Handwash Study 3Efficacy of VF481 _GOJO_ against two norovirus by linxiaoqin


									Why are Noroviruses such
 successful pathogens in
   healthcare settings?
Studies of Norovirus Infectivity,
 Persistence and Disinfection

      Christine L. Moe and Pengbo Liu
        Center for Global Safe Water
       Rollins School of Public Health
       Emory University, Atlanta, GA
Introduction to noroviruses
Trends in norovirus epidemiology
Studies of norovirus infectivity
Studies of norovirus persistence
   Environmental Surfaces
   Human Hands

Efficacy of various of handwash agents against
Summary of what we know so far
Group of small round
structured viruses 27-33
nanometer diameter
RNA viruses
Classified as Caliciviridae
  Two human genera: noroviruses
  and sapoviruses
  Two genogroups of human
     Genogroup I and Genogroup II    Norwalk virus
     Hundreds of different strains   S. Miller
Norovirus Phylogenetic Tree

                     Kageyama et al., 2004
       Norovirus Background
Most common cause of epidemic
acute gastroenteritis
  Est. 23 million cases in US annually
Paradigm since 1972
  Can not be grown outside the host
    Culture in 3-D rotating cell system
          Straub et al., 2007
  No animal model
                                                 B.V. Prasad
    Infection of germfree piglets
            Cheetham et al., 2006
  Two animal caliciviruses used as
  surrogates for human virus because they
  can be grown in cells outside the host
    Feline calicivirus - respiratory virus
    Murine norovirus - infects multiple organs
    Clinical Presentation of
      Norovirus Infection
Acute gastroenteritis with predominant
vomiting, nausea, abdominal cramps,
diarrhea ( Stomach Flu )
Incubation period: 24 - 72 hours
Duration: 12 - 72 hours
High attack rates: ave 45%
Trends in Norovirus
Transmission Mode and Settings of
    348 Norovirus Outbreaks:

                             MMWR, 2001
  Transmission mode of 103
Norovirus outbreaks reported in
         the literature

 Percent of Outbreaks

                        40.0%                                                 GI
                        30.0%                                                 GII

                                Food borne   Waterborne   Person to person

                                                          Honorat, 3rd Int Calicivirus Conf,2007
         Norovirus in the UK
Health Protection Agency and Public Health
Laboratory Service collect reports of lab-confirmed
cases since 1995
Typically 130-250 NoV outbreaks reported in
England and Wales per year - estimated 1 million
cases per year (Lopman et al., 2003)
Incidence in the UK appears to be increasing
Most common setting for NoV outbreaks in the
UK is healthcare facilities: hospitals (40%) and
residential care facilities (39%) (Lopman et al.,
 NoV Outbreaks in Healthcare
Settings in the UK: Risk Factors
Large proportion of highly susceptible
population (elderly)
Shared hospital wards
Staff contact with multiple patients
Inadequate hygiene or use of disinfectants

                                      Age-specific rates of hospital patients
           QuickTime and a
    are needed to see this picture.   affected in nosocomial outbreaks of
                                      gastroenteritis in Avon, England,
                                      2002 2003. Vertical bars, 95% CIs.
                                      Lopman et al., 2004
  NoV Outbreaks in Healthcare
Settings in the UK: Characteristics
Introduction of virus into the environment by fecal
contamination, projectile vomiting, contaminated food
Propagation of outbreak by direct and indirect contact
with patients and staff - via hands and contaminated
Outbreaks typically involve both staff and patients.
Reported attack rates of >50% (Chadwick et al., 2000)
Multiple units within hospital are often affected.
Molecular epidemiology studies indicate transmission
between hospital units (Lopman et al., 2006)
Result in ward closures, staff shortages and major
disruption of hospital activities.
       NoV Outbreaks in Healthcare
     Settings in the UK: Characteristics
    Compared to NoV outbreaks in other settings -
      Strong seasonality - winter peak
      Smaller in size (17.5 cases, geo mean)
      Most likely to be person-to-person transmission (95%)
      Higher mortality rates
      Prolonged duration (8.8 days, geo mean)

                                                             QuickTime and a
                                                      are needed to see this picture.

