Carl J. Mason

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.

Detection of Shigella by a PCR Assay Targeting the ipaH Gene Suggests Increased Prevalence of Shigellosis in Nha Trang, Vietnam

ABSTRACT Shigella spp. are exquisitely fastidious gram-negative organisms which frequently escape detection by traditional culture methods. To get a more complete understanding of the disease burden caused by Shigella in Nha Trang, Vietnam, real-time PCR was used to detect Shigella DNA. Randomly selected rectal swab specimens from 60 Shigella culture-positive patients and 500 Shigella culture-negative patients detected by population-based surveillance of patients seeking care for diarrhea were processed by real-time PCR. The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH), carried by all four Shigella species and enteroinvasive Escherichia coli. Shigella spp. could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea. IpaH was detected in 55 of 60 (93%) Shigella culture-positive specimens, whereas it was detected in 87 of 245 (36%) culture-negative patients free of dysentery (P < 0.001). The number of PCR cycles required to detect a PCR product was highest for culture-negative, nonbloody diarrheal specimens (mean number of cycles to detection, 36.6) and was lowest for children with culture-positive, bloody diarrheal specimens (mean number of cycles, 25.3) (P < 0.001). The data from real-time PCR amplification indicate that the culture-proven prevalence of Shigella among patients with diarrhea may underestimate the prevalence of Shigella infections. The clinical presentation of shigellosis may be directly related to the bacterial load.

HLA-DR-Promiscuous T Cell Epitopes from Plasmodium falciparum Pre-Erythrocytic-Stage Antigens Restricted by Multiple HLA Class II Alleles

Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-γ responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8.7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.

Fecal markers of intestinal inflammation and permeability associated with the subsequent acquisition of linear growth deficits in infants.

Enteric infections are associated with linear growth failure in children. To quantify the association between intestinal inflammation and linear growth failure three commercially available enzyme-linked immunosorbent assays (neopterin [NEO], alpha-anti-trypsin [AAT], and myeloperoxidase [MPO]) were performed in a structured sampling of asymptomatic stool from children under longitudinal surveillance for diarrheal illness in eight countries. Samples from 537 children contributed 1,169 AAT, 916 MPO, and 954 NEO test results that were significantly associated with linear growth. When combined to form a disease activity score, children with the highest score grew 1.08 cm less than children with the lowest score over the 6-month period following the tests after controlling for the incidence of diarrheal disease. This set of affordable non-invasive tests delineates those at risk of linear growth failure and may be used for the improved assessments of interventions to optimize growth during a critical period of early childhood.

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.

Traveler's Diarrhea in Thailand: Randomized, Double-Blind Trial Comparing Single-Dose and 3-Day Azithromycin-Based Regimens with a 3-Day Levofloxacin Regimen

BACKGROUND Travelers diarrhea in Thailand is frequently caused by Campylobacter jejuni. Rates of fluoroquinolone (FQ) resistance in Campylobacter organisms have exceeded 85% in recent years, and reduced fluoroquinolone efficacy has been observed. METHODS Azithromycin regimens were evaluated in a randomized, double-blind trial of azithromycin, given as a single 1-g dose or a 3-day regimen (500 mg daily), versus a 3-day regimen of levofloxacin (500 mg daily) in military field clinics in Thailand. Outcomes included clinical end points (time to the last unformed stool [TLUS] and cure rates) and microbiological end points (pathogen eradication). RESULTS A total of 156 patients with acute diarrhea were enrolled in the trial. Campylobacter organisms predominated (in 64% of patients), with levofloxacin resistance noted in 50% of Campylobacter organisms and with no azithromycin resistance noted. The cure rate at 72 h after treatment initiation was highest (96%) with single-dose azithromycin, compared with the cure rates of 85% noted with 3-day azithromycin and 71% noted with levofloxacin (P=.002). Single-dose azithromycin was also associated with the shortest median TLUS (35 h; P=.03, by log-rank test). Levofloxacins efficacy was inferior to azithromycins efficacy, except in patients with no pathogen identified during the first 24 h of treatment or in patients with levofloxacin-susceptible Campylobacter isolates, in whom it appeared to be equal to azithromycin. The rate of microbiological eradication was significantly better with azithromycin-based regimens (96%-100%), compared with levofloxacin (38%) (P=.001); however, this finding was poorly correlated with clinical outcome. A higher rate of posttreatment nausea in the 30 min after receipt of the first dose (14% vs. <6%; P=.06) was observed as a mild, self-limited complaint associated with single-dose azithromycin. CONCLUSIONS Single-dose azithromycin is recommended for empirical therapy of travelers diarrhea acquired in Thailand and is a reasonable first-line option for empirical management in general.

