Pathogens Associated With Linear Growth Faltering in Children With Diarrhea and Impact of Antibiotic Treatment: The Global Enteric Multicenter Study.

BACKGROUND: The association between childhood diarrheal disease and linear growth faltering in developing countries is well described. However, the impact attributed to specific pathogens has not been elucidated, nor has the impact of recommended antibiotic treatment. METHODS: The Global Enteric Multicenter Study enrolled children with moderate to severe diarrhea (MSD) seeking healthcare at 7 sites in sub-Saharan Africa and South Asia. At enrollment, we collected stool samples to identify enteropathogens. Length/height was measured at enrollment and follow-up, approximately 60 days later, to calculate change in height-for-age z scores (ΔHAZ). The association of pathogens with ΔHAZ was tested using linear mixed effects regression models. RESULTS: Among 8077 MSD cases analyzed, the proportion with stunting (HAZ below -1) increased from 59% at enrollment to 65% at follow-up (P < .0001). Pathogens significantly associated with linear growth decline included Cryptosporidium (P < .001), typical enteropathogenic Escherichia coli (P = .01), and untreated Shigella (P = .009) among infants (aged 0-11 months) and enterotoxigenic E. coli encoding heat-stable toxin (P < .001) and Cryptosporidium (P = .03) among toddlers (aged 12-23 months). Shigella-infected toddlers given antibiotics had improved linear growth (P = .02). CONCLUSIONS: Linear growth faltering among children aged 0-23 months with MSD is associated with specific pathogens and can be mitigated with targeted treatment strategies, as demonstrated for Shigella.

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.

Detection of typhoid carriers by duodenal fluid culture in a tertiary care hospital, Karachi: A cross-sectional study.

We aimed to detect typhoid carriers by performing duodenal fluid culture in patients in a tertiary care hospital in Pakistan. A cross-sectional study was conducted during 2017 at the Aga Khan University Hospital, Karachi. Patients who underwent upper gastrointestinal endoscopy were included. Participants were interviewed, and duodenal fluid samples were taken for culture to detect Salmonella typhi (S. typhi) and paratyphi. A polymerase chain reaction on 100 randomly selected sub-samples was also conducted. A total of 477 participants were enrolled. The mean age was 42.4±15.5 years. History of typhoid fever was present in 73 (15.3%) participants. Out of the 477 duodenal fluid cultures tested for various micro-organisms, 250 (52.4%) were positive. Neither S. typhi nor paratyphi were isolated. S. typhi was also not detected by PCR. To better detect S. typhi carriage in general population, future studies should target people with gall bladder diseases and screen them using culture and PCR based methods.

Ceftriaxone-resistant Salmonella Typhi Outbreak in Hyderabad City of Sindh, Pakistan: High Time for the Introduction of Typhoid Conjugate Vaccine.

BACKGROUND: The Aga Khan University clinical microbiology laboratory identified an outbreak of ceftriaxone-resistant Salmonella Typhi in Hyderabad, Pakistan, through antimicrobial resistance surveillance. An outbreak investigation was carried out to identify the risk factors and institute control measures. Here we report the preliminary findings of this outbreak investigation, using data collected from 30 November 2016 to 28 March 2017. METHODS: The design for the investigation was a case-control study that included identification of culture-proven ceftriaxone-resistant S. Typhi cases, suspected cases from the households or neighborhood of the confirmed cases, and enrollment of controls matched by age to identify the risk factors. Data were collected through face-to-face interviews using a structured questionnaire. Blood cultures were obtained from all suspected cases. Drinking water samples from each household of cases and controls were obtained for microbiological testing. Geographic Information System coordinates were obtained for all cases and controls. RESULTS: Only 2 subdistricts of Hyderabad (Latifabad and Qasimabad) were affected. A total of 101 confirmed cases of ceftriaxone-resistant S. Typhi had been reported in 4 months with the first case reported on 30 November 2016. Median age was 48 (interquartile range, 29-84) months. The majority (60% [61/101]) of the cases were 6-60 months old. More than half (56% [57/101]) of the cases were male. About 60% of the cases were admitted to hospital and treated as inpatient. More than half (57/101) of the patients developed complications related to typhoid. CONCLUSIONS: Community awareness was raised regarding chlorination of drinking water and sanitation measures in Hyderabad. These efforts were coordinated with the municipal water and sewage authority established to improve chlorination at processing plants and operationalize fecal sludge treatment plants. Outbreak investigation and control efforts have continued. Immunization of children with typhoid conjugate vaccine within Hyderabad city is planned.

Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation.

BACKGROUND: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. DISCUSSION: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. TRIAL REGISTRATION: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Direct Detection of Shigella in Stool Specimens by Use of a Metagenomic Approach.

The underestimation of Shigella species as a cause of childhood diarrhea disease has become increasingly apparent with quantitative PCR (qPCR)-based diagnostic methods versus culture. We sought to confirm qPCR-based detection of Shigella via a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: nine Shigella culture-positive and qPCR-positive (culture+ qPCR+) samples, nine culture-negative but qPCR-positive (culture- qPCR+) samples, and nine culture-negative and qPCR-negative (culture- qPCR-) samples. Fecal DNA was sequenced using paired-end Illumina HiSeq, whereby 3.26 × 108 ± 5.6 × 107 high-quality reads were generated for each sample. We used Kraken software to compare the read counts specific to "Shigella" among the three groups. The proportions of Shigella-specific nonhuman sequence reads between culture+ qPCR+ (0.65 ± 0.42%) and culture- qPCR+ (0.55 ± 0.31%) samples were similar (Mann-Whitney U test, P = 0.627) and distinct from the culture- qPCR- group (0.17 ± 0.15%, P < 0.05). The read counts of sequences previously targeted by Shigella/enteroinvasive Escherichia coli (EIEC) qPCR assays, namely, ipaH, virA, virG, ial, ShET2, and ipaH3, were also similar between the culture+ qPCR+ and culture- qPCR+ groups and distinct from the culture- qPCR- groups (P < 0.001). Kraken performed well versus other methods: its precision and recall of Shigella were excellent at the genus level but variable at the species level. In summary, metagenomic sequencing indicates that Shigella/EIEC qPCR-positive samples are similar to those of Shigella culture-positive samples in Shigella sequence composition, thus supporting qPCR as an accurate method for detecting Shigella.

Enteroaggregative Escherichia coli Subclinical Infection and Coinfections and Impaired Child Growth in the MAL-ED Cohort Study.

OBJECTIVE: We evaluated the impact of subclinical enteroaggregative Escherichia coli (EAEC) infection alone and in combination with other pathogens in the first 6 months of life on child growth. METHODS: Nondiarrheal samples from 1684 children across 8 Multisite Birth Cohort Study, Malnutrition and Enteric Diseases (MAL-ED) sites in Asia, Africa, and Latin America were tested monthly; more than 90% of children were followed-up twice weekly for the first 6 months of life. RESULTS: Children with subclinical EAEC infection did not show altered growth between enrollment and 6 months. Conversely, EAEC coinfection with any other pathogen was negatively associated with delta weight-for-length (P < 0.05) and weight-for-age (P > 0.05) z scores between 0 and 6 months. The presence of 2 or more pathogens without EAEC was not significantly associated with delta weight-for-length and weight-for-age. The most frequent EAEC coinfections included Campylobacter spp, heat-labile toxin-producing enterotoxigenic E coli, Cryptosporidium spp, and atypical enteropathogenic E coli. Myeloperoxidase levels were increased with EAEC coinfection (P < 0.05). EAEC pathogen codetection was associated with lower neopterin levels compared to those of no-pathogen control children (P < 0.05). Mothers of children with EAEC coinfections had lower levels of education, poorer hygiene and sanitation, lower socioeconomic status, and lower breast-feeding rates compared to mothers of children in whom no pathogen was detected (P < 0.05). CONCLUSIONS: These data emphasize the public health importance of subclinical EAEC infection in early infancy in association with other pathogens and the need for improved maternal and child care, hygiene, sanitation, and socioeconomic factors.

