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The Journal of Infectious Diseases
BRIEF REPORT
Detection of Measles Virus RNA in Air
and Surface Specimens in a Hospital
Setting
Werner E. Bischoff,1 Rebecca J. McNall,2 Maria W. Blevins,1 JoLyn Turner,1
Elena N. Lopareva,2 Paul A. Rota,2 and John R. Stehle Jr1
these data can assist in explaining the highly contagious nature
of the virus and may guide the refinement of infection prevention measures. This is the first report of the environmental burden of MeV detected in the air, on surfaces, and on respirators
used by healthcare providers (HCPs) during routine care of a
patient hospitalized with measles.
1
Infectious Diseases, Wake Forest Baptist Health, Winston-Salem, North Carolina; and
Measles, Mumps, Rubella, and Herpesviruses Laboratory Branch, Division of Viral Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia
2
METHODS
Patient Information
Measles virus (MeV) is known to be highly contagious, with an
infectious period lasting from 4 days before to 4 days after rash
onset. An unvaccinated, young, female patient with measles
confirmed by direct epidemiologic link was hospitalized on
day 5 after rash onset. Environmental samples were collected
over the 4-day period of hospitalization in a single room.
MeV RNA was detectable in air specimens, on surface specimens, and on respirators on days 5–8 after rash onset. This is
the first report of environmental surveillance for MeV, and
the results suggest that MeV-infected fomites may be present
in healthcare settings.
Keywords. measles; aerosol; exposure; infectious period;
transmission; infection prevention.
An unvaccinated, otherwise healthy, immunocompetent young
woman developed extreme fatigue and fever 11 days after exposure to a known measles case [5]. On day 13 after exposure, a
diffuse rash started on the trunk and face, eventually spreading
to extremities. She was admitted to the hospital to rule out
pneumonia on day 18 after exposure, 5 days after onset of rash.
She received measles, mumps, and rubella vaccine 4 days after
exposure to the known case. The patient was placed on contact
and airborne isolation during hospitalization. Patient activities
such as movement and coughing were noted by study personnel. The Wake Forest Baptist Medical Center institutional
review board waived approval requirements owing to the study’s
limited focus on environmental sampling.
Measles is one of the most contagious viral infections known to
infect humans [1]. The disease is caused by measles virus (MeV),
a single-stranded, negative-sense, enveloped RNA virus of the
genus Morbillivirus within the family Paramyxoviridae. Measles is
a systemic infection characterized by fever, a maculopapular rash,
cough, coryza, and conjunctivitis. The incubation period ranges
from 8 to 14 days, with an average of approximately 10 days.
Measles cases are infectious from 4 days before rash onset
until 4 days after rash onset, with the rash generally occurring
around 14 days after infection [1]. MeV and MeV RNA can be
routinely detected in clinical samples collected >7 days after
rash onset [2], and MeV RNA can be detected in samples from
patients with measles for up to 3 months, especially in cases of
immunosuppression [3, 4].
Nosocomial transmission of measles is frequent. However, to
date, the presence of MeV in the environment occupied by a
MeV infected patient has not been studied directly. Obtaining
Setting
Received 6 July 2015; accepted 16 September 2015; published online 19 September 2015.
Presented in part: ID Week, Philadelphia, Pennsylvania, October 2014. Abstract 1221.
Correspondence: W. E Bischoff, Wake Forest University School of Medicine, Department of
Internal Medicine, Section on Infectious Diseases, Medical Center Blvd, Winston-Salem, NC
27157-1042 ([email protected]).
The Journal of Infectious Diseases® 2016;213:600–3
© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society
of America. All rights reserved. For permissions, e-mail [email protected].
DOI: 10.1093/infdis/jiv465
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Wake Forest Baptist Medical Center is an 885-bed tertiary-care
teaching hospital. Sampling was conducted in a single negativepressure isolation room under turbulent airflows (6 total air
changes/hour specified) at approximately 20°C and 40% relative
humidity. The room underwent daily cleaning of high-touch
areas.
MeV Sampling
Daily air samples were collected using 6-stage Andersen air
sampling devices at 3 locations: the head of the bed/chair,
2 feet away from patient head; the middle of the bed/chair,
4 feet away from the head; and the foot of the bed/chair, 8 feet
away from head [6]. The samples were collected in viral transport medium (VTM), aliquoted, and either added to AVL buffer
with carrier RNA (Qiagen) or directly frozen at −80C.
