Laboratory-acquired dengue virus infection--a case report.

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Symposium
Laboratory-Acquired Dengue Virus Infection—A Case
Report
Sumudu Britton1*, Andrew F. van den Hurk2, Russell J. Simmons2, Alyssa T. Pyke2, Judith A. Northill2,
James McCarthy3,4, Joe McCormack1,4
1Department of Infectious Diseases, Mater Health Services, Brisbane, Australia, 2Public Health Virology, Queensland Health Forensic and Scientific Services, Brisbane,
Australia, 3Queensland Institute of Medical Research, Brisbane, Australia, 4University of Queensland, Brisbane, Australia
Introduction
The WHO estimates there may be 50
million dengue virus (DENV) infections
worldwide every year, with the disease
being endemic in more than 100 countries
[1]. There has been a dramatic rise in the
incidence of dengue in recent decades,
making this an arbovirus of major inter-
national public health concern. Dengue
viruses belong to the family Flaviviridae and
are transmitted between humans via
infected female Aedes mosquitoes, particu-
larly Aedes aegypti. In the state of Queens-
land, Australia, infected travellers from
overseas have facilitated numerous DENV
outbreaks [2,3]. However, these outbreaks
are limited to the far north of the state, the
only area of Australia where Ae. aegypti
occurs [4].
There have been case reports of non-
vector, healthcare-associated transmission
of DENVs—four cases of percutaneous
transmission via needlestick injuries, mu-
cocutaneous transmission through a blood
splash to the face, vertical transmission,
and transmission via bone marrow trans-
plant (summarised in [5]). We report the
first case to our knowledge of DENV
infection acquired by a laboratory scientist
conducting mosquito infection and trans-
mission experiments.
The Case
The patient, a scientist at a research
laboratory, was referred to a public
hospital emergency department by a
general practitioner after presenting with
fever, myalgia, and a rash. The patient
resided in an area of Australia where
Ae.aegypti has not been reported since the
mid-1950 s [4]. The patient had travelled
to Argentina 4 weeks earlier but did not
have recent contact with similarly unwell
persons or pets and had no other medical
history of clinical significance. Ten days
prior to hospital admission, the patient
had performed a routine laboratory ex-
periment involving the primary infection
of colony mosquitoes with DENV-type 2
(DENV-2) via an artificial membrane
feeding apparatus. During the procedure
the patient had worn personal protective
equipment commensurate with what is
required for working with DENV in
Australian laboratories, including gown,
gloves, and eye protection [6]. The patient
reported a bite from an escaped non-
bloodfed mosquito during that day but
denied needlestick injury or mucocutane-
ous contact with the blood/virus mixture.
Four days later, the patient developed high
fever associated with marked lethargy and
fatigue, which progressed to myalgias and
severe back pain over the subsequent
48 hours. Three days after the onset of
fever, a fine, macular, blanching rash
developed that was generalised and pru-
ritic. Later findings following hospital
admission demonstrated evidence of neu-
tropenia (neutrophil count 0.76109/L)
andthrombocytopenia
796109/L). Results of liver function tests
also revealed elevated levels of alanine
aminotransferase (578 U/L) and aspartate
aminotransferase (630 U/L). Ten days
following the onset of fever, DENV
infection was confirmed by detection of
specific DENV-2 nucleic acid by real-time
TaqMan reverse transcriptase polymerase
chain reaction (G. Smith, unpublished
data) and anti-DENV-2 IgM antibodies
[7] in the patient’s serum. Subsequent
testing of a convalescent phase sample
collected 17 days after the first specimen
further demonstrated the presence of anti-
DENV IgM antibodies. Of note, serocon-
version of anti-flavivirus IgG antibodies
was also detected, suggesting that this was
an acute infection.
(platelet count
In support for this infection having been
acquired in the laboratory, the antibody
response was to the same virus serotype as
was used during the laboratory experi-
ment, and nucleotide sequencing analysis
affirmed that the DENV-2 strain recov-
ered from the patient was 99.8% homol-
ogous and therefore an identical strain to
the virus that had been used (Figure 1).
