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

I.

National case surveillance

• II. Sentinel site

case surveillance

• III. Outbreaks

PulseNe t NARMS

Listeria Initiative

NNDSS- LEDS FoodNet FDOSS CaliciNet NVEAIS

Estimates of Foodborne Illness

More than 75 labs in the PulseNet network

October 13, 2011

PulseNet International in 82 Countries

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Bacterial Culture

PulseNet New Zealand

Laboratory-based Surveillance

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

Rapid Tests

Rapid Tests

Ra Rapid/non-culture t e tes ests Cul ultur ure Speed eed

F ast S low

Inf nfrastruc uctur ure need eeded ed

Minimal S ignificant

Exper ertise e req equired ed

Minimal S ignificant

Labor

• r c

cos

• st

Low High

Co Cost of m mater erial als

High Low

Rapid / Culture-Independent Tests versus Culture

Rapid / Culture-Independent Tests versus Culture

Culture o e or stan andar ard test sts ( s (e.g. micros

• scop
• py)

Rapid/cul ultur ure indep epen enden ent test sts Sensit itiv ivit ity Gold standard Low to high Specif ific icit ity High Low to high, almost always different Int nterpretation

• n of
• f

posit itiv ive f fin indin ings Usually straightforward S ignificant issues Range nge o

• f patho

hoge gens ns det etec ected ed All pathogens allowed by growth or test conditions Limited to specific pathogen tested Allo llows for s susceptib ibilit ility testing & ng & ge geno notyping? ng? Y es Generally no

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
• Burden
• Attribution
• Trends
• Isolate-requiring
• PulseNet / OutbreakNet
• NARMS
• 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  5/13 PCR negative specimens tested so far in a different Campylobacter-specific PCR assay. All are positive for Campylobacter.

Number er o

• f

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

 Patient Management  Public Health Programs

• Requiring accurate case counts
• Burden
• Attribution
• Trends
• Isolate-requiring
• PulseNet / OutbreakNet
• NARMS
• Subtype-based attribution studies

Impacts

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 Hazards of Inaction

Post-culture STEC Surveillance System

Germany; population 81,471,834 (July 2011 est.)

 >30 detected and investigated in 10 years  Relatively few cases  Investigation expertise developed  Stimulated regulatory focus

U.S. Sprout-Associated Outbreaks

May 24, 2011; Doug Powell Blog

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 Solutions

 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

Potential Benefits of New Approaches

The Surveillance Process

Laboratory Reporting Takes Time

Patient Eats Contaminated Food Stool Sample Collected Public Health Laboratory Receives Sample Patient Becomes Ill Salmonella Identified Case Confirmed as Part of Outbreak

1 – 3 days Contact with health care system: 1 – 5 days Diagnosis: 1 – 3 days Shipping: 0 – 7 days Serotyping & DNA fingerprinting: 2 – 10 days

The Surveillance Process

Laboratory Reporting Takes Time

Patient Eats Contaminated Food Stool Sample Collected Public Health Laboratory Receives Sample Patient Becomes Ill Salmonella Identified Case Confirmed as Part of Outbreak

1 – 3 days Contact with health care system: 1 – 5 days Diagnosis: 1 – 3 days Shipping: 0 – 7 days Serotyping & DNA fingerprinting: 2 – 10 days

 High probability, high impact issue  Risks of inaction and benefits of change are significant

Summary: Culture Independent Diagnostics Impact

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

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)

Up to 1011bacteria/ml; ~500 species Bacteria in Human Stools

Random Shotgun Metagenomics

Total Host & Microbial NA Clinical Sample Random Amplification & Sequencing

Pan genome

Core genome

Meta 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