Characteristics of Salmonella Recovered From Stools of Children Enrolled in the Global Enteric Multicenter Study.

BACKGROUND: The Global Enteric Multicenter Study (GEMS) determined the etiologic agents of moderate-to-severe diarrhea (MSD) in children under 5 years old in Africa and Asia. Here, we describe the prevalence and antimicrobial susceptibility of nontyphoidal Salmonella (NTS) serovars in GEMS and examine the phylogenetics of Salmonella Typhimurium ST313 isolates. METHODS: Salmonella isolated from children with MSD or diarrhea-free controls were identified by classical clinical microbiology and serotyped using antisera and/or whole-genome sequence data. We evaluated antimicrobial susceptibility using the Kirby-Bauer disk-diffusion method. Salmonella Typhimurium sequence types were determined using multi-locus sequence typing, and whole-genome sequencing was performed to assess the phylogeny of ST313. RESULTS: Of 370 Salmonella-positive individuals, 190 (51.4%) were MSD cases and 180 (48.6%) were diarrhea-free controls. The most frequent Salmonella serovars identified were Salmonella Typhimurium, serogroup O:8 (C2-C3), serogroup O:6,7 (C1), Salmonella Paratyphi B Java, and serogroup O:4 (B). The prevalence of NTS was low but similar across sites, regardless of age, and was similar among both cases and controls except in Kenya, where Salmonella Typhimurium was more commonly associated with cases than controls. Phylogenetic analysis showed that these Salmonella Typhimurium isolates, all ST313, were highly genetically related to isolates from controls. Generally, Salmonella isolates from Asia were resistant to ciprofloxacin and ceftriaxone, but African isolates were susceptible to these antibiotics. CONCLUSIONS: Our data confirm that NTS is prevalent, albeit at low levels, in Africa and South Asia. Our findings provide further evidence that multidrug-resistant Salmonella Typhimurium ST313 can be carried asymptomatically by humans in sub-Saharan Africa.

The Clinical Presentation of Culture-positive and Culture-negative, Quantitative Polymerase Chain Reaction (qPCR)-Attributable Shigellosis in the Global Enteric Multicenter Study and Derivation of a Shigella Severity Score: Implications for Pediatric Shigella Vaccine Trials.

BACKGROUND: Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. METHODS: We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score. RESULTS: Compared to culture-positive Shigella MSD cases (n = 745), culture-negative/qPCR-attributable Shigella cases (n = 852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There was no difference in dehydration, hospitalization, or severe classification from a modified Vesikari score. Twenty-two (1.8%) Shigella MSD cases died within the 14-days after presentation to health facilities, and 59.1% of these deaths were in culture-negative cases. Age <12 months, diarrhea duration prior to presentation, vomiting, stunting, wasting, and hospitalization were associated with mortality. A model-derived score assigned points for dehydration, hospital admission, and longer diarrhea duration but was not significantly better at predicting 14-day mortality than a modified Vesikari score. CONCLUSIONS: A composite severity score consistent with severe disease or dysentery may be a pragmatic clinical endpoint for severe shigellosis in vaccine trials. Reliance on culture for microbiologic confirmation may miss a substantial number of Shigella cases but is currently required to measure serotype specific immunity.

Terpyridine derivatives as "turn-on" fluorescence chemosensors for the selective and sensitive detection of Zn2+ ions in solution and in live cells.

A terpyridine based compound L1 was designed and synthesized as an "off-on" chemosensor for the detection of Zn2+. Chemosensor L1 showed excellent selectivity and sensitivity toward Zn2+ by exhibiting a large fluorescence enhancement (∼51-fold) at 370 nm whereas other competitive metal ions did not show any noticeable change in the emission spectra of chemosensor L1. The chemosensor (L1) was shown to detect Zn2+ ions down to 9.76 µM at pH 7.4. However, chemosensor L1 binds Zn2+ in a 1 : 2 ratio (receptor : metal) with an association constant of 1.85 × 104 (R2 = 0.993) and this 1 : 2 stoichiometric fashion is established on the basis of a Job plot and mass spectroscopy. DFT/TD-DFT calculations were carried out to understand the binding nature, coordination features and electronic properties of L1 and the L1-2Zn2+ complex. In addition, this "turn-on" fluorescence probe was effectively used to image intracellular Zn2+ ions in cultured MDA-MB-468 cells.

