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Abstracts / International Journal of Infectious Diseases 79(S1) (2019) 1–150
Methods & Materials: A 77-year-old patient was hospitalized in
the late summer of 2017 on the second day of the illness manifested
by a fever up to 38.6 ◦ C and diarrhea. The patient did not report
recent travel but recalled mosquito bites. Past medical history
included hypertension. At admission, routine laboratory tests, electrocardiogram (ECG) and chest x-ray were performed. In addition,
cerebrospinal fluid (CSF), urine and blood samples were collected
for a virological analysis.
Results: At admission, WBC count was 24.6 (reference range
3.4-9.7x10(9)/L) with neutrophilia (92%, range 44-72%) and very
high levels of cardiac enzymes: creatinine phosphokinase 1856
(range 17-153 U/I), lactate dehydrogenase 433 (range 2-241 U/L),
myoglobin 3116 (range 20-80 ug/L) and troponin I 17.640 (range
0.000-0.056 ug/L). ECG showed ST elevation. In the cardiac intensive care unit, an emergency coronary angiography was performed
which confirmed the coronary artery stenosis. The patient’s condition complicated on the 4th day of the illness by an altered level
of consciousness with progression to coma, accompanied by neck
stiffness and positive meningeal signs. Computed tomography of
the brain was normal. Cerebrospinal fluid (CSF) showed pleocytosis with 26 cells/mm3, predominantly mononuclears (73%) and
elevated protein level (1.151, range 0.170-0.370 g/L). Both CSF and
urine were positive for WNV RNA by real-time and nested RTPCR. Phylogenetic analysis showed WNV lineage 2. The patient
was initially treated with acyclovir, ampicillin and cefepime parenterally with supportive therapy (antiedematous, antiaggregation
and antihypertensive therapy). On the 8th day of the illness a respiratory insufficiency developed. The patient was intubated and
mechanically ventilated, but developed hypotension and low oxygen saturation in spite of an adequate respiratory support. Despite
the cardiopulmonary resuscitation, the patient died due to cardiopulmonary arrest.
Conclusion: Although cardiac involvement is not frequently
reported in the course of a WNV infection, physicians should be
aware of the possibility of a WNV-related myocardial infection.
https://doi.org/10.1016/j.ijid.2018.11.294
21.087
Isolation of Brucella Melitensis in Azerbaijan in
2014
S. Asadova ∗ , M. Jahanov, R. Ismayilova, S.
Shikhaliyeva, R.M. Abdullayev
Republican Anti-Plague Station, Baku/AZ
Purpose: Republican Anti-Plague Station (RAPS) in Azerbaijan
provides confirmatory tests for especially dangerous pathogens
(e.g.Yersinia pestis, Bacillus anthracis, Brucella spp., Francisella
tularensis). In Azerbaijan RAPS and its regional Anti-Plague Divisions (APDs) are responsible for testing human samples for
abovementioned pathogens. RAPS receives samples for confirmatory test from patients referred by Baku, regional hospitals and
APDs.The aim of this study was to isolate and identify Brucella
cultures from human blood samples.
Methods & Materials: In 2014, 1,137 blood samples were collected by RAPS from patients with clinical symptoms such as high
temperature, perspiration, chills, myalgia, arthralgia, headache,
fatigue, lack of appetite. All samples were tested using the Azerbaijan Ministry of Health (EDPs Laboratory Guidelines, 2013)
algorithm via bacteriology and serology tests. Blood serum was
tested for the presence of the antibodies of Brucella spp. via Huddleson reaction, Rose Bengal test and Wright’s reaction. Samples
positive for Brucella spp. per Huddleson and Rose-Bengal reactions
further were tested by Wright’s reaction. Positive blood samples by
Wright’s reaction with titers 1/200 and higher were then cultured.
Isolated pure cultures were examined by biochemical (Urease,
Oxidase, Catalase, TSI/H2 S, Dye sensitivities) and serology (Trypaflavine, Agglutination with specific polyvalent serum, anti -A and
anti -M monospecific sera) tests.
Results: Data showed that 54.3% from total number of serum
samples were positive by Huddleson reaction; 35.6% from total
were positive by Rose-Bengal reaction and 38.7% from total samples were positive by Wright’s reaction. Positive blood samples
with titer 1/200 and higher from first diagnosed patients (n = 89)
were cultured. All isolates were identified as Brucella melitensis (an
overall isolation rate was 16.9%). The reports about positive results
are sent to the Ministry of Health and State Veterinary Control
Service (SVCS) and entered into the Electronic Integrated Disease
Surveillance System (EIDSS) database of the Ministry of Health of
Azerbaijan. Data from the system allows tracking the incidence of
brucellosis and making operational decisions.
