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Transcript
1
TRANSFER OF MICROORGANISMS FROM FOMITES TO HANDS AND RISK
ASSESSMENT OF CONTAMINATED AND DISINFECTED SURFACES
by
Gerardo Urquijo Lopez
_______________________
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF SOIL, WATER AND ENVIRONMENTAL SCIENCE
In Partial Fulfillments of the Requirements for the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2013
2
THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the
dissertation
prepared by
Gerardo Urquijo Lopez
entitled TRANSFER OF MICROORGANISMS FROM FOMITES TO HANDS AND RISK
ASSESSMENT OF CONTAMINATED AND DISINFECTED SURFACES
and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of
Doctor in Philosophy
Charles P. Gerba, PhD
Date: December 14, 2012
Kelly A. Reynolds, PhD
Date: December 14, 2012
Ian L. Pepper, PhD
Date: December 14, 2012
Kelly R. Bright, PhD
Date: December 14, 2012
Final approval and acceptance of this dissertation is contingent upon the candidate’s
submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and
recommend that it be accepted as fulfilling the dissertation requirement.
Dissertation Director: Charles P. Gerba, PhD
Date: December 14, 2012
3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of the requirements for
an advanced degree at the University of Arizona and is deposited in the University
Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission,
provided that accurate acknowledgment of source is made. Request for permission for
extended quotation from or reproduction of this manuscript in whole or in part may be
granted by the head of the major department or the Dean of the Graduate College when in
his or her judgment the proposed use of the material is in interests of scholarship. In all
other instances, however, permission must be obtained by the author.
SIGNED: GERARDO URQUIJO LOPEZ_______
4
ACKNOWLEDGMENTS
I first would like to say thank you Dr. Charles Gerba for the opportunity you gave me to
study in your laboratory. It was a challenging and rewarding experience and I am forever
grateful to you. I would also like to express gratitude to Dr. Kelly Reynolds for your
support, advice, and for giving me the opportunity to collaborate with you on your
projects. Thank you Dr. Ian Pepper for taking the time to mentor me in preparation for
my life after graduate school. I am also grateful to Dr. Kelly Bright for the support and
opportunity to work on your projects.
I would also like to express gratitude to Dr. Masaaki Kitajima for all the support, advice,
and guidance he gave me. Your time and energy is greatly appreciated. Dr. Akrum
Tamimi, thank you for advising me on the transfer data analysis. Sheri Carlino thank you
for the technical assistance you provided. I also would like to recognize everyone in the
Gerba lab. You made my time in the lab enjoyable and I will cherish these memories.
I also would like to show gratitude to Dr. Joan Rose and the Center for Advancing
Microbial Risk Assessment (CAMRA) for funding. I am also grateful to the Alfred P.
SLOAN Foundation for their support in while pursuing my Doctorate. I also greatly
appreciate the support Dr. Maria Teresa Velez and her office staff provided throughout
my graduate studies. I also would like to thank Dr. Silvertooth and his office staff for the
support and guidance.
5
DEDICATION
This dissertation is dedicated to the people in my life who provided me with support and
encouragement through out this endeavor. I could not have accomplished this goal
without you’re backing and unwavering encouragement. I am forever grateful to all of
you for helping me obtain this Ph.D. degree.
To my wife Stephanie and daughter Lucia, my parents Leonardo Gonzalez Lopez and
Guadalupe Urquijo Lopez (†), my in laws Edward and Lucy Chavez, and my family and
friends.
6
TABLE OF CONTENTS
ABSTRACT...................................................................................................................... 15
INTRODUCTION ............................................................................................................ 17
Problem Definition .................................................................................................. 17
Literature Review .................................................................................................... 18
Role of Fomites in Disease Transmission ..................................................... 18
Viruses on fomites ............................................................................... 19
Bacteria on fomites .............................................................................. 20
Survival of Disease on Fomites and Fingers ................................................. 23
Survival of viruses on fomites and fingers ........................................... 23
Survival of bacteria on fomites and fingers ......................................... 25
Role of Hands in Disease Transmission ........................................................ 25
Hands ................................................................................................... 25
Viral transmission ................................................................................ 26
Bacterial transmission .......................................................................... 28
DISSERTATION FORMAT ............................................................................................ 31
PRESENT STUDY ........................................................................................................... 33
REFERENCES ................................................................................................................. 39
APPENDIX A: MINIREVIEW: TRANSFER OF BACTERIA AND VIRUSES TO
HANDS ............................................................................................................................. 59
ABSTRACT ............................................................................................................ 60
INTRODUCTION ................................................................................................... 61
7
TABLE OF CONTENTS - Continued
FACTORS INFLUENCING TRANSFER .................................................... 62
Relative Humidity ................................................................................ 62
Damp versus dry fomites or hands ....................................................... 62
Duration of drying time........................................................................ 63
Type of fomite material ........................................................................ 64
Type of Organism ................................................................................ 65
Level of fomite contamination ............................................................. 67
Hand washing ....................................................................................... 67
Direction of transfer ............................................................................. 68
Friction and pressure ............................................................................ 70
DISCUSSION ......................................................................................................... 71
CONCLUSIONS ..................................................................................................... 73
ACKNOWLEDGEMENTS .................................................................................... 74
REFERENCES ........................................................................................................ 75
FIGURES ................................................................................................................ 83
TABLE .................................................................................................................... 88
APPENDIX B: COMPARISON OF TWO APPROACHES IN DETERMINING
TRANSFER EFFICIENCY OF ESCHERICHIA COLI FROM NONPOROUS FOMITES
TO FINGERS.................................................................................................................... 89
ABSTRACT ............................................................................................................ 90
TEXT ....................................................................................................................... 90
8
TABLE OF CONTENTS - Continued
ACKNOWLEDGEMENTS .................................................................................... 94
REFERENCES ........................................................................................................ 95
TABLE .................................................................................................................. 102
APPENDIX C: TRANSFER EFFICIENCY OF BACTERIA AND VIRUSES FROM
POROUS AND NONPOROUS FOMITES TO FINGERS UNDER DIFFERENT
RELATIVE HUMIDITY ................................................................................................ 103
ABSTRACT .......................................................................................................... 104
INTRODUCTION ................................................................................................. 105
MATERIALS AND METHODS .......................................................................... 106
Subjects ........................................................................................................ 106
Bacteria, Virus and preparation of inocula .................................................. 106
Control wash and disinfection ..................................................................... 108
Relative Humidity Conditions and Temperature ......................................... 108
Fomites tested .............................................................................................. 109
Inoculation of fomites .................................................................................. 110
Fomite-to-finger transfer, sampling, and assays .......................................... 111
Transfer efficiency calculation and statistical analyses ............................... 112
RESULTS.............................................................................................................. 113
Influence of relative humidity on microbial transfer ................................... 113
Influence of fomite type on microbial transfer ............................................ 114
DISCUSSION ....................................................................................................... 115
9
TABLE OF CONTENTS - Continued
CONCLUSIONS ................................................................................................... 120
ACKNOWLEDGEMENTS .................................................................................. 120
REFERENCES ...................................................................................................... 122
TABLES ................................................................................................................ 131
FIGURES .............................................................................................................. 135
APPENDIX D: COMPARATIVE PERSISTENCE OF BACTERIA AND VIRUSES
UNDER DIFFERENT RELATIVE HUMIDITY ON POROUS AND NONPOROUS
FOMITES ....................................................................................................................... 138
ABSTRACT .......................................................................................................... 139
INTRODUCTION ................................................................................................. 140
MATERIALS AND METHODS .......................................................................... 141
Bacteria, Virus and preparation of inocula .................................................. 141
Relative Humidity Conditions and Temperature ......................................... 143
Fomites tested .............................................................................................. 144
Inoculation of fomites .................................................................................. 144
Surface sampling and assays ....................................................................... 145
Reduction and Statistical analysis ............................................................... 146
RESULTS.............................................................................................................. 147
Influence of relative humidity on microbial survival .................................. 147
Influence of fomite type on microbial survival ........................................... 147
DISCUSSION ....................................................................................................... 149
10
TABLE OF CONTENTS - Continued
ACKNOWLEDGEMENTS .................................................................................. 154
REFERENCES ...................................................................................................... 155
TABLES ................................................................................................................ 163
FIGURES .............................................................................................................. 167
APPENDIX E: THE EFFECT OF A DISINFECTANT WIPE ON MICROBIAL
TRANSFER .................................................................................................................... 170
ABSTRACT .......................................................................................................... 171
INTRODUCTION ................................................................................................. 172
MATERIALS AND METHODS .......................................................................... 175
Subjects ........................................................................................................ 175
Bacteria, endospores, and preparation of inocula ........................................ 175
Study organisms ................................................................................. 175
Gram negative and Gram positive inoculum preparation .................. 175
Endospore-forming bacteria inoculum preparation ........................... 176
Virus inoculum preparation................................................................ 176
Control wash and disinfection ..................................................................... 176
Fomites tested .............................................................................................. 177
Disinfectant wipe and Neutralizing Solution............................................... 178
Relative Humidity and Temperature ........................................................... 178
Inoculation of fomites .................................................................................. 179
11
TABLE OF CONTENTS - Continued
Layout of fomites ............................................................................... 179
Organism concentration ..................................................................... 179
Fomite sampling .......................................................................................... 179
Intervention application ............................................................................... 180
Transfer experiments ................................................................................... 180
Sampling the fingers .................................................................................... 181
Organism assays .......................................................................................... 181
Transfer efficiency, log10 reduction, and statistical analysis ....................... 182
Calculation of transfer efficiency ....................................................... 182
RESULTS.............................................................................................................. 183
Removal of microbial contamination from nonporous surfaces ................. 183
Influence of disinfectant wipes on Microbial Transfer................................ 184
DISCUSSION ....................................................................................................... 184
CONCLUSIONS ................................................................................................... 188
ACKNOWLEDGEMENTS .................................................................................. 188
REFERENCES ...................................................................................................... 189
TABLES ................................................................................................................ 198
APPENDIX F: RISK OF CAMPYLOBACTER JEJUNI INFECTION FROM
PREPARING RAW CHICKEN IN DOMESTIC KITCHENS AND CROSSCONTAMINATION REDUCTION FROM DISINFECTANT WIPES ....................... 201
ABSTRACT .......................................................................................................... 202
12
TABLE OF CONTENTS - Continued
1. INTRODUCTION ............................................................................................ 203
2. METHODS....................................................................................................... 206
2.1 The Exposure Model ............................................................................. 206
2.1.1 Input Distribution ...................................................................... 207
2.1.2 Fomite-to-finger Dose ............................................................... 209
2.1.3 Risk of Infection......................................................................... 209
2.1.4 Risk of Illness ............................................................................ 210
2.1.5 Risk of Death ............................................................................. 210
2.1.6 Annual Risk ............................................................................... 211
2.1.7 Percent Reduction ..................................................................... 211
2.1.8 Model Implementation .............................................................. 212
3. RESULTS.......................................................................................................... 213
3.1 Risk Comparison of Scenario 1 and 2 under Condition A .................... 213
3.1.1 Risks per person per event ........................................................ 213
3.1.2 Annual Risks per person per year ............................................. 213
3.2 Risk Comparison of Scenario 1 and 2 under Condition B .................... 213
3.2.1 Risks per person per preparation event .................................... 213
3.2.2 Annual Risks per person per year ............................................. 214
4. DISCUSSION ................................................................................................... 214
ACKNOWLEDGEMENTS .................................................................................. 219
REFERENCES ...................................................................................................... 220
13
TABLE OF CONTENTS - Continued
FIGURES .............................................................................................................. 226
TABLES ................................................................................................................ 227
APPENDIX G: SUPPLEMENTAL MATERIALS ........................................................ 232
TABLES ................................................................................................................ 233
REFERENCES ...................................................................................................... 252
APPENDIX H: SUPPLEMENTAL MATERIALS ........................................................ 254
ADDTIONAL DETAILS TO METHODS AND MATERIALS ......................... 255
ADDITIONAL DETAILS TO RESULTS ............................................................ 255
TABLE .................................................................................................................. 256
FIGURES .............................................................................................................. 257
REFERENCES ...................................................................................................... 259
APPENDIX I: SUPPLEMENTAL MATERIALS ......................................................... 260
TABLES ................................................................................................................ 261
FIGURES .............................................................................................................. 264
APPENDIX J: SUPPLEMENTAL MATERIALS ......................................................... 267
ADDITIONAL DETAILS TO METHODS AND MATERIALS ........................ 268
Calculation of percent reduction.................................................................. 268
ADDITIONAL DETAILS TO RESULTS ............................................................ 268
Removal of microbial contamination from nonporous surfaces ................. 268
TABLES ................................................................................................................ 270
FIGURES .............................................................................................................. 272
14
TABLE OF CONTENTS - Continued
APPENDIX K: SUPPLEMENTAL MATERIALS ........................................................ 275
TABLES ................................................................................................................ 276
15
ABSTRACT
It is now widely accepted that surface contamination plays an important role in the
transmission of both respiratory and gastrointestinal infections in the domestic
environment and community setting. The efficiency of transfer of a pathogen to the hand
from a fomite is important in modeling transmission in microbial risk assessment models.
The objective of this study was to use published literature to assess the role of fomites
and hands in disease transmission, and to conduct fomite-to-finger transfer studies from
various porous and nonporous fomites under different relative humidity condition using
non-pathogenic strains of Escherichia coli, Staphylococcus aureus, MS2 coliphage,
Bacillus thuringiensis spores, and poliovirus 1; to evaluate the persistence of bacteria and
viruses on surfaces; to examine bacteria and virus transfer from treated surfaces; and to
conduct a foodborne quantitative microbial risk assessment using Campylobacter jejuni
from the data obtained in these studies. It was found that numerous factors influence the
transfer efficiency of microorganisms, with moisture being the most important, with
greater transfer under humid conditions. Other factors influencing transfer include drying
time, contact time, pressure, friction, type of material, and porosity of the fomite. Percent
transfer was greater under high relative humidity for both porous and nonporous surfaces.
Most organisms on average had greater transfer under high relative humidity (40 – 65%)
compared to low relative humidity (15 – 32%). Relative humidity and fomite type
influenced the survival of all studied organisms; survival was greater on nonporous
surfaces than those for porous surfaces. Test organisms were reduced up to 99.997% on
16
the fomites after the surfaces were wiped with a disinfectant wipe. Microbial fomite-tofinger transfer from disinfectant wipe-treated surfaces were, lower than from non-treated
surfaces. The disinfectant-wipe intervention reduced the risk of Campylobacter infection,
illness, and death by 2 to 3 orders on all fomites. The disinfectant-wipe intervention
reduced the annual risk of illness below the reported national average of diagnosed
Campylobacteriosis cases 1.3E-04. This risk assessment demonstrates that the use of
disinfectant wipes to decontaminate surface areas after chicken preparation reduces the
risk of C. jejuni infections up to 99.2%.
17
INTRODUCTION
Problem Definition
The role of fomites in the transmission of infectious diseases has been a controversial
subject (1). However, this controversy is dimensioning through evidence from studies
showing contamination of inanimate surfaces occurs in homes and community settings
through cross-contamination from colonized or infected people, raw animal meat, or by
contaminated sponges and cleaning cloths who serve as reservoirs and vehicles in
microbial transmission (2-6). Indoor environments play a key role in the dissemination
of microbial pathogens (7, 8). The home environment, specifically the kitchen and
bathroom, serves as reservoir of large numbers of microorganisms, and infectious disease
transmission has been shown to occur in 6 – 60% of households in which one member is
ill (9). Scott (10) suggested that during outbreaks of infection in the home, the transfer
between animate and inanimate surfaces promotes a cycle of re-infection. The
community setting in hospitals, nursing homes, schools, and day-care centers are known
to harbor viral and bacterial pathogens. Health-care associated infections (HAI) remain a
major cause of patient morbidity and mortality (11). From these studies we have learned
the importance of environmental contamination cannot be sufficiently emphasized.
There is strong evidence that person-to-person transmission via the hands and
contaminated fomites plays a key role in the spread of viral infections (12). Contact with
contaminated surfaces varies for each person depending on age, personal habits, and type
of activities (13-15). Once contact with the host is achieved, microbial pathogens can
18
gain entry into the host through portals of entry, including the mouth, nasopharynx or
eyes (16-18). The objective of this study was to use existing published literature to assess
the role of fomites and hands in disease transmission; to conduct fomite-to-finger transfer
studies from various porous and nonporous fomites under different relative humidity
conditions; to evaluate the persistence of bacteria and viruses on surfaces; to examine
bacteria and virus transfer from treated surfaces; and to conduct a quantitative microbial
risk assessment using the findings of these transfer studies as input parameters in a Monte
Carlo 20,000 iteration simulation and determine the risk of Campylobacter jejuni
infection in household kitchens.
Literature Review
Role of Fomites in Disease Transmission
Fomites consist of animate or inanimate porous and nonporous surfaces that can become
contaminated with pathogenic microorganisms and serve as reservoirs of microbial
pathogens and vectors for cross-transmission in the domestic environment and in
community settings (14, 18, 19). Potential pathogens from sources such as raw foods,
infected persons and animals can be transferred between inanimate and animate surfaces
through either direct or indirect contact (10, 20-25). Environmental surfaces become
contaminated with virus, bacteria or parasites shed by infected or colonized individuals
by direct contact with body secretions such as blood, feces, urine, saliva, and nasal fluid
(17, 18, 26-29), contact with soiled hands, contact with aerosolized virus or bacteria
generated by talking, sneezing, coughing, or vomiting (12, 17, 18, 28, 30). Boyce and
19
Pittet (31) described that surfaces near or in close contact with individuals can even
become contaminated from colonized normal skin with S. aureus (4, 32-38),
Enterococcus facium and faecalis (2, 39-43), Klebsiella spp. (44-47) and with Gram –
negative bacteria (35, 44, 47-50). Patients who have gastrointestinal colonization with
Clostridium difficile may also serve as reservoirs and thus contaminate the surrounding
environment (51-56). Layton et al. (6) found that the major reservoirs of MRSA within
institutions are patients with either S. aureus infection or colonization. Zervos et al. (43)
reported that the potential hospital reservoirs for gentamicin-resistant enterococci could
include colonized as well as infected patients, hospital personnel, and the inanimate
hospital environment. After a fomite becomes contaminated, the transfer of infectious
virus or bacteria easily occur between hand to fomite, fomite to hand, hand to hand, hand
to fomite to hand, or fomite to hand to fomite (1, 13, 30, 57-68).
Viruses on fomites
Bellamy et al. (17) surveyed the home environment for the presence of viruses and body
fluids and found haemoglobin on 2% of surfaces (taps, washbasins, toilets bowls and
seats) and amylase, an indicator or saliva, sweat and urine. Reynolds et al. (18) found
fecal and total coliform bacteria, protein, and biochemical markers to be widespread in
public places. These studies indicate that surfaces may remain soiled for long periods of
times and may not be thoroughly cleaned. Studies conducted on home and community
settings have provided a better understanding of how infectious disease is spread in these
environments (16). Day care centers and elementary schools have been investigated for
20
the presence of influenza A virus and norovirus and it has been found that influenza A
virus was detected on over 50% of the fomites in homes and day care centers during
influenza season (14, 69, 70).
Bacteria on fomites
Several studies have identified bacteria prevalent on environmental surfaces both in
domestic environments and in community settings. In a hospital study, Bonten et al. (2)
reported that vancomycin-resistant enterococci (VRE) were recovered more frequently
from rooms with contaminated surfaces 60-70% in which there was a high proportion of
VRE-colonized body sites than from rooms with only a few colonized body sites. In
another study, patients with VRE who had diarrhea had 46% of environmental cultures
contaminated, compared to 15% of surfaces cultured in the rooms of VRE patients
without diarrhea (3). In a similar study, MRSA was recovered from 58.8% of surfaces in
rooms with patients with diarrhea, compared to 23.3% of surfaces in rooms of patients
without diarrhea (51). Boyce et al. (4) found that when patients had methicillin-resistant
Staphylococcus aureus (MRSA) in a wound or urine, 36% of surfaces were contaminated,
while when MRSA was isolated from sputum, blood, or conjunctivae, only 6% of
surfaces were contaminated. In the same study, Boyce et al. (4) reported environmental
contamination of MRSA occurred in rooms of 73% of infected patients and 69% of
colonized patients. McFarland et al. (53) described that patients with symptomatic C.
difficile resulted in 49% of their rooms were contaminated, compared to 29% of room
occupied by asymptomatic patients were contaminated. The frequency of positive
21
personnel hand contamination with C. difficile has been strongly correlated with the
intensity of environmental contamination in rooms (5, 54, 56, 71). One study reported
room site contamination ranging from 1% to >50%, with the most heavily contaminated
rooms having >90% of sites positive for C. difficile (54).
Environmental surveillance studies have investingated the prevalence of nosocomial
pathogens in intensive care units (ICUs) and the most commonly reported surfaces to be
contaminated with MRSA, VRE, Enterococcus spp., Enterobacter spp., C. difficile, and
Gram negative rods are the floor next to the bed, bathroom floor, bathroom hand rails,
mops, bed linens, patient gowns, healthcare worker’s (HCWs) gowns or uniforms, gloves,
drawsheet, overbed tables, bedside tables/stands, bedside rails, bedsheets, door handle,
bedside commodes, toilet seat, toilet rail, keyboard, computer table, faucet, tubs,
washbasins, chairs, dresser, nurse call button, TV remote, shelves, cabinet, and window
sill (2-4, 42, 51, 53, 54, 72-81). Medical equipment reported to have been contaminated
with MRSA, VRE, mupirocin-resistant S. aureus are infusion pump buttons,
electrocardiogram-EKG wires and monitor, intravenous-IV poles and fluid pump, pulse
oximeters, stethoscopes, monitors, oxygen flow meters, rectal and ear probe electronic
thermometers, blood-pressure cuff, blood glucose monitor, suction canisters, enteral feed,
ventilator tubing, automated medication dispenser, and urine container (2-4, 6, 42, 51, 72,
74-77, 82-85). Even paper-towel dispensers near wash basins have been known to be
contaminated with Staphylococci bacteria (86). Patients’ charts have also been shown to
be potential reservoirs and vectors for cross-contamination of pathogenic bacteria MRSA,
22
VRE and several other to HCW hands and to patients (87-90). One study reported 81.1%
of patients’ charts in a surgical ICU and surgical ward were contaminated by infectious
agents (90). Inanimate surfaces near or in close contact with affected patients as well as
those that are frequently touched by hospital personnel commonly become contaminated
with MRSA, VRE, and other pathogens and that the frequency of contamination is
affected by the body site at which patients are colonized or infected (4, 91). The
evidence that personnel may contaminate their gloves or possibly their hands, by
touching surfaces or medical equipment suggests that contaminated environmental
surfaces serve as microbial reservoirs and vehicles of transmission in hospitals (2-6).
Paper currency used in several countries has been reported to harbor bacteria and
parasites including E. coli, B. cereus, S. aureus, and Salmonella ranging from (0.1
CFU/cm2 to 128 CFU/cm2) (92) and has been identified as a reservoir and vehicle of
microbial transmission (92-94).
Several studies have demonstrated the potential for cross-contamination in the kitchen
after preparation of raw chicken, contaminated naturally with Campylobacter and
Salmonella (24, 95) or seeded with marker organisms to contaminate hands, counter tops,
sink basins, cutting boards, utensils, dishcloths, and sponges (20, 22, 68, 96-105). A
number of studies have investigated the domestic environment and have determined the
prevalence and quantity of bacterial contamination on surfaces (76, 106-111). The results
indicate bacteria to be found on kitchen surfaces (0.2 log10 CFU/ swab area to 8.4 CFU/
swab area), cleaning cloths and sponges (0.90 log10 CFU/ swab area or ml to 10.12 CFU/
23
swab area or ml) and in the bathroom (0.02 log10 CFU/ swab area to 8.5 CFU/ swab area).
Surface contamination has been shown to spread by using cleaning cloths that are
harboring viruses and bacteria (112). Public areas and homes have been investigated for
the occurrence of bacteria and biochemical markers (17, 18) and the results indicate that
they are prevalent. Reynolds et al. (18) found biochemical markers, i.e., hemoglobin
(blood marker), amylase (mucus, saliva, sweat, and urine marker), and urea (urine and
sweat marker) were detected on 3% (26/801); 15% (120/801), and 6% (48/801) of the
surfaces, respectively. Medrano-Felix et al. (107) found up to 3.27 log10 per 900 cm2 of
counter surface in kitchens.
Survival of Disease on Fomites and Fingers
Survival of viruses on fomites and fingers
Viruses outside a host may be considered inert particles, without an intrinsic metabolism,
they do not require any nutrients to persist (113). Nonetheless, they possess a degree of
robustness which allows them to remain infectious during the various conditions that they
may encounter between one host and another (113). The longer a virus can survive
outside a host, the greater are its chances for transmission (113). Virus and bacterial
survival on fomites is influenced by intrinsic factors which include fomite properties such
as hydrophobicity of the surface, virus and bacteria characteristics such as isoelectric
points and extrinsic factors including environmental temperature, humidity and other
factors (14).Virus are infectious at very low doses and have been shown to survive for
minutes to days on nonporous surfaces (17, 18). Both respiratory and enteric viruses
24
have been shown to survive on nonporous and porous fomites from a few minutes to
several days, with non-enveloped viruses such as rotavirus, adenovirus, poliovirus, herpes
simplex virus, and hepatitis A virus (HAV) having greater resistance to drying and thus
surviving longer and spreading more easily than enveloped viruses, such as influenza A
virus, coronaviruses, and respiratory syncytial virus (RSV) which are less stable in the
environment (12).
Enveloped viruses have been shown to survive on fomites. Respiratory viruses shown to
persist on fomites for 24 – 48 h are influenza A and B (nonporous stainless steel and
plastic surfaces) while surviving for < 8 – 12 h on cloth, paper, and tissues (114). Mahl
and Sadler (115) showed herpes simplex virus to survive on glass slides for 56 days,
while vaccinia virus for at least 14 days. Both of these viruses were also found to survive
in hospital floor sweepings for at least 14 days (115).
Non-enveloped viruses in general have been shown to survive longer (days to several
weeks) than enveloped viruses on fomites. Enteric viruses can persist on fomites for long
periods of time. Mahl and Sadler (115) showed adenovirus and poliovirus to survive on
glass slides for at least 56 days, while coxsackie virus for at least 14 days. Abad et al.
(116) found that hepatitis A virus (HAV) and human rotavirus were more resistant to
inactivation than enteric adenovirus (ADV) and poliovirus (PV), yet they all survived up
to 60 d. In a later study, Abad et al. (117) found that astroviruses can survive on
nonporous china (up to 60 d) and porous paper surfaces (up to 90 d) under different
25
environmental conditions. Both respiratory and enteric viruses have been shown to
survive on fingers or gloves from a few minutes to several hours and again nonenveloped viruses surviving longer than enveloped viruses. Influenza A and B has been
shown to survive on hands for up to 5 min (114).
Survival of bacteria on fomites and fingers
Both Gram negative and Gram positive bacteria have been shown to survive on
nonporous and porous fomites from a few minutes to several days. Noskin et al. (118)
showed vancomycin-resistant enterococci (VRE) to survive on several fomites. They
found E. faecalis and E. faecium surviving 5 days and 7 days on countertops, respectively,
24 h on bedrails, 60 min on telephone handpiece, and 30 min on the surface of
stethoscopes (118). Neely and Maley (119) reported the survival of MRSA and VRE on
five common hospital materials, clothing, towels, scrub suits and lab coats, privacy
drapes, and splash aprons to be from 1 day to as long as 90 days. VRE, E. faecium, and E.
faecalis have been shown to survive for at least 60 min on gloved and ungloved fingertips
(118).
Role of Hands in Disease Transmission
Hands
Multiple studies prove that environmental contamination in the domestic environment
and community setting plays an important role in the transmission of microbial pathogens.
Epidemiologic data have suggested for more than a century and a half that healthcare
26
workers (HCWs) spread microbes from patient to patient via contaminated hands causing
health care-associated infections (HAI) (11, 120). It is estimated that in the United States
there are 1.7 million HAI each year, with approximately 99,000 deaths (11). An
estimated 20% to 40% of these HAI have been attributed to cross infection via the hands
of health care personnel, that have become contaminated from direct contact with the
patient or indirectly by touching contaminated environmental surfaces (11). Touching
fomites animate (hands) or inanimate, such as counter tops, toys, eating utensils, towels
or doorknobs, inadvertently contaminated with secretions, vomit, or nasal mucosa from
infected person and then transferring the virus from the hands to the eyes, nose or mouth,
are further routes of spread (12). Transfer of gastrointestinal viruses to food and food
contact surfaces during handling and preparation is an important route of spread of viral
gastroenteritis (12). Most documented foodborne viral outbreaks can be traced to food
that has been manually handled by an infected foodhandler (28).
Viral transmission
Viruses are undoubtedly the most common cause of infectious disease acquired within
indoor environments and cause considerable morbidity (12). Close personal contact
within the home and community settings, such as daycare centers, schools, hospitals,
nursing homes, and cruise chips, make them ideal places for the rapid spread of viral
infections. A study by Jiang et al. (121) investigated virus transmission in childcare
facilities using modified cauliflower virus DNA as an environmental marker. The viral
DNA introduced through treated toy balls spread within a few hours of children handling
27
the toys, even after the treated balls were removed after one day, the viral DNA
continued to circulate in the day-care center for up to 2 weeks. The markers could also
be detected in the children’s homes, on the hands of family members, and on surfaces
within those homes (121).
Studies have shown that the home is conducive to the transmission of respiratory and
enteric viruses. A respiratory infected person will deposit virus onto their hands from
their nasal mucous and spread it to their surrounding environment where family members
can transfer the virus onto their hands and self-inoculate. This has been demonstrated
with natural and laboratory rhinovirus infections (7, 8, 65, 122, 123), and influenza virus
(114, 124). Bean et al. (114) showed influenza A and B viruses to transfer from stainless
steel surfaces to hands for 24 h and from tissues to hands for up to 15 min.
The spread of respiratory syncytial virus (RSV) from contaminated surfaces in the
hospital demonstrated that the infectious virus could be transferred to the hands of
hospital personnel touching the contaminated surfaces (26). The link between school and
day-care centers with homes of children has been identified as critical in the chain of
dissemination of rhinovirus (8), and influenza virus (69).
It has been estimated that 30-40% of infectious gastroenteritis cases in developed
countries are attributable to viruses (125). The presence of microorganisms associated
with diarrhea has been isolated from the household environment (25, 126). Enteric viral
28
infections such as rotavirus are shed from children and adults in large numbers with feces
containing greater than 1012 particles per gram and can excreted up to 34 d (12, 127, 128).
Common foodborne viruses for which we become reservoirs of while ill are norovirus
and HAV and some less common viruses are enteric adenovirus (types 40/41), rotavirus
sapovirus, astrovirus, coronavirus, and aichivirus (28, 113). Human norovirus (HuNoV)
transmission via contaminated fomites has been well documented and several outbreaks
have been reported in cruise ships (129), U.S. Navy ships (130), house boats (131), and
river rafters (132), long-term care facility (133) nursing homes (16). Norwalk-like
viruses are the number one cause of foodborne illness, accounting for much higher
incidence than that of the two highest bacterial etiologies combined (63). It is estimated
that vomiting associated with Norwalk-like viruses (NLVs) can have up to 3.0 x107
particles are distributed as an aerosol into the environment (12) and shedding can persist
up to 10 d. Noroviruses account for greater than 90% of nonbacterial and approximately
50% of all epidemic gastroenteritis (11). They are responsible for an estimated 267
million infections annually worldwide and 23 million infections annually in the United
States (11).
Bacterial transmission
There is substantial evidence to show that bacterial transmission occurs in both homes
and community settings. Studies have shown that the same strain of gentamicin-resistant
E. faecalis and MRSA has been isolated in two separate hospitals, suggesting that
interhospital spread of gentamicin-resistant enterococci may have been caused by
29
