Real-time PCR threshold cycle cutoffs help to identify agents causing acute childhood diarrhea in Zanzibar

Abstract

Molecular assays might improve the identification of causes of acute diarrheal disease but might lead to more frequent detection of asymptomatic infections. In the present study, real-time PCR targeting 14 pathogens was applied to rectal swabs from 330 children aged 2 to 59 months in Zanzibar, including 165 patients with acute diarrhea and 165 asymptomatic control subjects. At least one pathogen was detected for 94% of the patients and 84% of the controls, with higher rates among patients for norovirus genogroup II (20% versus 2.4%; P<0.0001), rotavirus (10% versus 1.8%; P=0.003), and Cryptosporidium (30% versus 11%; P<0.0001). Detection rates did not differ significantly for enterotoxigenic Escherichia coli (ETEC)-estA (33% versus 24%), ETEC-eltB (44% versus 46%), Shigella (35% versus 33%), and Campylobacter (35% versus 33%), but for these agents threshold cycle (CT) values were lower (pathogen loads were higher) in sick children than in controls. In a multivariate analysis, CT values for norovirus genogroup II, rotavirus, Cryptosporidium, ETEC-estA, and Shigella were independently associated with diarrhea. We conclude that this real-time PCR allows convenient detection of essentially all diarrheagenic agents and provides CT values that may be critical for the interpretation of results for pathogens with similar detection rates in patients and controls. The results indicate that the assessment of pathogen loads may improve the identification of agents causing gastroenteritis in children.

FIG 1

Box plot showing C_T values for agents targeted by real-time PCR among patients and controls. Boxes show the median (midline) and the 25th and 75th percentiles, and bars show the 10th and 90th percentiles. Numbers indicate the number of positive subjects for the respective agent.

FIG 2

Proportions of patients and asymptomatic controls younger or older than 18 months who tested positive by PCR. *, P < 0.10; **, P < 0.01; ***, P < 0.001.