Comparative Evaluation of the Check&Trace Salmonella Test

Transcription

1 Comparative Evaluation of the Check&Trace Salmonella Test Doris Mueller-Doblies, Bacteriology Department, APHA Weybridge Woodham Lane, New Haw, KT15 3NB, UK

2 Aim of the project Defra-funded project between 2009 and 2012 Aim: To evaluate alternative molecular methods of Salmonella detection with the aim of providing evidence for a rapid Salmonella alerting system Three arrays were tested: a luminex based array (LUM array), a linear probe based array (SGSA) and a SNPbased array (Check & Trace)

3 Possible uses of the assay Additional quick screening tool for regulated serovars in poultry? Use instead of WK-LM scheme in countries where WK- LM is not established? EU legislation regarding Salmonella control in poultry?

4 Check-Points: Infection Prevention Since 2002, based in Wageningen, NL Proprietary diagnostic system: assays and equipment Salmonella typing Check& Trace Salmonella (former Premi Test Salmonella) Introduced in 2008 International certifications: AOAC-RI and OIE Multidrug-resistance (MDR) Check-MDR product range First product introduced in 2009 Extend platforms to meet main market needs Distributors and sales in > 20 countries 4

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7 Principle of C&T Discriminates Salmonella serovars through differences in their DNA sequence Even non-typable isolates can be assigned to a serovar, thereby offering a greater discriminative power than conventional techniques Each position on the microarray represents a specific DNA marker associated with a unique Salmonella target sequence Spots only become visible if the DNA markers exactly match the corresponding DNA sequences of the Salmonella isolate The combination of present and absent markers yields a pattern

8 Check&Trace Salmonella Workflow Crude DNA extraction Probe arms hybridize to the target sequence Ligation Clean up step (Exonuclease) Amplification (PCR) Adding Biotin at the 5 Hybridization and Detection of the target on the microarray (3 samples/tube-zip code) 8 Wattiau et al. Int Journal of Food Microbiology 2008

9 9 How does C&T work

10 How are the images interpreted The pattern of primary markers defines the pattern, and therefore the serotype. Each spot of the primary set has a number, based on the binary system The secondary set of markers are indicated with a letter, as shown above. The secondary set is used for confirmation of the result from the primary marker set, eg when the primary set of markers does not show a unique pattern, or when the outcome can be multiple serotypes. If the CTS software uses the secondary set, it automatically adds the. sign to the genovar score, as well as the letter(shown in the software) 10

11 the differences Generic differences No prescreening > serotypes vs. > CTS spot patterns C&T Salmonella patterns automatically interpreted Presence/absence limited number of spots C&T Salmonella flexible system: sequences can be added (recently added an extra marker for FljB) Av. Price per test in high volume setting* : 25 *High volume is more than 5000 samples/ year and depending on the country 11

12 Salmonella isolates used in the study Total of 2135 isolates of known serovar, including: 1. Panel of 104 isolates. These have been serotyped many times 2. Field isolates, mainly collected in non-salmonella isolates (E. coli, Staph. epidermidis, Staph. aureus) All isolates were serotyped according to WK-LM scheme at APHA 171 different serovars covered

13 The 104 known Salmonella serovars is a panel collated by the APHA over a number of years and has been used extensively in the development of different technologies at many collaborating institutes, including the PT array, the C&T array, the Luminex array and the SGSA (Salmonella geno-serotyping array) in their initial development.

14 Results First run: 93.44% match with WK-LM results 4.4% of inaccuracies were due to human error or reaction failures and samples were repeated After repeating these samples, the match could be increased to 97.84% All samples containing Salmonella were identified as Salmonella All non-salmonella were identified as non-salmonella

15 Problems identified Salmonella Genovar result only More than one species Salmonella proposed or related species proposed (includes overlaps) Partially correct antigenic formulae proposed

16 Salmonella Genovar result only This included: Samples, that are not recognised by the C&T software and C&T database Isolates that are Salmonella non enterica subspecies Scores, that were found to be unique to the APHA data set and could be added to the C&T software. It does, however, still recognise that a Salmonella species is present due to the DNA recognition of the O and H antigens

