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1.  Microbiota That Affect Risk for Shigellosis in Children in Low-Income Countries 
Emerging Infectious Diseases  2015;21(2):242-250.
Co-infection with Shigella spp. and other microbes modifies diarrhea risk.
Pathogens in the gastrointestinal tract exist within a vast population of microbes. We examined associations between pathogens and composition of gut microbiota as they relate to Shigella spp./enteroinvasive Escherichia coli infection. We analyzed 3,035 stool specimens (1,735 nondiarrheal and 1,300 moderate-to-severe diarrheal) from the Global Enteric Multicenter Study for 9 enteropathogens. Diarrheal specimens had a higher number of enteropathogens (diarrheal mean 1.4, nondiarrheal mean 0.95; p<0.0001). Rotavirus showed a negative association with Shigella spp. in cases of diarrhea (odds ratio 0.31, 95% CI 0.17–0.55) and had a large combined effect on moderate-to-severe diarrhea (odds ratio 29, 95% CI 3.8–220). In 4 Lactobacillus taxa identified by 16S rRNA gene sequencing, the association between pathogen and disease was decreased, which is consistent with the possibility that Lactobacillus spp. are protective against Shigella spp.–induced diarrhea. Bacterial diversity of gut microbiota was associated with diarrhea status, not high levels of the Shigella spp. ipaH gene.
doi:10.3201/eid2101.140795
PMCID: PMC4313639  PMID: 25625766
shigellosis; Shigella; bacteria; polymicrobial infection; Escherichia coli; enteroinvasive E. coli; EIEC; Lactobacillus; rotavirus; viruses; co-occurring pathogens; enteropathogens; microbiota; ipaH gene; diarrhea; children; low-income countries
2.  Characterization of Immunological Cross-Reactivity between Enterotoxigenic Escherichia coli Heat-Stable Toxin and Human Guanylin and Uroguanylin 
Infection and Immunity  2014;82(7):2913-2922.
Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.
doi:10.1128/IAI.01749-14
PMCID: PMC4097616  PMID: 24778111
3.  Experimental infection of healthy volunteers with enterotoxigenic Escherichia coli wild-type strain TW10598 in a hospital ward 
BMC Infectious Diseases  2014;14(1):482.
Background
Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea in resource-limited regions. It is also an important cause of diarrhea in travellers to these areas.
To evaluate the protective efficacy of new ETEC vaccines that are under development, there is a need to increase the capacity to undertake Phase IIB (human challenge) clinical trials and to develop suitable challenge models.
Methods
An in-hospital study was performed where fasting adult volunteers were experimentally infected with 1 × 106 to 1 × 109 colony forming units (CFUs) of the wild-type ETEC strain TW10598, which had been isolated from a child with diarrhea in West Africa in 1997. We recorded symptoms and physical signs and measured serum immune response to the TW10598 bacterium.
Results
We included 30 volunteers with mean age 22.8 (range 19.8, 27.4) years. The most common symptoms were diarrhea (77%), abdominal pain (67%), nausea (63%), and abdominal cramping (53%). Seven subjects (23%) experienced fever, none were hypotensive. Most of the volunteers responded with a substantial rise in the level of serum IgA antibodies against the challenge strain.
Conclusions
We established the capacity and methods for safely undertaking challenge studies to measure the efficacy of ETEC vaccine candidates in a hospital ward. Strain TW10598 elicited both clinical symptoms and an immune response across the doses given.
doi:10.1186/1471-2334-14-482
PMCID: PMC4165915  PMID: 25190096
ETEC; Challenge; Experimental infection; Inpatient
4.  Effect of implementation of Integrated Management of Neonatal and Childhood Illness programme on treatment seeking practices for morbidities in infants: cluster randomised trial 
Objective To determine the effect of implementation of the Integrated Management of Neonatal and Childhood Illness strategy on treatment seeking practices and on neonatal and infant morbidity.
Design Cluster randomised trial.
Setting Haryana, India.
Participants 29 667 births in nine intervention clusters and 30 813 births in nine control clusters.
Main outcome measures The pre-specified outcome was the effect on treatment seeking practices. Post hoc exploratory analyses assessed morbidity, hospital admission, post-neonatal infant care, and nutritional status outcomes.
Interventions The Integrated Management of Neonatal and Childhood Illness intervention included home visits by community health workers, improved case management of sick children, and strengthening of health systems. Outcomes were ascertained through interviews with randomly selected caregivers: 6204, 3073, and 2045 in intervention clusters and 6163, 3048, and 2017 in control clusters at ages 29 days, 6 months, and 12 months, respectively.
Results In the intervention cluster, treatment was sought more often from an appropriate provider for severe neonatal illness (risk ratio 1.76, 95% confidence interval 1.38 to 2.24), for local neonatal infection (4.86, 3.80 to 6.21), and for diarrhoea at 6 months (1.96, 1.38 to 2.79) and 12 months (1.22, 1.06 to 1.42) and pneumonia at 6 months (2.09, 1.31 to 3.33) and 12 months (1.44, 1.00 to 2.08). Intervention mothers reported fewer episodes of severe neonatal illness (risk ratio 0.82, 0.67 to 0.99) and lower prevalence of diarrhoea (0.71, 0.60 to 0.83) and pneumonia (0.73, 0.52 to 1.04) in the two weeks preceding the 6 month interview and of diarrhoea (0.63, 0.49 to 0.80) and pneumonia (0.60, 0.46 to 0.78) in the two weeks preceding the 12 month interview. Infants in the intervention clusters were more likely to still be exclusively breast fed in the sixth month of life (risk ratio 3.19, 2.67 to 3.81).