transient carriage on the hands of personnel who rotate between the two hospitals or by
the transfer of patients from one facility to the other (91, 120). Muto et al. (120) reported
that there has even been evidence to suggest transmission of MRSA clones from one city
to city, from country to country, and even from continent to continent traced to the
transfer of patients infected or colonized with MRSA.
The hands of HCWs are a major source of transmission of nosocomial pathogens and
frequently acquire transient flora, which colonize the superficial layers of the skin (31,
134). Multiple studies have shown that HCWs hands or gloves frequently acquire
multidrug-resistant MRSA, VRE, Klebsiella spp., C. difficile and other pathogenic flora
during direct contact with patients or contact with contaminated surfaces found in close
proximity of infected or colonized patients (31, 40, 44, 54, 56, 81, 83, 118, 135-141).
MRSA and VRE have even contaminated gloves of personnel who had no direct contact
with patients, but had touched contaminated surfaces (4, 140). Tenorio et al. (140)
reported that HCWs who tended a patient with diarrhea resulted in 100% gloves positive
for VRE, as compared to 44% (12/27) HCWs gloves who tended to a patient without
diarrhea. Studies in hospitals have shown that transmission via the contaminated hands
of HCWs to be an important mechanism and risk factor in previously uncolonized
patients acquiring the same VRE strain as colonized patients in other rooms (3, 142).
Another study identified HCWs hands were becoming contaminated with MRSA while
tending to patients in their rooms and then possibly cross-contaminating hospital
computer keyboards used only by clinicians (143). Yet in another hospital outbreak of
30
gentamicin-resistant E. faecalis the HCWs hands were suggested as the cause of spread
due to frequent contact with a contaminated door handle leading out of the surgical ICU
(43). Duckro et al. (83) conducted a study to determine the frequency of VRE
transmission from VRE-colonized patients’s skin or contaminated surface to the HCWs
hands or gloves and then to a VRE-negative site, VRE was transferred 10.6% (16/151) to
negative sites. C. difficile has been recovered from the hands of infected patients and
HCWs, with one study reporting 59% (20.5/35) HCWs hands being positive after tending
a culture positive patient (56). In a similar study, Samore et al. (54) found 14% (10/73)
personnel hands were positive for C. difficile.
31
DISSERTATION FORMAT
This dissertation contains eleven appendices: six manuscripts and five supplemental
materials. Appendix A is a Minireview entitled “Minireview: Transfer Bacteria and
Viruses to Hands” that will be submitted for publication. This review assesses the
importance of factors affecting the transfer of bacteria and viruses, and the degree of
transfer, which might be expected under different environmental conditions. Appendix B
is a short form manuscript entitled “Comparison of Two Approaches in Determining
Transfer of Escherichia coli from Nonporous Fomites to Fingers” that will be submitted
for publication. This manuscript evaluates the fomite-to-finger percent transfer of E. coli
from fomites using two approaches; one determines the percent transfer based on control
fomite, while the other is based on the sum of bacteria recovered from a finger and donor
fomite. Appendix C is a long form manuscript entitled “Percent Transfer of Bacteria and
Viruses from Nonporous and Porous Fomites to Fingers Under Different Relative
Humidity” that will be submitted for publication. This research examines the effect of
low and high relative humidity on fomite-to-finger percent transfer of four model
organisms: Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis spores, MS2
coliphage, and poliovirus 1 from nine common inanimate surfaces (fomites). Appendix
D is a manuscript entitled “Comparative Persistence of Bacteria and Viruses under
different Relative Humidity on Porous and Nonporous Fomites” that will be submitted
for publication. This study evaluates the survival of several model organisms under low
and high relative humidity on various types of fomites to better understand
32
microorganism survival in modeling microbial exposure and risk assessment. Appendix
E is a manuscript entitled “The Effect of a Disinfectant Wipe on Microbial Transfer” that
will be submitted for publication. This research investigated the effects of a disinfectant
wipe on removing microorganisms from fomites and reducing microbial fomite-to-finger
transfer. Appendix F is a manuscript entitled “Risk of Campylobacter jejuni infection
from Preparing Raw Chicken in Domestic Kitchens and Cross-contamination Reduction
from Disinfectant Wipes” that will be submitted for publication. This manuscript
describes the Quantitative Microbial Risk Assessment conducted on the risk of infection
after the preparation of raw chicken fillet in a domestic kitchen and cross-contamination
to fomite-to-hand exposure and the reduction in risk by disinfectant wipes on the
contaminated surfaces. Appendices G through K provide additional materials for the
corresponding manuscripts.
33
PRESENT STUDY
The manuscript in Appendix A is a Minireview of the transfer of microorganisms to
hands from fomites and vice versa. This review assesses the importance of factors
affecting the transfer of bacteria and viruses, and the degree of transfer, which might be
expected under different environmental conditions. Such information is important to
reduce the uncertainty in quantitative microbial risk assess models, which can be used to
estimate the risk of infection from contact with contaminated surfaces and the impact of
interventions (disinfectants, hand sanitizers, self-sanitizing surfaces). It was found that
numerous factors influence the transfer efficiency of microorganisms, with moisture
being the most important, with greater transfer occurring in humid environments. Other
factors influencing transfer include drying time, contact time, pressure, friction type of
material, and nature of the fomite. The greater the pressure applied to a surface and
longer the contact time the greater the amount of transfer that can be expected. Much
greater transfer occurs with hard surfaces (stainless steel) than porous surfaces (paper,
cloth). Overall trends can be observed but transfer is organism dependent. Both Gram
negative and positive bacteria have a greater transfer from hand-to-fomite than virus.
Fomite-to-hand transfer is similar for both virus and bacteria. Hand-to-hand, hand-tofomite-to-hand and the fomite-to-hand-to-fomite transfer direction had an overall lower
transfer than hand-to-fomite and fomite-to-hand transfer. Hand-to-fomite-to-hand showed
the least transfer. Uncertainty in risk models can best be reduced by studies on specific
data on transfer from hard nonporous surfaces, since the greatest transfer for most
34
organisms occurs under these conditions. It is important that future studies on microbial
transfer include data on the important environmental factors that influence transfer for
better comparison among groups of organisms.
The manuscript in Appendix B evaluates the fomite-to-finger percent transfer of
Escherichia coli from fomites using two approaches: approach 1 defines transfer
efficiency (TE) as colony forming units (CFU) recovered from finger relative to CFU
recovered from control fomite, while approach 2 defines TE as the CFU recovered from
the finger (recipient) relative to the sum of the CFU recovered from the finger (recipient)
and the donor fomite. Fomite-to-finger transfer efficiency of microbial pathogens is
important in modeling the potential for transmission. Consistent approaches for
determining microbial transfer efficiencies are essential in producing accurate input
values for risk assessment modeling. Five fomites of different compositions were studied
representing nonporous surfaces. E. coli were placed on fomites in 10 µl drops and
allowed to dry for 15 to 32 min under low (20% to 34%) relative humidity. Fomite-tofinger transfers were performed using a 1.0 kg/cm2 of pressure for 10 ± 2 s. Index, middle,
and ring finger pads were sampled and E. coli was enumerated by the spread plate
technique and transfer efficiencies were calculated using the two approaches. There were
no statistical differences between transfer efficiencies determined by the two approaches
(P = 0.05), demonstrating that the transfer efficiencies determined by each approach are
comparable.
35
The manuscript in Appendix C examines the effect of low and high relative humidity on
fomite-to-finger transfer efficiency of four model organisms from several common
inanimate surfaces (fomites). The efficiency of transfer of a pathogen to the hand from
the fomite is important in modeling the potential for transmission. Nine fomites of
different compositions were studied representing porous and nonporous surfaces.
Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, MS2 coliphage, and
poliovirus 1 were placed on fomites in 10 µl drops and allowed to dry for 30 min under
low (15% to 32%) or high (40% to 65%) relative humidity. Fomite-to-finger transfers
were performed using a 1.0 kg/cm2 of pressure for 10 s. Index, middle, and ring finger
pads were sampled and organisms were enumerated and transfer efficiencies were
calculated. Transfer efficiencies were greater under high relative humidity for both
porous and nonporous surfaces. Most organisms on average had greater transfer
efficiencies under high relative humidity compared to low relative humidity. Nonporous
surfaces had a greater transfer efficiency (up to 57%) than porous surfaces (< 6.8%)
under low relative humidity, as well as under high relative humidity; nonporous (up to
79.5%) and porous (< 13.4%). Transfer efficiency also varied by fomite material and
organism type. These results better qualify the transfer efficiencies of several different
types of organisms under control conditions to provide data that could be used in
quantitative microbial risk assessment (QMRA) models.
The manuscript in Appendix D evaluates the survival of several surrogate pathogens
under low and high relative humidity on various types of fomites to better understand
36
microorganism survival in modeling microbial exposure and risk assessment. Nine
fomites of different compositions were studied representing porous and nonporous
surfaces. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, MS2
coliphage, and poliovirus 1 were placed on various fomites in 10 µl drops and allowed to
dry for 30 min under low (15% to 32%) or high (40% to 65%) relative humidity. Relative
humidity influenced organism survival on fomites; greater survival was observed under
high relative humidity for E. coli, S. aureus, MS2, and poliovirus 1 . B. thuringiensis
spores survived longer at low relative humidity. Fomite type did influence the survival of
all model organisms; survival was greater on nonporous surfaces than those for porous
surfaces. Organism survival was greatest with paper currency and least with cotton for
most pathogens under both low and high relative humidity. Generally most organisms
had greater survival under high relative humidity compared to low relative humidity with
B. thuringiensis spores showing the opposite effect. Survival was less on porous surfaces
compared to nonporous surfaces. The data obtained in our study can be used to better
model survival parameters in microbial risk assessment models.
The manuscript in Appendix E quantitatively assesses the percent microbial transfer to
and from various surfaces and the influence of a disinfectant intervention to inhibit such
transfers. Such data is needed for the development of quantitative microbial risk
assessment models to assess the impact of interventions on reduction in the risk of
infection. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis spores and
poliovirus 1 were inoculated onto ceramic tile, laminate and granite in 10 µl drops and
37
allowed to dry for 30 min under relative humidity of 15% - 32%. Non-treated control
fomites were sampled and remaining surfaces were wiped with a disinfectant wipe and
allowed to dry for 10 min. Disinfectant wipe-treated control fomites were sampled and
fomite-to-finger transfers were performed on the remaining surfaces using a 1.0 kg/cm2
pressure for 10 sec. The test organisms were reduced by 98.1% to 99.997% on the
fomites after the surfaces were wiped. Microbial fomite-to-finger transfer from
disinfectant wipe-treated surfaces were, lower (less than 0.5%) than from non-treated
surfaces (up to 36.3%). This study demonstrated a disinfectant wipe intervention to be
very effective in reducing bacterial contamination on several different types of fomites
found in homes, office building, and healthcare facilities. These findings also showed that
less fomite-to-finger transfer occurs when surfaces have been treated with disinfectant
wipes compared to non-treated surfaces (fomites).
The manuscript in Appendix F describes the QMRA modeling the probability of a C.
jejuni infection from a cross-contamination to the surface and a subsequent fomite-tofinger transfer then a hand-to-mouth transfer occurring resulting in a dose. The risk of
infection C. jejuni without and with the use of a disinfectant wipe is compared. A Monte
Carlo simulation was used to compare the risk of transferring C. jejuni strain, from
various surfaces to hands and subsequent transfer it to the mouth. Several assumptions
were used as input parameters along with α and N50 values in the classical Beta-Poisson
model to determine the risk of infection. The disinfectant-wipe intervention reduced the
risk of Campylobacter infection, illness, and death by 2 to 3 orders on all fomites. The
38
disinfectant-wipe intervention reduced the annual risk of illness below the reported
national average of diagnosed Campylobacteriosis cases 1.3E-04. This risk assessment
demonstrates that the use of disinfectant wipes to decontaminate surface areas after
chicken preparation reduces the risk of C. jejuni infections up to 99.2%.
39
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59
APPENDIX A: MINIREVIEW: TRANSFER OF BACTERIA AND VIRUSES TO
HANDS
Running title: Transfer of Microorganisms to Hands
Gerardo U. Lopez and Charles P. Gerba#
Department of Soil, Water and Environmental Science, University of Arizona, Tucson,
Arizona 85721
#
Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail:
[email protected]
A manuscript for
Applied and Environmental Microbiology
60
ABSTRACT
Fomites are known to play a role in the transmission of bacterial and viral pathogens.
Advances in quantitative microbial risk assessment have allowed for the development of
models, which can be used to determine the probability of infection via the hand and the
likely success of interventions. Critical to these models is information on the
effectiveness of organism transfer from fomites to hands and vice versa. Numerous
factors influence the transfer efficiency of microorganisms, with environmental humidity
being the most important factor with increased transfer. Other factors influencing transfer
include drying time, contact time, pressure, friction type of material, and nature of the
fomite. The greater the pressure applied to a surface and longer the contact time the
greater the amount of transfer that can be expected. Much greater transfer occurs with
hard surfaces (stainless steel) than porous surfaces (paper, cloth). Overall trends can be
observed but transfer is organism dependent. Both Gram negative and positive bacteria
have a greater transfer from hand-to-fomite than virus. Fomite-to-hand transfer is similar
for both virus and bacteria. Hand-to-hand, hand-to-fomite-to-hand and the fomite-tohand-to-fomite transfer direction showed an overall lower transfer than hand-to-fomite
and fomite-to-hand transfer. Hand-to-fomite-to-hand showed the least transfer.
61
INTRODUCTION
It is widely accepted that environmental contamination plays an important role in the
transmission of both respiratory and gastrointestinal infections in homes and in public
settings (1). Fomites found in indoor environments are known reservoirs of microbial
pathogens and a number of studies have shown human bodily fluids commonly
contaminate fomites (2-5) becoming vectors for direct or indirect transfer of pathogens (6,
7). Fomites found in day care centers (4, 8-12), schools (13), office building (14), homes
(15-22), public areas (23) and hospitals (24-30) have been identified as reservoirs and
vectors for secondary modes of transmission. The presence and survival of pathogens on
fomites found in indoor environments is an important factor in evaluating exposure
potential and in the development of quantitative microbial risk assessment models to
assess risk and the likely success of interventions (23, 31, 32). A number of studies have
shown that nosocomial and foodborne pathogens can persist on inanimate surfaces for
weeks or even months (26, 30, 33, 34). They can also remain viable up to several hours
on fingers (35-37). Bacterial and viral persistence on fomites has been shown to be
dependent on the numbers deposited, type of organism, and environmental conditions (26,
38, 39).
Routes of transmission studies using different transfer scenarios; hand-to-fomite, fomiteto-hand, hand-to-hand, hand-to-fomite-to-hand, and fomite-to-hand-to-fomite have been
conducted (Figure 1). The purpose of this review was to assess the importance of factors
affecting the transfer of bacteria and viruses, and the degree of transfer, under different
62
environmental conditions. Such information is important to reduce the uncertainty in
quantitative microbial risk assess models, which can be used to estimate the risk of
infection from contact with contaminated surfaces and the impact of interventions
(disinfectants, hand sanitizers, self-sanitizing surfaces) (40-42).
FACTORS INFLUENCING TRANSFER
Relative Humidity
Few studies have reported the influence of relative humidity on microbial transfer. The
recommended indoor relative humidity is 40 to 60% (43) although it may be as low as
10% during indoor heating and in arid regions (44, 45). Lopez et al., (submitted for
publication) investigated the transfer efficiency of Escherichia coli, Staphylococcus
aureus, Bacillus thuringiensis spores, coliphage MS2, and poliovirus type 1 from porous
and nonporous fomites under high (40% to 65%) and low (15% to 32%) relative humidity.
It was found that greater transfer typically occurs under high relative humidity (up to
79.5%) compared to low relative humidity (up to 57%) with a few exceptions. Poliovirus
transfer however was not affected by relative humidity.
Damp versus dry fomites or hands
A number of studies have shown that moist, damp fomites or hands result in greater
microbial transfer than dry surfaces. Reed (46) reported under dry conditions rhinovirus
transfer rates were less efficient than under damp conditions (Figures 2, 3, and 5). Damp
63
mucus was more efficient in transfer than mucous dried on fingers. Marples and Tower
(47) found 85% transfer of Staphylococcus saprophyticus from wet hands contaminated
after firmly grasping a bottle with a rectangle kitchen cloth taped to it (Figure 2). The
greater transfer efficiency was attributed to wet hands (47). Sattar et al. (48) found that
moist poly cotton fabric and all-cotton fabric had greater S. aureus transfer than transfer
from dry fabrics.
Duration of drying time
A number of studies have shown that drying time fomites or hands decreases microbial
transfer. This most likely do to inactivation of the organisms during drying of the surface
(14). Ansari et al. (35) found that longer drying times of rotavirus inoculum from 20 to 60
min on fingers or fomites resulted in lower transfer. Transfer of rotavirus from 20 and 60
min observed were (16.1% and 1.8% hand-to-fomite), (16.8% and 1.6% fomite-to-hand),
and (6.6% and 2.8% hand-to-hand), respectively (Figures 2, 3, and 5) (35). Similar
observations were observed by Marples and Towers (47) when fomite-to-hand transfers
were conducted using 200 cm2 rectangle kitchen cloths taped to glass bottles and
contaminated with S. saprophyticus and immediately grasped firmly by hands resulting in
a 9.8% and 6.2% transfer efficiencies using the palm-index finger and thumb wash
elution method, respectively (Figure 4). Compared to transfer efficiencies drying for 1.0
h showed 0.05% and 0.4% (Figure 4) (47). They also observed a decrease of S.
saprophyticus transfer from contaminated fabric to hands and then to a second donor
64
fabric when hands were not allowed to dry compared to when they were air dried for 15
min.
Scott and Bloomfield (49) found Staphylococcus aureus, Escherichia coli, Klebsiella
aerogenes, and Salmonella spp. to be consistent with Ansari et al. (35) observations with
a decrease in transfer from laminate to hands with a longer drying time from zero to 24 h
(Figure 4). Similar results were observed with cloth to hands however at the 24 and 48 h
the to numerous to count bacterial data suggested there was regrowth in the cleaning
cloth (Figure 4).
Mbithi et al. (36) performed a series of hepatitis A virus (HAV) transfer studies
examining the effects of drying time from 20 to 240 min. Mbithi et al. (36) reported a
decrease in HAV transfer after the longer drying time (Figures 2, 3, and 5). Paulson (50)
reported on a series of transfer studies using feline calicivirus strain F9 as surrogate for
noroviruses (Figure 2). Generally there was no difference in transfer between the 5 and
15 min drying with the spatula, fork, and the cutting board except for the doorknob where
there was a greater transfer at 15 min than at 5 min (50).
Type of fomite material
Several studies have shown that fomite type influences microbial transfer. Scott and
Bloomfield (49) found Staphylococcus aureus and Escherichia coli had a greater transfer
from nonporous fomite laminate to hands than porous cloth to hands (Figure 4). Similar
65
results were reported by Rusin et al. (51) for PRD 1, Micrococcus luteus, Serratia
rubidea observing transfer rates from hard, nonporous surfaces; faucet handles and phone
receivers to hands, to be greater than from porous surfaces; dishcloth, sponge, and
laundry (Figures 3 and 4). Despite the low percent transfer for the dishcloth and sponge
they had the highest contamination of hands compared to the other surfaces because of
the large numbers of bacteria on these fomites (51).
Lopez et al., (submitted for publication) also observed that E. coli, S. aureus, B.
thuringiensis, and MS2 had greater transfer efficiencies from ceramic tile, glass, granite
and other nonporous fomites than from nonporous fomites; cotton, polyester, and paper
currency (Figure 3 and 4). Transfer rates under high relative humidity for nonporous
fomites were greater (30% to 79.5%) compared to porous fomites (0.3% to 2.3%) and
under low relative humidity nonporous fomites were also greater (5.4% to 57.0%)
compared to porous (0.03% to 0.4%) (Figures 3 and 4).
Sattar et al. (48) found that 50% polyester 50% cotton had greater transfer of S. aureus to
fingers than 100% cotton. These findings are similar to that observed with cotton,
polyester, and paper currency (Figure 4) (Lopez et al., submitted for publication).
Type of Organism
A number of studies have shown that microorganism type does influence transfer. Ansari
et al. (2) compared the transfer of rhinovirus 14, a non enveloped virus, and human
66
parainfluenza virus 3 (HPIV), an enveloped virus, and found HPIV 3 to transfer less than
rhinovirus 14 who transferred (less than 1.0%) in hand-to-fomite and hand-to-hand
transfers (Figures 2 and 5). However, in fomite-to-hand transfer direction HPIV 3 had a
slightly greater transfer than rhinovirus 14 with 1.5% (Figure 3) (2).
Rusin et al. (51) reported the transfer of Micrococcus luteus a Gram positive bacterium,
Serratia rubidea a Gram negative bacterium, and PRD-1 coliphage from faucet handles
and phone receivers. They found PRD-1 to have a transfer of (33.47% and 65.8%)
(Figure 3), M. luteus to have a slightly greater transfer (40.03% and 41.81%) than S.
rubidea (27.59% and 38.47%), respectively (Figure 4). All three organisms had (< 1.0%)
transfer from porous objects to hands. In contrast M. luteus had the greatest transfer while
PRD-1 and S. rubidea were similar (Figure 2) (51). These transfer differences are
attributed to the ability of the virus to remain viable and tolerant of the environmental
conditions and the interaction with hand characteristics.
Mackintosh and Hoffman (52) observed a transfer difference between Staphylococcus
saprophyticus, a Gram positive bacteria, that had a greater fomite-to-hand transfer
(1.67%) than the Gram negative bacteria E. coli, Klebsiella aerogenes, Serratia
marcescens, and Pseudomonas aeruginosa with (0.29% to 0.5%), however Streptococcus
pyogens a Gram positive bacteria did not follow this observation (Figure 4).
67
Level of fomite contamination
Pancic et al. (53) found that increasing the amount of mucous containing rhinovirus
increased transfer from hand to fomite from 13.6% to 44.6% (Figure 2). However the
opposite was seen from fomite-to-hand transfer with the lower inoculum showing a
greater transfer (61.5%) (Figure 3) (53).
Hand washing
Marples & Tower (47) also tested the effects of various hand washing procedures on
transfer of organisms from fabrics. They found 70% ethanol to reduce transfer the most
compared to detergent and bar soap. Marples and Tower (47) also compared the effect of
rubbing hands with alcohol-impregnated wipes and with 80% ethanol and found less
transfer with hands rubbed with ethanol (21%). Mackintosh and Hoffman (52) also
investigated the influence of hand washing methods and found both the use of 70% or
80% alcohol and soap and water were effective in reducing transfer.
Julian et al. (54) found that hand washing prior to reduced transfer of the coliphages
ΦX174, fr, and MS2. The reduction in coliphage transfer was greater for finger-to-glass
than glass-to-finger transfer (Figure 2 and 3) (54).
Rheinbaben et al. (37) reported ΦX174 fomite-to-hand transfer efficiencies of 0.000005%
from washed hands and an average of 0.0004% for three individuals with unwashed
hands after 6 h (Figure 3). The low transfer was due to the rapid inactivation of the virus.
68
In the finger-to-finger hand washing transfer a 16.7% was determined (Figure 5) (37).
Virus re-isolated from the soap could have contributed to the transmission to recipient
fingers (37).
Direction of transfer
Direction of transfer may influence transfer but appears species dependent. Mackintosh
and Hoffman (52) observed a distinct difference in transfer of S. saprophyticus, S.
pyogens E. coli, K. aerogenes, S. marcescens, and P. aeruginosa from fabric to fabric via
the hands. Transfer stage fomite-to-hand (0.021% to 1.67%) (Figure 4), was 100 times
less than hand-to-fomite (17% to 88%) (Figure 2), overall fomite-to-hand-to-fomite
transfer was only (0.01% to 0.37%). The greater transfer rate in the hand to fomite is
consistent with Marples and Tower (47) observations of 85% transfer efficiency of
Staphylococcus saprophyticus contaminated hands to fabric taped to bottles (Figure 2).
Both studies also observed very low fomite-to-hand-to-fomite transfer of less than 0.5%.
Hubner et al. (55) demonstrated the transfer of E. coli by inoculating the index finger of a
volunteer, air-drying it and then touched a sterilized paper, then a second volunteer’s
index finger was moistened with saline to simulate licking the finger before turning pages
and then pressed on the contaminated swatch to simulate cross-contamination. They
found a 0.009% transfer rate (Figure 6) that is consistent with (Lopez et al., submitted for
publication) found with paper currency transfers to fingers (Table 3).
69
A transfer rate of 0.03% for M. luteus and 0.04% for S. marcescens was observed from
contaminated hands to paper-towel dispenser, while greater transfer rates were observed
from contaminated dispenser to hands for two separate towels for M. luteus (13.1% and
6.0%, respectively) and for S. marcescens (12.4% and 6.7%, respectively) (Figure 2 and
4) (56).
In contrast Pancic et al. (53) observed similar rhinovirus transfer from both hand-tofomite and fomite-to-hand transfer directions. The hand-to-fomite-to-hand transfer
directions for rhinovirus did show greater transfer than observed in the fomite-to-hand-tofomite by Mackintosh and Hoffman (52) and Marples and Tower (47).
The degree of virus transfer is dependent on virus type. Rhinovirus 14 was found to have
similar transfer regardless of direction (0.67% to 0.92%) while human parainfluenza
virus 3 (HPIV) showed very different transfer rates depending on the direction of transfer,
hand-to-fomite and hand-to-hand (2) (Figure 2, 3, and 5). Different transfer rates for
ΦX174, fr, and MS2 coliphage are influenced by direction of transfer and is specific to
viral species (54). All three types of coliphage had similar transfer rates from finger-tofomite (15% to 28%) and ΦX174 and MS2 coliphage had comparable transfers from
fomite-to-finger as observed from hand-to-fomite (Figure 2 and 3). However fr
coliphage had greater transfer (37% to 39%) from fomite-to-finger.
Coliphage ΦX174 could be horizontally transferred to 14 consecutive individuals with an
average percent transfer of 0.04% after touching a contaminated door handle allowed to
70
initially dry for 15 min and with 15 s intervals between each consecutive contact (37). In
the finger-to-finger transfer experiments they reported an average of 0.004% and 0.03%
for the horizontal (14 consecutive fingers) and vertical (finger-to-finger-to-finger)
transfers, respectively (Figure 5) (37). Even though the transfer efficiencies for the
horizontal and vertical transfers were < 1.0% and generally decreased after each contact,
a 2 to 3 log transfer of ΦX174 resulted (37). In contrast Pancic et al. (53) observed
greater rhinovirus hand-to-hand transfer (5.9%) (Figure 5) than observed by Rheinbaben
et al. (37) for ΦX174.
Friction and pressure
Contact time, friction, and pressure have been shown to influence transfer efficiency.
Transfer of S. aureus contaminated fabric to fingers is greater with friction than without
friction independent if the fabric was moist or dry (48). Friction increased the level of
transfer by two- to five fold from both types of fabric with a transfer range for the dry
fingerpad without friction from 0.01% to < 0.2%, and with friction 0.01% to 1%, while
the moistened fingerpad without friction 0.01% to < 2.%, and with friction 0.01% to <
3%. Friction also increases the transfer of viruses (36). The mean HAV transfer was
almost 3 times greater when friction was applied (36). The amount transferred to fingers
increased almost 5 times when the finger pressure was increased from 0.2 kg/cm2 to 1.0
kg/cm2 (36).
71
DISCUSSION
Factors that influence transfer are not independent of each other, but rather interact with
each other to either promote or reduce overall transfer. Microbial transfer is influenced
by environmental factors such as relative humidity, moisture, drying time, fomite
material, direction of microbial transfer as well as biological factors such as bacterial or
virus species, and the characteristics of the fingers washed or unwashed hands (Table 1).
Usually higher relative humidity conditions reduce the desiccation of the microbial
suspension and as a result greater bacterial and viral transfer occurs. Lower relative
humidity conditions allow the microbial suspension to dry faster and promote bacterial
and viral inactivation resulting in lower transfer of viable microorganisms. Longer drying
times have been shown to reduce transfer rates by promoting the desiccation of the
suspension medium resulting in microbial inactivation.
Moisture found on fomites or hands has been shown to increase transfer rates. Greater
microbial contamination levels found on donor or recipient fomites increases transfer.
Friction and pressure have also been shown to increase transfer rates of microbial
pathogens. Washing hands has been shown to reduce transfer compared to unwashed
hands. Relative humidity, moisture, level of contamination, friction, pressure, and drying
time interact with each other and in combination along with hand characteristics pH and
washed or unwashed hands influence microbial transfer rates.
72
Type of fomite material has been shown to have a significant impact on transfer rates.
Transfer rates between nonporous fomites have been shown to be much greater than from
porous fomites. Moisture in the form of microbial suspension is able to persist longer on
nonporous fomites compared to porous fomites that can absorb the suspension and
provide crevices for bacteria and viruses to attach to and reduce transfer.
Direction of transfer has also been indicated to influence transfer but is species dependent.
Bacterial and viral species have been shown to have different transfer rates due to the
unique interaction that a species has with the suspension medium, fomite, and
environmental conditions. In general Gram negative bacteria seem to be transferred less
readily than Gram positive bacteria.
Coliphage are generally transferred at similar rates, however they seem to be dependent
on the direction of the transfer, fomite-to-hand or hand-to-fomite. Enteric viruses usually
are more readily transferred than respiratory viruses with direction playing a major role.
There is plenty of evidence to suggest that the environment could contribute significantly
to contamination of healthcare workers hands, child care givers, and foodhandlers (57).
The long-term survival of viruses and bacteria on common surfaces found in many
environments, in addition to the laboratory, emphasizes the possible role of nonporous
and porous fomites in the transmission of viruses and bacteria (1, 34, 58). These studies
suggest that the transmission of virus from donors who are shedding large amounts of
73
virus through coughing, sneezing, or nasal mucosa can cross-contaminate surrounding
surfaces (58).
CONCLUSIONS
Transfer of pathogens in the indoor environment is complex being dependent on a
number of factors. Transfer is very organism dependent, with relative humidity and type
of fomite having the greatest influence on transfer. Obviously hard surfaces will present
a greater risk than porous surfaces. While general assumptions can be made on the degree
of transfer for bacteria and viruses, it is organism dependent. Uncertainty in risk models
can best be reduced by studies on specific data on transfer from hard nonporous surfaces,
since the greatest transfer for most organisms occur under these conditions. It is
important that future studies on microbial transfer include data on the important
environmental factors that influence transfer for better comparison among groups of
organisms.
The lack of a standard transfer method along with the different factors influencing
transfer makes evaluating transfer rates difficult. Future transfer studies should
standardize key factors such as pressure, moisture, contact time, and drying time.
Bacterial hand-to-hand transfer studies are still lacking to better understand the
transmission between individuals. There is also a need for hand-to-fomite-to-hand
bacterial and viral studies as well as fomite-to-hand-to-fomite viral studies.
74
ACKNOWLEDGEMENTS
This research was supported by the Center for Advancing Microbial Risk Assessment,
funded by the U.S. Environmental Protection Agency Science to Achieve Results
(STAR) program. And the U.S. Department of Homeland Security University Program
Grant R83236301. Special thanks to Sheri Maxwell for technical assistance.
75
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83
FIGURES
Hand
Fomite
Hand
Fomite
Portal
Entry
FIG 1 Transfer direction; hand-to-fomite, fomite-to-hand, hand-to-fomite-to-hand,
fomite-to-hand-to-fomite.
84
100
90
Transfer efficiency (%)
80
70
60
50
40
30
20
10
0
Nonporous
Nonporous
Nonporous
Rhinovirus
Human
Parainfluenza
virus-3
Rotavirus
Respiratory Viruses
Nonporous
Hepa
s A Virus
Enteric Viruses
Lips - skin
Nonporous
Nonporous
Nonporous
Porous
PRD-1
Φ-X174
fr
MS2
Staphylococcus
saprophy cus
Coliphage
Virus
FIG 2 Hand-to-fomite bacterial and viral transfer efficiency
Lips - skin
Nonporous
Micrococcus luteus
Porous
Porous
Porous
Nonporous
Lips - skin
Porous
Escherichia coli
Klebsiella
aerogenes
Psuedomonas
aeruginosa
Enterobacter
aerogenes
Serra a rubidea
Serra a
marcescens
Gram (+) bacteria
Gram (-) bacteria
Bacteria
85
100
90
Transfer efficiency (%)
80
70
60
50
40
30
20
10
0
Nonporous
Nonporous
Nonporous
Rhinovirus
Human Para
Influenza
Rotavirus
Respiratory Viruses
Nonporous
Hepa
s A Virus
Nonporous
Nonporous
Feline Calicivirus
Poliovirus type 1
Nonporous
Enteric Viruses
Nonporous
Nonporous
ΦX174
fr
Coliphage
Virus
FIG 3 Fomite-to-hand viral transfer efficiency
Porous
PRD-1
Nonporous
Porous
MS2
86
90
80
Transfer efficiency
70
60
50
40
30
20
10
0
Porous
Porous
Staphylococcus
saprophy cus
Streptococcus
pyogenes
Nonporous
Porous
Staphylococcus aureus
Nonporous
Porous
Micrococcus luteus
Nonporous
Porous
Escherichia coli
Porous
Porous
Klebsiella
aerogenes
Serra a
marcescens
Gram (+) bacteria
Porous
Serra a rubidea
Gram (-) bacteria
Bacteria
FIG 4 Fomite-to-hand bacterial transfer efficiency
Nonporous
Porous
Nonporous
Nonporous
Psuedomonas
aeruginosa
Enterobacter
aerogenes
Salmonella spp.
Nonporous
Porous
Bacillus thuringiensis
Endospores
87
Transfer efficiency (%)
40
35
30
25
20
15
10
5
0
Finger
Finger
Finger
Rhinovirus
Human Parainfluenza
virus-3
Rotavirus
Respiratory Viruses
Hepa
Enteric Viruses
Virus
FIG 5 Hand-to-hand viral transfer efficiency
Finger
s A Virus
Finger
ΦX174
Coliphage
88
TABLE
TABLE 1 Factors influencing transfer
Factor
Remarks
Relative Humidity
Usually greater transfer at high relative
humidity
Drying time
Greater transfer with short drying time
Contact time
Greater transfer with longer contact time
Pressure
Greater transfer with greater contact
pressure
Friction
Greater transfer with friction
Moisture
Greater transfer with moister than under
dry conditions
Contamination level
Greater contamination level the greater the
transfer
Transfer direction
Usually greater transfer from fomite-tohand than hand-to-fomite but is species
dependent
Type of fomite material
Greater transfer with nonporous than with
porous surfaces
Washed vs unwashed hands
Usually greater transfer with unwashed
hand than with washed hands
Finger vs hand
Similar transfer in relation to surface area
Bacteria, virues, and ensospores
Generally, Gram positive bacteria transfer
more readily, then viruses, Gram negative
bacteria and then endospores
Gram negative, Gram positive,
and endosprores
Generally, Gram positive bacteria transfer
more readily than Gram negative bacteria
and endospores
Enteric and respiratory viruses
Enteric viruses usually transfer more
readily than respiratory viruses
Coliphage transfer is similar among
different types but is dependent on
direction
Coliphage
89
APPENDIX B: COMPARISON OF TWO APPROACHES IN DETERMINING
TRANSFER EFFICIENCY OF ESCHERICHIA COLI FROM NONPOROUS
FOMITES TO FINGERS
Running title: Determining Bacterial Percent Transfer
Gerardo U. Lopez1, Masaaki Kitajima1, Kelly A. Reynolds2, and Charles P. Gerba1 #
1
Department of Soil, Water and Environmental Science, The University of Arizona,
Tucson, Arizona 85721 USA; 2The University of Arizona, Mel and Enid Zuckerman
College of Public Health, 1295 N. Martin Ave., Tucson, Arizona 85724, USA
#
Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail:
[email protected]
A manuscript for
Applied and Environmental Microbiology
90
ABSTRACT
Fomites were inoculated with Escherichia coli and fomite-to-finger transfer efficiency
was evaluated using two approaches: one determines the efficiency based on control
fomite; the other is based on the sum of bacteria recovered from a finger and donor
fomite. No statistical difference was observed between the two approaches.
TEXT
Inanimate surfaces (fomites) are reservoirs of microbial pathogens and play a critical role
in fomite-hand routes of transmission (1-4). Several studies have demonstrated that
fomites found in indoor environments such as day care centers and schools (5-11), homes
(12-19), public areas (20, 21) and hospitals (22-27) have been contaminated with
microbes and human bodily fluids (6-8, 13, 20, 21, 28-30) that persist on fomites for
hours to months (23, 31, 32) and contribute to secondary modes of transmission.
Fomite-to-finger transfer efficiency of microbial pathogens is important in modeling the
potential for transmission (1, 33-35). Consistent approaches for determining microbial
transfer efficiencies are essential in producing accurate exposure input values for risk
assessment modeling. In the present study, we compared the following two approaches in
determining fomite-to-finger transfer efficiency: approach 1 defines transfer efficiency
(TE) as colony forming units (CFU) recovered from finger relative to CFU recovered
from control fomite, while approach 2 defines TE as the CFU recovered from the finger
91
(recipient) relative to the sum of the CFU recovered from the finger (recipient) and the
donor fomite.
One subject conducted the fomite-to-finger transfer experiments. Project approval
obtained from the University of Arizona’s Office for Human Subjects Research.
Subject’s hands were washed, rinsed and sprayed with 70% ethanol. Study organism,
Escherichia coli C-3000 (ATCC 15597) obtained from the American Type Culture
Collection (ATCC Washington DC), was grown as previously described (36).
Temperature (19°C to 25°C) and relative humidity (20% to 34%) were monitored with a
High Accuracy Thermo – Hygro (VWR, Mississauga, ON). Five nonporous fomite
materials were tested with surface areas of 16 to 25 cm2; acrylic (poly-methyl
methacrylate) matte non-glare finish, ceramic tile porcelain (Home Depot, Atlanta, GA),
glass slides (VWR, Mississauga, ON), laminate various colors (Wilsonart International,
Temple, TX), and stainless steel 304 (AK Steel Corporation, West Chester, OH). Acrylic
was radiated under UV light for 30 min on each side, while the other fomites were
sprayed with 70% ethanol, washed, and autoclaved. Baseline control, transfer fomite, and
negative control swatch/coupons were placed on a laboratory bench.
A protocol adapted from previous publications (37, 38) was used. Briefly, approximately
107 to 109 CFU/cm2 of E. coli in 10 µl droplets were spread over approximately a 1.0 cm2
area on the center of each fomite using the tip of the pipet and allowed to air-dry (15 to
32 min) on the laboratory bench. Fomites were sampled using a cotton-tipped swab
92
applicator (Puritan Medical Products Company, Guilford, ME). Swabs were wet in 1.0
ml of phosphate buffer saline (PBS) (PBS, SIGMA, St. Louis, MO) and a 6.0 cm2 area
was swabbed with applicator pressed firmly against the surface before being placed back
into the remaining PBS and vortexed. To assess fomite-to-finger transfer, the fomite was
placed at the center of a scale with a digital readout and a finger transfer was performed
by placing the right hand index finger on the center covering the inoculated area of the
fomite for 10 ± 2 s with a 1.0 kg/cm2 (98.0665 kPa) of average pressure (range of 700 g –
1500 g) (FIG S1) (37, 38). Using a cotton-tipped swab applicator (Puritan Medical
Products Company, Guilford, ME), moistened in 1.0 ml of PBS, the index, middle, and
ring finger pads were sampled and placed in the PBS vial and vortexed directly after each
transfer event. E. coli was enumerated by the spread plate technique using MacConkey
agar (EMD Chemicals Inc., Gibbstown, NJ).
Transfer efficiency was calculated using two approaches; equations to calculate TE (%)
by approaches 1 and 2 can be expressed with the following equations (1) (39, 40) and (2)
(36, 41, 42), respectively.
 