Rotavirus Infection and Disease in a Multisite Birth Cohort: Results From the MAL-ED Study.

Background: In a multicountry birth cohort study, we describe rotavirus infection in the first 2 years of life in sites with and without rotavirus vaccination programs. Methods: Children were recruited by 17 days of age and followed to 24 months with collection of monthly surveillance and diarrheal stools. Data on sociodemographics, feeding, and illness were collected at defined intervals. Stools were tested for rotavirus and sera for antirotavirus immunoglobulins by enzyme immunoassays. Results: A total of 1737 children contributed 22646 surveillance and 7440 diarrheal specimens. Overall, rotavirus was detected in 5.5% (408/7440) of diarrheal stools, and 344 (19.8%) children ever had rotavirus gastroenteritis. Household overcrowding and a high pathogen load were consistent risk factors for infection and disease. Three prior infections conferred 74% (P < .001) protection against subsequent infection in sites not using vaccine. In Peru, incidence of rotavirus disease was relatively higher during the second year of life despite high vaccination coverage. Conclusions: Rotavirus infection and disease were common, but with significant heterogeneity by site. Protection by vaccination may not be sustained in the second year of life in settings with high burdens of transmission and poor response to oral vaccines.

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.

Age and Sex Normalization of Intestinal Permeability Measures for the Improved Assessment of Enteropathy in Infancy and Early Childhood: Results From the MAL-ED Study.

OBJECTIVES: The aim of the study was to describe changes in intestinal permeability in early childhood in diverse epidemiologic settings. METHODS: In a birth cohort study, the lactulose:mannitol (L:M) test was administered to 1980 children at 4 time points in the first 24 months of life in 8 countries. Data from the Brazil site with an incidence of diarrhea similar to that seen in the United States and no growth faltering was used as an internal study reference to derive age- and sex-specific z scores for mannitol and lactulose recoveries and the L:M ratio. RESULTS: A total of 6602 tests demonstrated mannitol recovery, lactulose recovery, and the L:M ratio were associated with country, sex, and age. There was heterogeneity in the recovery of both probes between sites with mean mannitol recovery ranging for 1.34% to 5.88%, lactulose recovery of 0.19% to 0.58%, and L:M ratios 0.10 to 0.17 in boys of 3 months of age across different sites. We observed strong sex-specific differences in both mannitol and lactulose recovery, with boys having higher recovery of both probes. Alterations in intestinal barrier function increased in most sites from 3 to 9 months of age and plateaued or diminished from 9 to 15 months of age. CONCLUSIONS: Alterations in recovery of the probes differ markedly in different epidemiologic contexts in children living in the developing world. The rate of change in the L:M-z ratio was most rapid and consistently disparate from the reference standard in the period between 6 and 9 months of age, suggesting that this is a critical period of physiologic impact of enteropathy in these populations.

Epidemiology and Impact of Campylobacter Infection in Children in 8 Low-Resource Settings: Results From the MAL-ED Study.

BACKGROUND: Enteropathogen infections have been associated with enteric dysfunction and impaired growth in children in low-resource settings. In a multisite birth cohort study (MAL-ED), we describe the epidemiology and impact of Campylobacter infection in the first 2 years of life. METHODS: Children were actively followed up until 24 months of age. Diarrheal and nondiarrheal stool samples were collected and tested by enzyme immunoassay for Campylobacter Stool and blood samples were assayed for markers of intestinal permeability and inflammation. RESULTS: A total of 1892 children had 7601 diarrheal and 26 267 nondiarrheal stool samples tested for Campylobacter We describe a high prevalence of infection, with most children (n = 1606; 84.9%) having a Campylobacter-positive stool sample by 1 year of age. Factors associated with a reduced risk of Campylobacter detection included exclusive breastfeeding (risk ratio, 0.57; 95% confidence interval, .47-.67), treatment of drinking water (0.76; 0.70-0.83), access to an improved latrine (0.89; 0.82-0.97), and recent macrolide antibiotic use (0.68; 0.63-0.74). A high Campylobacter burden was associated with a lower length-for-age Z score at 24 months (-1.82; 95% confidence interval, -1.94 to -1.70) compared with a low burden (-1.49; -1.60 to -1.38). This association was robust to confounders and consistent across sites. Campylobacter infection was also associated with increased intestinal permeability and intestinal and systemic inflammation. CONCLUSIONS: Campylobacter was prevalent across diverse settings and associated with growth shortfalls. Promotion of exclusive breastfeeding, drinking water treatment, improved latrines, and targeted antibiotic treatment may reduce the burden of Campylobacter infection and improve growth in children in these settings.