17 Examples - Salmonella Genovar result only Genovar score S. Agama, code specific to APHA data set, code seen throughout data set for S. Agama Genovar score 6176 S. Derby, code specific to APHA data set, code seen throughout data set for S. Derby Genovar score S. Kisarawe, code specific to APHA data set, code seen throughout data set for S. Kisarawe Genovar score S. London, code specific to APHA data set, code seen throughout data set for S. London

18 Mismatches S. Altona (8,20:r,[i]:z6) strain was typed as S. Poona (1,13,22:z:1,6:[z44] by C&T. Salmonella Binza (3,15: y : 1,5) is a known variant of S. Orion (3,10: y : 1,5) as determined by WK-LM. It would be considered an acceptable result using a microarray but noted that WK-LM can determine between the two. Salmonella California and Salmonella Banana are totally identical in structure; the difference between the two serotypes is determined by a biochemical reaction with inositol. The C&TS array can currently identify Banana but not California although this would also be considered an acceptable result.

19 Overlaps There are a known number of overlaps that have been seen with C&T They result from similarities between WK-LM antigenic formulae

20 Known overlaps S. Blegdam (9,12:g,m,q:-) with S. Enteritidis (1,9,12:g,m:-) S. Nitra (2,12:g,m:-) with S. Enteritidis (1,9,12:g,m:-) S. Kiel (1,2,12:g,p:-) with S. Dublin (1,9,12:g,p:-) S. Rostock (1,9,12:g,p,u:-) with S. Dublin (1,9,12:g,p:-)

21 Potential overlaps S. Kimuenza (1,4,12,27: l,v: e,n,x) with S. Bredeney (1,4,12,27: l,v: 1,7) S. Nagoya (6,8: b: 1,5) with S. Telelkibir (13,23: d: e,n,z 15 ) S. Sundsvall ( [1],6,14,[25]: z: e,n,x) with S. Telelkibir (13,23: d: e,n,z 15 ) Potential overlaps are those seen in the APHA data set; it may be possible to adjust the software to correct these as in some instances the software did not look at the secondary probe set.

22 Partially correct antigenic formulae proposed This includes strains that were partially typed using WK- LM, where C&T gave the nearest match E.g. O Rough:g,m:- was typed as S. Enteritidis by C&T PCR and sequencing of the amplicon would be required to verify the identity of this isolate

23 Commonly seen serovars Number of samples screened Overall Match S. Enteritidis % S. Dublin % S. Typhimurium % S. Derby % S. Kedougou % S. Virchow % S. Mbandaka % 4,5,12:i: % S. Java 1 100% S. Senftenberg % S. Montevideo % S. Tennessee 9 100% S. Agona 9 100% S. Havana 6 100% S. Ohio 7 100% Sub-total 1473

24 Discussion points C&T: Easy to perform test, less expertise needed compared to serotyping No need for antisera Result in one day Possibility of reaction failure and human error need to repeat samples Cheaper than KW: ~25 (= 18) No additional PCR needed to confirm monophasic STs Serotyping: Significant amount of expertise needed Time consuming: several days depending on serovar WK-LM serotyping in Weybridge: ~158 antisera produced ~60 somatic and 50 flagella antisera usually in stock ~50 antisera used every week Costs for 3 ml of antiserum vary between 6 and 120 (APHA) Shelf life in excess of 3 years Approximate costs across Europe: Additional tests necessary to confirm/exclude live vaccine strains

25 Discussion points (2) Would C&T be an alternative to WK-LM for developing countries who don t have any serotyping expertise at present? Vaccine strains need to be included and identified correctly Rough strains, e.g. O Rough:g,m:- Implications for poultry flocks under the Salmonella NCPs?

26 Special thanks to Meenaxi Sharma, Michaela Giles (APHA Weybridge) Rob Davies (APHA Weybridge) Wouter de Levita (C&T, The Netherlands)

27 Thank you for your attention