Session 272 Infections

Transcription

ARVO 2016 Annual Meeting Abstracts
272 Infections
Monday, May 02, 2016 3:45 PM–5:30 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 2334–2354/A0001–A0021
Organizing Section: Immunology/Microbiology
Program Number: 2334 Poster Board Number: A0001
Presentation Time: 3:45 PM–5:30 PM
Interrelationship of primary virus replication in the eye, level of
latency and time to reactivate in the trigeminal ganglia (TG) of
latently infected mice
Homayon Ghiasi1, Kevin Mott1, Sariah Allen1, Yasamin Ghiasi2,
Terrence Town2, Steven Wechsler3, Harry Matundan1.
1
Surgery -Ophthalmology, Cedars-Sinai Medical Center, Los Angeles,
CA; 2USC, Los Angels, CA; 3UCI, Irvine, CA.
Purpose: Very little is known regarding the inter-relationship
between primary virus replication in the eye, the level of latency in
the TG and the time to reactive of the mouse model. This study was
designed to answers these questions.
Methods: Female C57BL/6 mice were infected ocularly with virulent
(McKrae) or avirulent (KOS, RE) strains of HSV-1. Virus titers in the
eyes on days 3 and 5 post infection (PI), level of viral gB DNA in TG
on day 28 PI, and virus reactivation on day 28 PI were measured.
Results: Our results suggest that the avirulent strains of HSV-1 even
after corneal scarification had fewer viruses in the eye, less latency,
and a longer time to reactivate than virulent strains of HSV-1. The
time to explant reactivation of avirulent strains of HSV-1 was similar
to that of the virulent LAT-minus McKrae derived mutant. The viral
dose with the McKrae strains of HSV-1 affected level of viral DNA
and time to explant reactivation.
Conclusions: Our results point to the absence of any correlation
between the level of primary virus replication with the level of viral
DNA during latency and neither were an indicator of how rapidly the
virus reactivated following explant TG induced reactivation.
Commercial Relationships: Homayon Ghiasi, None; Kevin Mott,
None; Sariah Allen, None; Yasamin Ghiasi, None; Terrence Town,
None; Steven Wechsler, None; Harry Matundan, None
Support: This study was supported by Public Health Service NIH
grants RO1EY024649 and RO1EY013615.
Differential trafficking of adenovirus in corneal epithelial cells
and fibroblasts
Jaya Rajaiya, JiSun Lee, AshrafAli M. Ismail, JeongYoon Lee,
James Chodosh. Ophthalmology, Massachusetts Eye & Ear Infirmary,
Boston, MA.
Purpose: Ocular surface infection by viruses within
human adenovirus species D (HAdV-D) causes epidemic
keratoconjunctivitis, manifest by acute membranous
keratoconjunctivitis and delayed-onset stromal keratitis. We have
earlier shown that human adenovirus type D37 (HAdV-D37) uses
a lipid raft mediated caveolin-1 pathway to enter human corneal
fibroblasts (HCF), and that this pathway is negatively regulated
by dynamin-2. In the current study, we extend these studies to the
role of cellular microtubules in viral entry, and further determine
mechanisms of HAdV-D37 entry into tert-immortalized human
corneal epithelial (THE) cells.
Methods: HCF and THE cells were grown in standard media, and
pretreated with bafilomycin, cytochalasin, nocodazole, IPA-3, or
DMSO control for 30 min and then infected with HAdV-D37 for 1
hr. In other experiments, cells were transfected with siRNA against
dynamin-2, or scRNA, prior to infection. RNA was extracted
by Trizol for real-time qPCR or cells lysed for protein studies.
Viral titers were performed by the tissue culture infectious dose
assay. Alternately, cell cultures were fixed with paraformaldehyde
for confocal microscopy to visualize viral entry and changes in
intracellular microtubules.
Results: The endosomal acidification inhibitor, bafilomycin did
not impact viral entry in either cell type. The actin polymerization
inhibitor, cytochalasin D, inhibited viral entry in both cell types.
The microtubule inhibitor, nocodazole, repressed viral entry in
HCF, but only partially inhibited viral entry in THE cells. The
macropinocytosis inhibitor, IPA-3, significantly decreased adenoviral
infection in THE cells. Pretreatment with dynamin-2 siRNA increased
HAdV-D37 entry and replication, increased Src phosphorylation,
and caused a concurrent change in microtubule organizing center
localization in HCF, as shown by confocal immunomicroscopy with
antibody to pericentrin, but had no effect in THE cells.
Conclusions: Adenovirus trafficking differs among cell types
within the cornea. Understanding viral entry pathways in adenovirus
keratitis may lead to new, information-based molecular therapies
against infection.