Respiratory viruses associated with severe pneumonia in children under 2 years old in a rural community in Pakistan.

The objective of this study was to determine the incidence of respiratory viruses associated with severe pneumonia among children less than 2 years of age in the rural district of Matiari in Sindh, Pakistan. This study was a community-based prospective cohort active surveillance of infants enrolled at birth and followed for 2 years. Cases were identified using the World Health Organization's Integrated Management of Childhood Illnesses' definition of severe pneumonia. Nasopharyngeal swabs were obtained for assessment by multiplex RT-PCR for eight viruses and their subtypes, including RSV, influenza virus, human metapneumovirus, enterovirus/rhinovirus, coronavirus, parainfluenza virus, adenovirus, and human bocavirus. Blood cultures were collected from febrile participants. A total of 817 newborns were enrolled and followed with fortnightly surveillance for 2 years, accounting for a total of 1,501 child-years of follow-up. Of the nasopharyngeal swabs collected, 77.8% (179/230) were positive for one or more of the above mentioned respiratory viruses. The incidence of laboratory confirmed viral-associated pneumonia was 11.9 per 100 child-years of follow-up. Enterovirus/rhinovirus was detected in 51.7% patients, followed by parainfluenza virus type III (8.3%), and RSV (5.7%). Of the uncontaminated blood cultures, 1.4% (5/356) were positive. Respiratory viruses are frequently detected during acute respiratory infection episodes in children under 2 years old in a rural community in Pakistan. However, causal association is yet to be established and the concomitant role of bacteria as a co-infection or super-infection needs further investigation. J. Med. Virol. 88:1882-1890, 2016. © 2016 Wiley Periodicals, Inc.

Diagnostic methods to determine microbiology of postpartum endometritis in South Asia: laboratory methods protocol used in the Postpartum Sepsis Study: a prospective cohort study.

BACKGROUND: The South Asian region has the second highest risk of maternal death in the world. To prevent maternal deaths due to sepsis and to decrease the maternal mortality ratio as per the World Health Organization Millenium Development Goals, a better understanding of the etiology of endometritis and related sepsis is required. We describe microbiological laboratory methods used in the maternal Postpartum Sepsis Study, which was conducted in Bangladesh and Pakistan, two populous countries in South Asia. METHODS/DESIGN: Postpartum maternal fever in the community was evaluated by a physician and blood and urine were collected for routine analysis and culture. If endometritis was suspected, an endometrial brush sample was collected in the hospital for aerobic and anaerobic culture and molecular detection of bacterial etiologic agents (previously identified and/or plausible). DISCUSSION: The results emanating from this study will provide microbiologic evidence of the etiology and susceptibility pattern of agents recovered from patients with postpartum fever in South Asia, data critical for the development of evidence-based algorithms for management of postpartum fever in the region.

Detection of Typhoidal and Paratyphoidal Salmonella in Blood by Real-time Polymerase Chain Reaction.

