125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

## An Update on EPA’s Initiatives & Activities of 

## AOAC Expert Review Panel on Clostridium difficile : Is C. difficile becoming

Less Difficile? 

# Jafrul Hasan, PhD, MPH Team Leader, Office of Pesticide Programs Microbiology Laboratory Branch Environmental Protection Agency

& Chair, AOAC Expert Review Panel on

C. difficile125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# Outline
 Background on Clostridium difficile 

#  EPA Interim Guidance for C. difficile  Need for Standardized Methodology 

#  C. difficile Initiatives at EPA  AOAC Expert Review Panel (ERP)125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# Background: C. difficile
 

# Strict anaerobic spore - forming bacillus  

# Major cause of nosocomial (hospital - acquired) gastrointestinal infections in developed world; C. difficile Infection (CDI) range from uncomplicated diarrhea to sepsis and even death  

# Fecal - oral transmission through contaminated environment and hands of healthcare personnel  

# Exposure to antibiotics is major risk factor for disease; other risk factors: older age, hospitalization, antiulcer medications, etc. 3125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# Pathogenesis of C. difficile Infection (CDI)

 Ingestion (vegetative form or spores) 

#  Colonization in small intestine (spores germinate to vegetative form)  CDI can arise in large intestine if normal flora 

# disrupted by antibiotic therapy  C. difficile reproduces in intestinal crypts, releases toxins A (enterotoxin) and B 

# (cytotoxin) - leading to colitis, pseudomembrane formation and watery diarrhea Adapted from Sunenshine and Mcdonald, Cleveland Clin J Medicine, Vol 73, 2006
4125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 5 

## Yearly C. difficile–related Mortality by Listing on Death 

## (per million) Certificates, United States, 1999–2004. Source: Centers for Disease Control and Prevention

# of death per million125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

## C. Difficile Vegetative Cells 

## Courtesy: LS Wiggs , Public Health Information Library (#9999), CDC125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# Visualization of Pure C. difficile Spores

 ( a) Endospore stain of C. difficile culture showing the pink vegetative cells (veg ) and pink extracellular matrix (ex), with a few interspersed green spores (sp). (b) Purified C. difficile spores are stained green. (c) TEM of sectioned C. difficile spores, demonstrating the spore ultrastructure including the exosporium , coat, cortex, core, membrane, and ribosomes . Bar, 100 nm. (d) Magnified section of outer surface of spore. Bar, 50 nm.  Source: Lawley et., al., J. Bacteriol ., 2009, p. 5377, Vol. 191, No. 17 Copyright © 2009 , American Society for Microbiology .125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# EPA on C. difficile

 EPA‟s Office of Pesticide Programs (OPP) has the responsibility under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) to regulate the registration of antimicrobial products, including products designed to treat surfaces contaminated with spores of C. difficile . Under FIFRA, the registrant of a product with a public health claim is required to submit efficacy, or effectiveness data, to EPA in support of the product's registration.  EPA‟s Antimicrobial Testing Program (ATP) has been testing hospital sterilants , disinfectants, and tuberculocides since 1991 to help ensure that products in the marketplace meet stringent efficacy standards.  EPA issued interim guidance in 2009 for the efficacy evaluation of antimicrobial products labeled for use to treat hard non - porous surfaces in healthcare settings contaminated with spores of C. difficile .  EPA is conducting post- registration evaluations of products registered for claims against C. difficile .125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# EPA Interim Guidance for C. difficile Claims

 

### Antimicrobial Products ( dilutable liquids, powders, ready- to - use formulations, spray products, towelettes) to treat surfaces in health 

### care settings  

### Claim: Kills C. difficile spores on hard, non - porous surfaces in ≤10 minutes  

### Test Methods (Qualitative/Quantitative):

 

### AOAC Sporicidal Activity Test (AOAC SAT 966.04)  

### Quantitative Three Step Method (TSM, ASTM E2414)  

### Quantitative Carrier Test Method (QCT- 2, ASTM E2197)  

### Test Strains: ATCC 43598, ATCC 700792, ATCC 43599  

### Must achieve ≥6 log reduction of viable spores with control carriers >10 6 

### spores/carrier  

### No organic soil load; label must specify pre - clean step125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 10 

# C. difficile Initiatives at EPA 

# (Since September, 2008)  EPA‟s need for developing standardized 

# methodology and regulatory guidance related to C. difficile was due to requests for registration  Lacked standard method to generate spore 

