Environmental Assessment 

for the Presence of 

Influenza Viruses (2009 

Pandemic Influenza A 

H1N1 and Seasonal) in 

Dental Practices – Ohio 

Steven H. Ahrenholz, PhD, MS, CIH

Scott E. Brueck, MS, CIH

Marie A. de Perio, MD

Health Hazard Evaluation Report

HETA 2010-0019 & 2010-0021-3120

January 2011

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Centers for Disease Control and Prevention

Workplace

Safety and Health

 National Institute for Occupational 

Safety and Health

This Health Hazard Evaluation (HHE) report and any recommendations made herein are for the specific 

facility evaluated and may not be universally applicable. Any recommendations made are not to be 

considered as final statements of NIOSH policy or of any agency or individual involved. Additional HHE 

reports are available at http://www.cdc.gov/niosh/hhe/
The employer shall post a copy of this report 

for a period of 30 calendar days at or near 

the workplace(s) of affected employees. The 

employer shall take steps to insure that the 

posted determinations are not altered, defaced, 

or covered by other material during such 

period. [37 FR 23640, November 7, 1972, as 

amended at 45 FR 2653, January 14, 1980].
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page i

Appendix

Report Abbreviations.......................................................................ii

Highlights of the NIOSH Health Hazard Evaluation............ iii

Summary .............................................................................v

Introduction..........................................................................1

Assessment.........................................................................3

Results.................................................................................9

Discussion .........................................................................14

Conclusions.......................................................................20

Recommendations.............................................................20

References ........................................................................23

Contents

Methods.............................................................................26

Acknowledgments Acknowledgments and Availability of Report.....................31
Page ii Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

°C Degrees Celsius

°F Degrees Fahrenheit

μl Microliter

μm Micrometer

μM Micromole

ACGIH® American Conference of Governmental Industrial Hygienists

ACIP Advisory Committee on Immunization Practices

CDC Centers for Disease Control and Prevention

cm2 Centimeters squared

cDNA Complimentary deoxyribonucleic acid (DNA)

GSD Geometric standard deviation

HELD Health Effects Laboratory Division

HHE Health hazard evaluation

HHPC Hand Held Particle Counter (ART Instruments, Inc., Grants Pass, Oregon)

ILI Influenza-like illness

ID Internal diameter

ISO International Organization for Standardization

Lpm Liters per minute

mL Milliliter

mm Millimeter

NA Not applicable

NAICS North American Industry Classification System

NIOSH National Institute for Occupational Safety and Health

NP Nasopharyngeal

pH1N1 2009 pandemic influenza A (H1N1)

PPE Personal protective equipment

PTFE Polytetrafluoroethylene

qPCR Quantitative polymerase chain reaction

RNA Ribonucleic acid

RSV Respiratory syncytial virus

RT-PCR Reverse transcription polymerase chain reaction

SHEA Society for Healthcare Epidemiology of America

T1 First stage centrifuge tube of bioaerosol sampler

T2 Second stage centrifuge tube of bioaerosol sampler

WHO World Health Organization

Abbreviations
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page iii

What NIOSH Did

● We visited each dental practice four times over a 6-week 

period beginning in late January 2010.

● We collected air and surface samples in both dental practices 

on eight separate dates.

● We collected nasopharyngeal samples from dental staff.

● We tested all samples for pH1N1 and seasonal flu viruses.

● We administered a flu symptom survey to dental staff.

● We reviewed information about flu symptoms among 

patients. 

What NIOSH Found

● No pH1N1 virus was found in the air or surface samples at 

either dental practice.

● Seasonal flu virus was found in the air at the pediatric 

dentistry practice during one visit.

● Dental staff did not come to work while ill.

● Vaccination rates for both seasonal and pH1N1 were low for 

staff at both dental practices.

What Managers Can Do

● Encourage employees to get the seasonal flu vaccine every 

year.

● Develop procedures for tracking ill employees and excluding 

them from work.

● Screen patients for flu symptoms before their visits or at the 

time of check-in.

What Employees Can Do

● Get the seasonal flu vaccine every year.

● Self assess for flu symptoms.

● Report any symptoms to management as soon as possible.

● Do not report for work when ill. 

The National Institute for 

Occupational Safety and 

Health (NIOSH) received 

requests for health hazard 

evaluations from two 

dental practices (one 

general and one pediatric) 

in a metropolitan area 

of Ohio. Management 

at each dental practice 

was concerned about 

transmission of 2009 

pandemic influenza (also 

known as flu) A (H1N1) 

(pH1N1) virus to dental 

staff.

Highlights of the

NIOSH Health

Hazard Evaluation
Page iv Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

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Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page v

In October 2009, NIOSH received requests for HHEs from the 

management of two dental practices in a metropolitan area of 

Ohio. The dental practices were concerned about transmission 

of pH1N1 to dental employees. NIOSH investigators visited each 

dental practice four separate times over a 6-week period beginning 

in January 2010. During each site visit, we collected air and surface 

samples for pH1N1 and seasonal influenza virus. Air samples 

were collected with bioaerosol samplers that separated airborne 

particles into three size ranges: ≥ 4.1 μm; 1.0–4.1 μm; and ≤ 1.0 μm 

aerodynamic diameter. Air samples were collected near patients 

in exam chairs and at a distance of several feet from patients to 

distinguish between large and small airborne influenza particles. 

We also collected NP swabs from dental staff, administered a 

influenza symptom survey to dental staff, and reviewed symptom 

surveys that the dental practices had asked patients to complete.

No pH1N1 or seasonal influenza A virus was detected in the 48 air 

samples or 52 surface swab samples we obtained at Dental Practice 

A, a general dentistry practice. No pH1N1 virus was detected 

in the 48 air samples collected at Dental Practice B, a pediatric 

dentistry practice. However, we identified a seasonal influenza 

A strain (H3N2) (≤ 1μm in size) from air samples collected near 

patient exam chairs in three exam rooms at Dental Practice B on 

March 2, 2010. No pH1N1 or seasonal influenza A virus was found 

in the 54 surface swabs collected in Dental Practice B.

All NP samples collected from dental practice staff at both 

practices were negative for influenza A viruses. During our 

sampling dates, none of the dental staff who completed the 

influenza symptom survey at Dental Practice A had ILI in the 

prior 7 days. Only one dental staff member who completed the 

survey at Dental Practice B had ILI in the prior 7 days. Of those 

who completed the survey at Dental Practice A, only two patients 

or individuals accompanying patients had ILI in the prior 7 days. 

At Dental Practice B, six patients or individuals accompanying 

patients had ILI in the prior 7 days. For staff at Dental Office A, 

8% reported getting the pH1N1 vaccine and 25% reported getting 

the seasonal influenza vaccine. Of the staff at Dental Practice B, 

18% reported getting both the pH1N1 and seasonal influenza 

vaccine.

Exposure of dental staff at both practices to airborne pH1N1 and 

seasonal influenza A viruses during the sampling days appears to 

have been limited. This may be because the community influenza 

No 2009 pandemic 

influenza A H1N1 virus 

was found in any air or 

surface samples at two 

dental practices, but 

seasonal influenza A was 

found in the air of the 

pediatric dental practice. 

Influenza vaccination 

rates for staff at the 

dental practices were 

below national rates for 

healthcare personnel 

during the 2009–2010 

influenza season. We 

recommend that all staff 

get the seasonal influenza 

vaccine every year.

Summary
Page vi Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Summary (continued) incidence during the time of our evaluation was below typical 

seasonal influenza levels. The absence of positive surface swab 

samples for pH1N1 or seasonal influenza may be primarily due to 

the low number of patients reporting ILI.

We recommend that all employees get the seasonal influenza 

vaccine every year. Employees should self-assess for influenza 

symptoms and not report to work if ill. Dental practices 

should monitor and manage employees who are ill and absent. 