BACKGROUND: The gold standard for diagnosis of enteric fever caused by Salmonella Typhi or Salmonella Paratyphi A or B is bone marrow culture. However, because bone marrow aspiration is highly invasive, many hospitals and large health centers perform blood culture instead. As blood culture has several limitations, there is a need for novel typhoid diagnostics with improved sensitivity and more rapid time to detection. METHODS: We developed a clyA-based real-time polymerase chain reaction (qPCR) method to detect Salmonella Typhi and Salmonella Paratyphi A simultaneously in blood. The sensitivity and specificity of this probeset was first evaluated in vitro in the laboratory and then in a typhoid-endemic population, in Karachi, Pakistan, and in healthy US volunteers. RESULTS: We optimized a DNA extraction and real-time PCR-based method that could reliably detect 1 colony-forming unit/mL of Salmonella Typhi. The probe set was able to detect clinical Salmonella Typhi and Salmonella Paratyphi A strains and also diarrheagenic Escherichia coli, but not invasive E. coli or other invasive bacteria. In the field, the clyA qPCR diagnostic was 40% as sensitive as blood culture. However, when qPCR-positive specimens were considered to be true positives, blood culture only exhibited 28.57% sensitivity. Specificity was ≥90% for all comparisons and in the healthy US volunteers. qPCR was significantly faster than blood culture in terms of detection of typhoid and paratyphoid. CONCLUSIONS: Based on lessons learned, we recommend that future field trials of this and other novel diagnostics that detect typhoidal and nontyphoidal Salmonella employ multiple methodologies to define a "positive" sample.

Microbiologic methods utilized in the MAL-ED cohort study.

A central hypothesis of The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study is that enteropathogens contribute to growth faltering. To examine this question, the MAL-ED network of investigators set out to achieve 3 goals: (1) develop harmonized protocols to test for a diverse range of enteropathogens, (2) provide quality-assured and comparable results from 8 global sites, and (3) achieve maximum laboratory throughput and minimum cost. This paper describes the rationale for the microbiologic assays chosen and methodologies used to accomplish the 3 goals.

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.

Pandemic influenza A(H1N1)pdm09: an unrecognized cause of mortality in children in Pakistan.

The role of influenza virus as a cause of child mortality in South Asia is under-recognized. We aimed to determine the incidence and case fatality rate of influenza A(H1N1)pdm09 infections in hospitalized children in Karachi, Pakistan. Children less than 5 y old admitted with respiratory illnesses to the Aga Khan University Hospital, Karachi, from 17 August 2009 to 16 September 2011, were tested for influenza A(H1N1)pdm09 using a real-time reverse transcriptase polymerase chain reaction. Out of 2650 children less than 5 y old admitted with a respiratory illness during the study period, 812 (31%) were enrolled. Influenza A(H1N1)pdm09 virus was detected in 27 (3.3%) children. There were 4 deaths in children who tested positive for influenza A(H1N1)pdm09 (case fatality rate of 15%). Children with influenza A(H1N1)pdm09 were 5 times more likely to be admitted or transferred to the intensive care unit, 5.5 times more likely to be intubated, and 12.9 times more likely to die as compared to children testing negative for influenza A(H1N1)pdm09.

Simultaneous detection of six diarrhea-causing bacterial pathogens with an in-house PCR-luminex assay.

Diarrhea can be caused by a range of pathogens, including several bacteria. Conventional diagnostic methods, such as culture, biochemical tests, and enzyme-linked immunosorbent assay (ELISA), are laborious. We developed a 7-plex PCR-Luminex assay to simultaneously screen for several of the major diarrhea-causing bacteria directly in fecal specimens, including pathogenic Aeromonas, Campylobacter jejuni, Campylobacter coli, Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia. We included an extrinsic control to verify extraction and amplification. The assay was first validated with reference strains or isolates and exhibited a limit of detection of 10(3) to 10(5) CFU/g of stool for each pathogen as well as quantitative detection up to 10(9) CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested for Campylobacter by ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% for Aeromonas, 89% and 93% for Campylobacter, 96% and 95% for Salmonella, 94% and 94% for Shigella, 92% and 97% for Vibrio, and 100% and 100% for Yersinia, respectively. All discrepant results were further examined by singleplex real-time PCR assays targeting different gene regions, which revealed 89% (55/62 results) concordance with the PCR-Luminex assay. The fluorescent signals obtained with this approach exhibited a statistically significant correlation with the cycle threshold (C(T)) values from the cognate real-time PCR assays (P < 0.05). This multiplex PCR-Luminex assay enables sensitive, specific, and quantitative detection of the major bacterial causes of gastroenteritis.