James A. Platts-Mills

Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED)

BACKGROUND Most studies of the causes of diarrhoea in low-income and middle-income countries have looked at severe disease in people presenting for care, and there are few estimates of pathogen-specific diarrhoea burdens in the community. METHODS We undertook a birth cohort study with not only intensive community surveillance for diarrhoea but also routine collection of non-diarrhoeal stools from eight sites in South America, Africa, and Asia. We enrolled children within 17 days of birth, and diarrhoeal episodes (defined as maternal report of three or more loose stools in 24 h, or one loose stool with visible blood) were identified through twice-weekly home visits by fieldworkers over a follow-up period of 24 months. Non-diarrhoeal stool specimens were also collected for surveillance for months 1-12, 15, 18, 21, and 24. Stools were analysed for a broad range of enteropathogens using culture, enzyme immunoassay, and PCR. We used the adjusted attributable fraction (AF) to estimate pathogen-specific burdens of diarrhoea. FINDINGS Between November 26, 2009, and February 25, 2014, we tested 7318 diarrhoeal and 24 310 non-diarrhoeal stools collected from 2145 children aged 0-24 months. Pathogen detection was common in non-diarrhoeal stools but was higher with diarrhoea. Norovirus GII (AF 5·2%, 95% CI 3·0-7·1), rotavirus (4·8%, 4·5-5·0), Campylobacter spp (3·5%, 0·4-6·3), astrovirus (2·7%, 2·2-3·1), and Cryptosporidium spp (2·0%, 1·3-2·6) exhibited the highest attributable burdens of diarrhoea in the first year of life. The major pathogens associated with diarrhoea in the second year of life were Campylobacter spp (7·9%, 3·1-12·1), norovirus GII (5·4%, 2·1-7·8), rotavirus (4·9%, 4·4-5·2), astrovirus (4·2%, 3·5-4·7), and Shigella spp (4·0%, 3·6-4·3). Rotavirus had the highest AF for sites without rotavirus vaccination and the fifth highest AF for sites with the vaccination. There was substantial variation in pathogens according to geography, diarrhoea severity, and season. Bloody diarrhoea was primarily associated with Campylobacter spp and Shigella spp, fever and vomiting with rotavirus, and vomiting with norovirus GII. INTERPRETATION There was substantial heterogeneity in pathogen-specific burdens of diarrhoea, with important determinants including age, geography, season, rotavirus vaccine usage, and symptoms. These findings suggest that although single-pathogen strategies have an important role in the reduction of the burden of severe diarrhoeal disease, the effect of such interventions on total diarrhoeal incidence at the community level might be limited.

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.

Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study

BACKGROUND Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease. METHODS We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. FINDINGS The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043). INTERPRETATION Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. FUNDING Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.

Etiology of Diarrhea in Bangladeshi Infants in the First Year of Life Analyzed Using Molecular Methods

BACKGROUND Diarrhea causes enormous morbidity and mortality in developing countries, yet the relative importance of multiple potential enteropathogens has been difficult to ascertain. METHODS We performed a longitudinal cohort study from birth to 1 year of age in 147 infants in Dhaka, Bangladesh. Using multiplex polymerase chain reaction, we analyzed 420 episodes of diarrhea and 1385 monthly surveillance stool specimens for 32 enteropathogen gene targets. For each infant we examined enteropathogen quantities over time to ascribe each positive target as a probable or less-likely contributor to diarrhea. RESULTS Multiple enteropathogens were detected by the first month of life. Diarrhea was associated with a state of overall pathogen excess (mean number of enteropathogen gene targets (± SE), 5.6 ± 0.1 vs 4.3 ± 0.1 in surveillance stool specimens; P < .05). After a longitudinal, quantitative approach was applied to filter out less-likely contributors, each diarrheal episode still had an average of 3.3 probable or dominant targets. Enteroaggregative Escherichia coli, Campylobacter, enteropathogenic E. coli, rotavirus, and Entamoeba histolytica were the most frequent probable contributors to diarrhea. Rotavirus was enriched in moderate to severe diarrheal episodes. CONCLUSIONS In this community-based study diarrhea seemed to be a multipathogen event and a state of enteropathogen excess above a high carriage baseline.

Estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for the Global Burden of Disease Study 2015