Conclusion Implementation of the Integrated Management of Neonatal and Childhood Illness programme was associated with timely treatment seeking from appropriate providers and reduced morbidity, a likely explanation for the reduction in mortality observed following implementation of the programme in this study.
Trial registration Clinical trials NCT00474981; ICMR Clinical Trial Registry CTRI/2009/091/000715.
doi:10.1136/bmj.g4988
PMCID: PMC4148946  PMID: 25172514
5.  Growth effects of exclusive breastfeeding promotion by peer counsellors in sub-Saharan Africa: the cluster-randomised PROMISE EBF trial 
BMC Public Health  2014;14:633.
Background
In this multi-country cluster-randomized behavioural intervention trial promoting exclusive breastfeeding (EBF) in Africa, we compared growth of infants up to 6 months of age living in communities where peer counsellors promoted EBF with growth in those infants living in control communities.
Methods
A total of 82 clusters in Burkina Faso, Uganda and South Africa were randomised to either the intervention or the control arm. Feeding data and anthropometric measurements were collected at visits scheduled 3, 6, 12 and 24 weeks post-partum. We calculated weight-for-length (WLZ), length-for-age (LAZ) and weight-for-age (WAZ) z-scores. Country specific adjusted Least Squares Means with 95% confidence intervals (CI) based on a longitudinal analysis are reported. Prevalence ratios (PR) for the association between peer counselling for EBF and wasting (WLZ < −2), stunting (LAZ < −2) and underweight (WAZ < −2) were calculated at each data collection point.
Results
The study included a total of 2,579 children. Adjusting for socio-economic status, the mean WLZ at 24 weeks were in Burkina Faso −0.20 (95% CI −0.39 to −0.01) and in Uganda −0.23 (95% CI −0.43 to −0.03) lower in the intervention than in the control arm. In South Africa the mean WLZ at 24 weeks was 0.23 (95% CI 0.03 to 0.43) greater in the intervention than in the control arm. Differences in LAZ between the study arms were small and not statistically significant. In Uganda, infants in the intervention arm were more likely to be wasted compared to those in the control arm at 24 weeks (PR 2.36; 95% CI 1.11 to 5.00). Differences in wasting in South Africa and Burkina Faso and stunting and underweight in all three countries were small and not significantly different.
Conclusions
There were small differences in mean anthropometric indicators between the intervention and control arms in the study, but in Uganda and Burkina Faso, a tendency to slightly lower ponderal growth (weight-for-length z-scores) was found in the intervention arms.
Trial registration number
ClinicalTrials.gov: NCT00397150
doi:10.1186/1471-2458-14-633
PMCID: PMC4082276  PMID: 24950759
Exclusive breastfeeding promotion; Peer counselling; Child growth; Anthropometry; Stunting; Wasting; Underweight; Undernutrition; Community randomised trial
6.  Cobalamin and Folate Status in 6 to 35 Months Old Children Presenting with Acute Diarrhea in Bhaktapur, Nepal 
PLoS ONE  2014;9(3):e90079.
Background
Cobalamin and folate are essential micronutrients and are important in DNA and RNA synthesis, cell proliferation, growth, hematopoiesis, and cognitive function. However, data on cobalamin and folate status are lacking particularly from young children residing in low and middle income countries.
Objective
To measure cobalamin and folate status and identifies their predictors among 6 to 35 months old children presenting with acute diarrhea.
Design
This was a cross-sectional study in 823 children presenting with acute diarrhea. We measured plasma cobalamin, folate, methylmalonic acid and total homocysteine who sought treatment for acute diarrhea between June 1998 and August 2000.
Results
The mean (SD) plasma concentrations of cobalamin, folate, total homocysteine and methylmalonic acid were 206 (124) pmol/L, 55 (32) nmol/L, 11.4 (5.6) µmol/L and 0.79 (1.2) µmol/L, respectively. The prevalence of low plasma cobalamin (<150 pmol/L) was 41% but less than 2% (15) children had low folate concentration (<10 nmol/L). Plasma homocysteine and methylmalonic acid concentrations were negatively associated with cobalamin concentration but not associated with folate status. The prevalence of cobalamin deficiency was higher in breastfed than non-breastfed children (44% vs 24%; p = <0.001). The prevalence of hyperhomocysteinemia (>10 µmol/L) and elevated methylmalonic acid (>0.28 µmol/L) were 73% and 52%, respectively. In the regression analyses, the plasma cobalamin concentration was positively associated with age, and introduction of animal or formula milk.
Conclusions
Our study indicated that poor cobalamin status was common particularly among breastfed children. Folate deficiency was virtually none existent. Possible consequences of cobalamin deficiency in young children need to be explored.
doi:10.1371/journal.pone.0090079
PMCID: PMC3940712  PMID: 24594935
7.  Quantitative PCR for Detection of Shigella Improves Ascertainment of Shigella Burden in Children with Moderate-to-Severe Diarrhea in Low-Income Countries 
Journal of Clinical Microbiology  2013;51(6):1740-1746.
Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 104 ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 104 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥2.9 × 104 ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 104 ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 104 ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.
doi:10.1128/JCM.02713-12
PMCID: PMC3716050  PMID: 23536399
8.  The Global Enteric Multicenter Study (GEMS) of Diarrheal Disease in Infants and Young Children in Developing Countries: Epidemiologic and Clinical Methods of the Case/Control Study 
Background. Diarrhea is a leading cause of illness and death among children aged <5 years in developing countries. This paper describes the clinical and epidemiological methods used to conduct the Global Enteric Multicenter Study (GEMS), a 3-year, prospective, age-stratified, case/control study to estimate the population-based burden, microbiologic etiology, and adverse clinical consequences of acute moderate-to-severe diarrhea (MSD) among a censused population of children aged 0–59 months seeking care at health centers in sub-Saharan Africa and South Asia.
Methods. GEMS was conducted at 7 field sites, each serving a population whose demography and healthcare utilization practices for childhood diarrhea were documented. We aimed to enroll 220 MSD cases per year from selected health centers serving each site in each of 3 age strata (0–11, 12–23, and 24–59 months), along with 1–3 matched community controls. Cases and controls supplied clinical, epidemiologic, and anthropometric data at enrollment and again approximately 60 days later, and provided enrollment stool specimens for identification and characterization of potential diarrheal pathogens. Verbal autopsy was performed if a child died. Analytic strategies will calculate the fraction of MSD attributable to each pathogen and the incidence, financial costs, nutritional consequences, and case fatality overall and by pathogen.
Conclusions. When completed, GEMS will provide estimates of the incidence, etiology, and outcomes of MSD among infants and young children in sub-Saharan Africa and South Asia. This information can guide development and implementation of public health interventions to diminish morbidity and mortality from diarrheal diseases.
doi:10.1093/cid/cis753
PMCID: PMC3502307  PMID: 23169936
9.  Some Epidemiologic, Clinical, Microbiologic, and Organizational Assumptions That Influenced the Design and Performance of the Global Enteric Multicenter Study (GEMS) 
The overall aim of the Global Enteric Multicenter Study–1 (GEMS-1) is to identify the etiologic agents associated with moderate-to-severe diarrhea (MSD) among children <5 years of age, and thereby the attributable pathogen-specific population-based incidence of MSD, to guide investments in research and public health interventions against diarrheal disease. To accomplish this, 9 core assumptions were vetted through widespread consultation: (1) a limited number of etiologic agents may be responsible for most MSD; (2) a definition of MSD can be crafted that encompasses cases that might otherwise be fatal in the community without treatment; (3) MSD seen at sentinel centers is a proxy for fatal diarrheal disease in the community; (4) matched case/control is the appropriate epidemiologic design; (5) methods across the sites can be standardized and rigorous quality control maintained; (6) a single 60-day postenrollment visit to case and control households creates mini-cohorts, allowing comparisons; (7) broad support for GEMS-1 messages can be achieved by incorporating advice from public health spokespersons; (8) results will facilitate the setting of investment and intervention priorities; and (9) wide acceptance and dissemination of the GEMS-1 results can be achieved.
doi:10.1093/cid/cis787
PMCID: PMC3502315  PMID: 23169935
10.  Statistical Methods in the Global Enteric Multicenter Study (GEMS) 
The Global Enteric Multicenter Study (GEMS) is an investigation of the burden (number of cases and incidence) of moderate-to-severe diarrhea (MSD) in children <60 months of age at 7 sites in sub-Saharan Africa and South Asia. The population attributable fraction for a putative pathogen, either unadjusted or adjusted for other pathogens, is estimated using the proportion of MSD cases from whom the pathogen was isolated and the odds ratio for MSD and the pathogen from conditional logistic regression modeling. The adjusted attributable fraction, proportion of MSD cases taken to a sentinel health center (SHC), number of cases presenting to an SHC, and the site's population are used to estimate the annual number of MSD cases and MSD incidence rate attributable to a pathogen or group of pathogens. Associations with death and nutritional outcomes, ascertained at follow-up visits to case and control households, are evaluated both in MSD cases and in the population.
doi:10.1093/cid/cis788
PMCID: PMC3502316  PMID: 23169937
11.  Case/Control Studies With Follow-up: Constructing the Source Population to Estimate Effects of Risk Factors on Development, Disease, and Survival 
If individuals in a case/control study are subsequently observed as a cohort of cases and a cohort of controls, weighted regression analyses can be used to estimate the association between the exposures initially recorded and events occurring during the follow-up of the 2 cohorts. Such analyses can be conceptualized as being undertaken on a reconstructed source population from which cases and controls stem. To simulate this population, the cohort of cases is added to the cohort of controls expanded with the reciprocal of the case disease incidence odds (the sampling weight) to include all individuals in the source population who did not develop the case disease. We use a simulated dataset to illustrate how weighted generalized linear model regression can be used to estimate the association between an exposure captured during the case/control study component and an outcome that occurs during follow-up.
doi:10.1093/cid/cis802
PMCID: PMC3502318  PMID: 23169939
12.  Lopinavir/Ritonavir versus Lamivudine peri-exposure prophylaxis to prevent HIV-1 transmission by breastfeeding: the PROMISE-PEP trial Protocol ANRS 12174 
BMC Infectious Diseases  2012;12:246.