Commercial Relationships: Jaya Rajaiya, None; JiSun Lee,
None; AshrafAli M. Ismail, None; JeongYoon Lee, None;
James Chodosh, None
Support: NIH grants EY013124, EY021558, and P30EY014104, a
Senior Scientific Investigator Award (to JC) from Research to Prevent
Blindness, the Massachusetts Lions Eye Research Fund, and the Falk
Foundation
Differential cytokine expression by ocular surface cells infected
with adenovirus
James Chodosh, Xiaohong Zhou, Emma Materne,
AshrafAli M. Ismail, Jaya Rajaiya. Ophthalmology, Mass Eye & Ear
Infirmary/HMS, Boston, MA.
Purpose: Eye infections with human adenovirus species D type
37 (HAdV-D37) result in severe, ocular surface inflammation,
recognized clinically as epidemic keratoconjunctivitis. Our previous
work focused on the role of intracellular signaling and downstream
chemokine expression in response to HAdV-D37 infection of human
corneal fibroblasts. We now elucidate differential patterns of cytokine
expression across a broad range of ocular surface cell types and
show a relationship between cytokine expression and intracellular
trafficking by the virus.
Methods: Human ocular surface cells, including epithelial cells,
stromal cells, and bone marrow derived cells isolated from peripheral
blood, were infected with cesium chloride gradient purified
HAdV-D37 (MOI of 5 for 4 hours), empty HAdV-D37 viral capsid,
or buffer control (mock infection), and incubated in Brefeldin A (20
µg/ml), followed by intracellular staining with antibodies against
IL6, IL1β, CXCL10, TNFα, CXCL8, and CCL2, fixation in 2%
paraformaldehyde, and quantification by flow cytometry, with
statistical analysis by ANOVA. Intracellular localization of Cy3labeled HAdV-D37 was studied by the Streptolysin O (SLO) method,
in which cells infected for one hour are permeabilized with SLO, and
stained with anti-Cy3 antibody.
Results: Each cell type tested except tert-immortalized, human
corneal epithelial cells, showed a significant increase in one or more
of the cytokines tested above mock infection levels. In particular,
in conventional dendritic cells, all the cytokines were increased by
infection except CXCL10; intact virus was a stronger inducer of IL6
and CCL2 than empty capsid. On its own, empty capsid also elicited
increased IL6 and CCL2 expression. In contrast, in plasmacytoid
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dendritic cells, only IL6 expression was increased by infection. At
one hour post infection, SLO analysis showed HAdV-D37 in the
cytosol of corneal epithelial cells but in the endosomes of other cells
tested.
Conclusions: The absence of cytokine expression by HAdV-D37
infected human corneal epithelial cells is consistent with prior
observations. These data further confirm cell type specific cytokine
responses upon infection, and suggest that endosomal retention of
virus is associated with relatively greater proinflammatory responses
by an infected cell.
Commercial Relationships: James Chodosh, None;
Xiaohong Zhou, None; Emma Materne, None;
AshrafAli M. Ismail, None; Jaya Rajaiya, None
Support: NIH grants EY013124, EY021558, and P30EY014104, a
Senior Scientific Investigator Award (to JC) from Research to Prevent
Blindness, the Massachusetts Lions Eye Research Fund, and the Falk
Foundation
Is Benzalkonium Chloride (BAK) an Effective Antiviral against
Adenovirus?
Eric G. Romanowski, Kathleen A. Yates, Robert M. Shanks,
Regis P. Kowalski. The Charles T. Campbell Ophthalmic
Microbiology Laboratory, UPMC Eye Center, Ophthalmology and
Visual Sciences Research Center, Eye and Ear Institute, Department
of Ophthalmology, University of Pittsburgh School of Medicine,
Pittsburgh, PA.
Purpose: Benzalkonium chloride (BAK) is a common preservative
used in ophthalmic medications and is the active ingredient in some
skin disinfectants and hand sanitizers. BAK is known to be effective
in killing bacteria and enveloped viruses. However, its activity
against non-enveloped viruses is unknown. The goal of the current
study was to determine whether BAK, at concentrations contained
in ophthalmic medications, skin disinfectants and hand sanitizers, is
effective in reducing titers of common ocular types of non-enveloped
adenovirus (Ad) in vitro.
Methods: The direct in vitro inactivating activity of BAK was
determined in duplicate trials by incubating high titer stocks of
clinical isolates of Ad3, Ad4, Ad5, Ad7a, Ad8, Ad19, and the ATCC
reference Ad37 isolate with BAK concentrations of 0.001%, 0.003%,
0.005%, 0.01% (concentrations found in ophthalmic medications),
0.1% (concentration found in skin disinfectants and hand sanitizers),
and 0% (control media) for 1h at 33oC. Following incubation,
standard plaque assays were performed on the reaction mixtures
to determine the adenovirus titers after BAK or control treatment.
Adenovirus titers were Log10 converted and Log10 reductions in titers
from the control were calculated. Decreases in titers of ≥ 3 Log10
were considered virucidal while decreases in titers of < 1 Log10 were
considered ineffective.