Conclusion: Future genetic analyses of these isolates can help
identify subtypes of Brucella melitensis and establish the origin of
B.melitensis circulating in the Azerbaijan territory and compare the
results with those of neighboring countries.
https://doi.org/10.1016/j.ijid.2018.11.295
21.088
Generation, lyophilisation and epitope
modification of high titre filovirus pseudotyped
lentiviruses for use in antibody neutralisation
assays
M. Mayora-Neto 1,∗ , E. Bentley 2 , E. Wright 3 , N.
Temperton 1 , I. Ploemen 4 , P. Soema 4 , R. Ten
Have 4 , S. Masterson 5 , M. Michaelis 5 , M. Wass 5 ,
S.D. Scott 1
1 University of Kent, Viral Pseudotype Unit,
Chatham/UK
2 National Institute of Biological Standards and
Control (NIBSC), Potters Bar/UK
3 University of Sussex, Brighton/UK
4 Intravacc, Bilthoven/NL
5 University of Kent, School of Biosciences,
Canterbury/UK
Purpose: Filoviruses, such as Ebolavirus, are zoonotic pathogens
causing disease outbreaks with high mortality rates, requiring
scarce high containment facilities for research. Nevertheless, pseudotyped viruses (PV), consisting of a lentiviral core (plus luciferase
reporter) and the envelope glycoprotein (GP), allow basic and
translational virology to be conducted under low containment.
Consequently, filovirus PVs were generated and viability assessed
after lyophilisation and long-term storage. Next, antibody neutralisation tests were performed using native and hybrid GPs to assess
differentiation between genera and species.
Methods & Materials: PVs were produced using a 3-plasmid
transfection system (representing core, reporter and envelope) in
HEK293T/17 cells, and supernatant titrated. Supernatants were
then lyophilised in sucrose cryoprotectant solution, stored under
various conditions, reconstituted and titrated. For antibody neutralisation tests, serially diluted, polyclonal convalescent sera
(NIBSC, UK) or anti-GP monoclonal antibodies (Xiangguo Qiu, PHA,
Canada; Erica Saphire, Scripps, USA) were incubated with PV for
1 h at 37 ◦ C, prior to titration. To create artificial GP antigens, EBOV
neutralising epitopes were inserted into the GP of another genus
(Cuevavirus; LLOV) by mutagenesis, PVs generated and infectivity
and neutralisation assessed.
Abstracts / International Journal of Infectious Diseases 79(S1) (2019) 1–150
Results: High titre PVs were produced with titres between
∼1 × 108 RLU/mL (Ebolavirus/Cuevavirus)and ∼1 × 1010 RLU/mL
(Marburgvirus).
Lyophilised PV titres remained constant stored at −20 ◦ C and
◦
4 C for 12 months, while PVs kept at room temperature (22.5 ◦ C)
demonstrated titre decreases of up to 3 orders of magnitude after
6 months. At 37 ◦ C, five log (Marburgvirus) or three log (Ebolavirus
and Cuevavirus) decreases occurred after one month.
Zaire Ebolavirus (EBOV) antibodies showed no cross reactivity
with native LLOV PVs. Furthermore, EBOV epitopes inserted into
the LLOV GP and expressed on PVs had no significant impact on
PV infectivity, and EBOV neutralising epitopes were successfully
reconstituted in these chimeric antigens
Conclusion: In this study, high titre PVs were generated and
found to be amenable to lyophilisation and long-term storage.
Reconstituted PVs retained their function in neutralisation assays
suggesting their structure is not compromised during freezedrying. Insertion of epitopes in heterologous GPs did not impact
infectivity or functionality. This data suggests a PV-based serological kit could be utilised in resource-limited countries for serological
studies, after simple refrigeration storage.
https://doi.org/10.1016/j.ijid.2018.11.296
21.089
Tula virus phylogeography
V. Cirkovic 1 , G. Stamenkovic 2 , M. Siljic 3 , A.
Gligic 4 , M. Stanojevic 1,∗
1 University of Belgrade School of Medicine, Belgrade,
Serbia, Belgrade/RS
2 University of Belgrade, Institute for Biological
Research “Siniša Stanković”, Belgrade/RS
3 University of Belgrade School of Medicine,
Belgrade/RS
4 Institute of Virology, Vaccines and Sera Torlak,
Belgrade, Serbia, Belgrade/RS
Purpose: Tula hantavirus (TULV) is zoonotic virus widespread
across Eurasia, where numerous small mammal species have been
shown to be its reservoirs. In the Balkans, Serbia is the first country
where TULV was detected, in European pine vole, M. subterraneus
trapped in 1987. Although TULV is not considered pathogenic for
humans, cases of human infection have been reported on several
occasions so far.
