- Email: [email protected]
Molecular epidemiology and environmental contamination during an outbreak of parainfluenza virus 3 in a haematology ward
Accepted Manuscript Molecular epidemiology and environmental contamination during an outbreak of parainfluenza virus 3 in a hematology ward Taeeun Kim, Choong Eun Jin, Heungsup Sung, Bonhan Koo, Joungha Park, Sun-Mi Kim, Ji Yeun Kim, Yong Pil Chong, Sang-Oh Lee, Sang-Ho Choi, Yang Soo Kim, Jun Hee Woo, Jung-Hee Lee, Je-Hwan Lee, Kyoo-Hyung Lee, Yong Shin, Sung-Han Kim PII:
S0195-6701(17)30493-0
DOI:
10.1016/j.jhin.2017.09.003
Reference:
YJHIN 5217
To appear in:
Journal of Hospital Infection
Received Date: 26 June 2017 Revised Date:
0195-6701 0195-6701
Accepted Date: 5 September 2017
Please cite this article as: Kim T, Jin CE, Sung H, Koo B, Park J, Kim S-M, Kim JY, Chong YP, Lee S-O, Choi S-H, Kim YS, Woo JH, Lee J-H, Lee J-H, Lee K-H, Shin Y, Kim S-H, Molecular epidemiology and environmental contamination during an outbreak of parainfluenza virus 3 in a hematology ward, Journal of Hospital Infection (2017), doi: 10.1016/j.jhin.2017.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Molecular epidemiology and environmental contamination during an outbreak of
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parainfluenza virus 3 in a hematology ward
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Taeeun Kim1,2§, Choong Eun Jin3§, Heungsup Sung4, Bonhan Koo3, Joungha Park5, Sun-Mi
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Kim1, Ji Yeun Kim1, Yong Pil Chong1, Sang-Oh Lee1, Sang-Ho Choi1, Yang Soo Kim1, Jun
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Hee Woo1, Jung-Hee Lee6, Je-Hwan Lee6, Kyoo-Hyung Lee6, Yong Shin3†, Sung-Han
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Kim1†
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Departments of Infectious Diseases1, Convergence Medicine3, Laboratory Medicine4, Medicine5, and Hematology6, Asan Medical Center, University of Ulsan College of
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Medicine, 88 Olympicro-43gil, Songpa-gu, Seoul, Republic of Korea.
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University Hospital, 79, Gangnam-ro, Jinju, Gyeongsangnam-do, 52727, Republic of
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Korea
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Division of Infectious Diseases, Department of Internal Medicine, Gyeongsang National
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Co-corresponding authors:
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These authors contributed equally to this work.
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Sung-Han Kim, MD. Department of Infectious Diseases, Asan Medical Center,
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University of Ulsan College of Medicine, 88 Olympicro-43gil, Songpa-gu, Seoul, Republic
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of Korea (e-mail: [email protected])
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Tel: 82-2-3010-3305
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Fax: 82-2-3010-6970
Yong Shin, PhD. Department of Convergence Medicine, Asan Medical Center,
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University of Ulsan College of Medicine & Biomedical Engineering Research Center, Asan
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Institute for Life Sciences, Asan Medical Center, 88 Olympicro-43gil, Songpa-gu, Seoul, 1
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Republic of Korea (e-mail: [email protected])
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Tel: 82-2-3010-4193
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Running Title: An hPIV-3 outbreak in a hematology ward (40 characters – limit 40
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characters including spaces)
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Words count of abstract- 250 (limit 250)
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Words count of text- 3494 (limit 4000)
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Number of Tables- 1, Number of Figures- 4
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Number of References- 27
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ACCEPTED MANUSCRIPT Summary (250 words)
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Background: Although fomites or contaminated surfaces have been considered as
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transmission routes, the role of environmental contamination by human parainfluenza virus
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type 3 (hPIV-3 ) in healthcare settings is not established.
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Aim: To describe an hPIV-3 nosocomial outbreak and the results of environmental sampling
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to elucidate the source of nosocomial transmission and the role of environmental
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contamination
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Methods: During an hPIV-3 outbreak between May and June 2016, we swabbed
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environmental surfaces in contact with clustered patients and used respiratory specimens
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from infected patients and epidemiologically unlinked controls. We investigated the
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epidemiologic relatedness of hPIV-3 strains by sequencing of the hemagglutinin-
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neuraminidase and fusion protein genes.
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Results: Of 19 hPIV-3 infected patients, 8 were hematopoietic stem cell recipients and 1 was
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health care worker (HCW). In addition, 4 had upper and 12 had lower respiratory tract
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infections. Of the 19 patients, 6 (32%) were community-onset (symptom onset within <7
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days of hospitalization) and 13 (68%) were hospital-onset infections (≥7 days of
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hospitalization). Phylogenetic analysis identified two major clusters: 5 patients, and 3 patients
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plus one HCW. Therefore, 7 (37%) were classified as nosocomial transmissions. hPIV-3 was
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detected in 21 (43%) of 49 environmental swabs up to 12 days after negative respiratory PCR
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conversion.
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Conclusion: Our data demonstrated that at least one-third of a peak season nosocomial hPIV-
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3 outbreak originated from nosocomial transmission, with multiple importations of hPIV-3
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from the community, providing experimental evidence for extensive environmental hPIV-3
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contamination. Direct contact with the contaminated surfaces and fomites or indirect
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transmission from infected HCWs could be responsible for nosocomial transmission.
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Key Words: human parainfluenza virus type 3; nosocomial outbreak; hematology unit;
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phylogenetic analysis; environmental contamination
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Human parainfluenza viruses (hPIV) are, along with human respiratory syncytial virus (RSV),
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are well-recognized respiratory pathogens in infants and young children, ranging from mild
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upper respiratory tract (URI) symptoms to croup and pneumonia [1, 2]. In the
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immunocompetent adult, URIs are mild and self-limiting in nature, and re-infections can
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occur due to incomplete immunity to hPIV. However, in the immunocompromised host,
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especially in hematopoietic stem cell transplant (HSCT) recipients and patients with
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hematologic malignancies, hPIV causes a significant morbidity and mortality with prolonged
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virus shedding [3-5]. Of the five known human serotypes, human parainfluenza virus type 3
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(hPIV-3) is the most common pathogen [4]. Epidemics of hPIV are seasonal, with the peak of
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activity usually between May and June each year in South Korea, accounting for about 20-
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35% of detected respiratory virus agents in the community [6].
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There are several reports of hPIV-3 nosocomial outbreaks in HSCT recipients or patients with
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hematologic malignancy [7-14]. In these reports, while some outbreaks were due to a single
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strain circulating within the unit [7, 12], others originated from multiple hPIV-3 strains
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introduced from the community [11, 14]. Sequencing and phylogenetic analysis of hPIV-3
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strains allowed for verification of whether a single strain or multiple strains were responsible
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for the outbreak. In addition, in some of these reports, infection control, including
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symptomatic surveillance and isolation, were often ineffective in terminating transmission,
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suggesting asymptomatic viral shedding among patients, staff, or outside visitors or
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environmental contamination as a possible explanation [9]. Although fomites or contaminated
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surfaces have been considered as possible transmission routes of hPIV-3 [15, 16], in addition
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to droplets or close personal contact; the role of environmental contamination by hPIV-3 in
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healthcare settings has not been well defined. In the present study, we describe an hPIV-3
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nosocomial outbreak in a hematology ward and the results of environmental sampling to
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elucidate the source of nosocomial transmission and the role of environmental contamination.
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Hospital setting and patient population
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This study was conducted at a hematology unit in a 2,700-bed tertiary care hospital in Seoul,
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South Korea. The hematology unit, which serves chemotherapy for patients with hematologic
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malignancy and hematopoietic stem cell transplantation (HCT), consists of 2 wards located
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on different floors. One ward (A) has 42 beds; 12 of these are for patients undergoing HCT.
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The other ward (B) has 50 beds, including a mixture of single-, two-, five-, and six-bedded
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rooms, communal showers, and a visiting room for patients on post-HCT care or cytotoxic
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chemotherapy. These wards routinely allow limited visitation due to the
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immunocompromised status of admitted patients.
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In mid-June 2016, an increased number of hPIV-3 positive cases were identified, clustered in
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a six-bed room on ward B. By the end of June, one or two more cases were confirmed each
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day, leading to the notification of an outbreak in the hematology unit by the Infection Control
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team. After thorough review, all laboratory-confirmed hPIV-3 patients and symptomatic
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healthcare workers (HCWs) in the hematology ward during this period were enrolled in this
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study. Since the incubation period of hPIV-3 infection in adults has been estimated to be 2-6
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days [17-19], an infection was considered to be hospital-onset if the patient had been
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hospitalized for ≥7 days before the onset of respiratory symptoms. All rooms occupied by the
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original cluster of diagnosed patients, including the six-bedded room, the adjacent room, and
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the isolation room were selected for the environmental study. This study was approved by the
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institutional review board of Asan Medical Center.