Results: A BAK concentration of 0.1% was virucidal for Ad3,
Ad5, Ad7a, Ad19, and Ad37. 0.1% BAK reduced titers >1 Log10
but < 3 Log10 for Ad4 and Ad8. A >1 Log10 decrease in titers was
demonstrated for BAK concentrations of 0.003%, 0.005% and 0.01%
for Ad5 only. Decreases in titers for the other adenovirus types for
those concentrations were ≤ 0.53 Log10. 0.001% BAK produced
decreases in titers of ≤ 0.17 Log10 for all adenovirus types.
Conclusions: BAK, at concentrations used in common ophthalmic
medications, demonstrated variable efficacy in reducing titers of
the adenovirus types tested. However, 0.1% BAK was effective
in reducing titers of all of the adenovirus types tested. BAK, at
concentrations used in common ophthalmic medications, was not
consistently effective as an agent against adenovirus, but higher
concentrations should be further investigated as a topical treatment
for adenoviral ocular infections, as long as ocular toxicity is not an
issue.
Commercial Relationships: Eric G. Romanowski, None;
Kathleen A. Yates, None; Robert M. Shanks; Regis P. Kowalski,
None
Support: NIH Core Grant P30 EY008098, RPB, Eye & Ear
Foundation
Diagnosis cytomegalovirus anterior uveitis/endothelitis in
immunocompetent patients
Laure E. Caspers2, 4, Joelle Antoun1, Jolanda de Groot-Mijnes5,
Elie Motulsky2, 3, N.H.ten Dam-van Loon5, François Willermain1, 3,
Lia Judice Relvas1, 3. 1Univ of Brussels-St Pierre Hosp, Brussels,
Belgium; 2Ophthalmology, CHU St Pierre, Brussels, Belgium;
3
Ophthalmology, CHU Brugmann, Brussels, Belgium; 4Université
Libre de Bruxelles, Brussels, Belgium; 5University Medical Center
Utrecht, Utrecht, Netherlands.
Purpose:
Purpose: To evaluate the diagnostic methods and clinical signs
leading the diagnosis of CMV anterior uveitis (AU) and/or
endothelitis from 2 uveitis tertiary referral center in Brussels
(Belgium) and in Utrecht (Nederland).
Methods: A retrospective study of patients with a clinical diagnosis
of CMV AU and endothelitis with positive polymerase chain reaction
(PCR) and or Goldmann-Witmer coefficient (GWc) positive.
Results: Results: We report a series of 19 patients presenting
clinical characteristics of CMV AU and /or endothelitis including
ring-shaped (coin-shaped) KPs, Posner-Shlossman syndrome and
Fuchs heterochromic iridocyclitis with a positive PCR and/or
GWc for CMV. There was 13/19 (68.4 %) males, 2 Asian (10.5%).
At diagnosis, the mean was 37.0 years [14-62], all (100%) were
unilateral and 16/18 (88.9%) had elevated intraocular pressure (IOP)
> 25 mm Hg (mean elevated IOP: 41.6 mm Hg). PCR was tested in
all the 19 patients while GWc was only tested in 8 patients. PCR was
positive for CMV in 14/19 patients (73.7 %) and GWc was positive
for CMV in 7/8 patients (87.5%), both PCR and GW were positive
for CMV in 2 patients. Aqueous tap was repeated in 8 patients (2
times in 5 cases, 3 times in 2 cases) to be able to confirm the clinical
diagnosis of CMV anterior uveitis/endothelitis.
Conclusions: Conclusion: the biological confirmation by PCR of
clinical diagnosis of CMV AU/ endothelitis remains difficult and can
be improved by repeated aqueous taps and GWc measurement..
Commercial Relationships: Laure E. Caspers, None;
Joelle Antoun; Jolanda de Groot-Mijnes, None; Elie Motulsky,
None; N.H.ten Dam-van Loon, None; François Willermain, None;
Lia Judice Relvas, None
QTL Based Virulence Determinant Mapping of the HSV-1
Genome in Murine Ocular Infection Reveals Genes Involved in
Viral Regulatory and Innate Immune Networks that Contribute
to Virulence
Curtis R. Brandt1, Aaron W. Kolb1, Kyubin Lee2, Mark W. Craven2.
1
Ophthal & Visual Sci, Univ of Wisconsin-Madison, Madison,
WI; 2Dept of Biostatistics and Medical Informatics, UW-Madison,
Madison, WI.
Purpose: Herpes simplex virus type 1 causes is the leading cause of
infectious blindness in the United States. Animal studies have shown
that the severity of HSV-1 ocular disease is influenced by three main
factors; innate resistance, host immune response and viral strain. We
previously showed that mixed infection with two avirulent HSV-1
strains (OD4 and CJ994) resulted in recombinants that exhibited a
range of disease phenotypes from severe to avirulent, suggesting
epistatic interactions were involved. The goal of this study was to
develop a quantitative trait locus (QTL) analysis of HSV-1 ocular
virulence determinants and to identify virulence associated SNPs.
