding groups and we demonstrated a dose response of the zinc level in the diet on growth. The induced zinc deficiency had no discernible effect on the antipneumococcal polysaccharide immunoglobulin M (IgM) response following immunization with the pneumococcal vaccine. Although zinc depletion has a detrimental effect on the immune system, the murine T-cell-independent response to antigens such as those in the pneumococcal polysaccharide capsule does not seem to be affected.

Pneumococcal pulmonary infection, septicaemia and survival in young zinc-depleted mice.

The aim of the present study was to explore whether mice fed a diet low in Zn (2.0 mg Zn/kg diet) for a relatively short period of time were more prone to severe Streptococcus pneumoniae infection than mice fed a normal diet (25 mg elemental Zn/kg). The Zn-deficient mice were compared with mice in two Zn-adequate control groups; one pair-fed and another with free access to the diet. After 2 weeks feeding, the mice were infected intranasally under anaesthesia with a suspension containing about 10(7) pneumococci. Clinical status was observed every day and blood samples were examined for S. pneumoniae every second day for a week. All infected mice examined carried the infecting strain intranasally. The survival time and time before positive blood culture were significantly shorter in the Zn-depleted group than in the pair-fed Zn-adequate group (hazard ratios 15.6 and 3.2, and respectively). At the end of the observation period, ten of the twelve mice in the Zn-deficient group were dead while one of twelve and two of twelve were dead in the two Zn-adequate control groups. This study shows that even acutely-induced Zn deficiency dramatically increases the risk of serious pneumococcal infection in mice.

Genotypic detection of enterotoxigenic Escherichia coli colonisation factors.

We have developed a nonradioactive colony hybridisation assay for the detection of enterotoxigenic Escherichia coli (ETEC) that harbor the structural genes for CFA/I, CS1, CS2, CS4, CS17, or PCFO166. Thus, a polynucleotide probe derived from the colonisation factor antigen I (CFA/I) operon hybridised under very low stringency conditions to total DNA from CFA/I-producing (CFA/I), coli-surface antigen 1 and 3 (CS1 CS3-), CS2 CS3-, CS4 CS6-, CS17-, and putative colonisation factor O166 (PCFO166)-producing enterotoxigenic Escherichia coli (ETEC). The probe did not hybridise to DNA from CS3, CFA/III CS6, CS5 CS6, CS6, CS7, or PCFO159 ETEC. Visual registration of colour intensity could be used to differentiate between CFA/I, CS4 and PCFO166-positive strains on the one hand and strains with the genetic potential to express CS1, CS2, or CS17 on the other. As a confirmatory test, restriction fragment patterns obtained from Sau3AI-digested ETEC plasmid DNA could be used to distinguish between CFA/I, CS1, CS4, CS17, and PCFO166 ETEC in nonradioactive Southern blot hybridisation. The simultaneous genotypic detection of several ETEC colonisation factors will prove useful in vaccine-oriented studies of ETEC disease.

Micronutrient deficiency in children.

Malnutrition increases morbidity and mortality and affects physical growth and development, some of these effects resulting from specific micronutrient deficiencies. While public health efforts must be targeted to improve dietary intakes in children through breast feeding and appropriate complementary feeding, there is a need for additional measures to increase the intake of certain micronutrients. Food-based approaches are regarded as the long-term strategy for improving nutrition, but for certain micronutrients, supplementation, be it to the general population or to high risk groups or as an adjunct to treatment must also be considered. Our understanding of the prevalence and consequences of iron, vitamin A and iodine deficiency in children and pregnant women has advanced considerably while there is still a need to generate more knowledge pertaining to many other micronutrients, including zinc, selenium and many of the B-vitamins. For iron and vitamin A, the challenge is to improve the delivery to target populations. For disease prevention and growth promotion, the need to deliver safe but effective amounts of micronutrients such as zinc to children and women of fertile age can be determined only after data on deficiency prevalence becomes available and the studies on mortality reduction following supplementation are completed. Individual or multiple micronutrients must be used as an adjunct to treatment of common infectious diseases and malnutrition only if the gains are substantial and the safety window sufficiently wide. The available data for zinc are promising with regard to the prevention of diarrhea and pneumonia. It should be emphasized that there must be no displacement of important treatment such as ORS in acute diarrhea by adjunct therapy such as zinc. Credible policy making requires description of not only the clinical effects but also the underlying biological mechanisms. As findings of experimental studies are not always feasible to extrapolate to humans, the biology of deficiency as well as excess of micronutrients in humans must continue to be investigated with vigour.

