AFLP analysis of Bacillus cereus, Clostridium perfringens, Clostridium novyi from drug users, Jim McLauchlin PHLS, London.

Transcription

1 AFLP analysis of Bacillus cereus, Clostridium perfringens, Clostridium novyi from drug users, Jim McLauchlin PHLS, London.

2 AFLP (amplified fragment length polymorphism) Restriction enzyme restriction site Genomic DNA DIGESTION Restriction fragments LIGATION OF ADAPTERS selective nucleotides adapter Ligated restriction fragments Selective primer AMPLIFICATION GEL ANALYSIS OF AMPLIFIED FRAGMENTS

3 AFLP analysis of B.cereus from drug users, single primer (HI-A) analysis. Source C try Date Serotype AFLP type 1 blood E June 2000 NT I 2 aspirate S June 2000 NT II 3 tissue S June III 4 tissue S June 2000 NT II -1 5 Heroin E June 2000 NT IV 6 Heroin S June 2000 NT II Culture number Culture number

4 AFLP typing of B.cereus Six isolates tested, Four from drug users two from heroin Four AFLP patterns identified Three AFLP types recovered from four patients Heroin and corresponding patient plus second patient s isolates all indistinguishable by AFLP Unique AFLP type from heroin seized in England (?unrelated to outbreak)

5 AFLP gels C.perfringens

6 AFLP Analysis of Clostridium perfringens, single primer (HI-G) analysis

7 AFLP Analysis of C.perfringens C P A F L P

8 AFLP Analysis of C. perfringens from drug users 40 isolates tested from 13 drug users one used syringe contents two heroin samples 19 isolates serotypable, 9 types identified serotyping and AFLP results corresponded

9 AFLP Analysis of C.perfringens A: multiple isolates from a sample of heroin A D C B-D: multiple isolates from three patients B

10 AFLP Analysis of C. perfringens from drug users 16 AFLP patterns distinguished same AFLP type identified in 5 of 8 sources where multiple isolates were recovered Multiple AFLP types were recovered from three patients (1 with three and two with two types) One AFLP type common to two patients and the syringe One AFLP type common to two patients AFLP types recovered from two heroin samples differed and were unique

11 C.novyi AFLP analysis EC-A EC-C EC-T HI-C HI-G AFLP pattern associated with drug users in 2000

12 AFLP analysis of 43 C.novyi isolates using multiple primer (HI-C, HI-G, EC-A, EC-C, EC-T) 23 from 22 drug users ( ) 1 in 1999, 1 in from wounds, 1 from blood and 1 from a leg abrasion (1988,1991,1995, 2000) 3 NCTC cultures (1920, 1944, NK) 1 isolate from food (1989) 7 isolates from the Wellcome collection (1918, 1941, NK) 5 isolates from animals

13 AFLP Analysis of C.novyi IDU 2000 (22) Wellcome collection (7) Other human infections (4) NCTC collection (3) Animals (5) Food (1)

14 AFLP analysis of C.novyi Method reproducible 23 AFLP types distinguished unique types were found in 4 drug users» 3 in England (1999, 2000 and 2001)» 1 in the Republic of Ireland (2000) 3 NCTC cultures 2 wound and blood isolates 1 isolate from food 4 isolates from animals 7 Wellcome isolates

15 AFLP analysis of C.novyi AFLP type A recovered from 18 drug users 3 in England (Feb - May 2000) 13 in Scotland (Apr-Aug 2000) 1 Republic of Ireland (June 2000) 1 in Norway (Aug 2000) 1 other patient (UK 2000)

16 Clinical presentation: Cases in drug injectors who presented to hospital with: Case Classification Possible Probable Definite abscess or other significant inflammation at an injecting site YES YES YES severe inflammatory process (large abscess/extensive oedema) at or around the injecting site NO YES/NO YES severe systemic inflammatory reaction with evidence of multi-organ failure and a high white cell count NO NO/YES YES

17 AFLP analysis of C.novyi: Clinical presentation amongst drug users AFLP Type A 12 cases definite 6 cases probable Other AFLP types 2 cases probable 2 cases possible

18 AFLP analysis of B.cereus, C.perfringens C.novyi from drug users B.cereus Heterogeneous group Link between from heroin and blood of a patient Possible link with second patient C.perfringens Heterogeneous group No or limited evidence of commonality C.novyi homogeneous group of isolates from 2000 drug users differed from isolates from other sources except for 1 patient in 2000

19 C.novyi AFLP type A Possible explanations: Common source contaminated Clonal selection adaptive advantage in spore germination, survival or growth following the drug preparation and injection, production of a more severe clinical presentation.

20 Acknowledgements PHLS Central Public Health Laboratory CFL Amar, MM Brett, RC George, O Mapamugo, V Mithani PHL Wales J Salmon, J Brazier Other colleagues elsewhere who provided cultures, clinical material, data or other contributions