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study.

BACKGROUND: Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). METHODS: GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. FINDINGS: We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2-96·0) at the population level, compared with 51·5% (48·0-55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6-80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. INTERPRETATION: A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. FUNDING: Bill & Melinda Gates Foundation.

Shigella isolates from the global enteric multicenter study inform vaccine development.

BACKGROUND: Shigella, a major diarrheal disease pathogen worldwide, is the target of vaccine development. The Global Enteric Multicenter Study (GEMS) investigated burden and etiology of moderate-to-severe diarrheal disease in children aged <60 months and matched controls without diarrhea during 3 years at 4 sites in Africa and 3 in Asia. Shigella was 1 of the 4 most common pathogens across sites and age strata. GEMS Shigella serotypes are reviewed to guide vaccine development. METHODS: Subjects' stool specimens/rectal swabs were transported to site laboratories in transport media and plated onto xylose lysine desoxycholate and MacConkey agar. Suspect Shigella colonies were identified by biochemical tests and agglutination with antisera. Shigella isolates were shipped to the GEMS Reference Laboratory (Baltimore, MD) for confirmation and serotyping of S. flexneri; one-third of isolates were sent to the Centers for Disease Control and Prevention for quality control. RESULTS: Shigella dysenteriae and S. boydii accounted for 5.0% and 5.4%, respectively, of 1130 Shigella case isolates; S. flexneri comprised 65.9% and S. sonnei 23.7%. Five serotypes/subserotypes comprised 89.4% of S. flexneri, including S. flexneri 2a, S. flexneri 6, S. flexneri 3a, S. flexneri 2b, and S. flexneri 1b. CONCLUSIONS: A broad-spectrum Shigella vaccine must protect against S. sonnei and 15 S. flexneri serotypes/subserotypes. A quadrivalent vaccine with O antigens from S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 can provide broad direct coverage against these most common serotypes and indirect coverage against all but 1 (rare) remaining subserotype through shared S. flexneri group antigens.

Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study.

BACKGROUND: Diarrhoeal diseases cause illness and death among children younger than 5 years in low-income countries. We designed the Global Enteric Multicenter Study (GEMS) to identify the aetiology and population-based burden of paediatric diarrhoeal disease in sub-Saharan Africa and south Asia. METHODS: The GEMS is a 3-year, prospective, age-stratified, matched case-control study of moderate-to-severe diarrhoea in children aged 0-59 months residing in censused populations at four sites in Africa and three in Asia. We recruited children with moderate-to-severe diarrhoea seeking care at health centres along with one to three randomly selected matched community control children without diarrhoea. From patients with moderate-to-severe diarrhoea and controls, we obtained clinical and epidemiological data, anthropometric measurements, and a faecal sample to identify enteropathogens at enrolment; one follow-up home visit was made about 60 days later to ascertain vital status, clinical outcome, and interval growth. FINDINGS: We enrolled 9439 children with moderate-to-severe diarrhoea and 13,129 control children without diarrhoea. By analysing adjusted population attributable fractions, most attributable cases of moderate-to-severe diarrhoea were due to four pathogens: rotavirus, Cryptosporidium, enterotoxigenic Escherichia coli producing heat-stable toxin (ST-ETEC; with or without co-expression of heat-labile enterotoxin), and Shigella. Other pathogens were important in selected sites (eg, Aeromonas, Vibrio cholerae O1, Campylobacter jejuni). Odds of dying during follow-up were 8·5-fold higher in patients with moderate-to-severe diarrhoea than in controls (odd ratio 8·5, 95% CI 5·8-12·5, p<0·0001); most deaths (167 [87·9%]) occurred during the first 2 years of life. Pathogens associated with increased risk of case death were ST-ETEC (hazard ratio [HR] 1·9; 0·99-3·5) and typical enteropathogenic E coli (HR 2·6; 1·6-4·1) in infants aged 0-11 months, and Cryptosporidium (HR 2·3; 1·3-4·3) in toddlers aged 12-23 months. INTERPRETATION: Interventions targeting five pathogens (rotavirus, Shigella, ST-ETEC, Cryptosporidium, typical enteropathogenic E coli) can substantially reduce the burden of moderate-to-severe diarrhoea. New methods and accelerated implementation of existing interventions (rotavirus vaccine and zinc) are needed to prevent disease and improve outcomes. FUNDING: The Bill & Melinda Gates Foundation.