# suspensions of high titer (>8 log 10

# /mL) & quality (≥90% spores/resistance to acid, etc)  Research at EPA addressed following attributes: ● Strains ● Sporulation Media ● Recovery Medium ● Incubation Conditions ● Monitoring of sporulation ● Anaerobic Jar vs Chamber125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 11 

# Findings - EPA Research

 Strains: Non- toxigenic (700057) and Toxigenic (700792, 43598, 43599, 9689)



### – 700792, 43598 and 43599 part of interim guidance.

– 43598 (Toxin B + only) was advanced based on use by 

### industry, ease of production of spores, and safety  Sporulation Media : • Liquid : Reinforced Clostridial Medium, Columbia Broth, Brain Heart Infusion broth, and Cooked meat medium • Solid: TSA with 5% blood , and CDC Anaerobic Blood Agar (CABA) plates



### – CABA found to be most effective sporulation medium

 Recovery Media: TSA with 5% blood , CDC agar, Egg Yolk Agar, Columbia Agar, Cycloserine - Cefoxitin Fructose Agar- w/ horse blood and taurocholate (CCFA- HT)

– CCFA – HT selected as most efficient recovery medium
 Explored the most appropriate length of incubation for spore generation:

– 7 - 10 days optimum incubation / monitored sporulation rate using phase contrast microscopy
 Optimized incubation conditions in Anaerobic Jar and Anaerobic Chamber125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# EPA Method Journal of AOAC I, Jan - Feb, 2011, vol 94 (1), p 259 - 272 Spread 100 μL of actively growing C. difficile (ATCC 43598) in



### RCM onto 10 pre- reduced CABA plates Incubate plates inverted anaerobically for 7- 10 days at 36 ± l ° C Harvest spores from plates using ST - 80/heat inactivate at 65 ±2 ° C for 10 min Purify spores using HistoDenz, a density gradient column. Determine spore titer by plating on CCFA- HT, and perform quantitative acid resistance test125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

## Monitoring percent sporulation of C. difficile (ATCC 43598) during 

## anaerobic incubation at 36 ± 1oC under phase contrast microscopy

(magnification 1000 × )

13 

### Day 2 Day 8 Day 10125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

### A: Non - purified C. difficile spores (ATCC 43598) B. purified (≥99% ) spores by a density gradient medium, free from vegetative cells and cell fragments 

# A B125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

### C. Separated vegetative cells, and, D. Separated cell fragments upon purification by HistoDenz TM 

### , a density gradient medium

15 

## C D125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# Product of EPA Research: New ASTM Standard Method E -2839 - 11  ASTM Standard Method E2839 - 11: Debuts First Standard Method for sporulation for C. difficile (ATCC 43598)  Spores are repeatedly being generated using EPA Method (ASTM Method E2839 - 11)  EPA uses spore production procedure to launch 

# efficacy testing of products registered to treat surfaces contaminated with spores of C. difficile (post - registration evaluation adapted for 

# registrant study).125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# AOAC ERP - C. difficile

 Under EPA‟s Office of Pesticide Programs contract with AOACI, an AOAC Expert Review Panel (ERP) on C. 

## difficile was formed in January, 2010. Upon a “Call for Experts” was issued by AOAC, members selected from regulatory, academia and industry.  ERP was charged to develop and address the issues identified in the “Fitness for Purpose” Statement  Panel Members are:

– Judith Noble - Wang, CDC

– Dave Rottjakob, ATS Laboratories

– Syed Sattar, Univ of Ottawa

– Lynne Sehulster, CDC



### – Jafrul Hasan (Chair, EPA) 17125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# ERP: Fitness for Purpose (cont.) Methodology should include: 1) Culture and maintenance of C. difficile 2) Generation of high titer spore suspensions 3) Inoculation of carriers and enumeration of spore load 4) Product neutralization and confirmation 5) Addition of soil load to the inoculum . Assays such as resistance to acid (e.g., as per AOAC Method 2008.05) or sodium hypochlorite may be utilized to qualify spore preparations for use. 

