Culture-Independent Diagnostic Testing: Implications for Public - PowerPoint PPT Presentation

Culture-Independent Diagnostic Testing: Implications for Public Health John Besser, PhD, MS Deputy Chief, Enteric Diseases Laboratory Branch Division of Foodborne, Waterborne, and Environmental Diseases National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention Collaborative Food Safety Forum November 3, 2011 National Center for Emerging and Zoonotic Infectious Diseases Division of Foodborne, Waterborne, and Environmental Diseases

Major Foodborne Illness Surveillance Systems Major Categories National case I. surveillance PulseNe FoodNet t II. Sentinel site NNDSS- FDOSS LEDS case surveillance III. Outbreaks NARMS CaliciNet Listeria Initiative NVEAIS

Estimates of Foodborne Illness

More than 75 labs in the PulseNet network

October 13, 2011

/wEPDwU true false Browse Browse 0x9DAE6 none False 0 /wEPDwU true false Browse Browse 0x9DAE6 none False 0 PulseNet International in 82 Countries

Bacterial Culture PulseNet New Zealand

Laboratory-based Surveillance

Rapid Tests http://www.meridianbioscience.com/diagnostic-products/foodborne

Rapid Tests

Rapid / Culture-Independent Tests versus Culture Rapid/non-culture t Ra e tes ests Cul ultur ure F ast S low Speed eed Inf nfrastruc uctur ure Minimal S ignificant need eeded ed Exper ertise e Minimal S ignificant req equired ed Low High Labor or c cos ost High Low Co Cost of m mater erial als

Rapid / Culture-Independent Tests versus Culture Culture o e or stan andar ard Rapid/cul ultur ure test sts ( s (e.g. indep epen enden ent test sts micros oscop opy) Sensit itiv ivit ity Gold standard Low to high Low to high, almost Specif ific icit ity High always different Int nterpretation on of of Usually S ignificant issues posit itiv ive f fin indin ings straightforward All pathogens allowed Range nge o of patho hoge gens ns Limited to specific by growth or test det etec ected ed pathogen tested conditions Allo llows for s susceptib ibilit ility Y es Generally no testing & ng & ge geno notyping? ng?

Demise of GC Culture • Rapid (hours) • Urine specimen (vs urethral swab) • Includes Chlamydia trachomatis • High sensitivity/specificity • No susceptibility data • Specimen incompatible with culture • Expensive

Impacts  Patient Management  Public Health Programs  Requiring accurate case counts o Burden o Attribution o Trends  Isolate-requiring o PulseNet / OutbreakNet o NARMS o Subtype-based attribution studies

Possible Solutions: Burden, Attribution, Trends  Understand extent of issue  Study test performance  Redefine case definitions

Preliminary Results Multi-State Campylobacter Diagnostics Study  A total of 3.1% (87/2772) of specimens were positive by culture Number er o of culture posit itiv ive sp specimens( s(n=87) Prem emier er ProspecT ICS CS XpecT cT PCR CR 60 P P P P P(n=56), Neg (n=3*) 13 Neg Neg Neg Neg P(n=4), Neg (n=8*) 2 Neg P Neg Neg Neg* 1 P Neg Neg Neg P 5 P P Neg Neg P 1 Neg P Neg P P 2 P P Neg P P 2 P P P Neg P 1 P Neg P P P Nos. of false 16 15 24 23 13 negatives  5/13 PCR negative specimens tested so far in a different Campylobacter -specific PCR assay. All are positive for C ampylobacter .

Impacts  Patient Management  Public Health Programs  Requiring accurate case counts o Burden o Attribution o Trends  Isolate-requiring o PulseNet / OutbreakNet o NARMS o Subtype-based attribution studies

Hazards of Inaction

Hazards of Inaction  Diminished ability to detect or respond to outbreaks  Significantly reduced pressure on industry to produce safe food  Less ability to guide regulatory focus  Less accurate data to determine burden / attribution

Post-culture STEC Surveillance System Germany; population 81,471,834 (July 2011 est.)