Summary Background The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 (GBD 2015) provides an up-to-date analysis of the burden of diarrhoeal diseases. This study assesses cases, deaths, and aetiologies spanning the past 25 years and informs the changing picture of diarrhoeal disease worldwide. Methods We estimated diarrhoeal mortality by age, sex, geography, and year using the Cause of Death Ensemble Model (CODEm), a modelling platform shared across most causes of death in the GBD 2015 study. We modelled diarrhoeal morbidity, including incidence and prevalence, using a meta-regression platform called DisMod-MR. We estimated aetiologies for diarrhoeal diseases using a counterfactual approach that incorporates the aetiology-specific risk of diarrhoeal disease and the prevalence of the aetiology in diarrhoea episodes. We used the Socio-demographic Index, a summary indicator derived from measures of income per capita, educational attainment, and fertility, to assess trends in diarrhoeal mortality. The two leading risk factors for diarrhoea—childhood malnutrition and unsafe water, sanitation, and hygiene—were used in a decomposition analysis to establish the relative contribution of changes in diarrhoea disability-adjusted life-years (DALYs). Findings Globally, in 2015, we estimate that diarrhoea was a leading cause of death among all ages (1·31 million deaths, 95% uncertainty interval [95% UI] 1·23 million to 1·39 million), as well as a leading cause of DALYs because of its disproportionate impact on young children (71·59 million DALYs, 66·44 million to 77·21 million). Diarrhoea was a common cause of death among children under 5 years old (499 000 deaths, 95% UI 447 000–558 000). The number of deaths due to diarrhoea decreased by an estimated 20·8% (95% UI 15·4–26·1) from 2005 to 2015. Rotavirus was the leading cause of diarrhoea deaths (199 000, 95% UI 165 000–241 000), followed by Shigella spp (164 300, 85 000–278 700) and Salmonella spp (90 300, 95% UI 34 100–183 100). Among children under 5 years old, the three aetiologies responsible for the most deaths were rotavirus, Cryptosporidium spp, and Shigella spp. Improvements in safe water and sanitation have decreased diarrhoeal DALYs by 13·4%, and reductions in childhood undernutrition have decreased diarrhoeal DALYs by 10·0% between 2005 and 2015. Interpretation At the global level, deaths due to diarrhoeal diseases have decreased substantially in the past 25 years, although progress has been faster in some countries than others. Diarrhoea remains a largely preventable disease and cause of death, and continued efforts to improve access to safe water, sanitation, and childhood nutrition will be important in reducing the global burden of diarrhoea. Funding Bill & Melinda Gates Foundation.

Detection of Campylobacter in Stool and Determination of Significance by Culture, Enzyme Immunoassay, and PCR in Developing Countries

ABSTRACT Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level.

Association Between Stool Enteropathogen Quantity and Disease in Tanzanian Children Using TaqMan Array Cards: A Nested Case-Control Study

Etiologic studies of diarrhea are limited by uneven diagnostic methods and frequent asymptomatic detection of enteropathogens. Polymerase chain reaction-based stool pathogen quantification may help distinguish clinically significant infections. We performed a nested case-control study of diarrhea in infants from a community-based birth cohort in Tanzania. We tested 71 diarrheal samples and pre-diarrheal matched controls with a laboratory-developed TaqMan Array Card for 19 enteropathogens. With qualitative detection, no pathogens were significantly associated with diarrhea. When pathogen quantity was considered, rotavirus (odds ratio [OR] = 2.70 per log10 increase, P < 0.001), astrovirus (OR = 1.49, P = 0.01), and Shigella/enteroinvasive Escherichia coli (OR = 1.47, P = 0.04) were associated with diarrhea. Enterotoxigenic E. coli (0.15 SD decline in length-for-age z score after 3 months per log10 increase, P < 0.001) and Campylobacter jejuni/C. coli (0.11 SD decline, P = 0.003) in pre-diarrheal stools were associated with poor linear growth. Quantitative analysis can help refine the association between enteropathogens and disease in endemic settings.

Update on the burden of Campylobacter in developing countries

Purpose of review Recent work has added to the understanding of the burden of Campylobacter jejuni, C. coli, and non-jejuni/coli Campylobacter strains in children living in the developing world. Recent findings New diagnostic modalities and carefully designed field studies are demonstrating that the burden of Campylobacter diarrhea in children in the developing world has been greatly underestimated. Furthermore, there is emerging recognition of an association between Campylobacter infection and malnutrition. Important progress has been made toward a Campylobacter jejuni vaccine. Finally, evidence of antibiotic resistance continues to be an important issue that is accentuated by the realization that the burden of disease is greater than previously recognized. Summary Additional research is needed to refine our understanding of the epidemiology of Campylobacter infections in developing countries, in particular to improve estimates of the burden of Campylobacter diarrhea in endemic settings, to determine the impact of recurrent Campylobacter infections on child development, and to describe the prevalence and clinical significance of non-jejuni/coli Campylobacter infections. Progressive antibiotic resistance of isolates argues for augmented and expanded control measures of antibiotics in livestock. Continued work in vaccine development is warranted as is the extension of data available on the serotypes related to burden in different areas of the world and the relationship of serotypes to disease severity.

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

In a multisite birth cohort study, we document a high burden of Campylobacter infection using enzyme immunoassay, demonstrate an association between Campylobacter and linear growth shortfalls and both increased intestinal permeability and intestinal and systemic inflammation, and identify potential interventions.

Norovirus Infection and Acquired Immunity in 8 Countries: Results From the MAL-ED Study

This longitudinal, multisite study reports a high prevalence of norovirus infection and norovirus-positive diarrhea and describes patterns of age acquisition, disease severity, genogroup-specific immunity, and relationships between norovirus and undernutrition in the first 2 years of life.