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

Immunogenicity and efficacy following sequential parenterally-administered doses of <i>Salmonella</i> Enteritidis COPS:FliC glycoconjugates in infant and adult mice

<div><p>In sub-Saharan Africa, invasive nontyphoidal <i>Salmonella</i> (iNTS) infections with serovars <i>S</i>. Enteritidis, <i>S</i>. Typhimurium and I 4,[5],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of <i>S</i>. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian <i>S</i>. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of <i>S</i>. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. <i>S</i>. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against <i>S</i>. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa.</p></div

Survival of adult immunized with 3 doses of native FliC and challenged with <i>S</i>. Enteritidis R11.

<p>Kaplan-Meier survival curves for adult-primed mice immunized with 3 doses of either PBS (black squares) or FliC (grey circles) after challenge with 1x10⁶ CFU/mL of <i>S</i>. Enteritidis R11 (<i>n</i> = 20/group). Survival curves were compared using log rank analysis. For comparisons between vaccinated mice and PBS controls, P-values ≤ 0.05 were considered to be statistically significant. *<i>P</i> ≤ 0.0005.</p

Survival for adult mice immunized with 1 or 2 doses of <i>S</i>. Enteritidis COPS:FliC adjuvanted with MPL and challenged with <i>S</i>. Enteritidis R11 <i>∆fliC</i>.

<p>Kaplan-Meier survival curves for adult mice immunized with 1 or 2 doses of PBS (black squares) or COPS:FliC (grey circles) after challenge with 1x10⁶ CFU/mL of <i>S</i>. Enteritidis R11 <i>∆fliC</i> (<i>n</i> = 10–20/group). Survival curves were compared using log rank analysis. For comparisons between vaccinated mice and PBS controls, P-values ≤ 0.05 were considered to be statistically significant. ns, not significant; *<i>P</i> ≤ 0.005.</p

Survival for adult and infant mice immunized with 1, 2, or 3 doses of <i>S</i>. Enteritidis COPS:FliC adjuvanted with MPL and challenged with <i>S</i>. Enteritidis R11.

<p>Kaplan-Meier survival curves for adult-primed (A) or infant-primed (B) mice immunized with 1, 2, or 3 doses of PBS (black squares) or COPS:FliC (circles; grey and white color indicate adults and infants, respectively) after challenge with 1x10⁶ CFU/mL of <i>S</i>. Enteritidis R11 (<i>n</i> = 13–20/group). Survival curves were compared using log rank analysis. For comparisons between vaccinated mice and PBS controls, P-values ≤ 0.05 were considered to be statistically significant. ns, not significant; *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.0005.</p

Anti-COPS IgG responses in adult and infant mice after immunization with <i>S</i>. Enteritidis COPS:FliC alone or formulated with different adjuvants.

<p>(A) Infant and adult mice (<i>n</i> = 16–20/group) were immunized as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006522#pntd.0006522.g001" target="_blank">Fig 1</a>. Serum anti-COPS IgG titers taken 12–14 days after each dose were determined by ELISA. Each point represents an individual mouse. Red squares indicate mice that succumbed to subsequent challenge. Bars represent the GMT for adults (grey) and infants (white), and were compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. ns, not significant. *<i>P</i> ≤ 0.05; **<i>P</i> ≤ 0.005; ***<i>P</i> ≤ 0.0005 for indicated comparisons. (B) Reverse cumulative distribution curves for post 3^rd immunization anti-COPS IgG titers for adults (grey circles) and infants (white circles) are depicted. Dotted lines indicate the cut-off for seroconversion (50 EU/mL), which represents a 4-fold rise over the anti-COPS IgG GMT for PBS controls.</p

Isotype and avidity of anti-COPS IgG in adult mice after immunization with <i>S</i>. Enteritidis COPS:FliC formulated with MPL.

<p>(A) The avidity of anti-COPS IgG after the 3^rd immunization (<i>n</i> = 6) was determined by ELISA. (B) Post 3^rd immunization serum COPS-specific IgG1 and IgG2b titers were determined by ELISA. The ratio of the two titers is plotted. Each point represents an individual mouse. The bar represents the median.</p

Anti-FliC IgG responses in adult and infant mice after immunization with <i>S</i>. Enteritidis COPS:FliC alone or formulated with different adjuvants.

<p>Infant and adult mice (<i>n</i> = 16–20/group) were immunized with PBS or <i>S</i>. Enteritidis COPS:FliC formulated alone (ɸ), adsorbed to alum, or admixed with MPL. (A) Serum anti-FliC IgG titers taken 12–14 days after each dose were determined by ELISA. Each point represents an individual mouse. Red squares indicate mice that succumbed to subsequent challenge. Bars represent the GMT for adults (grey) and infants (white), and were compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. ns, not significant. *<i>P</i> ≤ 0.05; **<i>P</i> ≤ 0.005; ***<i>P</i> ≤ 0.0005 for indicated comparisons. (B) Reverse cumulative distribution curves for post 3^rd immunization anti-FliC IgG titers for adults (grey circles) and infants (white circles) are depicted.</p

Isotype and avidity of anti-FliC IgG in adult and infant mice after immunization with <i>S</i>. Enteritidis COPS:FliC alone or formulated with different adjuvants.

<p>(A) The avidity of anti-FliC IgG in sera taken after each immunization (<i>n</i> = 8–10) was determined by sensitivity to urea treatment in an ELISA format. (B) Post 3^rd immunization FliC-specific serum IgG1 and IgG2b titers were determined by ELISA (<i>n</i> = 9–10). The ratio of the two titers is plotted. Each point represents an individual mouse. Bars represent the median for adults (grey) and infants (white) that was compared using a two-tailed Mann-Whitney U test. Adjustments for multiple comparisons were not made. P-values ≤ 0.05 were considered to be statistically significant; ns, not significant. *<i>P</i> ≤ 0.05; **<i>P</i> ≤ 0.005; ***<i>P</i> ≤ 0.0005 for indicated comparisons.</p