The laboratory diagnosis of food-borne zoonoses

Transcription

1 8/3/212 The laboratory diagnosis of food-borne zoonoses Current practice and pitfalls Here s the groceries, Marge. I'm not gonna risk EATING anything until this food poisoning scare is over. Estimated Burden of Diarrhoeal & Foodborne Illness OzFoodNet Survey (circa 2) Annual no. diarrhoeal illnesses: 17.2 million / year Foodborne: Foodborne illness: 32% (multiple modes of transmission) 5.4 million / year Episodes per person:.3 / year Known Pathogens: 28% Hospitalisations: Deaths: 15, / year 8 / year EID 25; 11: Number of Cases Selected Enteric Pathogens Salmonella Shigella Cryptosporidium Australian National Notifiable Diseases Surveillance System Number of Cases Selected Enteric Pathogens Typhoid Cholera Australian National Notifiable Diseases Surveillance System GE due to known pathogens in Australia in a typical year circa 2* Agent Cases % Food related Median no. foodborne non- E. coli % 563, spp % 28, Salmonella spp % 81, Aeromonas spp % 9,8 3 65% 1,9 spp % 1,62 Vibrio parahaemolyticus 18 71% 74 Shigella spp. 32 1% % 446, Rotavirus 241 2% 4,7 Astrovirus/adenovirus 19 1% 17,5 Cryptosporidium parvum 271 1% 25, Giardia lamblia 43 5% 2,4 Clostridium perfringens 43 1% 43, Staphylococcus aureus 141 1% 14,2 Bacillus cereus 69 1% 6,9 OzFoodNetSurvey EID 25;8:

2 8/3/212 Estimated Food Related Cases of Selected Pathogens Agent Median no. foodborne (%) non- E. coli 563, 39% Clostridium perfringens 43, 3% Staphylococcus aureus 14,2 1% Bacillus cereus 6,9 % spp. 28, 14% Salmonella spp. 81, 6% Cryptosporidium parvum 25, 2% Giardia lamblia 2,4 1% Aeromonas spp. 9,8 1% Shigella spp. 3 % 446, 31% Astrovirus/adenovirus 17,5 1% Rotavirus 4,7 % 1,9 % spp. 1,62 % Vibrio parahaemolyticus 74 % NOT TESTED ROUTINE ON REQUEST OzFoodNetSurvey EID 25;8: MBS Schedule Culture ± microscopy without concentration techniques of faeces using at least 2 selective or enrichment media and culture in at least 2 different atmospheres including (if performed): (a) pathogen identification and antibiotic susceptibility testing (b) the detection of clostridial toxins; and - 1 examination in any 7 day period Fee: $53.25 Benefit: 75% = $ % = $ Microscopy of faeces for ova, cysts and parasites that must include a concentration technique, and the use of fixed stains or antigen detection for cryptosporidia and giardia 1 of this item in any 7 day period Fee: $33.65 Benefit: 75% = $ % = $ Microscopy of faeces for ova, cysts and parasites using concentration techniques examined subsequent to item on a separately collected and identified specimen collected within 7 days of the examination described in examination in any 7 day period Fee: $19.25 Benefit: 75% = $ % = $ MBS Schedule Detection of Clostridium difficile or Clostridium difficile toxin (except if a service described in items 69345, has been performed) Fee: $28.85 Benefit: 75% = $ % = $24.55 Service area: 2,,5 km % of all and Salmonella notifications Detection of a virus or microbial antigen or microbial nucleic acid Fee: $28.85 Benefit: 75% = $ % = $ tests described in item Fee: $36.1 Benefit: 75% = $ % = $ tests described in item Fee: $43.35 Benefit: 75% = $ % = $ No. Tested MCS2-8% Faeces MCS 1 & 2: n=259,632 samples Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec $ Supply collection information and containers Ensure appropriate transport Data entry Laboratory testing Complex algorithms Significant isolate identification Antibiotic susceptibility Issue report Pathologist input education, Dx, Rx Reference Laboratory Isolates Salmonella, Shigella,,,, Rotavirus positive faeces Researchers - isolates Notify significant results (fax/hardcopy) Denominator data Ensure QC and QAP 2

