William C. Blackwelder

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.

Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans

Recent outbreaks of avian influenza in humans have demonstrated the need for vaccines for influenza viruses with pandemic potential. Recombinant hemagglutinins are an attractive option for such vaccines because they do not require handling potentially highly pathogenic influenza viruses for vaccine production. In order to evaluate the immunogenicity, optimum dosing and timing of administration of a recombinant baculovirus-expressed H5 HA (rH5) in humans, 147 healthy adults were assigned randomly to receive intramuscular rH5 as two doses of 25, 45 or 90 microg each, one dose of 90 microg followed by a dose of 10 microg, or two doses of placebo, at intervals between doses of 21, 28 or 42 days. All doses of rH5 were well tolerated. The rH5 vaccine was modestly immunogenic at high dose. Neutralizing antibody responses to a titer of 1:80 or greater were seen in 23% (14/60) of individuals after a single dose of 90 microg, and in 52% (15/29) after two doses of 90 microg. Varying intervals between doses from 21 to 42 days had no significant effect on antibody responses to vaccination. These results suggest that baculovirus-expressed H5 HA can induce functional antibody in individuals who have not had prior exposure to H5 viruses, but that further studies to improve the immunogenicity of the vaccine are needed.

In vitro assay for neutralizing antibody to hepatitis C virus: Evidence for broadly conserved neutralization epitopes

Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.

The Global Enteric Multicenter Study (GEMS) of Diarrheal Disease in Infants and Young Children in Developing Countries: Epidemiologic and Clinical Methods of the Case/Control Study

Background. Diarrhea is a leading cause of illness and death among children aged <5 years in developing countries. This paper describes the clinical and epidemiological methods used to conduct the Global Enteric Multicenter Study (GEMS), a 3-year, prospective, age-stratified, case/control study to estimate the population-based burden, microbiologic etiology, and adverse clinical consequences of acute moderate-to-severe diarrhea (MSD) among a censused population of children aged 0–59 months seeking care at health centers in sub-Saharan Africa and South Asia. Methods. GEMS was conducted at 7 field sites, each serving a population whose demography and healthcare utilization practices for childhood diarrhea were documented. We aimed to enroll 220 MSD cases per year from selected health centers serving each site in each of 3 age strata (0–11, 12–23, and 24–59 months), along with 1–3 matched community controls. Cases and controls supplied clinical, epidemiologic, and anthropometric data at enrollment and again approximately 60 days later, and provided enrollment stool specimens for identification and characterization of potential diarrheal pathogens. Verbal autopsy was performed if a child died. Analytic strategies will calculate the fraction of MSD attributable to each pathogen and the incidence, financial costs, nutritional consequences, and case fatality overall and by pathogen. Conclusions. When completed, GEMS will provide estimates of the incidence, etiology, and outcomes of MSD among infants and young children in sub-Saharan Africa and South Asia. This information can guide development and implementation of public health interventions to diminish morbidity and mortality from diarrheal diseases.

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.

Pre-clinical immunogenicity and efficacy trial of a recombinant hepatitis E vaccine

We have demonstrated that recombinant hepatitis E vaccine suitable for clinical evaluation was highly immunogenic and efficacious in preventing hepatitis E and even infection in rhesus macaques following intravenous challenge with three different genotypes of hepatitis E virus (HEV). Two doses of vaccine were essential for optimal protection; the two-dose regimen was more important than the formulation of the vaccine for achieving efficacy. The titers of anti-HEV that were protective in this study were quantified against a World Health Organization (WHO) standard. This permits direct comparison of the results with other studies. The results of this pre-clinical trial of a candidate hepatitis E vaccine strongly suggest that it will be highly efficacious for preventing hepatitis E in the field trial of this vaccine that is currently in progress in Nepal.

Shigella Isolates From the Global Enteric Multicenter Study Inform Vaccine Development

Shigella case isolates from the Global Enteric Multicenter Study were serotyped to guide vaccine development. A quadrivalent vaccine that includes O antigens from S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 should provide broad protection.