To test for MeV on environmental surfaces, sterile swabs
were moistened with VTM, and a 6.45-cm2 area was swabbed
once at 3 high-touch locations daily: the head of the bed hand
rail, the middle of the food tray table, and a table at the back of
the room (approximately 3 m from the foot of the bed). The
swabs were vortexed for 30 seconds and processed as described
for air samples.
Respirators (N-95) worn by HCPs were collected daily, and a
12.9-cm2 midsection of the respirator was cut out, using sterile
scissors. The cutouts were placed in 2 mL of VTM and frozen.
After thawing, samples were vortexed/agitated and further processed as described for air samples.
cytopathic effect was observed after 3 passages, the sample was
considered negative.
RNA Sample Preparation
RESULTS
RNA extraction was performed on samples containing AVL,
using the QIAmp Viral RNA Mini Extraction Kit (catalog
no. 52 906; Qiagen) per the manufacturer’s instructions. To
increase sample detection, the samples were concentrated by
extracting 3 replicates of each sample and loading them onto
1 QIAmp Mini column.
Real-Time Quantitative Reverse-Transcription Polymerase Chain
Reaction (qRT-PCR) Analysis
Real-time qRT-PCR analysis was used to detect MeV in samples, using primers targeting the nucleoprotein gene [7]. RNA
extracted from samples (2.5 µL) was used in each 25 µL reaction. Real-time qRT-PCR reactions were performed in duplicate, using the SuperScript III Platinum One-Step qRT-PCR
kit (catalog no. 11732-020; Life Technologies) on the ABI
7500 real-time instrument for 40 cycles.
Samples were considered positive for MeV if the threshold
cycle (Ct) value for both duplicates was <38 and negative if no
Ct value (denoting an undetermined result) was produced.
Samples that had discordant duplicates (Ct values that differed
by >1.5 or in which 1 duplicate had a negative result), and samples that had Ct values of 38–40 were considered to have indeterminate results and were repeated. Positive samples were
subjected to sequence analysis [8].
Tissue Culture Infectivity Testing
Vero/hSLAM cells were inoculated with 0.5 mL of sample and
maintained in Dulbecco’s modified Eagle’s medium supplemented with 2% fetal bovine serum, 100 µ/mL penicillin, 100 µg/mL
streptomycin, and 0.4 mg/mL G418 sulfate [9]. Cells were observed by light microscopy on a daily basis to look for cytopathic
effect. Three passages of infected cells were performed. If no
Figure 1.
The patient spent the first 48 hours mostly in bed sleeping, followed by being alert and sitting up in bed during sampling. She
had minor coughing episodes on day 5, which intensified on
days 6 and 7 after rash onset.
MeV RNA was detected in aerosol samples on days 5 (420
MeV RNA copies/10-L respiratory volume/minute; 1 positive
sample) and 7 (1517 MeV RNA copies/10-L respiratory volume/minute; 3 positive samples) after rash onset at all sampling
locations. Particle size distribution changed with distance to the
patient’s head, with particles >4.7 µm detected only close to the
patient’s head and small particles <4.7 µm detected at the middle and foot of the bed (Figure 1).
Surface samples tested positive for MeV RNA on all 4 study
days, with higher copy numbers on days 6 and 7 after rash onset
(Figure 2). The bed rail at the head of the bed showed the highest average copy number, with 564736 MeV RNA copies/cm2
(4 positive samples), followed by the bedside table (13 MeV
RNA copies/cm2; 1 positive sample) and a second table 3 m
away (1 MeV RNA copies/cm2; 1 positive sample).
Out of 134 samples from respirators, 4 (3%) tested positive
for MeV RNA, all of which were worn on day 6 after rash
onset. The RNA copy numbers ranged from 123 to 570 MeV
RNA copies/cm2, with an average of 333 MeV RNA copies/cm2.
Sequencing was attempted on 8 of the positive samples, including all 6 positive surface swabs, the strongest positive air
sample, and the strongest positive respirator sample. Sequences
were obtained for 4 of these samples (the respirator sample and
3 surface samples), and all sequences were identical and members of measles genotype D8. These sequences were also identical to sequences obtained from clinical specimens collected
Spatial distribution of the average aerosol concentrations of measles virus (MeV) over a 20-minute sampling period in the patient’s room.
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Figure 2.