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Sample collection
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Respiratory samples, including nasopharyngeal swabs/aspirates and bronchoalveolar lavage 8
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outbreak, respiratory samples were tested for respiratory viruses including hPIV types 1-4,
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adenovirus, influenza A and B virus, rhinovirus, human metapneumovirus, bocavirus,
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coronavirus 229E, NL63, and OC43, enterovirus, and RSV types A and B by real-time
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multiplex RT-PCR as a routine screening of any patient or healthcare worker presenting with
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respiratory symptoms and/or fever. Additional microbiological investigations performed on
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sputum or BAL fluid included Gram stain, acid-fast stain, and cultures for conventional
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bacteria, mycobacteria, and fungi, as needed. Samples were also tested for galactomannan
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and CMV DNA load (CMV qPCR) on blood and BAL fluid, if invasive aspergillosis or CMV
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disease was suspected. Once the hPIV-3 infection was diagnosed, respiratory samples for
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hPIV-3 testing were taken weekly until negative or patient discharge. Thus, we did not take
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further respiratory samples at the time of environmental sampling.
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Dacron swabs pre-moistened with viral transport medium were used to aseptically swab
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surfaces that were frequently touched by patients or healthcare workers. The following types
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of surface were swabbed: (1) fomites (ie, stethoscopes, infusion regulator, had sanitizer tip,
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nasal prongs or masks, pillows, curtains and TV remote control); (2) fixed structures in the
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rooms and their associated restrooms (ie, doorknobs, bed side rails, toilet seats, call button,
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telephone buttons, hand sanitizing dispensor, and light switch). All environmental samples
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were collected after routine cleaning or periodic linen change. The standard cleaning
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procedures of the rooms included routine bed cleaning (e.g., bed rails, control panel, call bell,
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bedside locker, switches, telephone, and main door knob) and toilet cleaning at least daily;
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and discharge bed cleaning after the patients moved or were discharge.
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Eleven isolates that were concurrently circulating in the community were obtained from 9
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respiratory specimens of epidemiologically unlinked patients from different wards of our
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institution during the same period to be used as controls.
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Real-time RT-PCR and hPIV-3 strain sequencing
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Viral RNA was extracted from all respiratory or environmental specimens using QIAmp®
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Viral RNA Mini Kit (Qiagen, Germany). The respiratory specimens from 19 patients with
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confirmed hPIV-3 and environmental samples were included in the subsequent
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epidemiological investigation, involving partial sequence analysis of the hemagglutinin-
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neuraminidase (HN) gene. In addition, 11 hPIV-3 isolates that were obtained from respiratory
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specimens of epidemiologically unlinked patients on different wards of our institution during
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the same period were also sequenced as controls. RT-PCR was performed on the extracts
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using primers F (5’-ATT ACT CGA GGT TGC CAG GA-3’) and R (5’-CCG CGA CAC
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CCA GTT GTG-3’) covering 450 bp of the HN gene. The OneStep RT-PCR Kit (Qiagen,
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Germany) was used according to the manufacturer’s instructions, with 5 µL of extract being
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amplified in a final volume of 25µL, containing 5X one-step RT-PCT buffer, 0.25 mM
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deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of one-step RT-PCR
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Enzyme Mix. Thermal cycling conditions were as follows: 30 min at 50 °C, followed by 15
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min at 95 °C and 50 cycles of 30 s at 95 °C, 30 s at 50 °C, and 30 s at 72 °C, and a final
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elongation step at 72 °C 10 min. Sequences were aligned with the MEGA 7.0 software using
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the Clustal W method. Phylogenetic analysis was carried out using the MEGA 7.0 software
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maximum likelihood method, while the kimura 2-parameter was selected as an evolution
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model, and the bootstrap value was 1,000. We further analyzed the sequence of fusion protein
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(F) gene for clarifying the epidemiologic links within an equivocal cluster. The OneStep RT-
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PCR Kit (Qiagen, Germany) was also used with primers F (5’-CTT TGG AGG GGT AAT
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TGG AAC TA-3’) and R (5’-ATG ATG TGG CTG GGA AGA GG-3’) covering 621 bp of F
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gene. Thermal cycling conditions were followed as previously described. A positive result
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was confirmed by agarose gel electrophoresis and visualization by UV transilluminator.
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Statistical analysis
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The Kaplan-Meier method was used to construct survival curves for the time period during
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which patients had positive RT-PCR results. As flock swab samples were not obtained at
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discharge from patients who recovered, we defined these patients who did not undergo
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follow-up flock swab sampling, as “censored” patients. All statistical analyses were
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performed using SPSS version 21.0 (IBM Co., Armonk, NY).
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hPIV-3 outbreak
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From May 19, 2016 through June 30, 2016, 19 patients with hPIV-3, including 8 HCT
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recipients and 1 HCW, were identified in hematology ward B (Figure 1). Table 1 shows the
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clinical characteristics of these patients. The mean age of the patients was 44 years. The
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majority of the patients were lymphopenic (12 [64%]). Six of the 8 HCT recipients had
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undergone transplantation < 12 months before hPIV-3 infection developed. Of the 19 infected
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patients, 4 had upper and 12 had lower respiratory tract infections, and 5 (26%) died of hPIV-
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3 infection. Of the 19 patients, 6 (32%) were community-onset infection (symptom onset
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within < 7 days of hospitalization) and 13 (68%) were hospital-onset infection (symptom
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onset within ≥7 days of hospitalization). Fourteen patients (74%) had other serious concurrent
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infections, including invasive fungal infections (8 patients). Median length (IQR) of viral
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shedding from respiratory specimen was 18 days (14-19 days).
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Molecular analysis
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Phylogenetic analysis of the HN gene of the 42 hPIV-3 strains relevant to the 19 infected
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patients, 11 control patients unrelated to this outbreak, and 21 environmental samples of 49
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swab specimens are shown in Figure 2. Two major clusters were identified: cluster #1,
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including 30 identical hPIV-3 strains (relevant to patient A, E, B, C, D, and 2 control patients),
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and cluster #5, including 17 identical hPIV-3 strains (relevant to patient F, G, H, I, J, K, and 3
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control patients). Further analysis of the F gene within cluster #5 (17 hPIV-3 strains relevant
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for the 6 patients and 3 control patients) was performed because the HN gene had low
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discriminate power within cluster #5. The F gene analysis revealed the sub-cluster of 8
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identical hPIV-3 strains (relevant to patient H, I, J, K, the last hPIV-3 strains from patient F,
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and 1 control patient). Other clusters consisted of 3 or 4 identical hPIV-3 strains isolated
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from1 patient and one or two control patients.
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Epidemiological analysis
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The temporal and spatial relationships among the 11 outbreak cases of hPIV-3 belonging to
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the two identified major clusters (ie, cluster #1 and #5) are shown in Figure 3. These analyses
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revealed that 7 (37%) cases including one re-infection case were classified as nosocomial
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transmissions. The 6 cases except one re-infection case were initially classified as hospital-
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onset infection (symptom onset within ≥7 days of hospitalization). Genetically identical
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strains are identified by the same inner circle colors; likewise different rooms are indicated by
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different box colors. Within cluster #1, patient A, the index case, had positive hPIV-3 PCR
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results over 10 days and infected 4 patients. Of these 4 patients, 3 were hospitalized in the
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same room with patient A at the time of hPIV-3 diagnosis. Finally, patient A moved to a
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single room (room 5) for effective isolation. After isolation of patient A, the same nursing
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team cared for the patients in room 5 (patient A) and room 34 (patient D).
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Within cluster #5, patient F, G, I, and J presented symptoms associated with hPIV-3 infection
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or positive hPIV-3 PCR results on admission. According to the additional F-gene sequence
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analysis, initial hPIV-3 strains isolated from these 4 patients were distinctive community-
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acquired strains. Patient I, who had acquired hPIV-3 in the community, was admitted through
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the emergency department and transmitted hPIV-3 to patient H, F (reinfection), and K (HCW).
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Even though the HCW (patient K) was positive for hPIV-3 by PCR on 28 June, she
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developed symptoms two weeks prior. During her symptomatic period, she cared for the
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patients in room 34, including patient F. The last (6th) hPIV-3 isolate from patient F was a
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different strain from the other isolates from patient F, and the same strain as that of the sub-
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cluster. Patient F succumbed to re-infection by the last hPIV-3 strain in a hospital ward, either
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from other patients (ie, patient I or H) or the HCW (patient K). The pairwise comparison
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among all 6 strains from patient F and I also support our explanation of re-infection in patient
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F (Supplemental Figure 1).