Community-based controlled trial of dietary management of children with persistent diarrhea: sustained beneficial effect on ponderal and linear growth.

BACKGROUND: Uncontrolled hospital-based studies in developing countries have reported promising results of dietary rehabilitation of children with persistent diarrhea. OBJECTIVE: The objective was to determine the immediate and long-term effects of a dietary supplement and micronutrients given to children with persistent diarrhea during the episode and for 1 wk during convalescence. DESIGN: The study was open, controlled, and community-based and was conducted in a periurban area in Guinea-BISSAU: Children <3 y of age with persistent diarrhea were identified during weekly household visits. The children randomly assigned to the treatment and control groups were examined by a physician and all medical conditions were treated. The children in the treatment group were offered home-based dietary treatment consisting of locally available foods and micronutrient supplements. RESULTS: There were 141 episodes of persistent diarrhea during the study: 70 in the treatment group (in 58 children) and 71 in the control group (in 62 children). During the intervention period (median: 17 d), weight gain in the treatment group exceeded that of the control group by 61.5 g/wk (95% CI: 49.2, 73.8), whereas there was no significant difference in linear growth on the basis of knee-heel length. At a median follow-up period of 6.6 mo after the intervention was stopped, weight gain in the treatment group exceeded that of the control group by 12.5 g/wk (95% CI: 7.7, 17.3); knee-heel length was 7.5 mm/y (4.8, 10.2) greater and total length was 0.65 cm/y (0.11, 1.19) greater in the treatment group. CONCLUSION: Therapeutic feeding and micronutrient supplementation had an immediate and sustained beneficial effect on growth in children with persistent diarrhea.

Measurement of specific IgA in faecal extracts and intestinal lavage fluid for monitoring of mucosal immune responses.

Currently available methods for the evaluation of antigen-specific immune responses in the intestine, i.e. measurement of IgA in intestinal lavage and antibody secreting cells (ASC) in peripheral blood, are not applicable to large-scale immunogenicity studies or to kinetic studies where repeated sampling is required. Simple and reliable methods need to be developed. Intestinal lavage and faecal samples were collected from 12 mice on days 0, 14, 21, 28 and 35 following initial immunization with four doses of cholera toxin (CT) by the gastric or rectal routes. The concentrations of anti-CT IgA in the faecal extracts showed a high level of correlation with those in the lavage samples (Spearman's correlation coefficient=0.85, P<0. 0001) regardless of the route of CT administration. Moreover, the kinetics of the immune response as reflected in the faecal extracts mirrored those in the lavage samples regardless of immunization route. As compared to gastric immunization, rectal administration of CT yielded higher levels of anti-CT IgA in both intestinal lavage fluids and in faecal extracts. The use of rectal immunization and the measurement of IgA in faecal extracts for monitoring mucosal immune responses may be relevant for the development of effective enteric vaccines.

Genotype profiles of rotavirus strains from children in a suburban community in Guinea-Bissau, Western Africa.

The P (VP4) and G (VP7) genotypes of 167 group A rotavirus strains obtained during the period 1996 to 1998 from 149 children living in a suburban community in Guinea-Bissau, western Africa, were determined by the reverse transcription-PCR technique. A total of nine combinations including five different P types and five different G types were identified. The globally common genotype pairs P[8], G1; P[4], G2; P[8], G3 and P[8], G4 were underrepresented in this study area. We found a substantial year-to-year variation in the occurrence of the genotype combinations. In 1996 and 1997, P[6], G2 was the most frequent, whereas P[8], G1 was more common in 1998. The unusual type P[9], G3 and a few mixed infections were detected. Sixteen percent of the rotavirus-positive samples were nontypeable.

Community-based randomized controlled trial of reduced osmolarity oral rehydration solution in acute childhood diarrhea.