Environmental surface burden of measles virus (MeV) during hospitalization (cumulative totals).
during the outbreak in North Carolina [5]. Tissue culture infectivity testing did not detect MeV in a selection of PCR-positive
samples.
DISCUSSION
Endemic measles transmission was declared in 2000 to have
been eliminated in the United States, because of an effective
vaccination program that has maintained very high coverage
rates. In 2011, the Pan American Health Organization verified
that surveillance parameters are adequate to ensure the maintenance of measles elimination in the United States [10]. However, measles is endemic in many parts of the world, and imported
cases continue to cause outbreaks in the United States. Outbreaks are often associated with transmission in healthcare settings [11]. Understanding the risk of exposure to MeV could
help to develop more-effective infection control procedures.
This study attempted to assess the environmental burden of
MeV in a hospital setting during routine patient care. To our
knowledge, this is the first study to direct detect evidence of airborne transmission of MeV.
Current Centers for Disease Control and Prevention guidelines for healthcare workers recommend airborne isolation for
all patients suspected of having measles in healthcare settings
[12]. The patient described in our report had a known epidemiologic link to a laboratory-confirmed measles case and displayed the classical symptoms; the patient was hospitalized 5
days after rash onset. Beginning on the first day of hospitalization, MeV RNA was detectable in the air up to 2.43 m from the
patient, on surfaces within the hospital room, and on respirators
worn by visitors and HCPs. MeV RNA detection patterns were
associated with increased patient coughing and movement.
MeV RNA was detected up to 2.43 m from the patient, with
larger droplets closer to the patient’s head and smaller droplet
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nuclei further away. Transmission via air has been associated
with the explosive character of measles outbreaks, reflected in
high basic reproductive rates of 15–20 [1]. Entry routes of the
virus are thought to be through target cells in both alveolar
spaces and the mucocilliary epithelium of the mid and upper
respiratory tract [13]. This makes transmission by exposure to
the broad range of particle sizes and the long distance, as detected in this study, possible.
Transmission of MeV may occur not only through air but
also through contact with surfaces in the direct-care environment of a patient. MeV survives on surfaces for <2 hours
[14]. MeV RNA was detected in high concentrations on several
high-touch areas, making transmission via indirect contact
possible. Virus transfer through touching and settling of MeVassociated particles out of the air probably led to contamination
of the areas in the close-care environment. However, MeV RNA
was also detected on a rarely touched table 3 m from the patient,
making deposition of MeV solely by air more likely.
Patient activities such as coughing have been associated with
increased levels of influenza virus detection [6]. The patient
with measles coughed more frequently during days 6 and 7, coinciding with the highest frequency of detection of viral RNA.
Experimental infection of rhesus macaques showed large numbers of MeV-infected epithelial and lymphoid cells in the upper
respiratory tract, and coughing and sneezing led to the shedding
of MeV-infected cells, cell debris, and cell-free virus [15]. This
suggests that there are at least 2 possible mechanisms of MeV
transmission: from close contact with cell-associated virus
and from cell-free MeV in small particles. Cell-associated transmission via large particles (>4.7 μm) may also be more effective
than cell-free transmission, by providing greater protection
against environmental hazards such as temperature, humidity,
and UV radiation than cell-free transmission.
MeV RNA was also detected on N95 respirators worn by
HCPs. Four of 134 masks tested positive for MeV RNA, matching the high level of air and surface detection observed on day
6. Testing was limited to the center portion of the respirator exposed to the air currents of the wearer. While contamination
due to touching cannot be excluded, correct donning and removal usually do not expose the midsection of the mask. This
novel detection method provides a direct assessment of the individual exposure to airborne viruses.
Of course, this study is limited to one patient, and shedding patterns are likely to vary considerably between patients. In this case,
environmental testing was not initiated until day 5 after rash onset
and did not occur during the postulated infectious period. Testing
of PCR-positive samples in cell cultures failed to detect viable
MeV, making it impossible to assess the potential of these samples
to serve as a source of infection. However, these results show that
environmental surveillance for MeV is possible and suggest that
additional studies to measure shedding of MeV may provide insight into understanding the highly infectious nature of MeV.
Notes
Disclosure. The findings and conclusions in this report are those of the
authors and do not necessarily represent the official position of the Centers
for Disease Control and Prevention.
Potential conflicts of interest. All authors: No reported conflicts. All
authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content
of the manuscript have been disclosed.
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