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Environmental contamination
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On July 5th, environmental samples were collected in three rooms of 4 patients (patient A, B,
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C, and D) within cluster #1. By this day, all but 1 patient, who was discharged just before
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environmental sampling, had negative PCR results from their respiratory specimens. Of the
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49 swab samples collected, 21 samples tested positive for hPIV-3 by RT-PCR and HN
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sequencing (Supplemental Table 1). In particular, 11 of 20 fomite swabs and 10 of 29 fixed-
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structure swabs were positive for hPIV-3. Room 5 (Figure 4B) was the single-bed room used
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for the isolation of patient A, the index case of cluster #1. Out of 15 swab samples, 12 tested
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positive for viral RNA despite being swabbed, 12 days after the patient’s last positive PCR
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and 5 days after a negative PCR for hPIV-3.
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Room 33 (Figure 4C), the six-bedded room, was where it was speculated that the hPIV-3
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outbreak began. Six of 15 swab samples were positive for hPIV-3, 17 days after patient B’s
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last positive PCR and 12 days after a negative PCR. Her symptoms had resolved by the time
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of environmental sampling. Patient C only had mild rhinorrhea at the time of environmental
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swab. Of 11 swab samples, 2 were positive for viral RNA, 8 days after the patient C’s last
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positive PCR and 1 day after negative PCR. Patient E was discharged 5 days prior to
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environmental sampling. In room 34 (Figure 4D), the five-bedded room, only the stethoscope
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tested positive for viral RNA. Environmental sampling of this room was performed
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immediately after cleanup following patient D’s discharge. The RT-PCR results of the
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environmental specimens revealed the fluid infusion regulator (3 of 3 specimens), TV remote
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hPIV-3. Detailed information of all tested environmental samples is depicted in Figure 4. The
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virus strains isolated from the environmental samples were consistently found to be identical
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to their source patients (Figure 2).
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Our data demonstrated that an hPIV-3 outbreak in a hematology unit caused considerable
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morbidity and mortality in a substantial portion of patients with hematologic malignancies,
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and that at least one-third of an hPIV-3 outbreak resulted from nosocomial transmission,
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originating from multiple importations of hPIV-3 strains from the community during its peak
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epidemic season. In addition, we found that the extensive environmental contamination of
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hPIV-3 occurred in patients with negative PCR conversion from his/her respiratory specimen
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despite the routine disinfection procedures. Therefore, our findings provide important
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evidence for the probable routes for nosocomial transmission and emphasize the importance
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of strict adherence to infection control precautions and the limited visitation during periods of
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high hPIV-3 activity in the community.
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This study clearly showed that both multiple importations from the community and
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subsequent nosocomial transmission equally contributed to this hPIV-3 outbreak. As the
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vulnerability of our healthcare system, such as patients occupying rooms with many beds,
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communal showers or a visiting room, and the visitation to hospitalized patients by friends
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and family members, was shown in the large Middle East respiratory syndrome outbreak in
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South Korea, we would like to elucidate which transmission route is important in our
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complex situation during an hPIV-3 outbreak. Previous studies have shown that molecular
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investigations are useful in discerning routes of transmission in hPIV-3 outbreaks [7-11, 14,
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20, 21]. One study demonstrated that both community-acquired and nosocomial transmitted
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infections occurred during one hematology unit outbreak [8], while others reported that
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nosocomial infections play a more significant role than multiple importations from the
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community [7, 14, 21]. This discrepancy among studies arises from the differences in terms
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of hPIV-3 activity in the community. It has been accepted that the epidemiology of
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community respiratory viral infections, including hPIV-3, in inpatients often mirrors the
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epidemiology in the outpatient population [22]. In addition, differences in infection control
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procedure, the presence of multi-patient rooms, and visits to hospitalized patients by friends
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and family members might contribute to the conflicting results of previous studies.
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The current scientific evidence suggests that fomites and contaminated surfaces are more
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concerning routes of hPIV transmission compared to aerosol [2, 23]. By air sampling, hPIV-1
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was isolated from air obtained in only 1 of 150 infected children at a distance of 60 cm [24].
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However, nasal secretions can travel a distance greater than 10 feet to contaminate
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surrounding fomites [23]. An experimental study demonstrated that hPIV can survive in
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stainless steel for 10 hours [16]. Although the transfer of hPIV-3 from finger to finger or
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finger to metal disk did not occur, hPIV-3 could be transferred from contaminated disks to
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clean hands [25]. Therefore, person-to-person spread by direct hand contact appears to be an
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unlikely mode of transmission, whereas contaminated surfaces may lead to direct self-
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inoculation. To date, only these pieces of indirect experimental evidence have supported
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contaminated surfaces as a mode of hPIV-3 transmission. In this context, the extensive
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environmental contaminations and prolonged environmental presence of hPIV-3 were
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obviously demonstrated, and these findings may partially explain why hPIV-3 is easily spread
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in the hematology ward. In addition, this emphasizes the importance of disinfection of
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environmental surfaces. It is noteworthy that hPIV-3 infection was documented in one
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minimally symptomatic HCW who obtained this virus from the hPIV-3-infected patients.
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Although it is not exactly known whether asymptomatic or mildly symptomatic HCWs can
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transmit hPIV-3, asymptomatic hPIV-3 viral shedding in the nasopharynx among
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immunocompetent hosts [17, 26] and the previous outbreak reports suggest the potential role
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of asymptomatic healthy carriers in nosocomial transmission [9]. In our data, we could not
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define the precise role of none-to-minimal symptomatic HCWs in nosocomial transmission.
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However, given that the same nursing team cared for the index patients and the lastly infected
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cases of cluster #1, and 1 of the nursing team members (patient K in cluster #5) had
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documented hPIV-3 infection, HCWs may have a role in nosocomial transmission. Further
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studies are needed on this issue.
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In the immunocompromised host, hPIV is associated with significant morbidity and mortality.
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We observed a mortality rate of 26% during a single hPIV-3 outbreak episode. In previous
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reports, the mortality rate of hPIV-3 infection varied, ranging from 3%-47% according to the
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type of transplant, conditioning and immunosuppression therapy, and post-transplant
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complications [4, 8, 10, 11]. However, in the majority cases, it is difficult to verify the cause
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of death as hPIV-3 infection, and thus only an association of hPIV-3 infection and death can
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be documented. Particularly in HSCT recipients, LRTI with hPIV significantly increased the
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risk of airflow decline (odds ratio, 17.9 [95% confidence interval, 2.0-160]) [27], and this
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complication is associated with increased mortality risk. In our study, one of 5 patient deaths
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had a post-HSCT pulmonary complication and died due to pulmonary function deterioration
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following hPIV-3 pneumonia. Considering the morbidity and mortality associated with hPIV-
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3 infection in the immunocompromised hosts and the absence of effective antiviral agents or
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vaccination, prevention via more thorough infection control is essential. Interestingly, we
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clearly demonstrated that hPIV-3 re-infection occurred during the hospital stay (patient F,
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Figure 3 and Supplemental Figure 1). There is a need for more careful and comprehensive
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prevention guidelines during the outbreak in hematology unit.
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This study has a few limitations. First, samples positive for viral RNA by RT-PCR may not 18
ACCEPTED MANUSCRIPT contain live virus with infectivity. However, the molecular analysis showed extensive and
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prolonged environmental viral contamination surrounding clustered hPIV-3 infected patients
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by almost same strain. Particularly, the fomites, easily overlooked during routine daily
337
cleaning (i.e., TV remote control) or frequently manipulated by HCWs (i.e., the fluid infusion
338
regulator and stethoscope), were consistently positive during environmental sampling and
339
might have served as the episource during this outbreak. Therefore, these data provide more
340
direct insight into the possible routes of nosocomial transmission of hPIV-3. Second, we did
341
not perform air sampling. A previous study showed the limited role of aerosol as a mode of
342
hPIV transmission [23]. Lastly, in the phylogenetic analysis of the HN gene, differences in a
343
small number of nucleotide (nt) (1-3 bp) within the given 450 bp were grouped together.
344
Although one might postulate that even more than 3 nt differences may not represent
345
different strains of viruses but rather reflect errors introduced during RT-PCR, the obvious
346
epidemiologic link supports the grouping in cluster #1. On the contrary, in cluster #5, due to
347
the low discriminative ability of the HN gene analysis and uncertain epidemiologic link, we
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further analyzed the F gene, another variable region, and carried out sequence alignments for
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hPIV-3 amplicons in some strains.