Background
Postnatal transmission of HIV-1 through breast milk remains an unsolved challenge in many resource-poor settings where replacement feeding is not a safe alternative. WHO now recommends breastfeeding of infants born to HIV-infected mothers until 12 months of age, with either maternal highly active antiretroviral therapy (HAART) or peri-exposure prophylaxis (PEP) in infants using nevirapine. As PEP, lamivudine showed a similar efficacy and safety as nevirapine, but with an expected lower rate of resistant HIV strains emerging in infants who fail PEP, and lower restrictions for future HIV treatment. Lopinavir/ritonavir (LPV/r) is an attractive PEP candidate with presumably higher efficacy against HIV than nevirapine or lamivudine, and a higher genetic barrier to resistance selection. It showed an acceptable safety profile for the treatment of very young HIV-infected infants. The ANRS 12174 study aims to compare the risk of HIV-1 transmission during and safety of prolonged infant PEP with LPV/r (40/10 mg twice daily if 2-4 kg and 80/20 mg twice daily if >4 kg) versus Lamivudine (7,5 mg twice daily if 2-4 kg, 25 mg twice daily if 4-8 kg and 50 mg twice daily if >8 kg) from day 7 until one week after cessation of BF (maximum 50 weeks of prophylaxis) to prevent postnatal HIV-1 acquisition between 7 days and 50 weeks of age.
Methods
The ANRS 12174 study is a multinational, randomised controlled clinical trial conducted on 1,500 mother-infant pairs in Burkina Faso, South Africa, Uganda and Zambia. We will recommend exclusive breastfeeding (EBF) until 26th week of life and cessation of breastfeeding at a maximum of 49 weeks in both trial arms.
HIV-uninfected infants at day 7 (± 2 days) born to HIV-1 infected mothers not eligible for HAART who choose to breastfeed their infants.
The primary endpoint is the acquisition of HIV-1 (as assessed by HIV-1 DNA PCR) between day 7 and 50 weeks of age. Secondary endpoints are safety (including resistance, adverse events and growth) until 50 weeks and HIV-1-free survival until 50 weeks.
Discussion
This study will provide a new evidence-based intervention to support HIV-1-infected women not eligible for HAART to safely breastfeed their babies.
Trial registration number ( http://www.clinicaltrials.gov)
NCT00640263
doi:10.1186/1471-2334-12-246
PMCID: PMC3482558  PMID: 23039034
13.  The high burden of infant deaths in rural Burkina Faso: a prospective community-based cohort study 
BMC Public Health  2012;12:739.
Background
Infant mortality rates (IMR) remain high in many sub-Saharan African countries, especially in rural settings where access to health services may be limited. Studies in such communities can provide relevant data on the burden of and risk factors for infant death. We measured IMR and explored risk factors for infant death in a cohort of children born in Banfora Health District, a rural area in South-West Burkina Faso.
Methods
A prospective community-based cohort study was nested within the PROMISE-EBF trial (NCT00397150) in 24 villages of the study area. Maternal and infant baseline characteristics were collected at recruitment and after birth, respectively. Home visits were conducted at weeks 3, 6, 12, 24 and 52 after birth. Descriptive statistics were calculated using robust standard errors to account for cluster sampling. Cox multivariable regression was used to investigate potential risk factors for infant death.
Results
Among the 866 live born children included in the study there were 98 infant deaths, yielding an IMR of 113 per 1000 live births (95% CI: 89–143). Over 75% of infant deaths had occurred by 6 months of age and the post neonatal infant mortality rate was 67 per 1000 live births (95% CI: 51–88). Infections (35%) and preterm births complications (23%) were the most common probable causes of death by 6 months. Multivariable analyses identified maternal history of child death, polygyny, twin births and poor anthropometric z-scores at week-3 as factors associated with increased risk of infant death.
Conclusions
We observed a very high IMR in a rural area of Burkina Faso, a country where 75% of the population lives in rural settings. Community-based health interventions targeting mothers and children at high risk are urgently needed to reduce the high burden of infant deaths in these areas.
doi:10.1186/1471-2458-12-739
PMCID: PMC3489611  PMID: 22947029
Infant mortality; Risk factors; Rural areas; Burkina Faso
14.  Risk Factors for Extended Duration of Acute Diarrhea in Young Children 
PLoS ONE  2012;7(5):e36436.
Objective and Background
We sought to identify predictors of extended duration of diarrhea in young children, which contributes substantially to the nearly 1 1/2 million annual diarrheal deaths globally.
Methods
We followed 6-35 month old Nepalese children enrolled in the placebo-arm of a randomized controlled trial with 391 episodes of acute diarrhea from the day they were diagnosed until cessation of the episode. Using multiple logistic regression analysis, we identified independent risk factors for having diarrhea for more than 7 days after diagnosis.
Results
Infants had a 17 (95% CI 3.5, 83)-fold and toddlers (12 to 23 month olds) a 9.9 (95% CI 2.1, 47)-fold higher odds of having such illness duration compared to the older children. Not being breastfed was associated with a 9.3 (95% CI 2.4, 35.7)-fold increase in the odds for this outcome. The odds also increased with increasing stool frequency. Furthermore, having diarrhea in the monsoon season also increased the risk of prolonged illness.