Seasonality of Norovirus outbreaks in residential
homes and hospitals compared to all other settings,
England and Wales, 1992 2000.
Lopman et al., 2003
  Widespread environmental contamination
   with Norwalk-like viruses detected in a
prolonged hotel outbreak of gastroenteritis -
         Cheesebrough et al., 2000
 Jan - May 1996
 Large hotel (500 beds) in NW England
 850 cases (out of 4291 guests) from Jan 15 - March 15
 Outbreak investigation did not show any association with
 March - closed hotel for 1 week for deep clean
 (Washed hard surfaces with water and detergents,
 shampooed and vacuumed carpets. Did not use
Outbreak Curve, Jan - May 1996

                      Cheesbrough et al., 2000
        Environmental Sampling Indicates
       Multiple Areas of NoV Contamination

144 environmental swabs collected before March deep clean . Tested for NoV RNA by PCR.
Carpet cleaned by water, detergent and vacuuming before sampling.

                                                             Cheesbrough et al., 2000
  Why are noroviruses such
   successful pathogens?
Highly infectious?
Widespread host susceptibility?
No protective immunity?
Environmental persistence?
Resistance to disinfection?
Prolonged viral shedding?
High numbers of virus shed in stool?
Studies of Norovirus Infectivity
    in Human Volunteers
             Norovirus Inocula
Norwalk Virus:                Snow Mountain Virus:
  Genogroup I.1 norovirus      Genogroup II.2 norovirus
   prototype Norovirus
                               Associated with a
  Associated with a school     waterborne outbreak in a
  outbreak in Norwalk, Ohio
  in 1968                      resort in Colorado in 1976
  First seen by electron       Few previous human
  microscopy by Kapikian in    challenge studies
  1972                         Sequenced by Hardy
  Several previous human
  challenge studies
  Sequenced by Jiang and
  Estes in 1990
  Molecular detection by
  RT-PCR developed 1992
Summary of Human Challenge Studies by Moe Research Team
                               Inoculum      # of subjects   # infected
                              Dose Range
 Norwalk virus                8FIIa (NIH)               45            19
 Dose-range                   10 - 107 PDU
 Norwalk virus                8FIIa (NIH)               31                3
 Low Dose                     0.1 10 PDU
 Norwalk virus                8FIIb                     33            16
 Ondansetron trial            107 108 PDU
 Snow Mountain virus          SMV (NIH)                 15                9
 Dose-range                   10 - 105 PDU
 Norwalk virus                8FIIb                     13            10
 Persistence in water         >107 genomic
 Norwalk virus                8FIIb                  In Progress
 Inactivation in oysters by
 high hydrostatic pressure
 Safety-        Healthy
  tested        Adult           Collect serum, saliva, stool, emesis,
   NoV          Volunteer                   symptoms

                               d1      d2      d3       d4       d5

                                 5 Day Stay at GCRC (Inpatient)

                               d8     d14      d21     d28     d35

                                 5 Outpatient Follow-up visits

Determine Infection Status:
Test stool samples by RT-PCR to determine viral shedding
Test sera samples by ELISA to determine NV seroconversion
Norwalk Virus vs. Snow Mountain
        Virus Infectivity
Both NV and SMV are HIGHLY infectious
NV was more infectious at low doses than SMV
NV Flatter curve. More variation in dose
SMV Steeper curve. More subjects were
susceptible to infection
SMV infection more likely to cause illness than NV
SMV symptoms lasted longer than NV symptoms
     Key Findings of Human
       Challenge Studies
Noroviruses are extremely infectious - most
infectious agent ever described (Teunis et al.,
2008) ID50 = 18 virus genomes
Symptoms typically last 2-5 days
20-30% of infected subjects did not have
Virus can be shed in feces for up to 35 days in a
normal infected person
Virus can be shed in high titers - up to 1012
genomic copies per gram feces (Atmar et al. 2008)
Norovirus Persistence in the
  Indoor Environmental Surfaces
  Human Hands
Studies of Norovirus Persistence
 Examine Norwalk Virus (NV) and Snow
 Mountain Virus (SMV) persistence in
 water, on environmental surfaces and
 Examine Norovirus reduction by various
 handwash agents
RT-PCR Detection of Norovirus
Because it is not feasible to measure human
norovirus by culture outside of the human host,
we detect and measure norovirus by RT-PCR
RT-PCR is a molecular method that amplifies
and detects the viral RNA
  Very sensitive and specific test
Presence of RNA does not always mean that the
virus is still infectious
  Conservative estimate of virus risk
Absence of RNA means that there is NO virus
Safety-tested                               Collect serum, saliva, stool, emesis,
                             Volunteer                  symptoms