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In conclusion, this study suggested that an apparently single nosocomial outbreak was
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equally attributed to multiple importations of hPIV-3 strains from the community and
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subsequent nosocomial transmission. Furthermore, these data provide further direct
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experimental evidence for extensive and prolonged viral contamination of materials
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surrounding hPIV-3 patients and/or from minimally symptomatic HCWs as probable
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nosocomial transmission routes. The findings of the present study indicate and reinforce the
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idea that preventive measures should consist of the isolation of infected patients, routine hand
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washing with ethanol-based disinfectants by HCWs, patients, and visitors, meticulous
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environmental cleaning, and limited patient visitation.
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Acknowledgements
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We thank Hye-Won Jung (e-medical contents team, Asan Medical Center, Seoul, South
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Korea) for the excellent artwork; Vanessa Topping (Scientific publications team, Asan
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Medical Center, Seoul, South Korea) for assistance and thoughtful comments.
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Description of Conflicts of Interest
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There are no potential conflicts of interest for any authors.
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Funding
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This work was supported by a grant of the Korea Health Technology R&D Project through
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Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &
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Welfare, Republic of Korea (grant no. HI15C2774).
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Ansari SA, Springthorpe VS, Sattar SA, Rivard S, Rahman M. Potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus 3
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community respiratory viruses. J Infect Dis 2006; 193:1619-25.
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Figure Legends
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Figure 1. Number of hPIV-3 cases detected during outbreak in ward B.
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Figure 2. Phylogenetic analysis of the 65 identified hPIV-3 strains. A. Sequence comparison
454
of PIV3 sequenced in the hemagglutinin-neuraminidase (HN) gene of 450bp. B. Sequence
455
comparison of PIV3 sequenced in the fusion protein (F) gene from cluster 5. GenBank
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accession numbers are KP690792 for hPIV-3 strain 11-s-83, GU732139 for hPIV-3 strain
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BJ/036/08, KT898932 for hPIV-3 strain Pavia/15215/2014, and KJ672616 for hPIV-3 strain
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PER/FLU8925/2007.
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Figure 3. Chronological appearance of the hPIV-3 strains of the 11 infected patients in cluster
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#1 and cluster #5 during the outbreak.
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Figure 4. A. Floor plan of the hematology unit. Room numbers are indicated in red stars. B.
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Results of viral PCR of swabs from patient room No.5. Patient A was a 48-year-old woman
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with pneumonia on noninvasive ventilation. She moved from room No. 33 to room No. 5 on
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day 12 from after the first positive hPIV-3 PCR. C. Results of viral PCR of swabs from
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patient room No.33. Patient B was a 41-year-old woman with upper respiratory infection, and
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patient C was a 19-year-old woman with pneumonia. D. Results of viral PCR of swabs from
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patient room No.34. Patient D was a 34-year-old woman without definite symptoms. The
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solid lines radiating from the large ovals indicate the angles of observation used for drawing
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the illustrations of the patients’ rooms.
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ACCEPTED MANUSCRIPT Table 1. Characteristic of 19 patients infected with parainfluenza virus 3 (PIV3) Total n = 19 (%)
Mean age, years (± SD)
44 ± 19
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Patient characteristics
Male gender (%)
7 (37)
Underlying malignancya (%)
18 (95)
Leukemiab
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Lymphomac Multiple myeloma Myelodysplasia Hemophagocytic lymphohistiocytosis Neutropenia (<0.5 x 109 cells/L) (%)
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Lymphopenia (<1.5 x 109 cells/L) (%)
Hematopoietic stem cell transplant (%)
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Allogeneic
2 2
1 6 (32) 12 (64) 8 (42) 7 6 (5)
More than 12 months ago
2
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Within 12 months (allogeneic)
Admission with PIV3 infection (%)
2 (11)
Associated symptoms/signs Asymptomatic
1
Upper respiratory tract infection
4
Lower respiratory tract infection
12
Fever only
3
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Neutropenic sepsis Co-infection (%)
14 (74) 5
Viral co-infectione
2
Fungal co-infectionf
8
Length of PIV3 excretion, (days ± SD)g
5 (26)
One patient was a nurse working in ward B, who was previously healthy.
Four acute lymphoblastic, seven acute myeloid, and one aggressive natural killer cell
leukemias.
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18 ± 6
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Mortality (%) a
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Bacterial co-infectiond
c
One diffuse large B cell lymphoma.
d
Bacterial co-infection included vancomycin-resistant enterococcal bacteremia, methicillin-
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susceptible Staphylococcus aureus, Escherichia coli and Aeromonas hydrophila bacteremia, and Clostridium difficile infection.
Virus co-infection included CMV pneumonia and CMV antigenemia.
f
Fungal co-infection included 5 invasive pulmonary aspergilloses, 2 Pneumocystis jirovecii
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g
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pneumonia, and 1 chronic disseminated candidiasis. The Kaplan-Meier method was used to construct survival curves for the time period during
which patients had positive RT-PCR results. Once the hPIV-3 infection was diagnosed, respiratory samples for hPIV-3 testing were taken weekly until negative conversion or patient discharge.
Date 14-07-2016
12-07-2016
10-07-2016
08-07-2016
06-07-2016
04-07-2016
02-07-2016
30-06-2016
28-06-2016
26-06-2016
24-06-2016
22-06-2016
20-06-2016
18-06-2016
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Outbreak was detected.
Isolation area was created.
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2
16-06-2016
D
3
14-06-2016
12-06-2016
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1
10-06-2016
08-06-2016
0 06-06-2016
04-06-2016
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02-06-2016
31-05-2016
29-05-2016
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27-05-2016
25-05-2016
23-05-2016
21-05-2016
19-05-2016
Number of hPIV-3 cases 4
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A B
Community acquired
(Different inner color means the different strains of hPIV-3)
Hospital acquired PCR negative period (dashed box)
, PCR positive period
(Line color means the room No.)
Room No. 33 E
Room No. 5
B
A
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Probable transmission Suspected transmission
Same HCW? Fomite?
June-14
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C
June-13
June-18
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Cluster 1
D
June-24
June-25 ↑Patient A moved to room No. 5
June-30
Room No. 34
July-5
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Cluster 5
ICU
Room No. 34
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Room No. 2
F
Room No. 6 → 1
G
I
ICU
H
Room No. 8
D
Room No. 6
Room No. 3
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K (HCW)
May-24
June-8
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May-19
June-10
J
June-11~12, JUN-14
June-25
↑ During these days, patient K has
worked
in
D
team
including Room No. 3 and her symptom was started.
Room No. 33
June-28
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33 34
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A
5
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B Positive from viral PCR
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Negative from viral PCR
• 48/F with pneumonia on noninvasive
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Patient A
Fomites not depicted in the Figure
Nasal prong, mask for the noninvasive ventilator, stethoscope, remote
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ventilation
• Time of sampling: 23 days after first positive
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PCR from respiratory specimen (negative respiratory PCR at the time of sampling) • 6 of 7 fomites swabs and 6 of 8 fixed-
structure swabs were positive for RT-PCR.
control of TV Hand washer tip Fixed structure not depicted in the Figure Call button, door knob in the inside room
Telephone button, exit door knob in the common corridor
Positive from viral PCR
Fomites not depicted in the Figure
Negative from viral PCR
Infusion regulator, curtains
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C
Fomites not depicted in the Figure Infusion regulator, remote control of TV
Patient C
Hand washer tip Fixed structure not depicted in the Figure
Hand washer tip, curtains
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Fixed structure not depicted in the Figure
Side rail of the bed, call button, telephone button
Side rail of the bed, call button, telephone
Patient B
Door knob in the room, door knob in the rest room,
button
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standing hand washer in common corridor, light switch
00 Patient C
• 41/F with upper respiratory infection
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Patient B
• 19/F with pneumonia • Time of sampling: 11 days after first positive PCR from
respiratory specimen (negative respiratory PCR at the
respiratory specimen (negative respiratory PCR at the
time of sampling)
time of sampling)
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• Time of sampling: 17 days after first positive PCR from
• 2 of 4 fomites and 4 of 11 fixed-structure swabs were positive for RT-PCR.
• 2 of 5 fomites and none of 6 fixed-structure swabs were positive for RT-PCR.
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D Positive from viral PCR
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Negative from viral PCR
• 34/F without symptoms
Fomites not depicted in the Figure
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• Time of sampling: 6 days after first positive PCR from respiratory specimen (discharged
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before next respiratory sample)
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Patient D
• 1 of 4 fomites swabs and none of 4 fixedstructure swabs were positive for RT-PCR.