Additionally, dental practices should consider developing 

mechanisms to screen patients for ILI symptoms prior to their visits 

or at the time of check-in. Dental practices should advise patients 

to consider postponing visits and procedures that are not urgent 

until 24 hours after the patient is free of fever, especially in times 

of high influenza activity within the community.

Keywords: NAICS 621210 (Offices of Dentists), dental practice, 

dentist, seasonal influenza, H1N1, pandemic influenza, swine flu, 

bioaerosol
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 1

Introduction

We received requests for HHEs from the management of two 

dental practices in a metropolitan area of Ohio. The management 

at both dental practices was concerned about transmission of 

pH1N1 virus to dental employees.

Because the methods and issues were the same for both dental 

practices, the investigations are discussed together in this report. 

Our primary objective for these evaluations was to assess dental 

practice employees’ potential for exposure to pH1N1 and seasonal 

influenza viruses at work. The questions we sought to answer were:

● Are pH1N1 or seasonal influenza viruses present in the 

workplace?

● Are workers potentially exposed to airborne pH1N1or 

seasonal influenza viruses (size range < 4.1 μm aerodynamic 

diameter) in their breathing zone or in the immediate 

proximity of patients (approximately 3 feet or less)?

● Are influenza viruses, if present within the workplace, 

capable of being collected as an airborne particulate < 4.1 μm 

aerodynamic diameter at distances beyond the immediate 

proximity of the source (greater than 3 feet)? (Note that the 

maximum distance at which droplet transmission may end 

and the minimum distance at which airborne transmission is 

considered to occur remains unresolved [Siegel et al. 2007]).

The methods available to us at the time of this evaluation allowed 

for the identification of influenza viruses in the work environment 

but could not determine if these viruses were viable and capable 

of causing illness. Presence of the influenza virus does not by itself 

indicate risk of infection. Additionally, the amount of virus found 

cannot be used by itself to assess the likelihood of infection or 

illness. However, if viruses are detected, this indicates the potential 

for exposure.

 The focus of this evaluation was to determine if pH1N1 and/

or seasonal influenza viruses are present in a workplace such as 

a dental practice, which does not perform aerosol generating 

healthcare procedures. Aerosol generating procedures are 

considered to increase the risk of influenza transmission to 

employees. Dental practice employees are unlikely to encounter 

individuals seeking care for influenza symptoms or complications, 

although these individuals may visit the dental practice despite 

having influenza symptoms.
Page 2 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Introduction 

(continued) Background on the Dental Practices 

Dental practice A provides general dentistry services to adults and 

children over the age of three. The majority of patients are adults. 

The practice employs two dentists, four dental hygienists, three 

dental assistants, and two administrative staff. Dental practice B 

provides pediatric dentistry services for children ages 1 to 18 years 

of age. A majority of patients are in the 1 to 10 year age range. This 

practice employs two dentists, three dental hygienists, six dental 

assistants, and three administrative staff.

Background on 2009 Pandemic Influenza 

A (H1N1) Virus

The pH1N1 virus, also referred to initially as “swine flu,” was 

first detected in humans in the United States in April 2009. On 

June 11, 2009, the WHO signaled that a pandemic of pH1N1 was 

underway. CDC estimated that, between April 2009 and April 

2010, 43–89 million cases of pH1N1, 195,000–403,000 pH1N1-

related hospitalizations, and 8,870–18,300 pH1N1-related deaths 

occurred [CDC 2010a].

Spread of the pH1N1 virus is thought to occur in the same way 

that seasonal influenza spreads [CDC 2009a]. Influenza viruses 

are spread mainly through droplet transmission though evidence 

for airborne transmission and transmission via direct contact also 

exists [CDC 2009a].

The symptoms of pH1N1 infection include fever, cough, sore 

throat, runny or stuffy nose, body aches, headache, chills, and 

fatigue. Some patients have had vomiting and diarrhea, while 

some patients have respiratory symptoms without a fever. Illness 

with the pH1N1 virus has ranged from mild to severe. While 

most people who have been sick have recovered without needing 

medical treatment, hospitalizations and deaths from infection with 

this virus have occurred. Many people who have been hospitalized 

with this pH1N1 virus have had one or more medical conditions 

previously recognized as placing people at “high risk” of serious 

seasonal influenza-related complications, including pregnancy, 

diabetes, heart disease, asthma, and kidney disease [CDC 2009a].
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 3

Introduction (continued)

Assessment

Background on Influenza Vaccines 

The pH1N1 vaccine became available in the United States in 

October 2009. In July 2009, CDC’s ACIP recommended that 

certain groups of the population receive the pH1N1 vaccine 

first [CDC 2009b]. These target groups included all healthcare 

personnel, including employees in outpatient clinics such as dental 

care settings. In July 2009, CDC’s ACIP updated its longstanding 

recommendations for seasonal influenza vaccination [CDC 2009c]. 

The groups targeted for this vaccine were similar to those for the 

2009 pH1N1 vaccine and also included all healthcare personnel, 

including employees in outpatient clinics such as dental care 

settings.

The assessment methods are described in general below. 

Additional details are found in the appendix to this report.

Environmental Assessment 

We visited each dental practice on 4 days over a period of 6 weeks 

beginning in January 2010. Each week we asked both practices to 

identify their busiest days for the upcoming week. We selected the 

days for our evaluation that involved greater patient scheduling and 

the presence of most of the dental practices’ staff.

We collected aerosols with a two-stage cyclone bioaerosol sampler 

(Figure 1) developed and used by other NIOSH investigators to 

identify the presence and size fraction of airborne pH1N1 and 

seasonal influenza viruses and viral fragments [Lindsley et al. 

2006; Blachere et al. 2009]. The bioaerosol samplers allowed for 

the collection of pH1N1 and seasonal influenza virus and viral 

particles across three size fractions: ≥ 4.1 μm; 1.0–4.1 μm; and < 1.0 

μm aerodynamic diameter.

We collected air samples in five patient rooms and the receptionist 

area over 4 days between January 27, 2010, and February 24, 

2010, at Dental Practice A. At Dental Practice B, we collected air 

samples in six patient rooms and the reception desk area on 4 

days between January 26, 2010, and March 2, 2010. We collected 

environmental samples at each practice 1 day per week and limited 

sampling duration to between 4 and 5 hours because of concerns 
Page 4 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Assessment (continued)

Figure 1. Close-up of NIOSH two-stage cyclone bioaerosol sampler 

mounted on dental light fixture.

about excessive desiccation of viruses during longer sample periods. 

Samples were collected during the first half of the workday on 

the days considered by the practices to be their busiest. RT-PCR 

analyses were used to identify the presence of pH1N1 and seasonal 

influenza virus and viral particles collected by the air samplers. 

RT-PCR analyses for the presence of RSV were added for the last 

3 days of environmental sampling at Dental Practice B. RSV is a 

common respiratory illness that occurs in children and has been 

evaluated previously along with air sampling for influenza virus 

[Lindsley et al. 2010].

The bioaerosol samplers were mounted in two locations within 

patient rooms. One was positioned about 2 to 3 feet from the 

potential source (patient) of pH1N1 or seasonal influenza virus, 

depending upon where the dental staff positioned the exam light 

while they were working, and was usually located on the dental 

exam light support structure (Figure 2). This placed the sampler 

within approximately 3 feet of dental staffs’ breathing zone when 

they did exams and procedures. Dental staff positioned the exam 

light closer while working with a patient and pushed it up and 

away when finished. The objective of using this spacing was to 

determine if we could find influenza virus in the area where 

droplet and large aerosol transmission is thought to occur (within 

approximately 3 feet) [Siegel et al. 2007].
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 5

Assessment (continued)

Figure 2. Two-stage NIOSH cyclone bioaerosol sampler attached to 

support above dental exam light at Dental Practice B.