Conclusion
We found that high stool frequency, not being breastfed, young age and acquiring diarrhea in the rainy season were risk factors for prolonged diarrhea. In populations such as ours, breastfeeding may be the most important modifiable risk factor for extended duration of diarrhea.
doi:10.1371/journal.pone.0036436
PMCID: PMC3348155  PMID: 22590543
15.  Distribution of Classical and Nonclassical Virulence Genes in Enterotoxigenic Escherichia coli Isolates from Chilean Children and tRNA Gene Screening for Putative Insertion Sites for Genomic Islands▿†  
Journal of Clinical Microbiology  2011;49(9):3198-3203.
Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea. Three adhesins (Tia, TibA, EtpA), an iron acquisition system (Irp1, Irp2, and FyuA), a GTPase (LeoA), and an autotransporter (EatA) are ETEC virulence-related proteins that, in contrast to the classical virulence factors (enterotoxins and fimbrial colonization factors) have not heretofore been targets in characterizing isolates from epidemiological studies. Here, we determined the occurrence of these nonclassical virulence genes in 103 ETEC isolates from Chilean children with diarrhea and described their association with O serogroups and classical virulence determinants. Because tia, leoA, irp2, and fyuA are harbored by pathogenicity islands inserted into the selC and asnT tRNA genes (tDNAs), we analyzed the regions flanking these loci. Ten additional tDNAs were also screened to identify hot spots for genetic insertions. Associations between the most frequent serogroups and classical colonization factor (CF)-toxin profiles included O6/LT-STh/CS1-CS3-CS21 (i.e., O6 serogroup, heat-labile [LT] and human heat-stable [STh] enterotoxins, and CFs CS1, -3 and -21), O6/LT-STh/CS2-CS3-CS21, and O104-O127/STh/CFAI-CS21. The eatA and etpA genes were detected in more than 70% of the collection, including diverse serogroups and virulence profiles. Sixteen percent of the ETEC strains were negative for classical and nonclassical adhesins, suggesting the presence of unknown determinants of adhesion. The leuX, thrW, and asnT tDNAs were disrupted in more than 65% of strains, suggesting they are hot spots for the insertion of mobile elements. Sequences similar to integrase genes were identified next to the thrW, asnT, pheV, and selC tDNAs. We propose that the eatA and etpA genes should be included in characterizations of ETEC isolates in future epidemiological studies to determine their prevalence in other geographical regions. Sequencing of tDNA-associated genetic insertions might identify new ETEC virulence determinants.
doi:10.1128/JCM.02473-10
PMCID: PMC3165568  PMID: 21775541
16.  A Comparative Genomic Analysis of Diverse Clonal Types of Enterotoxigenic Escherichia coli Reveals Pathovar-Specific Conservation▿ †  
Infection and Immunity  2010;79(2):950-960.
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in children less than 5 years of age in low- and middle-income nations, whereas it is an emerging enteric pathogen in industrialized nations. Despite being an important cause of diarrhea, little is known about the genomic composition of ETEC. To address this, we sequenced the genomes of five ETEC isolates obtained from children in Guinea-Bissau with diarrhea. These five isolates represent distinct and globally dominant ETEC clonal groups. Comparative genomic analyses utilizing a gene-independent whole-genome alignment method demonstrated that sequenced ETEC strains share approximately 2.7 million bases of genomic sequence. Phylogenetic analysis of this “core genome” confirmed the diverse history of the ETEC pathovar and provides a finer resolution of the E. coli relationships than multilocus sequence typing. No identified genomic regions were conserved exclusively in all ETEC genomes; however, we identified more genomic content conserved among ETEC genomes than among non-ETEC E. coli genomes, suggesting that ETEC isolates share a genomic core. Comparisons of known virulence and of surface-exposed and colonization factor genes across all sequenced ETEC genomes not only identified variability but also indicated that some antigens are restricted to the ETEC pathovar. Overall, the generation of these five genome sequences, in addition to the two previously generated ETEC genomes, highlights the genomic diversity of ETEC. These studies increase our understanding of ETEC evolution, as well as provide insight into virulence factors and conserved proteins, which may be targets for vaccine development.
doi:10.1128/IAI.00932-10
PMCID: PMC3028850  PMID: 21078854
17.  Malaria Parasitaemia among Infants and Its Association with Breastfeeding Peer Counselling and Vitamin A Supplementation: A Secondary Analysis of a Cluster Randomized Trial 
PLoS ONE  2011;6(7):e21862.
Background
Malaria is the second highest contributor to the disease burden in Africa and there is a need to identify low cost prevention strategies. The objectives of this study were to estimate the prevalence of malaria parasitaemia among infants and to measure the association between peer counselling for exclusive breastfeeding (EBF), vitamin A supplementation, anthropometric status (weight and length) and malaria parasitaemia.
Methods
A cluster randomized intervention trial was conducted between 2006 and 2008 where 12 of 24 clusters, each comprising one or two villages, in Eastern Uganda were allocated to receive peer counselling for EBF. Women in their third trimester of pregnancy (based on the last normal menstrual period) were recruited in all 24 clusters and followed up until their children's first birthday. Blood was drawn from 483 infants between 3 and 12 months of age, to test for malaria parasitaemia.