OBJECTIVE: The standard oral rehydration solution (ORS) recommended by WHO and UNICEF does not reduce the volume or frequency of stools or the length of the episode. Hospital-based studies from developing and developed countries and intestinal perfusion studies suggest a beneficial effect on water and sodium absorption with reduced osmolarity ORS as compared with standard ORS. We conducted a community-based study comparing the efficacy of reduced osmolarity ORS (224 mmol/l) with standard ORS (311 mmol/l) in acute childhood diarrhea in a West African community. METHODS: Infants and toddlers age 0 to 30 months having 738 episodes of diarrhea identified by weekly household visits were randomly assigned to treatment with either standard ORS (n = 376) or reduced osmolarity ORS (n = 362). The children were followed by daily home visits to assess ORS intake and clinical characteristics. Duration of diarrhea was compared by proportional hazards regression analysis, the hazard ratio being interpreted as the relative recovery rate between the children receiving the two types of ORS. Because earlier reports have suggested that weaning status might be an important modifier for the performance of reduced osmolarity ORS, the effect was assessed overall and as an interaction between type of ORS and weaning status and age. Maternal satisfaction was assessed in a paired analysis among mothers whose children participated at least twice in the study. RESULTS: In the overall analysis reduced osmolarity ORS was as efficacious as standard ORS as assessed by duration of diarrheal episode and total number of stool evacuations on Days 1 and 2. Non-breast-fed toddlers (i.e. children ages 12 to 30 months) treated with reduced osmolarity ORS had significantly shorter diarrheal episodes [1.14 days vs. 1.78 days with standard ORS; hazard ratio, 1.50; 95% confidence interval (CI), 1.07 to 2.09] and lower total number of stool evacuations on Days 1 and 2 (3.9 stool evacuations vs. 5.0 stool evacuations with standard ORS; ratio of geometric means, 0.77; 95% CI 0.60 to 1.01). No significant difference was found for breast-fed toddlers or for infants. There was no statistically significant difference in the ORS intake between the two treatment groups. The odds ratio for the mother preferring reduced osmolarity ORS to standard ORS was 1.92 (95% CI 0.97 to 3.85). CONCLUSIONS: Reduced osmolarity ORS was as efficacious as standard ORS. Non-breast-fed children treated with reduced osmolarity ORS had significantly shorter diarrheal episodes and a tendency toward lower stool frequency. These findings may be of importance, especially in developing countries where early weaning is common.

Expression of colonization factor antigen I fimbriae by enterotoxigenic Escherichia coli; influence of growth conditions and a recombinant positive regulatory gene.

Enterotoxigenic Escherichia coli (ETEC) may spontaneously lose the positive regulatory cfaR gene and thereby the capacity to express colonization factor antigen I (CFA/I). A recombinant plasmid harbouring the cfaR gene was transformed into cfaR-negative mutant ETEC strains. CFA/I expression of wild-type and cfaR-transformed ETEC cultivated in different liquid media was quantified. At 37 degrees C, a high level of CFA/I expression from wild-type and cfaR-transformed strains was observed after growth in CFA broth. Transformation enhanced CFA/I expression only marginally. The transformant cultures showed a considerable variation in CFA/I expression which was paralleled by the proportion of individual bacteria producing CFA/I. This heterogeneity could be explained by a variable tendency to structural CFA/I gene loss among individual cfaR-transformed bacteria.

A new putative fimbrial colonization factor, CS19, of human enterotoxigenic Escherichia coli.

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.

Colonization factors of enterotoxigenic Escherichia coli isolated from children in north India.

Colonization factor antigens (CFAs) mediate attachment of enterotoxigenic Escherichia coli (ETEC) to the intestinal mucosa and induce protective immunity against ETEC diarrhea. ETEC strains (n = 111) isolated from North Indian children from 1985 to 1989 were examined for CFAs and putative colonization factors (PCFs). CFA/IV was the most common factor (26%), followed by coli surface antigen 17 (CS17) (19%), CFA/I (14%), PCFO166 (7%), and CFA/II (5%), while 24% of the isolates were negative for CFAs and PCFs. Among the strains producing heat-stable and heat-labile toxin (ST+LT+ strains), the STaI gene was strongly associated with the absence of known CFAs and PCFs, making the STaI+LT+ isolates an interesting target for the identification of previously undescribed factors. Repetitive sequence--based polymerase chain reaction revealed that the CS17+ strains, although clonally related, represented endemically circulating strains with a diversity greater than that of the CFA/I+ strains, which showed a substantial clonal clustering.

Identification and characterization of CS20, a new putative colonization factor of enterotoxigenic Escherichia coli.

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.

Genotypic and phenotypic identification of coli surface antigen 6-positive enterotoxigenic Escherichia coli.

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.

Enteroaggregative Escherichia coli may be a new pathogen causing acute and persistent diarrhea.

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.

Evaluation of DNA-DNA hybridization for the direct detection of enterotoxigenic Escherichia coli in stool blots.

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.