The challenges and successes of implementing a sustainable antimicrobial resistance surveillance programme in Nepal.

BACKGROUND: Antimicrobial resistance (AMR) is a major global public health concern and its surveillance is a fundamental tool for monitoring the development of AMR. In 1998, the Nepalese Ministry of Health (MOH) launched an Infectious Disease (ID) programme. The key components of the programme were to establish a surveillance programme for AMR and to develop awareness among physicians regarding AMR and rational drug usage in Nepal. METHODS: An AMR surveillance programme was established and implemented by the Nepalese MOH in partnership with the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) from 1998 to 2003. From 2004 to 2012, the programme was integrated and maintained as a core activity of the National Public Health Laboratory (NPHL) and resulted in an increased number of participating laboratories and pathogens brought under surveillance. The main strategies were to build national capacity on isolation, identification and AMR testing of bacterial pathogens, establish laboratory networking and an External Quality Assessment (EQA) programme, promote standardised recording and reporting of results, and to ensure timely analysis and dissemination of data for advocacy and national policy adaptations. The programme was initiated by nine participating laboratories performing AMR surveillance on Vibrio cholerae, Shigella spp., Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. RESULTS: The number of participating laboratories was ultimately increased to 13 and the number of pathogens under surveillance was increased to seven (Salmonella spp. was added to the surveillance programme in 2002 and extended spectrum ß-lactamase producing Escherichia coli in 2011). From 1999 to 2012, data were available on 17,103 bacterial isolates. During the AMR programme, we observed changing trends in serovars/species for Salmonella spp., Shigella spp. and V. cholerae and changing AMR trend for all organisms. Notably, N. gonorrhoeae isolates demonstrated increasing resistance to ciprofloxacin. Additionally, the performance of the participating laboratories improved as shown by annual EQA data evaluation. CONCLUSIONS: This Nepalese AMR programme continues and serves as a model for sustainable surveillance of AMR monitoring in resource limited settings.

Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.

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 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥ 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) 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 × 10(4) 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.

Diagnostic microbiologic methods in the GEMS-1 case/control study.

To understand the etiology of moderate-to-severe diarrhea among children in high mortality areas of sub-Saharan Africa and South Asia, we performed a comprehensive case/control study of children aged <5 years at 7 sites. Each site employed an identical case/control study design and each utilized a uniform comprehensive set of microbiological assays to identify the likely bacterial, viral and protozoal etiologies. The selected assays effected a balanced consideration of cost, robustness and performance, and all assays were performed at the study sites. Identification of bacterial pathogens employed streamlined conventional bacteriologic biochemical and serological algorithms. Diarrheagenic Escherichia coli were identified by application of a multiplex polymerase chain reaction assay for enterotoxigenic, enteroaggregative, and enteropathogenic E. coli. Rotavirus, adenovirus, Entamoeba histolytica, Giardia enterica, and Cryptosporidium species were detected by commercially available enzyme immunoassays on stool samples. Samples positive for adenovirus were further evaluated for adenovirus serotypes 40 and 41. We developed a novel multiplex assay to detect norovirus (types 1 and 2), astrovirus, and sapovirus. The portfolio of diagnostic assays used in the GEMS study can be broadly applied in developing countries seeking robust cost-effective methods for enteric pathogen detection.