 %       100
(1)
 
 %         100
(2)
93
To assess the statistical significance of untransformed percent transfer efficiencies
between the two approaches, a Student’s t-test (two-tailed) was conducted using
Microsoft Excel 2011 and the software package StatPlus: mac, 2009, (AnalystSoft).
Transfer efficiency for E. coli was determined for a total of 72 fomite-to-finger transfer
events. Transfer efficiencies were slightly greater with approach 1 (6.3% to 27.3%) than
with approach 2 (3.8% to 20.7%) (Table 1). E. coli recovered on fingers and fomites are
in (Table 1S) and (FIG S2) provides transfer efficiency in log10 scale and geometric mean
in order to give the range and variability of the data. The greatest transfer efficiency was
obtained with glass (27.3%) with approach 1, while glass and acrylic (20.7%) with
approach 2. Laminate showed the least transfer efficiency for approach 1 and 2 (6.3% and
3.8%, respectively). There were no statistical differences between transfer efficiencies
determined by the two approaches (P = 0.05), demonstrating that the transfer efficiencies
determined by each approach are comparable (Table 1).
Approach 1 has been previously used to investigate the transfer of Staphylococcus
saprophyticus from fabric to hands (39) and to examine the transfer of feline calicivirus
from various fomites to foodhandler gloves (40). Variations of approach 1 have been
previously used to evaluate the transmission of microbial pathogens immediately
following inoculation of fomites (42-46). A limitation to approach 1 is that transfer
efficiencies greater than 100% can result and would need to be truncated to 100% as done
94
with glass and ceramic tile (Table 1) in order to prevent skewing the data. Approach 2
has also been used by several studies for determining transfer efficiency of both bacteria
and viruses from fomites to fingers (36-38, 41, 47).
To the best of our knowledge, this is the first study directly comparing the two
approaches that are widely used to calculate microbial transfer efficiencies and
demonstrating that the efficiencies determined by the two approaches are comparable
showing no statistical difference. The data obtained in the present study can be used as
input values for E. coli transfer efficiency parameters in quantitative microbial risk
assessment (QMRA) to assess risk of infection associated with fomite-to-finger
transmissions and relative exposure from different types of nonporous fomites.
ACKNOWLEDGEMENTS
This research was supported by the Center for Advancing Microbial Risk Assessment,
funded by the U.S. Environmental Protection Agency Science to Achieve Results
(STAR) program. And the U.S. Department of Homeland Security University Program
Grant R83236301.
95
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102
TABLE
TABLE 1: E. coli fomite-to-finger transfer efficiency
Approach 1a
Approach 2b
Transfer efficiency (%)
Transfer efficiency (%)
(n) Average ± SD (Range)
(n) Average ± SD (Range)
Acrylic
(15) 22.2 ± 21.1 (1.8 - 74.8)
(15) 20.7 ± 24.4 (1.4 - 93.9)
0.856
Glass
(15) 27.3 ± 32.3 (3.6 - 100)d
(15) 20.7 ± 22.6 (< 70.6)c
0.526
Ceramic Tile
(21) 13.3 ± 28.3 (1.3 - 100)d
(21) 7.8 ± 8.4 (0.5 - 29.8)
0.405
Laminate
(15) 6.3 ± 4.8 (1.5 - 18.0)
(15) 3.8 ± 3.0 (0.8 - 9.6)
0.096
Stainless Steel
(6) 17.1 ± 15.2 (0.2 - 37.4)
(6) 16.3 ± 16.0 (0.5 - 44.9)
0.928
Surface Type
a
Transfer efficiency (%) = (CFU finger/CFU baseline control fomite) X 100 using untransformed data
b
Transfer efficiency (%) = (CFU finger/CFU finger + CFU donor fomite) X 100 using untransformed data
c
E. coli recovery from fomite or finger were below the detectible limit of 10 CFU/2-cm2 indicated by <
d
Transfer event was > 100% and was truncated to 100%
Student t-test
P-value
103
APPENDIX C: TRANSFER EFFICIENCY OF BACTERIA AND VIRUSES
FROM POROUS AND NONPOROUS FOMITES TO FINGERS UNDER
DIFFERENT RELATIVE HUMIDITY
Running title: Fomite to Finger Microbial Transfer
Gerardo U. Lopez1, 3, Charles P. Gerba1, 3#, Akrum Tamimi1, Masaaki Kitajima1, Sheri
Maxwell1, and Joan B. Rose2, 3
1
Department of Soil, Water and Environmental Science, The University of Arizona,
Tucson, Arizona 85721 USA; 2Department of Fisheries and Wildlife, and 3Center for
Advancing Microbial Risk Assessment, Michigan State University, East Lansing,
Michigan
#
Corresponding author. Tel: (520) 621-6906, Fax (520) 621-6366, E-mail:
[email protected]
Manuscript for
Applied and Environmental Microbiology
104
ABSTRACT
Fomites can serve as routes of transmission for both enteric and respiratory pathogens.
The present study examined the effect of low and high relative humidity on fomite-tofinger transfer efficiency of four model organisms from several common inanimate
surfaces (fomites). Nine fomites of different compositions were studied representing
porous and nonporous surfaces. Escherichia coli, Staphylococcus aureus, Bacillus
thuringiensis, MS2 coliphage, and poliovirus 1 were placed on fomites in 10 µl drops
allowed to dry for 30 min under low (15% to 32%) or high (40% to 65%) relative
humidity. Fomite-to-finger transfers were performed using 1.0 kg/cm2 of pressure for 10
s. Transfer efficiencies were greater under high relative humidity for both porous and
nonporous surfaces. Most organisms on average had greater transfer efficiencies under
high relative humidity compared to low relative humidity. Nonporous surfaces had a
greater transfer efficiency (up to 57%) than porous surfaces (< 6.8%) under low relative
humidity, as well as under high relative humidity; nonporous (up to 79.5%) and porous
(< 13.4%). Transfer efficiency also varied with fomite material and organism type. The
data generated can be used in quantitative microbial risk assessment models to assess risk
of infection from fomite transmitted pathogens to humans and relative exposure from
different types of fomites and microorganisms.
105
INTRODUCTION
Inanimate objects, or fomites, are a potential reservoir in the transmission of pathogens
either directly, by surface-to-mouth contact, or indirectly, by contamination of fingers
and subsequent hand-to-mouth, hand-to-eye, or hand-to-nose contact (1-5). Bodily fluids
such as saliva, mucus, nasal secretions, blood, urine, and feces may all potentially contain
pathogens that can be transmitted via fomites (6-9). A number of studies have shown that
enteric and respiratory pathogens are capable of surviving from hours to months on
fomites, depending on the numbers deposited, the type of microorganism, and the
variable environmental conditions (10-12). Several studies have shown that inanimate
surfaces found in day care centers (8, 13-17), schools (18), office buildings (19), homes
(20-27), public areas (28), or hospitals (12, 29-33) can be reservoirs for secondary modes
of transmission with contaminated hands playing a critical role as a route of exposure.
The efficiency of transfer of a pathogen to the hand from the fomite is important in
modeling the potential for its transmission (11, 34-36). This information can be used to
understand the spread of disease in indoor environments, and the potential for designing
surfaces that reduce transfer efficiency and /or are antimicrobial (5). The purpose of this
work was to better elucidate the transfer efficiencies of several different types of
organisms under control conditions to provide data that may be used in quantitative
microbial risk assessment (QMRA) models.
106
MATERIALS AND METHODS
Subjects
A single subject conducted the fomite-to-finger transfer experiments. Permission was
obtained from the University of Arizona’s Office for Human Subjects Research prior to
the study.
Bacteria, Virus and preparation of inocula
Study organisms. Escherichia coli (ATCC 15597), Staphylococcus aureus (ATCC
25923), Bacillus thuringiensis (ATCC 10792), and coliphage MS2 (ATCC 15597-B1)
were obtained from the American Type Culture Collection (ATCC Manassas, VA).
Poliovirus 1 (PV-1; strain LSc-2ab) was obtained from the Department of Virology and
Epidemiology at the Baylor College of Medicine (Houston, TX). These organisms were
selected as model organisms for pathogenic Gram negative and Gram positive bacteria,
spore-forming bacteria, and viruses.
Gram negative and Gram positive bacteria inoculum preparation. Frozen aliquots of E.
coli and S. aureus were transferred into separate flasks containing100 to 150 ml of
Tryptic Soy Broth, (TSB, EMD, Gibbstown, NJ) incubated for 18 ± 2 h at 37°C on an
orbital shaker (150 to 180 rpm), and streaked for isolation onto Tryptic Soy Agar plates
(TSA, EMD Gibbstown, NJ). The bacteria were then subcultured in a flask of TSB and
incubated for 18 ± 2 h at 37°C on an orbital shaker (150 to 180 rpm) (37).
107
Endospore-forming bacteria inoculum preparation. B. thuringiensis spores were
prepared as previously described with minor modifications (38). Briefly, spores were
suspended in Difco sporulation media with supplements (DSM + S; Becton, Dickinson
and Company, Sparks, MD), cultivated for 24 h at 37°C on an orbital shaker (150 to 180
rpm), and resuspended in fresh DSM + S to obtain a final OD600 of 0.1 (Spectronic
Genesys 5, Milton Roy, Ontario Canada).
Virus inoculum preparation. MS2 coliphage were prepared as previously described with
minor modifications (37). Briefly, 0.1 ml of phage suspension and 0.5 ml of a log phase E.
coli 15597 (host bacterium) culture 0.5 ml were added to top agar, melted, and
maintained at 48°C. The inoculated top agar was mixed and poured over the TSA. The
solidified agar overlay plates were then inverted and incubated at 37°C for 24 h. TSB
was then added to each plate and maintained at room temperature for 2 h. The TSB eluent
was aspirated and centrifuged at (1,989 X g for 10 min), after which, the supernatant was
filtered through 0.22 µm pore size Acrodisc® Syringe Filters (Pall Corporation, Ann
Arbor, MI) pre-moistened with 3% beef extract (Bacto™, Becton, Dickinson and
Company, Sparks, MD), the virus tittered, and stored at 4°C.
PV-1 propagation and plaque-forming assays were conducted as described previously (39,
40). Briefly, PV-1 was propagated on buffalo green monkey kidney (BGM; ATCC CCL81; American Type Culture Collection, Manassas, VA) cell line monolayers with
minimal essential media (MEM) containing 5% calf serum (HyClone Laboratories,
108
Logan, UT) at an incubation temperature of 37°C with 5% CO2. Plaque-forming assays
were performed using six-well plates with confluent monolayers of the BGM cells.
Control wash and disinfection
Prior to all experiments, the subject’s hands were washed with warm water and nonantibacterial soap (Liquid Joy; Procter and Gamble, Cincinnati, OH) for 45 s, rinsed with
water, and dried with paper towels. Each hand was then sprayed twice with 70% ethanol,
rubbing the alcohol thoroughly over the hands and wrists for 15 s, and subsequently airdried. After conducting fomite-to-finger transfer experiments with the prepared inocula,
fingers were disinfected twice with 70% ethanol, wrapped with a 70% ethanol saturated
paper towel for 30 s, then washed and rinsed using warm water and Softsoap® antibacterial liquid hand soap (Colgate-Palmolive, Morristown, NJ) for 45 s, and then dried
with paper towels. After sampling fingers for B. thuringiensis spores and PV-1, fingers
were placed in 10% bleach (The Clorox Company, Oakland, CA) for 15 s and then
neutralized with 10% sodium thiosulfate (EMD, Gibbstown, NJ). The hands were then
washed as described above to prepare for subsequent trials.
Relative Humidity Conditions and Temperature
The study consisted of two relative humidity conditions - high (40% to 65%) and low
(15% to 32%) relative humidity. To achieve both humidity conditions, two separate
incubators were turned off and used. Incubator temperatures thus reflected room
temperature ranges of 19°C to 25°C. The temperature and relative humidity were
109
monitored with a High Accuracy Thermo – Hygro (VWR, Mississauga, ON). During
days with higher ambient relative humidity in the laboratory, t.h.e.® Desiccant (EMD,
Gibbstown, NJ) and Drierite desiccant (Drierite, Xenia, OH) were utilized in the
incubator to decrease the relative humidity to the low relative humidity range (15% to
32%). During laboratory ambient lower relative humidity conditions a BIONAIRE®
humidifier (Milford, MA) was used in the specific incubator to increase the relative
humidity to the high relative humidity range (40% to 65%).
Fomites tested
Nine different types of fomite materials were tested ranging in surface areas of 16 to 25
cm2 six nonporous and three porous (Table 1). With the exception of acrylic, all fomites
were sterilized by autoclave whereas acrylic was radiated under UV light (254 nm) for 30
min on each side. After fomite-to-finger transfers with E. coli, S. aureus, and MS2,
nonporous fomites were sprayed three times with 70% ethanol and allowed to dry for 10
min. Nonporous fomites used in the finger transfers with B. thuringiensis spores and PV1 were disinfected with 10% bleach (The Clorox Company, Oakland, CA), then allowed
to sit for 10 min, and subsequently neutralized in 10% sodium thiosulfate (EMD,
Gibbstown, NJ). Fomites were then washed under warm running water with nonantibacterial soap (Liquid Joy, Procter and Gamble, Cincinnati, OH), rubbed with a wet
paper towel on the surface area of inoculation, rinsed thoroughly with RO treated water,
air-dried, and autoclaved. Cotton and polyester fomites were discarded after use. Paper
currency was autoclaved and reused.
110
Inoculation of fomites
Layout of fomites. For each of the six nonporous and three porous fomites, seven
swatches/coupons were evenly spaced on the middle shelf of an incubator. Each trial
consisted of three control swatches/coupons, three fomite-to-finger transfer
swatches/coupons, and one negative control swatch/coupon to ensure that the fomites
were not previously contaminated.
Organism concentration. The concentration of organisms added to the fomites was
approximately 107 to 108 colony forming units (CFU)/cm2 of E. coli in (TSB, EMD,
Gibbstown, NJ), 108 to 109 CFU/cm2 of S. aureus in TSB, 106 to 107 CFU/cm2 of B.
thuringiensis spores in (DSM + S; Becton, Dickinson and Company, Sparks, MD), 109 to
1011 plaque forming units (PFU)/cm2 of MS2 in TSB, and 108 PFU/cm2 of PV-1 in (PBS,
Sigma-Aldrich, St. Louis, MO) in 10 µl droplets. Using a pipet tip, the 10 µl inoculum
droplets were spread over approximately a 1.0 cm2 area on the center of each fomite. The
paper currency were divided into four 24 cm2 sections; two one dollar bills were used to
make the seven swatches. With paper money for each set of transfer experiments, a new
1.0 cm2 area on each of the 24 cm2 sections were inoculated. Using an ink marker, an
identifying spot was placed near the inoculated area on cotton, polyester, and paper
money due to the absorbance. The fomites were allowed to dry for 30 min.
111
Fomite-to-finger transfer, sampling, and assays
Fomite sampling. Fomites were sampled using a cotton-tipped swab applicator (Puritan
Medical Products Company, Guilford, ME), when inoculated with E. coli, S. aureus, B.
thuringiensis, and MS2. In the case of PV-1, a polyester fiber-tipped applicator swab
(Falcon, Becton Dickinson and Company, Cockeysville, MD) was used. Swabs were wet
in 1.0 ml of phosphate buffer saline (PBS, Sigma-Aldrich, St. Louis, MO), and then a 6.0
cm2 area on the fomite was swabbed using firm sweeping and rotating motions. The
swab was then placed back into the remaining PBS and vortexed for 5 s.
Transfer experiment. One transfer trial consisted of three separate fomite-to-finger
transfer events using the index, middle, and ring fingers of the right hand for each surface
type. Two transfer trials were conducted, resulting in six transfers total for each fomite
under both low and high relative humidity. A protocol adapted from Ansari et al. (41)
and Mbithi et al. (42) was used to perform the fomite-to-finger transfer of test organisms
from the previously mentioned nonporous and porous fomites to hands after a 30 min
drying time. To assess transfer, the fomite was placed at the center of a scale with a
digital readout, and a finger transfer was performed by placing the right hand finger on
the center, covering the inoculated area of the fomite, for 10 s with 1.0 kg/cm2 (98.0665
kPa) of average pressure (range of 700 g – 1500 g) (41, 42).
Sampling the fingers. Using a cotton-tipped swab applicator (Puritan Medical Products
Company, Guilford, ME) moistened in 1.0 ml of PBS (PBS, SIGMA, St. Louis, MO), the
112
index, middle, and ring finger pads were sampled using a sweeping, rotating motion and
placed in the PBS vial and vortexed directly after each transfer event. A polyester-tipped
swab (Puritan Medical Products Company, Guilford, ME) was used to sample PV-1.
Organism assays. E. coli, S. aureus, and B. thuringiensis spores were enumerated using
the spread plate technique on MacConkey (EMD Chemicals Inc, Gibbstown, NJ),
Mannitol salt agar (MSA, EMD Chemicals Inc, Gibbstown, NJ), and TSA (EMD
Chemicals Inc., Gibbstown, NJ) agar plates, respectively. The plates were incubated at
37°C for 18 ± 2 h. B. thuringiensis spore samples were heat shocked at 81 ± 2°C for 10
min prior to spread plating, to stimulate germination. The MS2 plaque assay was
conducted using the double agar overlay method using TSA (EMD Chemicals Inc.,
Gibbstown, NJ) (39, 43). PV-1 titrations were performed using 10-fold serial dilution
plaque-forming assays as described previously (39, 40). All dilutions were assayed in
duplicate.
Transfer efficiency calculation and statistical analyses
Calculation of transfer efficiency. Bacterial colonies and viral plaques were enumerated
and the transfer efficiencies were calculated using Equation (1) below (44, 45). The
transfer efficiency (TE) is defined as the number of CFU or PFU recovered from finger
relative to the CFU or PFU recovered from the control fomite. If bacteria or phage were
not recovered from the finger pad, the lower detection limit of 10 CFU or PFU was used
as an estimate for the microorganism recovered as previously described (46, 47). A less
113
than value (<) is used to indicate that the transfer efficiency was lower than the least
detectable limit. Finger transfers greater than 100% were truncated to 100%.
   