A second sampler was located approximately 4 to 8 feet (Dental 

Practice A: average 6.6 feet; Dental Practice B: average 5.9 feet) 

from the potential source. This distance would allow us to assess 

whether influenza virus spreads farther than the immediate 3-foot 

vicinity of the source. Influenza virus collected by the distant 

sampler would more likely be present in small (< 4.1 μm) airborne 

particulates that remain suspended within the room and carried 

beyond the region where droplet transmission typically occurs. 

Depending on the room configuration, we placed the second 

sampler on a tripod in the corner of the room, on a short wall 

between rooms, on top of a small wall cabinet, or on an elevated 

location above and behind the patient exam chair (Figure 3). The 

size of the patient rooms and configuration of equipment was such 

that maintaining minimum distances for our second samplers of at 

least 6 feet was not always possible.

Figure 3. Two-stage bioaerosol sampler mounted on a tripod in the 

corner of an exam room at Dental Practice A. (Note: Additional 

sampler on support arm of exam light above dental chair.)
Page 6 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Assessment (continued)

Figure 4. NIOSH investigator obtains a surface swab sample on a 

dental equipment support structure.

We collected surface swab samples (swabs – Copan Diagnostics, 

Corona, California; phosphate buffer to moisten swabs – 

Invitrogen, Aukland, New Zealand) in each room where we took 

air samples to evaluate whether pH1N1 or seasonal influenza 

virus was present on surfaces in close proximity to patients and on 

work surfaces used by the dental staff. Other investigators have 

previously identified the presence of influenza A virus on up to 

50% of household and daycare center fomites and classroom 

surfaces tested during influenza season [Boone and Gerba 

2005; Bright et al. 2010]. All environmental samples were stored 

and shipped on dry ice at -20oC to -70oC to NIOSH HELD, 

Morgantown, West Virginia. The samples were analyzed for the 

presence of pH1N1 and seasonal influenza viruses using real-time 

RT-PCR methodology. Additional information about our surface 

swab sampling is in the appendix. Figures 4–6 show some of the 

surfaces on which we collected swab samples.
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 7

Assessment (continued)

Figure 5. NIOSH investigator obtains a surface swab sample on the 

counter next to a sink used by patients during tooth brushing.

Figure 6. NIOSH investigator obtains a surface swab sample on a 

dental chair armrest.
Page 8 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Assessment (continued) We submitted spiked positive controls for seasonal influenza A 

to the analytic laboratory to determine if there were potential 

problems with field sampling or lab analysis procedures. Positive 

controls indistinguishable from our field samples were submitted 

to the lab and handled the same way as our field samples and field 

blanks.

We obtained additional environmental information for the patient 

areas using two HHPC samplers (ART Instruments, Grants Pass, 

Oregon). We placed HHPC units alongside two of the aerosol 

samplers during each day we sampled for pH1N1 and seasonal 

influenza. The HHPC provides a total count for particles falling 

into one of six size ranges: 0.3–0.5 μm, 0.5–1.0 μm, 1.0–3.0 μm, 

3.0–5.0 μm, 5.0–10.0 μm, and > 10.0 μm. The HHPC results are 

not specific for viral particles. They identify the presence of all 

particles in the air in different size categories regardless of particle 

composition or source. The particle size cutoff for each of the 

HPPC six size ranges differs from the three particle size cutoffs of 

the bioaerosol sampler. We also measured indoor temperature and 

relative humidity data for the days we did environmental sampling.

Medical Assessment

We invited all employees at both practices working on the HHE 

sampling dates to participate in the evaluation. We handed out a 

brief survey to all employee participants on each day of sampling. 

The survey addressed recent history of symptoms of ILI and recent 

influenza vaccination. Our definition for ILI was fever >100°F 

along with a cough and/or sore throat (http://www.cdc.gov/flu/

weekly/fluactivity.htm), which was the CDC definition.

After obtaining informed consent, we obtained NP samples from 

each participating employee on each sampling date. The samples 

were analyzed at the NIOSH HELD analytical laboratory for the 

presence of pH1N1 and seasonal influenza viruses using real-time 

RT-PCR methodology. NP samples collected on February 8, 2010; 

February 23, 2010; and March 2, 2010, were also analyzed for RSV 

using the same methodology. Following each week of sampling, we 

informed each employee participant in writing of the results of his 

or her nasopharyngeal swab test and its significance.

We also developed a brief survey for patients. As part of the patient 

registration process, dental practice staff handed out this survey to 
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 9

Assessment (continued) all patients seeking dental care (or to their parents if the patients 

were less than 18 years of age) on the HHE sampling dates. The 

survey was voluntary and addressed recent history of influenza 

symptoms and recent influenza vaccination of the patient and 

any accompanying individuals. Dental practice staff collected the 

completed surveys and placed them in the individual patient’s 

dental record. We then abstracted the information (without 

personal identifiers) from the patient’s dental record. We did not 

have direct contact with patients during this process, and we did 

not collect nasopharyngeal samples from patients.

Exposure Assessment 

Dental Practice A 

No pH1N1 or seasonal influenza A virus was detected using RTPCR on the 48 air samples or 52 surface swab samples we obtained 

at Dental Practice A. Airborne particles within the size ranges 

we would have expected to find influenza viruses were present 

during the days we sampled, based upon the real-time HHPC data 

collected alongside two of our bioaerosol samplers located about 

6 feet from the dental chair headrest. (Note the HHPC and twostage bioaerosol sampler sitting on top of the half-wall at the left 

side of Figure 2.) Figure 7 presents an example of the HHPC total 

particle counts for six size fractions obtained on January 27, 2010, 

at Dental Practice A.

Note that the top two lines in Figure 7 present particle counts for 

particle sizes (≤ 1μm) that are also collected in the final stage of 

the NIOSH bioaerosol sampler. The particle counts presented on 

the third and fourth lines down from the top in Figure 7 overlap 

with the 1—4.1 μm size particles collected by the second stage of 

the bioaerosol sampler. The bottom three lines plotting airborne 

particle size in Figure 7 correspond with the particle sizes captured 

in the first stage of the bioaerosol sampler, (all particles ≥ 4.1μm in 

size).

Results
Page 10 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Results (continued)

Figure 7. Plot of HHPC data of total particle count versus sample time for six airborne 

particle size fractions obtained January 27, 2010, Dental Practice A, room 4.

1

10

100

1000

10000

100000

10:0410:2110:3710:5411:1111:2811:4512:0212:1812:3512:5213:0913:2613:4313:59

Log 10 Particle Count

Time of Day

C 0.3-0.5um

C 0.5-1.0um

C 1.0-3.0um

C 3.0-5.0um

C 5.0-10.0um

C >10.0um

Dental Practice B 

No pH1N1 was identified in the 48 air samples collected at Dental 

Practice B. However, we identified seasonal influenza A on the 

filter stage only (≤ 1μm in size) of the bioaerosol samplers mounted 

on the dental exam light in rooms three, five, and six on March 

2, 2010. In room three, the positive seasonal influenza A response 

on the bioaerosol sampler filter was subtyped as influenza H3N2. 

Analytical limitations prevented any further subtyping of the 

seasonal influenza A virus particles found on the other two positive 

samples. No seasonal influenza A virus was identified in the first 

two stages for any of these three samplers (particle sizes ≥ 4.1 μm 

and 1–4.1 μm). However, other airborne particles within these size 

ranges were identified using the HPPC located alongside two of 

our bioaerosol samplers (Figure 8).
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 11

Results (continued)

Figure 8. Plot of HHPC data of total particle count versus sample time for six airborne 

particle size fractions obtained March 2, 2010, Dental Practice B, on half-wall between 

Rooms 3 and 4. (See left side of Figure 2.)

1

10

100

1000

10000

100000

7:367:538:108:278:439:009:179:349:51

10:0810:2510:4210:5811:1511:3211:49

Log 10 Partcle Count

Time of Day

C 0.3-0.5um

C 0.5-1.0um

C 1.0-3.0um

C 3.0-5.0um

C 5.0-10.0um

C >10.0um

The 54 surface samples obtained at Dental Practice B in the 

same rooms where air samples were collected were all negative for 

pH1N1 and seasonal influenza A viruses.