Methods: Blepharitis, stromal keratitis, and neovascularization
quantitative scores were characterized for 40 OD4:CJ994
recombinants and viral titers in the eye were also measured. Virulence
quantitative trait locus mapping (vQTLmap) was performed using
the Lasso, Random Forest, and Ridge regression methods to identify
significant phenotypically meaningful regions for each ocular disease
parameter. The best-fit Ridge regression model identified several
SNPs for blepharitis, vascularization and stromal keratitis.
Results: Notably, phenotypically meaningful nonsynonymous
variations were detected in the UL24, UL29 (ICP8), UL41 (VHS),
UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and
gG genes. Network analysis revealed that many of these genes were
in viral regulatory (IE genes) networks and viral genes that affect
innate resistance mechanisms. For the first time QTL based analysis
has been used on HSV-1 genomes to identify ocular virulence
gene networks. Several genes previously implicated in virulence
were identified, while other genes were novel. Several novel
polymorphisms were identified in these genes.
Conclusions: This approach provides a framework that will be useful
for identifying virulence genes in other pathogenic viruses and to
resolve protein-protein interactions and epistatic effects that affect
HSV-1 ocular virulence.
Commercial Relationships: Curtis R. Brandt, None;
Aaron W. Kolb, None; Kyubin Lee, None; Mark W. Craven, None
Support: NIH Grant P30 EY016665 and NIH R01 EY023292
In vitro evaluation of anti HSV-1 siRNAs and in vivo evaluation
of electroporation to transfect siRNAs on murine cornea
Antoine Rousseau1, 3, Virgine Escriou2, Pierre Roy4,
Nolwenn Poccardi3, Julie Takissian3, Yves Gaudin3, Pascal Bigey2,
Marc Labetoulle3, 1. 1Ophthalmology, Bicêtre Hospital, Le Kremlin
Bicetre, France; 2Pharmacologie moléculaire et génétique, Unité
des Technologies Chimiques et Biologiques de la Santé CNRS
UMR 8258- Inserm U1022, Paris, France; 3Virologie, Institut de
Biologie Intégrative de la cellule, Gif-Sur-Yvette, France; 4OPIA SAS
Technologies, Paris, France.
Purpose: HSV-1 keratitis (HSK) is a leading cause of infectious
blindness in developed countries. Available treatments rely on
nucleosidic DNA polymerase inhibitors, which are used curatively
or prophylactically. Massive use of these treatments may favor the
emergence of resistance. Anti-HSV1 small interfering RNAs (siRNA)
may be efficient to overcome this issue, but their transfection into
corneal cells remains a challenge. The purpose of this study was
to assess the in vitro efficacy of siRNA targeting HSV-1 DNA
polymerase to reduce HSV-1 replication, and the in vivo efficacy of
electroporation to transfect siRNA into corneal cells on the murine
cornea.
Methods: Three different anti HSV-1 DNA polymerase siRNAs
(S1-S3) and one control siRNA were transfected into vero cells using
cationic lipids, which were secondarily infected with the SC16 strain
of HSV-1. Efficacy on viral replication was assessed using flow
cytometry, quantitative PCR (qPCR) and plaque assay technique. On
murine cornea, fluorescent siRNAs were injected subconjunctivally
and electroporation was performed with custom made electrodes
applied on the conjunctiva. The eyes were enucleated, and observed
under fluorescence microscopy.
Results: The three siRNAs were able to inhibit viral replication.
Compared to the control siRNA, S3 was the most efficient siRNA,
decreasing by 60% the number of infected cells as measured with
flow cytometry, by 59% the number of plaques and by 75% the
viral load estimated with qPCR. Electroporation improved siRNA
penetration into the corneal epithelium compared to subconjunctival
injection alone.
Conclusions: These results demonstrate that siRNA directed against
HSV-1 DNA polymerase efficiently inhibits HSV-1 replication,
suggesting that siRNA based antiviral strategy may be a potential
therapeutic alternative to treat HSK. Besides, intracorneal penetration
may be facilitated by electroporation.
Commercial Relationships: Antoine Rousseau; Virgine Escriou,
None; Pierre Roy, None; Nolwenn Poccardi, None; Julie Takissian,
None; Yves Gaudin, None; Pascal Bigey, None; Marc Labetoulle,
None
Human Leukocyte Antigen Associations with Cytomegalovirus
Anterior Uveitis
Jay J. Siak1, 2, Nobuyo Yawata2, Anne Jansen1,
Samanthila Waduthantri1, 2, Anita Sook Yee Chan1, 2,
Gemmy Cheung1, 2, Soon-Phaik Chee1, 2. 1Singapore National Eye
Centre, Singapore, Singapore; 2Singapore Eye Research Institute,
Singapore, Singapore.