 %        100
(1)
Statistical analyses. Data were entered in Microsoft Excel 2010 and the software
package StatPlus:mac, 2009, (AnalystSoft) to compute the descriptive statistic measures
of mean percent transfer efficiency, the standard deviation, and statistical significance.
To assess the statistical significance at the (P = 0.05) level, the percent transfer
efficiencies between low and high relative humidity and nonporous and porous fomites
were compared in a Student’s t-test.
RESULTS
Influence of relative humidity on microbial transfer
The percent transfer efficiency was determined for 468 finger transfers; 234 transfer
events were performed for both low and high relative humidity conditions. Tables 2, 3,
and 4 summarize the fomite to finger percent transfer efficiency results. Relative
humidity influenced the transfer rate of pathogens from fomites to fingers except for PV1. Most organisms had greater transfer efficiencies under high relative humidity (< 0.1%
to 79.5%) compared to low relative humidity (0.03% to 57%) with a few exceptions
(Table 2 and 3). E. coli and MS2 had greater transfer efficiencies under high relative
humidity for all the fomites. S. aureus had greater transfer rates under high relative
114
humidity except for cotton and paper currency. B. thuringiensis spores had greater
transfer rates under high relative humidity except for paper currency. PV-1 seemed not
to be influenced by relative humidity with transfer efficiencies for ceramic tile, laminate,
and granite under low relative humidity of 23.1%, 36.3%, 33.8%, respectively compared
to 29.2%, 25.5%, 25.9% under high relative humidity (Table 4). Figures 1, 2, and 3 show
the distribution of each of the six transfer efficiencies in logarithmic scale along with the
geometric mean to show the variability in the transfer efficiencies.
The statistical significance (P = 0.05) between low and high transfer efficiencies for each
microorganism are indicated on (Tables 2 and 3). No statistical significance (P = 0.05)
between low and high transfer efficiencies for PV-1 were found (Table 4).
Influence of fomite type on microbial transfer
Fomite type did influence the transfer efficiency of all model organisms except PV-1. In
general, the transfer efficiencies were greater from nonporous surfaces (0.04% to 57%
under low relative humidity; 12.8% to 79.5% under high relative humidity) than those for
porous surfaces (< 6.8% under low relative humidity and < 13.4% under high relative
humidity) (Tables 2 and 3). Under low relative humidity, acrylic provided the highest
transfer rate for E. coli, B. thuringiensis, and MS2 coliphage (40.7%, 57%, and 21.7%,
respectively), while S. aureus had the highest transfer efficiency from glass (20.3%)
(Table 2). Under high relative humidity, glass provided the highest transfer rate for E.
coli (78.6%), laminate for S. aureus (61.9%), and acrylic for B. thuringiensis and MS2
115
coliphage (65.6% and 79.5%, respectively) (Table 3). PV-1 transfer did not seem to be
influenced by the type of nonporous surfaces (Table 4). The transfer efficiency was least
with paper currency under both low and high relative humidity for E. coli, S. aureus, and
B. thuringiensis (< 0.05%, 0.2%, < 0.1% under low relative humidity; 0.1%, 0.2%, <
0.1% under high relative humidity, respectively). Cotton produced the lowest transfer
efficiencies for MS2 under both low and high relative humidity (0.03% and 0.3%,
respectively).
DISCUSSION
The goal of this study was to obtain detailed quantitative information on fomite-to-finger
transfer that could be used to model the probability of infection from exposure to various
types of pathogens, a parameter needed for quantitative microbial risk assessments (1, 5,
48, 49). Unfortunately, there are no standard methods for quantifying transfer rates,
making it difficult to compare the results from various studies. Our results indicate that
while transfer is influenced by the relative humidity and fomite type, different organisms
vary greatly in the relative influence of these environmental factors. The relative
humidity greatly influenced the transfer for most of the microorganisms except for PV-1;
however, on average, most had greater transfer rates at a high relative humidity. Under
low relative humidity, E. coli, MS2, and PV-1 had the greatest transfer efficiencies. No
substantial difference of transfer efficiencies between different organisms was observed
at a high relative humidity, although E. coli and MS2 exhibited slightly greater transfer
rates than the other organisms. B. thurigiensis spores were poorly transferred under low
116
relative humidity conditions from all surfaces except acrylic. Desiccation did not seem to
account for these differences since E. coli is more influenced by desiccation than S.
aureus and endospores (50). These differences may also reflect differences in the
composition of the cell or endospore outer surface, and the hydrophobicity or other
chemical/structural characteristics of the organisms. Generally, it would appear that the
smoother surfaces provide greater transfer efficiencies.
As might be expected, porous surface had much lower transfer rates than the nonporous
surfaces. Porous surfaces may entrap organisms within their matrix and provide a much
greater surface area for attachment. Overall, acrylic tended to exhibit greater transfer,
especially under low relative humidity. Acrylic (poly-methyl methacrylate) is a
transparent thermoplastic, often used as a lightweight or shatter-resistant alternative to
glass and paints. S. aureus and MS2 tended to have greater transfer from the porous
surfaces than the other organisms.
PV-1 transfer rates were similar to the ranges (16% to 32%) reported by Mbithi et al. (42)
for hepatitis A virus; Ansari et al. (41) for rotavirus; and Paulson (45) for feline
calicivirus. However it is important to note that different methods were used to assess
these transfer efficiencies.
Rheinbaben et al. (51) found lower transfer of ΦX174 coliphage from door handles to
volunteers hands (0.001 to 0.4%) than that of MS2 from stainless steel (37.4%) under
117
high relative humidity observed in the present study. Rusin et al. (37) found PRD 1
phage to have a transfer efficiency of 33.5% and 65.8% from faucet handle and phone
receiver, respectively. Julian et al. (47) investigated the effects of washed and unwashed
hands on the transfer rates of MS2, ΦX174, and fr coliphages from glass slides to fingers
and fingers to glass slides and found similar transfer rates of 25%, 21%, 37% for
unwashed hands and 26%, 11%, 39% for washed hands, respectively. The fomite-tofinger transfer efficiency of MS2 observed in our study agrees with the findings of these
previous reports. Transfer rates for MS2 under high relative humidity for glass was
greater (67.3%) than under low relative humidity (19.3%) (Tables 2 and 3). Transfer rates
for porous fomites, cotton, polyester, and paper currency under low and high relative
humidity were also comparable to the PRD 1 transfer rates found by Rusin et al. (37).
Previous studies have used the 1.0 kg/cm2 pressure which has been estimated to be
equivalent to the pressure applied in a handshake without friction, or to opening a door
with a door handle with friction (6, 41, 42). Other studies have used lower levels of
contact pressure to simulate children handling and grasping objects (47), or to represent
ordinary touching of environmental surfaces (42). Mbithi et al. (42) found a significant
difference in the amount of pressure and friction applied in determining the amount of
hepatitis A virus transferred from stainless steel disks to fingers.
Another factor that might influence the results is drying time. We selected a 30 min
drying time based on our preliminary studies. The seeded suspension medium on various
118
surfaces became visibly dry between 15 min to 32 min under a relative humidity range of
20% to 34%, which allowed us to compare the effects of low and high relative humidity
on fomite-to-finger transfer efficiencies. Other investigators have used drying times
varying from 5 min to 48 h and contact time varying from 5 s to 30 s (6, 37, 41, 42, 45-47,
50-55). Based on these previous studies, we can expect the transfer rates to be lower over
longer drying periods, allowing the seeded suspension medium to become drier, resulting
in lower transfer rates. Different contact pressures and contact times could have been
examined, providing a more broad view of the transfer rates during variable contact times
and pressures. Incorporating more volunteer hands could also better characterize the
effects of individual hand characteristics such as pH (56).
The transfer efficiencies observed in our study for S. aureus from nonporous surfaces
under high relative humidity (39.6% to 61.9%) were greater compared with (9% to 43%)
reported by Scott and Bloomfield (50). The difference in contact time and drying time
could have influenced the different transfer rates. Our low relative humidity transfer
efficiencies seem to agree with Scott and Bloomfield (50) transfer efficiency at the 24 h
drying time of 9.05%. Transfer efficiencies for porous fomites, under low and high
relative humidity were lower than those reported by Scott and Bloomfield (50) and Sattar
et al. (57) with the exception of polyester at a high relative humidity (5% transfer). Sattar
et al. (57) observed the same greater transfer efficiency of S. aureus from poly-cotton
fabric than from 100% cotton and explained that this might be due to the higher
hydrophobic nature of polyester material than cotton. The hydrophobic nature reduced
119
the ability of the bacterial cells to penetrate deeper into individual fibers. Data reported
by Hubner et al. (58) study on the hand-to-paper-to-hand transfer cycle of E. coli
(0.009%) agree with our fomite-to-finger observations of E. coli with paper currency
under low relative humidity.
A possible reason of higher transfer efficiency under high relative humidity observed in
our study was that the high humidity prevented the inoculum from drying, resulting in
greater transfer efficiencies. Previous studies have shown that virus transfer efficiencies
between contaminated surfaces were greater when the seeded suspension medium was
not completely dry (4, 7, 41, 42, 55, 59). This was seen with coliphage MS2, which had
the most significant difference between low and high relative humidity with the
nonporous inanimate surfaces, and to a lesser degree with the porous surfaces (Fig. 1, 2,
and 3). It was also seen with Gram negative E. coli, Gram positive S. aureus, and spores
of B. thuringiensis. In most cases, there was a significant difference (P ≤ 0.05) between
nonporous and porous surfaces between organism transfer efficiencies.
The transfer efficiency for each organism on a specific surface can be influenced by the
seeded suspension medium along with the physicochemical properties of the species and
how it interacts with the physical properties of the environmental surfaces (60-63). Both
the isolectric point and the hydrophobicity of the surface can influence the interactions
between the fomite and the organism. MS2 has a relatively low isoelectric point of pH
3.9 compared to many non-enveloped enteric viruses and is often less attracted to
120
common fomites (62, 64). In the case of the low transfer efficiencies seen with B.
thuringiensis spores and S. aureus from nonporous surfaces under low relative humidity,
a possible explanation could be that the spores and the Gram positive bacteria had
stronger electrostatic interactions through van der Waals forces, resulting in a stronger
attachment to the fomites (61).
CONCLUSIONS
In the present study, a number of different fomites were tested, providing a broad data set
to estimate the distributions in the transfer efficiency that depend on the type of fomite
and the microorganism. Our results highlight the importance of relative humidity in the
organism fomite-to-finger transfer efficiency rates. Most species on average had greater
transfer efficiencies under high relative humidity in comparison to low relative humidity.
The fomite type was found to influence the transfer efficiency, with nonporous surfaces
having greater transfer efficiency than porous surfaces. The data obtained in our study
can be used as input values for transfer efficiency exposure parameters in microbial risk
assessment models.
ACKNOWLEDGEMENTS
This research was supported by the Center for Advancing Microbial Risk Assessment,
funded by the U.S. Environmental Protection Agency’s Science to Achieve Results
(STAR) program and the U.S. Department of Homeland Security (Grant R83236301). A
121
special thanks goes to Elena Almada Quijada and Aldo Robles Arevalo for assisting in
the bacteriophage assays.
122
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TABLES
TABLE 1 Fomites tested
Fomite
Nonporous
Notes
Manufactures
Acrylic
Poly-methyl methacrylate, matte nonglare finish
Home Depot, Atlanta, GA
Glass
Slides
VWR, Mississauga, ON
Ceramic Tile
Porcelain
Home Depot, Atlanta, GA
Laminate
Various colors
Wilsonart International, Temple, TX
Stainless Steel
Gage 304
AK Steel Corporation, West Chester, OH
Granite
One from India and two from Brazil
GRANITE Kitchen & Bath Countertops,
Tucson, AZ
Cotton
100% fabric
Polyester
100% fabric
Paper Currency
Cotton-based one dollar bills
Home Trends Brand, Walmart,
Bentonville, AR
Home Trends Brand, Walmart,
Bentonville, AR
United States
Porous
132
TABLE 2 Fomite-to-finger transfer efficiencya of microorganisms under low relative humidity 15% - 32%
E. coli
S. aureus
B. thuringiensis
MS-2
(n) Average (%) ± SD (Range)
(n) Average (%) ± SD (Range)
(n) Average (%) ± SD (Range)
(n) Average (%) ± SD (Range)
Acrylic
(6) 40.7 ± 37.7 (6.4 - 93.5)
(6) 3.4 ± 2.5 (0.9 - 8.0)c
(6) 57.0 ± 12.0 (45.8 - 74.8)
(6) 21.7 ± 15.0 (3.0 - 40.6)c
Glass
(6) 5.1 ± 5.4 (0.7 - 15.1)c
(6) 20.3 ± 33.4 (0.6 - 85.4)
(6) < 0.5 ± 0.2 (< 0.3 - 0.9)b, c
(6) 19.3 ± 13.2 (2.9 - 40.5)c
Ceramic Tile
(6) 11.6 ± 11.8 (0.1 - 33.3)c
(6) 2.7 ± 2.3 (0.8 - 6.7)c
(6) < 0.2 ± 0.1 (< 0.1 - 0.4)b
(6) 7.1 ± 4.0 (3.8 - 15.0)c
Laminate
(6) 21.7 ± 23.9 (5.2 - 66.5)
(6) 4.3 ± 2.4 (1.3 - 7.4)c
(6) 0.2 ± 0.1 (< 0.1 - 0.3)b, c
(6) 5.4 ± 3.6 (1.0 - 10.0)c
Stainless Steel
(6) 3.8 ± 2.5 (1.5 - 7.1)c
(6) 4.0 ± 4.0 (1.1 - 11.9)c
(6) < 0.5 ± 0.2 (< 1.0)b, c
(6) 6.9 ± 8.9 (1.4 - 24.2)c
Granite
(6) 7.3 ± 10.6 (< 0.1 - 28.0)b
(6) 3.9 ± 5.0 (0.7 - 13.9)
(6) 0.04 ± 0.03 (< 0.02 - 0.1)b
(6) 10.2 ± 5.0 (4.8 - 16.9)
Cotton
(6) < 6.8 ± 7.0 (< 15.4)b
(6) < 1.0 ± 0.6 (< 1.9)b
(6) < 0.6 ± 0.1 (< 0.8)b
(6) 0.03 ± 0.02 (0.01 - 0.1)
Polyester
(6) < 0.37 ± 0.28 (< 0.9)b
(6) 0.37 ± 0.48 (0.04 - 1.3)b
(6) < 0.6 ± 0.6 (< 1.7)b
(6) 0.3 ± 0.2 (0.1 - 0.7)c
Paper Currency
(6) < 0.05 ± 0.04 (< 0.02 - 0.1)b
(6) 0.2 ± 0.1 (0.1 - 0.4)
(6) < 0.1 ± 0.1 (< 0.02 - 0.2)b
(6) 0.4 ± 0.4 (0.1 - 0.9)
Surface Type
Nonporous
Porous
a
Transfer efficiency (%) = (CFU or PFU finger/CFU or PFU control fomite) X 100
b
Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 indicated by <
c
There was a statistical difference (Student t-test P = 0.05) of the transfer efficiency between low and high relative humidity
133
TABLE 3 Fomite-to-finger transfer efficiencya of organisms under high relative humidity 40% - 65%
E. coli
S. aureus
B. thuringiensis
MS-2
(n) Average (%) ± SD (Range)
(n) Average (%) ± SD (Range)
(n) Average (%) ± SD (Range)
(n) Average (%) ± SD (Range)
Acrylic
(6) 53.3 ± 27.5 (30.4 - 98.0)
(6) 47.2 ± 17.9 (24.4 - 67.3)d
(6) 65.6 ± 15.9 (48.8 - 94.9)
(6) 79.5 ± 21.2 (54.1 - 100)c, d
Glass
(6) 78.6 ± 27.1 (38.0 - 100)c, d
(6) 45.5 ± 15.5 (25.7 - 65.5)
(6) 33.8 ± 24.0 (< 4.3 - 65.9)b, d
(6) 67.3 ± 25.0 (37.4 - 96.9)d
Ceramic Tile
(6) 60.7 ± 45.4 (3.7 - 100)c, d
(6) 54.7 ± 18.8 (27.7 - 77.6)d
(6) 21.2 ± 28.2 (< 1.3 - 76.4)b
(6) 41.2 ± 18.8 (18.7 - 74.7)d
Laminate
(6) 27.4 ± 30.2 (1.9 - 77.0)
(6) 61.9 ± 24.7 (30.9 - 89.8)d
(6) 53.5 ± 19.6 (33.8 - 79.0)d
(6) 63.5 ± 24.0 (36.2 - 100)c, d
Stainless Steel
(6) 54.1 ± 23.5 (29.4 - 99.0)d
(6) 48.3 ± 25.4 (16.6 - 85.5)d
(6) 57.0 ± 9.7 (47.5 - 71.4)d
(6) 37.4 ± 16.0 (19.5 - 62.4)d
Granite
(6) 36.5 ± 39.3 (0.3 - 100)c
(6) 39.6 ± 41.5 (1.3 - 100)c
(6) 12.8 ± 19.8 (0.1 - 42.7)
(6) 30.0 ± 24.3 (4.9 - 59.3)
Cotton
(6) < 13.4 ± 11.7 (< 33.3)b
(6) 0.5 ± 0.5 (0.1 - 1.3)b
(6) < 3.5 ± 3.5 (< 10.0)b
(6) 0.3 ± 0.3 (0.04 - 0.6)
Polyester
(6) < 0.7 ± 0.8 (< 2.2)b
(6) 5.0 ± 6.9 (0.1 - 15.5)
(6) < 4.6 ± 6.1 (< 16.3)b
(6) 2.3 ± 0.8 (1.2 - 3.2)d
Paper Currency
(6) 0.1 ± 0.3 (< 0.01 - 0.7)b
(6) 0.2 ± 0.1 (0.1 - 0.3)
(6) < 0.1 ± 0.1 (< 0.2)b
(6) 0.7 ± 0.5 (0.1 - 1.5)
Surface Type
Nonporous
Porous
a
b
Transfer efficiency (%) = (CFU or PFU finger/CFU or PFU control fomite) X 100
Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 indicated by <
c
Transfer of organism from fomite-to-fingers for one or more transfer events were > 100% and were truncated to 100%
d
There was a statistical difference (Student t-test P = 0.05) of the transfer efficiency between low and high relative humidity
134
TABLE 4 Fomite-to-finger transfer efficiencya of poliovirus 1
Surface Type
(n) Average (%) ± SD (Range)
Low RH (15% - 32%)
Ceramic Tile
(6) 23.1 ± 24.0 (0.4 - 52.7)
Laminate
(6) 36.3 ± 8.7 (24.1 - 50.0)
Granite
(6) 33.8 ± 40.4 (0.4 - 100)b
High RH (40% - 65%)
a
b
Ceramic Tile
(6) 29.2 ± 6.4 (19.4 - 35.4)
Laminate
(6) 25.5 ± 15.5 (3.4 - 50.0)
Granite
(6) 25.9 ± 4.1 (19.7 - 32.1)
Transfer efficiency (%) = (PFU finger/PFU control fomite) X 100
Transfer event was > 100% and was truncated to 100%
135
FIGURES
A
B
Geometric Mean
Geometric Mean
100
% Transfer Efficiency
% Transfer Efficiency
100
10
1
0.1
0.01
10
1
0.1
0.01
Acrylic
Glass
Ceramic Laminate Stainless Granite
Tile
Steel
Co on Polyester
Nonporous
Paper
Currency
Acrylic
Glass
Porous
Ceramic Laminate Stainless Granite
Tile
Steel
Co on Polyester
Nonporous
C
Porous
D
Geometric Mean
Geometric Mean
100
100
% Transfer Efficiency
% Transfer Efficiency
Paper
Currency
10
1
0.1
0.01
10
1
0.1
0.01
Acrylic
Glass
Ceramic Laminate Stainless
Tile
Steel
Granite
Nonporous
Co on
Polyester
Porous
Paper
Currency
Acrylic
Glass
Ceramic Laminate Stainless
Tile
Steel
Nonporous
Granite
Co on
Polyester
Paper
Currency
Porous
FIG 1 Transfer efficiency (%) of (A) E. coli 15597, (B) S. aureus 25923, (C) B. thuringiensis spores, and (D) MS2 under low
relative humidity (15% - 32%).
136
A
B
Geometric Mean
Geometric Mean
100
% Transfer Efficiency
% Transfer Efficiency
100
10
1
0.1
0.01
10
1
0.1
0.01
Acrylic
Glass
Ceramic Laminate Stainless
Tile
Steel
Granite
Co on
Nonporous
Polyester
Paper
Currency
Acrylic
Glass
Porous
Ceramic Laminate Stainless
Tile
Steel
Granite
Co on
Nonporous
C
Polyester
Paper
Currency
Porous
D
Geometric Mean
Geometric Mean
100
% Transfer Efficiency
% Transfer Efficiency
100
10
1
0.1
0.01
10
1
0.1
0.01
Acrylic
Glass
Ceramic Laminate Stainless
Tile
Steel
Granite
Nonporous
Co on
Polyester
Porous
Paper
Currency
Acrylic
Glass
Ceramic Laminate Stainless
Tile
Steel
Nonporous
Granite
Co on
Polyester
Paper
Currency
Porous
FIG 2 Transfer efficiency (%) of (A) E. coli 15597, (B) S. aureus 25923, (C) B. thuringiensis spores, and (D) MS2 under high
relative humidity (40% - 65%).
137
A
B
Geometric Mean
100
10
10
% Transfer Efficiency
% Transfer Efficiency
Geometric Mean
100
1
0.1
1
0.1
0.01
0.01
Ceramic Tile
Laminate
Nonporous
Granite
Ceramic Tile
Laminate
Granite
Nonporous
FIG 3 Transfer efficiency (%) of Poliovirus 1 under (A) low relative humidity (15% - 32%) and (B) high relative humidity
(40% - 65%).
138
APPENDIX D: COMPARATIVE PERSISTENCE OF BACTERIA AND VIRUSES
UNDER DIFFERENT RELATIVE HUMIDITY ON POROUS AND NONPOROUS
FOMITES
Abbreviated running headline: Persistence of Microbes on Fomites
Gerardo U. Lopez, Masaaki Kitajima, Akrum Tamimi, Charles P. Gerba#
Department of Soil, Water and Environmental Science, University of Arizona, Tucson,
Arizona 85721 USA
#
Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail:
[email protected]
A manuscript for
Journal of Applied Microbiology
139
ABSTRACT
Aims: Fomites can play a role in the transmission of many types of human pathogens.
Information on the persistence of bacteria and viruses on fomites is needed for the
development of quantitative microbial risk assessment models to reduce uncertainty in
predicting risk from contact with contaminated fomites. Survival of bacteria and virus on
several common fomites under different relative humidity conditions was determined.
Methods and Results: Nine fomites of different compositions were studied representing
porous and nonporous surfaces. Escherichia coli, Staphylococcus aureus, Bacillus
thuringiensis, MS2 coliphage, and poliovirus 1 were placed on various fomites in 10 µl
drops allowed to dry for 30 min under low (15% to 32%) or high (40% to 65%) relative
humidity. Relative humidity influenced organism survival on fomites; greater survival
was observed under high relative humidity for E. coli, S. aureus, MS2, and PV-1. B.
thuringiensis spores survived longer at low relative humidity. Fomite type did influence
the survival of all model organisms; survival was greater on nonporous surfaces than
those for porous surfaces. Organism survival was greatest with paper currency and least
with cotton for most pathogens under both low and high relative humidity.
Conclusions: Generally most organisms had greater survival under high relative
humidity compared to low relative humidity with B. thuringiensis spores showing the
opposite effect. Survival was less on porous surfaces compared to nonporous surfaces.
Significance and Impact of Study: This data can be used to predict microorganism log10
reduction parameters in microbial risk assessment models, in order to be able to develop
140
more sophisticated and realistic models taking into account bacterial and virus survival
on various fomites under different relative humidity.
Keywords: Bacilus thuringiensis, Staphylococcus aureus, relative humidity, nonporous,
porous, fomites, surfaces, survival
INTRODUCTION
Fomites can serve as reservoirs of microbial pathogens and contribute to direct or indirect
transmission of infectious agents (Haas et al., 1999, Nicas and Sun, 2006). Several
studies have shown that environmental surfaces found in indoor environments are
contaminated with human bodily fluids indicating the potential presence of infectious
agents (Ansari et al., 1991, Barker et al., 2001, Barker et al., 2004, Hall et al., 1980). A
number of indoor fomites in day care centers (Barker et al., 2001, Boone and Gerba, 2005,
Butz et al., 1993, Keswick et al., 1983a, Keswick et al., 1983b, Laborde et al., 1993),
schools (Bright et al., 2010), office building (Boone and Gerba, 2010), homes (Bellamy
et al., 1998, Fekety et al., 1981, Josephson et al., 1997, Medrano-Félix et al., 2010, Rusin
et al., 1998, Scott et al., 1982, Sinclair and Gerba, 2011, Speirs et al., 1995), public areas
(Reynolds et al., 2005) and hospitals (Huslage et al., 2010, Kramer et al., 2006, Otter et
al., 2011, Pittet et al., 2006, Pittet et al., 1999, Weber et al., 2010) have been identified as
reservoirs and vectors for secondary modes of transmission. In order for a microbial
pathogen to be spread via contaminated fomites the bacteria or virus needs to maintain
infectivity long enough to come into contact with a susceptible host. A number of studies
141
have shown that microbial pathogens can persist from hours to months on fomites
depending on the numbers deposited, type of organism and environmental conditions
(Abad et al., 2001, Boone and Gerba, 2007, Kramer et al., 2006). However, few studies
have examined the effects of relative humidity on microbial survival. Data on the survival
of bacteria and viruses on fomites is important in modeling the efficiency of transfer of a
pathogens from fomite to hands in risk assessment models (Atkinson and Wein, 2008,
Boone and Gerba, 2007, Brankston et al., 2007, Nicas and Best, 2008).
The purpose of the present study was to evaluate the survival of five model organisms
under low and high relative humidity on various types of fomites to better understand
microorganism survival in modeling microbial exposure and risk assessment.
MATERIALS AND METHODS
Bacteria, Virus and preparation of inocula
Escherichia coli (ATCC 15597), Staphylococcus aureus (ATCC 25923), Bacillus
thuringiensis (ATCC 10792), and coliphage MS2 (ATCC 15597-B1) were obtained from
the American Type Culture Collection (ATCC Manassas, VA). Poliovirus 1 (PV-1;
strain LSc-2ab) was obtained from the Department of Virology and Epidemiology at the
Baylor College of Medicine (Houston, TX). These organisms were selected as model
organisms for pathogenic Gram negative, Gram positive bacteria, spore-forming bacteria,
and viruses.
142
Frozen aliquots of E. coli and S. aureus were transferred into separate flasks containing
100 to 150 ml of Tryptic Soy Broth, (TSB, EMD, Gibbstown, NJ) incubated for 18 ± 2 h
at 37°C on an orbital shaker (150 to 180 rpm), and streaked for isolation onto Tryptic Soy
Agar plates (TSA, EMD Gibbstown, NJ), the bacteria were then subcultured in a flask of
TSB, and incubated for 18 ± 2 h at 37°C on a orbital shaker (150 to 180 rpm) (Rusin et al.,
2002).
B. thuringiensis spores were prepared as previously described with minor modification
(Nicholson and Setlow, 1990). Briefly, spores were suspended in Difco sporulation media
with supplements (DSM + S; Becton, Dickinson and Company, Sparks, MD), cultivated
for 24 h at 37°C on an orbital shaker (150 to 180 rpm), and resuspended in fresh DSM +
S to obtain a final OD600 of 0.1 (Spectronic Genesys 5, Milton Roy, Ontario Canada).
MS2 coliphage were prepared as previously described with minor modifications (Rusin et
al., 2002). Briefly, 0.1 ml of phage suspension and 0.5 ml of a log phase E. coli 15597
(host bacterium) culture 0.5 ml were added to top agar, melted, and maintained at 48°C.
The inoculated top agar was mixed and poured over the TSA. The solidified agar overlay
plates were then inverted and incubated at 37°C for 24 h. TSB was then added to each
plate and maintained at room temperature for 2 h. The TSB eluent was aspirated and
centrifuged at (1,989 X g for 10 min), after which, the supernatant was filtered through
0.22 µm pore size Acrodisc® Syringe Filters (Pall Corporation, Ann Arbor, MI) pre-
143
moistened with 3% beef extract (Bacto™, Becton, Dickinson and Company, Sparks, MD),
the virus tittered, and stored at 4°C.
PV-1 propagation and plaque-forming assays were conducted as previously described
(Bidawid et al., 2003, Ikner et al., 2011). Briefly, poliovirus 1 was propagated on buffalo
green monkey kidney (BGM; ATCC CCL-81); obtained from the American Type Culture
Collection (ATCC), Manassas, VA) cell line monolayers with minimal essential media
(MEM) containing 5% calf serum (HyClone Laboratories, Logan, UT) at an incubation
temperature of 37°C with 5% CO2. Plaque-forming assays were performed using sixwell plates with confluent monolayers of the BGM cells.
Relative Humidity Conditions and Temperature
Two relative humidity ranges were studied, high (40% to 65%) and low (15% to 32%)
relative humidity. To achieve both humidity conditions, two separate incubators were
turned off and used. Incubator temperatures thus reflected room temperature ranges of
19°C to 25°C. The temperature and relative humidity were monitored with a High
Accuracy Thermo – Hygro (VWR, Mississauga, ON). During days with higher ambient
relative humidity in the laboratory, t.h.e.® Desiccant (EMD, Gibbstown, NJ) and Drierite
desiccant (Drierite, Xenia, OH) were utilized in the incubator to decrease the relative
humidity to the low relative humidity range (15% to 32%). During laboratory ambient
lower relative humidity conditions a BIONAIRE® humidifier (Milford, MA) was used in
144
the specific incubator to increase the relative humidity to the high relative humidity range
(40% to 65%).
Fomites tested
Nine different types of fomite materials were tested ranging in surface areas of 16 to 25
cm2 six nonporous and three porous (Table 1). With the exception of acrylic, all fomites
were sterilized by autoclave whereas acrylic was radiated under UV light (254 nm) for 30
min on each side. After fomite-to-finger transfers with E. coli, S. aureus, and MS2,
nonporous fomites were sprayed three times with 70% ethanol and allowed to dry for 10
min. Nonporous fomites used in the finger transfers with B. thuringiensis spores and PV1 were disinfected with 10% bleach (The Clorox Company, Oakland, CA), then allowed
to sit for 10 min, and subsequently neutralized in 10% sodium thiosulfate (EMD,
Gibbstown, NJ). Fomites were then washed under warm running water with nonantibacterial soap (Liquid Joy, Procter and Gamble, Cincinnati, OH), rubbed with a wet
paper towel on the surface area of inoculation, rinsed thoroughly with RO treated water,
air-dried, and autoclaved. Cotton and polyester fomites were discarded after use. Paper
currency was autoclaved and reused.
Inoculation of fomites
For each of the six nonporous and three porous fomites, seven swatches/coupons were
evenly spaced on the middle shelf of an incubator. Each trial consisted of three control
145
swatches/coupons, three fomite-to-finger transfer swatches/coupons, and one negative
control swatch/coupon to ensure that the fomites were not previously contaminated.
The concentration of organisms added to the fomites was approximately 107 to 108
colony forming units (CFU)/cm2 of E. coli in (TSB, EMD, Gibbstown, NJ), 108 to 109
CFU/cm2 of S. aureus in TSB, 106 to 107 CFU/cm2 of B. thuringiensis spores in (DSM +
S; Becton, Dickinson and Company, Sparks, MD), 109 to 1011 plaque forming units
(PFU)/cm2 of MS2 in TSB, and 108 PFU/cm2 of PV-1 in (PBS, Sigma-Aldrich, St. Louis,
MO) in 10 µl droplets. Using a pipet tip, the 10 µl inoculum droplets were spread over
approximately a 1.0 cm2 area on the center of each fomite. The paper currency were
divided into four 24 cm2 sections; two one dollar bills were used to make the seven
swatches. With paper money for each set of transfer experiments, a new 1.0 cm2 area on
each of the 24 cm2 sections were inoculated. Using an ink marker, an identifying spot
was placed near the inoculated area on cotton, polyester, and paper money due to the
absorbance. The fomites were allowed to dry for 30 min.
Surface sampling and assays
Fomites were sampled using a cotton-tipped swab applicator (Puritan Medical Products
Company, Guilford, ME), when inoculated with E. coli, S. aureus, B. thuringiensis, and
MS2. In the case of PV-1, a polyester fiber-tipped applicator swab (Falcon, Becton
Dickinson and Company, Cockeysville, MD) was used. Swabs were wet in 1.0 ml of
phosphate buffer saline (PBS, Sigma-Aldrich, St. Louis, MO), and then a 6.0 cm2 area on
146
the fomite was swabbed using firm sweeping and rotating motions. The swab was then
placed back into the remaining PBS and vortexed for 5 s.
E. coli, S. aureus, and B. thuringiensis spores were enumerated using the spread plate
technique on MacConkey (EMD Chemicals Inc, Gibbstown, NJ), Mannitol salt agar
(MSA, EMD Chemicals Inc, Gibbstown, NJ), and TSA (EMD Chemicals Inc.,
Gibbstown, NJ) agar plates, respectively. The plates were incubated at 37°C for 18 ± 2 h.
B. thuringiensis spore samples were heat shocked at 81 ± 2°C for 10 min prior to spread
plating, to stimulate germination. The MS2 plaque assay was conducted using the double
agar overlay method using TSA (EMD Chemicals Inc., Gibbstown, NJ) (Adams, 1959,
Ikner et al., 2011). PV-1 titrations were performed using 10-fold serial dilution plaqueforming assays as described previously (Bidawid et al., 2003, Ikner et al., 2011). All
dilutions were assayed in duplicate.
Reduction and Statistical analysis
Log10 reduction was calculated using Eqn (1) and is described as the negative Log10 of
the value of CFU or PFU concentration recovered from fomite divided by CFU or PFU of
the stock titer.
     
  

   