Samples collected on February 8, 2010; February 23, 2010; and 

March 2, 2010, at Dental Practice B were also analyzed for RSV. 

One air and one surface swab sample, both obtained in room two 

on March 2, 2010, were positive for RSV. RSV was detected in the 

first stage (≥ 4.1 μm size particles) of the aerosol sampler located 

on the dental exam light above the patient dental chair. The RSV 

positive surface swab was collected on the right armrest of the 

same chair. All air and surface sampling results for both dental 

practices are summarized in Table 1.
Page 12 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Results (continued)

Table 1. Summary Sampling Results for Viruses in Airborne and Surface Swab Samples Obtained at Two 

Dental Practices from January 26 to March 2, 2010

Dental 

Practice Sample Type

Total No. 

Samples

# pH1N1

Positive

# Seasonal 

Influenza 

Positive

# RSV

Positive (Practice B)

Dental 

Practice A

Bioaerosol size 

fraction:

≥ 4.1 μm 48 0 0 NA*

1.0–4.1 μm 48 0 0 NA

≤ 1 μm 48 0 0 NA

Surface swabs 52 0 0 NA

Dental 

Practice B

Bioaerosol size 

fraction:

≥ 4.1 μm 48 0 0 1

1.0–4.1 μm 48 0 0 0

≤ 1 μm 48 0 3 0

Surface swabs 54 0 0 1

Total samples 

(%)

Bioaerosol samples 

(air)

Surface swabs

96 (100%)

106 (100%)

0

0

3 (3%)

0

1/36 (3%)†

 (Practice B)

1/42 (2%)† (Practice B)

*NA = RSV analyses for Dental Practice A not requested, not applicable. 

†Qualitative analyses of air and wipe samples for RSV were obtained for samples collected at Dental Practice 

B only for the sampling dates of February 8, 2010; February 23, 2010; and March 2, 2010; resulting in the lower 

total number of samples evaluated for the presence of RSV.

Medical Assessment

All employees working at both dental practices completed surveys 

on the sampling dates. Participation rates of employees in the NP 

sampling ranged from 55%–100% at Dental Practice A and ranged 

from 73%–100% at Dental Practice B on the sampling dates). All 

NP samples collected from dental practice staff at both practices 

were negative for influenza A viruses. All NP samples collected 

from employees at Dental Practice B on February 8, 2010; February 

23, 2010; and March 2, 2010; were also negative for RSV.

During sampling visits on January 27, 2010; February 2, 2010; and 

February 24, 2010, at Dental Practice A, none of the employees, 

patients, or individuals accompanying patients who completed 
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 13

Results (continued) symptom surveys reported symptoms consistent with ILI in the 

prior 7 days. During our third sampling visit on February 17, 

2010, none of the employees reported symptoms consistent with 

ILI. However, one of the 32 patients and one of the 8 individuals 

accompanying patients did report symptoms consistent with ILI. 

Thus, a total of 2 of 185 (1%) reported ILI symptoms on the 

sampling dates at Dental Practice A.

During our first sampling visit to Dental Practice B on January 26, 

2010, one of the 14 employees and three of the 14 patients who 

completed symptom surveys reported symptoms consistent with 

ILI in the prior 7 days. None of the individuals accompanying 

patients on this date who completed symptoms surveys reported 

ILI symptoms. During our second sampling visit on February 8, 

2010, none of the employees or individuals accompanying patients 

reported ILI symptoms. However, one of the 15 patients did report 

ILI symptoms. During our third sampling visit on February 23, 

2010, none of the employees reported ILI symptoms. However, 

one of the 14 patients and one of the 16 individuals accompanying 

patients reported ILI symptoms. During our fourth sampling visit 

on March 2, 2010, none of the employees, patients, or individuals 

accompanying patients reported ILI symptoms. Thus, a total of 

seven of 158 (4%) individuals reported ILI symptoms on the 

sampling dates at Dental Practice B, the majority of whom were 

patients.

Self-reported vaccination rates for employees at Dental Practice 

A through February 24, 2010, were 8% (1 of 12 employees) for 

pH1N1 and 25% (3 of 12 employees) for seasonal influenza. 

Self-reported vaccination rates for employees at Dental Practice B 

through March 2, 2010 were 18% (3 of 17 employees) for pH1N1 

and 18% (3 of 17 employees) for seasonal influenza (Table 2).

Self-reported vaccination rates for patients at Dental Practice A on 

the sampling dates were 20% (26 of 130 patients) for pH1N1 and 

42% (55 of 130 patients) for seasonal influenza. Parent-reported 

vaccination rates for pediatric patients at Dental Practice B on the 

sampling dates were 64% (32 of 50 patients) for pH1N1 and 48% 

(24 of 50 patients) for seasonal influenza (Table 2).
Page 14 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Results (continued)

Dental employees providing direct patient care have the potential 

for close contact with patients during dental procedures. 

Additionally, employees in a dental practice may encounter 

patients and colleagues who could be shedding influenza virus 

with or without accompanying influenza symptoms. Dental 

employees do not perform aerosol generating procedures identified 

by SHEA to be “high risk” [CDC 2010b, SHEA 2009]. However, 

they perform procedures, such as tooth preparation with a rotary 

instrument or air abrasion, use of an air-water syringe or ultrasonic 

scaler, and air polishing, that may result in aerosolization of 

influenza viruses because the mouth may harbor bacteria and 

viruses from the nose, throat, and respiratory passages [Harrel and 

Molinari 2004].

Influenza infections occur through three primary modes of 

exposure: droplet transmission, contact transmission, and aerosol 

transmission. The relative contribution of these three modes 

of transmission is unknown and remains a subject of debate 

[Atkinson and Wein 2008]. The generally accepted primary mode 

of transmission for influenza is via droplet spread [SHEA 2009]. 

Previous measurement for airborne influenza virus in a hospital 

emergency department identified 53% of the detectable influenza 

virus particles within the respirable aerosol (≤ 4.1 μm size) fraction 

[Blachere et al. 2009]. An observational study of influenza virus in 

human exhaled breath reported that > 99% of exhaled particles 

were < 5.0 μm [Fabian et al. 2008]. Researchers evaluating the size 

distribution of droplets in the exhaled breath of healthy human 

subjects from mouth breathing, nose breathing, coughing, and 

talking found that the majority of particles were ≤ 1μm [Papineni 

and Rosenthal 1997].

Discussion

Table 2. Self-reported pH1N1 and Seasonal Influenza Vaccination Rates Among Employees and Patients

Dental Practice Employees

pH1N1 (%)

Employees

Seasonal (%)

Patients

pH1N1 (%)

Patients

Seasonal (%)

A 1/12 (8) 3/12 (25) 26/130 (20) 55/130 (55)

B 3/17 (18) 3/17 (18) 32/50 (64) 24/50 (48)
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 15

Discussion (continued) The peak month for seasonal influenza cases in the Northern 

hemisphere from 1976–1977 through 2008–2009 has been 

February followed by January, March, and December [CDC 

2009c]. However, the 2009–2010 influenza season did not follow 

this pattern. The pH1N1 influenza virus first appeared in late 

April 2009, with resurgence in September and October 2009. 

Uncertainty existed regarding whether a third resurgence would 

occur. We initiated our exposure assessment during the last week 

of January 2010 when influenza activity was below typical seasonal 

influenza levels; no resurgence or “third wave” of pH1N1 occurred. 