Purpose: Cytomegalovirus (CMV) infection is associated with
recurrent acute or chronic anterior uveitis / endotheliitis in
immunocompetent individuals, especially among Asians. Major
Histocompatibility Complex Class I restricted CD8+ T-cell response
represents one of the major immune response that controls CMV,
and previous studies suggested that different human leukocyte
antigen (HLA) class I alleles can confer different levels of CD8+Tcell protection against human CMV reactivation. We hypothesize
that these individuals may harbor an impaired immune defense
against CMV. Therefore, we compared HLA class I genotypes of
immunocompetent Chinese individuals with CMV anterior uveitis
against that of Chinese healthy controls without ocular inflammation
to identify potential HLA allele associations.
Methods: Genomic DNA was isolated from venous blood samples of
13 immunocompetent Chinese individuals with aqueous humor PCR
proven CMV anterior uveitis against that of 28 age-matched Chinese
healthy controls without ocular inflammation. HLA class I A, B, C
typing was performed by Next Generation Sequencing on a MiSeq
using Conexio’s Capture HLATM protocol and Assign MPS (Conexio
Genomics PTE LTD, Perth, Australia).
Results: HLA-B*1502 is significantly associated with CMV anterior
uveitis compared to controls (Frequencies 38.5% versus 7.1%,
Odds Ratio 8.13, (95% Confidence Interval 1.32-50.21)). The other
common alleles among our Chinese subjects with CMV anterior
uveitis include A*1101 (61.5%), A*2402 (53.8%), B*5801 (38.5%),
C*0801 (38.5%) and C*0302 (38.5%). These genotype frequencies
are higher than the previous reported frequencies among Singapore
Chinese population.
Conclusions: Our data suggested that certain HLA haplotypes such
as HLA-B*1502 may be associated with CMV anterior uveitis. These
alleles are dominant in Chinese populations compared to Caucasian
populations. Further studies are needed to validate our observation.
Commercial Relationships: Jay J. Siak, None; Nobuyo Yawata,
None; Anne Jansen, None; Samanthila Waduthantri, None; Anita
Sook Yee Chan, None; Gemmy Cheung, None; Soon-Phaik Chee,
None
Support: Singapore National Medical Research Council Grant
(NMRC/CNIG/1113/2013), Singapore National Eye Centre Health
Research Endowment Fund (R1043/58/2013)
Reducing the concentration and irradiation time of rose bengalmediated photodynamic antimicrobial therapy for inhibition of
fungal keratitis isolates
Alejandro Arboleda1, Nidhi Relhan1, Heather A. Durkee1,
Mariela C. Aguilar1, Karam A. Alawa1, Francisco Halili1,
Cornelis Rowaan1, Guillermo Amescua2, Harry W. Flynn2,
Darlene Miller3, Jean-Marie A. Parel1, 4. 1Ophthalmic Biophysics
Center, Department of Ophthalmology, Bascom Palmer Eye Institute,
University of Miami Miller School of Medicine, Miami, FL;
2
Anne Bates Leach Eye Hospital, Department of Ophthalmology,
Bascom Palmer Eye Institute, University of Miami Miller School of
Medicine, Miami, FL; 3Ocular Microbiology Laboratory, Department
of Ophthalmology, Bascom Palmer Eye Institute, University of
Miami Miller School of Medicine, Miami, FL; 4CHU Sart-Tillman,
Department of Ophthalmology, University of Liege, Liege, Belgium.
Purpose: Fungal keratitis is a potentially blinding condition
that is difficult to manage clinically with the currently available
antimycotics. One emerging alternate treatment is photodynamic
antimicrobial therapy (PDAT). In this study we explore the efficacy
of different parameters for rose bengal-mediated PDAT to inhibit
growth of four fungal isolates from patients with infectious keratitis.
Methods: Four fungi (Fusarium solani, Candida albicans,
Purpureocillium lilacinum (Paecilomyces lilacinus), and
Pseudoallescheria boydii) were isolated from patients with confirmed
fungal keratitis and grown on Sabouraud-Dextrose agar plates. A
suspension of each isolate was made using sterile, deionized water
and was subsequently mixed with appropriate photosensitizer
solution according to experimental group and plated in triplicate onto
nutrient agar. Water was used as a control to compare to experimental
groups treated with rose bengal at varying concentrations (0.1%,
0.05%, & 0.01%) and irradiated for 5.4 J/cm2 total exposure. An
additional test was performed with 0.1% rose bengal irradiated for a
half the time resulting in a reduced exposure of 2.7 J/cm2. Green light
irradiation was performed using a circular array (47 mm diameter) of
518 nm light emitting diodes. Plates were placed in a 30°C non-CO2
incubator and photographed at 72 hours. Fungi growth was evaluated
using Labview-based software.
Results: At 72 hours, Rose bengal-mediated PDAT with 0.05%
concentration completely inhibited the growth of the four fungal
isolates within central 47mm area. 0.01% rose bengal PDAT
inhibited for Pseudoallescheria boydii and incompletely for Candida
albicans and Purpureocillium lilacinum. All four fungal isolates
showed central inhibition zone corresponding to the diameter of the
irradiation source head in 0.1% rose bengal reduced time PDAT (2.7
J/cm2).