(1)
147
Data were entered in Microsoft Excel 2010 and the software package StatPlus:mac, 2009,
(AnalystSoft) to compute the descriptive statistic measures of mean percent transfer
efficiency, the standard deviation, and statistical significance. To assess the statistical
significance at the (P = 0.05) level, the percent transfer efficiencies between low and high
relative humidity and nonporous and porous fomites were compared in a Student’s t-test.
RESULTS
Influence of relative humidity on microbial survival
In general relative humidity influenced organism survival. S. aureus had greater survival,
less log10 reduction (Table 2 and 3) (Fig. 1 and 2), under high relative humidity on all
fomites except granite. PV-1 had slightly greater survival under high relative humidity
for the three fomites tested (Table 4) and (Fig. 3). E. coli survived better under high
relative humidity for most fomites except for granite, cotton, and polyester (Table 2 and
3) (Fig. 1 and 2). MS2 was less influenced by relative humidity with the exception of
stainless steel, polyester, and paper currency where MS2 survived better under high
relative humidity (Table 2 and 3) (Fig.1and 2). B. thuringiensis spores on the other hand
seemed to be more stable under low relative humidity with most fomites except for glass,
stainless steel, and paper currency (Table 2 and 3) (Fig. 1 and 2).
Influence of fomite type on microbial survival
Fomite type also influenced survival. In general, survival was greater for nonporous
surfaces (less than 2.6 reduction log10 CFU or PFU/2-cm2 under both low and high
148
relative humidity) than those for porous surfaces (up to 5.0 reduction log10 CFU or
PFU/2-cm2 under low and high relative humidity) (Fig. 1, 2, and 3). Granite showed the
least reduction between low and high relative humidity for all microorganisms (less than
0.05 reduction log10 CFU or PFU/2-cm2 except with PV-1 (Fig. 3). Cotton showed the
greatest reduction for all organisms under low and high relative humidity, while paper
currency showed the least reduction. MS2 also showed a greater PFU recovery than the
initial titer on ceramic tile, stainless steel, polyester, and paper currency resulting in a
negative reduction (Table 2 and 3) (Fig. 2 and 3).
The statistical significance (Student t-test P = 0.05) between low and high transfer
efficiencies for each microorganism are indicated on (Tables 2, 3, and 4). No statistical
significance (P = 0.05) between low and high transfer efficiencies for PV-1 were found
(Table 4).
A statistical difference (Student t-test P = 0.05) between high and low relative humidity
log10 reduction at 30 min (Fig. 1, 2, and 3) occurred with E. coli between acrylic, glass,
ceramic tile, and laminate; S. aureus for acrylic, glass, ceramic tile, laminate, stainless
steel, cotton, and polyester; B. thuringiensis for glass, ceramic tile, laminate, stainless
steel, and cotton; MS2 coliphage for all fomites except laminate and granite; poliovirus
for the three fomites tested. No statistical difference between high and low relative
humidity log10 reduction at 30 min was seen with PV-1 (Fig. 3).
149
DISCUSSION
The objective of the present study was to quantitate the survival of several surrogate
pathogens in order to determine log10 reduction under low and high relative humidity on
various types of fomites to better quantify microorganism survival in modeling microbial
exposure and risk assessment. Our results indicate that relative humidity as well as fomite
type influenced survival on various surfaces. The different organisms studied varied
greatly among the influence of these environmental factors. Over all microorganisms
were more stable resulting in a lower reduction under high relative humidity however, did
seem to be fomite dependent. Greater organism reduction was observed with porous
fomites than with nonporous fomites. The observed reduction difference between the
relative humidity ranges was up to 2.5 reduction log10 CFU or PFU/2-cm2.
Figures 1, 2, and 3 compares the distribution of the microbial reduction log10 CFU or
PFU/2-cm2 and the geometric mean between low and high relative humidity for each
organism. Reduction plotted below the zero axis indicated that there was a greater
recovery of the organism than the initial titer. Such results were seen more often under
high relative humidity and could be due to aggregated bacteria cells and virus particles
that were not as easily able to break apart under low relative humidity, while at high
relative humidity the drying process was inhibited by the relative humidity conditions
which was conducive for the organisms to break apart.
150
Our observations with both bacteria and viruses were consistent with Abad et al. (1994)
that found viruses to have a lower survival on porous fomites than nonporous surfaces
with up to a four log difference in survival under different relative humidity and
temperatures over 60 days. A recent study by Hubner et al. (2011) also showed that
several strains of bacteria that have been found on medical records can survive on paper
for several days and can be transferred to healthcare professions leading to crosscontamination to patients and themselves. Our study looked at both bacteria and virus
survival on mostly different nonporous and porous fomites than used in these previous
studies under different relative humidity at the same temperature range. Even at a short
time period overall organisms survived least on cotton, even compared to polyester than
on nonporous fomites. Organisms survived the greatest on paper currency which had
similar results as nonporous fomites indicating that bacteria and viruses can persist on
paper currency. Studies have found the presence of organisms on paper currency (Khin
Nwe and al., 1989, Uneke and Ogbu, 2007, Vriesekoop et al., 2010) suggesting that
dollar bills can be reservoirs for potential pathogens. The overall lower survival observed
in porous fomites over time? could be due to entrapment of organisms within their matrix
over time or they provide a much greater surface for attachment.
Most organisms showed a statistical significance (P = 0.05) between low and high
relative humidity survival measured in reduction (Fig. 1, 2, and 3). Even though the
reduction for most organisms was on average less than 1.0 to 2.0 log10 CFU or PFU/2-
151
cm2. This statistical difference might have reflected the small standard deviation within
each group under the different relative humidity conditions.
Poliovirus has been reported to survive 4 h to < 8 days (Kramer et al., 2006) on stainless
steel disks. In the present study poliovirus seemed to be less influenced by relative
humidity, however still showing a lower reduction at 40% to 65% relative humidity (Fig.
3). These results agree with (Mbithi et al., 1991) and (Abad et al., 1994) observations of
PV-1 surviving better on nonporous fomites at 95% and 85% relative humidity,
respectively. Greater stability under higher relative humidity was also reported by (Sattar
et al., 1988) for enterovirus 70 (EV-70) and Abad et al. (1994) reported no effects of
relative humidity on enteric adenovirus (ADV). These findings support the general idea
that nonenveloped viruses are more stable at relative humidity levels higher than 80%
(Buckland and Tyrrell, 1962, Sattar et al., 1988). On the other hand other studies reported
contradictory results for other nonenveloped viruses; Mbithi et al. (1991) indicated
hepatitis A virus (HAV) to be more stable at lower relative humidity (25% ± 5%), while
Abad et al. (1994) demonstrated HAV to have greater stability at higher relative humidity.
Similar contrasting observations were reported for human rotavirus (HRV) where Moe
and Shirley (1982) showed greater HRV survival under both low and high relative
humidity, while Sattar et al. (1986) reported more stability at low or medium level, and
Abad et al. (1994) observed greater stability at high relative humidity. Explanation for
this variableness in virus survival among different studies is unknown. Never the less the
present study shows that the lower relative humidity results in increased poliovirus
152
survival while higher relative humidity enhances poliovirus stability on nonporous
surfaces. Such results indicate that the influence of relative humidity is very virus specific.
Even though the relative humidity tested in this study did not have a great effect on MS2
coliphage survival (Tables 2 and 3) (Fig. 1 and 2) a slightly lower reduction was observed
under high relative humidity. Under both low and high relative humidity conditions MS2
was very stable, however, there was a greater reduction under low relative humidity
particularly with porous fomites (Figure 1 and 2). Using feline calicivirus as a surrogate
for norovirus foodborne outbreaks (Paulson, 2005) found that feline calicivirus can
persist on various fomites after a 15 min drying time with little inactivation.
B. thuringiensis spores were more stable under low relative humidity. Bacterial spores
such as Clostridium difficile are known to be very stable in the environment and have
been reported to persist on fomites for several months (Kim et al., 1981). Persons
infected with acute diarrhea can excrete 107 to 109 micro-organisms per gram of feces
resulting in wide spread contamination of the surrounding surfaces with spores (Mutters
et al., 2008). Up to 3.5 log10 CFU of C. difficile spores on surfaces in rooms of patients
positive with C. difficile infections have been reported (Mutters et al., 2008). B. anthracis
and B. thuringiensis strains have been shown to survive better (> 6.5 log10 CFU) at lower
temperatures (60°C) and lower relative humidity (60%) than at higher temperatures and
relative humidity (Buhr et al., 2012) and (Van Cuyk et al., 2011) found B. thuringiesis
subsp. Kurstaki to persist in urban environments for at least 4 years. Even though we did
153
not test the effects of temperature we also observed similar results showing that B.
thuringiensis is less stable at higher relative humidity.
E. coli has been described to persist on inanimate surfaces from 1.5 hours to 16 months
(Kramer et al., 2006). A study by (Abrishami et al., 1994) found a log10 reduction 0.06
after 24 h of drying on plastic cutting boards in a laminar flow hood. Whereas S. aureus
has been reported to persist for 7 days to 7 months on dry inanimate surfaces (Kramer et
al., 2006). In the present study we found S. aureus to be more stable with a 0.4 reduction
log10 CFU/2-cm2 on nonporous surfaces under high relative humidity than E. coli (2.4
reduction log10 CFU/2-cm2). Our results are consistent with (Scott and Bloomfield,
1990) observations where S. aureus a Gram positive bacteria survives better than E. coli a
Gram negative bacteria on laminate.
In conclusion, most organisms in this study generally had greater survival under high
relative humidity compared to low relative humidity with B. thuringiesis showing the
opposite. The present study also demonstrated that fomite type influenced survival with
microorganisms having the least reduction from nonporous surfaces. The data obtained in
our study can be used to better model survival parameters in microbial risk assessment
models (Gerba, 2001, Haas et al., 2005, Nicas and Best, 2008).
154
ACKNOWLEDGEMENTS
This research was supported by the Center for Advancing Microbial Risk Assessment,
funded by the U.S. Environmental Protection Agency’s Science to Achieve Results
(STAR) program and the U.S. Department of Homeland Security (Grant R83236301). A
special thanks goes to Elena Almada Quijada and Aldo Robles Arevalo for assisting in
the bacteriophage assays.
155
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163
TABLES
TABLE 1 Fomites tested
Fomite
Nonporous
Notes
Manufactures
Acrylic
Poly-methyl methacrylate, matte nonglare finish
Home Depot, Atlanta, GA
Glass
Slides
VWR, Mississauga, ON
Ceramic Tile
Porcelain
Home Depot, Atlanta, GA
Laminate
Various colors
Wilsonart International, Temple, TX
Stainless Steel
Gage 304
AK Steel Corporation, West Chester, OH
Granite
One from India and two from Brazil
GRANITE Kitchen & Bath Countertops,
Tucson, AZ
Cotton
100% fabric
Polyester
100% fabric
Paper Currency
Cotton-based one dollar bills
Home Trends Brand, Walmart,
Bentonville, AR
Home Trends Brand, Walmart,
Bentonville, AR
United States
Porous
164
TABLE 2 Survival of organisms on fomites under low relative humidity 15% - 32%
E. coli
Surface Type
S. aureus
2
B. thuringiensis
2
MS-2
2
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean PFU/2-cm2 log10 ±
SD (Range)
Acrylic
(6) 4.8 ± 0.4 (4.5 - 5.4)a
(6) 6.2 ± 0.1 (6.1 - 6.4)a
(6) 4.8 ± 0.1 (4.6 - 4.9)
(6) 8.5 ± 0.2 (8.3 - 8.7)a
Glass
(6) 5.0 ± 0.4 (4.4 - 5.4)
(6) 6.2 ± 0.2 (5.9 - 6.4)a
(6) 4.4 ± 0.2 (4.1 - 4.5)a
a
a
Nonporous
a
(6) 6.2 ± 0.2 (6.0 - 6.4)
(6) 4.9 ± 0.1 (4.9 - 5.0)
(6) 8.5 ± 0.1 (8.3 - 8.6)
(6) 8.8 ± 0.1 (8.6 - 9.0)a
Ceramic Tile
(6) 4.9 ± 0.2 (4.5 - 5.2)
Laminate
(6) 4.3 ± 0.3 (4.1 - 4.9)a
(6) 6.2 ± 0.1 (6.1 - 6.3)a
(6) 4.9 ± 0.05 (4.8 - 5.0)
(6) 8.8 ± 0.1 (8.7 - 9.0)a
Stainless Steel
(6) 4.5 ± 0.6 (3.8 - 5.2)a
(6) 6.1 ± 0.2 (5.8 - 6.4)a
(6) 4.3 ± 0.1 (4.2 - 4.4)a
(6) 7.1 ± 0.9 (6.2 - 8.6)a
Granite
(6) 4.5 ± 0.3 (4.3 - 5.0)
(6) 6.1 ± 0.2 (5.9 - 6.3)
(6) 4.7 ± 0.1 (4.7 - 4.8)
(6) 8.1 ± 0.2 (7.7 - 8.3)
Cotton
(6) 2.5 ± 0.7 (1.8 - 3.5)
(6) 3.1 ± 0.2 (2.7 - 3.4)a
(6) 3.2 ± 0.1 (3.1 - 3.3)a
(6) 7.0 ± 0.3 (6.6 - 7.4)a
Polyester
(6) 3.6 ± 0.4 (3.1 - 4.1)
(6) 4.1 ± 0.3 (3.7 - 4.4)a
(6) 3.3 ± 0.4 (2.8 - 3.8)a
(6) 7.2 ± 0.4 (6.9 - 7.9)
(6) 6.0 ± 0.4 (5.6 - 6.6)
(6) 4.6 ± 0.1 (4.5 - 4.8)
(6) 7.3 ± 0.2 (7.0 - 7.4)a
Porous
Paper Currency
a
(6) 4.6 ± 0.2 (4.4 - 4.8)
a
There was a statistical difference (Student t-test P = 0.05) of the organism survival between low and high relative humidity
165
TABLE 3 Survival of organisms on fomites under high relative humidity 40% - 65%
E. coli
Surface Type
S. aureus
2
B. thuringiensis
2
MS-2
2
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean PFU/2-cm2 log10 ±
SD (Range)
Acrylic
(6) 6.7 ± 0.2 (6.3 - 6.9)a
(6) 6.8 ± 0.2 (6.6 - 7.1)a
(6) 4.8 ± 0.1 (4.6 - 4.9)
(6) 7.0 ± 0.2 (6.8 - 7.2)a
Glass
(6) 5.0 ± 0.3 (4.7 - 5.5)
(6) 6.9 ± 0.3 (6.6 - 7.1)a
(6) 4.9 ± 0.3 (4.4 - 5.3)a
a
(6) 4.7 ± 0.2 (4.4 - 5.0)
a
Nonporous
a
(6) 6.5 ± 0.2 (6.2 - 6.8)
(6) 8.4 ± 0.2 (8.2 - 8.7)
(6) 8.6 ± 0.2 (8.3 - 8.7)a
Ceramic Tile
(6) 5.7 ± 0.3 (5.5 - 6.2)
Laminate
(6) 6.7 ± 0.2 (6.4 - 7.0)a
(6) 6.7 ± 0.2 (6.4 - 6.9)a
(6) 4.8 ± 0.2 (4.6 - 4.9)
(6) 8.6 ± 0.2 (8.3 - 8.8)a
Stainless Steel
(6) 5.9 ± 0.3 (5.6 - 6.4)a
(6) 6.8 ± 0.2 (6.6 - 7.1)a
(6) 5.0 ± 0.2 (4.7 - 5.2)a
(6) 8.8 ± 0.1 (8.7 - 9.1)a
Granite
(6) 4.4 ± 1.6 (3.8 - 5.2)
(6) 6.4 ± 0.3 (6.1 - 6.7)
(6) 4.8 ± 0.1 (4.7 - 4.8)
(6) 8.3 ± 0.1 (8.1 - 8.5)
Cotton
(6) 2.5 ± 0.4 (1.5 - 2.6)
(6) 3.7 ± 0.4 (3.2 - 4.3)a
(6) 2.6 ± 0.4 (2.0 - 3.1)a
(6) 6.3 ± 0.1 (6.1 - 6.5)a
Polyester
(6) 3.4 ± 0.5 (2.7 - 4.2)
(6) 5.0 ± 0.2 (4.8 - 5.4)a
(6) 2.9 ± 0.1 (2.8 - 3.2)a
(6) 7.1 ± 0.2 (6.9 - 7.3)
(6) 6.3 ± 0.1 (6.1 - 6.4)
(6) 4.6 ± 0.2 (4.3 - 4.9)
(6) 6.9 ± 0.2 (6.7 - 7.2)a
Porous
Paper Currency
a
(6) 5.0 ± 0.2 (4.8 - 5.3)
a
There was a statistical difference (Student t-test P = 0.05) of the organism survival between low and high relative humidity
166
TABLE 4 Survival of PV-1on fomites
Surface Type
(n) Mean PFU/2-cm2 log10 ±
SD (Range)
Low RH (15% - 32%)
Ceramic Tile
(6) 5.5 ± 0.3 (5.1 - 5.9)a
Laminate
(6) 5.8 ± 0.2 (5.4 - 6.0)a
Granite
(6) 5.1 ± 0.6 (4.4 - 5.8)a
High RH (40% - 65%)
a
Ceramic Tile
(6) 6.2 ± 0.1 (6.0 - 6.4)
Laminate
(6) 6.5 ± 0.4 (6.3 - 7.3)
Granite
(6) 6.2 ± 0.1 (6.0 - 6.3)
There was a statistical difference (Student t-test P = 0.05) of
the organism survival between low and high relative humidity
167
FIGURES
B
3.0
1.4
2.5
1.2
Reduc on log10 CFU/2-cm2
Reduc on log10 CFU/2-cm2
A
2.0
1.5
1.0
0.5
1.0
0.8
0.6
0.4
0.2
0.0
0.0
-0.5
-0.2
LRH
HRH
Acrylic
LRH
HRH
Glass
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Stainless Steel
LRH
LRH
HRH
Average
C
LRH
HRH
Glass
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Stainless Steel
LRH
HRH
Granite
D
1.2
2.0
1.0
Reduc on log10 PFU/2-cm2
Reduc on log10 CFU/2-cm2
HRH
Acrylic
Average
Granite
0.8
0.6
0.4
0.2
0.0
1.5
1.0
0.5
0.0
-0.5
-1.0
-0.2
LRH
HRH
Acrylic
LRH
HRH
Glass
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Stainless Steel
LRH
HRH
Granite
Average
LRH
HRH
Acrylic
LRH
HRH
Glass
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Stainless Steel
LRH
HRH
Granite
Average
Figure 1 Reduction of organisms from nonporous fomites after 30 min drying time (A) E. coli 15597, (B) S. aureus 25923, (C)
B. thuringiensis spores, and (D) MS2 under low (15% - 32%) and high (40% - 65%) relative humidity. Reduction = -log10
(CFU or PFU fomite/(CFU or PFU titer/100)).
168
A
B
4.5
4.0
5.0
Reduc on log10 CFU/2-cm2
Reduc on log10 CFU/2-cm2
6.0
4.0
3.0
2.0
1.0
2.5
2.0
1.5
1.0
0.5
0.0
0.0
LRH
HRH
LRH
Co on
HRH
Polyester
LRH
LRH
HRH
C
HRH
Co on
Paper Currency
LRH
HRH
Polyester
LRH
HRH
Paper Currency
Average
Average
D
3.5
3.5
3.0
3.0
Reduc on log10 PFU/2-cm2
Reduc on log10 CFU/2-cm2
3.5
3.0
2.5
2.0
1.5
1.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
0.5
-1.0
0.0
LRH
HRH
Co on
LRH
HRH
Polyester
LRH
LRH
HRH
Average
HRH
Co on
Paper Currency
LRH
HRH
Polyester
LRH
HRH
Paper Currency
Average
Figure 2 Reduction of organisms from porous fomites after 30 min drying time (A) E. coli 15597, (B) S. aureus 25923, (C) B.
thuringiensis spores, and (D) MS2 under low (15% - 32%) and high (40% - 65%) relative humidity. Reduction = -log10 (CFU
or PFU fomite/(CFU or PFU titer/100)).
169
Reduc on log10 PFU/2-cm2
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Granite
Average
Figure 3 Reduction of Poliovirus 1 from nonporous fomites after 30 min drying time under low (15% - 32%) and high (40% 65%) relative humidity. Reduction = -log10 (CFU or PFU fomite/(CFU or PFU titer/100)).
170
APPENDIX E: THE EFFECT OF A DISINFECTANT WIPE ON MICROBIAL
TRANSFER
Gerardo U. Lopez1, Masaaki Kitajima1, Aaron Havas1, and Kelly A. Reynolds2
1
Department of Soil, Water and Environmental Science, The University of Arizona,
Tucson,
Arizona 85721, USA
2
Mel and Enid Zuckerman College of Public Health, The University of Arizona, 1295 N.
Martin Ave., Tucson, Arizona 85724, USA
Corresponding author. Tel: (520) 626-8230, Fax (520) 626-8009, E-mail:
[email protected]
A manuscript for
International Journal of Hygiene and Environmental Health
171
ABSTRACT
Inanimate environmental surfaces, or fomites, can serve as routes of transmission for
microbial pathogens. This study investigated the effects of a disinfectant wipe on
removing microorganisms from fomites and reducing microbial fomite-to-finger transfer
rates. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis spores and
poliovirus 1 were inoculated onto ceramic tile, laminate and granite in 10 µl drops and
allowed to dry for 30 min under relative humidity of 15% - 32%. Non-treated control
fomites were sampled and remaining surfaces were wiped with a disinfectant wipe and
allowed to dry for 10 min. Fomite-to-finger transfers were then performed on the
remaining surfaces using a 1.0 kg/cm2 pressure for 10 sec. Test organisms were reduced
by 98.1% to 99.997% on the fomites after the surfaces were wiped. Microbial fomite-tofinger transfer from disinfectant wipe-treated surfaces were, lower (less than 0.5%) than
from non-treated surfaces (up to 36.3%). This is the first study providing data on the
reduction of fomite-to-finger microbial transfer with a disinfectant wipe.
Keywords: Disinfectant wipes, Fomites, Fomite-to-finger transfer, Escherichia coli,
Staphylococcus aureus, B. thuringiensis spores
172
INTRODUCTION
Inanimate objects and surfaces (fomites) are known to play a role as a reservoir for the
transmission of pathogens in the environment either directly, by surface-to-mouth contact,
or indirectly, by contamination of fingers and subsequent hand-to-mouth, hand-to-eye,
hand-to-nose contact and even through cuts in the skin (Haas et al., 1999, Nicas and Best,
2008).
Studies have been conducted in laboratory settings to model food preparation in domestic
kitchens (De Jong et al., 2008, Van Asselt et al., 2008) to better understand crosscontamination of foodborne pathogens. In addition the occurrence and spread of
pathogens throughout the home have been conducted to better understand the role of
fomites in exposure (Fekety et al., 1981, Josephson et al., 1997, Medrano-Félix et al.,
2010, Rusin et al., 1998, Scott et al., 1982, Sinclair and Gerba, 2011, Speirs et al., 1995).
It is estimated that up to 87% of reported foodborne outbreaks are associated with food
prepared or consumed in the home (Van Asselt et al., 2008). In the United States,
foodborne pathogens cause an estimated 48 million cases of illnesses, 128,000
hospitalizations and 3,000 deaths per year (CDC, 2011).
Microbial contaminated surfaces (fomites) in hospitals and nursing homes contribute to
healthcare-acquired infections (HAI) (Bhalla et al., 2004, Bonten et al., 1996, Boyce et al.,
1997, Hayden et al., 2006, Ray et al., 2002, Weber and Rutala, 2011, Weber et al., 2010).
Fomites have been implicated in the transmission of hospital-acquired infections (HAIs)
173
because they are in close proximity to the patient and are frequently touched by hands
(Bhalla et al., 2004, Dancer, 2009, Dancer et al., 2008, Kramer et al., 2006, Murphy et al.,
2012, Sexton et al., Siani et al., 2011, Weber et al., 2010, White et al., 2008). In addition,
HAIs caused by methicillin-resistant Staphylococcus aureus (MRSA), methicillinsusceptible S. aureus (MSSA), and Clostridium difficile are associated with high
morbidity and mortality (Cosgrove et al., 2005, Humphreys, 2009, Murphy et al., 2012,
Schweickert et al., 2011, Siani et al., 2011, Weber et al., 2010, Williams et al., 2007,
Williams et al., 2009).
Pathogen presence and survival on fomites found in domestic homes, public places,
hospitals, and other healthcare facilities are important factors in evaluating human
exposure potentials (Reynolds et al., 2005). Most nosocomial and foodborne pathogens
can persist on fomites for weeks or even months (Kramer et al., 2006, Masago et al.,
2008, Neely and Maley, 2000, Weber et al., 2010) and up to several hours on fingers
(Ansari et al., 1988, Mbithi et al., 1992, Rheinbaben et al., 2000).
Hand and environmental hygiene are crucial in preventing the spread of infectious
disease in homes, healthcare facilities, and public places. Numerous studies have focused
specifically on hand hygiene interventions (Bloomfield et al., 2008, Boyce and Pittet,
2002, Muto et al., 2003, Sattar et al., 2000, Scott et al., 2010, Weber et al., 2010).
Including specific studies of healthcare workers’ hand known as vehicles of nosocomial
pathogen transmission (Boyce and Pittet, 2002, Farrington et al., 1990, Laborde et al.,
174
1993, Mermel et al., 1997, Muto et al., 2003). Publications by the Centers for Disease
Control and Prevention (CDC), such as The Guidelines for Hand Hygiene in Health-Care
Settings and other organizations recommend that healthcare workers routinely sanitize or
wash their hands after contact with inanimate objects in the immediate vicinity of all
patients (Bhalla et al., 2004, Bloomfield et al., 2008, Boyce and Pittet, 2002).
Improved cleaning and disinfection of the environment has been shown to reduce
transmission of pathogens (Bhalla et al., 2004, Bloomfield et al., 2008, Boyce et al., 2010,
Hayden et al., 2006, Kramer et al., 2006, Mayfield et al., 2000, Muto et al., 2003).
Several studies have examined the efficacy of surface cleaning and hygiene to reduce
pathogen exposure in households (Bloomfield et al., 2008, Medrano-Félix et al., 2010,
Scott et al., 2010), hospitals (Bhalla et al., 2004, Bloomfield et al., 2008, Boyce et al.,
2010, Dancer, 2009, Dancer et al., 2008, Hayden et al., 2006, Kramer et al., 2006,
Mayfield et al., 2000, Muto et al., 2003, Weber et al., 2010, White et al., 2008, Williams
et al., 2007, Williams et al., 2009), and nursing homes (Murphy et al., 2012).
However, few studies have quantitatively assessed the efficiency of microbial transfer to
and from various surfaces or the efficacy of a disinfectant intervention to inhibit such
transfers. Such data is needed for the development of quantitative microbial risk
assessment models to assess the impact of interventions on reduction in the risk of
infection (Atkinson and Wein, 2008, Boone and Gerba, 2007, Brankston et al., 2007,
Gerba et al., 1996, Nicas and Best, 2008). This study provides information to reduce the
175
uncertainty in risk models and aids to better quantitatively predict the public health
benefit of targeted hygiene interventions.
MATERIALS AND METHODS
Subjects
One subject conducted the fomite-to-finger transfer experiments. Permission was
obtained from the University of Arizona Office for Human Subjects Research prior to the
study.
Bacteria, endospores, and preparation of inocula
Study organisms
Escherichia coli C-3000 (ATCC 15597), Staphylococcus aureus (ATCC 25923), and
Bacillus thuringiensis (ATCC 10792) were obtained from the American Type Culture
Collection (ATCC Manassas, VA). Poliovirus 1 (PV-1; strain LSc-2ab) was obtained
from the Department of Virology and Epidemiology at the Baylor College of Medicine
(Houston, TX). These organisms were selected as model organisms Gram negative and
Gram positive bacteria, spore-forming bacteria, and viruses.
Gram negative and Gram positive inoculum preparation
Frozen aliquots of E. coli and S. aureus were transferred into separate 150 ml volumes of
tryptic soy broth, (TSB) (EMD, Gibbstown, NJ) incubated for 18 ± 2 h at 35 ± 2°C on an
orbital shaker (180 rpm), and streaked for isolation onto tryptic soy agar (TSA) (EMD
176
Gibbstown, NJ). The bacteria were then subcultured to a new flask of TSB, and incubated
as before (Rusin et al., 2002).
Endospore-forming bacteria inoculum preparation
B. thuringiensis spores were prepared as previously described with slight modification
(Nicholson and Setlow, 1990). Briefly, spores were suspended in Difco sporulation media
with supplements (DSM + S; Becton, Dickinson and Company, Sparks, MD), cultivated
for 24 h at 37°C on an orbital shaker (150 to 180 rpm), and resuspended in fresh DSM +
S to obtain a final OD600 of 0.1 (Spectronic Genesys 5, Milton Roy, Ontario Canada).
Virus inoculum preparation
PV-1 propagation and plaque-forming assays were conducted as described previously
(Bidawid et al., 2003, Ikner et al., 2011). Briefly, poliovirus 1 was propagated on buffalo
green monkey kidney (BGM; ATCC CCL-81; American Type Culture Collection,
Manassas, VA) cell line monolayers with minimal essential media (MEM) containing 5%
calf serum (HyClone Laboratories, Logan, UT) at an incubation temperature of 37°C with
5% CO2. Plaque-forming assays were performed using six-well plates with confluent
monolayers of the BGM cells.
Control wash and disinfection
Prior to all experiments volunteer’s hands were washed with warm water and nonantibacterial soap (Liquid Joy, Procter and Gamble, Cincinnati, OH) for 45 s, rinsed with
177
water, and dried with paper towels. Each hand was then sprayed twice with 70% ethanol,
rubbing the alcohol thoroughly over hands and wrists for 15 s, and allowing them to airdry. After seeded experiments with E. coli and S. aureus, fingers were disinfected twice
with 70% ethanol and wrapped with a 70% ethanol saturated paper towel for 30 s, then
washed and rinsed using warm water and Softsoap® liquid hand soap (Colgate-Palmolive,
Morristown, NJ) for 45 s, and then dried with paper towels. After seeded experiments
with B. thuringiensis spores and PV-1, fingers were placed in 10% sodium hypochlorite
solution (The Clorox Company, Oakland, CA) for 15 s and then neutralized in 10%
sodium thiosulfate (EMD, Gibbstown, NJ). The hands were then washed as described
above to prepare for subsequent trials.
Fomites tested
Three types of fomite materials were tested ranging in surface areas of 16 to 25 cm2:
ceramic tile porcelain (Home Depot, Atlanta, GA), laminate (Wilsonart International.
Temple, TX), and three different types of granite, one from India and two from Brazil
(GRANITE Kitchen & Bath Countertops, Tucson AZ). After fomite to finger transfers
with E. coli, and S. aureus, nonporous fomites were sprayed three times with 70%
ethanol and allowed to sit for 10 min. After seeded experiments with B. thuringiensis
spores and PV-1, were disinfected with 10% sodium hypochlorite solution (The Clorox
Company, Oakland, CA) allowed to sit for 10 min and subsequently neutralized in 10%
sodium thiosulfate (EMD, Gibbstown, NJ). All fomites were then washed under warm
running water with non-antibacterial soap (Liquid Joy, Procter and Gamble, Cincinnati,
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OH), rubbed with a wet paper towel on the surface area of inoculation, rinsed thoroughly
with RO treated water, air-dried, autoclaved, and reused.
Disinfectant wipe and Neutralizing Solution
The disinfectant tested in this study was a ready-to-use disinfectant wipe CLOROX
Disinfecting Wipes (The Clorox Company, Oakland, CA) with a square measure of 20.5
cm x 18 cm. The active ingredients were: n-Alkyl (C14, 60%; C16, 30%; C12, 5%; C18,
5%;) Dimethyl Benzl Ammonium Chloride 0.184%, n-Alkyl (C12, 68%; C14, 32%)
Dimethyl Ethylbenzyl Ammonium Chloride 0.184%. Other ingredients 99.632%. D/E
neutralizing medium was used to neutralize the bactericidal effects of the disinfectant
wipe (D/E, EMD, Gibbstown, NJ).
Relative Humidity and Temperature
The study was conducted at a relative humidity of 15% - 32% (typical indoor relative
humidity in Tucson, AZ) and temperature of 19°C to 25°C (i.e., room temperature).
Temperature and relative humidity were monitored with a High Accuracy Thermo –
Hygro (VWR, Mississauga, ON). During high relative humidity in the laboratory, t.h.e.®
Desiccant (EMD, Gibbstown, NJ) and Drierite desiccant (Drierite, Xenia, OH) were used
to decrease the relative humidity to the low relative humidity range (15% to 32%).
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Inoculation of fomites
Layout of fomites
For each of the three nonporous fomites ten fomite test coupons were evenly spaced in
three rows on the middle shelf of an incubator. Each trial consisted of three control
coupons without the disinfectant wipe application, three control coupons with
disinfectant wipe use, three fomite-to-finger transfer coupons with disinfectant wipe use,
and one negative control coupon to ensure fomites were not previously contaminated.
Organism concentration
The concentration of organisms added to the fomites was approximately 108 to 109
colony forming units (CFU)/cm2 of E. coli in (TSB, EMD, Gibbstown, NJ), 109 CFU/cm2
of S. aureus in TSB, 107 to 108 CFU/cm2 of B. thuringiensis spores in (DSM + S; Becton,
Dickinson and Company, Sparks, MD), and 108 plaque forming units (PFU)/cm2 of PV-1
in (PBS, Sigma-Aldrich, St. Louis, MO) in 10 µl droplets. Using a pipet tip, the 10 µl
inoculum droplets were spread over approximately 1.0 cm2 area on the center of each
fomite. Fomites were seeded in time intervals to provide a consistent 30 min drying time
to each coupon.
Fomite sampling
Fomites inoculated with E. coli, S. aureus, and B. thuringiensis were sampled using a
cotton-tipped swab applicator (Puritan Medical Products Company, Guilford, ME). In
the case of poliovirus a polyester fiber-tipped applicator swab (Falcon, Becton Dickinson
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and Company, Cockeysville, MD) was used. Swabs were moistened with 1.0 ml of D/E
neutralizing medium (D/E, EMD, Gibbstown, NJ), a 6.0 cm2 area was swabbed with the
applicator firmly against the surface and in a rotating motion before placed back into the
remaining D/E and vortexed for 5 s. Control fomites without the disinfectant wipe
application were sampled as described after a 30 min drying time.
Intervention application
A protocol adapted from William et al. (2007) and Siani et al. (2011) were used for the
use of the disinfectant wipe to assess bacterial and viral removal from contaminated
surfaces. Three control coupons and three fomite-to-finger transfer coupons were
removed one at a time from the incubator placed on the counter and held stationary with
the left hand. Coupons were treated with disinfectant wipes using a surface pressure of
approximately 300 – 500 g ± 50 applied in one sweeping motion from the far corner of
the coupon towards the proximal corner as previously described (Williams et al., 2009).
The disinfectant wipe was then discarded and the fomite was placed back into the
controlled humidity and room temperature incubator for an additional 10 min of drying.
Transfer experiments
The three control fomites with disinfectant wipe application were sampled as described
above at the end of 10 min. Fomite-to-finger transfer experiments were then conducted
with the remaining three swatches/coupons using the index, middle, and ring fingers of
the right hand. The transfer was done using a method adapted from Ansari et al. (1988)
181
and Mbithi et al. (1992) by placing the fomite at the center of a scale with a digital
readout and performing a finger transfer by placing the right hand index finger on the
center covering the inoculated area of the fomite, for ten seconds with a 1.0 kg/cm2
(98.0665 kPa) of average pressure (range of 700 g to 1500 g). Finger transfers with the
middle and ring fingers were conducted in the same fashion to complete one trial.
Sampling the fingers
Using a cotton-tipped swab applicator (Puritan Medical Products Company, Guilford,
ME), moistened in 1.0 ml of D/E neutralizing medium (D/E, EMD, Gibbstown, NJ), the
index finger pad was sampled by a sweeping, rotating motion and placed into the
remaining D/E vial and vortexed. Middle and ring fingers were sampled in the same way.
To sample PV-1 to finger transfers, a polyester fiber-tipped applicator swab (Falcon,
Becton Dickinson and Company, Cockeysville, MD) was used.
Organism assays
Organisms were eluted from the swab with 100 µl of D/E neutralizing medium (D/E,
EMD, Gibbstown, NJ), diluted, and quantitated for E. coli, S. aureus, and B. thuringiensis
spores using the spread plate technique on MacConkey, mannitol salt agar (MSA), and
tryptic soy agar (TSA) (EMD Chemicals Inc, Gibbstown, NJ), respectively. Agar plates
were incubated at 35 ± 2°C for 18 ± 2 h. B. thuringiensis spore samples were heat
shocked at 81 ± 2°C for 10 min prior to spread plating, to stimulate germination. PV-1
titrations were performed using 10-fold serial dilution plaque-forming assay previously
182
described (Bidawid et al., 2003, Ikner et al., 2011). Dilutions and plating were done in
duplicate.
Transfer efficiency, log10 reduction, and statistical analysis
Calculation of transfer efficiency
Bacterial colonies and plaques were enumerated and transfer efficiencies were calculated
using Eqn (1) (Marples and Towers, 1979, Paulson, 2005). Transfer efficiency (TE) is
defined as CFU or PFU recovered from finger relative to CFU or PFU recovered from
control fomite without intervention. If bacteria or phage were not recovered from the
finger pad, the lower detection limit of 10 CFU or PFU was used as an estimate for the
microorganism recovered as previously described (Chen et al., 2001, Julian et al., 2010).
A less than value (<) was used to indicate the transfer efficiency was less than the least
detectable limit. Finger transfers greater than 100% were truncated to 100%.
   