Because we did not find any pH1N1 and very little seasonal 

influenza viruses in our environmental samples, we suspended our 

field evaluation after completing four weeks of sampling. Figure 9 

shows that the weekly percent of visits for ILI continued to decline 

below the Region 5 seasonal ILI baseline of 1.7% of outpatient 

visits [National Center for Infectious Diseases 2010]. The annual 

influenza season normally starts during the first full week of 

October. Our first week of sampling began on week 4 of the 

calendar year. Our last 2 weeks of sampling occurred during weeks 

8 and 9. Figure 9 shows that by this time the second wave of peak 

pH1N1 activity was over and the likelihood of additional pH1N1 

infections continued to decline.

Considering that only 3% (3 of 96) of our air samples for influenza 

A viruses were positive and none were positive for pH1N1, 

exposure of dental staff at both practices to airborne influenza A 

viruses during the sampling days appears to have been limited. The 

HHPC results indicated that even though we saw no influenza A 

viruses, particles in the size range where we would expect to find 

influenza viruses were present in both dental practices. The HHPC 

results indicated that even though influenza A viruses were not 

present, it was not because particulates within the size range which 

would include influenza viruses were not present in the patient 

rooms.
Page 16 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Discussion (continued)

Figure 9. Region 5 weekly percent of visits for ILI for the 2009–2010 influenza season 

through May 8, 2010 (week 18) [National Center for Infectious Diseases 2010].

It is not surprising that we only had a few positive results for 

seasonal influenza virus strains in the environmental samples. 

During a period similar to our sampling dates (January 24–

March 6, 2010), CDC reported that 99%–100% of subtyped 

influenza A viruses reported to CDC were pH1N1 viruses. This 

demonstrates the low circulating activity of non-pH1N1 influenza 

virus strains [CDC 2009b]. The continuing decline of ILI within 

the community meant that it was less likely dental staff would 

encounter patients, patient family members, or coworkers shedding 

pH1N1 or seasonal influenza viruses. Because of the low level of 

pH1N1 and seasonal influenza activity, we are unable to draw 

conclusions about general risk of exposure for dental staff during 

periods of higher influenza activity.
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 17

Discussion (continued) The absence of positive surface swab samples for influenza A 

viruses may be primarily due to the low number of patients 

reporting ILI. Additionally, the surface disinfection procedures 

used by dental staff between patients may have also effectively 

removed surface contamination by influenza A virus, if present. 

Direct contact surfaces in the patient rooms at both dental 

practices are wiped down with disinfectant spray or wipes after each 

patient. As mentioned earlier in this report, other investigators 

have identified the presence of influenza A virus on up to 50% 

of surfaces tested during the influenza season [Boone and Gerba 

2005; Bright et al. 2010].

The negative results from our environmental samples during 

the first days of sampling reinforced our decision to prepare and 

submit (to the analyst) unidentified positive samples along with our 

field samples and blanks. We wanted to determine that we didn’t 

have a problem with the handling and shipping of our completed 

field samples prior to their arrival at the laboratory. The analyst 

identified all of the positive control samples we submitted.

We observed dental staff at both practices using face shields, safety 

glasses, procedure masks, and disposable gloves when working with 

patients. Because of the low levels of exposure we found, we are 

unable to assess the adequacy of the personal protective equipment 

used by the dental staff. The three influenza A positive air samples 

that we did find were all associated with the smallest size fraction 

of airborne particulate (≤ 1 μm) and were all obtained at a location 

close to where dental staff work (immediately above the dental 

exam light). Procedure masks are not considered respiratory 

protection and are not designed to filter this size of particulate 

[Brosseau et al. 1997]. CDC recommended the use of NIOSHcertified respiratory protection that was at least as protective as a 

fit-tested disposable N95 respirator for healthcare personnel who 

were in close contact with patients with suspected or confirmed 

pH1N1. This recommendation applied uniquely to the special 

circumstances of the pH1N1 pandemic during the fall and winter 

of 2009—2010 [CDC 2010c]. Facemasks that have been cleared for 

marketing by the U.S. Food and Drug Administration have been 

tested for their ability to resist blood and body fluids, and generally 

provide a physical barrier to droplets that are expelled directly at 

the user. Although they do not efficiently filter small particles from 

the air and they allow leakage around the mask, they are a barrier 

to splashes, droplet sprays, and autoinoculation of influenza virus 

from the hands to the nose and mouth.
Page 18 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Discussion (continued) All NP samples collected from employees at both dental practices 

on all sampling dates were negative for influenza A viruses. These 

results were anticipated, as none of the employees at Dental 

Practice A reported ILI symptoms, and only one employee at 

Dental Practice B reported ILI symptoms on just one sampling 

date. Our survey results demonstrate the low prevalence of ILI 

symptoms, 1% at Dental Practice A and 4% at Dental Practice B, 

among employees and patients during this 5-week period. This is 

consistent with the CDC reports at the time, which documented 

low influenza and ILI activity. In addition, these results suggest 

that dental staff did not come to work when ill and very few 

patients visited the dental practice when ill on the sampling dates.

pH1N1 vaccination rates for employees were 8% for Dental 

Practice A and 18% for Dental Practice B. Though CDC’s 

ACIP recommended that healthcare personnel be among the 

target groups to receive the pH1N1 vaccine first [CDC 2009e], 

vaccination rates among these dental employees were suboptimal. 

These rates are also well below the estimated pH1N1 vaccination 

rate among employees in ambulatory, outpatient, and dental 

practices nationwide as of mid-January, which was 39% [CDC 

2010d].

Seasonal influenza vaccination rates for employees were 25% for 

Dental Practice A and 18% for Dental Practice B. CDC’s ACIP 

has had longstanding recommendations that target groups for 

seasonal influenza vaccination include all healthcare personnel 

[CDC 2009a]. Despite this, seasonal influenza vaccination rates 

among the dental employees were also suboptimal. These rates are 

also well below the estimated seasonal influenza vaccination rate 

of 64% among employees in ambulatory, outpatient, and dental 

practices nationwide as of mid-January [CDC 2010d].

The self-reported pH1N1 vaccination rate for Dental Practice A 

patients, consisting of mostly adults, was 20%. This rate was similar 

to the estimated pH1N1 vaccination rate among adults in the 

state of Ohio, which was 18% as of the end of January 2010 [CDC 

2010e]. The parent-reported pH1N1 vaccination rate for patients 

(all children) at Dental Practice B was 67%. This is higher than the 

estimated pH1N1 vaccination rate among children in the state of 

Ohio, which was 34% as of the end of January 2010 [CDC 2010e]. 

Our findings are consistent with CDC’s findings that vaccination 

coverage was higher among children than adults, largely thought 

to be due to school-based vaccination programs. The higher than 

expected pH1N1 vaccination rates among Dental Practice A and 
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 19

Discussion (continued) B patients may have been one of the factors that contributed to 

the fact that pH1N1 virus was not detected in the air and surface 

sampling.

The self-reported seasonal influenza vaccination rate for Dental 

Practice A patients was 42%. This rate was similar to the estimated 

seasonal influenza vaccination rate among adults in the state of 

Ohio, which was 41% as of the end of January, 2010 [CDC 2010f]. 

The parent-reported rate for Dental Practice B patients was 48%. 

This is higher than the estimated seasonal influenza vaccination 

rate among children in the state of Ohio, which was 39% as of the 

end of January, 2010 [CDC 2010f]. These relatively high rates of 

seasonal influenza vaccination may have been one of the factors 

that contributed to the fact that seasonal influenza virus was not 

detected in the air or environmental samples.

Our evaluation was subject to some limitations. The continuing 

decline in influenza activity below seasonal levels may have 

contributed to the absence of pH1N1 and seasonal influenza 

during our exposure assessment. The identification of influenza 

and respiratory viruses collected using the NIOSH two-stage 

bioaerosol sampler does not provide any indication of the collected 

virus’ viability or infectivity. (Note that determining the viability of 

collected airborne viruses was not an objective of this evaluation.)