Stethoscope Hand washer tip, curtains
Fixed structure not depicted in the Figure Side rail of the bed, call button, telephone button
S0195-6701(17)30493-0
DOI:
10.1016/j.jhin.2017.09.003
Reference:
YJHIN 5217
To appear in:
Journal of Hospital Infection
Received Date: 26 June 2017 Revised Date:
0195-6701 0195-6701
Accepted Date: 5 September 2017
Please cite this article as: Kim T, Jin CE, Sung H, Koo B, Park J, Kim S-M, Kim JY, Chong YP, Lee S-O, Choi S-H, Kim YS, Woo JH, Lee J-H, Lee J-H, Lee K-H, Shin Y, Kim S-H, Molecular epidemiology and environmental contamination during an outbreak of parainfluenza virus 3 in a hematology ward, Journal of Hospital Infection (2017), doi: 10.1016/j.jhin.2017.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Molecular epidemiology and environmental contamination during an outbreak of
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parainfluenza virus 3 in a hematology ward
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Taeeun Kim1,2§, Choong Eun Jin3§, Heungsup Sung4, Bonhan Koo3, Joungha Park5, Sun-Mi
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Kim1, Ji Yeun Kim1, Yong Pil Chong1, Sang-Oh Lee1, Sang-Ho Choi1, Yang Soo Kim1, Jun
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Hee Woo1, Jung-Hee Lee6, Je-Hwan Lee6, Kyoo-Hyung Lee6, Yong Shin3†, Sung-Han
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Kim1†
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Departments of Infectious Diseases1, Convergence Medicine3, Laboratory Medicine4, Medicine5, and Hematology6, Asan Medical Center, University of Ulsan College of
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Medicine, 88 Olympicro-43gil, Songpa-gu, Seoul, Republic of Korea.
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2
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University Hospital, 79, Gangnam-ro, Jinju, Gyeongsangnam-do, 52727, Republic of
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Korea
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Division of Infectious Diseases, Department of Internal Medicine, Gyeongsang National
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†
Co-corresponding authors:
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These authors contributed equally to this work.
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Sung-Han Kim, MD. Department of Infectious Diseases, Asan Medical Center,
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University of Ulsan College of Medicine, 88 Olympicro-43gil, Songpa-gu, Seoul, Republic
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of Korea (e-mail: [email protected])
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Tel: 82-2-3010-3305
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Fax: 82-2-3010-6970
Yong Shin, PhD. Department of Convergence Medicine, Asan Medical Center,
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University of Ulsan College of Medicine & Biomedical Engineering Research Center, Asan
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Institute for Life Sciences, Asan Medical Center, 88 Olympicro-43gil, Songpa-gu, Seoul, 1
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Republic of Korea (e-mail: [email protected])
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Tel: 82-2-3010-4193
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Running Title: An hPIV-3 outbreak in a hematology ward (40 characters – limit 40
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characters including spaces)
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Words count of abstract- 250 (limit 250)
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Words count of text- 3494 (limit 4000)
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Number of Tables- 1, Number of Figures- 4
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Number of References- 27
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ACCEPTED MANUSCRIPT Summary (250 words)
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Background: Although fomites or contaminated surfaces have been considered as
40
transmission routes, the role of environmental contamination by human parainfluenza virus
41
type 3 (hPIV-3 ) in healthcare settings is not established.
42
Aim: To describe an hPIV-3 nosocomial outbreak and the results of environmental sampling
43
to elucidate the source of nosocomial transmission and the role of environmental
44
contamination
45
Methods: During an hPIV-3 outbreak between May and June 2016, we swabbed
46
environmental surfaces in contact with clustered patients and used respiratory specimens
47
from infected patients and epidemiologically unlinked controls. We investigated the
48
epidemiologic relatedness of hPIV-3 strains by sequencing of the hemagglutinin-
49
neuraminidase and fusion protein genes.
50
Results: Of 19 hPIV-3 infected patients, 8 were hematopoietic stem cell recipients and 1 was
51
health care worker (HCW). In addition, 4 had upper and 12 had lower respiratory tract
52
infections. Of the 19 patients, 6 (32%) were community-onset (symptom onset within <7
53
days of hospitalization) and 13 (68%) were hospital-onset infections (≥7 days of
54
hospitalization). Phylogenetic analysis identified two major clusters: 5 patients, and 3 patients
55
plus one HCW. Therefore, 7 (37%) were classified as nosocomial transmissions. hPIV-3 was
56
detected in 21 (43%) of 49 environmental swabs up to 12 days after negative respiratory PCR
57
conversion.
58
Conclusion: Our data demonstrated that at least one-third of a peak season nosocomial hPIV-
59
3 outbreak originated from nosocomial transmission, with multiple importations of hPIV-3
60
from the community, providing experimental evidence for extensive environmental hPIV-3
61
contamination. Direct contact with the contaminated surfaces and fomites or indirect
62
transmission from infected HCWs could be responsible for nosocomial transmission.
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Key Words: human parainfluenza virus type 3; nosocomial outbreak; hematology unit;
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phylogenetic analysis; environmental contamination
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ACCEPTED MANUSCRIPT Introduction
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Human parainfluenza viruses (hPIV) are, along with human respiratory syncytial virus (RSV),
69
are well-recognized respiratory pathogens in infants and young children, ranging from mild
70
upper respiratory tract (URI) symptoms to croup and pneumonia [1, 2]. In the
71
immunocompetent adult, URIs are mild and self-limiting in nature, and re-infections can
72
occur due to incomplete immunity to hPIV. However, in the immunocompromised host,
73
especially in hematopoietic stem cell transplant (HSCT) recipients and patients with
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hematologic malignancies, hPIV causes a significant morbidity and mortality with prolonged
75
virus shedding [3-5]. Of the five known human serotypes, human parainfluenza virus type 3
76
(hPIV-3) is the most common pathogen [4]. Epidemics of hPIV are seasonal, with the peak of
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activity usually between May and June each year in South Korea, accounting for about 20-
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35% of detected respiratory virus agents in the community [6].
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There are several reports of hPIV-3 nosocomial outbreaks in HSCT recipients or patients with
81
hematologic malignancy [7-14]. In these reports, while some outbreaks were due to a single
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strain circulating within the unit [7, 12], others originated from multiple hPIV-3 strains
83
introduced from the community [11, 14]. Sequencing and phylogenetic analysis of hPIV-3
84
strains allowed for verification of whether a single strain or multiple strains were responsible
85
for the outbreak. In addition, in some of these reports, infection control, including
86
symptomatic surveillance and isolation, were often ineffective in terminating transmission,
87
suggesting asymptomatic viral shedding among patients, staff, or outside visitors or
88
environmental contamination as a possible explanation [9]. Although fomites or contaminated
89
surfaces have been considered as possible transmission routes of hPIV-3 [15, 16], in addition
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to droplets or close personal contact; the role of environmental contamination by hPIV-3 in
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healthcare settings has not been well defined. In the present study, we describe an hPIV-3
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nosocomial outbreak in a hematology ward and the results of environmental sampling to
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elucidate the source of nosocomial transmission and the role of environmental contamination.
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Hospital setting and patient population
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This study was conducted at a hematology unit in a 2,700-bed tertiary care hospital in Seoul,
98
South Korea. The hematology unit, which serves chemotherapy for patients with hematologic
99
malignancy and hematopoietic stem cell transplantation (HCT), consists of 2 wards located
100
on different floors. One ward (A) has 42 beds; 12 of these are for patients undergoing HCT.
101
The other ward (B) has 50 beds, including a mixture of single-, two-, five-, and six-bedded
102
rooms, communal showers, and a visiting room for patients on post-HCT care or cytotoxic
103
chemotherapy. These wards routinely allow limited visitation due to the
104
immunocompromised status of admitted patients.
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In mid-June 2016, an increased number of hPIV-3 positive cases were identified, clustered in
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a six-bed room on ward B. By the end of June, one or two more cases were confirmed each
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day, leading to the notification of an outbreak in the hematology unit by the Infection Control
109
team. After thorough review, all laboratory-confirmed hPIV-3 patients and symptomatic
110
healthcare workers (HCWs) in the hematology ward during this period were enrolled in this
111
study. Since the incubation period of hPIV-3 infection in adults has been estimated to be 2-6
112
days [17-19], an infection was considered to be hospital-onset if the patient had been
113
hospitalized for ≥7 days before the onset of respiratory symptoms. All rooms occupied by the
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original cluster of diagnosed patients, including the six-bedded room, the adjacent room, and
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the isolation room were selected for the environmental study. This study was approved by the
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institutional review board of Asan Medical Center.