 %  /   / .  100
(1)
Log10 reduction was calculated using Eqn (2) and is described as the negative Log10 of
the value of CFU or PFU concentration recovered from the control fomite with
intervention divided by CFU or PFU recovered from fomite without intervention.
  / .
       / .
(2)
183
Data were entered into the software package StatPlus:mac, 2009, (AnalystSoft) to
compute descriptive statistical measures of mean survival, standard deviation, minimum,
maximum and statistical significance. Student’s t-test was used to assess the statistical
significance (P = 0.05) of the CFU/2-cm2, CFU/finger, and percent transfer efficiency
between the treated and non-treated surfaces.
RESULTS
Removal of microbial contamination from nonporous surfaces
E. coli, S. aureus, B. thuringiensis spores and poliovirus recovered from disinfectant
wipe-treated fomites was compared to concentrations recovered from non-treated fomites.
Greater numbers of test organisms were recovered from non-treated surfaces (4.5 – 6.9
log10 CFU or PFU/2-cm2) than from disinfectant wipe-treated surfaces (< 1.2 – 2.7 log 10
CFU or PFU/2-cm2) (Table 1).
There were significantly fewer (Student’s t-test, P < 0.05) E. coli, S. aureus, B.
thuringiensis spores, and poliovirus recovered from ceramic tile, laminate, and granite
following the application of a disinfectant wipe. Reduction of B. thuringiensis spores was
always lower than the other microorganisms (Table 1), indicating that B. thuringiensis
spores are less efficiently removed/killed by the disinfectant wipe.
184
Influence of disinfectant wipes on Microbial Transfer
Transfer of E. coli, S. aureus, B. thuringiensis and PV-1 from disinfectant wipe-treated
fomites to fingers were compared to microbial transfer from non-treated fomites to
fingers reported by Lopez et al., (submitted for publication) (Table 2 and 3). All model
microorganisms had lower transfer and recovery to fingers from disinfectant wipe-treated
fomites (< 0.0002% to 0.1%) and (< 1.0 – 1.7 log10 CFU or PFU/2-cm2), respectively
than from non-treated fomites (< 0.04% to 36.3%) and (1.2 – 5.3 log10 CFU or PFU/2cm2), respectively (Tables 2 and 3).
Transfer from disinfectant wipe-treated fomites to fingers was significantly lower than
that from non-treated fomites for E. coli for ceramic tile, S. aureus, B. thuringiensis, and
PV-1 for ceramic tile and laminate (Student t-test, P ≤ 0.05). There was also a statistical
difference (Stuent’s t-test, P ≤ 0.05) for E. coli, S. aureus and PV-1 recovered from
fingers in contact with all disinfectant wipe-treated surfaces and B. thuringiensis for
ceramic tile and laminate than those recovered from fingers in contact with non-treated
surfaces.
DISCUSSION
The purpose of this study was to determine the impact on fomite-to-finger transfer of
various types of microorganisms from surfaces contaminated treated with a disinfectant
wipe. Such information can be used to assess this type of intervention in reducing
exposure (Gibson et al., 1999, Haas et al., 1999, Nicas and Sun, 2006, Wein and
185
Atkinson, 2009). As expected the present study showed that the use of disinfectant wipes
greatly reduced the number of bacteria and virus on contaminated surfaces that are
commonly found in indoor environments (Table 1). Our results demonstrate that the use
of disinfectant wipes on surfaces greatly reduced the potential transfer of E. coli, S.
aureus, B. thuringiensis spores, and PV-1 to fingers (Tables 2 and 3). S. aureus was
reduced by four orders, while E. coli and PV-1 was reduced by three. B. thuringiensis
spores are the most resistant to wipe disinfectants and were therefore the most resistant to
removal.
Williams et al. (2007) using a three-stage efficacy protocol tested grapefruit extractcontaining surface wipes for the removal of several different strains of methicillinresistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) on stainless
steel discs (step 1). Williams et al. (2007) inoculated stainless steel discs with 20 uL of
test suspension 6-7 log10 cfu/disk and dried at 37°C for 25 min. Test suspension consisted
of bovine serum albumin (BSA) concentration to mimic clean (0.3 g/L) and dirty (3 g/L)
conditions. Cell removal was assessed by the attachment of a disinfectant-containing
wipe to a steel rod to allow mechanical rotation with a drill using an exerting weight
between the disc and the wipe. They used these parameters to follow the observation of
staff using the wipes in Intensive Therapy Units (Williams et al., 2007) and found cell
removal of 3 – 6.73 log10 CFU/disc and 2.55 – 4.66 log10 CFU/disc under clean and dirty
conditions, respectively. Our study observed similar result with a bacterial removal for S.
aureus (4.2 – 5.7 log10, 4.4 – > 6.3 log10, and 3.4 – > 6.2 log10) for ceramic tile, laminate,
186
and granite, respectively, (data not shown) using a wipe containing quaternary
ammonium compounds (QAC), even though we used different methodologies and
parameters than (Williams et al., 2007).
Our study used a recommendation by Williams et al. (2009) of a “one wipe – one
application – one direction” approach to wipe the surface, with a surface pressure similar
to Siani et al. (2011) of 300 – 500 g. This wiping method is possibly a more reasonable
application of the disinfectant wipe. A 10 min drying time followed the application of the
intervention wipe. The additional 10 min drying time after the intervention was applied
allowed any residual quaternary ammonium compounds (QAC) remaining on the fomite,
to interact with the bacteria and spores resulting in greater bactericidal and sporicidal
affects. As expected the present study showed an effective cell removal with S. aureus
showing a % reduction (up to 99.997%) and a viability loss of a log10 reduction (up to 5.0
log10 CFU/2-cm2) for the nonporous fomites (Table 1).
In a similar study Siani et al. (2011) examined the efficacy of nine sporicidal wipes on
two strains of Clostridium difficile spores. Using a modified three-stage protocol
described by Williams et al. (2007) stainless steel discs were inoculated with C. difficile
suspension medium and dried for 1.0 h at 37°C. Siani et al. (2011) reported bacterial
removal for both C. difficile strains to be < 2.5 log10 CFU/disk for most wipes tested. Our
model bacteria for C. difficile was B. thuringiensis spores. B. thuringiensis was the most
resistant organism to the intervention with a % reduction up to 99.4%, a log10 reduction
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of up to 2.5 log10 CFU/2-cm2, and spore removal of > 6 log10 CFU/2-cm2 on nonporous
surfaces.
The influence of disinfectant wipes on E. coli and poliovirus from ceramic tile, laminate,
and granite was similarly observed with the other microorganisms resulting in a percent
reduction, log10 reduction, and a total cell removal for E. coli of > 99.97%, > 4.0 log10
CFU/2-cm2, > 6.2 log10 CFU/2-cm2 and for PV-1 of up to 99.98%, 4.0 log10 PFU/2-cm2,
5.0 log10 PFU/2 cm2, respectively (Table 1). This present study demonstrated that there
were significantly fewer (Student’s t-test, P ≤ 0.05) E. coli, S. aureus, B. thuringiensis
spores, and poliovirus recovered from treated surfaces than non-treated and while this is
an expected outcome, quantitative reduction levels are necessary to detail future
intervention efficacy can be mathematically modeled.
To our knowledge, this is the first study that quantitates microbial transfer rates after the
use of disinfectant wipes as an intervention (Table 2). Fomite-to-finger transfer
efficiencies from non-treated surfaces compared to treated surfaces were: E. coli (7.3 to
21.7% and < 0.004), S. aureus (2.7 to 4.3% and < 0.002), B. thuringiensis spores (0.04 to
0.2% and < 0.03), and poliovirus type 1 (23.1 to 36.3% and < 0.004), respectively. S.
aureus, B. thuringiensis, and poliovirus had a significant difference (Student t-test, P ≤
0.05) with ceramic tile and laminate. All tested organisms did not show any significant
difference in pre/post intervention reduction with granite. This could be attributed to the
more porous nature of granite surface, it was sealed by the manufacturer, or specific
188
differences, in microbial attachment mechanisms to various surfaces. Unlike the transfer
efficiencies observed by the other model organisms from non-treated fomites B.
thuringiensis was transferred less than the other organisms (Table 2 and 3).
These results showed the importance of disinfectant wipes in reducing fomite-to-finger
transfer of microorganisms. The fomite-to-finger transfer will improve model accuracy
where this information could not previously be modeled to predict disinfectant efficacy
and exposure following intervention use and public health benefit of the intervention
(Boone and Gerba, 2007, Nicas and Best, 2008). With this data, we can mathematically
predict these outcomes, which will help to make cost benefit decisions based on the wipe
methods efficacy and the interventions role in reducing human exposures and risk.
CONCLUSIONS
The present study showed disinfectant wipes to be very effective in reducing fomite-tofinger bacterial transfer in indoor environments. Our findings also showed that less
fomite-to-finger transfer occurs when surfaces have been treated with disinfectant wipes
compared to non-treated surfaces (fomites). Our findings will provide exposure
assessment transfer rate input data in modeling risk assessment following the use of
disinfectant wipe intervention strategies.
ACKNOWLEDGEMENTS
The research was supported in part by the Clorox Company.
189
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TABLES
TABLE 1 Recovered microorganisms from fomites pre and post disinfectant wipe interventionb
Surface Type
E. coli
Mean ± SD (Range)
S. aureus
c
Mean ± SD (Range)
B. thuringiensis
c
Mean ± SD (Range)
Poliovirus Type 1
c
Mean ± SD (Range)c
Ceramic Tile
Non-treated
5.1 ± 0.4 (4.8 - 5.5)
Treated
< 1.6 ± 0.9 (< 1.0 - 3.2)
Reduction
3.6 ± 0.9 (2.3 - > 4.5)
a
6.8 ± 0.1 (6.7 - 6.8)
4.6 ± 0.2 (4.4 - 4.8)
5.6 ± 0.4 (5.1 - 6.2)
2.3 ± 0.6 (1.5 - 2.9)
2.7 ± 0.4 (2.2 - 3.4)
1.6 ± 0.4 (1.0 - 2.1)
4.5 ± 0.6 (3.7 - 5.3)
1.9 ± 0.5 (1.1 - 2.5)
3.9 ± 0.8 (3.0 - > 5.2)
Laminate
Non-treated
5.1 ± 0.9 (4.2 - 6.6)
Treated
< 1.2 ± 0.4 (< 1.0 - 1.9)
Reduction
4.0 ± 0.9 (3.2 - > 5.6)
6.9 ± 0.1 (6.8 - 7.1)
a
< 1.9 ± 0.8 (< 1.0 - 2.9)
4.6 ± 0.1 (4.5 - 4.7)
a
5.0 ± 0.8 (4.0 - > 5.8)
< 2.1 ± 0.6 (< 1.0 - 2.6)
5.6 ± 0.1 (5.5 - 5.7)
a
2.5 ± 0.7 (1.9 - > 3.6)
< 1.8 ± 0.5 (< 1.0 - 2.5)a
3.8 ± 0.5 (3.2 - > 4.7)
Granite
Non-treated
5.4 ± 0.1 (5.3 - 5.6)
Treated
< 1.9 ± 1.0 (< 1.0 - 3.2)
Reduction
3.5 ± 0.9 (2.4 - > 4.3)
6.8 ± 0.3 (6.4 - 7.1)
a
< 2.5 ± 1.2 (< 1.0 - 3.8)
4.4 ± 1.1 (3.1 - > 5.7)
4.5 ± 0.3 (4.1 - 4.9)
a
< 2.4 ± 0.8 (< 1.0 - 3.2)
2.1 ± 0.9 (1.3 - > 3.8)
5.5 ± 0.2 (5.0 - 5.7)
a
< 1.8 ± 0.8 (< 1.0 - 2.8)a
3.7 ± 0.9 (2.8 - > 4.5)
a
Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2
b
There were six replicates for each organism
c
Mean ± SD (Range) are expressed as CFU or PFU/2-cm2 log10
199
TABLE 2 Fomite-to-finger percent transfer of microorganismsd
E. coli
S. aureus
B. thuringiensis
Poliovirus Type 1
Transfer (%) ± SD (Range)
Transfer (%) ± SD (Range)
Transfer (%) ± SD (Range)
Transfer (%) ± SD (Range)
Without Interventionb
11.6 ± 11.8 (0.1 - 33.3)
2.7 ± 2.3 (0.8 - 6.7)
< 0.2 ± 0.1 (< 0.1 - 0.4)a
23.1 ± 24.0 (0.4 - 52.7)
With Intervention
< 0.01 ± 0.01 (< 0.003 - < 0.02)a
< 0.003 ± 0.01 (< 0.0001 - < 0.02)a
< 0.04 ± 0.03 (< 0.02 - < 0.1)a
< 0.01 ± 0.02 (< 0.003 - 0.05)
21.7 ± 23.9 (5.2 - 66.5)
4.3 ± 2.4 (1.3 - 7.4)
< 0.2 ± 0.1 (< 0.1 - 0.3)a
Surface Type
Ceramic Tile
Laminate
Without Interventionb
a
36.3 ± 8.7 (24.1 - 50.0)
< 0.02 ± 0.03 (< 0.0002 - < 0.1)
< 0.0002 ± 0.0001 (< 0.0001 - < 0.0004)
< 0.03 ± 0.01 (< 0.02 - < 0.03)
< 0.004 ± 0.004 (< 0.002 - 0.01)a
Without Interventionb
< 7.3 ± 10.6 (< 0.1 - 28.0)a
3.9 ± 5.0 (0.7 - 13.9)
< 0.04 ± 0.03 (< 0.02 - 0.1)a
33.8 ± 40.4 (0.4 - 100)c
With Intervention
< 0.004 ± 0.001 (< 0.002 - < 0.01)a
< 0.001 ± 0.001 (< 0.0002 - < 0.004)a
< 0.1 ± 0.1 (< 0.01 - < 0.2)a
< 0.01 ± 0.01 (< 0.002 - 0.02)a
With Intervention
a
a
Granite
a
Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2
b
Percent transfer data from (Lopez et al., submitted for publication)
c
Transfer event was > 100% and was truncated to 100%
d
There were six replicates for each organism
200
TABLE 3 Recovered microorganims from fingersc
Surface Type
E. coli
Mean ± SD (Range)
S. aureus
d
Mean ± SD (Range)
B. thuringiensis
d
Mean ± SD (Range)
Poliovirus Type 1
d
Mean ± SD (Range)d
Ceramic Tile
Without Interventionb
3.6 ± 0.7 (2.4 - 4.1)
4.5 ± 0.3 (4.1 - 4.9)
< 2.1 ± 0.2 (< 2.0 - 2.5)a
4.4 ± 0.6 (3.4 - 5.0)
With Intervention
< 1.0 ± 0.0 (< 1.0 - < 1.0)a
< 1.7 ± 0.7 (< 1.0 - 2.9)a
< 1.1 ± 0.2 (< 1.0 - < 1.4)a
< 1.6 ± 0.5 (< 1.0 - 2.3)a
3.5 ± 0.4 (2.8 - 3.9)
4.7 ± 0.3 (4.2 - 5.0)
< 2.1 ± 0.2 (< 2.0 - 2.4)a
5.3 ± 0.2 (5.0 - 5.6)
Laminate
Without Interventionb
With Intervention
< 1.0 ± 0.0 (< 1.0 - < 1.0)
a
< 1.2 ± 0.3 (< 1.0 - 1.5)
a
< 1.0 ± 0.0 (< 1.0 - < 1.0)
a
< 1.1 ± 0.2 (< 1.0 - 1.5)a
Granite
Without Interventionb
With Intervention
< 2.9 ± 0.7 (< 2.2 - 3.7)a
< 1.0 ± 0.0 (< 1.0 - < 1.0)
< 1.2 ± 0.3 (< 1.0 - 1.6)a
4.5 ± 0.3 (4.1 - 5.0)
a
< 1.5 ± 0.6 (< 1.0 - 2.5)
a
< 1.2 ± 0.2 (< 1.0 - 1.5)
a
a
Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2
b
Recovery of organism without intervention reported in (Lopez et al., submitted for publication)
c
There were six replicates for each organism
d
Mean ± SD (Range) are expressed as CFU or PFU/2-cm2 log10
4.1 ± 1.3 (2.5 - 5.5)
< 1.3 ± 0.2 (< 1.0 - 1.4)a
201
APPENDIX F: RISK OF CAMPYLOBACTER JEJUNI INFECTION FROM
PREPARING RAW CHICKEN IN DOMESTIC KITCHENS AND CROSSCONTAMINATION REDUCTION FROM DISINFECTANT WIPES
Gerardo U. Lopez1, 2, Masaaki Kitajima1, Jonathan Sexton1, 2, Charles P. Gerba1, and
Kelly A. Reynolds2*
1
Department of Soil, Water and Environmental Science, University of Arizona, Tucson,
Arizona 85721, USA
2
The University of Arizona, Mel and Enid Zuckerman College of Public Health, 1295 N.
Martin Ave., Tucson, Arizona 85724, USA
*
Corresponding author. Tel: (520) 626-8230, Fax (520) 626-8009, E-mail:
[email protected]
A manuscript for
Risk Analysis
202
ABSTRACT
Chicken bought at a retail store has a 71% chance of being contaminated with
Campylobacter jejuni. Using a new Consumer Phase Model (CPM) we conducted a
quantitative microbial risk assessment on the risk of infection following the preparation
of raw chicken filet in a domestic Kitchen. Using a Monte Carlo simulation of the risk of
transferring C. jejuni strain A3249, from various surfaces to hands and subsequently
transferring it to the mouth was forecasted. The use of a disinfectant wipe intervention to
disinfect contaminated surface area was also assessed. Several assumptions were used as
input parameters in the classical Beta-Poisson model to determine the risk of infection.
The disinfectant-wipe intervention reduced the risk of Campylobacter infection, illness,
and death by 2 to 3 orders on all fomites. The disinfectant-wipe intervention reduced the
annual risk of illness below the reported national average of diagnosed
Campylobacteriosis cases 1.3E-04. This risk assessment demonstrates that the use of
disinfectant wipes to decontaminate surface areas after chicken preparation reduces the
risk of C. jejuni infections up to 99.2%.
KEY WORDS: Campylobacter jejuni, Quantitative Microbial Risk Assessment,
disinfectant wipes, nonporous surfaces, fomite-to-hand transfer
1. INTRODUCTION
Foodborne infection remains a significant worldwide problem (1-3). Foodborne
pathogens cause a broad range of diarrhoeal to flu like symptoms (4). It is estimated that
1.8 million childhood deaths are associated with disease-causing organisms acquired
from food consumption with developing countries having the greatest number of cases (5).
In addition more than 130 million people in European countries and over 17 million
people in Australia suffer from foodborne diseases (6). In the United States, foodborne
pathogens cause an estimated 48 million cases of illnesses, 128,000 hospitalizations and
3,000 deaths per year (7). The annual cost of foodborne illnesses caused by the four most
common bacterial pathogens alone (Salmonella strains, Shigella, Campylobacter species,
and Escherichia coli has been estimated at 6.9 billion (8).
There is growing awareness of the foodborne illness transmission from household
exposures (6, 8). Even though the most common cause of foodborne acquired infections
has been attributed to the global food distribution and international travels, in European
countries such as England, Wales and the Netherlands, 80% of Salmonella and
Campylobacter infections are acquired in the home (3, 4). According to Van Asselt (9) up
to 87% of reported foodborne outbreaks are associated with food prepared or consumed
in the home. Several studies conducted in homes and in the laboratory show that
Campylobacter jejuni, Clostridium perfringes, Escherichia coli 0157:H7, Listeria
monocytogenes, non-typhoid Salmonella and Staphylococcus aureus are the primary
pathogens of concern (1, 4, 10-14).
204
Raw food, whether from vegetable or uncooked/undercooked animal meat sources can be
contaminated with human pathogens and transmitted to humans (4, 13-17). The food with
the greatest potential risk is raw poultry meat. Handling of poultry in domestic kitchens
has been shown to widely disseminate bacteria throughout the kitchen (18, 19). The
potential for cross-contamination in the kitchen via counters, cutting boards, and other
hand and food contact surfaces, after preparation of raw chicken contaminated either
naturally or seeded with marker organisms, has been demonstrated by several studies.
Dewit et al. (10) found that 60 families were each given a frozen chicken artificially
contaminated with Escherichia coli K12, they were able to recover the indicator
bacterium from many sites within the kitchen, often after routine cleaning. Another study
isolated Campylobacter jejuni from 170 (61%) of 279 chicken products, while
Salmonella was isolated from 44 (54%) of 81 chicken products(18). Several studies have
demonstrated that the preparation and cooking of chicken resulted in the frequent transfer
of Campylobacter and Salmonella to various surfaces throughout the kitchen (1, 20, 21). A
study conducted by Humphrey et al. (11) found that Salmonella enteritidis PT4 was
recovered kitchen utensils, from fingers, and work surfaces after breaking of artificially
contaminated eggs.
Potential pathogens from sources such as raw foods, humans, and animals can be
transferred between inanimate and animate surfaces through either direct or indirect
contact (9). During outbreaks of infection within the home, this transfer between animate
and inanimate surfaces may even give rise to cyclic infection (3). According to Kagan et al.
(2)
, secondary infectious disease transmission occur between 6 to 60% of the time within
205
households in which one member is ill. Evidence from cross-contamination studies, both
in the field and in simulated laboratory scenarios exists for several areas in the home,
including toilets, kitchen counters, and cutting boards, together with other hand and food
contact surfaces, kitchen sponges and cloths, and household laundry (12, 22). In a review of
consumer phase models (CPM) on quantitative microbial risk assessment (QMRA),
Nauta et al. (23) found that cross-contamination from fresh chicken meat to other foods
and hands, is the dominant route of exposure in all risk assessments and not
undercooking.
This risk assessment study focuse specifically on forecasting the exposure to C.
jejuni contaminated surfaces and the transfer of C. jejuni to hands and then finally to the
mouth. Therefore, the risk of infection from C. jejuni was evaluated after a consumer
prepared chicken filets at home and C. jejuni cross-contaminated the surface and is
transferred to a household members fingers after touching the contaminated surface
(fomite). We also investigate how using disinfectant wipe intervention to clean the
possible contaminated work area decreases the risk. We used a CPM on C. jejuni strain
A3249 for quantitative microbial risk assessment in order to compare the risk of infection
under two different scenarios. Both scenarios involve the preparation of a chicken filet
dinner resulting in cross-contamination to three different surface (fomite) materials
(ceramic tile, laminate, and granite) where the chicken was prepared prior to cooking. In
one scenario the food preparation surface is not wiped with a disinfectant wipe, where as
in the second scenario the work area is wiped with a disinfectant wipe. In both scenarios
the food preparer touches the disinfectant wipe, treated or non-treated surfaces and then
206
touches their mouth transferring. An assumption made in this study is that 100% of C.
jejuni that transfers to the mouth is capable of causing infection.
2. METHODS
2.1 The Exposure Model
Our CPM exposure scenario considers input parameters that predict the risk in
transferring C. jejuni from treated and non-treated surfaces to fingers. Input parameters
are shown in Table 1. The exposure assessment begins in the domestic kitchen where the
consumer is preparing a chicken dinner using a package of raw chicken filets purchased
at a retail store (Fig. 1). The probability that the consumer purchased C. jejuni
contaminated chicken meat is considered (input 1a). C. jejuni concentration is variable
and is also considered (input 1b). These two variables combined predict the concentration
of C. jejuni on the chicken filet portions. The consumer begins to prepare the chicken
filet by opening the package and placing the chicken portions on a cutting board. Portion
size is not considered in the scenario for simplification. Raw chicken juice contaminated
with C. jejuni spills (input 2) onto a certain surface area (input 3) of the kitchen counter
(fomite); ceramic tile, laminate, and granite. The consumer or a household member
touches the contaminated surface with their finger (input 4) and transfers C. jejuni to their
fingers (input 5). At this point the consumer touches their mouth with their finger. The
mouth is assumed to be an absorbing state as, described by Nicas and Sun (24). Thus we
assume that the C. jejuni concentration in contact with the mouth is absorbed into the
body as a total dose.
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2.1.1 Input Distribution
Concentrations of C. jejuni on raw chicken filet, Cchicken, are not static. Rather
two variable parameters were used to forecast the concentration on the chicken meat.
The forecast of the C. jejuni contamination parameter, Pcontamination, was based on the data
reported from Zhao et al. (25) where 71% of chicken samples randomly collected from
retail stores in the Washington, D.C. area were contaminated with C. jejuni. Based on
these findings a Yes-No distribution was used to forecast the probability that a consumer
purchasing a chicken filet package would have a 71% chance (Yes) of preparing chicken
meat contaminated with C. jejuni. There would then remain a 29% chance that the
consumer would purchase a chicken package that was not contaminated (Table I). C.
jejuni concentrations on raw chicken meat are variable and are given a Lognormal
distribution (mean 1.5, standard deviation 1.2), Cdistribution, provided by Nauta et al. (26),
(Table I). We convert the output value produced after the 20,000 iterations from the
Monte Carlo simulation back to actual CFU before multiplying by the probability of
contamination.
    
(1)
The volume assumed to have spilled, Vspilled, while opening and preparing the
chicken filet is 1 ml and the assumed spilled area, Afomite, on the fomite is 100 cm2. The
surface area in contact between the fomite and the finger, Sfomite/finger, is assumed to be 2
208
cm2 based on Nicas and Best (27) the average surface area of a finger tip (Table I). We
also assumed that every time the individual touches the surface they also then touch their
mouth.
Fomite-to-finger transfer efficiencies, TEFomite  Finger, of C. jejuni were based on
Gram negative Escherichia coli transfer rates found by Lopez et al., (submitted for
publication) under both treated and non-treated transfer scenarios (Table I). Transfer rates
from non-treated ceramic tile and laminate were assumed to have a lognormal
distribution based on the best-fit distribution on transfer efficiencies of E. coli from
Lopez et al. (submitted for publication). Lognormal distributions had the mean and
standard deviation as input parameters. Transfer rates from non-treated granite as well as
the transfer rates from treated surfaces were assumed to have a triangular distribution in
order to take into account the least detectable limit of 10 CFU/cm2 seen in some transfers.
The triangular distribution considers the minimum (0.0), likeliest (half of the maximum),
and maximum as distribution parameters.
The hand-to-fomite transfer efficiency, TEFinger  Mouth, parameter of 34% is a
point estimate based on Rusin et al. (22) using Gram negative Serratia rubidea hand-tomouth transfer data (Table I). Decay rates are not considered in this model because
transfer rates are based on a 30 min survival time of E. coli. C. jejuni exposure is
assumed to take place within or at the 30 min exposure window.
The surface area in contact between the fomite and the finger Sfomite/finger, was
assumed to be 2 cm2 based on Nicas and Best (27) previous assumptions of the average
surface area of a finger tip. Condition A, used one contact between the fomite and the
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finger Sfomite/finger equaling 2 cm2 surface area and condition B, used 4 contacts between
the fomite and the finger Sfomite/finger equaling 8 cm2 surface area as an input parameter
(Table I). The number of times an individual touches their mouth was based on the
observations reported by Nicas and Best (27). who found a total of 24 contacts in 3 hours
with an average contact of 8 h-1. Since our transfer data (Lopez et al., submitted for
publication) is based on a 30 min drying time and decay rate is not considered we
assumed 4 contacts per 30 min-1 equaling Sfomite/finger 8 cm2 with the surface for condition
b (Table I). We also assume that each surface contact resulted in a hand-to-mouth
transfer and, as Nicas and Sun (24) previously described, the mouth is assumed to be an
absorbing state. Based on this assumption we consider the C. jejuni in contact with the
mouth to be absorbed into the body as a dose.
2.1.2 Fomite-to-finger Dose
This fomite-to-finger CPM is developed to show the probability distribution of
the ingested dose of C. jejuni (CFU/finger). A single Dose (D) can be represented with:
 
  

 /        
(2)
2.1.3 Risk of Infection
The C. jejuni risk of infection (Pinfection) was determined by modeling the doseresponse relationship. The dose-response relationship in risk assessment describes the
210
relationship between exposure, expressed as the number of Camplobacter bacteria
ingested, and the probability of a response. The Beta-Poisson equation (3) suggested by
the QMRA wiki (28) and the parameters provided were used in modeling this risk
assessment. The QMRA wiki provides the N50 of 8.9E+02 and α 0.144 (28) for the BetaPoisson model.


  1  1  
 


(3)
2.1.4 Risk of Illness
The C. jejuni risk of illness (Pillness) was calculated by using equation (4).
Morbidity ratio was determined by dividing the 2.4 million people estimated by the CDC
(29)
to be affected by Campylobacteriosis by the United States population estimated to be
315,000,000 people (30). Therefore the morbidity ratio is estimated to be 7.62E-03.
    
(4)
2.1.5 Risk of Death
The C. jejuni risk of death (Pdeath) was calculated by using equation (5). Mortality
ratio was determined by dividing the estimated 124 deaths per year from Campylobacter
infections reported by the CDC (29) by the 2.4 million people estimated to be affected by
Campylobacteriosis (29). Therefore the mortality ratio is estimated to be 5.17E-05.
211
    
(5)
2.1.6 Annual Risk
Annual risk of infection (Pinfection), illness (Pillness), and death (Pdeath) were
determined by using equation 6 and the results of a National Chicken Council frequency
of eating chicken survey (31). The survey did not distinguish between cooked and raw
chicken meat so the total biweekly average was used for each year. Consumers
purchasing no chicken filets, cooked or raw, were included in the average. The 2010,
2011, and 2012 survey found that the average people purchased chicken from a retail
grocery store was 3.6, 3.6, and 3.4, respectively within the previous two weeks from the
time of the survey. The three year average was 3.53 multiplied by 26 biweekly periods
resulted in 91.87 ≈ 92 times people purchased chicken filets and prepared a meal at home
in a year (Table I).
  1  1  
(6)
2.1.7 Percent Reduction
Percent reduction was calculated using equation (7) and is defined as the risk of
touching a non-treated surface minus the risk touching a treated surface relative to the
risk from the non-treated surface.
212
 %  
   –   
  