Importance of Complement Source in Measuring Meningococcal Bactericidal Titers

ABSTRACT Complement-mediated bactericidal antibodies in serum confer protection against meningococcal disease. The minimum protective titer is estimated to be between 1:4 and 1:8 when measured by the Goldschneider assay performed with human complement, the assay used in the 1960s to establish the correlation between bactericidal antibodies and protection. A more recently described bactericidal assay standardized by an international consortium uses rabbit complement, which is known to augment bactericidal titers. To define a protective titer measured by the standardized assay, we compared bactericidal titers against serogroup C strains measured by this assay to titers measured by the assay described by Goldschneider et al. A titer of ≥1:128 measured by the standardized assay was needed to predict with ≥80% certainty a positive titer of ≥1:4 as measured by the Goldschneider assay. However, the majority of samples with titers of 1:4 measured by the Goldschneider assay had titers of <1:128 when measured by the standardized assay. Therefore, by the results of the standardized assay such persons would be falsely categorized as being susceptible to disease. In conclusion, high bactericidal titers measured with the standardized assay performed with rabbit complement are predictive of protection, but no threshold titer is both sensitive and specific for predicting a positive titer measured by the Goldschneider assay using human complement. Up to 10% of the U.S. adult population lacks intrinsic bactericidal activity against serogroup C strains in serum and can serve as complement donors. Therefore, use of the Goldschneider assay or an equivalent assay performed with human complement is preferred over assays that use rabbit complement.

Ty21a Live Oral Typhoid Vaccine and Prevention of Paratyphoid Fever Caused by Salmonella enterica Serovar Paratyphi B

In randomized, controlled field trials in Area Norte and Area Occidente of Santiago, Chile, 2 (Norte) or 3 (Occidente) doses of live oral typhoid vaccine Ty21a in enteric-coated capsules conferred protection against confirmed Salmonella enterica serovar Typhi disease (53% efficacy in Norte; 67% efficacy in Occidente) during 3 years of follow-up. There was also a trend in each trial showing protection against S. enterica serovar Paratyphi B disease (56% efficacy in Norte; 42% efficacy in Occidente). To enhance statistical power, an analysis was performed using pooled data from the 2 trials; this pooling of data was justified by the following facts: epidemiologic surveillance and microbiological methods were identical, the trials overlapped during 22 of the 36 months of follow-up in each trial, the estimates of efficacy against paratyphoid B fever in the 2 trials were roughly similar, and the ratio of follow-up of vaccine recipients to control subjects in both trials was ~1 : 1. In the pooled analysis, Ty21a conferred significant protection against paratyphoid B fever (efficacy, 49%; 95% confidence interval, 8%-73%; P=.019).

Age- and Serotype-Specific Pediatric Invasive Pneumococcal Disease: Insights from Systematic Surveillance in Santiago, Chile, 1994–2007

BACKGROUND We monitored pediatric invasive pneumococcal disease (IPD) in Santiago, Chile, from 1994 to 2007. METHODS Three related data sets were generated: (1) IPD cases requiring hospitalization, 1994--2007; (2) cases of bacteremia detected among febrile patients aged 0-35 months seen in emergency departments, 2000--2007; and (3) nasopharyngeal carriage of pneumococcal serotypes, determined from repetitive culturing, among 524 newborns followed up through age 23 months. RESULTS Of 2369 IPD cases requiring hospitalization, 1878 (79.3%) occurred in those aged 0-59 months, and 1200 (50.7%) occurred in those aged 6-35 months. Among infants aged 0-5 months, meningitis and sepsis comprised 48.4% of all IPD cases (serotype 5 predominated); among those 6-35 months old, 522 (43.5%) of 1200 cases were bacteremic pneumonia (serotype 14 predominated). Serotype 1 peritonitis was common among 5-14-year-old girls. Meningitis and sepsis exhibited high case fatality rates (14%-29%) among all ages. Remarkably, 34 (28.8%) of 118 children with sepsis died, versus 1 fatality (0.4%) among 276 children hospitalized with bacteremia without a focus (P < .001, Fishers exact test). Serotype 5 was significantly more common among hospitalized patients < 36 months of age, whereas serotype 18C was overrepresented among ambulatory patients. The annual incidence of serotype 14 was stable; those of serotypes 1 and 5 fluctuated markedly. Serotypes 14, 5, and 1 were overrepresented among invasive compared with nasopharyngeal isolates. CONCLUSIONS Clinical syndromes of IPD and predominant serotypes vary with age.