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Sample collection
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Respiratory samples, including nasopharyngeal swabs/aspirates and bronchoalveolar lavage 8
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121
outbreak, respiratory samples were tested for respiratory viruses including hPIV types 1-4,
122
adenovirus, influenza A and B virus, rhinovirus, human metapneumovirus, bocavirus,
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coronavirus 229E, NL63, and OC43, enterovirus, and RSV types A and B by real-time
124
multiplex RT-PCR as a routine screening of any patient or healthcare worker presenting with
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respiratory symptoms and/or fever. Additional microbiological investigations performed on
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sputum or BAL fluid included Gram stain, acid-fast stain, and cultures for conventional
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bacteria, mycobacteria, and fungi, as needed. Samples were also tested for galactomannan
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and CMV DNA load (CMV qPCR) on blood and BAL fluid, if invasive aspergillosis or CMV
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disease was suspected. Once the hPIV-3 infection was diagnosed, respiratory samples for
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hPIV-3 testing were taken weekly until negative or patient discharge. Thus, we did not take
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further respiratory samples at the time of environmental sampling.
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Dacron swabs pre-moistened with viral transport medium were used to aseptically swab
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surfaces that were frequently touched by patients or healthcare workers. The following types
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of surface were swabbed: (1) fomites (ie, stethoscopes, infusion regulator, had sanitizer tip,
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nasal prongs or masks, pillows, curtains and TV remote control); (2) fixed structures in the
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rooms and their associated restrooms (ie, doorknobs, bed side rails, toilet seats, call button,
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telephone buttons, hand sanitizing dispensor, and light switch). All environmental samples
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were collected after routine cleaning or periodic linen change. The standard cleaning
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procedures of the rooms included routine bed cleaning (e.g., bed rails, control panel, call bell,
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bedside locker, switches, telephone, and main door knob) and toilet cleaning at least daily;
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and discharge bed cleaning after the patients moved or were discharge.
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Eleven isolates that were concurrently circulating in the community were obtained from 9
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respiratory specimens of epidemiologically unlinked patients from different wards of our
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institution during the same period to be used as controls.
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Real-time RT-PCR and hPIV-3 strain sequencing
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Viral RNA was extracted from all respiratory or environmental specimens using QIAmp®
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Viral RNA Mini Kit (Qiagen, Germany). The respiratory specimens from 19 patients with
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confirmed hPIV-3 and environmental samples were included in the subsequent
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epidemiological investigation, involving partial sequence analysis of the hemagglutinin-
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neuraminidase (HN) gene. In addition, 11 hPIV-3 isolates that were obtained from respiratory
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specimens of epidemiologically unlinked patients on different wards of our institution during
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the same period were also sequenced as controls. RT-PCR was performed on the extracts
156
using primers F (5’-ATT ACT CGA GGT TGC CAG GA-3’) and R (5’-CCG CGA CAC
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CCA GTT GTG-3’) covering 450 bp of the HN gene. The OneStep RT-PCR Kit (Qiagen,
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Germany) was used according to the manufacturer’s instructions, with 5 µL of extract being
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amplified in a final volume of 25µL, containing 5X one-step RT-PCT buffer, 0.25 mM
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deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of one-step RT-PCR
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Enzyme Mix. Thermal cycling conditions were as follows: 30 min at 50 °C, followed by 15
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min at 95 °C and 50 cycles of 30 s at 95 °C, 30 s at 50 °C, and 30 s at 72 °C, and a final
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elongation step at 72 °C 10 min. Sequences were aligned with the MEGA 7.0 software using
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the Clustal W method. Phylogenetic analysis was carried out using the MEGA 7.0 software
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maximum likelihood method, while the kimura 2-parameter was selected as an evolution
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model, and the bootstrap value was 1,000. We further analyzed the sequence of fusion protein
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(F) gene for clarifying the epidemiologic links within an equivocal cluster. The OneStep RT-
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PCR Kit (Qiagen, Germany) was also used with primers F (5’-CTT TGG AGG GGT AAT
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TGG AAC TA-3’) and R (5’-ATG ATG TGG CTG GGA AGA GG-3’) covering 621 bp of F
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gene. Thermal cycling conditions were followed as previously described. A positive result
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was confirmed by agarose gel electrophoresis and visualization by UV transilluminator.
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Statistical analysis
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The Kaplan-Meier method was used to construct survival curves for the time period during
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which patients had positive RT-PCR results. As flock swab samples were not obtained at
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discharge from patients who recovered, we defined these patients who did not undergo
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follow-up flock swab sampling, as “censored” patients. All statistical analyses were
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performed using SPSS version 21.0 (IBM Co., Armonk, NY).
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hPIV-3 outbreak
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From May 19, 2016 through June 30, 2016, 19 patients with hPIV-3, including 8 HCT
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recipients and 1 HCW, were identified in hematology ward B (Figure 1). Table 1 shows the
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clinical characteristics of these patients. The mean age of the patients was 44 years. The
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majority of the patients were lymphopenic (12 [64%]). Six of the 8 HCT recipients had
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undergone transplantation < 12 months before hPIV-3 infection developed. Of the 19 infected
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patients, 4 had upper and 12 had lower respiratory tract infections, and 5 (26%) died of hPIV-
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3 infection. Of the 19 patients, 6 (32%) were community-onset infection (symptom onset
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within < 7 days of hospitalization) and 13 (68%) were hospital-onset infection (symptom
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onset within ≥7 days of hospitalization). Fourteen patients (74%) had other serious concurrent
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infections, including invasive fungal infections (8 patients). Median length (IQR) of viral
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shedding from respiratory specimen was 18 days (14-19 days).
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Molecular analysis
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Phylogenetic analysis of the HN gene of the 42 hPIV-3 strains relevant to the 19 infected
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patients, 11 control patients unrelated to this outbreak, and 21 environmental samples of 49
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swab specimens are shown in Figure 2. Two major clusters were identified: cluster #1,
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including 30 identical hPIV-3 strains (relevant to patient A, E, B, C, D, and 2 control patients),
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and cluster #5, including 17 identical hPIV-3 strains (relevant to patient F, G, H, I, J, K, and 3
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control patients). Further analysis of the F gene within cluster #5 (17 hPIV-3 strains relevant
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for the 6 patients and 3 control patients) was performed because the HN gene had low
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discriminate power within cluster #5. The F gene analysis revealed the sub-cluster of 8
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identical hPIV-3 strains (relevant to patient H, I, J, K, the last hPIV-3 strains from patient F,
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and 1 control patient). Other clusters consisted of 3 or 4 identical hPIV-3 strains isolated
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from1 patient and one or two control patients.
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Epidemiological analysis
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The temporal and spatial relationships among the 11 outbreak cases of hPIV-3 belonging to
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the two identified major clusters (ie, cluster #1 and #5) are shown in Figure 3. These analyses
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revealed that 7 (37%) cases including one re-infection case were classified as nosocomial
211
transmissions. The 6 cases except one re-infection case were initially classified as hospital-
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onset infection (symptom onset within ≥7 days of hospitalization). Genetically identical
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strains are identified by the same inner circle colors; likewise different rooms are indicated by
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different box colors. Within cluster #1, patient A, the index case, had positive hPIV-3 PCR
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results over 10 days and infected 4 patients. Of these 4 patients, 3 were hospitalized in the
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same room with patient A at the time of hPIV-3 diagnosis. Finally, patient A moved to a
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single room (room 5) for effective isolation. After isolation of patient A, the same nursing
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team cared for the patients in room 5 (patient A) and room 34 (patient D).
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Within cluster #5, patient F, G, I, and J presented symptoms associated with hPIV-3 infection
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or positive hPIV-3 PCR results on admission. According to the additional F-gene sequence
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analysis, initial hPIV-3 strains isolated from these 4 patients were distinctive community-
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acquired strains. Patient I, who had acquired hPIV-3 in the community, was admitted through
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the emergency department and transmitted hPIV-3 to patient H, F (reinfection), and K (HCW).
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Even though the HCW (patient K) was positive for hPIV-3 by PCR on 28 June, she
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developed symptoms two weeks prior. During her symptomatic period, she cared for the
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patients in room 34, including patient F. The last (6th) hPIV-3 isolate from patient F was a
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different strain from the other isolates from patient F, and the same strain as that of the sub-
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cluster. Patient F succumbed to re-infection by the last hPIV-3 strain in a hospital ward, either
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from other patients (ie, patient I or H) or the HCW (patient K). The pairwise comparison
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among all 6 strains from patient F and I also support our explanation of re-infection in patient
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F (Supplemental Figure 1).