  100
(7)
2.1.8 Model Implementation
The CPM simulations were conducted with Monte Carlo (Oracle Crystal Ball,
Fusion Edition, Redwood Shores, CA) and Excel Mac version (Microsoft Excel 2011).
Monte Carlo simulations consisted of 20,000 iterations to predict the risk of infection
from non-treated and treated surfaces. The Monte Carlo sampling generates model
parameter inputs from defined probability distributions, previously mentioned, allowing
the incorporation of parameter uncertainty. Each simulation produced an input value
based on the assumption of the distribution for the input 1a probability for contamination,
input 1b concentration distribution of C. jejuni contamination on the chicken portions,
and input 5 fomite-to-finger transfer efficiency.
The output values produced for distribution assumptions 1a and 1b were used to
calculate concentrations on chicken (Equation 1). This output value along with the
previously mentioned point estimates and input value 5 were used in calculating the dose
(Equation 2). In turn the dose output value was an input value in the Beta Poisson model
(Equation 3), considered a forecast cell in the Monte Carlo simulation to determine the
risk of infection (Pinfection). The risk of infection output value was then an input value in
(Equation 4), considered then the forecast cell in the Monte Carlo simulation to determine
the risk of illness (Pillness). In turn the risk of illness output value was an input value in
(Equation 5), considered the new forecast cell in the Monte Carlo simulation to determine
213
the risk of death (Pdeath). The Monte Carlo simulation was performed the same way for
determining the annual risk of infection, illness, and death.
3. RESULTS
3.1 Risk Comparison of Scenario 1 and 2 under Condition A
3.1.1 Risks per person per event
Scenario 2, fomites treated with the disinfectant-wipe intervention resulted in a >
99% reduction for ceramic tile, laminate, and granite for risk of infection, illness, and
death, compared to scenario 1, non-treated fomites. Condition A, depicted one fomite to
hand contact and subsequently a hand to mouth transfer. Risk of infection, illness, and
death was 2 to 3 orders greater under non-treated fomites, scenario 1, than treated fomites
(Table II).
3.1.2 Annual Risks per person per year
The percent reduction of annual risk of infection, illness, and death were all >
than 99% except for a laminate for risk of infection. The annual risk of the infection was
up to 1.67E-01 for scenario 1, non-treated fomites, compared to as low as 1.00E-03 for
scenario 2, treated fomites. Annual risk of death was up to 2.9E-07 with non-treated
fomites, while with treated fomites as low as 2.2E-10 (Table III).
3.2 Risk Comparison of Scenario 1 and 2 under Condition B
3.2.1 Risks per person per preparation event
Condition B, describes 4 fomite to hand contacts with 4 subsequent hand to mouth
transfers. Risk of infection under scenario 1, non-treated surfaces, was an up to 1.8E-02,
214
while treated fomites, scenario 2, showed 2 to 3 orders lower risk (Table IV). Most
fomites showed a 99% reduction. Risk of illness and death were also 2 to 3 orders lower
with the use of the disinfectant-wipe intervention.
3.2.2 Annual Risks per person per year
The annual risks under Condition B were all greater than under Condition A. The
percent reduction of the annual risk of infection, illness, and death were all > than 95%.
The annual risk of infection was up to 2.71E-01 for scenario 1, non-treated fomites,
compared to as low as 2.00E-03 for scenario 2, treated fomites. Annual risk of illness
was up to 1.21E-02 with non-treated laminate, while with treated laminate 1.38E-04
(Table V).
4. DISCUSSION
A number of QMRA have been conducted using Consumer Phase Models (CPM)
showing the cross-contamination of C. jejuni and the risk associated with preparing raw
poultry in domestic kitchens (23, 26). However, this is the first QMRA modeling the
probability of a C. jejuni infection from a cross-contamination to the surface and a
subsequent fomite-to-finger transfer and then a hand-to-mouth transfer occurring
resulting in a dose. Further we provide a novel comparison of the risk of infection with
and without the use of a disinfectant wipe.
The probability of infection given the CPM presented in this QMRA is relatively
high. With a 71% chance that chicken purchased at a retail store would be contaminated
with C. jejuni (25), along with the other assumptions, resulted in a higher mean risk of
215
infection per person per preparation event for scenario 1, non-treated surfaces, compared
to scenario 2, disinfectant-wipe treated surfaces, under both Condition A and B. Under
Condition A, 1-fomite to hand to mouth transfers, the risk of infection with scenario 1
non-treated surfaces was (5.6E-03 to 8.00E-03), while with scenario 2 treated surfaces
(1.5E-05 to 6.00E-06) (Table II). As expected the risk of infection per person per
preparation event increased by 2 to 3 orders under Condition B, 4-fomite to hand to
mouth contacts, scenario 1 non-treated surfaces (1.23E-02 to 1.80E-02), compared with
scenario 2 treated fomites (2.17E-04 to 4.6E-05) (Table IV).
The annual risk of infection per person per year (92 preparation events) under
Condition A resulted in (1.26E-01 to 1.67E-01) under scenario 1, compared to (1.00E-03
to 4.00E-03) under scenario 2 (Table III). As expected the annual risk of infection per
person per year under Condition B showed (2.21E-01 to 2.71E-01) for scenario 1 nontreated fomites, compared to (2.00E-03 to 1.2E-02) for scenario 2, disinfectant-wipe
treated fomites (Table V). The annual risk of illness under Condition A for scenario 1
non-treated surfaces (3.80E-03 to 5.50E-03) is greater than scenario 2 treated surfaces
(4.9E-05 to 7.9E-06) (Table III). The same trend is seen under Condition B with 4fomite to hand to mouth transfers, where risk of illness is greater without the use of the
disinfectant wipe and lower with the use of the disinfectant-wipe intervention. The same
pattern is observed with the risk of death for both Conditions and scenarios.
The FoodNet surveillance data shows 13 cases are diagnosed each year for each
100,000 persons in the population (29), which means 1.3E-04, 1.3 in 10,000 persons
become ill with Campylobacteriosis annually. The risk of illness with scenario 1 non-
216
treated surfaces under Condition A, resulted in 38.0E-04 to 55.0E-04, while under
scenario 2 treated fomites was 0.49E-04 to 0.079E-04 (Table III). Compared to the risk
of illness under Condition B scenario 1, 8.50E-03 to 1.05E-02 (85.0E-04 to 105.0E-04),
while scenario 2 showed 1.38E-04 to 2.92E-05 (1.38E-04 to 29.2E-04) (Table V).
Sensitivity analysis showed the probability of contamination having the greatest
influence on the probability of infection. Most Monte Carlo simulations reported a
greater than 70% influence on the outcome of infection with concentration most of the
time having a 25% influence. Transfer efficiency showed the least influence compared to
the other two assumption distributions. The probability of contamination had a Yes-No
distribution, which meant that 29% of the time the output was zero, which gave a final
dose of zero. This means one-third of the 20,000 iterations resulted in a zero risk. This
input parameter seems to have caused the annual risk of infection, illness and death to be
up to 30 times greater than the national average. A retail study by Gupta et al. (32) found
44% of the chicken products samples where contaminated with Campylobacter. This
Yes-No distribution will be used on future QMRA studies to see if the risk is closer to the
national average. It is important to compare surveillance, diagnosed cases from a region
with the retail survey study to see if Campylobacter contamination on chicken products
transfer over to reported cases. Our study clearly showed an over estimation of
Campylobacteriosis in the Washington, DC area where the retail study was conducted (25).
The surface area in contact between the fomite and the finger Sfomite/finger, was
assumed to be 2 cm2 by Nicas and Best (27) the average surface area of a finger tip.
Condition A, used one contact between the fomite and the finger Sfomite/finger equaling 2
217
cm2 surface area as an input parameter and condition B, used 4 contacts between the
fomite and the finger Sfomite/finger equaling 8 cm2 surface area as an input parameter (Table
I). The surface area between the fomite and finger and the number of times an individual
touches their mouth was based on the observations reported by Nicas and Best (27), who
found a total of 24 contacts in 3 hours with an average contact of 8 h-1. Since our transfer
data (Lopez et al., submitted for publication) is based on a 30 min drying time and decay
rate is not considered we assumed 4 contacts per 30 min-1 equaling Sfomite/finger 8 cm2 with
the surface for condition b (Table I). Nicas and Best (27) described that the hand contact
with environmental surfaces depends on the individual and the activities being performed,
they assumed 1 min-1 in their influenza QMRA model. We also assume that each surface
contact resulted in a hand-to-mouth transfer and, as Nicas and Sun (24) previously
described, the mouth is assumed to be an absorbing state. Based on this assumption we
consider the C. jejuni in contact with the mouth to be absorbed into the body as a dose.
The classic Beta-Poisson model was used to describe the probability of infection
based on the feeding study by Black et al. (33) fitted by Medema et al. (34) and Teunis and
Havelaar (35). The maximum likelihood estimates for the Beta-Poisson model parameters
based on these data are α = 0.145 and β = 7.59 (34). The Beta-Poisson equation (3)
suggested by the QMRA wiki (28) and the parameters provided were used in modeling this
risk assessment. Even though there are updated parameters for C. jejuni developed based
on milk outbreaks by Teunis et al. (36) the parameters used in this risk assessment are well
accepted and do not seem to over estimate the probability risk of infection (37). These
218
input parameters are more often used in C. jejuni risk assessments models, with some
variation in the model equation (23, 26).
The results of this QMRA study show that the use of a disinfectant wipe
intervention lowers the risk of infection, illness, and death by 2 to 3 orders of magnitude,
percent reduction 95% to >99%. The risk of infection and illness is over estimated
compared to the national average of 1.3 in 10,000 persons with Campylobacteriosis each
year. However, FoodNet surveillance has shown that risk of illness is greater in some
surveillance locations up to 6.4E-04 (38). Since the probability of contamination showed
to have the greatest influence on the probability of infection another retail study isolated
Campylobacter from 44% (80/180) chicken products (32) probability of contamination
will be used compared to the 71% (130/184) used in this study (25). The “farm-to-fork”
approach to food safety places a responsibility on the consumer to protect both
themselves and those for whom they prepare food. Proper precaution and effective home
hygiene practices should be practiced in order to prevent cross-contamination of
pathogenic microbes to our foods and hands. This risk assessment shows that the use of
disinfectant wipes after the preparation of raw chicken meat could reduce the risk of C.
jejuni infection.
In conclusion, the QMRA study show that the use of disinfectant wipes reduces
the risk of Campylopacter infection, illness, and death by 2 to 3 orders of magnitude. The
disinfectant-wipe intervention reduced the annual risk of illness below the reported
national average of diagnosed Campylobacteriosis cases. The present study also found
that risk increase with the number of fomite to hand to mouth transfers. The sensitivity
219
analysis showed the Yes-No distribution had the greatest influence. Based on these
results future studies will use retail surveillance of the probability of chicken product
contamination with FoodNet surveillance states in order to reduce the disparity in the risk
to the actual cases. This risk assessment shows that the use of disinfectant wipes to
decontaminate surface areas after chicken preparation reduces the risk of C. jejuni
infections up to 99.2%.
ACKNOWLEDGEMENTS
This research was supported by the Center for Advancing Microbial Risk
Assessment, funded by the U.S. Environmental Protection Agency Science to Achieve
Results (STAR) program and the U.S. Department of Homeland Security (Grant
R83236301), and the Clorox Company.
220
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Cogan TA, Slader J, Bloomfield SF, Humphrey TJ. Achieving hygiene in the
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Nicas M, Sun G. An Integrated Model of Infection Risk in a Health-Care
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226
FIGURES
Chicken Fillet
Cu ng Board
Surface (fomite)
Ceramic Tile, Laminate, Granite
Interven on
Disinfectant wipe
Hands (fingers)
Mouth (Lips)
Fig. 1. Describes the Consumer Phase Model and direction of cross-contamination.
227
TABLES
Table I. In put parameters used in CPM
Variable Description
C. jejuni chicken concentration
Symbol
Probability of contamination
Pcontamination
Distribution of concentration
Cdistribution
Chicken concentration
Units
Log
2
Input Parameters
Source/Reference
Yes (0.71), No (0.29)
(Zhao et al., 2001)
Lognormal (1.5, 1.2)
(Nauta and Christensen, 2011)
Cchicken
CFU/cm
Forecaset
Monte Carlo simulation
Vspilled
ml
1
Assumption
Afomite
cm2
100
Assumption
Sfomite/finger
cm2
2
(Nicas and Best 2008)
Sfomite/finger
2
8
(Nicas and Best 2008)
(Lopez et al. submitted for
publication)
Chicken package juice
Volume spilled
Total contaminated
Area of fomite
Surface area Between fomite/finger
Condition A
Condition B
cm
Transfer rate from non-treated surfaces
Ceramic Tile to finger
TEceramic tile --> finger
Lognormal (0.12, 0.12)
Laminate to finger
TElaminate --> finger
Lognormal (0.22, 0.24)
Granite to finger
TEgranite --> finger
Triangular (0.0, 0.14, 0.28)
Ceramic Tile to finger
TEceramic tile --> finger
Triangular (0.0, 0.0001, 0.0002)
Laminate to finger
TElaminate --> finger
Triangular (0.0, 0.0005, 0.001)
Granite to finger
TEgranite --> finger
Triangular (0.0, 0.00005, 0.0001)
Transfer rate from treated surfaces
TEfinger --> mouth
0.34
(Rusin et al. 2002)
Frequency of eating chicken survey
Fpreparation
92
(National Chicken Council 2012)
Beta Piosson Parameters
α and N50
0.144 and 8.9E+02
(CAMRA QMRA Wiki, 2012)
Dose
D
Forecast
Monte Carlo simulation
Transfer rate from treated surfaces
CFU/finger
(Lopez et al. submitted for
publication)
228
Table II Risks associated with one preparation of chicken filet (/person/event): Condition A
Intervention
Ceramic tile
Laminate
Granite
Fomite
Non-treatment
Risk of Infection
5.60E-03
Risk of Illness
4.30E-05
Risk of Death
2.20E-09
Treatment
1.50E-05
7.30E-08
5.20E-12
Percent Reduction
99.73
99.83
99.76
Non-treatment
8.00E-03
6.00E-05
3.10E-09
Treatment
6.60E-05
4.01E-07
2.10E-11
Percent Reduction
99.18
99.33
99.32
Non-treatment
6.00E-03
4.80E-05
2.40E-09
Treatment
6.00E-06
4.50E-08
2.20E-12
Percent Reduction
99.90
99.91
99.91
229
Table III Annual Risk (/ person/year): Condition A
Intervention
Ceramic tile
Laminate
Granite
Fomite
Non-treatment
Risk of Infection
1.26E-01
Risk of Illness
3.80E-03
Risk of Death
1.90E-07
Treatment
1.00E-03
7.90E-06
3.90E-10
Percent Reduction
99.21
99.79
99.79
Non-treatment
1.67E-01
5.50E-03
2.90E-07
Treatment
4.00E-03
4.90E-05
2.30E-09
Percent Reduction
97.60
99.11
99.21
Non-treatment
1.51E-01
4.50E-03
2.40E-07
Treatment
1.00E-03
7.90E-06
2.20E-10
Percent Reduction
99.34
99.82
99.91
230
Table IV Risks associated with one preparation of chicken filet (/person/event): Condition B
Intervention
Ceramic tile
Laminate
Granite
Fomite
Non-treatment
Risk of Infection
1.23E-02
Risk of Illness
1.03E-04
Risk of Death
5.20E-09
Treatment
4.60E-05
3.52E-07
1.61E-11
Percent Reduction
99.63
99.66
99.69
Non-treatment
1.80E-02
1.39E-04
6.80E-09
Treatment
2.17E-04
1.54E-06
8.20E-11
Percent Reduction
98.79
98.89
98.79
Non-treatment
1.50E-02
1.21E-04
6.10E-09
Treatment
2.70E-05
1.92E-07
9.60E-12
Percent Reduction
99.82
99.84
99.84
231
Table V Annual Risk (/ person/year): Condition B
Intervention
Ceramic tile
Laminate
Granite
Fomite
Non-treatment
Risk of Infection
2.21E-01
Risk of Illness
8.50E-03
Risk of Death
4.90E-07
Treatment
4.00E-03
2.92E-05
1.89E-09
Percent Reduction
98.19
99.66
99.61
Non-treatment
2.71E-01
1.21E-02
6.90E-07
Treatment
1.20E-02
1.38E-04
7.80E-09
Percent Reduction
95.57
98.86
98.87
Non-treatment
2.57E-01
1.05E-02
5.70E-07
Treatment
2.00E-03
1.69E-05
9.10E-10
Percent Reduction
99.22
99.84
99.84
232
APPENDIX G: SUPPLEMENTAL MATERIALS
Transfer of Bacteria and Viruses to Hands
Running title: Transfer of Microorganisms to Hands
Gerardo U. Lopez and Charles P. Gerba#
Department of Soil, Water and Environmental Science, University of Arizona, Tucson,
Arizona 85721
#
Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail:
[email protected]
A manuscript for
Applied and Environmental Microbiology
Supplemental Material Not Included in Publication
233
TABLES
TABLE 1 Hand-to-Fomite Transfer Efficiency (%)
Organism Type
Respiratory
Viruses
Species (Reference)
Rhinovirus (1)
Pen, SSb
Pen, SS
Rhinovirus (2)
Rhinovirus 14 (3)
Human Parainfluenza virus-3 (3)
Enteric Viruses
Fomite
Rotavirus (4)
Hepatitis A Virus (5)
Brass Door
Knob
Faucet handle
Brass Door
Knob
Stainless Steel
Stainless Steel
Stainless Steel
N/D
Drying time
(min)
Dryd
Natural
Contact
Time (s)
Hand to
Fomite (%)
3 min
Undetected
3 min
14
N/D
0
e
c
S/B/F
N/D
13.6
N/D
0e
S/B/F
N/D
35.7
N/D
0
f
S/B/F
N/D
44.6
50 ± 5
20g
1
5
0.92
50 ± 5
20
g
1
5
Undetected
20
g
1
10
16.1
g
1
10
1.8
50
Damp
d
Contact
Pressure
kg/cm2
Natural
Stainless Steel
50
60
Stainless Steel
45 ± 5
20g
0.2
10
26.8
45 ± 5
g
Stainless Steel
Stainless Steel
Stainless Steel
Stainless Steel
a
Relative
Humidity
(%)
N/Da
45 ± 5
45 ± 5
45 ± 5
60
0.2
10
21.2
120
g
0.2
10
14.1
180
g
0.2
10
3.5
240
g
0.2
10
1.6
N/D - Not determined; b SS - Stainless Steel spoons; c S/B/F - Sliding back and forth; d Inoculum (ml) ≤ 0.005; e Inoculum (ml) 0.05; f Inoculum (ml) 0.1; g
Inoculum (ml) 0.01
234
TABLE 1 Continued
Organism Type
Coliphage
Species (Reference)
Fomite
Drying time
(min)
N/Da
30 sb
Contact
Pressure
kg/cm2
N/D
Contact
Time (s)
Hand to
Fomite (%)
10
33.9
PRD-1 (6)
Lips - skin
Φ-X174 (7)
Glass cover slip
45 - 60
Visibly dryb
0.2 - 0.4
10
26c
Glass cover slip
45 - 60
Visibly dryb
0.2 - 0.4
10
17d
Glass cover slip
45 - 60
Visibly dryb
0.2 - 0.4
10
28c
Glass cover slip
45 - 60
Visibly dryb
0.2 - 0.4
10
20d
Glass cover slip
45 - 60
Visibly dryb
0.2 - 0.4
10
24c
Glass cover slip
45 - 60
Visibly dryb
0.2 - 0.4
10
15d
fr (7)
MS2 (7)
a
Relative
Humidity (%)
N/D - Not Determined; b Inoculum (ml) 0.005;
c
Unwashed hands; d Washed hands
235
TABLE 1 Continued
Organism Type
Gram (+) bacteria
Staphylococcus saprophyticus (8)
Kitchen cloth
N/Da
0d
Contact
Pressure
kg/cm2
G/Fb
Staphylococcus saprophyticus (9)
Fabric
N/D
0d
G/F
10
17c
Micrococcus luteus (6)
Lips - skin
N/D
30 se
Species (Reference)
Fomite
Micrococcus luteus (10)
Gram (-) bacteria
Paper-towel Disp.
Drying time
(min)
N/D
Contact
Time (s)
Hand to
Fomite (%)
N/D
85
N/D
10
40.99
f
N/D
Natural
0.03
d
G/F
10
88c
0
Escherichia coli (9)
Fabric
N/D
0
Klebsiella aerogenes (9)
Fabric
N/D
0d
G/F
10
86c
Psuedomonas aeruginosa (9)
Fabric
N/D
0d
G/F
10
76c
Enterobacter aerogenes (11)
Spigot
N/D
0g
N/D
N/D
1.02
N/D
10
33.97
N/D
Natural
0.04
Serratia rubidea (6)
Lips - skin
Serratia marcescens (10)
a
Relative
Humidity (%)
N/D - Not Determined; b G/F - Grasped firmly;
Contamination
Paper-towel Disp.
c
N/D
N/D
e
30 s
0
f
Considered second stage of transfer; d Inoculum (ml) 1.0; e Inoculum (ml) 0.005; f 1.0x108 CFU; g Cross-
236
TABLE 2 Fomite-to-Hand Transfer Efficiency (%)
Organism Type
Respiratory
Viruses
Species (Reference)
Rhinovirus (1)
Rhinovirus (2)
Pen, Table, SSb
Relative
Humidity
(%)
N/Da
Pen, Table, SS
N/D
Fomite
Brass Door Knob
Faucet Handle
Brass Door Knob
Rhinovirus 14 (3)
Human Parainfluenza virus-3 (3)
Enteric Viruses
Rotavirus (4)
Hepatitis A Virus (5)
Stainless Steel
Stainless Steel
Stainless Steel
Dampf
F/R
Fomite to
Hand (%)
15e
15
15e
21
10
d
S/B/F
N/D
61.5
10
g
S/B/F
N/D
16.3
N/D
10
h
S/B/F
N/D
32.9
50 ± 5
20i
1
5
0.67
50 ± 5
20
i
1
5
1.5
20
i
1
10
16.8
i
1
10
1.6
0.2
10
22.1
N/D
N/D
50
50
60
Stainless Steel
45 ± 5
20i
45 ± 5
i
Stainless Steel
Contact
Time (s)
g
Stainless Steel
Stainless Steel
a
Dryf
Contact
Pressure
kg/cm2
F/Rc
Drying time
(min)
45 ± 5
60
0.2
10
14.6
120
i
0.2
10
9.0
i
0.2
10
4.6
0.2
10
0
Stainless Steel
45 ± 5
180
Stainless Steel
45 ± 5
240i
N/D - Not determined; b SS - Stainless Steel spoons; c F/R - Firmly rubbing table top or fingers; d S/B/F - Sliding back and forth; e Contact with table top 15 s
and 3 min with plastic pen and stainless steel spoon; f Inoculum (ml) ≤ 0.005; g Inoculum (ml) 0.05; h Inoculum (ml) 0.1; i Inoculum (ml) 0.01
237
TABLE 2 Continued
Organism Type
Enteric Viruses
Relative
Humidity (%)
Drying time
(min)
Spatula
N/Da
5b
Contact
Pressure
kg/cm2
0.2 - 0.4
Spatula
N/D
15b
Species (Reference)
Feline Calicivirus, Strain F9
(12)
Fomite
Fork
Fork
Cutting Board
N/D
5
b
Cutting Board
N/D
15b
N/D
5
b
b
N/D
15
Stainless Steel
N/D
5
b
Stainless Steel
N/D
15b
Ceramic Tile
Ceramic Tile
15 - 32
40 - 65
Fomite to
Hand (%)
5 - 10
31.62
0.2 - 0.4
5 - 10
31.62
0.2 - 0.4
5 - 10
25.12
0.2 - 0.4
5 - 10
25.12
0.2 - 0.4
5 - 10
25.12
0.2 - 0.4
5 - 10
25.12
0.2 - 0.4
5 - 10
6.31
0.2 - 0.4
5 - 10
12.59
0.2 - 0.4
5 - 10
19.95
0.2 - 0.4
5 - 10
12.59
30
b
1
10
23.1
30
b
1
10
29.2
b
1
10
36.3
Laminate
15 - 32
30
Laminate
40 - 65
30b
1
10
25.5
15 - 32
30
b
1
10
33.8
30
b
1
10
25.9
Granite
Granite
N/D - Not Determined; b Inoculum (ml) 0.01
b
15
Door Knob
a
5
N/D
Door Knob
Poliovirus Type 1 (Lopez et al.,
submitted for publication)
N/D
b
Contact
Time (s)
40 - 65
238
TABLE 2 Continued
Organism Type
Enteric Viruses
Species (Reference)
Poliovirus Type 1 (Lopez et al.,
submitted for publication)
Relative
Humidity
(%)
15 - 32
Fomite
Ceramic Tile
Laminate
Granite
Coliphage
PRD-1 (6)
Faucet handle
a
10
< 0.004
15 - 32
N/D
30 + 10
Contact
Time (s)
Fomite to
Hand (%)
10
0.01
1
10
0.01
1
f
N/D
30
33.47
1
f
N/D
30
65.8
1
g
N/D
10
0.03
h
N/D
10
0.02
N/D
N/D
< 0.01
Sponge
N/D
1
Laundry 100%b
N/D
1i
N/D
h
Laundry 50:50%
MS2 (7)
1
e
N/D
b
fr (7)
30 + 10e
N/D
Dishcloth
ΦX174 (7)
15 - 32
a
Phone receiver
ΦX174 (13)
30 + 10e
Contact
Pressure
kg/cm2
1
Drying time
(min)
1
N/D
N/D
< 0.01
j
N/D
Natural
0.04
Door handles
N/D
15
Door handles
N/D
15j
N/D
Natural
0.000005d
Door handles
N/D
15j
N/D
Natural
0.0004c
Glass cover slip
45 - 60
Visibly dryk
0.2 - 0.4
10
21c
Glass cover slip
45 - 60
Visibly dryk
0.2 - 0.4
10
11d
Glass cover slip
45 - 60
Visibly dryk
0.2 - 0.4
10
37c
Glass cover slip
45 - 60
Visibly dryk
0.2 - 0.4
10
39d
Glass cover slip
45 - 60
Visibly dryk
0.2 - 0.4
10
25c
Glass cover slip
45 - 60
Visibly dryk
0.2 - 0.4
10
26d
N/D - Not Determined; b Laundry 100% - cotton, Laundry 50% polyester: 50% cotton; c Unwashed hands; d Washed hands; e Inoculum (ml) 0.01; f Inoculum
N/D; g Inoculum (ml) 50; h Inoculum (ml) 100; i Inoculum (ml) 200; j Inoculum (ml) 0.4; k Inoculum (ml) 0.005
239
TABLE 2 Continued
Organism Type
Coliphage
Relative
Humidity (%)
Drying time
(min)
15 - 32
30a
Acrylic
40 - 65
30
a
Glass
15 - 32
40 - 65
Species (Reference)
MS2 (Lopez et al., submitted
for publication)
Fomite
Acrylic
Glass
Ceramic Tile
Fomite to
Hand (%)
10
21.7
1
10
79.5
30a
1
10
19.3
30
a
1
10
67.3
30
a
1
10
7.1
a
1
10
41.2
40 - 65
30
Laminate
15 - 32
30a
1
10
5.4
40 - 65
30
a
1
10
63.5
30
a
1
10
6.9
a
1
10
37.4
Stainless Steel
15 - 32
Stainless Steel
40 - 65
30
Granite
15 - 32
30a
1
10
10.2
40 - 65
30
a
1
10
30.0
30
a
1
10
0.03
a
1
10
0.3
Granite
Cotton
15 - 32
Cotton
40 - 65
30
Polyester
15 - 32
30a
1
10
0.3
40 - 65
30
a
1
10
2.3
30
a
1
10
0.4
30
a
1
10
0.7
Polyester
Paper Currency
Paper Currency
Inoculum (ml) 0.01
Contact Time
(s)
Ceramic Tile
Laminate
a
15 - 32
Contact
Pressure
kg/cm2
1
15 - 32
40 - 65
240
TABLE 2 Continued
Organism Type
Species (Reference)
Gram (+) bacteria
Staphylococcus saprophyticus (8)
Relative
Humidity (%)
Drying time
(min)
N/Da
0e
Fabric
N/D
0
e
Fabric
26
26
Fomite
Fabric
Fabric
Fomite to
Hand (%)
N/D
9.8
G/F
N/D
6.2
60e
G/F
N/D
0.05
e
60
G/F
N/D
0.4
e
G/F
10
1.67c
G/F
10
0.021c
Fabric
N/D
0
Streptococcus pyogenes (9)
Fabric
N/D
0e
Staphylococcus aureus (14)
Laminate
40 - 45
0f
25.24
N/D
30
43.81
f
1h
2h
N/D
30
30.48
Laminate
40 - 45
24 hf
N/D
30
9.05
Cloth
c
30
40 - 45
Cloth
N/D - Not Determined; b G/F - Grasped firmly;
0.1; g Inoculum (ml) 3.0
40 - 45
N/D
f
Laminate
Cloth
a
Contact Time
(s)
Staphylococcus saprophyticus (9)
Laminate
Contact
Pressure
kg/cm2
G/Fb
60
60
60
0
g
N/D
30
3.6
g
N/D
30
3.86
g
1h
2h
N/D
30
2.49
g
Cloth
60
24 h
N/D
30
TNTCd
Cloth
60
48 hg
N/D
30
TNTC
Considered first stage of transfer; d TNTC - To Numerous To Count; e Inoculum (ml) 1.0; f Inoculum (ml)
241
TABLE 2 Continued
Organism Type
Species (Reference)
Gram (+) bacteria
Staphylococcus aureus (Lopez et
al., submitted for publication)
Fomite
Acrylic
Acrylic
Glass
15 - 32
30a
40 - 65
30
a
30
a
a
15 - 32
Contact
Pressure
kg/cm2
1
Contact
Time (s)
Fomite to
Hand (%)
10
3.4
1
10
47.2
1
10
20.3
1
10
45.5
40 - 65
30
Ceramic Tile
15 - 32
30a
1
10
2.7
40 - 65
30
a
1
10
54.7
a
1
10
4.3
Laminate
15 - 32
30
Laminate
40 - 65
30a
1
10
61.9
15 - 32
30
a
1
10
4
30
a
1
10
48.3
30
a
1
10
3.9
a
1
10
39.6
Stainless Steel
Stainless Steel
Granite
40 - 65
15 - 32
Granite
40 - 65
30
Cotton
15 - 32
30a
1
10
< 1.0
40 - 65
30
a
1
10
0.5
a
1
10
0.37
Cotton
Polyester
15 - 32
30
Polyester
40 - 65
30a
1
10
5
15 - 32
30
a
1
10
0.2
30
a
1
10
0.2
Paper Currency
Paper Currency
Inoculum (ml) 0.01
Drying time
(min)
Glass
Ceramic Tile
a
Relative
Humidity (%)
40 - 65
242
TABLE 2 Continued
Organism Type
Species (Reference)
Gram (+) bacteria
Staphylococcus aureus (Lopez et
al., submitted for publication)
Micrococcus luteus (6)
Relative
Humidity
(%)
15 - 32
Fomite
Ceramic Tile
30 + 10c
N/D
10
< 0.003
< 0.0002
15 - 32
N/D
Fomite to
Hand (%)
10
Granite
Faucet handle
Contact
Time (s)
1
30 + 10
a
Contact
Pressure
kg/cm2
1
c
15 - 32
1
10
0.001
1
d
N/D
30
40.03
1
d
N/D
30
41.81
e
N/D
10
0.04
Dishcloth
N/D
1
Sponge
N/D
1f
N/D
10
0.03
N/D
g
N/D
ND
0.13
f
N/D
ND
0.06
h
N/D
Natural
13.1
N/D
Natural
6.0
b
Laundry 100%
b
Laundry 50:50%
a
30 + 10c
Laminate
Phone receiver
Micrococcus luteus (10)
Drying time
(min)
N/D
1
1
Paper-towel Disp.
N/D
0
Paper-towel Disp.
N/D
0h
N/D - Not Determined; b Laundry 100% - cotton, Laundry 50% polyester: 50% cotton; c Inoculum (ml) 0.01; d Inoculum N/D; e Inoculum (ml) 50.0; f
Inoculum (ml) 100.0; g Inoculum (ml) 200.0; h Inoculum (ml) 1.0x108 CFU/hand
243
TABLE 2 Continued
Organism Type
Gram (-) bacteria
Species (Reference)
Fomite
Escherichia coli (9)
Fabric
Escherichia coli (14)
Laminate
Laminate
0e
40 - 45
0f
40 - 45
60
f
60
30
22.69
30
23.79
N/D
30
1.4
N/D
30
0.67
g
120
N/D
N/D
g
60
0.47c
25.95
24 hf
60
10
30
40 - 45
0
Fomite to
Hand (%)
N/D
Laminate
60
Contact
Time (s)
f
120
Cloth
c
N/Da
Contact
Pressure
kg/cm2
G/Fb
40 - 45
Cloth
N/D - Not Determined; b G/F - Grasped firmly;
(ml) 0.1; g Inoculum (ml) 3.0
Drying time
(min)
Laminate
Cloth
a
Relative
Humidity (%)
N/D
30
0.61
g
N/D
30
0.36
g
Cloth
60
24 h
N/D
30
TNTCd
Cloth
60
48 hg
N/D
30
TNTC
Considered first stage of transfer; d TNTC - To Numerous To Count; e Inoculum (ml) 1.0; f Inoculum
244
TABLE 2 Continued
Organism Type
Species (Reference)
Gram (-) bacteria
Escherichia coli (Lopez et al.,
submitted for publication)
Relative
Humidity (%)
Drying time
(min)
Acrylic
15 - 32
30a
Contact
Pressure
kg/cm2
1
Acrylic
40 - 65
30a
15 - 32
30
a
30
a
30
a
a
Fomite
Glass
Glass
Ceramic Tile
10
40.7
1
10
53.3
1
10
5.1
1
10
78.6
1
10
11.6
1
10
60.7
40 - 65
30
Laminate
15 - 32
30a
1
10
21.7
40 - 65
30
a
1
10
27.4
a
1
10
3.8
Stainless Steel
15 - 32
30
Stainless Steel
40 - 65
30a
1
10
54.1
15 - 32
30
a
1
10
7.3
30
a
1
10
36.5
30
a
1
10
< 6.8
a
1
10
< 13.4
Granite
Granite
Cotton
40 - 65
15 - 32
Cotton
40 - 65
30
Polyester
15 - 32
30a
1
10
< 0.37
40 - 65
30
a
1
10
< 0.7
a
1
10
< 0.05
1
10
0.1
Polyester
Inoculum (ml) 0.01
15 - 32
Fomite to
Hand (%)
Ceramic Tile
Laminate
a
40 - 65
Contact
Time (s)
Paper Currency
15 - 32
30
Paper Currency
40 - 65
30a
245
TABLE 2 Continued
Organism Type
Species (Reference)
Gram (-) bacteria
Escherichia coli (Lopez et al.,
submitted for publication)
Relative
Humidity (%)
Drying time
(min)
15 - 32
30 + 10e
15 - 32
30 + 10
e
15 - 32
30 + 10
e
Acrylic
21 - 27
25 - 32
e
Glas
23 - 34
29 - 30
Fomite
Ceramic Tile
Laminate
Granite
Escherichia coli (Lopez et al.,
submitted for publication)
Ceramic Tile
Fomite to
Hand (%)
10
< 0.01
1
10
< 0.02
1
10
< 0.004
1
13 ± 2
22.17
20 - 26e
1
13 ± 2
27.26
15 - 29
e
1
13 ± 2
13.26
e
1
13 ± 2
6.28
20 - 24
27 - 30
Stainless Steel
21 - 32
30e
f
Klebsiella aerogenes (9)
Fabric
N/D
0
Klebsiella aerogenes (14)
Cloth
60
0g
Cloth
a
Contact
Time (s)
Laminate
a
N/D - Not Determined; b G/F - Grasped firmly;
1.0; g Inoculum (ml) 3.0
c
60
Contact
Pressure
kg/cm2
1
60
g
1
13 ± 2
26.67
b
G/F
10
0.29c
N/D
30
9.8
N/D
30
6.58
g
N/D
30
4.55
Cloth
60
120
Cloth
60
24 hg
N/D
30
TNTCd
Cloth
60
48 hg
N/D
30
TNTC
Considered first stage of transfer; d TNTC - To Numerous To Count; e Inoculum (ml) 0.01; f Inoculum (ml)
246
TABLE 2 Continued
Organism Type
Gram (-) bacteria
Species (Reference)
Relative
Humidity
(%)
N/Da
Fomite
Serratia marcescens (9)
Fabric
Serratia marcescens (10)
Paper-towel Disp.
Paper-towel Disp.
Serratia rubidea (6)
Faucet handle
Phone receiver
N/D
0f
N/D
Natural
12.4
N/D
0f
N/D
Natural
6.7
N/D
1
g
N/D
30
27.59
1
g
N/D
30
38.47
h
N/D
10
< 0.01
N/D
Fomite to
Hand (%)
10
0.46c
N/D
1
Sponge
N/D
1i
N/D
10
< 0.01
N/D
1
j
N/D
N/D
< 0.01
1
i
N/D
N/D
< 0.01
e
G/F
10
0.36
N/D
N/D
10.08
N/D
30
13.05
Laundry 100%
d
Laundry 50:50%
N/D
Psuedomonas aeruginosa (9)
Fabric
N/D
0
Enterobacter aerogenes (11)
Spigot
N/D
0k
Salmonella spp.Wild type strains
(14)
Contact
Time (s)
Dishcloth
d
a
0e
Contact
Pressure
kg/cm2
G/Fb
Drying time
(min)
Laminate
Laminate
40 - 45
40 - 45
0
l
60
l
N/D
30
17.15
l
N/D
30
1.17
N/D
30
0.54
Laminate
40 - 45
120
Laminate
40 - 45
24 hl
N/D - Not Determined; b G/F - Grasped firmly; c Considered first stage of transfer; d Laundry 100% - cotton, Laundry 50% polyester: 50% cotton; e
Inoculum (ml) 1.0; f Inoculum 1.0x108 CFU/hand; g Inoculum N/D; h Inoculum (ml) 50.0; i Inoculum (ml) 100.0; j Inoculum (ml) 200.0; k CrossContamination; l Inoculum (ml) 0.1
247
TABLE 2 Continued
Organism Type
Species (Reference)
Endospores
Bacillus thuringiensis (Lopez et
al., submitted for publication)
Relative
Humidity (%)
Drying time
(min)
15 - 32
30a
Acrylic
40 - 65
30
a
Glass
15 - 32
40 - 65
Fomite
Acrylic
Glass
Ceramic Tile
Fomite to
Hand (%)
10
57.0
1
10
65.6
30a
1
10
< 0.5
30
a
1
10
33.8
30
a
1
10
< 0.2
a
1
10
21.2
40 - 65
30
Laminate
15 - 32
30a
1
10
0.2
40 - 65
30
a
1
10
53.5
30
a
1
10
< 0.5
a
1
10
57.0
Stainless Steel
15 - 32
Stainless Steel
40 - 65
30
Granite
15 - 32
30a
1
10
0.04
40 - 65
30
a
1
10
12.8
30
a
1
10
< 0.6
a
1
10
< 3.5
Granite
Cotton
15 - 32
Cotton
40 - 65
30
Polyester
15 - 32
30a
1
10
< 0.6
40 - 65
30
a
1
10
< 4.6
30
a
1
10
< 0.1
30
a
1
10
< 0.1
Polyester
Paper Currency
Paper Currency
Inoculum (ml) 0.01
Contact
Time (s)
Ceramic Tile
Laminate
a
15 - 32
Contact
Pressure
kg/cm2
1
15 - 32
40 - 65
248
TABLE 2 Continued
Organism Type
Endospores
Species (Reference)
Bacillus thuringiensis (Lopez
et al., submitted for
publication)
Fomite
Ceramic Tile
Laminate
Granite
a
Inoculum (ml) 0.01
Relative
Humidity (%)
Drying time
(min)
15 - 32
30 + 10a
15 - 32
30 + 10
a
30 + 10
a
15 - 32
Contact
Pressure
kg/cm2
1
Contact
Time (s)
Fomite to
Hand (%)
10
< 0.04
1
10
< 0.03
1
10
0.1
249
TABLE 3 Hand-to-Hand Transfer Efficiency (%)
Organism Type
Respiratory
Viruses
Species (reference)
Rhinovirus (1)
Rhinovirus (2)
Rhinovirus 14 (3)
Enteric Viruses
Fingerb
Relative
Humidity
(%)
N/Da
Fingerb
N/D
Fomite
Finger-skin
Finger
F/R
N/D
g
d
Hand to
Hand (%)
15e
17
15e
34
G/R
N/D
5.9
20
h
1
5
0.71
h
1
5
Undetected
Finger
50 ± 5
20
Rotavirus (4)
Finger
50
20h
1
10
6.6
50
60
h
1
10
2.8
20
h
0.2
10
23.5
h
0.2
10
23.5
Hepatitis A Virus (5)
Fingerpad
45 ± 5
60
Fingerpad
45 ± 5
120h
0.2
10
16.9
45 ± 5
180
h
0.2
10
5.1
240
h
Fingerpad
ΦX174 (13)
45 ± 5
Fingerpad
Fingerpad
45 ± 5
0.2
10
0.9
i, j
N/D
N/D
0.004
Finger tip skin
N/D
15
Finger tip skin
N/D
15i, k
N/D
N/D
0.03
N/D
i, l
N/D
N/D
16.7
Finger tip skin
a
50 ± 5
Dampf
Contact
Time (s)
Human Parainfluenza virus-3 (3)
Finger
Coliphage
N/D
Dryf
Contact
Pressure
kg/cm2
F/Rc
Drying time
(min)
15
N/D - Not determined; b Finger, back of hand skin; c F/R - Firmly rubbing table top or fingers; d Gently rubbing; e Contact with table top 15 s and 3 min with
plastic pen and stainless steel spoon; f Inoculum (ml) ≤ 0.005; g Inoculum (ml) 0.05; h Inoculum (ml) 0.01; i Inoculum (ml) 0.1; j Horizontal transmission with
14 other fingers; k Vertical transmission finger-to-finger-to-finger; l Hand washing with bar soap then touching fingers
250
TABLE 4 Hand-to-Fomite-to-Hand Transfer Efficiency (%)
Organism Type
Respiratory Virus
Gram (-) bacteria
a
Relative
Humidity (%)
Drying time
(min)
N/Da
10c, d
Faucet handle
N/D
10
c, d
Brass Door Knob
N/D
10c, e
Species (reference)
Rhinovirus (2)
Escherichia coli (15)
Fomite
Brass Door Knob
Paper
N/D
f
Air-dry
Contact
Pressure
kg/cm2
S/B/Fb
N/D
Hand to
Fomite to
Hand (%)
11.2
S/B/F
N/D
5.63
S/B/F
N/D
12.6
N/D
30
0.009
Contact
Time (s)
N/D - Not determined; b S/B/F - Sliding back and forth; e 10 min drying on the fomite before recipeint handles fomite; d Inoculum (ml) 0.05; e Inoculum
(ml) 0.1; f Inoculum (ml) 0.025
251
TABLE 5 Fomite-to-Hand-to-Fomite Transfer Efficiency (%)
G/F
N/D
4.29d
15j
G/F
N/D
1.57d
N/D
0j
G/F
N/D
1.62e
Fabric
N/D
15j
G/F
N/D
4.58e
Fabric
N/D
0j
G/F
N/D
0.01f
Fabric
N/D
15j
G/F
N/D
0.0f
Fabric
N/D
0j
G/F
N/D
31g
Fabric
N/D
0j
G/F
N/D
21h
Staphylococcus saprophyticus (9)
Fabric
N/D
0j
G/F
10
0.37i
Streptococcus pyogenes (9)
Fabric
N/D
0j
G/F
10
0.01i
Escherichia coli (28)
Fabric
N/D
0j
G/F
10
0.018i
Klebsiella aerogenes (28)
Fabric
N/D
0j
G/F
10
0.076i
Serratia marcescens (28)
Fabric
N/D
0j
G/F
10
0.11i
Psuedomonas aeruginosa (28)
Fabric
N/D
0j
G/F
10
0.14i
Species (reference)
Gram (+) bacteria
Staphylococcus saprophyticus (8)
Gran (-) bacteria
a
N/D
Fomite to
Hand to
Fomite (%)
0.04c
Organism Type
Relative
Humidity (%)
Drying time
(min)
N/Da
0j
Fabric
N/D
0
j
Fabric
N/D
Fabric
Fomite
Fabric
Contact
Pressure
kg/cm2
G/Fb
Contact Time
(s)
N/D - Not Determined; b G/F - Grasped firmly; c Average transfer efficiency from multiple handling methods of the recipient fabric; d Hand washing with
detergent; e Hand washing with bar soap; f Hand washing with70% ethanol; g Rubbing procedure using alcohol-impregnated wipes; h Rubbing procedure
using 0.2 ml 80% ethanol; i Considered a complete transfer; j Inoculum (ml) 1.0
252
REFERENCES
1.
Reed SE. 1975. An Investigation of the Possible Transmission of Rhinovirus Colds
through Indirect Contact. J. Hyg. 75:249-258.
2.
Pancic F, Carpentier DC, Came PE. 1980. Role of Infectious Secretions in the
Transmission of Rhinovirus. J. Clin. Microbiol. 12:567-571.
3.
Ansari SA, Springthorpe VS, Sattar SA, Rivard S, Rahman M. 1991. Potential role of
hands in the spread of respiratory viral infections: studies with human parainfluenza virus
3 and rhinovirus 14. J. Clin. Microbiol. 29:2115-2119.
4.
Ansari SA, Sattar SA, Springthorpe VS, Wells GA, Tostowaryk W. 1988. Rotavirus
survival on human hands and transfer of infectious virus to animate and nonporous
inanimate surfaces. J. Clin. Microbiol. 26:1513-1518.
5.
Mbithi JN, Springthorpe VS, Boulet JR, Sattar SA. 1992. Survival of hepatitis A virus
on human hands and its transfer on contact with animate and inanimate surfaces. J. Clin.
Microbiol. 30:757-763.
6.
Rusin P, Maxwell S, Gerba C. 2002. Comparative surface-to-hand and fingertip-tomouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage. J.
Appl. Microbiol. 93:585-592.
7.
Julian TR, Leckie JO, Boehm AB. 2010. Virus transfer between fingerpads and fomites.
J. Appl. Microbiol. 109:1868-1874.
8.
Marples RR, Towers AG. 1979. A Laboratory Model for the Investigation of Contact
Transfer of Micro-Organisms. J. Hyg. 82:237-248.
253
9.
MacKintosh CA, Hoffman PN. 1984. An Extended Model for Transfer of MicroOrganisms via the Hands: Differences between Organisms and the Effect of Alcohol
Disinfection. J. Hyg. 92:345-355.
10.
Harrison WA, Griffith CJ, Ayers T, Michaels B. 2003. Bacterial transfer and crosscontamination potential associated with paper-towel dispensing. Am. J. Infect. Control
31:387-391.
11.
Chen Y, Jackson KM, Chea FP, Schaffner DW. 2001. Quantification and variability
analysis of bacterial cross-contamination rates in common food service tasks. J. Food
Prot. 64:72-80.
12.
Paulson DS. 2005. The Transmission of Surrogate Norwalk Virus - From Inanimate
Surfaces to Gloved Hands: Is It a Threat? Food Prot. Trends 25:450-454.
13.
Rheinbaben Fv, Schunemann S, Grob T, Wolff MH. 2000. Transmission of viruses
via contact in ahousehold setting: experiments using bacteriophage 0X174 as a model
virus. J. Hosp. Infect. 46:61-66.
14.
Scott E, Bloomfield SF. 1990. The survival and transfer of microbial contamination via
cloths, hands and utensils. J. Appl. Microbiol. 68:271-278.
15.
Hubner N-O, Hubner C, Kramer A, Assadian O. 2011. Survival of Bacterial
Pathogens on Paper and Bacterial Retrieval from Paper to hands: Preliminary Results.
Am. J. Nurs. 111:30-34.
254
APPENDIX H: SUPPLEMENTAL MATERIALS
Comparison of two approaches in determining transfer efficiency of Escherichia coli
from nonporous fomites to fingers
Running title: Determining Bacterial Percent Transfer
Gerardo U. Lopez1, Masaaki Kitajima1, Kelly A. Reynolds2 and Charles P. Gerba1, #
1
Department of Soil, Water and Environmental Science, The University of Arizona,
Tucson, Arizona 85721 USA; 2The University of Arizona, Mel and Enid Zuckerman
College of Public Health, 1295 N. Martin Ave., Tucson, Arizona 85724, USA
#
Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail:
[email protected]
A manuscript for
Applied and Environmental Microbiology
255
ADDTIONAL DETAILS TO METHODS AND MATERIALS
Figure S1 shows a diagram of the experimental design of the method used in assessing
the fomite-to-finger transfer efficiency. The donor fomite was placed on the scale as
previously described (1, 2). After the transfer event the donor fomite and the finger were
swabbed and assayed. The baseline control fomite was also swabbed and assayed.
Approach 1 used E. coli recovered on the finger relative to E. coli recovered from
baseline control fomite, where as approach 2 used E. coli recovered on the finger relative
to E. coli recovered on the donor fomite.
ADDITIONAL DETAILS TO RESULTS
Table S1 shows the log10 CFU/2-cm2 of E. coli recovered from fingers after the transfer
event with the donor fomite (Fig. S1) and E. coli recovered from the baseline control
fomite (Fig. S1). E. coli recovered from fomites are based on 2-cm2 area as used by Nicas
and Best (3) in their modeling of quantifying hand-to-face contact rate. The inoculum
was spread with the tip of a pipet over a 1.0 cm2 area and dried on laboratory bench for
30 min. The index, middle, and ring fingers each had an average 2.0 cm2 surface area that
covered the entire seeded inoculum.
Figure S2 provides the transfer efficiency in log10 scale and geometric mean. Transfer
efficiencies calculated by both approaches show considerable variability that has been
observed in previous studies (2, 4, 5).
256
TABLE
TABLE S1: E. coli recovered from fomites and fingers
Approach 1
Approach 2
Recovered from finger
Recovered from baseline control fomite
Recovered from donor fomite
Mean CFU/2-cm2 log10 ± SD (Range)
Mean CFU/2-cm2 log10 ± SD (Range)
Mean CFU/2-cm2 log10 ± SD (Range)
Acrylic
3.4 ± 0.5 (2.5 - 4.4)
4.3 ± 0.5 (3.5 - 4.7)
4.2 ± 0.6 (3.2 - 5.2)
Glass
4.0 ± 0.4 (3.4 - 4.8)
4.8 ± 0.5 (4.2 - 5.3)
4.8 ± 0.6 (< 3.7 - 5.6)
Ceramic Tile
3.9 ± 0.6 (3.0 - 4.9)
5.3 ± 0.7 (4.3 - 6.2)
5.2 ± 0.5 (4.4 - 5.9)
Laminate
4.1 ± 0.3 (3.7 - 4.6)
5.5 ± 0.1 (5.3 - 5.6)
5.7 ± 0.2 (5.3 - 5.9)
Stainless Steel
4.5 ± 0.7 (3.2 - 5.2)
5.6 ± 0.3 (5.1 - 5.9)
5.5 ± 0.2 (5.3 - 5.7)
Surface Type
a
E. coli recovered from fomite were below the detectible limit of 10 CFU/2-cm2 indicated by <
257
FIGURES
FIG S1 Diagram showing experimental design.
258
A
B
100
% Transfer efficiency
% Transfer efficiency
100
10
1
0.1
Acrylic
Geometric Mean
10
1
0.1
Glass
Ceramic Tile
Laminate
Stainless Steel
Acrylic
Geometric Mean
Glass
Ceramic Tile
Laminate
Stainless Steel
FIG S2 Transfer efficiency log scale (%) of E. coli determined with (A) approach 1 and (B) approach 2.
259
REFERENCES
1.
Ansari SA, Sattar SA, Springthorpe VS, Wells GA, Tostowaryk W. 1988.
Rotavirus survival on human hands and transfer of infectious virus to animate and
nonporous inanimate surfaces. J. Clin. Microbiol. 26:1513-1518.
2.
Mbithi JN, Springthorpe VS, Boulet JR, Sattar SA. 1992. Survival of hepatitis
A virus on human hands and its transfer on contact with animate and inanimate
surfaces. J. Clin. Microbiol. 30:757-763.
3.
Nicas M, Best D. 2008. A Study Quantifying the Hand-to-Face Contact Rate and
Its Potential Application to Predicting Respiratory Tract Infection. J. Occup.
Environ. Hyg. 5:347-352.
4.
Chen Y, Jackson KM, Chea FP, Schaffner DW. 2001. Quantification and
variability analysis of bacterial cross-contamination rates in common food service
tasks. J. Food Prot. 64:72-80.
5.
Paulson DS. 2005. The Transmission of Surrogate Norwalk Virus - From
Inanimate Surfaces to Gloved Hands: Is It a Threat? Food Prot. Trends 25:450454.
260
APPENDIX I: SUPPLEMENTAL MATERIALS
Transfer efficiency of bacteria and viruses from porous and nonporous fomites to
fingers under different relative humidity
Running title: Fomite to Finger Microbial Transfer
Gerardo U. Lopez1, 3, Charles P. Gerba1, 3#, Akrum Tamimi1, Masaaki Kitajima1, Sheri
Maxwell1, and Joan B. Rose2, 3
1
Department of Soil, Water and Environmental Science, The University of Arizona,
Tucson, Arizona 85721 USA; 2Department of Fisheries and Wildlife, and 3Center for
Advancing Microbial Risk Assessment, Michigan State University, East Lansing,
Michigan
#
Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail:
[email protected]
Manuscript for
Applied and Environmental Microbiology
Supplemental Material Not Included in Publication
261
TABLES
TABLE S1 Recovered organisms from fingers under low relative humidity 15% - 32%
E. coli
Surface Type
S. aureus
2
B. thuringiensis
2
MS-2
2
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm2 log10 ±
SD (Range)
Acrylic
(6) 4.2 ± 0.5 (3.7 - 5.1)
(6) 4.6 ± 0.3 (4.3 - 5.0)
(6) 4.5 ± 0.04 (4.5 - 4.6)
(6) 7.7 ± 0.4 (7.1 - 8.1)
Glass
(6) 3.4 ± 0.3 (2.9 - 3.8)
(6) 4.9 ± 0.7 (4.2 - 6.0)
(6) < 2.1 ± 0.2 (< 2.0 - 2.4)
(6) 7.7 ± 0.3 (7.0 - 7.9)
Ceramic Tile
(6) 3.6 ± 0.7 (2.4 - 4.1)
(6) 4.5 ± 0.3 (4.1 - 4.9)
(6) < 2.1 ± 0.2 (< 2.0 - 2.5)
(6) 7.6 ± 0.2 (7.4 - 8.0)
Laminate
(6) 3.5 ± 0.4 (2.8 - 3.9)
(6) 4.7 ± 0.3 (4.2 - 5.0)
(6) 2.1 ± 0.2 (< 2.0 - 2.4)
(6) 7.5 ± 0.3 (7.0 - 7.7)
Stainless Steel
(6) 3.0 ± 0.7 (2.0 - 4.0)
(6) 4.5 ± 0.4 (3.9 - 5.2)
(6) < 2.0 ± 0.1 (< 2.0 - 2.2)
(6) 5.8 ± 1.2 (4.6 - 7.5)
(6) 4.5 ± 0.3 (4.1 - 5.0)
(6) 1.2 ± 0.3 (< 1.0 - 1.6)
(6) 7.1 ± 0.2 (6.9 - 7.3)
(6) < 1.1 ± 0.1 (< 1.3)a
(6) < 1.0 ± (< 1.0)a
(6) 3.3 ± 0.2 (3.1 - 3.6)
Nonporous
Granite
(6) 2.9 ± 0.7 (< 2.2 - 3.7)
a
Porous
Cotton
Polyester
Paper Currency
a
(6) < 1.0 ± (< 1.0)a
(6) < 1.0 ± 0.1 (< 1.2)
a
(6) < 1.1 ± 0.3 (< 1.0 - 1.7)
(6) 1.4 ± 0.3 (1.0 - 1.9)
a
(6) 3.3 ± 0.4 (2.8 - 4.0)
a
(6) < 1.0 ± (< 1.0)
a
(6) < 1.2 ± 0.3 (< 1.0 - 1.8)
(6) 4.5 ± 0.5 (3.9 - 5.1)
a
(6) 4.7 ± 0.3 (4.5 - 5.3)
Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 indicated by <
262
TABLE S2 Recovered organisms from fingers under high relative humidity 40% - 65%
E. coli
Surface Type
S. aureus
2
B. thuringiensis
2
MS-2
2
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm log10 ±
SD (Range)
(n) Mean CFU/2-cm2 log10 ±
SD (Range)
Acrylic
(6) 6.4 ± 0.2 (6.2 - 6.7)
(6) 6.4 ± 0.2 (6.1 - 6.6)
(6) 4.6 ± 0.1 (4.5 - 4.7)
(6) 7.0 ± 0.3 (6.6 - 7.3)
Glass
(6) 5.0 ± 0.5 (4.2 - 5.6)
(6) 6.5 ± 0.1 (6.3 - 6.7)
(6) 4.3 ± 0.4 (< 3.7 - 4.8)
(6) 8.2 ± 0.1 (8.0 - 8.3)
Ceramic Tile
(6) 5.4 ± 0.4 (4.8 - 5.8)
(6) 6.3 ± 0.2 (6.0 - 6.5)
(6) 3.7 ± 0.6 (< 3.0 - 4.4)
(6) 8.1 ± 0.2 (7.8 - 8.3)
Laminate
(6) 5.8 ± 0.8 (5.0 - 6.6)
(6) 6.5 ± 0.3 (6.1 - 6.8)
(6) 4.5 ± 0.2 (4.1 - 4.7)
(6) 8.4 ± 0.1 (8.2 - 8.5)
Stainless Steel
(6) 5.6 ± 0.3 (5.3 - 6.1)
(6) 6.5 ± 0.3 (6.0 - 6.8)
(6) 4.7 ± 0.2 (4.5 - 5.0)
(6) 8.4 ± 0.2 (8.1 - 8.5)
Granite
(6) 3.6 ± 1.1 (2.3 - 5.0)
(6) 5.6 ± 0.9 (4.2 - 6.6)
(6) 2.7 ± 1.3 (1.7 - 4.4)
(6) 7.6 ± 0.6 (6.8 - 8.3)
Cotton
(6) < 1.0 ± (< 1.0)a
(6) 1.2 ± 0.3 (< 1.0 - 1.9)a
(6) < 1.0 ± (< 1.0)a
(6) 3.5 ± 0.5 (3.0 - 4.2)
Polyester
(6) < 1.1 ± 0.1 (< 1.2)a
(6) 3.0 ± 1.0 (1.9 - 4.0)
(6) < 1.3 ± 0.5 (< 2.0)a
Nonporous
Porous
Paper Currency
a
(6) < 1.6 ± 0.8 (< 1.0 - 3.2)
a
(6) 3.4 ± 0.3 (2.9 - 3.8)
(6) < 1.5 ± 0.5 (< 1.0 - 2.0)
(6) 5.4 ± 0.2 (5.2 - 5.7)
a
(6) 4.6 ± 0.4 (4.2 - 5.2)
Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 indicated by <
263
TABLE S3 Recovered poliovirus 1 from fingers
Surface Type
(n) Mean CFU/2-cm2 log10 ± SD
(Range)
Low RH (15% - 32%)
Ceramic Tile
(6) 4.4 ± 0.6 (3.4 - 5.0)
Laminate
(6) 5.3 ± 0.2 (5.0 - 5.6)
Granite
(6) 4.1 ± 1.3 (2.5 - 5.5)
High RH (40% - 65%)
Ceramic Tile
(6) 5.7 ± 0.04 (5.6 - 5.7)
Laminate
(6) 5.8 ± 0.2 (5.6 - 6.2)
Granite
(6) 5.6 ± 0.1 (5.4 - 5.7)
264
FIGURES
B
8.0
Recovered from fingers log10 CFU/2-cm2
Recovered from fingers log10 CFU/2-cm2
A
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
LRH
HRH
Acrylic
Geometric Mean
LRH
HRH
Glass
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Stainless Steel
LRH
6.8
5.8
4.8
3.8
2.8
1.8
0.8
-0.2
HRH
LRH
Granite
C
HRH
Acrylic
Geometric Mean
LRH
HRH
Glass
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Stainless Steel
LRH
HRH
Granite
D
6.0
Recovered from fingers log10 PFU/2-cm2
Recovered from fingers log10 CFU/2-cm2
7.8
5.0
4.0
3.0
2.0
1.0
0.0
LRH
HRH
Acrylic
Geometric Mean
LRH
HRH
Glass
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Stainless Steel
LRH
HRH
Granite
11.0
9.0
7.0
5.0
3.0
1.0
-1.0
LRH
HRH
Acrylic
Geometric Mean
LRH
HRH
Glass
LRH
HRH
Ceramic Tile
LRH
HRH
Laminate
LRH
HRH
Stainless Steel
LRH
HRH
Granite
FIG S1 Organisms recovered from fingers after contact with nonporous fomites (A) E. coli 15597, (B) S. aureus 25923, (C) B.
thuringiensis spores, and (D) MS2 under low (15% - 32%) and high (40% - 65%) relative humidity.
265
B
3.5
Recovered from fingers log10 CFU/2-cm2
Recovered from fingers log10 CFU/2-cm2
A
3.0
2.5
2.0
1.5
1.0
0.5
0.0
LRH
HRH
LRH
Co on
Geometric Mean
HRH
Polyester
LRH
HRH
2.5
2.0
1.5
1.0
0.5
0.0
HRH
LRH
Co on
Geometric Mean
HRH
Polyester
LRH
HRH
Paper Currency
D
Recovered from fingers log10 PFU/2-cm2
2.5
Recovered from fingers log10 CFU/2-cm2
3.5
3.0
LRH
Paper Currency
C
4.5
4.0
2.0
1.5
1.0
0.5
0.0
LRH
Co on
Geometric Mean
HRH
LRH
HRH
Polyester
LRH
HRH
Paper Currency
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
-1.0
LRH
Co on
Geometric Mean
HRH
LRH
HRH
Polyester
LRH
HRH
Paper Currency
FIG S2 Organisms recovered from fingers after contact with porous fomites (A) E. coli 15597, (B) S. aureus 25923, (C) B.
thuringiensis spores, and (D) MS2 under low (15% - 32%) and high (40% - 65%) relative humidity.
Recovered from fingers log10 PFU/2-cm2
266
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
LRH
HRH
Ceramic Tile
Geometric Mean
LRH
HRH
Laminate
LRH
HRH
Granite
FIG 3 Poliovirus 1 recovered from fingers after contact with nonporous fomites under low
(15% - 32%) and high (40% - 65%) relative humidity.
267
APPENDIX J: SUPPLEMENTAL MATERIALS
The Effect of a Disinfectant Wipe on Microbial Transfer
Gerardo U. Lopez1, Masaaki Kitajima1, Aaron Havas1, and Kelly A. Reynolds2
1
Department of Soil, Water and Environmental Science, The University of Arizona,
Tucson,
Arizona 85721, USA
2
Mel and Enid Zuckerman College of Public Health, The University of Arizona, 1295 N.
Martin Ave., Tucson, Arizona 85724, USA
Corresponding author. Tel: (520) 626-8230, Fax (520) 626-8009, E-mail:
[email protected]
A manuscript for
International Journal of Hygiene and Environmental Health
Supplemental Material Not Included in Publication
268
ADDITIONAL DETAILS TO METHODS AND MATERIALS
Calculation of percent reduction
Percent reduction was calculated using Eqn (1) and is defined as the CFU or PFU
recovered from control fomite without intervention minus CFU or PFU recovered from
control fomite with intervention relative to CFU or PFU recovered from control fomite
without intervention.
 %  
  / . –   / .
  / .
  100
(1)
Log10 organism removal from fomite surface was calculated using Eqn (2) by subtracting
the mean Log10 CFU or PFU recovered from the control fomite with intervention from
the Log10 CFU or PFU inoculated onto the fomite factoring the 10 ul inoculum volume .
     
