Detection of Salmonella Typhi in Bile by Quantitative Real-Time PCR

Abstract

In countries where the incidence of typhoid fever is high, fecal material from short-term carriers of Salmonella Typhi contaminates inadequately treated water supplies. As treated water supplies and improved sanitation become available, chronic (mainly gallbladder) carriers of S. Typhi become important. The objective of this study was to develop a method for detection of S. Typhi in bile by quantitative real-time PCR (qPCR) in patients undergoing cholecystectomy. We evaluated sensitivity and specificity of probesets that target oriC, viaB, fliC-d, STY0201, and stoD. We optimized DNA extraction from bile and compared the sensitivity of culture and our qPCR method to detect S. Typhi in bile samples containing various cephalosporins. With the use of an optimized DNA extraction technique, our limit of detection of S. Typhi in spiked human bile samples was 7.4 × 10² CFU/mL. We observed that S. Typhi could be detected by qPCR in samples containing cefazolin, cefotaxime, or ceftriaxone whereas culture could only detect Typhi in samples containing cefazolin but not cefotaxime or ceftriaxone. Our qPCR detection method for S. Typhi in bile should be preferred in areas where antibiotic usage is common. IMPORTANCE New Salmonella Typhi conjugate vaccines have been deployed, which will potentially lead to a fall in incidence rates of typhoid fever in endemic areas. Identification of chronic carriers of S. Typhi will be important as these individuals can be a potential source of transmission to susceptible persons. To address this public health concern, we have developed a novel method to detect S. Typhi in bile using real-time PCR. Our method can be used to identify carriers of S. Typhi among patients undergoing cholecystectomy (gallbladder removal surgery). The sensitivity of our molecular-based assay was superior to culture when performed in the presence of antibiotics commonly used during surgery. Our methodology will complement efforts to eliminate typhoid disease.

Keywords: bile; carrier; detection; gall bladder; typhoid.

FIG 1

Sensitivity of quantitative real-time PCR (qPCR) probesets with purified genomic DNA. Probesets were tested for sensitivity using decreasing concentrations of purified S. Typhi Ty2 gDNA. (A) STY0201. (B) oriC. (C) stoD. Data are shown as means ± standard deviations (SDs) from three biological replicates. Cq, quantification cycle.

FIG 2

Sensitivity of qPCR probesets on DNA extracted from spiked human bile samples. Human bile samples were spiked with varying concentrations of S. Typhi strain Ty2. DNA was extracted from bile samples using the optimized protocol, and qPCR was completed using either the oriC probeset (A), the STY0201 probeset (B), or the stoD probeset (C). Data are shown as means ± SDs from duplicate technical replicates.

FIG 3

Variability in qPCR sensitivity between human bile samples. Bile samples from human subjects in the United States (filled circles) or Chile (open circles) were spiked with 10⁵ CFU mL⁻¹ of S. Typhi. Quantitative PCR was run on DNA extracted from spiked samples in duplicate using each of the four test probesets. Data are shown as means ± SDs; *, P < 0.05.