U.S. Sprout-Associated Outbreaks  >30 detected and investigated in 10 years  Relatively few cases  Investigation expertise developed  Stimulated regulatory focus

May 24, 2011; Doug Powell Blog

Potential Solutions  Short term: process changes to preserve isolates  Intermediate term: develop culture- independent, pathogen-specific subtyping/virulence targets  Longer-term: high-tech solutions (e.g. single cell sequencing and/or metagenomics

Potential Benefits of New Approaches  Less time to cluster detection  Less time to interview / tracebacks  Higher proportion of successful investigations  Some new technology (e.g. metagenomics) will allow….  Better understanding of disease causation and microbial interactions  Potential for studying host factors

The Surveillance Process Laboratory Reporting Takes Time Patient Eats 1 – 3 days Contaminated Food Contact with health care system: 1 – 5 days Patient Becomes Ill Diagnosis: 1 – 3 days Stool Sample Collected Shipping: 0 – 7 days Salmonella Identified Serotyping & DNA fingerprinting: 2 – 10 days Public Health Laboratory Receives Sample Case Confirmed as Part of Outbreak

The Surveillance Process Laboratory Reporting Takes Time Patient Eats 1 – 3 days Contaminated Food Contact with health care system: 1 – 5 days Patient Becomes Ill Diagnosis: 1 – 3 days Stool Sample Collected Shipping: 0 – 7 days Salmonella Identified Serotyping & DNA fingerprinting: 2 – 10 days Public Health Laboratory Receives Sample Case Confirmed as Part of Outbreak

Summary: Culture Independent Diagnostics Impact  High probability, high impact issue  Risks of inaction and benefits of change are significant

Adapted from Daryl Cagle, MSNBC: http://cagle.com/news/BirdFlu05/main.asp

Bacteria in Human Stools Up to 10 11 bacteria/ml; ~500 species Bacteroides fragilis Clostridium putrificum Streptococcus sp. (S. equinus) Bacteroides vulgatus Clostridium sp. (C. cadaveris) Streptococcus sp. (S. pyogenes) Bacteroides eggerthii Clostridium difficile Enterococcus faecalis Bacteroides sp. (B. fragilis) Eubacterium tenue Enterococcus gallinarum Bacteroides sp. (B. thetaiotaomicron) Clostridium bifermentans Lactobacillus acidophilus Bacteroides sp. (B. vulgatus) Clostridium sp. (C. sordellii) Weissella kandleri Bacteroides sp. (B. eggerthii) Peptostreptococcus (P. anaerobius) Lactobacillus fermentum Bacteroides sp. (B. uniformis) Fusobacterium nucleatumd Vagococcus fluvialis Cytophaga xylanolytica Eubacterium plautii Bifidobacterium infantis Bacteroides distasonis Eubacterium sp. (E. cylindroides) Bifidobacterium dentium Bacteroides sp. (B. distasonis) Streptococcus sanguis Bifidobacterium sp. (B. longum) Clostridium oroticum Streptococcus oralis Bifidobacterium adolescentis Clostridium sp. (C. nexile) Streptococcus intermedius Bifidobacterium pseudolongum Ruminococcus hansenii Lactococcus lactis subsp. cremoris Escherichia coli Ruminococcus productus Streptococcus sp. (S. mitis) Carnobacterium divergens Eubacterium ventriosum Leuconostoc lactis Lactobacillus maltaromicus Clostridium sp. (C. clostridiiforme) Streptococcus sp. (S. bovis) Salmonella sp. (S. typhi) Clostridium histolyticum Streptococcus sp. (S. equi subsp. equi) Enterobacter sp. (E. aerogenes) Clostridium sp. (C. beijerinckii) Streptococcus mutans Serratia sp. (S. marcescens) Clostridium sp. (C. butyricum) Streptococcus sp. (S. sanguis) Proteus sp. (P. vulgaris) Clostridium sp. (C. perfringens) Streptococcus sp. (S. salivarius) Klebsiella sp. (K. pneumoniae)

Random Shotgun Metagenomics Clinical Sample Total Host & Random Amplification Microbial NA & Sequencing

Meta genome Pan genome Core genome

Metagenomic Approach  Sequence all genetic material in sample  Assemble and identify contigs  Extract and analyze sequences of interest

Metagenomics: Potential Benefits  Fast, culture-independent  More pathogens / combinations of pathogens detected  Better understanding of microbial interactions  Potential for understanding host factors