 

     / .  (2)
ADDITIONAL DETAILS TO RESULTS
Removal of microbial contamination from nonporous surfaces
Percent reduction from ceramic tile, laminate, and granite for E. coli (> 99.8%, > 99.97%,
99.9%), S. aureus (99.9%, 99.997%, 99.98%), B. thuringiensis (99.4%, 99.4%, 98.1%),
and Poliovirus (99.98%, 99.8%, 99.9%) respectively (Tables S1 and S2). Calculated
using Eqn 1.
269
The removal from ceramic tile, laminate, and granite for E. coli was (> 5.8, > 6.2, 5.1
log10 CFU/2 cm2), S. aureus (4.9, 5.3, 4.7 log10 CFU/2 cm2), B. thuringiensis (2.5, 3.1,
3.8 log10 CFU/2 cm2), and for Poliovirus was (5.0, 4.9, 4.9 log10 PFU/2 cm2) respectively
(Tables S1 and S2). Calculated using Eqn 2.
Figure S1 shows recovery of E. coli, S. aureus, B. thuringiensis spores, and poliovirus 1
from control non-treated and intervention treated fomites. Figure S2 shows fomite-tofinger transfer efficiency % of E. coli, S. aureus, B. thuringiensis spores, and poliovirus 1
from control non-treated (Lopez et al., submitted for publication) and intervention treated
fomites. Figure S3 shows recovery of E. coli, S. aureus, B. thuringiensis spores, and
poliovirus 1 from fingers in contact with non-treated (Lopez et al., submitted for
publication) and intervention treated fomites.
270
TABLES
Table S1. Recovered microorganisms from fomitesa
E. coli
S. aureus
Mean CFU/2-cm2 log10 ± SD (Range)
Mean CFU/2-cm2 log10 ± SD (Range)
% Reduction
> 99.8 ± 0.3 (> 99.5 - > 99.997)
99.9 ± 0.01 (99.98 - 99.9995)
Organism Removal
> 5.8 ± 0.9 (> 4.4 - > 6.6)
4.9 ± 0.6 (4.2 - 5.7)
% Reduction
> 99.97 ± 0.02 (> 99.9 - > 99.9998)
99.997 ± 0.004 (99.99 - 99.9999)
Organism Removal
> 6.2 ± 0.4 (> 5.7 - > 6.6)
5.3 ± 0.8 (4.4 - > 6.3)
% Reduction
99.9 ± 0.2 (99.6 - > 99.995)
99.98 ± 0.03 (99.9 - > 99.9998)
Organism Removal
5.1 ± 0.9 (3.9 - > 5.8)
4.7 ± 1.2 (3.4 - > 6.2)
Surface Type
Ceramic Tile
Laminate
Granite
a
There were six replicates for each organism
271
Table S2. Recovered microorganisms from fomitesa
B. thuringiensis
Poliovirus Type 1
Mean CFU/2-cm2 log10 ± SD (Range)
Mean CFU/2-cm2 log10 ± SD (Range)
% Reduction
99.4 ± 0.4 (91.8 - 99.7)
99.98 ± 0.01 (99.9 - > 99.999)
Organism Removal
2.5 ± 0.3 (2.0 - 2.8)
5.0 ± 0.4 (4.5 - 5.7)
% Reduction
99.4 ± 0.5 (98.7 - > 99.98)
99.97 ± 0.02 (99.9 - > 99.998)
Organism Removal
3.1 ± 0.5 (2.6 - > 4.0)
4.9 ± 0.5 (4.2 - 5.7)
% Reduction
98.1 ± 1.7 (95.3 - > 99.98)
99.94 ± 0.1 (99.8 - > 99.997)
Organism Removal
3.8 ± 1.3 (2.5 - > 6.0)
4.9 ± 0.8 (3.9 - 5.7)
Surface Type
Ceramic Tile
Laminate
Granite
a
There were six replicates for each organism
272
FIGURES
A
B
log10 Reduction
3.6
3.9
log10 Reduction
3.5
Recovery of bacteria CFU /2-cm 2
Recovery of bacteria CFU/2-cm2
4.5
5.0
Ceramic Tile
Laminate
4.4
8
8
7
6
5
4
3
2
1
7
6
5
4
3
2
1
0
0
Ceramic Tile
Control non-treatment log10 CFU/2-cm2
Laminate
Granite
Surfaces
Interven on treatment log10 CFU/2-cm2
C
Control non-treatment log10 CFU/2-cm2
Granite
Surfaces
Interven on treatment log10 CFU/2-cm2
D
1.9
2.5
2.1
8
log10 Reduction
Recovery of virus PFU/2cm 2
Recovery of spores CFU/2cm2
log10 Reduction
7
6
5
4
3
2
3.9
3.8
3.7
8
7
6
5
4
3
2
1
1
0
0
Ceramic Tile
Laminate
Granite
Ceramic Tile
Control non-treatment log10 CFU/2cm2
Interven on treatment log10 CFU/2cm2
Laminate
Granite
surfaces
surfaces
Control non-treatment log10 PFU/2-cm2
Interven on treatment log10 PFU/2-cm2
Fig. S1. Recovery of (A) E. coli, (B) S. aureus, (C) B. thuringiensis spores, and (D) poliovirus 1 from control non-treated and
intervention treated fomites
273
A
B
Geometric Mean
100
100
10
10
% Transfer Efficiency
% Transfer Efficiency
Geometric Mean
1
0.1
0.01
1
0.1
0.01
0.001
0.001
0.0001
0.0001
Ceramic Tile
Laminate
Granite
Ceramic Tile
Without Interven on
Laminate
Granite
Ceramic Tile
With Interven on
Laminate
Granite
Ceramic Tile
Without Interven on
C
Laminate
D
Geometric Mean
Geometric Mean
100
100
10
10
% Transfer Efficiency
% Transfer Efficiency
Granite
With Interven on
1
0.1
0.01
0.001
1
0.1
0.01
0.001
0.0001
0.0001
Ceramic Tile
Laminate
Without Interven on
Granite
Ceramic Tile
Laminate
With Interven on
Granite
Ceramic Tile
Laminate
Granite
Ceramic Tile
Without Interven on
Laminate
Granite
With Interven on
Fig. S2. Transfer efficiency % of (A) E. coli, (B) S. aureus, (C) B. thuringiensis spores, and poliovirus 1 from control nontreated (Lopez et al., submitted for publication) and intervention treated fomites
274
A
B
Geometric Mean
Recovery of S. aureus log10 CFU/2-cm 2
Recovery of E. coli log 10 CFU/2-cm 2
Geometric Mean
6
5
4
3
2
1
0
Ceramic Tile
Laminate
Granite
Ceramic Tile
Without Interven on
Laminate
5
4
3
2
1
0
Ceramic Tile
Granite
Laminate
Granite
Ceramic Tile
Without Interven on
With Interven on
C
Laminate
Granite
With Interven on
D
Geometric Mean
Geometric Mean
6
6
Recovery of virus log 10 PFU/2-cm 2
Recovery of spores log10 CFU/2-cm 2
6
5
4
3
2
1
0
Ceramic Tile
Laminate
Without Interven on
Granite
Ceramic Tile
Laminate
With Interven on
Granite
5
4
3
2
1
0
Ceramic Tile
Laminate
Granite
Ceramic Tile
Without Interven on
Laminate
Granite
With Interven on
Fig. S3. Recovery of (A) E. coli, (B) S. aureus, (C) B. thuringiensis spores, and poliovirus 1 from fingers in contact with nontreated (Lopez et al., submitted for publication) and intervention treated fomites.
275
APPENDIX K: SUPPLEMENTAL MATERIALS
Risk of Campylobacter jejuni Infection From in Preparing Raw Chicken in Domestic
Kitchens and Cross-contamination Reduction From Disinfectant Wipes
Gerardo U. Lopez1, 2, Masaaki Kitajima1, Jonathan Sexton1, 2, Charles P. Gerba1, and
Kelly A. Reynolds2*
1
Department of Soil, Water and Environmental Science, University of Arizona, Tucson,
Arizona 85721, USA
2
The University of Arizona, Mel and Enid Zuckerman College of Public Health, 1295 N.
Martin Ave., Tucson, AZ 85724, USA
*
Corresponding author. Tel: (520) 626-8230, Fax (520) 626-8009, E-mail:
[email protected]
A manuscript for
Risk Analysis
Supplemental Material Not Included in Publication
276
TABLES
Table SI Risk of Infection (/person/event): Condition A
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition A
5.60E-03
Standard Deviation
2.62E-02
Treatment
1.50E-05
5.76E-04
Non-treatment
8.00E-03
3.30E-02
Treatment
6.60E-05
1.03E-03
Non-treatment
6.00E-03
2.70E-02
Treatment
6.00E-06
7.40E-05
Table SII Risk of Illness (/person/event): Condition A
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition A
4.30E-05
Standard Deviation
2.03E-04
Treatment
7.30E-08
6.74E-07
Non-treatment
6.00E-05
2.42E-04
Treatment
4.01E-07
5.13E-06
Non-treatment
4.80E-05
1.99E-04
Treatment
4.50E-08
5.65E-07
277
Table SIII Risk of Death (/person/event): Condition A
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition A
2.20E-09
Standard Deviation
1.02E-08
Treatment
5.20E-12
1.22E-10
Non-treatment
3.10E-09
1.22E-08
Treatment
2.10E-11
3.49E-10
Non-treatment
2.40E-09
1.04E-08
Treatment
2.20E-12
2.49E-11
Table SIV Annual Risk of Infection (/person/year): Condition A
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition A
1.26E-01
Standard Deviation
2.55E-01
Treatment
1.00E-03
1.20E-02
Non-treatment
1.67E-01
2.96E-01
Treatment
4.00E-03
3.10E-02
Non-treatment
1.51E-01
2.78E-01
Treatment
1.00E-03
1.20E-02
278
Table SV Annual Risk of Illness (/person/year): Condition A
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition A
3.80E-03
Standard Deviation
1.68E-02
Treatment
7.90E-06
1.11E-04
Non-treatment
5.50E-03
2.09E-02
Treatment
4.90E-05
1.01E-03
Non-treatment
4.50E-03
1.84E-02
Treatment
7.90E-06
5.29E-04
Table SVI Annual Risk of Death (/person/year): Condition A
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition A
1.90E-07
Standard Deviation
9.00E-07
Treatment
3.90E-10
4.23E-09
Non-treatment
2.90E-07
1.17E-06
Treatment
2.30E-09
4.12E-08
Non-treatment
2.40E-07
9.90E-07
Treatment
2.20E-10
3.23E-09
279
Table SVII Risk of Infection (/person/event): Condition B
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition B
1.23E-02
Standard Deviation
4.11E-02
Treatment
4.60E-05
5.88E-04
Non-treatment
1.80E-02
5.20E-02
Treatment
2.17E-04
2.33E-03
Non-treatment
1.50E-02
4.60E-02
Treatment
2.70E-05
6.39E-04
Table SVIII Risk of Illness (/person/event): Condition B
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition B
1.03E-04
Standard Deviation
3.46E-04
Treatment
3.52E-07
4.92E-06
Non-treatment
1.39E-04
4.12E-04
Treatment
1.54E-06
1.44E-05
Non-treatment
1.21E-04
3.63E-04
Treatment
1.92E-07
2.96E-06
280
Table SIX Risk of Death (/person/event): Condition B
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition B
5.20E-09
Standard Deviation
1.70E-08
Treatment
1.61E-11
1.55E-10
Non-treatment
6.80E-09
2.01E-08
Treatment
8.20E-11
1.43E-09
Non-treatment
6.10E-09
1.87E-08
Treatment
9.60E-12
2.00E-10
Table SX Annual Risk of Infection (/person/year): Condition B
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition B
2.21E-01
Standard Deviation
3.38E-01
Treatment
4.00E-03
2.80E-02
Non-treatment
2.71E-01
3.68E-01
Treatment
1.20E-02
6.70E-02
Non-treatment
2.57E-01
3.60E-01
Treatment
2.00E-03
1.90E-02
281
Table SXI Annual Risk of Illness (/person/year): Condition B
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition B
8.50E-03
Standard Deviation
2.72E-02
Treatment
2.92E-05
3.12E-04
Non-treatment
1.21E-02
3.42E-02
Treatment
1.38E-04
1.19E-03
Non-treatment
1.05E-02
3.16E-02
Treatment
1.69E-05
2.37E-04
Table SXII Annual Risk of Death (/person/year): Condition B
Fomite
Ceramic tile
Laminate
Granite
Intervention
Non-treatment
Condition B
4.90E-07
Standard Deviation
1.58E-06
Treatment
1.89E-09
3.78E-08
Non-treatment
6.90E-07
2.01E-06
Treatment
7.80E-09
8.29E-08
Non-treatment
5.70E-07
1.71E-06
Treatment
9.10E-10
1.99E-08