Results
The prevalence of malaria parasitaemia was 11% in the intervention areas and 10% in the control areas. The intervention did not seem to decrease the prevalence of malaria (PR 1.7; 95% CI: 0.9, 3.3). After controlling for potential confounders, infants not supplemented with Vitamin A had a higher prevalence for malaria compared to those who had been supplemented (PR 6.1; 95% CI: 2.1, 17.6). Among children supplemented with vitamin A, every unit increase in length-for-age Z (LAZ) scores was associated with a reduced prevalence in malaria (PR 0.5; 95% CI:0.4, 0.6). There was no association between LAZ scores and malaria among children that had not been supplemented.
Conclusion
Peer counselling for exclusive breastfeeding did not decrease the prevalence of malaria parasitaemia. Children that had not received Vitamin A supplementation had a higher prevalence of malaria compared to children that had been supplemented.
Trial registration
Clinicaltrials.gov: NCT00397150.
doi:10.1371/journal.pone.0021862
PMCID: PMC3131393  PMID: 21760916
18.  Vaccination coverage and timeliness in three South African areas: a prospective study 
BMC Public Health  2011;11:404.
Background
Timely vaccination is important to induce adequate protective immunity. We measured vaccination timeliness and vaccination coverage in three geographical areas in South Africa.
Methods
This study used vaccination information from a community-based cluster-randomized trial promoting exclusive breastfeeding in three South African sites (Paarl in the Western Cape Province, and Umlazi and Rietvlei in KwaZulu-Natal) between 2006 and 2008. Five interview visits were carried out between birth and up to 2 years of age (median follow-up time 18 months), and 1137 children were included in the analysis. We used Kaplan-Meier time-to-event analysis to describe vaccination coverage and timeliness in line with the Expanded Program on Immunization for the first eight vaccines. This included Bacillus Calmette-Guérin (BCG), four oral polio vaccines and 3 doses of the pentavalent vaccine which protects against diphtheria, pertussis, tetanus, hepatitis B and Haemophilus influenzae type B.
Results
The proportion receiving all these eight recommended vaccines were 94% in Paarl (95% confidence interval [CI] 91-96), 62% in Rietvlei (95%CI 54-68) and 88% in Umlazi (95%CI 84-91). Slightly fewer children received all vaccines within the recommended time periods. The situation was worst for the last pentavalent- and oral polio vaccines. The hazard ratio for incomplete vaccination was 7.2 (95%CI 4.7-11) for Rietvlei compared to Paarl.
Conclusions
There were large differences between the different South African sites in terms of vaccination coverage and timeliness, with the poorer areas of Rietvlei performing worse than the better-off areas in Paarl. The vaccination coverage was lower for the vaccines given at an older age. There is a need for continued efforts to improve vaccination coverage and timeliness, in particular in rural areas.
Trial registration number
ClinicalTrials.gov: NCT00397150
doi:10.1186/1471-2458-11-404
PMCID: PMC3126743  PMID: 21619642
19.  Perinatal Mortality in Eastern Uganda: A Community Based Prospective Cohort Study 
PLoS ONE  2011;6(5):e19674.
Background
To achieve a child mortality reduction according to millennium development goal 4, it is necessary to considerably reduce neonatal mortality. We report stillbirth and early neonatal mortality risks as well as determinants of perinatal mortality in Eastern Uganda.
Methods
A community-based prospective cohort study was conducted between 2006 and 2008. A total of 835 pregnant women were followed up for pregnancy outcome and survival of their children until 7 days after delivery. Mother's residence, age, parity, bed net use and whether delivery took place at home were included in multivariable regression analyses to identify risk factors for perinatal death.
Results
The stillbirth risk was 19 per 1,000 pregnancies and the early neonatal death risk 22 per 1,000 live births. Overall, the perinatal mortality risk was 41 [95%CI: 27, 54] per 1,000 pregnancies. Of the deaths, 47% followed complicated deliveries and 24% preterm births. Perinatal mortality was 63/1,000 pregnancies among teenage mothers, 76/1,000 pregnancies among nulliparous women and 61/1,000 pregnancies among women delivering at home who, after controlling for potential confounders, had a 3.7 (95%CI: 1.8, 7.4) times higher perinatal mortality than women who gave birth in a health facility. This association was considerably stronger among nulliparous women [RR 8.0 (95%CI: 2.9, 21.6)] than among women with a previous live birth [RR 1.8 (95%CI: 0.7, 4.5)]. All perinatal deaths occurred among women who did not sleep under a mosquito net. Women living in urban slums had a higher risk of losing their babies than those in rural areas [RR: 2.7 (95%CI: 1.4, 5.3)].
Conclusion
Our findings strengthen arguments for ensuring that pregnant women have access to and use adequate delivery facilities and bed nets.
doi:10.1371/journal.pone.0019674
PMCID: PMC3090412  PMID: 21573019
20.  Ancestral Lineages of Human Enterotoxigenic Escherichia coli▿ †  
Journal of Clinical Microbiology  2010;48(8):2916-2924.
Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among children living in and among travelers visiting developing countries. Human ETEC strains represent an epidemiologically and phenotypically diverse group of pathogens, and there is a need to identify natural groupings of these organisms that may help to explain this diversity. Here, we sought to identify most of the important human ETEC lineages that exist in the E. coli population, because strains that originate from the same lineage may also have inherited many of the same epidemiological and phenotypic traits. We performed multilocus sequence typing (MLST) on 1,019 ETEC isolates obtained from humans in different countries and analyzed the data against a backdrop of MLST data from 1,250 non-ETEC E. coli and eight ETEC isolates from pigs. A total of 42 different lineages were identified, 15 of which, representing 792 (78%) of the strains, were estimated to have emerged >900 years ago. Twenty of the lineages were represented in more than one country. There was evidence of extensive exchange of enterotoxin and colonization factor genes between different lineages. Human and porcine ETEC have probably emerged from the same ancestral ETEC lineage on at least three occasions. Our findings suggest that most ETEC strains circulating in the human population today originate from well-established, globally widespread ETEC lineages. Some of the more important lineages identified here may represent a smaller and more manageable target for the ongoing efforts to develop effective ETEC vaccines.
doi:10.1128/JCM.02432-09
PMCID: PMC2916599  PMID: 20534806
21.  Maternal education is associated with vaccination status of infants less than 6 months in Eastern Uganda: a cohort study 
BMC Pediatrics  2010;10:92.
Background
Despite provision of free childhood vaccinations, less than half of all Ugandan infants are fully vaccinated. This study compares women with some secondary schooling to those with only primary schooling with regard to their infants' vaccination status.
Methods
A community-based prospective cohort study conducted between January 2006 and May 2008 in which 696 pregnant women were followed up to 24 weeks post partum. Information was collected on the mothers' education and vaccination status of the infants.
Results
At 24 weeks, the following vaccinations had been received: bacille Calmette-Guérin (BCG): 92%; polio-1: 91%; Diphteria-Pertussis-Tetanus-Hepatitis B-Haemophilus Influenza b (DPT-HB-Hib) 3 and polio-3: 63%. About 51% of the infants were fully vaccinated (i.e., had received all the scheduled vaccinations: BCG, polio 0, polio 1, DPT-HB-Hib1, polio 2, DPT-HB-Hib 2, polio 3 and DPT-HB-Hib 3). Only 46% of the infants whose mothers' had 5-7 years of primary education had been fully vaccinated compared to 65% of the infants whose mothers' had some secondary education. Infants whose mothers had some secondary education were less likely to miss the DPT-HB-Hib-2 vaccine (RR: 0.5, 95% CI: 0.3, 0.8), Polio-2 (RR: 0.4, 95%CI: 0.3, 0.7), polio-3 (RR: 0.5, 95%CI: 0.4, 0.7) and DPT-HB-Hib-3 (RR: 0.5, 95%CI: 0.4, 0.7). Other factors showing some association with a reduced risk of missed vaccinations were delivery at a health facility (RR = 0.8; 95%CI: 0.7, 1.0) and use of a mosquito net (RR: 0.8; 95%CI: 0.7, 1.0).
Conclusion
Infants whose mothers had a secondary education were at least 50% less likely to miss scheduled vaccinations compared to those whose mothers only had primary education. Strategies for childhood vaccinations should specifically target women with low formal education.
doi:10.1186/1471-2431-10-92
PMCID: PMC3019133  PMID: 21159193
22.  Heat-Stable Enterotoxin of Enterotoxigenic Escherichia coli as a Vaccine Target ▿  
Infection and Immunity  2010;78(5):1824-1831.
Enterotoxigenic Escherichia coli (ETEC) is responsible for 280 million to 400 million episodes of diarrhea and about 380,000 deaths annually. Epidemiological data suggest that ETEC strains which secrete heat-stable toxin (ST), alone or in combination with heat-labile toxin (LT), induce the most severe disease among children in developing countries. This makes ST an attractive target for inclusion in an ETEC vaccine. ST is released upon colonization of the small intestine and activates the guanylate cyclase C receptor, causing profuse diarrhea. To generate a successful toxoid, ST must be made immunogenic and nontoxic. Due to its small size, ST is nonimmunogenic in its natural form but becomes immunogenic when coupled to an appropriate large-molecular-weight carrier. This has been successfully achieved with several carriers, using either chemical conjugation or recombinant fusion techniques. Coupling of ST to a carrier may reduce toxicity, but further reduction by mutagenesis is desired to obtain a safe vaccine. More than 30 ST mutants with effects on toxicity have been reported. Some of these mutants, however, have lost the ability to elicit neutralizing immune responses to the native toxin. Due to the small size of ST, separating toxicity from antigenicity is a particular challenge that must be met. Another obstacle to vaccine development is possible cross-reactivity between anti-ST antibodies and the endogenous ligands guanylin and uroguanylin, caused by structural similarity to ST. Here we review the molecular and biological properties of ST and discuss strategies for developing an ETEC vaccine that incorporates immunogenic and nontoxic derivatives of the ST toxin.
doi:10.1128/IAI.01397-09
PMCID: PMC2863518  PMID: 20231404
23.  Perinatal mortality in rural Burkina Faso: a prospective community-based cohort study 
Background
There is a scarcity of reliable data on perinatal mortality (PNM) in Sub-Saharan Africa. The PROMISE-EBF trial, during which we promoted exclusive breastfeeding, gave us the opportunity to describe the epidemiology of PNM in Banfora Health District, South-West in Burkina Faso.
