MLVA Update. Eija Trees, Ph.D., D.V.M. PulseNet Methods Development and Reference Unit Enteric Diseases Laboratory Branch CDC, Atlanta, GA
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1 MLVA Update Eija Trees, Ph.D., D.V.M. PulseNet Methods Development and Reference Unit Enteric Diseases Laboratory Branch CDC, Atlanta, GA
2 Presentation outline Status of the S. Enteritidis MLVA protocol External validation of the S. Typhimurium MLVA protocol MLVA training and certification MLVA testing of culture negative samples
3 Eight VNTR loci included in the proposed MLVA protocol for Salmonella Enteritidis VNTR Repeat size (bp) No. of repeats No. of alleles Diversity Minimum Maximum VNTR-1* VNTR-2* VNTR VNTR-5* VNTR VNTR VNTR VNTR-11* * Loci included in MLVA scheme developed by Dr. Bjorn-Arne Lindstedt (unpublished)
4 JEGX VNTR loci Strain ID DOC State VNTR_vals VNTR_cmp VNTR_cmp VN VN VN VN VN VN VN VN CDC_ SC CDC_ SC CDC_ SC CDC_ SC CDC_ SC CDC_ SC CDC_ SC CDC_ SC _07E _07E _07E _07E _07E _07E CDC_ CDC_ CDC_ CDC_ CDC_ CDC_ CDC_ JEG-1c (Raw Eggs) CDC_ CDC_ CDC_ CDC_ _07E _07E _07E _07E _07E _07E _07E _07E _07E
5 Most Common SE MLVA Pattern Out of 263 isolates tested 62 isolates clustered in one MLVA pattern JEGX JEGX JEGX JEGX JEGX JEGX PFGE patterns included in this cluster: JEGX (Includes isolates from 5 separate outbreak investigations) JEGX JEGX JEGX JEGX JEGX
6 External validation of S. Typhimurium MLVA protocol 14 labs participated (7 CEQ, 7 ABI) Lessons learned: All thermocyclers are not equal Calculated (tube) vs. block temperature control PCR tubes vs. plates Calibration issues Lid engineering problems Degraded primers cause inaccurate sizing in locus 3 Well optimized primer concentrations essential for ABI Fluorescence carry-over Inaccurate sizing
7 External validation ABI fragment sizes compared to CEQ Locus ABI 3130xl ABI 3100 ST3 +12 bp +12 bp ST5 Small: -2 bp Large: +2 bp Small: -2 bp Large: +2 bp ST bp +3-4 bp STTR10pl Small: +3 bp Large: -2 bp Small: +2 bp Large: -6 bp ST2 +12 bp +11 bp ST6 ST8 Small: +3 bp Large: +4 bp Small: bp Large: +/-1 bp Small: +4 bp Large: +6 bp Small: +/-1 bp Large: bp
8 How to fix the CEQ-ABI sizing discrepancy? VNTR copy number = Amplicon size (offset 1 + offset 2) repeat size
9 MLVA training CEQ training 3 bench training workshops in ; 12 individuals trained Internal training for CDC and APHL personnel in 2007 One-on-one training for international collaborators Plans for 2008?? ABI training Plans for 2008: 2 bench training workshops in fall 2008; max 4 individuals each
10 MLVA SOPs STEC O157 and S. Typhimurium lab for Beckman CEQ: PNL19 and PNL21 STEC O157 and S. Typhimurium analysis for Beckman CEQ: PND14 and PND15 MLVA certification: PNQ05
11 MLVA certification STEC O157 and S. Typhimurium peak files (lab) only Request certification sets by ing CDC will provide PCR primers, three control isolates and eight certification isolates Certified individuals will have to pass a yearly proficiency testing Same isolates used for both MLVA and PFGE PT