                                            d1     d2      d3       d4       d5
                0, 7, 14, 21, 28, 61 days
                                             5 Day Stay at GCRC (Inpatient)
                Room temp
   Norwalk                                  d8    d14      d21     d28     d35
  Inoculum                                   5 Outpatient Follow-up visits

   Determine Infection Status:
   Test stool samples by RT-PCR to determine viral shedding
   Test sera samples by ELISA to determine NV seroconversion
Norovirus Persistence on Environmental Surfaces
               20% Norovirus-positive stool suspension

                  Ceramic     Formica    Stainless Steel
                                                  (Room temperature 24oC, 40% humidity)

        Serial Time Points (~4 weeks for NV, ~ 6 weeks for SMV)

                       Elute virus from surface

                          Concentrate virus

                  Store at -80oC until RT-PCR assay
     Summary of Environmental
        Surfaces Results
Norwalk virus in a stool suspension gradually
declines over time on common indoor surfaces
   1.5 to 3 logs (99.9%) over 28 days).
Snow Mountain virus seems more stable than
Norwalk virus
Noroviruses persist longer on surfaces at cooler
Naked NV RNA degrades after 7 days on stainless
steel and formica surfaces

                          Liu et al., Food and Envr Virol, In Press
Norovirus Persistence on
    Human Hands
          Norwalk Virus and Snow Mountain Virus
              Persistence on Human Hands

                                Virus: 20% NV- or SMV-positive
              30 min                   stool suspension
     60 min
                       15 min
120 min                         Subjects: 6 volunteers, both hands
Summary of Norovirus Persistence on
   Human Hands and in Water

Noroviruses are stable on human hands.
Almost no change in NoV RNA levels
after more than two hours on human
Norwalk virus maintained infectivity in
water at room temperature for at least 60
     Viral shedding in feces

Surfaces                 Hands


       New Susceptible Host
  Efficacy of Various Handwash
   Agents against Noroviruses

Handwash Study 1: Efficacy of standard benchmark
alcohol-based sanitizer PURELL (62% Ethanol)

Handwash Study 2: Efficacy of VF447 (70% ethanol with

Handwash Study 3: Efficacy of VF481 (70% ethanol with
  Handwash Study 1: Efficacy of Benchmark
PURELL and Liquid Soap against Norwalk Virus
  Virus ---- 20% NV-positive stool sample

  Subject ---- 5 volunteers

  ASTM Standard Method

  Replicate Trials ----
    Both left and right hands,
    Day 1 and Day 2

  Test products
  Hand sanitizer (from grocery store): 62% ethanol
  Liquid soap (lab): antibacterial, Triclosan
  Water rinse: tap water
American Standard Test Method for in vivo evaluation of
the activity of handwash agents using the fingerpad
(ASTM E 1838-02)
              Hand sanitizer
                                     Study procedures
    Liquid soap
                      Dry control
Water rinse

                             Input control

                                             Virus dried

                                             Elute virus from
                     Fingerpad Method
1. Wash hands, dry, mark fingerpads                2. Add virus suspension to fingerpad

                                      3. Allow virus to dry on fingerpads
                     Fingerpad Method
4. Expose fingerpad to handwash agent        5. Handwash agent removes and/or inactivates virus

                                        6. Expose finger to buffer to remove any remaining virus
              Fingerpad Method
7. Test buffer by RT-PCR to measure amount of remaining
8. Compare to input level of virus on control finger to calculate
virus reduction
Summary of Handwash Study 1
Benchmark alcohol-based hand sanitizer
(62% ethanol) is less effective than liquid
soap or water at reducing Norwalk virus on
Implications for health care settings and
food preparation establishments
  Settings where norovirus outbreaks
  commonly occur
  Settings where alcohol-based sanitizers are
  commonly used
       Handwash Study 2: Efficacy of
      VF447 (GOJO) against Norovirus

VF 447
 70% ethanol with additional ingredients (polyquaternium and citric acid)
that serve as alcohol potentiators
 Data from animal calicivirus surrogates indicate that synergistic action
provides better virus removal/inactivation
     Handwash Study 3: Efficacy of VF481
    (GOJO) against two norovirus strains -
        Norwalk virus and GII.4 virus