Conclusions: Rose bengal-mediated PDAT inhibited Fusarium
solani, Candida albicans, Purpureocillium lilacinum, and
Pseudoallescheria boydii keratitis isolates in the irradiated area.
These in vitro results demonstrate the potential strength of rose
bengal-mediated PDAT as an alternative treatment modality of fungal
keratitis.
Rose bengal-mediated PDAT using reduced concentrations of
photosensitizing agent.
Rose bengal-mediated PDAT using 0.1% rose bengal and reduced
energy
Commercial Relationships: Alejandro Arboleda,
None; Nidhi Relhan, None; Heather A. Durkee, None;
Mariela C. Aguilar, None; Karam A. Alawa, None;
Francisco Halili, None; Cornelis Rowaan, None;
Guillermo Amescua, None; Harry W. Flynn, None;
Darlene Miller, None; Jean-Marie A. Parel, None
Support: This work was supported in part by: Florida Lions Eye
Bank, Edward D. and Janet K. Robson Foundation. Drs. KR Olsen
and ME Hildebrandt, NIH center grant P30EY14801, Drs. Raksha
Urs and Aaron Furtado, Research to Prevent Blindness, Brien Holden
Vision Institute, Henri and Flore Lesieur Foundation (JMP).Technical
support provided by: Alex Gonzalez, Juan Silgado, Victor Hernandez,
Carolina de Freitas, and Adriana Henao-Pink.
Photodynamic Antimicrobial Therapy to inhibit Purpureocillium
lilacinum, Pseudallescheria boydii and Cochliobolus lunatus
isolates
Nidhi Relhan1, Alejandro Arboleda1, Heather A. Durkee1,
Mariela C. Aguilar1, Karam A. Alawa1, Cornelis Rowaan1,
Guillermo Amescua2, Darlene Miller3, Harry W. Flynn2,
Jean -Marie A. Parel1, 4. 1Ophthalmic Biophysics Center, Department
of Ophthalmology, Bascom Palmer Eye Institute, University of
Miami Miller School of Medicine, Miami, FL; 2Anne Bates Leach
Eye Hospital, Department of Ophthalmology, Bascom Palmer Eye
Institute, University of Miami Miller School of Medicine, Miami, FL;
3
Ocular Microbiology Laboratory, Department of Ophthalmology,
Bascom Palmer Eye Institute, University of Miami Miller School
of Medicine, Miami, FL; 4CHU Sart-Tillman, Department of
Ophthalmology, University of Liege, Liege, Belgium.
Purpose: To assess the use of rose bengal- and riboflavin-mediated
photodynamic antimicrobial therapy (PDAT) to inhibit the growth of
three fungal isolates that cause infectious keratitis.
Methods: Three fungi, Purpureocillium lilacinum (Paecilomyces
lilacinus), Pseudallescheria boydi, Cochliobolus lunatus (Curvularia
lunata), were isolated from patients with confirmed fungal keratitis
and grown on Sabouraud-Dextrose agar plates. Using the method we
published previously (Arboleda et al, AJO 2014 Jul;158(1):64-70),
triplicate test plates were separated into 7 groups: (1) control (fungal
isolate only), (2) green irradiation only, (3) 0.1% rose bengal only, (4)
0.1% rose bengal-mediated PDAT (rose bengal + green irradiation),
(5) UV irradiation only, (6) 0.1 % riboflavin only, and (7) 0.1%
riboflavin-mediated PDAT (riboflavin + UV-A irradiation). Irradiation
was performed using either a 47mm diameter 518nm green light
emitting diode (LED) array or a 37mm diameter 375nm UV-A LED
array for a final energy density of 5.4 J/cm2. After treatment, plates
were placed in a 30°C non-CO2incubator and observed for growth.
Plates were photographed at day 3 to document fungal growth and
images were analyzed using a Labview program created in our
laboratory.
Results: Rose bengal-mediated PDAT successfully inhibited the
growth of Purpureocilium lilacinum and Pseudallescheria boydii.
For these two fungi, rose bengal-mediated PDAT showed complete
inhibition within the central 47mm area corresponding to the
diameter of the light source at day 3. Cochliobolus lunatus showed
minimal inhibition in the central region, but no clear inhibition zone.
No other groups demonstrated any inhibitory effect on the three
fungal isolates.
Conclusions: Rose bengal-mediated PDAT inhibited Purpureocilium
lilacinum and Pseudallescheria boydii keratitis isolates within the
irradiated area; however it was not effective for Cochliobolus lunatus.
Rose bengal-mediated PDAT has the potential to be a treatment
option for fungal keratitis.
Inhibition of three fungal species: (Row 1) Control, (Row 2) Green
Irradiation Only, (Row 3) 0.1% Rose Bengal Only, (Row 4) 0.1%
Rose bengal-mediated PDAT.