Epidemiology and risk factors of incident hepatitis E virus infections in rural Bangladesh.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis in the world. Most of South Asia is HEV endemic, with frequent seasonal epidemics of hepatitis E and continuous sporadic cases. This author group's epidemiologic work and clinical reports suggest that Bangladesh is HEV endemic, but there have been few population-based studies of this country's HEV burden. The authors calculated HEV infection rates, over an 18-month interval between 2003 and 2005, by following a randomly selected cohort of 1,134 subjects between the ages of 1 and 88 years, representative of rural communities in southern Bangladesh. Baseline prevalence of antibody to hepatitis E virus (anti-HEV) was 22.5%. Seroincidence was 60.3 per 1,000 person-years during the first 12 months and 72.4 per 1,000 person-years from >12 to 18 months (during the monsoon season), peaking by age 50 years and with low rates during childhood. Few of the seroconverting subjects reported hepatitis-like illness. Overall incidence was calculated to be 64 per 1,000 person-years, with 1,172 person-years followed. No significant associations were found between anti-HEV incidence and demographic or socioeconomic factors for which data were available. This is the first study to document annual HEV infection rates among "healthy" and very young to elderly subjects in a rural Bangladeshi population.

Diarrhoeal disease and subsequent risk of death in infants and children residing in low-income and middle-income countries: analysis of the GEMS case-control study and 12-month GEMS-1A follow-on study.

BACKGROUND: The Global Enteric Multicenter Study (GEMS) was a 3-year case-control study that measured the burden, aetiology, and consequences of moderate-to-severe diarrhoea (MSD) in children aged 0-59 months. GEMS-1A, a 12-month follow-on study, comprised two parallel case-control studies, one assessing MSD and the other less-severe diarrhoea (LSD). In this report, we analyse the risk of death with each diarrhoea type and the specific pathogens associated with fatal outcomes. METHODS: GEMS was a prospective, age-stratified, matched case-control study done at seven sites in Africa and Asia. Children aged 0-59 months with MSD seeking care at sentinel health centres were recruited along with one to three randomly selected matched community control children without diarrhoea. In the 12-month GEMS-1A follow-on study, children with LSD and matched controls, in addition to children with MSD and matched controls, were recruited at six of the seven sites; only cases of MSD and controls were enrolled at the seventh site. We compared risk of death during the period between enrolment and one follow-up household visit done about 60 days later (range 50-90 days) in children with MSD and LSD and in their respective controls. Approximately 50 pathogens were detected using, as appropriate, classic bacteriology, immunoassays, gel-based PCR and reverse transcriptase PCR, and quantitative real-time PCR (qPCR). Specimens from a subset of GEMS cases and controls were also tested by a TaqMan Array Card that compartmentalised probe-based qPCR for 32 enteropathogens. FINDINGS: 223 (2·0%) of 11 108 children with MSD and 43 (0·3%) of 16 369 matched controls died between study enrolment and the follow-up visit at about 60 days (hazard ratio [HR] 8·16, 95% CI 5·69-11·68, p<0·0001). 12 (0·4%) of 2962 children with LSD and seven (0·2%) of 4074 matched controls died during the follow-up period (HR 2·78, 95% CI 0·95-8·11, p=0·061). Risk of death was lower in children with dysenteric MSD than in children with non-dysenteric MSD (HR 0·20, 95% CI 0·05-0·87, p=0·032), and lower in children with LSD than in those with non-dysenteric MSD (HR 0·29, 0·14-0·59, p=0·0006). In children younger than 24 months with MSD, infection with typical enteropathogenic Escherichia coli, enterotoxigenic E coli encoding heat-stable toxin, enteroaggregative E coli, Shigella spp (non-dysentery cases), Aeromonas spp, Cryptosporidium spp, and Entamoeba histolytica increased risk of death. Of 61 deaths in children aged 12-59 months with non-dysenteric MSD, 31 occurred among 942 children qPCR-positive for Shigella spp and 30 deaths occurred in 1384 qPCR-negative children (HR 2·2, 95% CI 1·2-3·9, p=0·0090), showing that Shigella was strongly associated with increased risk of death. INTERPRETATION: Risk of death is increased following MSD and, to a lesser extent, LSD. Considering there are approximately three times more cases of LSD than MSD in the population, more deaths are expected among children with LSD than in those with MSD. Because the major attributable LSD-associated and MSD-associated pathogens are the same, implementing vaccines and rapid diagnosis and treatment interventions against these major pathogens are rational investments. FUNDING: Bill & Melinda Gates Foundation.

