Evaluation of Salmonella Typhi Detection Methods

Primary influent wastewater grab samples were collected from a local wastewater treatment plant in Seattle, WA, that processes 90 million gallons per day (mgd) during the dry season and can process more than 300 mgd during the rainy season,⁴⁰ resulting in matrix variability. Grab samples (7–14 L per carboy) were stored at 4°C until processing (conducted within 72 hours of collection). Varying amounts of Ty21a or Ty2 were seeded to 10 mL 1× phosphate-buffered saline, vortexed (30 seconds), and seeded into varying volumes of wastewater to reach the target concentrations. The final concentration of Ty2 or Ty21a in the seeded wastewater varied depending on the experiment and ranged from approximately 0.001 to 10,000 colony-forming units (CFU) per milliliter (Supplemental Figure 1). Methods tested at the same concentration level used primarily the same initial wastewater matrix with replicates of three or six to enable comparison between the methods. The seeded concentrations were assessed in parallel for each experiment via spread plating of 100 µL of relevant dilutions on LB-Miller agar (Ty21a) or LB-Miller agar with a supplemental aromatic amino acid mix and 50 ng/mL ferrioxamine E (Ty2). The seeded wastewater was thoroughly mixed and then distributed using a peristaltic pump while continuously shaken for processing by 1) filter cartridge, 2) differential centrifugation, 3) grab enrichment, 4) membrane filtration, and 5) Moore swab methods (Figure 1). The methods were evaluated in a laboratory setting, and factors considered included feasibility of performing these methods in other settings and Salmonella Typhi detection by qPCR. Feasibility was based on processing time, equipment, and supplies needed. Active time was defined as the total hands-on time required to conduct an individual method, excluding time in which personnel could complete other tasks. These periods could include incubation, shaking, and filtration.