The DENV-2 strain used had been
originally isolated during an outbreak in
Townsville in 1993. After 3 days in
hospital, the patient was discharged and
within 48 hours all symptoms had resolved
and the results of laboratory tests had
returned to normal.
Conclusions
There have been numerous reports of
personnel acquiring incidental infections
during manipulation of arboviruses within
the laboratory [5,8,9]. However, to our
knowledge, this is the first reported case
where exposure during laboratory-based
mosquito infection and transmission ex-
periments has resulted in an acute DENV
infection. In this instance, the experiments
involved exposing colony-reared uninfect-
ed mosquitoes to an artificial blood meal
containing DENV-2 via a membrane
feeding apparatus. The high sequence
homology and phylogenetic relatedness
between the virus obtained from the
patient and the virus used during the
vector competence experiments confirms
that they were identical strains and
strongly suggests that the patient acquired
the infection during the course of this
procedure. Furthermore, these findings
Citation: Britton S, van den Hurk AF, Simmons RJ, Pyke AT, Northill JA, et al. (2011) Laboratory-Acquired
Dengue Virus Infection—A Case Report. PLoS Negl Trop Dis 5(11): e1324. doi:10.1371/journal.pntd.0001324
Editor: Maria G. Guzman, Tropical Medicine Institute Pedro Kourı ´, Cuba
Published November 29, 2011
Copyright: ? 2011 Britton et al. This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
Funding: No funding resources were required for the preparation of this manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: kannangaras@yahoo.com.au
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provide substantial evidence that the
patient was not infected while travelling
in Argentina, where DENVs do circulate.
In any case, this is highly unlikely given
the fact that the patient had returned from
Argentina 4 weeks prior to developing
dengue, an interval that greatly exceeds
the normal incubation period for DENVs
(between 3 and 14 days [10]). Finally,
although north Queensland was experi-
encing concurrent outbreaks involving all
four DENV serotypes at the time [11], the
patient had not travelled to this region
prior to developing dengue.
Upon notification of the case, the
research facilities were independently in-
spected to assess the potential for further
laboratory-acquired cases and the risk to
the general public as well as to determine
the potential route of virus exposure.
Inspectors observed that the research
facility and associated procedures adhered
to the regulations as they apply to physical
containment 2 (PC2) laboratories, the
containment level required for experimen-
tation with DENVs in Australia [6].
Importantly, it was concluded that the
PC2 level of containment within the
facilities significantly reduced the risk of
exposure to other laboratory workers or
the general public via experimentally
infected mosquitoes. Furthermore, given
that Ae. aegypti does not occur in southeast
Queensland, the region where the patient
resided, the risk of local transmission was
considered to be negligible.
The investigations carried out in re-
sponse to the case did not conclusively
determine the specific route by which the
patient was infected with DENV-2. It was
noted that, although the majority of
proceduresinvolving
performed in a class II biological safety
cabinet(BSC),some
experiments were, by necessity, conduct-
ed on the bench outside the BSC. It was
during this process that the patient may
have been exposed to the virus within
aerosolized blood droplets, resulting in
mucocutaneous transmission, or perhaps
may have become infected via contact
through an unrecognized dermal abra-
sion. Indeed, mucocutaneous exposure
was previously incriminated as the source
of DENV infection in a health worker in
the United States [12]. Alternatively, the
route of exposure may have been via
mosquito bite, as the patient reported
being bitten by an unengorged mosquito
that had escaped during the feeding
period. Even if the mosquito had only
probed the blood/virus mixture without
feeding, or had even imbibed a small
amount of the mixture, there would not
have been sufficient time for the virus to
replicate in the mosquito before transmis-
sion. Nonetheless, mechanical transmis-
sion of the virus via contaminated mos-
quito mouthparts cannot be completely
excluded, as this phenomenon has been
demonstrated previously with DENVs
[13].