The incidence, aetiology, and adverse clinical consequences of less severe diarrhoeal episodes among infants and children residing in low-income and middle-income countries: a 12-month case-control study as a follow-on to the Global Enteric Multicenter Study (GEMS).

BACKGROUND: Diarrheal diseases remain a leading cause of illness and death among children younger than 5 years in low-income and middle-income countries. The Global Enteric Multicenter Study (GEMS) has described the incidence, aetiology, and sequelae of medically attended moderate-to-severe diarrhoea (MSD) among children aged 0-59 months residing in censused populations in sub-Saharan Africa and south Asia, where most child deaths occur. To further characterise this disease burden and guide interventions, we extended this study to include children with episodes of less-severe diarrhoea (LSD) seeking care at health centres serving six GEMS sites. METHODS: We report a 1-year, multisite, age-stratified, matched case-control study following on to the GEMS study. Six sites (Bamako, Mali; Manhiça, Mozambique; Basse, The Gambia; Mirzapur, Bangladesh; Kolkata, India; and Bin Qasim Town, Karachi, Pakistan) participated in this study. Children aged 0-59 months at each site who sought care at a sentinel hospital or health centre during a 12-month period were screened for diarrhoea. New (onset after ≥7 diarrhoea-free days) and acute (onset within the previous 7 days) episodes of diarrhoea in children who had sunken eyes, whose skin lost turgor, who received intravenous hydration, who had dysentery, or who were hospitalised were eligible for inclusion as MSD. The remaining new and acute diarrhoea episodes among children who sought care at the same health centres were considered LSD. We aimed to enrol the first eight or nine eligible children with MSD and LSD at each site during each fortnight in three age strata: infants (aged 0-11 months), toddlers (aged 12-23 months), and young children (aged 24-59 months). For each included case of MSD or LSD, we enrolled one to three community control children without diarrhoea during the previous 7 days. From patients and controls we collected clinical and epidemiological data, anthropometric measurements, and faecal samples to identify enteropathogens at enrolment, and we performed a follow-up home visit about 60 days later to ascertain vital status, clinical outcome, and interval growth. Primary outcomes were to characterise, for MSD and LSD, the pathogen-specific attributable risk and population-based incidence values, and to assess the frequency of adverse clinical consequences associated with these two diarrhoeal syndromes. FINDINGS: From Oct 31, 2011, to Nov 14, 2012, we recruited 2368 children with MSD, 3174 with LSD, and one to three randomly selected community control children without diarrhoea matched to cases with MSD (n=3597) or LSD (n=4236). Weighted adjusted population attributable fractions showed that most attributable cases of MSD and LSD were due to rotavirus, Cryptosporidium spp, enterotoxigenic Escherichia coli encoding heat-stable toxin (with or without genes encoding heat-labile enterotoxin), and Shigella spp. The attributable incidence per 100 child-years for LSD versus MSD, by age stratum, for rotavirus was 22·3 versus 5·5 (0-11 months), 9·8 versus 2·9 (12-23 months), and 0·5 versus 0·2 (24-59 months); for Cryptosporidium spp was 3·6 versus 2·3 (0-11 months), 4·3 versus 0·6 (12-23 months), and 0·3 versus 0·1 (24-59 months); for enterotoxigenic E coli encoding heat-stable toxin was 4·2 versus 0·1 (0-11 months), 5·2 versus 0·0 (12-23 months), and 1·1 versus 0·2 (24-59 months); and for Shigella spp was 1·0 versus 1·3 (0-11 months), 3·1 versus 2·4 (12-23 months), and 0·8 versus 0·7 (24-59 months). Participants with both MSD and LSD had significantly more linear growth faltering than controls at follow-up. INTERPRETATION: Inclusion of participants with LSD markedly expands the population of children who experience adverse clinical and nutritional outcomes from acute diarrhoeal diseases. Since MSD and LSD have similar aetiologies, interventions targeting rotavirus, Shigella spp, enterotoxigenic E coli producing heat-stable toxin, and Cryptosporidium spp might substantially reduce the diarrhoeal disease burden and its associated nutritional faltering. FUNDING: Bill & Melinda Gates Foundation.