Inhibition of three fungal species: (Row 1) Control, (Row 2) UV-A
Irradiation Only, (Row 3) 0.1% Riboflavin Only, (Row 4) 0.1%
Riboflavin-mediated PDAT.
Commercial Relationships: Nidhi Relhan, None;
Alejandro Arboleda, None; Heather A. Durkee,
None; Mariela C. Aguilar, None; Karam A. Alawa,
None; Cornelis Rowaan, None; Guillermo Amescua,
None; Darlene Miller, None; Harry W. Flynn, None;
Jean -Marie A. Parel, None
Support: Supported in part by the Florida Lions Eye Bank, Edward
D. and Janet K. Robson Foundation, Drs. KR Olsen and ME
Hildebrandt, Dr. Raksha Urs and Aaron Furtado, NIH center grant
P30EY14801, Research to Prevent Blindness, and the Henri and
Flore Lesieur Foundation (JMP). The authors are grateful to Alex
Gonzalez, Juan Silgado, Victor Hernandez, and Adriana Henao-Pink
for their technical contributions.
Cereolysin O influences TLR4-dependent retinal gene expression
during Bacillus cereus endophthalmitis
Phillip S. Coburn1, Frederick C. Miller3, 4, Craig Land2,
Austin L. LaGrow1, Michelle Callegan1. 1Ophthalmology, The Univ
of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2Microbiology and
Molecular Genetics, Oklahoma State University, Stillwater, OK; 3Cell
Biology, The Univ of Oklahoma Hlth Sci Cntr, Oklahoma City, OK;
4
Family and Preventative Medicine, The Univ of Oklahoma Hlth Sci
Cntr, Oklahoma City, OK.
Purpose: To determine whether cereolysin O, a member of the
bacterial cholesterol-dependent cytolysin family, alters TLR4dependent retinal gene expression in response to B. cereus infection
early during the course of experimental murine endophthalmitis.
Methods: C57BL/6J or TLR4-/- mice were intravitreally injected
with 100 CFU of B. cereus ATCC 14579 or an isogenic cereolysin
O mutant. Fellow eyes served as uninfected controls. At 4 h postinfection, total RNA was isolated from retinas and subjected to qPCR.
The threshold cycle (CT) was used to determine relative amounts of
transcripts between RNA samples from infected and uninfected eyes.
Fold increases were calculated by subtracting CT values of infected
samples from CT values of uninfected samples. That value as a power
of 2 yielded the fold increase of the transcript from infected samples
relative to uninfected samples.
Results: We previously identified a subset of genes related to the
acute inflammatory response, inflammatory cell recruitment, cytokine
signaling, photoreceptor survival, and pathogen recognition and
clearance which were significantly upregulated 5-fold or greater in B.
cereus-infected retinas at 4 h post-infection. These included CXCL1,
CXCL2, CXCL10, CCL2, and CCL3, IL6, ICAM-1, SOCS3, LIF,
PTGS2/COX-2, and PTX3. Analysis of the 4-h transcriptome in B.
cereus-infected TLR4-/- retinas showed no change in the expression
of these genes. Intravitreal infection of C57BL/6J mice with the
isogenic cereolysin O mutant of B. cereus ATCC 14579 resulted
in significantly reduced expression of CXCL1, CXCL2, CXCL10,
CCL2, and CCL3, IL6, ICAM-1, and LIF. There were no changes
in the expression of SOCS3 and PTX3, and PTGS2/COX-2 was
downregulated 2-fold relative to uninfected mice.
Conclusions: These results suggest that cereolysin O plays a role
in the activation of TLR4-dependent genes important for the acute
inflammatory response and neutrophil recruitment, as well as genes
related to photoreceptor survival and pathogen recognition and
clearance. Future studies will evaluate the retinal and global ocular
inflammatory responses over the course of B. cereus endophthalmitis
to identify pathway-based anti-inflammatory targets, specifically
those that are regulated by TLR4, and to determine whether
cereolysin O/TLR4 interactions contribute to the uniquely explosive
course of B. cereus endophthalmitis.
Commercial Relationships: Phillip S. Coburn, None;
Frederick C. Miller, None; Craig Land; Austin L. LaGrow, None;
Michelle Callegan, None
Support: NIH/NEI R01 EY024140, NIH P30EY21725, Research to
Prevent Blindness
Microbial Keratitis in North Texas: Public and Private Patient
Populations
David Truong, H D. Cavanagh. UT Southwestern Medical Center,
Dallas, TX.
Purpose: To compare the epidemiology, risk factors, microbiologic
spectrum, and treatment of microbial keratitis at an urban public
hospital to an adjacent private practice with the same physicians.
Methods: Retrospective chart review in the 5-year interval 2009
through 2014. Primary outcome measures included best-corrected
visual acuity (BCVA), risk factors, culture and sensitivities,
treatment, and complication rates.
Results: 331 eyes with microbial keratitis have been identified.
