Evaluation of a Multiplex Real-Time PCR Assay for Detecting Major Bacterial Enteric Pathogens in Fecal Specimens: Intestinal Inflammation and Bacterial Load Are Correlated in Campylobacter Infections

Abstract

ABSTRACT A total of 1,056 native or Cary-Blair-preserved stool specimens were simultaneously tested by conventional stool culturing and by enteric bacterial panel (EBP) multiplex real-time PCR for Campylobacter jejuni , Campylobacter coli , Salmonella spp., and shigellosis disease-causing agents ( Shigella spp. and enteroinvasive Escherichia coli [EIEC]). Overall, 143 (13.5%) specimens tested positive by PCR for the targets named above; 3 coinfections and 109 (10.4%) Campylobacter spp., 17 (1.6%) Salmonella spp., and 20 (1.9%) Shigella spp./EIEC infections were detected. The respective positive stool culture rates were 75 (7.1%), 14 (1.3%), and 7 (0.7%). The median threshold cycle ( C T ) values of culture-positive specimens were significantly lower than those of culture-negative ones ( C T values, 24.3 versus 28.7; P < 0.001), indicating that the relative bacterial load per fecal specimen was significantly associated with the culture results. In Campylobacter infections, the respective median fecal calprotectin concentrations in PCR-negative/culture-negative ( n = 40), PCR-positive/culture-negative ( n = 14), and PCR-positive/culture-positive ( n = 15) specimens were 134 mg/kg (interquartile range [IQR], 30 to 1,374 mg/kg), 1,913 mg/kg (IQR, 165 to 3,813 mg/kg), and 5,327 mg/kg (IQR, 1,836 to 18,213 mg/kg). Significant differences were observed among the three groups ( P < 0.001), and a significant linear trend was identified ( P < 0.001). Furthermore, the fecal calprotectin concentrations and C T values were found to be correlated ( r = −0.658). Our results demonstrate that molecular screening of Campylobacter spp., Salmonella spp., and Shigella spp./EIEC using the BD Max EBP assay will result in timely diagnosis and improved sensitivity. The determination of inflammatory markers, such as calprotectin, in fecal specimens may aid in the interpretation of PCR results, particularly for enteric pathogens associated with mucosal damage and colonic inflammation.