Colonization factors among enterotoxigenic Escherichia coli isolates from children with moderate-to-severe diarrhea and from matched controls in the Global Enteric Multicenter Study (GEMS).

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) encoding heat-stable enterotoxin (ST) alone or with heat-labile enterotoxin (LT) cause moderate-to-severe diarrhea (MSD) in developing country children. The Global Enteric Multicenter Study (GEMS) identified ETEC encoding ST among the top four enteropathogens. Since the GEMS objective was to provide evidence to guide development and implementation of enteric vaccines and other interventions to diminish diarrheal disease morbidity and mortality, we examined colonization factor (CF) prevalence among ETEC isolates from children age <5 years with MSD and from matched controls in four African and three Asian sites. We also assessed strength of association of specific CFs with MSD. METHODOLOGY/PRINCIPAL FINDINGS: MSD cases enrolled at healthcare facilities over three years and matched controls were tested in a standardized manner for many enteropathogens. To identify ETEC, three E. coli colonies per child were tested by polymerase chain reaction (PCR) to detect genes encoding LT, ST; confirmed ETEC were examined by PCR for major CFs (Colonization Factor Antigen I [CFA/I] or Coli Surface [CS] antigens CS1-CS6) and minor CFs (CS7, CS12, CS13, CS14, CS17, CS18, CS19, CS20, CS21, CS30). ETEC from 806 cases had a single toxin/CF profile in three tested strains per child. Major CFs, components of multiple ETEC vaccine candidates, were detected in 66.0% of LT/ST and ST-only cases and were associated with MSD versus matched controls by conditional logistic regression (p≤0.006); major CFs detected in only 25.0% of LT-only cases weren't associated with MSD. ETEC encoding exclusively CS14, identified among 19.9% of 291 ST-only and 1.5% of 259 LT/ST strains, were associated with MSD (p = 0.0011). No other minor CF exhibited prevalence ≥5% and significant association with MSD. CONCLUSIONS/SIGNIFICANCE: Major CF-based efficacious ETEC vaccines could potentially prevent up to 66% of pediatric MSD cases due to ST-encoding ETEC in developing countries; adding CS14 extends coverage to ~77%.

Coordination Polymers Based on Phthalic Acid and Aminopyrazine Ligands: On the Importance of N⁻H···π Interactions.

Two new Co(II) and Cu(II) coordination polymers, {Co(HL1)2(µ-L2)(H2O)2}n (1) and {[Cu(HL1)2(µ-L2)H2O]·H2O}n (2) (H2L1 = Phthalic acid and L2 = 2-aminopyrazine), have been synthesized by slow evaporation of solvent and characterized by IR spectroscopic, elemental, single-crystal X-ray diffraction and thermal analysis. X-ray results indicate that in both the polymers, phthalate acts as a monodentate ligand and the aminopyrazine ligand is responsible for the formation of the infinite one-dimensional chain structure. The solid-state structures are stabilized through hydrogen bonds and N‒H···π interactions by generating two-dimensional layered structures. Finally, the non-covalent interactions have been studied energetically and using Bader's theory of atoms in molecules by means of Density Functional Theory (DFT) calculations. The influence of the metal coordination on the strength of the interaction has been studied using molecular electrostatic potential surface calculations.

Enhanced Photosensitive Schottky Diode Behavior of Pyrazine over 2-Aminopyrimidine Ligand in Copper(II)-Phthalate MOFs: Experimental and Theoretical Rationalization.

