Coinfection with Enteric Pathogens in East African Children with Acute Gastroenteritis-Associations and Interpretations

Abstract

Enteric coinfections among children in low-income countries are very common, but it is not well known if specific pathogen combinations are associated or have clinical importance. In this analysis, feces samples from children in Rwanda and Zanzibar less than 5 years of age, with (N = 994) or without (N = 324) acute diarrhea, were analyzed by real-time polymerase chain reaction targeting a wide range of pathogens. Associations were investigated by comparing co-detection and mono-detection frequencies for all pairwise pathogen combinations. More than one pathogen was detected in 840 samples (65%). A negative association (coinfections being less common than expected from probability) was observed for rotavirus in combination with Shigella, Campylobacter, or norovirus genogroup II, but only in patients, which is statistically expected for agents that independently cause diarrhea. A positive correlation was observed, in both patients and controls, between Ct (threshold cycle) values for certain virulence factor genes in enteropathogenic Escherichia coli (EPEC) (eae and bfpA) and toxin genes in enterotoxigenic E. coli (eltB and estA), allowing estimation of how often these genes were present in the same bacteria. A significant positive association in patients only was observed for Shigella and EPEC-eae, suggesting that this coinfection might interact in a manner that enhances symptoms. Although interaction between pathogens that affect symptoms is rare, this work emphasizes the importance and difference in interpretation of coinfections depending on whether they are positively or negatively associated.

Figure 1.

Number of pathogens detected by real-time polymerase chain reaction in patients and controls.

Figure 2.

Heat map showing degree of correlation for all pathogen combinations, comparing their detection as coinfection vs. mono-infection. The color represents the correlation coefficient (r), as indicated in the legend in the lower right corner. Correlations in patients are shown in the lower left part and those in controls in the upper right part. Statistical significance is indicated by asterisks: *P < 0.05, **P < 0.01, and ***P < 0.001. The corresponding details (counts and P values) are described in Supplemental Table 2.

Figure 3.

Threshold cycle (Ct) values for 84 samples that were positive for enteropathogenic E. coli (EPEC)-eae and EPEC-bfpA (upper) and for 153 samples that were positive for enterotoxigenic E. coli (ETEC)-eltB and ETEC-estA (lower). Pearson’s correlation coefficient (ρ) was 0.79 for eae and bfpA, and 0.61 for eltB and estA (P < 0.0001 for both). Red dots represent samples with less than 3.3 cycles difference in Ct value, suggesting that the target genes were present in the same bacterial strain (72 with bfpA and eae; 82 with eltB and estA). Samples with ≥ 3.3 cycles difference are shown as blue dots (12 bfpA and eae; 71 eltB and estA). Fifty-four samples that were positive for EPEC-bfpA alone, 177 for EPEC-eae alone, 303 for ETEC-eltB alone, and 94 for ETEC-estA alone were given a Ct value of 45 and are shown as black circles.