12 MLVA testing of culture negative samples STEC O157 in GN broth DNA template Spin down 500 µl of GN broth Resuspend the pellet in 100 µl dh 2 O Boil for 10 min Undiluted template and 1:10, 1:100 and 1:1000 dilutions tested by PCR
13 STEC O157:H7 cluster 0708MLEXH-1c MLVA_ VNTR loci Strain ID XbaI BlnI Source MLVA_composite CDC_K5294 EXHX CDC_K5297_100 CDC_K5342 EXHX CDC_K5343 EXHX UT_ EXHX CAOC_SB EXHX KY_ EXHX TX_TXB07BE EXHX CDC_K5344 EXHX EXHA EXHA EXHA EXHA EXHA EXHA EXHA EXHA NC/stool NC/stool culture neg NC/stool NC/stool UT/stool CAOC/stool IN/stool TX/stool NC/stool CDC_K5294 CDC_K5342 PFGE-XbaI
14 STEC O157:H7 cluster 0710NYEXH-1mlc (EXHX /EXHA ) MLVA VNTR loci Strain ID Source DOC MLVA_composite CDC_K5418 CDC_K5419 CDC_K5471 CDC_K5472 CDC_K5477 CDC_K5478 CDC_K5489 CDC_K5490 CDC_K5491 CDC_K5492 CDC_K5493 CDC_K5494 CDC_K5495 CDC_K5498 CDC_K5499 CDC_K5500 CDC_K5501 CDC_K5502 CDC_K5504 CDC_K5505 CDC_K5506 CDC_K5497_100 HI/stool HI/stool AL/Stool AL/Stool MD/Stool MD/Stool IL/Stool WI/Stool WI/Waukesha/Stool WI/Outagamie/Stool WI/Outagamie/Stool WI/Waukesha/Ground beef WI/Waukesha/Ground beef MN/stool MN/Washington/Stool MN/Dakota/Stool MN/Stool MN/stool MN/Hamburger MN/Hamburger MN/Hamburger WI/stool culture neg 9/5/2007 9/10/2007 9/21/2007 9/25/2007 9/16/2007 9/28/2007 9/27/2007 9/27/2007 9/26/ /1/ /8/ /10/ /10/2007 9/25/ /1/ /1/2007 9/26/2007
15 MLVA testing of culture negative samples S. Typhimurium in water DNA template Incubate wash filters overnight in peptone broth Spin down 10 ml of broth and resuspend the pellet in ~ 1 ml of broth remaining in the tube Transfer to microfuge tube, spin down and resuspend the pellet in 200 µl of BioRad Insta-Gene matrix Follow the purification protocol by the manufacturer with the exception of 4 min incubation times Undiluted templates and 1:50 dilutions tested by PCR
16 S. Typhimurium cluster 0803COJPX-1c VNTRcom VNTRcomp VNTR loci Strain ID XbaI Source DOC CDC_ /17/ CDC_ CDC_ CDC_ CDC_ Water 3/15/2008 3/16/2008 3/17/2008 CDC_ CDC_ CDC_ CDC_ /15/2008 3/17/2008 3/15/2008 3/17/2008 CDC_ CDC_ CDC_ CDC_ /16/2008 3/15/2008 3/16/2008 3/15/2008 CDC_ CDC_ CDC_ CDC_ Water 3/16/2008 3/17/2008 3/16/2008 CDC_K5861 CDC_K CDC_K5862 CDC_K5864 Water CDC_K5865 CDC_K CDC_K5867 Water CDC_K5868 CDC_K5870 CDC_K5871 CDC_K5873 CDC_K5874
17 Acknowledgements CDC Enteric Diseases Laboratory Branch Peter Gerner-Smidt Efrain Ribot Mary Ann Fair Kara Cooper Duncan MacCannell Patti Lafon Nancy Garrett Jessica Halpin Patti Fields John McQuiston Matt Mikoleit Susan Van Duyne Cheryl Bopp Kathy Greene PFGE Data Administration Team APHL Shari Shea Heather Green Kristy Kubota Applied Math Paul Vauterin State Public Health Laboratories of: CAOC CO DE FLA HI MA ME NC OH OK WA WI VA PulseNet Canada
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