Two novel Cu(II)-based metal-organic frameworks [C40H34Cu2N6O18 (1) and C20H18CuN2O10 (2)] have been synthesized using 2-aminopyrimidine or pyrazine ligands and phthalate ion and characterized spectroscopically and by X-ray single-crystal diffraction. Both 1 and 2 show electrical conductivity and photosensitivity, evidencing their potentiality in optoelectronic device applications. Experimental and theoretical investigations revealed that the electrical conductivity under irradiation of visible light increases compared to that under dark condition (photosensitive Schottky barrier diode behavior), especially in complex 2. Both 1 and 2 have been successfully applied in technologically challenging thin-film active devices.

A multicentre study of Shigella diarrhoea in six Asian countries: disease burden, clinical manifestations, and microbiology.

BACKGROUND: The burden of shigellosis is greatest in resource-poor countries. Although this diarrheal disease has been thought to cause considerable morbidity and mortality in excess of 1,000,000 deaths globally per year, little recent data are available to guide intervention strategies in Asia. We conducted a prospective, population-based study in six Asian countries to gain a better understanding of the current disease burden, clinical manifestations, and microbiology of shigellosis in Asia. METHODS AND FINDINGS: Over 600,000 persons of all ages residing in Bangladesh, China, Pakistan, Indonesia, Vietnam, and Thailand were included in the surveillance. Shigella was isolated from 2,927 (5%) of 56,958 diarrhoea episodes detected between 2000 and 2004. The overall incidence of treated shigellosis was 2.1 episodes per 1,000 residents per year in all ages and 13.2/1,000/y in children under 60 months old. Shigellosis incidence increased after age 40 years. S. flexneri was the most frequently isolated Shigella species (1,976/2,927 [68%]) in all sites except in Thailand, where S. sonnei was most frequently detected (124/146 [85%]). S. flexneri serotypes were highly heterogeneous in their distribution from site to site, and even from year to year. PCR detected ipaH, the gene encoding invasion plasmid antigen H in 33% of a sample of culture-negative stool specimens. The majority of S. flexneri isolates in each site were resistant to amoxicillin and cotrimoxazole. Ciprofloxacin-resistant S. flexneri isolates were identified in China (18/305 [6%]), Pakistan (8/242 [3%]), and Vietnam (5/282 [2%]). CONCLUSIONS: Shigella appears to be more ubiquitous in Asian impoverished populations than previously thought, and antibiotic-resistant strains of different species and serotypes have emerged. Focusing on prevention of shigellosis could exert an immediate benefit first by substantially reducing the overall diarrhoea burden in the region and second by preventing the spread of panresistant Shigella strains. The heterogeneous distribution of Shigella species and serotypes suggest that multivalent or cross-protective Shigella vaccines will be needed to prevent shigellosis in Asia.

Genome diversity of Shigella boydii.

ITALIC! Shigella boydiiis one of the four ITALIC! Shigellaspecies that causes disease worldwide; however, there are few published studies that examine the genomic variation of this species. This study compares genomes of 72 total isolates; 28 ITALIC! S. boydiifrom Bangladesh and The Gambia that were recently isolated as part of the Global Enteric Multicenter Study (GEMS), 14 historical ITALIC! S. boydiigenomes in the public domain and 30 ITALIC! Escherichia coliand ITALIC! Shigellareference genomes that represent the genomic diversity of these pathogens. This comparative analysis of these 72 genomes identified that the ITALIC! S. boydiiisolates separate into three phylogenomic clades, each with specific gene content. Each of the clades contains ITALIC! S. boydiiisolates from geographic and temporally distant sources, indicating that the ITALIC! S. boydiiisolates from the GEMS are representative of ITALIC! S. boydii.This study describes the genome sequences of a collection of novel ITALIC! S. boydiiisolates and provides insight into the diversity of this species in comparison to the ITALIC! E. coliand other ITALIC! Shigellaspecies.

Genomic diversity of EPEC associated with clinical presentations of differing severity.