Study objectives
To measure the perinatal mortality rate (PNMR) in the PROMISE-EBF cohort in Banfora Health District and to identify potential risk factors for perinatal death.
Methods
We used data collected prospectively during the PROMISE-EBF-trial to estimate the stillbirth rate (SBR) and early neonatal mortality rate (ENMR). We used binomial regression with generalized estimating equations to identify potential risk factors for perinatal death.
Results
895 pregnant women were enrolled for data collection in the EBF trial and followed-up to 7 days after birth. The PNMR, the SBR and the ENMR, were 79 per 1000 (95% CI: 59-99), 54 per 1000 (95% CI: 38-69) and 27 per 1000 (95% CI: 9-44), respectively. In a multivariable analysis, nulliparous women (RR = 2.90, 95% CI: 1.6-5.0), primiparae mothers (RR = 2.20, 95% CI: 1.2-3.9), twins (RR = 4.0, 95% CI: 2.3-6.9) and giving birth during the dry season (RR = 2.1 95% CI: 1.3-3.3) were factors associated with increased risk of perinatal death. There was no evidence that risk of perinatal death differed between deliveries at home and at a health centre
Conclusion
Our study observed the highest PNMR ever reported in Burkina. There is an urgent need for sustainable interventions to improve maternal and newborn health in the country.
doi:10.1186/1471-2393-10-45
PMCID: PMC2931454  PMID: 20716352
24.  RNA viruses in community-acquired childhood pneumonia in semi-urban Nepal; a cross-sectional study 
BMC Medicine  2009;7:35.
Background
Pneumonia is among the main causes of illness and death in children <5 years of age. There is a need to better describe the epidemiology of viral community-acquired pneumonia (CAP) in developing countries.
Methods
From July 2004 to June 2007, we examined nasopharyngeal aspirates (NPA) from 2,230 cases of pneumonia (World Health Organization criteria) in children 2 to 35 months old recruited in a randomized trial of zinc supplementation at a field clinic in Bhaktapur, Nepal. The specimens were examined for respiratory syncytial virus (RSV), influenza virus type A (InfA) and B (InfB), parainfluenza virus types 1, 2 and 3 (PIV1, PIV2, and PIV3), and human metapneumovirus (hMPV) using a multiplex reverse transcriptase polymerase chain reaction (PCR) assay.
Results
We identified 919 virus isolates in 887 (40.0%) of the 2,219 NPA specimens with a valid PCR result, of which 334 (15.1%) yielded RSV, 164 (7.4%) InfA, 129 (5.8%) PIV3, 98 (4.4%) PIV1, 93 (4.2%) hMPV, 84 (3.8%) InfB, and 17 (0.8%) PIV2. CAP occurred in an epidemic pattern with substantial temporal variation during the three years of study. The largest peaks of pneumonia occurrence coincided with peaks of RSV infection, which occurred in epidemics during the rainy season and in winter. The monthly number of RSV infections was positively correlated with relative humidity (rs = 0.40, P = 0.01), but not with temperature or rainfall. An hMPV epidemic occurred during one of the three winter seasons and the monthly number of hMPV cases was also associated with relative humidity (rs = 0.55, P = 0.0005).
Conclusion
Respiratory RNA viruses were detected from NPA in 40% of CAP cases in our study. The most commonly isolated viruses were RSV, InfA, and PIV3. RSV infections contributed substantially to the observed CAP epidemics. The occurrence of viral CAP in this community seemed to reflect more or less overlapping micro-epidemics with several respiratory viruses, highlighting the challenges of developing and implementing effective public health control measures.
doi:10.1186/1741-7015-7-35
PMCID: PMC2727531  PMID: 19635124
25.  Clonal Relatedness of Enterotoxigenic Escherichia coli Strains Isolated from a Cohort of Young Children in Guinea-Bissau 
Journal of Clinical Microbiology  2004;42(7):3100-3107.
In a cohort study of 200 young children in Guinea-Bissau, it was previously found that some enterotoxigenic Escherichia coli (ETEC) strains were more pathogenic than others, depending on the type of toxin that they could produce, and that natural ETEC infections induced substantial protection against new infections with ETEC strains that had the same combination of toxins and colonization factors (CFs). We wanted to describe the clonal relatedness of the ETEC strains isolated during this study and to investigate whether the protective antigens and the virulence factors that were responsible for the pathogenic traits were common to strains that were clonally closely related or whether they were more likely to be encoded by the ETEC virulence plasmids that normally encode the toxins and the CFs. By performing repetitive sequence-based PCR analysis of strains representing 452 infections, we found that strains that had the same toxin-CF profile were usually closely related, although a few were unrelated. Strains that did not have the same toxin-CF profiles but that were positive for a given toxin or for a given CF were not consistently more closely related to each other than to strains that were negative for the same toxin or CF. Our results indicate that the pathogenic traits of ETEC were mainly attributed to genes carried on the ETEC virulence plasmids. Because most strains that had the same toxin-CF profile were closely related, it seems likely that the toxin-CF-specific protection was clonal and was not targeting antigens encoded by the virulence plasmids. These results are of relevance to the ETEC vaccine development effort.
doi:10.1128/JCM.42.7.3100-3107.2004
PMCID: PMC446293  PMID: 15243067

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