The absence of influenza viruses in the work environment we 

evaluated prevented looking at near versus distant samplers’ ability 

to collect influenza viruses. Our results do not provide any insight 

into whether exposure is limited to close proximity to the potential 

source (patient) or if the virus may also be airborne at a distance 

at or beyond 6 feet. Air sampler spacing distances and location 

were limited by room and equipment configurations and do not 

represent individual dental staff breathing zone exposures.

HHPC data does not correlate exactly with the size fractions 

collected by the two-stage bioaerosol sampler and it does not 

provide any information on the composition or source of 

particulates making up the six different size fractions reported by 

the HPPC for samples collected at both dental practices.

We did not identify the primary dental procedures performed for 

each patient within a room during the sampling period. Our results 

cannot provide information about the potential for exposure to 

influenza virus from the patients under current working conditions 

in the dental practices evaluated.
Page 20 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Discussion (continued)

Conclusions

We did not achieve 100% participation in the collection of 

nasopharyngeal samples among employees at both practices 

during all sampling dates. However, 100% of employees working 

on those dates did complete surveys, and none of the employees 

who declined to provide a nasopharyngeal sample reported ILI 

symptoms in the previous seven days.

Also, we were informed that some parents of patients in Dental 

Practice B declined to complete the surveys passed out by dental 

practice staff upon registration. This limited our potential to detect 

the source of non-pH1N1 virus found in air samples at Dental 

Practice B on March 2, 2010, and the source of RSV found in swab 

samples on the same date. It should be noted that the survey asked 

employees and patients about symptoms specific for ILI and did 

not include some of the symptoms of RSV infection.

Our vaccination rates were based on self and parents reports and 

may have been subject to recall bias. Vaccination was not validated 

by medical records, and persons may have confused receipt of the 

pH1N1 and seasonal influenza vaccinations.

Because of low incidence of ILI among dental staff, patients, 

and within the community, we could not successfully evaluate 

the potential for exposure of dental staff to pH1N1 or seasonal 

influenza. We found no pH1N1 in any of the air samples and we 

only found influenza A in three samples at Dental Practice B on 

one day. Neither dental staff nor patients were coming in with 

ILI symptoms. Immunization rates among dental staff for both 

pH1N1 and seasonal influenza were below the rates reported for 

the 2009–2010 influenza season among health care workers. They 

were also lower than the influenza immunization rates reported by 

their patients. Both dental practices followed standard precautions 

regarding infection control procedures and use of PPE in dental 

practices.

Based on our findings, we recommend the actions listed below 

to create a more healthful workplace. We encourage the dental 

practices to discuss the recommendations in this report with all 

employees and jointly develop an action plan. Those involved in 

the work can best set priorities and assess the feasibility of our 

recommendations for their dental practices. Our recommendations 

Recommendations
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 21

Recommendations

(continued) are based on the occupational safety and health hierarchy of 

controls approach. This approach groups actions by their likely 

effectiveness in reducing or removing hazards. In most cases, 

the preferred approach is to eliminate hazardous materials or 

processes and install engineering controls to reduce exposure or 

shield employees. Until such controls are in place, or if they are 

not effective or feasible, administrative measures and/or personal 

protective equipment may be needed.

Elimination and Substitution 

Eliminating the potential source of exposure is a highly effective 

means for reducing hazards and ranks highest in the hierarchy of 

controls.

1. Develop procedures for monitoring and managing 

employees who are ill and absent. 

a. Encourage employees to self-assess for ILI symptoms and 

report symptoms to their supervisor.

b. Exclude employees with ILI symptoms from work 

according to the most recent CDC guidance, which can 

be found on the CDC website at http://www.cdc.gov/

flu/professionals/infectioncontrol/healthcaresettings.

htm.

2. Consider developing mechanisms to screen patients 

for ILI symptoms prior to their visits, such as through 

the appointment reminder system. Consider requesting 

symptomatic patients to postpone elective visits and 

procedures until 24 hours after they are free of fever, 

especially in times of high influenza activity.

Administrative Controls

Administrative controls are management-dictated work practices 

and policies to reduce or prevent exposures to workplace hazards. 

The effectiveness of administrative changes in work practices 

for controlling workplace hazards is dependent on management 

commitment and employee acceptance. Regular monitoring and 

reinforcement is necessary to ensure that control policies and 

procedures are not circumvented in the name of convenience or 

production.
Page 22 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Recommendations

(continued) 1. Encourage all employees to receive the annual seasonal 

influenza vaccine. The 2010–2011 seasonal influenza 

vaccine will protect against pH1N1 and two other influenza 

viruses. Consider instituting an employer requirement of 

influenza vaccine as part of a comprehensive influenza 

infection control strategy. This has been associated with 

higher rates of seasonal influenza vaccination compared 

with rates among health care providers whose employers 

neither required nor recommended seasonal influenza 

vaccination [CDC 2010g]. The WHO continues to 

encourage vaccination as an important preventive measure 

to reduce morbidity and mortality caused by influenza 

viruses during the post-pandemic period of pH1N1 

announced August 10, 2010 [World Health Organization 

2010]. Explore the feasibility of offering seasonal influenza 

vaccination to employees at the worksite [CDC 2006].

2. Identify patients with ILI by symptom screen and/

or temperature check at check-in and reschedule those 

presenting for non-urgent care. If evaluating for urgent 

care, offer the patient a facemask to wear prior to dental 

procedures and separate ill patients from others whenever 

possible.

Personal Protective Equipment 

PPE use in dental offices is a part of routine general infection 

control practices [CDC 2010h]. However, PPE use for influenza 

transmission prevention is the least effective means for controlling 

employee exposure and should not be relied upon as the sole 

method for limiting employee exposures. Proper use of PPE 

requires a comprehensive program, and calls for a high level of 

employee involvement and commitment to be effective. The 

latest CDC PPE recommendations for healthcare personnel who 

are in close contact with patients with confirmed or suspected 

influenza can be found at http://www.cdc.gov/flu/professionals/

infectioncontrol/healthcaresettings.htm.
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 23

Atkinson MP, Wein LM [2008]. Quantifying the routes of 

transmission for pandemic influenza. Bull Math Biol 70(3):820–

867.

Blachere FM, Lindsley WG, Pearce TA, Anderson SE, Fisher 

M, Khakoo R, Meade BJ, Lander O, Davis S, Thewlis RE, Celik 

I, Chen BT, Beezhold DH [2009]. Measurement of airborne 

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characteristics. Atlanta, GA:U.S. Department of Health and 

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surveillanceqa.htm]. Date accessed: September 2010.

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2010.

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cases, hospitalizations and deaths in the United States, April 2009 

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h1n1.htm]. Date accessed: December 2010.

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faq/protective_equipment.htm]. Date accessed: December 2010.

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Chan KH, Leung GM, Milton DK [2008]. Influenza virus in 
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References (continued) human exhaled breath: An observational study.PLoS ONE[serial 

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Page 26 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Environmental Sampling 

Aerosol Sampling 

Air samples to evaluate the presence of pH1N1 and seasonal influenza viruses/viral fragments were 

collected using two-stage cyclone bioaerosol samplers. These samplers collect size segregated airborne 

particles in two standard centrifuge tubes (15 and 1.5 mL tubes, Becton Dickinson, Franklin Lakes, 

New Jersey) and on a PFTE filter (SKC, Inc., Eighty Four, Pennsylvania) (Figure 10) [Lindsley et al. 

2006]. Aerosol samples were collected once a week at each dental facility for 4 weeks (a total of 8 sample 

collection days). Samples were collected for approximately 4–5 hours on each sample collection day. 

Sampling duration was limited to this shorter time period rather than a full work shift due to a reported 

decline in virus collection that occurs during longer sample periods. Air samples started with the first 

appointments of the day and ran continuously until 4–5 hours of time elapsed. For each day of sampling, 

we collected 12 air samples (96 total samples). We selected the sampling dates in consultation with the 

dental practices based on expected patient load, with preference given to busier days.