Enteropathogenic Escherichia coli (EPEC) are diarrhoeagenic E. coli, and are a significant cause of gastrointestinal illness among young children in developing countries. Typical EPEC are identified by the presence of the bundle-forming pilus encoded by a virulence plasmid, which has been linked to an increased severity of illness, while atypical EPEC lack this feature. Comparative genomics of 70 total EPEC from lethal (LI), non-lethal symptomatic (NSI) or asymptomatic (AI) cases of diarrhoeal illness in children enrolled in the Global Enteric Multicenter Study was used to investigate the genomic differences in EPEC isolates obtained from individuals with various clinical outcomes. A comparison of the genomes of isolates from different clinical outcomes identified genes that were significantly more prevalent in EPEC isolates of symptomatic and lethal outcomes than in EPEC isolates of asymptomatic outcomes. These EPEC isolates exhibited previously unappreciated phylogenomic diversity and combinations of virulence factors. These comparative results highlight the diversity of the pathogen, as well as the complexity of the EPEC virulence factor repertoire.

Evolution of atypical enteropathogenic E. coli by repeated acquisition of LEE pathogenicity island variants.

Atypical enteropathogenic Escherichia coli (aEPEC) is an umbrella term given to E. coli that possess a type III secretion system encoded in the locus of enterocyte effacement (LEE), but lack the virulence factors (stx, bfpA) that characterize enterohaemorrhagic E. coli and typical EPEC, respectively. The burden of disease caused by aEPEC has recently increased in industrialized and developing nations, yet the population structure and virulence profile of this emerging pathogen are poorly understood. Here, we generated whole-genome sequences of 185 aEPEC isolates collected during the Global Enteric Multicenter Study from seven study sites in Asia and Africa, and compared them with publicly available E. coli genomes. Phylogenomic analysis revealed ten distinct widely distributed aEPEC clones. Analysis of genetic variation in the LEE pathogenicity island identified 30 distinct LEE subtypes divided into three major lineages. Each LEE lineage demonstrated a preferred chromosomal insertion site and different complements of non-LEE encoded effector genes, indicating distinct patterns of evolution of these lineages. This study provides the first detailed genomic framework for aEPEC in the context of the EPEC pathotype and will facilitate further studies into the epidemiology and pathogenicity of EPEC by enabling the detection and tracking of specific clones and LEE variants.

Aeromonas-Associated Diarrhea in Children Under 5 Years: The GEMS Experience.

We report the clinical findings, epidemiology, and risk factors for moderate-to-severe diarrhea (MSD) associated with Aeromonas species in children 0-59 months of age, from the Global Enteric Multicenter Study, conducted at three sites in south Asia and four sites in sub-Saharan Africa. Children with MSD were enrolled along with controls matched for age, gender, and neighborhood. Pooled, age-stratified conditional logistic regression models were applied to evaluate the association of Aeromonas infection controlling for coinfecting pathogens and sociodemographic variables. A pooled, age-stratified, multivariate logistic regression analysis was done to identify risk factors associated with Aeromonas positivity in MSD cases. A total of 12,110 cases and 17,291 matched controls were enrolled over a period of 48 months. Aeromonas was identified as a significant pathogen in 736 cases of MSD in Pakistan and Bangladesh (22.2%). Aeromonas remained a significant pathogen even after adjustment for the presence of other pathogens and sociodemographic factors. Odds ratio (OR) for Aeromonas were higher in the presence of Shigella (matched OR: 6.2, 95% confidence interval [CI]: 1.9-20.2). Cases of Aeromonas were likely to present with dysentery, particularly in the 0-11 months (OR: 1.4, 95% CI 1.0-2.0) and 12-23 months (OR: 1.8, 95% CI: 1.3-2.5) age group. The odds of Aeromonas increased with increasing degree of stunting, being highest for severe stunting (OR: 10.1, 95% CI: 3.6-28.9). Aeromonas is a significant pathogen for MSD in Pakistan and Bangladesh. Presence of dysentery and co-occurrence with other pathogens, notably Shigella spp. are significant features of Aeromonas-associated diarrhea.