3 8/3/212 Severe diarrhoea Prolonged diarrhoea Fever or dysentery Young, Elderly Immunosuppressed Outbreak situations Who to test Routine Macroscopic Description Direct Microscopy Leuc RBCs Ova Cysts Parasites Concentrate Microscopy Ova Cysts Parasites EIA : Giardia Cryptosporidium Positive Negative Salmonella Aeromonas Shigella Cryptosporidium, Giardia Confirmation Modified Acid Fast Stain Giardia trophozoite Cryptosporidium acid fast stain Dientamoeba fragilis Faecal Vibrio /TCBS /CIN /CCFA /Frasers O:157 histolytica 17 3

4 8/3/ Bacterial Enteropathogens ,212/254,94 (7.5%) isolation rate No. of isolates Salmonella Aeromonas 23% 2% Other 4% 53% Shigella Pleisiomonas Arcobacter Edwardsiella EHEC Rotavirus: 1,469 / 2,766 : 563 / 18,483 4 Rotavirus 9. No. detected % Positive D.fragilis (QH) s (eae eha stx1 stx *) 17/952 Samples 25 eae ehe stx1 stx2 3 No. positive s notifications by State 14 ACT NSW NT Qld SA Tas Vic WA 12 No. tested eha eae stx 2 stx 1 No. of notifications Our last remaining gel based PCR Australian National Notifiable Diseases Surveillance System 4

5 8/3/212 PCR N = 2,545/24,543 (1.4%) Faecal Parasites ,299/255,18 (12.3%) No. of PCR positive Stool Community Hospital Community % Positive Hospital % Positive % Positive No. Detected Giardia 18% Blastocystis 64% Cryptosporidium 7% D.fragilis 3% Other 7% Endolimax nana coli histolytica Iodamoeba butschlii Enterobius vermicularis Hymenolepsis nana ova Chilomastix Cyclospora Taenia sp ova larvae Ascaris spp Trichurus ova Faecal Concentrate: Mini PARASEP SF EIA Broth: (Rotavirus,, ) Mol Path Broth:, ) (Modified Trichrome); (AFBs); Culture XLD, Aeromonas, Campy Selenite: Broth Sal ChromAgar Smear (Trichome, Fe Haematoxylin) Additional plateson request: : CIN Vibrio: Broth TCBS : ChromAgar : SorbitolMacConkeyAgar : Fraser broth Oxford & Blood Agar Complex Algorithms for Identification Challenges Increasing number of potential pathogens Emerging pathogens Those not detectable - toxin mediated foodborne disease, E.coli infections Low sensitivity viral antigen kits Rationalisation in order to make ends meet Complex algorithms Difficult to individualise testing Labour intensive Many staff involved in single specimen processing (inefficient) Reference laboratories making more and more demands for isolate characterisation Slow turnaround 72 hours + 5