We located the sampling pump with the aerosol sampler on the structural support arm immediately 

above the dental exam light. This placed the sampler within approximately 3 feet of the patient chair. 

These samples acted as surrogates for personal samples. We collected an additional area sample in dental 

patient rooms where we had placed a sampler above the dental exam light. The selection of patient rooms 

targeted those rooms scheduled with a full patient load on the day of sampling. Depending on the room 

dimensions, configuration, and work activities, area samples were collected at a distance of 6 feet or more 

from the typical patient location. When room configurations allowed it, area samplers were mounted 

on tripods at a height of approximately 5 feet from the floor to represent standing height. Alternatively, 

we placed samplers on top of shelves or cabinets. In addition to samples collected in patient rooms, area 

samples were obtained near the patient check in location.

All air sampling pumps (SKC Aircheck XR5000, Eighty Four, Pennsylvania) were calibrated using a 

volumetric flow calibrator (TSI Model 4146D, Shoreview, Minnesota) to operate at a flow rate of 3.5 Lpm. 

At a flow rate of 3.5 Lpm, the first centrifuge tube (T1) collects particles ≥ 4.1 μm (GSD 1.51), the second 

tube (T2) collects 1.0 to 4.1 μm particles (GSD 1.59) and the filter collects particles ≤ 1 μm. At 3.5 Lpm, 

the sampler conforms to the ACGIH/ISO criteria for respirable particle sampling [ISO 1995]. In addition 

to bioaerosol samples, characterization of total particle sizes in two patient rooms was performed using an 

optical particle counter (ART Instruments, Inc., HHPC-6, Grants Pass, Oregon). The particle counters 

were placed adjacent to an area bioaerosol sampler during data collection (See Figure 2 in the report text).

The arrows show the direction of air flow through the sampler. Air is drawn through the sampler using 

a small portable vacuum pump. Air flows into the 15 mL first stage centrifuge tube (T1) and swirls in a 

cyclonic motion. Particles larger than 4.1 μm are thrown against the wall of the tube by centrifugal force, 

where they stick. The air and remaining particles exit the first stage, flow around a bend, and enter the 

1.5 mL second stage centrifugal tube (T2). Since the second stage inlet and collection tube diameter are 

smaller than the first, air flow is accelerated and higher centrifugal forces are generated. Particles in the 

size range 1.0 to 4.1 μm are captured on the wall of the second tube. Finally, the air flows out of the 

second stage and passes through a 37 millimeter PTFE filter with 2.0 μm pore size (housed in a black

Appendix: Methods
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 27

Appendix: Methods (continued)

Figure 10. Schematic of the NIOSH two-stage cyclone aerosol sampler.

polypropylene conductive cassette), which captures particles smaller than 1.0 μm. The first stage has a 3 

mm ID inlet, a 6.1 mm ID outlet and a 6 mm vortex finder length. The second stage has a 1.3 mm ID 

inlet, 3 mm vortex finder length and a 2.5 mm ID outlet. The first and second stage inlets are at a 40° 

angle from the horizontal. The sampler is a modified version of that described previously [Lindsley et al. 

2006].

The two direct-reading, real-time instruments provided a qualitative assessment characterizing total 

airborne particulate levels in the dental practices across the size fraction of particles collected by the twostage cyclone aerosol samplers. The HHPC-6 hand-held particle counter (ART Instruments, Grants Pass, 

Oregon) determined the airborne particle counts based on optical counting principles using laser light 

scattering. This instrument measured the total number of particles per liter (particles/L) of air across six 

specific size cut points: 0.3–0.5 μm, 0.5–1.0 μm, 1.0–3.0 μm, 3.0–5.0 μm, 5.0–10.0 μm, and > 10.0 μm 

[Art Instruments, Inc. 2002]. Compared to the two-stage cyclone aerosol sampler, the first two size bins 

(smallest particle size ranges) of the HHPC identified the total particulate count for all particulate per liter 

of air sampled (viral and non-viral) that could be captured on the filter (Figure 10.) of the two-stage cyclone 
Page 28 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Appendix: Methods (continued)

aerosol sampler. Particles counted by the HHPC for 1.0–3.0 μm and 3.0–5.0 μm size fractions would be 

collected on the second stage collection tube of the two-stage cyclone aerosol sampler. HHPC counts for 

3.0–5.0 μm, 5.0–10.0 μm, and > 10.0 μm airborne particles reflect the particle sizes that could be collected 

by the first stage of the two-stage cyclone aerosol sampler. The HHPC counts all particles in the specified 

size ranges regardless of composition or source that are present in each 1 liter air sample. The HHPC 

was set to collect three samples per minute. The HHPCs were operated during the same time period that 

the two-stage cyclone aerosol samples were run to collect influenza virus. The two HHPCs were located 

adjacent to two separate two-stage cyclone aerosol samplers (See Figure 2). While the particulate size 

fractions collected by the HHPC did not align exactly with those of the two-stage cyclone aerosol sampler 

they provided a qualitative indication of the total number of particles present in the different size ranges 

from which the two-stage cyclone aerosol samplers were sampling for pH1N1 and seasonal influenza virus 

particles.

Air samples were transported back to the NIOSH laboratory upon completion of sampling. We added 

0.6 mL of lysis binding solution to the 15 and 1.5 mL centrifuge tubes from the samplers. The filters were 

transferred into a new 15 mL centrifuge tube and 0.5 mL of lysis binding solution added. All tubes were 

vortexed to distribute lysis binding solution throughout the sampling medium. These samples were also 

stored at -20°C and shipped on dry ice along with the surface samples to the NIOSH HELD analytical 

laboratory.

Surface Sampling

We did surface sampling of common high-contact non-porous surfaces to identify the presence of pH1N1 

and seasonal influenza virus. Samples were collected from some or all of the following surfaces on each 

day of air sampling: patient check-in/check-out countertop, doorknobs, patient room countertops, arms 

of patient chairs, handle of reusable dental tools (before cleaning), computer keyboard and mouse, 

and sink faucet handles. These samples were obtained in the same rooms where the air samples were 

collected. Surface samples were collected by swabbing the surfaces with sterile nylon flocked swabs (Copan 

Diagnostics, Corona, California) moistened with sterile phosphate buffered saline (Invitrogen, Aukland, 

New Zealand). For large flat surfaces a 10 cm x 10 cm template was used to collect a 100 cm2 surface area 

sample. For non-flat surfaces, 100 cm2 was estimated or the entire surface sampled (e.g., doorknob, dental 

tool handles, sink faucet handles) and the area estimated based on surface dimensions.

Immediately following collection, surface samples were placed into a 15 mL centrifuge tube (Becton 

Dickinson, Franklin Lakes, New Jersey) containing 0.5 mL of lysis binding solution (Ambion Diagnostics/

Applied Biosystems, Austin, Texas). Samples were stored at -20°C, then shipped on dry ice to the NIOSH 

HELD analytical laboratory.

Nasopharyngeal Sampling 

For NP sampling, a flexible flocked swab with a nylon tip (Copan Diagnostics, Inc., Murrieta, California) 

was inserted through each participant’s nose into the nasopharynx until resistance was encountered. The 

swab was rubbed gently and rolled for several seconds to absorb secretions before removing. Samples from 
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 29

Appendix: Methods (continued)

both nostrils were collected using the same swab. The swab was then immediately placed into a sterile vial 

containing 0.5 mL of lysis/binding solution concentrate. The swab stick was cut off to permit tightening 

of the cap. The NP samples were stored and shipped at -20°C to -70°C to the NIOSH HELD analytical 

laboratory.

Laboratory Analysis

Viral RNA Extraction

Immediately following collection, stages T1 and T2 aerosol samples were suspended in 500 μl of lysis 

binding solution concentrate (Ambion, Austin, Texas). Back-up filters were transferred to 50 mL 

polypropylene conical tubes (Becton Dickinson Labware, Franklin Lakes, New Jersey) containing 500 μl of 

the lysis binding solution concentrate. Nasopharyngeal swabs were placed in 15 mL polypropylene conical 

tubes (Becton Dickinson Labware, Franklin Lakes, New Jersey) containing 500 μl of the lysis binding 

solution concentrate. All aerosol and swab samples were stored at -20°C. Upon thawing, to enhance RNA 

extraction, each sample was spiked with carrier RNA (Ambion, Austin, Texas). Likewise, random samples 

were spiked with XenoRNA (Applied Biosystems, Austin, Texas), a qPCR internal control. Samples were 

subsequently transferred to designated wells in MagMax Express Microtiter 96-Deep Well Plates (Applied 

Biosystems, Austin, Texas). To complete the Lysis/Binding Solution preparation, 500 μl of isopropanol 

was added to each sample well. Total RNA was extracted from all samples using Applied Biosystems 

MagMaxTM Express-96, an automated Nucleic Acid Extractor, in conjunction with the MagMAXTM -96 

Viral RNA Isolation Kit (Ambion, Austin, Texas). Following isolation, RNA was immediately transcribed 

into cDNA.

cDNA Transcription

Complimentary DNA (cDNA) was generated by reverse transcription of the isolated RNA with use of 

the High Capacity RNA to cDNA Master Mix (Applied Biosystems). Samples were transcribed in an 

Eppendorf Mastercycler ep under the following conditions: 25°C for 5 minutes, 42°C for 30 minutes and 

85°C for 5 minutes. All samples were stored at -20°C until qPCR analysis.

qPCR Analysis

For real-time detection of seasonal influenza A and novel H1N1influenza, primers and probes from the 

CDC Swine Influenza Virus Real-Time RT-PCR Detection Panel (CDC REF. # FLUSW01, Lot # 904303) 

were used. Briefly, 20 μl of TaqMan Fast Universal Master Mix (Applied Biosystems, Austin, Texas) 

containing either influenza A or novel H1N1 specific primers and probes at a final concentration of 0.8 

μM and 0.2 μM, respectively, was added to 5 μl of sample cDNA. Reactions were performed and analyzed 

in the Applied Biosystems 7500 Fast Real-Time PCR System under the following cycling conditions: 95°C 

for 20 seconds followed by 50 cycles at 95°C for 15 seconds and 55°C for 30 seconds.
Page 30 Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120

Appendix: Methods (continued)

To determine the relative genome copy from seasonal influenza A-positive aerosol samples, a standard 

curve was generated from 10-fold serial dilutions of a plasmid-encoded influenza M1 matrix gene and 

analyzed concurrently with all qPCR reactions. A negative control without template was also included in 

all RT–PCR reactions. All reactions were run in duplicate.

Comparable to the analysis of influenza, real-time detection of RSV-positive aerosol samples was 

performed and analyzed in the Applied Biosystems 7500 Fast Real-Time PCR System using the following 

matrix specific primers and probe: Forward 5’ GGA AAC ATA CGT GAA CAA GCT TCA 3’, Reverse 5’ 

CAT CGT CTT TTT CTA AGA CAT TGT ATT GA 3’, Probe 6FAM- TGT GTA TGT GGA GCC TTMGBNFQ (Applied Biosystems, Austin, Texas).

To ensure that RT-PCR inhibitors were not present, real-time PCR detection of the Xeno RNA internal 

control was performed separately using the Xeno RNA Control TaqMan Gene Expression Assay from the 

TaqMan Cells to Ct Control Kit (Applied Biosystems, Austin, Texas). Cycling conditions were as follows: 

95°C for 20 seconds followed by 40 cycles at 95°C 3 seconds and 60°C for 30 seconds.

Positive Controls

The use of positive controls for the laboratory analyses confirmed that we would see pH1N1 seasonal 

influenza viruses in our samples if they were present. The high specificity of PCR methods for detecting 

pre-selected biological agents, in this case seasonal influenza A and pH1N1, allowed us to look for the 

virus of interest in the presence of other particulates. The seasonal qPCR assay (which selects for Matrix 

gene, segment 7 in the influenza A viruses) detects all influenza A strains. All samples were analyzed first 

using the qPCR assay for the presence of influenza A virus strains. Positive influenza A virus samples 

were further sub typed for seasonal and novel H1N1 (pH1N1) influenza viruses using an H1 and H3 sub 

typing qPCR assay. Tests for RSV on samples from Dental Practice B did identify this common respiratory 

virus encountered with children and often co-circulating with influenza viruses. Positive control samples 

prepared and handled the same as the field samples and blank came back identifying the presence of 

seasonal influenza. All field blanks for air and swab samples were negative for pH1N1and seasonal 

influenza. Additionally, all field blanks (swab and air sample) for Dental Practice B were negative for RSV.

References

ART Instruments, Inc. [2002]. Hand held particle counter (HHPC-6) User manual. Grants Pass, Oregon.

ISO [1995]. Air quality - Particle size fraction definitions for health-related sampling. Geneva, Switzerland: 

International Organization for Standardization, p. 9.

Lindsley WG, Schmechel D, Chen BT [2006]. A two-stage cyclone using microcentrifuge tubes for 

personal bioaerosol sampling. J Environ Monit 8(11):1136–1142.
Health Hazard Evaluation Report 2010-0019 & 2010-0021-3120 Page 31

Acknowledgments and

Availability of Report

The Hazard Evaluations and Technical Assistance Branch 

(HETAB) of the National Institute for Occupational Safety and 

Health (NIOSH) conducts field investigations of possible health 

hazards in the workplace. These investigations are conducted 

under the authority of Section 20(a)(6) of the Occupational Safety 

and Health Act of 1970, 29 U.S.C. 669(a)(6) which authorizes 

the Secretary of Health and Human Services, following a written 

request from any employer or authorized representative of 

employees, to determine whether any substance normally found 

in the place of employment has potentially toxic effects in such 

concentrations as used or found. HETAB also provides, upon 

request, technical and consultative assistance to federal, state, and 

local agencies; labor; industry; and other groups or individuals to 

control occupational health hazards and to prevent related trauma 

and disease.

The findings and conclusions in this report are those of the 

authors and do not necessarily represent the views of NIOSH. 

Mention of any company or product does not constitute 

endorsement by NIOSH. In addition, citations to websites 

external to NIOSH do not constitute NIOSH endorsement of 

the sponsoring organizations or their programs or products. 

Furthermore, NIOSH is not responsible for the content of these 

websites. All Web addresses referenced in this document were 

accessible as of the publication date.

This report was prepared by Steven H. Ahrenholz, Scott E. Brueck, 

and Marie A. de Perio of HETAB, Division of Surveillance, 

Hazard Evaluations and Field Studies. Analytical support was 

provided by Francoise Blachere and Don Beezhold, NIOSH Health 

Effects Laboratory Division. Health communication assistance 

was provided by Stefanie Evans. Editorial assistance was provided 

by Ellen Galloway. Desktop publishing was performed by Robin 

Smith.

Copies of this report have been sent to employee and 

management representatives at both dental practices, the state 

health department, and the Occupational Safety and Health 

Administration Regional Office. This report is not copyrighted and 

may be freely reproduced. The report may be viewed and printed 

at www.cdc.gov/niosh/hhe/. Copies may be purchased from the 

National Technical Information Service at 5825 Port Royal Road, 

Springfield, Virginia 22161.
Below is a recommended citation for this report:

NIOSH 2011. Health hazard evaluation report: environmental assessment for the 

presence of influenza viruses (2009 pandemic influenza A H1N1 and seasonal) 

in dental practices – Ohio. By Ahrenholz SH, Brueck SE, de Perio MA. Cincinnati, 

OH: U.S. Department of Health and Human Services, Centers for Disease Control 

and Prevention, National Institute for Occupational Safety and Health, NIOSH 

HETA No. 2010-0019 & 2010-0021-3120.

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