Molecular profiling of bacterial isolates: Examples of PFGE in support to the epidemiology of VTEC human infections and source attribution European Reference Laboratory for E. coli including VTEC
The Italian HUS Registry Pediatric Nephrology Units (Italian Society for Pediatric Nephrology) ISS Active since 1988 Cases notified to the Registry with clinical and epidemiological information Stool and serum samples submitted to the NRL for E. coli for laboratory diagnosis of VTEC infection
HUS cases reported to the Italian Registry, by year - 1988-2015 Total HUS cases recorded: 963 Mean HUS cases per year: 32 (range 12 67) Age (Median): 26 months (range 0 15 yrs) Mean annual incidence rate: 0.35 cases per 100,000 (0-15 yrs) 0.92 cases per 100,000 (<5 yrs.) - RR=2.7
The Registry network and the information flow Nephrology Units ISS Reporting of HUS cases Sampling for VTEC investigation Pediatric Nephrology Unit Epidemiological investigation Italian HUS Registry ISS Diagnostic results EU and National Reference Laboratory for E. coli Investigation on HUS cases and outbreaks Connection with the Vet network with units all over the country and the regional competent authorities
Investigation on an HUS case (Food) January 31, 2013 a HUS case in North East Italy was reported to the national HUS registry Laboratory diagnosis: Isolation of a STEC O157 stx2+, eae+ Epi questionnaire: A food source suspected Salami- March 01, 2013 a second HUS case in the same area was reported to the national HUS registry Laboratory diagnosis: Isolation of a STEC O157 stx2+, eae+ Epi questionnaire: A food source suspected Salami- March 10, 2013 Sampling in the families house: Salami leftover brought to the laboratory Laboratory testing: Isolation of eight STEC O157 stx2+, eae+ strains
Molecular profiling of isolates PFGE_XbaI PFGE_XbaI 80 90 100 1500 800.00 600.00 500.00 350.00 300.00 250.00 200.00 150.00 100.00 40.00 20.00 83.9 96.6 92.0. O ED 157 882 Food ED 882 (Salami). O ED 157 879 Food ED 879 (Salami). O157 ED 880 Food ED 880 (Salami). O ED 157 878 Food ED 878 (Salami). O157 ED 873 HUS ED 873. O157 ED 881 Food 569/7 (Salami) ED 881. O157 ED 879 Food 569/5 (Salami) ED 879. O157 ED 876 Food ED 876 (Salami). O157 ED 887 Food ED 887 (Salami) FOOD (SAL. FOOD (SAL. Food (salame1) food (cotechino) human FOOD (SAL. FOOD (SAL. FOOD SOP. FOOD SOP. V V. O157 ED 883 HUS ED 883 HUMAN 72.4 81.4 91.9 87.7. O157 ED 873 HUS ED 873 HUMAN. O157 ED 875 Food 569/1 (Salami) ED 875 FOOD (SAL.. O157 ED 716 Farm ED 716 Environment abbeveratoio. O157 ED 589 Deer ED 589 cervo V 71.2. O ED 157 526 HUS ED 526. O157 ED 851 HUS ED 851. O157 ED 587 Deer ED 587 HUMAN HUMAN cervo V
Benefits of the joint analysis The PFGE-aided Epi investigation allowed to: Identify an unexpected source of VTEC O157 infections (pig meat); Recall the concerned product from the market; Reduce the number of possible cases.
Investigation on an HUS case (Animal contact) June 27, 2015 a girl with HUS was admitted to Hospital Isolation of a STEC O26 stx2+, eae+ Her sister was asymptomatic but PCR positive for O26, stx2 and eae genes
Investigation on an HUS case (Animal contact) Epidemioloigcal investigation The family spent holidays in the Alps from 13 June to 20 June. On 16 June the family visited a didactic farm where children had contact with animals and their environment and were directly involved in a demonstration of cheese production The farm was visited on 7 July. Faecal samples collected from 20 cows, 20 sheep, 3 pigs. 3 bulk milk and 3 cheese samples (bovine, ovine, caprine). Sheep faeces positive for stx2, eae and O26 genes. Three STEC O26 isolated!
Investigation on an HUS case (Animal contact) Molecular profiling of isolates PFGE_XbaI PFGE_XbaI 100 1500 800.00 600.00 500.00 400.00 250.00 200.00 150.00 100.00 40.00 20.00. ED 1032 O 26 Sheep ANIMAL (ovino). ED 1031 O 26 Sheep ANIMAL (ovino). ED 1027 O 26 HUS HUMAN New samples from 2016: isolation of the same strain (sharing the same PFGE profile) after one year
Benefits of the joint analysis The PFGE-aided Epi investigation allowed to implement hygiene measures in the didactic farm: Facilities for handwashing; Specific pathways for children to avoid contact with faeces soiled environment; Pasteurization of milk used for cheese preparation
Investigation on an HUS Outbreak Exclusion of a suspected food item
Outbreak Investigation: Community acquired Summer 2013, Puglia region o o o o o o STEC O26, vtx2a, eae+ 20 HUS cases (resident and non-resident) All children No secondary cases observed among households No evident Epi link between cases Suspected transmission: Food-dairy
Molecular profiling of isolates
Benefits of the joint analysis The PFGE-aided Epi investigation allowed to: Identify a multiple aetiology outbreak that couldn t be discerned by other methods such as serotyping; Avoid an unnecessary recall of food products leading to commercial disputes and affecting consumers confidence.
Towards the EU preparedness Actions undertaken by the EU RL for E. coli to improve the skill of EU NRLs on PFGE profiling of pathogenic E. coli strains Training sessions EQA
Proficiency tests on PFGE PT-PFGE 1 (with PT10, January-March 2013) 16 EU NRLs + Norway and Switzerland 11 E.coli strains Conducted jointly with the ECDC-FWD network PT-PFGE 2 (with PT11, June-September 2013) 23 EU NRLs + Norway and Switzerland 6 E. coli strains PT-PFGE 3 (with PT13, June-September 2014) 25 EU NRLs + Norway and Switzerland 7 E. coli strains PT-PFGE 4 (with PT15, April-June 2015) 28 EU NRLs + Norway, FYROM and Switzerland 10 E. coli strains PT-PFGE 5 (with PT18, November-December 2016) 17 EU NRLs + Turkey and Norway 10 E. coli strains PT-PFGE 6 foreseen for November 2017
PFGE Proficiency testing (EQA) Increase in number of participants and performance 30 25 20 N of NRLs 15 10 EXCELLENT GOOD FAIR POOR 5 0 PT1 PT2 PT3 PT4 PFGE PT rounds