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Environmental contamination
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On July 5th, environmental samples were collected in three rooms of 4 patients (patient A, B,
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C, and D) within cluster #1. By this day, all but 1 patient, who was discharged just before
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environmental sampling, had negative PCR results from their respiratory specimens. Of the
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49 swab samples collected, 21 samples tested positive for hPIV-3 by RT-PCR and HN
239
sequencing (Supplemental Table 1). In particular, 11 of 20 fomite swabs and 10 of 29 fixed-
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structure swabs were positive for hPIV-3. Room 5 (Figure 4B) was the single-bed room used
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for the isolation of patient A, the index case of cluster #1. Out of 15 swab samples, 12 tested
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positive for viral RNA despite being swabbed, 12 days after the patient’s last positive PCR
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and 5 days after a negative PCR for hPIV-3.
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Room 33 (Figure 4C), the six-bedded room, was where it was speculated that the hPIV-3
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outbreak began. Six of 15 swab samples were positive for hPIV-3, 17 days after patient B’s
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last positive PCR and 12 days after a negative PCR. Her symptoms had resolved by the time
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of environmental sampling. Patient C only had mild rhinorrhea at the time of environmental
249
swab. Of 11 swab samples, 2 were positive for viral RNA, 8 days after the patient C’s last
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positive PCR and 1 day after negative PCR. Patient E was discharged 5 days prior to
251
environmental sampling. In room 34 (Figure 4D), the five-bedded room, only the stethoscope
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tested positive for viral RNA. Environmental sampling of this room was performed
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immediately after cleanup following patient D’s discharge. The RT-PCR results of the
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environmental specimens revealed the fluid infusion regulator (3 of 3 specimens), TV remote
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hPIV-3. Detailed information of all tested environmental samples is depicted in Figure 4. The
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virus strains isolated from the environmental samples were consistently found to be identical
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to their source patients (Figure 2).
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Our data demonstrated that an hPIV-3 outbreak in a hematology unit caused considerable
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morbidity and mortality in a substantial portion of patients with hematologic malignancies,
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and that at least one-third of an hPIV-3 outbreak resulted from nosocomial transmission,
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originating from multiple importations of hPIV-3 strains from the community during its peak
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epidemic season. In addition, we found that the extensive environmental contamination of
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hPIV-3 occurred in patients with negative PCR conversion from his/her respiratory specimen
266
despite the routine disinfection procedures. Therefore, our findings provide important
267
evidence for the probable routes for nosocomial transmission and emphasize the importance
268
of strict adherence to infection control precautions and the limited visitation during periods of
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high hPIV-3 activity in the community.
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This study clearly showed that both multiple importations from the community and
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subsequent nosocomial transmission equally contributed to this hPIV-3 outbreak. As the
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vulnerability of our healthcare system, such as patients occupying rooms with many beds,
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communal showers or a visiting room, and the visitation to hospitalized patients by friends
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and family members, was shown in the large Middle East respiratory syndrome outbreak in
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South Korea, we would like to elucidate which transmission route is important in our
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complex situation during an hPIV-3 outbreak. Previous studies have shown that molecular
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investigations are useful in discerning routes of transmission in hPIV-3 outbreaks [7-11, 14,
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20, 21]. One study demonstrated that both community-acquired and nosocomial transmitted
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infections occurred during one hematology unit outbreak [8], while others reported that
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nosocomial infections play a more significant role than multiple importations from the
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community [7, 14, 21]. This discrepancy among studies arises from the differences in terms
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of hPIV-3 activity in the community. It has been accepted that the epidemiology of
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community respiratory viral infections, including hPIV-3, in inpatients often mirrors the
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epidemiology in the outpatient population [22]. In addition, differences in infection control
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procedure, the presence of multi-patient rooms, and visits to hospitalized patients by friends
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and family members might contribute to the conflicting results of previous studies.
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The current scientific evidence suggests that fomites and contaminated surfaces are more
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concerning routes of hPIV transmission compared to aerosol [2, 23]. By air sampling, hPIV-1
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was isolated from air obtained in only 1 of 150 infected children at a distance of 60 cm [24].
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However, nasal secretions can travel a distance greater than 10 feet to contaminate
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surrounding fomites [23]. An experimental study demonstrated that hPIV can survive in
294
stainless steel for 10 hours [16]. Although the transfer of hPIV-3 from finger to finger or
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finger to metal disk did not occur, hPIV-3 could be transferred from contaminated disks to
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clean hands [25]. Therefore, person-to-person spread by direct hand contact appears to be an
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unlikely mode of transmission, whereas contaminated surfaces may lead to direct self-
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inoculation. To date, only these pieces of indirect experimental evidence have supported
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contaminated surfaces as a mode of hPIV-3 transmission. In this context, the extensive
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environmental contaminations and prolonged environmental presence of hPIV-3 were
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obviously demonstrated, and these findings may partially explain why hPIV-3 is easily spread
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in the hematology ward. In addition, this emphasizes the importance of disinfection of
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environmental surfaces. It is noteworthy that hPIV-3 infection was documented in one
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minimally symptomatic HCW who obtained this virus from the hPIV-3-infected patients.
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Although it is not exactly known whether asymptomatic or mildly symptomatic HCWs can
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transmit hPIV-3, asymptomatic hPIV-3 viral shedding in the nasopharynx among
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immunocompetent hosts [17, 26] and the previous outbreak reports suggest the potential role
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of asymptomatic healthy carriers in nosocomial transmission [9]. In our data, we could not
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define the precise role of none-to-minimal symptomatic HCWs in nosocomial transmission.
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However, given that the same nursing team cared for the index patients and the lastly infected
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cases of cluster #1, and 1 of the nursing team members (patient K in cluster #5) had
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documented hPIV-3 infection, HCWs may have a role in nosocomial transmission. Further
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studies are needed on this issue.
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In the immunocompromised host, hPIV is associated with significant morbidity and mortality.
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We observed a mortality rate of 26% during a single hPIV-3 outbreak episode. In previous
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reports, the mortality rate of hPIV-3 infection varied, ranging from 3%-47% according to the
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type of transplant, conditioning and immunosuppression therapy, and post-transplant
320
complications [4, 8, 10, 11]. However, in the majority cases, it is difficult to verify the cause
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of death as hPIV-3 infection, and thus only an association of hPIV-3 infection and death can
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be documented. Particularly in HSCT recipients, LRTI with hPIV significantly increased the
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risk of airflow decline (odds ratio, 17.9 [95% confidence interval, 2.0-160]) [27], and this
324
complication is associated with increased mortality risk. In our study, one of 5 patient deaths
325
had a post-HSCT pulmonary complication and died due to pulmonary function deterioration
326
following hPIV-3 pneumonia. Considering the morbidity and mortality associated with hPIV-
327
3 infection in the immunocompromised hosts and the absence of effective antiviral agents or
328
vaccination, prevention via more thorough infection control is essential. Interestingly, we
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clearly demonstrated that hPIV-3 re-infection occurred during the hospital stay (patient F,
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Figure 3 and Supplemental Figure 1). There is a need for more careful and comprehensive
331
prevention guidelines during the outbreak in hematology unit.
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This study has a few limitations. First, samples positive for viral RNA by RT-PCR may not 18
ACCEPTED MANUSCRIPT contain live virus with infectivity. However, the molecular analysis showed extensive and
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prolonged environmental viral contamination surrounding clustered hPIV-3 infected patients
336
by almost same strain. Particularly, the fomites, easily overlooked during routine daily
337
cleaning (i.e., TV remote control) or frequently manipulated by HCWs (i.e., the fluid infusion
338
regulator and stethoscope), were consistently positive during environmental sampling and
339
might have served as the episource during this outbreak. Therefore, these data provide more
340
direct insight into the possible routes of nosocomial transmission of hPIV-3. Second, we did
341
not perform air sampling. A previous study showed the limited role of aerosol as a mode of
342
hPIV transmission [23]. Lastly, in the phylogenetic analysis of the HN gene, differences in a
343
small number of nucleotide (nt) (1-3 bp) within the given 450 bp were grouped together.
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Although one might postulate that even more than 3 nt differences may not represent
345
different strains of viruses but rather reflect errors introduced during RT-PCR, the obvious
346
epidemiologic link supports the grouping in cluster #1. On the contrary, in cluster #5, due to
347
the low discriminative ability of the HN gene analysis and uncertain epidemiologic link, we
348
further analyzed the F gene, another variable region, and carried out sequence alignments for
349
hPIV-3 amplicons in some strains.
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In conclusion, this study suggested that an apparently single nosocomial outbreak was
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equally attributed to multiple importations of hPIV-3 strains from the community and
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subsequent nosocomial transmission. Furthermore, these data provide further direct
354
experimental evidence for extensive and prolonged viral contamination of materials
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surrounding hPIV-3 patients and/or from minimally symptomatic HCWs as probable
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nosocomial transmission routes. The findings of the present study indicate and reinforce the
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idea that preventive measures should consist of the isolation of infected patients, routine hand
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washing with ethanol-based disinfectants by HCWs, patients, and visitors, meticulous
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environmental cleaning, and limited patient visitation.
