Candy Rutherford Hamilton Regional Lab Medicine Program St Joseph s Healthcare, Hamilton

Transcription

1 Candy Rutherford Hamilton Regional Lab Medicine Program St Joseph s Healthcare, Hamilton

2 Using the BD Max and a Lab Developed Test

3 } Hektoen Enteric Agar 24hrs 37 o C } CIN Agar 24 hours 30 o C } Campy Agar 48 hours 42 o C } SMAC 24 hours 37 o C } Tube tests TSI, Urea, ONPG to screen for Shigella } Vitek MS for others } Subculture to blood agar for serology Salmonella, Shigella, Escherichia coli O157, } 48 hours for a negative report

4 } Lab developed for the usual suspects } Added stx and O157 specific primers/probes Salmonella ttr6 tetrathionate respiration Malorny B et al Applied and Environmental Microbiology Dec 2004 Shigella ipah invasive plasmid antigen H Kathy Luinstra Yersinia foxa ferrioxamine receptor Wang et al. JCM 2014 enterocolitica Campylobacter jejuni ccon cytochrome oxidase Toplak et al Journal of Applied Microbiology Campylobacter coli ceue iron ion transport Best et al FEMS Microbiology Letters stx 1 and stx 2 stx1 and stx2 genes Dr P Jayaratne O157 Somatic antigen Dr P Jayaratne

5 1. Manually added master mix 1 into position 2 green 2. Manually added master mix 2 into position 4 blue 3. Manually added user-defined buffer to position 3 (neut buffer from strip) TNA Strip Elution 12.5µl MM1 load top MM2 load bottom 4. Mixing of DNA with buffer (final volume 32.5 µl) 5. Mixing with MM : 12.5µl of TNA transferred to MM position 2 and 12.5µl of TNA transferred to MM position 4 2x MM 12.5 µl Wash 20 µl 2x MM buffer 12.5 µl Elution Neut. waste 6. Load PCR cartridge (~12.5µl) from each MM

6 } 20ul Lambda DNA Internal control } 5ul stool (loop) in TNA2- vortex 1 minute } Default extraction parameters } 25ul Max Neutralization buffer in snap-in 3 } Tube 1 master mix in green snap-in } Tube 2 master mix in blue snap-in } Luna Universal Probe qpcr master mix (M3004 New England BioLabs) } Tube 1 Salm, Shig, Yers, C.jejuni, IC } Tube 2 STX 1&2, C.coli, O157

7 } Gold Standard=Culture } Discordant resolution=bd Max IVD Extended Enteric panel } Optimization done on Rotorgene with easymag eluates } Adjusted and transferred assay to BD Max } LOD done using ATCC strains } LOD ~750 CFU/mL

8 Species (n=132 of 263) Culture Sensitivity PCR Sensitivity PCR Specificity Salmonella sp. (49) 93.8% 97.9% 99.5% Campylobacter sp. (54) 100% 100% 100% Shigella sp. (11) 63.6% 100% 99.2% Yersinia sp. (7) 57.1% 100% 100% STEC (11)* 20.0% 100% 99.6% * Culture only looks for E. coli O157

9 } Went live June 4, 2018 } June 4 to Nov 18: #tested = 2795 (same number tested in same period in 2017) } Started seeing non-o157 STEC

10 Salmonella Shigella 4 13 (isolated 5) Yersinia 5 13 Campylobacter E coli O157 STEC 4 6 (1 SF) O157 STEC neg 0 3 (1 not typed, O157:H25, O undetermined:h12) Non O157 STEC 0* 20 Other 2 (O157:nm) *SMAC culture only 2017

11 } S.boydii 22.7 (stx1 pos) grew on STEC plate } S.flexneri 18.6 } S.sonnei 20.5, 15.7, 18.3 } Not isolated:

12 } Confirmed LDT with BD Max IVD } Sent stool to TPHL for stx confirmation } Needed to isolate } CHROMAgar (Colorex STEC) produced by Dalynn in Canada } Grows O157 STEC and the Big Six } O26, O45, O103, O111, O121, O145 } Mauve to reddish colonies } Not much else except KES/Citro

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17 } Max IVD confirmed 20/20 } TPHL confirmed 19/20 } Stx1 = 13 } Stx2 = 3 } Stx 1 and 2 = 3 } Typing, hly and eae at NML for 4 O118:H2 hly+, eae+ O26:H11 hly+, eae+ (2 patients) O113:H4 hly+, eae-

18 } Colorex STEC Isolated 14 of 20 non O157 STEC (3 were very old before cultured) } Confirmed ID with ycct PCR for E. coli } Confirmed stx with uniplex assays (isolates) } No growth on STEC agar were cultured to Urine Orientation agar (BBL) } Isolated 3 more } TPHL isolated another (O113:H4) } Total 18/20

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21 } Working on an O typing PCR now } Primers from USDA paper } Ana Cabrera (LHS) helped with the design of the assay } Hoping it works with SYBR but may need probes especially group 2

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24 } Testing for STEC provides potentially relevant clinical information about the cause of diarrhea in the patient } non-o157 STEC were more prevalent than O157 STEC } CPHLN recommendation 2018 test for STEC } Need a better plate for Shigella

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