### 6) Efficacy test methods should demonstrate adequate repeatability (within lab variability) and reproducibility (between labs variability) based on performance of comparable antimicrobial test methods. Validated methodology is highly desirable. 18125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# Summary of ERP Activities (2010 - 2011)

 Held ~12 teleconferences and one „Face - to - Face‟ meeting (Aug, 

### 2010)  Developed “Fitness for Purpose”  Conducted literature search and reviewed 42 methods  Down selected 17 relevant methods for in - depth review and 

### deliberation (publications/submitted manuscript/protocol)  Assembled 90 pages of reviews from panel members  1 st 

### „Face - to - Face‟ meeting (Aug, 2010): Presented/discussed reviews in depth. Concurred on Phase I and II approach. 

###  √Conducted Pre - Phase 1 evaluation for optimal recovery medium  Phase I: Completed 7 labs collaborative for comparing two sporulation protocols  2 nd 

### „Face - to - Face‟ meeting held Nov 8 - 9, 2011 

###  Two Publications in the horizon: A white paper on ERP activities/Collaborative study comparing two sporulation protocols 19125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

### Pre - Phase 1 Study: Five C. difficile agar media were evaluated by ATS Lab, CDC and EPA.



### Colonies of C. difficile (ATCC 43598) after 48 hr incubation at 36 ° C

20 

## √CCFA- HT 

## √ BHIY - LT CDBA-T


## √ BHIY - HT CCFA- L125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# Update on Phase I Study ● Evaluated EPA and University of Ottawa methods; three replications per method. ● Major attributes compared for repeatability and reproducibility: 

### Spore titer, spore purity and HCL resistance. Feedback evaluated.

- Participating labs:  ATS Lab , Study Director: Dave Rottjakob  Bioscience Lab , Study Director: John Mitchell  CDC, Study Director: Judith Noble - Wang  EcoLab , Study Director: Lisa Hellickson  EPA , Study Director: Jafrul Hasan  Lonza , Study Director: Milady Brutofsky  STERIS , Study Director: Dan Klein  University of Ottawa , Study Director: Syed Sattar 

### ● Data collection completed: October 31, 2011 

### ● ERP Face-to -face meeting completed: Nov 8- 9, 2011125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

## Phase I collaborative: Comparison of Two



## Sporulation Methods EPA Ref.: J. of AOAC I, Jan - Feb, 2011, vol. 94 (1), p 259 -272.  Stock Culture : Frozen vegetative stock ≤ - 70 ° C  √Sporulation/Incubation (using solid medium). Freshly grown culture onto Commercially available CDC Anaerobic Blood Agar (CABA) plates/Incubate 7 - 10 days/Ensure ≥90% spores  Harvest: Scraping followed by centrifugation (4,500 × g) in saline tween , pre - purification heating at 65 ±5 ° C for 10 min  √Purification: Physical separation by c ommercially available Density Gradient medium.  Qualify: by HCl resistance  Enumeration: BHIY- HT, >8 log 10 ; ≥90% spores 

## Univ. of Ottawa

 Stock Culture : Liquid spore stock at 4 ° C  √Sporulation/Incubation (using liquid medium) Transfer culture successively from Columbia Broth to 500 mL Clospore liquid medium/Incubate 5 days/Ensure ≥60% spores  Harvest: Centrifugation (4,000 × g), wash in distilled water

 √Purification: Enzymatic digestion by trypsin/ lysozyme. Post - purification heating at 70 ° C for 10 min.  Qualify by HCl resistance.  Enumeration: BHIY- HT, 8.9 - 9.7 log 10 ; ≥90% spores Ref: J. of AOAC I, Mar -Apr, 2011, vol. 94 (2), p 1 - 9.125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# ERP Face -to -face Meeting on Nov 8 -9, 2011 at EPA

• Discussed Phase I multi - lab collaborative data for the two sporulation methods for titer (LD/mL), % purity and resistance to HCL. • Discussed statistical analyses including the repeatability and reproducibility. • Analyzed responses to questionnaire. • Discussed/Demonstrated One Step Method (OSM), modified from Three Step Method (AOAC Method 2008.05), for future consideration by the ERP for efficacy testing (?). • The two methods were found to result in spore suspensions of similar titer, purity, and resistance to HCl . • More data to be developed on relative resistance of spore suspensions from both methods to AI (e.g., NaOCl and H 2 O 2/ Peracetic acid). • More resistant spore suspensions from one method (preferable) may be advanced for efficacy testing (?) • Call for Laboratories; Proposed Dates: Feb - May, 2012125th AOAC Annual Meeting & Exposition September 18 -21, 2011 New Orleans, Louisiana 

# Acknowledgements

 EPA • Dr. Stephen Tomasino • Susan Lawrence • Knoxley Japal • Erick Christensen 

###  AOAC Expert Review Panel

– Dr. Judith Noble - Wang, CDC

– Dave Rottjakob , ATS Laboratories

– Dr. Syed Sattar, Univ of Ottawa

– Dr. Lynne Sehulster , CDC 

###  AOAC INTERNATIONAL • Jennifer Diatz • Maria Nelson
24