VF 481
 70% ethanol with additional ingredients (polyquaternium and copper
gluconate) that serve as alcohol potentiators
Summary of Handwash Results
Significant differences in hand sanitizer performance.
Benchmark alcohol-based hand sanitizer was not
effective against norovirus
VF447 was effective at reducing Norwalk Virus on
human hands but was not effective against Snow
Mountain Virus (data not shown)
VF481 was very effective for both NV and GII.4
Because we tested norovirus RNA titers (which may
include dead virus) in all studies, the actual efficacies of
the hand sanitizers were probably underestimated.
Experiments with different RNA extraction methods
showed similar trends but reduced log reduction.
      What do we know about
      Norovirus epidemiology?
Data from CDC and HPA surveillance indicate
increase in norovirus outbreaks
  Due in part to better PCR detection methods that
  are more widely available
  Emergence of new GII.4 strains
     More virulent?
     More susceptible hosts?
Majority of reported norovirus outbreaks in the UK
are associated with healthcare settings
       What do we know about
        Norovirus infections?
Data from human challenge studies confirm that
noroviruses are highly infectious
Prolonged virus shedding, high titers of virus in stool,
and infections without symptoms make it easier to
transmit virus
  Need simple diagnostic tests to identify who is infected and
  who is still shedding virus
Development of norovirus vaccines is in very early
research stages. Healthcare workers are an
important target group for norovirus vaccines.
What do we know about Norovirus
  stability in the environment?
  RT-PCR data indicates that Norwalk virus RNA
  is stable in groundwater for at least 70 days.
  Human challenge study indicates that Norwalk
  virus remains infectious in water for at least 61
  Norovirus RNA can persist on common
  environmental surfaces for at least 3-6 weeks
    Need EFFECTIVE cleaning and disinfection agents
    and protocols [Sodium hypochlorite (bleach)]
    What do we know about
    Noroviruses on hands?
Norovirus RNA is very stable on human hands
for at least two hours
NoV are less susceptible to alcohol and
detergents because no lipid envelope
Good hand hygiene with effective handwash
agents is critical for preventing transmission
Some new products used alone (VF481) or in
combination appear to be effective at reducing
noroviruses on hands
Need to continue to develop more effective
products for reducing noroviruses on hands to
reduce risks of person-to-person transmission
Moe Research Team      Dr. Lee-Ann Jaykus (NC State Univ)
   Dr. Pengbo Liu
   Dr. Juan Leon
                       Dr. Kellogg Schwab
   Marina Fernandez
                       (Johns Hopkins Univ)
   Esther Wong         Dr. Kim Green (NIH)
   Brynn Beaver        Dr. Michele Hardy (Montana State
   Hui-Mien Hsiao      Univ)
   Elisabeth Honorat
   Melissa Dowd
   Kizee Etienne       USEPA Coop Agreement
   Dr. Yu-Wen Chien    FDA
   Yvonne Yuen         USDA
   Laura Kovalchick
                       GOJO Industries
   Allison King
                          Dr. Dave Macinga
   Hannah Cluck
                          Dr. James Arbogast
Dr. Marshall Lyon
                          Carrie Zapka
Gwen Abdulhafid
Emory GCRC
Inocula                        Dose-Response Modeling
    John Herrmann                 Peter Teunis
    Neil Blacklow                 Paul Stewart
    John Treanor                  Burton Levine, Michelle Harb, Sharon
    Mark Sobsey                   Murray, Lauren Lindblad

Recombinant viral proteins     UNC GCRC
   Mary Estes                    Susan Pusek
   Ralph Baric                   William Heizer, Cathy Kapoor, James
   Boyd Yount                    Trogden, Bruce Gilliam, Meera
   Robert Johnston               Kashkari, David Wohl, Walter Straus
   Nancy Davis                   GCRC Nursing Staff

Electron Microscopy            RT-PCR and serology
    Sarah Miller                  Frances Tseng
                                  Erin-Joi Collins McNeal
                                  Lisa Lindesmith
                                  Deanne Rhodes, QingHong Yang,
                                  Cornelia Kaydos, Thao Vo, Amica
 Use of Animal Caliciviruses as
      Surrogates for Human
Feline calicivirus (FCV) and
norovirus (MNV) can be grown in cell culture
- measure infectivity
Used as model for human norovirus
Uncertain how closely the characteristics of
these viruses match the characteristics of
human noroviruses
Differences between FCV and MNV in pH,
alcohol and temperature sensitivity

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