Novel self-sampling culture method to monitor excretion of live, oral Shigella flexneri 2a vaccine SC602 during a community-based phase 1 trial

Abstract

A culture technique for assessing the excretion of live enteric vaccines was developed and verified during an outpatient safety trial of the Shigella flexneri 2a SC602 vaccine. Preliminary studies showed that SC602 could be recovered on Hektoen enteric (HE) agar plates that had been inoculated with seeded stools in one quadrant, held for up to 48 hours, streaked for isolation, and incubated for 24 ± 6 hours. Recovery results on HE plates held at 4°C and 25°C were comparable; however, 4°C better inhibited overgrowth before streaking. To prepare for a community-based vaccine trial, volunteers were trained to self-sample fresh stool and to swabinoculate a single quadrant of HE agar. The trial began with 36 volunteers ingesting 2.5 × 104 CFU of SC602 in bicarbonate buffer. During the study, volunteers inoculated HE plates with fresh stool, stored the plates at 4°C, and delivered them to the laboratory within 48 hours. A microbiologist then streaked the HE for isolation, incubated the plates at 35°C ± 2°C for 24 ± 6 hours, and identified presumptive S. flexneri colonies by slide agglutination with poly-group B antiserum. The attenuating genetic signature of SC602 was confirmed on selected isolates with the polymerase chain reaction with two specific DNA primer sets. Vaccine was detected from 20% of volunteers on day 1, increasing to 86% by day 4, and all but one vaccinee had excreted SC602 at least once by day 7. The latest initial SC602 detection occurred on day 7, the longest excretion occurred in one vaccinee on day 33, and excretion throughout the trial was intermittent. The trial was terminated by ciprofloxacin treatment on day 35. Volunteer compliance with self-sampling and HE plating was excellent because of the convenience of the method, and the advantage of immediate “bedside” plating was evident in the high recovery rate of excreted vaccine. This method can be applied in other trials of live enteric vaccines that require accurate sampling of excreted organisms.