Presented by: Elise B. Smith, B.S., MT(ASCP) Virginia Division of Consolidated Laboratory Services

Transcription

1 Clostridium botulinum PFGE Update 13 th Annual PulseNet Update Meeting; Snowbird, UT Session V; September 24, 2009 Presented by: Elise B. Smith, B.S., MT(ASCP) Virginia Division of Consolidated Laboratory Services Prepared by: Susan Maslanka National Botulism Laboratory Preparedness Team, Lead

2 Botulism is rare in the US Background Only about 25 foodborne cases per year; mostly due to home prepared food products Foodborne botulism usually occurs locally as a result of mishandling by the consumer Most US cases are infant botulism; ~100 per year Foodborne botulism is a public health emergency Widely distributed contaminated product could effect many patients Many foods are distributed across state lines and sometimes internationally Intentional contamination of food products is of concern to the Department of Homeland Security Most foodborne botulism is caused by botulinum toxin type A Toxin type can t be used to link cases that have no direct common o exposure such as sharing a meal. Molecular Subtyping will be the only way to link patients during a multi-state outbreak

3 Carrot Juice Outbreak, cases GA; 1 case FL; 2 cases Canada All Type A botulinum toxin GA cases consumed CR1 FL case consumed CR2 Unable to compare Canada strains with US since standard d PFGE protocol not available at the time - GA Stool #1 - GA Stool #2 - GA Stool #3 - CR1 - CR2 CR1 is indistinguishable from all 3 GA stool isolates. CR2 is distinct from CR1 and the GA clinical isolates.

4 Hot Dog Chili Sauce Outbreak, cases TX; 2 cases IN; 3 cases OH All cases confirmed type A botulinum toxin and reported consumption of hot dog chili sauce Additional 6 cases of type A botulism in the US within the same time frame 2 reported as having consumed the product 4 with less definitive food history Some reports of product consumption were not considered reliable First outbreak associated with a commercially canned product in 33 years Investigations were conducted in different states Some states had only positive clinical samples; no confirmed food source Standard PFGE method may have helped link cases in this multistate outbreak

5 Protocol Status In Final Review

6 Enzyme Info Sma I (10U/μl stock concentration) Xho I (20U/μl stock concentration) Xba I (20U/μl stock concentration) Reagent μl/plug Slice Reagent μl/plug Slice Reagent μl/plug Slice Sterile Ultrapure Water 10X Buffer 4 Enzyme (50 U/sample) Total Volume Incubate 175 μl 20 μl 5 μl 200 μl 25 o C 4 hours Sterile Ultrapure Water 10X Buffer 4 10X BSA Enzyme (100 U/sample) Total Volume Incubate 155 μl 20 μl 20 μl 5 μl 200 μl 37 o C 3 hours Sterile Ultrapure Water 10X Buffer H 10X BSA Enzyme (60 U/sample) Total Volume Incubate 157 μl 20 μl 20 μl 3 μl 200 μl 37 o C 2 hours Primary Secondary Standard

7 Running Conditions Select the following conditions on the CHEF MAPPER: Auto Algorithm low MW - 30 kb high MW kb Initial switch time: 0.5 sec Final switch time: 40 sec Run time: 10 well gel hours 15 well gel - 19 hours Note: The electrophoresis run times recommended d above are based on the equipment and reagents used at the CDC. Run times may be different in your laboratory and will have to be optimized for your gels, so that the lowest band in the S. ser. Braenderup H9812 standard migrates cm from the bottom of the gel.

8 Example Gel Sma I

9 Example Gel XhoI

10 Database Status We have a database!!! Currently working to populate database with the 200 or so patterns available

11 Select Agent Limitations As a Select Agent C. botulinum testing is restricted to NSAR approved facilities and personnel Viable organisms are present until electrophoresis. All material must be handled in accordance with Department of Health and Human Services, 42 CFR Part 72 and 73; Possession, Use, and Transfer of Select Agents and Toxins; Final Rule. Civil monetary penalties may be imposed for non-compliance; 42 CFR Part A single spore is considered a Select Agent. PFGE must be performed in an approved space with restricted access. Only authorized personnel can perform PFGE through to the electrophoresis portion of the protocol, at that point, the gel may be stained, imaged and analyzed in the regular PFGE laboratory. Stored plugs must be handled in compliance with Select Agent Inventory requirements. (As the organism remains viable within the plug until electrophoresis is completed.) Cultures or plugs cannot be transferred into a non-approved space or handled by non-approved staff even within the same facility.

12 Where do we go from here? The CDC C. botulinum PFGE Team, welcomes any labs that are interested in using this method How training will be conducted depends on the number of labs interested Certification procedure should be completed within the next couple of months CDC will accept the following isolate types for PFGE: Strains from any foodborne botulism investigation to assist states with their investigations (high priority testing). Any strain from other botulism cases (infant, wound, or foodborne) either current or archive strains to be used to populate the database with PFGE patterns which will represent US distribution of C. botulinum.

13 CDC CDC C. botulinum PFGE Team Carolina Luquez Botulism Outbreak Investigations Unit, Chief PFGE Point of Contact Susan Maslanka National a Botulism Laboratory ato Preparedness ed ess Team, Lead sht5@cdc.gov Lavin Joseph PFGE Testing Lead gyu4@cdc.gov Loretta McCroskey PFGE Method Developer FastTrack Drugs and Biologics, LLC lmm1@cdc.gov Steven Stroika Database Manager fru3@cdc.gov

14 Acknowledgement CDC Bot lab Loretta McCroskey Carolina Luquez Lavin Joseph PulseNet Methods Development and Reference Unit for thoughtful critique of method PulseNet Database Unit- for establishing the database and for their patience in guiding the bot lab into this new territory Virginia Division of Consolidated d Laboratories Denise Toney Elise Smith For all the hard work in improving the protocol including the umpteenth iteration. Identifying the Select Agent issue. Preparing a detailed protocol. Running all those strains AND, giving this presentation

15 Thank you for your attention The findings and conclusions in this presentation are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention