Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries

Abstract

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥ 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10(4) ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.

Fig 1

(A) Standard curves for set 1 (n = 877). For each plate, a standard curve was constructed by plotting the cycle threshold (C_T) values on the y axis and the known log-dilution of DNA of 1.0 to 0.00032 ng per ml in 5-fold dilutions on the x axis. (B) Decile graph for set 1 (n = 877). The quantities of ipaH gene copies were split into deciles with 88 or 87 samples per decile. The proportion of cases, samples positive for Shigella by culture, and samples positive for blood were plotted for each decile.

Fig 2

(A) ROC curve for model case-control status versus qPCR ipaH gene quantities from set 1 (n = 877) (B) ROC curve for model Shigella culture status versus qPCR ipaH gene quantities from set 1 (n = 877). Curves were plotted by calculating the sensitivity and 1-specificity of qPCR compared to case-control status (panel A) or Shigella culture status (panel B) at each unit increment in qPCR ipaH gene quantity. Squares indicate the points on the curve cutpoint of approximately 29,000 ipaH copies. The diamond in panel A indicates the values for sensitivity and 1 − specificity for culture.

Fig 3

qPCR results from spiked experiment. Two stool samples of 200 mg were each spiked with 10⁷ or 10⁵ CFU of Shigella flexneri. After DNA isolation, the samples were diluted and amplified using the primers for ipaH.