This case provides a timely reminder of
the risk of arbovirus infection acquired by
laboratory personnel through either vec-
tor or non-vector modes of transmission.
It also highlights the importance of
appropriate laboratory practices for con-
taining infected mosquitoes and prevent-
ing contact with potentially infectious
material, including the generation of
potentially infectious aerosols. The use
of personal protective equipment, includ-
ing face mask and eye protection, and
where possible, conducting all manipula-
tions using live virus within a class II
BSC, would be appropriate for preven-
tionoflaboratory-acquired
infections.
The patient has consented to publica-
tion of this case report.
liveviruswere
aspectsof the
arbovirus
Figure 1. DENV-2 phylogenetic tree based on partial M and E gene nucleotide
sequences depicting the relationship between the virus acquired by the patient and
that used in laboratory experiments (highlighted). These are compared with other recent
DENV-2 isolates such as Townsville 1993 that were imported or resulted in outbreaks in
Queensland. Also shown are the genotypes of DENV-2 and representative strains from each
grouping.
doi:10.1371/journal.pntd.0001324.g001
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References
1. WHO (2009) Dengue and dengue haemorrhagic
fever. Fact sheet no 117, March 2009.
2. Hanna JN, Ritchie SA (2009) Outbreaks of
dengue in north Queensland, 1990–2008. Com-
mun Dis Intell 33: 32–33.
3. Ritchie SA (2009) Dengue: Australia’s other
pandemic. Microbiology Australia 30: 114–117.
4. Russell RC, Currie BJ, Lindsay MD, Mackenzie
JS, Ritchie SA, et al. (2009) Dengue and climate
change in Australia: predictions for the future
should incorporate knowledge from the past.
Med J Aust 190: 265–268.
5. Chen L, Wilson M (2005) Non-Vector transmis-
sion of dengue and other mosquito-borne flavivi-
ruses. Dengue Bulletin 29: 18–31.
6. Standards ANZ (2003) Standards Australia/
Standards New Zealand. Safety in laboratories.
Part 3: microbiological aspects and containment
facilities. AS/NZS 2243.3:2010. Sydney and
Wellington: Standards Australia International
Ltd.
7. Taylor C, Simmons R, Smith I (2005) Develop-
ment of immunoglobulin M capture enzyme-
linked immunosorbent assay to differentiate
human flavivirus infections occurring in Australia.
Clin Diagn Lab Immunol 12: 371–374.
8. Tomori O, Monath TP, O’Connor EH, Lee VH,
Cropp CB (1981) Arbovirus infections among
laboratory personnel in Ibadan, Nigeria.
Am J Trop Med Hyg 30: 855–861.
9. Hanson RP, Sulkin SE, Beuscher EL, Ham-
mon WM, McKinney RW, et al. (1967) Arbovi-
rus infections of laboratory workers. Extent of
problem emphasizes the need for more effective
measures to reduce hazards. Science 158:
1283–1286.
10. George R, Lum L (1997) Clinical spectrum of
dengue infection. In: Gubler DJ, Kuno G, eds.
Dengue and dengue hemorrhagic fever. Oxon
(United Kingdom): CAB International.
11. Queensland Health (2 May 2009) Cairns dengue
epidemic now officially largest in 50 years [press
release]. Brisbane (Queensland): Queensland
Health, Available: http://www.health.qld.gov.
au/dengue/documents/media_release_050509.
pdf. Accessed 7 November 2011.
12. Chen L, Wilson M (2004) Transmission of dengue
virus without a mosquito vector: nosocomial
mucocutaneous transmission and other routes of
transmission. Clin Infect Dis 39: e56–e60.
13. Simmons JS, St John JH, Reynolds FHK (1931)
Experimental studies of dengue. Monograph of
the Phillipines Bureau of Science, No. 29.
www.plosntds.org3 November 2011 | Volume 5 | Issue 11 | e1324