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Acknowledgements
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We thank Hye-Won Jung (e-medical contents team, Asan Medical Center, Seoul, South
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Korea) for the excellent artwork; Vanessa Topping (Scientific publications team, Asan
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Medical Center, Seoul, South Korea) for assistance and thoughtful comments.
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Description of Conflicts of Interest
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There are no potential conflicts of interest for any authors.
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Funding
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This work was supported by a grant of the Korea Health Technology R&D Project through
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Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &
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Welfare, Republic of Korea (grant no. HI15C2774).
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Figure Legends
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Figure 1. Number of hPIV-3 cases detected during outbreak in ward B.
452
Figure 2. Phylogenetic analysis of the 65 identified hPIV-3 strains. A. Sequence comparison
454
of PIV3 sequenced in the hemagglutinin-neuraminidase (HN) gene of 450bp. B. Sequence
455
comparison of PIV3 sequenced in the fusion protein (F) gene from cluster 5. GenBank
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accession numbers are KP690792 for hPIV-3 strain 11-s-83, GU732139 for hPIV-3 strain
457
BJ/036/08, KT898932 for hPIV-3 strain Pavia/15215/2014, and KJ672616 for hPIV-3 strain
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PER/FLU8925/2007.
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Figure 3. Chronological appearance of the hPIV-3 strains of the 11 infected patients in cluster
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#1 and cluster #5 during the outbreak.
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Figure 4. A. Floor plan of the hematology unit. Room numbers are indicated in red stars. B.
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Results of viral PCR of swabs from patient room No.5. Patient A was a 48-year-old woman
465
with pneumonia on noninvasive ventilation. She moved from room No. 33 to room No. 5 on
466
day 12 from after the first positive hPIV-3 PCR. C. Results of viral PCR of swabs from
467
patient room No.33. Patient B was a 41-year-old woman with upper respiratory infection, and
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patient C was a 19-year-old woman with pneumonia. D. Results of viral PCR of swabs from
469
patient room No.34. Patient D was a 34-year-old woman without definite symptoms. The
470
solid lines radiating from the large ovals indicate the angles of observation used for drawing
471
the illustrations of the patients’ rooms.
AC C
EP
463
24
ACCEPTED MANUSCRIPT Table 1. Characteristic of 19 patients infected with parainfluenza virus 3 (PIV3) Total n = 19 (%)
Mean age, years (± SD)
44 ± 19
RI PT
Patient characteristics
Male gender (%)
7 (37)
Underlying malignancya (%)
18 (95)
Leukemiab
SC
12
Lymphomac Multiple myeloma Myelodysplasia Hemophagocytic lymphohistiocytosis Neutropenia (<0.5 x 109 cells/L) (%)
M AN U
1
TE D
Lymphopenia (<1.5 x 109 cells/L) (%)
Hematopoietic stem cell transplant (%)
EP
Allogeneic
2 2
1 6 (32) 12 (64) 8 (42) 7 6 (5)
More than 12 months ago
2
AC C
Within 12 months (allogeneic)
Admission with PIV3 infection (%)
2 (11)
Associated symptoms/signs Asymptomatic
1
Upper respiratory tract infection
4
Lower respiratory tract infection
12
Fever only
3
ACCEPTED MANUSCRIPT 3
Neutropenic sepsis Co-infection (%)
14 (74) 5
Viral co-infectione
2
Fungal co-infectionf
8
Length of PIV3 excretion, (days ± SD)g
5 (26)
One patient was a nurse working in ward B, who was previously healthy.
Four acute lymphoblastic, seven acute myeloid, and one aggressive natural killer cell
leukemias.
M AN U
b
18 ± 6
SC
Mortality (%) a
RI PT
Bacterial co-infectiond
c
One diffuse large B cell lymphoma.
d
Bacterial co-infection included vancomycin-resistant enterococcal bacteremia, methicillin-
TE D
susceptible Staphylococcus aureus, Escherichia coli and Aeromonas hydrophila bacteremia, and Clostridium difficile infection.
Virus co-infection included CMV pneumonia and CMV antigenemia.
f
Fungal co-infection included 5 invasive pulmonary aspergilloses, 2 Pneumocystis jirovecii
EP
e
g
AC C
pneumonia, and 1 chronic disseminated candidiasis. The Kaplan-Meier method was used to construct survival curves for the time period during
which patients had positive RT-PCR results. Once the hPIV-3 infection was diagnosed, respiratory samples for hPIV-3 testing were taken weekly until negative conversion or patient discharge.
Date 14-07-2016
12-07-2016
10-07-2016
08-07-2016
06-07-2016
04-07-2016
02-07-2016
30-06-2016
28-06-2016
26-06-2016
24-06-2016
22-06-2016
20-06-2016
18-06-2016
RI PT
Outbreak was detected.
Isolation area was created.
M AN US C
2
16-06-2016
D
3
14-06-2016
12-06-2016
TE
1
10-06-2016
08-06-2016
0 06-06-2016
04-06-2016
EP
02-06-2016
31-05-2016
29-05-2016
AC C
27-05-2016
25-05-2016
23-05-2016
21-05-2016
19-05-2016
Number of hPIV-3 cases 4
AC C EP TE D
SC
M AN U
RI PT
A B
Community acquired
(Different inner color means the different strains of hPIV-3)
Hospital acquired PCR negative period (dashed box)
, PCR positive period
(Line color means the room No.)
Room No. 33 E
Room No. 5
B
A
M AN US C
Probable transmission Suspected transmission
Same HCW? Fomite?
June-14
AC C
EP
TE
D
C
June-13
June-18
RI PT
Cluster 1
D
June-24
June-25 ↑Patient A moved to room No. 5
June-30
Room No. 34
July-5
RI PT
Cluster 5
ICU
Room No. 34
M AN US C
Room No. 2
F
Room No. 6 → 1
G
I
ICU
H
Room No. 8
D
Room No. 6
Room No. 3
EP
TE
K (HCW)
May-24
June-8
AC C
May-19
June-10
J
June-11~12, JUN-14
June-25
↑ During these days, patient K has
worked
in
D
team
including Room No. 3 and her symptom was started.
Room No. 33
June-28
AC C EP TE
33 34
D
M AN US C
RI PT
A
5
RI PT
B Positive from viral PCR
M AN US C
Negative from viral PCR
• 48/F with pneumonia on noninvasive
TE
D
Patient A
Fomites not depicted in the Figure
Nasal prong, mask for the noninvasive ventilator, stethoscope, remote
EP
ventilation
• Time of sampling: 23 days after first positive
AC C
PCR from respiratory specimen (negative respiratory PCR at the time of sampling) • 6 of 7 fomites swabs and 6 of 8 fixed-
structure swabs were positive for RT-PCR.
control of TV Hand washer tip Fixed structure not depicted in the Figure Call button, door knob in the inside room
Telephone button, exit door knob in the common corridor
Positive from viral PCR
Fomites not depicted in the Figure
Negative from viral PCR
Infusion regulator, curtains
RI PT
C
Fomites not depicted in the Figure Infusion regulator, remote control of TV
Patient C
Hand washer tip Fixed structure not depicted in the Figure
Hand washer tip, curtains
M AN US C
Fixed structure not depicted in the Figure
Side rail of the bed, call button, telephone button
Side rail of the bed, call button, telephone
Patient B
Door knob in the room, door knob in the rest room,
button
D
standing hand washer in common corridor, light switch
00 Patient C
• 41/F with upper respiratory infection
EP
TE
Patient B
• 19/F with pneumonia • Time of sampling: 11 days after first positive PCR from
respiratory specimen (negative respiratory PCR at the
respiratory specimen (negative respiratory PCR at the
time of sampling)
time of sampling)
AC C
• Time of sampling: 17 days after first positive PCR from
• 2 of 4 fomites and 4 of 11 fixed-structure swabs were positive for RT-PCR.
• 2 of 5 fomites and none of 6 fixed-structure swabs were positive for RT-PCR.
RI PT
D Positive from viral PCR
M AN US C
Negative from viral PCR
• 34/F without symptoms
Fomites not depicted in the Figure
EP
• Time of sampling: 6 days after first positive PCR from respiratory specimen (discharged
AC C
before next respiratory sample)
TE
D
Patient D
• 1 of 4 fomites swabs and none of 4 fixedstructure swabs were positive for RT-PCR.
Stethoscope Hand washer tip, curtains
Fixed structure not depicted in the Figure Side rail of the bed, call button, telephone button