Teska J D, Coster T, Byrne W R, Colbert J R, Taylor D, Venkatesan M, Hale T L
Geo-Centers Inc, Fort Detrick, Maryland, USA.
J Lab Clin Med. 1999 Aug;134(2):141-6. doi: 10.1016/s0022-2143(99)90118-x.
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 degrees C and 25 degrees C were comparable; however, 4 degrees C better inhibited overgrowth before streaking. To prepare for a community-based vaccine trial, volunteers were trained to self-sample fresh stool and to swab-inoculate a single quadrant of HE agar. The trial began with 36 volunteers ingesting 2.5 x 10(4) CFU of SC602 in bicarbonate buffer. During the study, volunteers inoculated HE plates with fresh stool, stored the plates at 4 degrees C, and delivered them to the laboratory within 48 hours. A microbiologist then streaked the HE for isolation, incubated the plates at 35 degrees C +/- 2 degrees 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.
在福氏志贺菌2a SC602疫苗的门诊安全性试验期间，开发并验证了一种用于评估活肠溶疫苗排泄情况的培养技术。初步研究表明，SC602可在赫氏肠道（HE）琼脂平板上回收，该平板的一个象限接种了接种粪便，保存长达48小时，划线分离，并在24±6小时内培养。在4℃和25℃保存的HE平板上的回收结果相当；然而，4℃能更好地抑制划线前的过度生长。为准备基于社区的疫苗试验，对志愿者进行了培训，使其能够自行采集新鲜粪便并在HE琼脂的单个象限进行拭子接种。试验开始时，36名志愿者在碳酸氢盐缓冲液中摄入2.5×10⁴CFU的SC602。在研究期间，志愿者用新鲜粪便接种HE平板，将平板保存在4℃，并在48小时内送至实验室。然后，微生物学家对HE进行划线分离，在35℃±2℃下将平板培养24±6小时，并通过与多价B抗血清的玻片凝集鉴定推定的福氏志贺菌菌落。用两种特异性DNA引物组通过聚合酶链反应在选定的分离株上确认了SC602的减毒遗传特征。第1天从20%的志愿者中检测到疫苗，到第4天增加到86%，到第7天除一名疫苗接种者外所有接种者都至少排泄过一次SC602。最晚首次检测到SC602发生在第7天，最长排泄发生在一名疫苗接种者的第33天，整个试验期间的排泄是间歇性的。试验在第35天用环丙沙星治疗终止。由于该方法的便利性，志愿者对自行采样和HE平板接种的依从性极佳，并且在排出疫苗的高回收率中，即时“床边”平板接种的优势明显。该方法可应用于其他需要准确采样排出微生物体的活肠溶疫苗试验。