Previousy, we have shown the evidence of recombinantion
events in studied TULV lineages from Serbia. In this study we
applied Bayesian phylogeography framework to reconstruct the
spatial and temporal dynamics of TULV based on sequences isolated
from different geographical areas.
Methods & Materials: The analyzed dataset was made of 137
TULV S segment sequences existing in the database, including two
sequences from Serbia; in total, 70 isolation sites within Europe
and Asia, covering time span of isolation of 28 years (1987-2015)
were included.
Sequences were aligned using CLUSTAL W implemented in
MEGA 6 and then manually edited. The best fit nucleotide substitution model was determined by jModeltest 0.1.1 using all 88
proposed models. TreePuzzle was employed to investigate the
phylogenetic signal of each sequence in the dataset. To explore
temporal structure of the dataset, root-to-tip analysis was done by
TempEst. The phylogeny, including phylogeographic distribution
was co-estimated in a Bayesian framework using a Markov Chain
Monte Carlo (MCMC) method implemented in the Beast package v
1.8.4
121
Results: Studied TULV strains formed three well supported
clades matching the geographical origin: the clade closest to the
tree root consisted of sequences from Russia and Kazahstan; the
second clade contained strains originating from western and central Europe; the third clade consisted of sequences from western
and southeast Europe. The place of origin was assessed to be in
Kazakhstan, with posterior probability of 1. The routes of viral
spread included local distribution across Kazakhstan and Russia
but also Europe, with the complex pattern of local viral migration
further on.
Conclusion: The place of TULV origin was assessed to be in
Kazakhstan, with westward spread leading to single introduction
of TULV to Europe.
https://doi.org/10.1016/j.ijid.2018.11.297
21.090
Serological study of leptospirosis in dogs from
French Guiana
C. Roqueplo 1 , B. Davoust 2 , J.-L. Marié 3,∗ , A.
Kodjo 4
1 French Forces Medical Service, Animal
epidemiology working group, Toulon/FR
2 IHU Méditerranée Infection, MEPHI, Marseilles/FR
3 Animal Working Group on Animal Epidemiology,
Regional Medical Command, Toulon, France,
Toulon/FR
4 VetAgro Sup, Leptospires Laboratory, Lyon/FR
Purpose: Leptospirosis is the most common zoonosis in the
world and is considered as an emerging human disease. In French
Guiana, recent epidemiological data indicate a significant increase
in human cases since 2012, with an annual incidence in 2014
of 39 cases per 100,000 inhabitants, making it one of the major
hotspots in the world. Considering this situation, the main goal of
the present study was to investigate the incidence of the infection in dogs which are possible reservoirs or maintenance hosts for
leptospires.
Methods & Materials: A serological survey was conducted in
Guiana in 2016 on 95 dogs from Cayenne and Kourou. Location,
race, sex, age, health and vaccination status were recorded for each
dog. Sera obtained were tested for 27 serovars of pathogenic Leptospira species by the microscopic agglutination test. The results
were interpreted according to the decision tree used in the VetAgro
Sup leptospires laboratory, Lyon, France.
Results: Among the 95 samples, 59 showed agglutination at the
cut-off point (1:20) for one or more pathogenic leptospiral serovars.
Focusing on high titres (≥1:160), seroprevalence was 11.6%. No statistically significant difference of prevalence due respectively to sex
and age was observed (p > 0.05). Icterohaemorhagiae (40%), Australis (33.3%) and Canicola (27%) were the most frequently observed
serogroups.
Conclusion: Dogs are not usually considered as a reservoir for
Leptospira, except for Canicola, thus, the high prevalence found
in this study in unvaccinated dogs probably results from a heavy
exposure. However, the cut-off points selected and the absence of
kinetic serology do not allow, in most cases, to conclude in favor of
a current active infection. Dogs are highly exposed to pathogenic
leptospires and humans living in the same environment are also
at risk of infection. Thus, dogs could be considered as sentinels for
human exposure to this zoonotic pathogen. In French Guiana, 98%
of which is covered by equatorial rainforest, all the conditions are in
place for the development of leptospirosis, particularly the climate
which is characterized by abundant rainfalls and high temperatures