6 8/3/212 Micro Gone Molecular A Revolution in the Microbiology Laboratory Commercial Tests Appearing DiarrheagenicE. coli/shigella: lt/st(etec) bfpa(epec) eae(epec, ) E. colio157:h7 ipah(eiec/shigella) paaplasmid (EAEC) stx2() stx1(, S. dysenteriae Considerations EasyScreen Enteric Bacteria Detection Kit Salmonella,, Shigella,, and C. difficile. Salmonella, Shigella, Aeromonas,, and EasyScreen Enteric Parasite Detection Kit E. Histolytica, Dientameoba, Cryptosporidium, Giardia, Blastocystis and. EasyScreen Enteric Virus Detection Kit I/II, Astrovirus,, Sapovirus and Rotavirus Cost Through put stool extractions slow and laborious Inhibitors in faecal samples Requirement for faecal concentration in MBS Increased detection rate including multiple pathogens in the same specimen Significance of increased truly positive cases Economic modelling suggests rapid technologies + additional culturing unlikely to be cost effective when testing one organism at a time 1 Extra investigations when pathogen not targeted 1. Health Technology Assessment NHSW R&D HTA Programmes Sept 27 Proposed workflow Stool received Portion to PBS -> TNA extraction Cluster tubes incubated : Cary-Blair media Campy Broth Selenite Broth High density PCR for 8 enteropathogen targets Report Viral results Report Parasite results Report Negative Bacterial results Report Presumptive Bacterial Positives Reflex culture/sensitivities Fewer staff, Quicker TAT, increased range of pathogens, increased sensitivity 24hrs PCR Pos Bacteria Presumptive Some future issues Loss of isolates for characterisation in Reference Laboratories Antibiotic susceptibility New typing methods More epidemiology for reference laboratories & public health units Surveillance must take into account changing techniques Bioinformatic expertise to process the data A solution? 2 tier remuneration Non culture techniques Cultures referred for public health analysis diagnostic laboratories receive some of the public health funding Viruses, Parasites, Neg Bacteria ID/sensitivities Jones TF et al Emerging Infectious Diseases Vol. 18, No. 3, March Stigi KA et al Emerg Infect Dis. 212;18: M ikanatha NM, Emerg Infect Dis. 212;18:

7 8/3/212 Pathogens involved Financial constraints Current methods Manual Complex algorithms Limitations Future directions Issues The Humble Faeces Lactobacillus fermentum(pcr control) Salmonella enterica Salmonella spp. spp. jejuni coli Arcobacterspp. Brachyspirahyodysenteriae Brachyspirapilosicoli Shigellaspp. Shigelladysenteriae Shigellaflexneri Shigellasonnei enterocolitica monocytogenes Vibriocholera Vibrioparahaemolyticus Escherichia coli EHEC Escherichia coli EPEC Escherichia coli ETEC Escherichia coli EIEC Escherichia coli EaggEC Aeromonashydrophila/spp. Plesiomonasshigeloides Clostridium difficile Targets Rotavirus A/B/C F 4/41 GI/GII/GIV GI GII Sapovirus Mamastrovirus/Astrovirus Picobirnavirus Aichivirus Coronavirus Parechovirus Enterovirus histolytica dispar moshkovskii coli Iodamoebabutschlii Endolimaxnana hartmanni Giardialamblia Dientamoebafragilis Chilomastixmesnili Retortamonasintestinalis Cryptosporidium spp. Cryptosporidium parvum Cryptosporidium hominis Blastocystishominis Cyclosporacayetanensis Isosporabelli Isosporanatalensis Sarcocystisspp. Enterobiusvermicularis Ascarislumbricoides Ancylostomaduodenale Necatoramericanus Trichostronglyus spp. stercoralis Trichuristrichiura Diphyllobothrium latum Taeniasolium Taeniasaginata Hymenolepsis nana Hymnenolepsis diminuta Dipylidium caninum Fasciolopsisbuski Echinostomailocanum Heterophyesheterophyes Metagonimus yokogawai Clonorchissinensis Fasciolahepatica Opisthorchisviverrini Paragonimus westermani Paragonimus mexicanus Schistosomaspp. (incl mekongi, intercalatum ) Schistosomamansoni Schistosomajaponicum Quanstudio/OpenArray Workflow Overview (post sample RT) At Life Technologies The custom assays are spotted onto OpenArray plates in California At Sullivan Nicolaides Load your samples with Master Mix onto the OpenArray plate Time to results: 3h OpenArray plates staked into cases Place upto 4 OpenArray plates in Quantstudio and cycle The only user intervention step Brisbane Stocks maintained at SNP and LT Brisbane Results Private Pathology Australia 212 St John of God WA St John of God Vic St Vincents Pathology Other 15% Healthscope 2% Sonic 35% Douglass Hanley Moir Barret & Smith Southern IML Sullivan Nicolaides Pathology Melbourne Pathology Clinpath Clinipath Capital Healthscope Victoria Healthscope NSW Healthscope Tasmaniia Primary 3% Queensland Medical Laboratories Laverties Dorevitch Abbott Pathology Westerns Pathology 7