Reducing stunting among children: the potential contribution of diagnostics

Stunting affects ~ 147 million children in developing countries. Studies have pointed to a relationship between stunting and different pathogens that are associated with diarrhoeal illness. New easy-to-use tools for diagnosing these pathogens could help to identify children at risk for growth shortfall, and reduce the prevalence of stunting and the large burden of disease associated with it.PREFACE The numbers of acquired immunodeficiency syndrome (AIDS)-related deaths among infants in developing countries are exceptionally high, largely because human immunodeficiency virus (HIV) infection remains undiagnosed in many cases. Current HIV testing methods are either impractical for developingcountry settings or inaccurate for use in infants. There is an urgent need to develop and deploy a new, easy-to-use HIV test, which could transform the management of paediatric HIV/AIDS in developing countries and avert millions of infant deaths.

Typhoid Fever: A Massive, Single-Point Source, Multidrug-Resistant Outbreak in Nepal

BACKGROUND In the summer of 2002, a total of 5963 cases of typhoid fever were recorded in Bharatpur, Nepal (population, 92,214) during a 7-week period. A team from the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, and the CIWEC Travel Medicine Clinic (Kathmandu, Nepal) assisted the Nepal National Public Health Laboratory (Kathmandu, Nepal) in the further investigation of this large, explosive febrile disease outbreak. METHODS Investigators conducted a thorough epidemiologic and laboratory investigation to assess the size and scope of the outbreak. In addition to subculturing of previously collected samples, blood samples were obtained from 100 febrile patients, and culture and susceptibility testing were done by standard laboratory methods. Pulsed field gel electrophoresis (PFGE) and plasmid analysis were done. RESULTS The majority of the isolates, including 1 from the municipal water supply, were multidrug resistant. The minimum inhibitory concentrations (MICs) of ciprofloxacin ranged from 0.19 microg/mL to 0.125 microg/mL. With use of PFGE, all isolates, including isolates from the water supply, showed an analytical similarity of 96%-100%. Multidrug-resistant isolates had a plasmid encoding for resistance, and those with resistance to nalidixic acid had a single-point mutation. CONCLUSIONS To the best of our knowledge, this outbreak is the largest single-point source outbreak of multidrug-resistant typhoid fever yet reported, and it was molecularly traced to the citys single municipal water supply. Isolates were uniformly resistant to nalidixic acid, there was a decrease in their susceptibility as measured by MIC of fluoroquinolones, and 90% of isolates obtained were resistant to >1 antibiotic.

Assessment of Environmental Enteropathy in the MAL-ED Cohort Study: Theoretical and Analytic Framework

Individuals in the developing world live in conditions of intense exposure to enteric pathogens due to suboptimal water and sanitation. These environmental conditions lead to alterations in intestinal structure, function, and local and systemic immune activation that are collectively referred to as environmental enteropathy (EE). This condition, although poorly defined, is likely to be exacerbated by undernutrition as well as being responsible for permanent growth deficits acquired in early childhood, vaccine failure, and loss of human potential. This article addresses the underlying theoretical and analytical frameworks informing the methodology proposed by the Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study to define and quantify the burden of disease caused by EE within a multisite cohort. Additionally, we will discuss efforts to improve, standardize, and harmonize laboratory practices within the MAL-ED Network. These efforts will address current limitations in the understanding of EE and its burden on children in the developing world.

Etiology of diarrhea in young children and patterns of antibiotic resistance in Cambodia.

Background: Little is known about diarrhea etiology and antibiotic resistance in developing countries where diarrhea is a major public health problem. Methods: To describe diarrhea etiology and antibiotic resistance patterns in Cambodia, 600 children aged 3 months to 5 years with acute diarrhea (cases) and 578 children without diarrhea (controls) were enrolled from a hospital in Phnom Penh. Stool samples were collected, and pathogens and antibiotic resistance patterns were described. Results: The most frequently isolated pathogens in these cases were enteroaggregative Escherichia coli (20%) and rotavirus (26%). Enterotoxigenic E. coli, enteroaggregative E. coli, Shigella, Aeromonas, rotavirus, and adenovirus were statistically significantly associated with diarrhea. Among cases, vomiting was associated with viral infections, whereas bloody stool was associated with Shigella. Enterotoxigenic E. coli isolates were highly resistant to ampicillin, sulfonamides, and tetracycline. Approximately 50% of Campylobacter coli and 30% of Campylobacter jejuni isolates were resistant to nalidixic acid and ciprofloxacin. Over 33% of Salmonella isolates were resistant to ampicillin and tetracycline, and almost 100% of Shigella isolates were resistant to trimethoprim/sulfamethoxazole. Conclusions: These data on the etiology of diarrhea and antibiotic resistance patterns in Cambodia will have significant effect on local public health policies and on local resource prioritization practices.