Contact lens wear, ocular trauma, and ocular surface diseases
were the most common risk factors. Gram-positive organisms
represented 46%, gram-negative organisms 39%, fungal organisms
15%, and Acanthamoeba <1% of corneal isolates. No common
corneal pathogens were resistant to aminoglycosides or vancomycin.
48% of cases were initially treated with fortified antibiotics, 43%
with fluoroquinolone monotherapy, and 6% with antifungals. At
resolution, average BCVA was 20/82 [logMAR 0.61] with 8% of
cases resulting in light perception or worse vision. The perforation
rate was 8%. 6% of cases underwent urgent penetrating keratoplasty
and 4% of cases underwent urgent enucleation or evisceration. The
public hospital population was (1) younger, (2) more likely to have
used contact lenses, (3) more likely to be admitted for inpatient
treatment, (4) more likely to be treated with combination fortified
antibiotics, and (5) less likely to harbor antibiotic-resistant pathogens.
The complication rates were similar between the two populations.
Conclusions: Microbial keratitis remains a clinical challenge in
the urban public hospital setting and may represent a distinct entity
requiring a tailored approach.
Commercial Relationships: David Truong, None; H D. Cavanagh,
None
Support: EY020799 and an unrestricted grant from Research to
Prevent Blindness
Global gene expression in ocular isolates of Escherichia coli with
potential to form biofilm
Shivaji Sisinthy1, Ranjit Konduri1, Arunasri Kotakonda2,
Reddy Satyanaryana Gundlapally2, Savitri Sharma1. 1L.V.Prasad Eye
Institute, Hyderabad, India; 2CCMB, Hyderabad, India.
Purpose: To establish the biofilm potential of ocular isolates of
E.coli, to ascertain their antibiotic susceptibility and to identify genes
that are responsible for biofilm formation and drug resistance.
Methods: Biofilm formation was assessed in 12 ocular isolates of E.
coli using the congo red agar method and tissue culture plate method.
The minimum inhibitory concentration (MIC) of eight antibiotics was
determined against E. coli strain L-1216 in the biofilm and planktonic
phases. To identify genes that may be differentially regulated during
biofilm formation, ocular E. coli was allowed to form biofilm for 72 h
and then used as the source for mRNA, cDNA preparation and DNA
microarray. Ocular E. coli (L-1339) which did not form a biofilm was
used as a control. All experiments were repeated thrice. Only genes
that deviated from the control by more than 2 fold and with a p value
<0.05 were considered as significantly differentially expressed.
Results: Six of the 12 ocular isolates were biofilm positive. E.
coli L-1216 was the best biofilm forming isolate. The MIC of all
antibiotics in the biofilm state was >5mg and ≤0.032mg in the
planktonic phase. DNA microarray data indicated that in the biofilm
forming E. coli 426 and 866 genes were up and down regulated
respectively compared to the non-biofilm forming cells. The heat map
and the Principal Component Analysis also showed that the biofilm
cells were more related to one other and were different from the
non-biofilm cells. The differentially expressed genes in the biofilm
forming cells could be classified as those involved in metabolic
processes, in organization of cellular components and in cellular
activities. In each of these categories, genes relevant to biofilm
formation were present like genes coding for EPS synthesis, genes
that facilitate adhesion, genes that respond to environmental signals
and those that are related to drug resistance.
Conclusions: Ocular E. coli have the potential to form biofilm
and cells in the biofilm are more resistant to antimicrobials.
Several virulent genes like those coding for pili/fimbriae,
lipopolysacccharides and exopolysaccharides synthesis, efflux
pumps, environmental signals etc. are upregulated. This is the
first study on DNA microarray analysis of an ocular E. coli with a
potential to form biofilm.
Commercial Relationships: Shivaji Sisinthy, None;
Ranjit Konduri; Arunasri Kotakonda, None;
Reddy Satyanaryana Gundlapally, None; Savitri Sharma, None
Role of Pseudomonas aeruginosa Condensins in Corneal Infection
Michelle Callegan1, 2, Phillip S. Coburn1, Hang Zhao3,
Valentin V. Rybenkov3. 1Ophthalmology, University of Oklahoma
Health Sciences Center, Oklahoma City, OK; 2Microbiology/
Immunology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK; 3Chemistry and Biochemistry, University of
Oklahoma, Norman, OK.
Purpose: Bacterial infections of the cornea can cause significant
vision loss. The spread of multidrug-resistant bacteria necessitates
the development of new antibiotics. Bacterial condensins govern
chromosomal organization and thereby influence growth and
virulence. In this study, we examined the P. aeruginosa condensins
MksB and SMC as plausible therapeutic targets in corneal infections.
Methods: Corneas of C57BL/6J mice were scarified and infected
with approximately 106 CFU of P. aeruginosa PAO1, an MksBdeficient mutant (Δmks), an SMC-deficient mutant (Δsmc), or a
double mutant deficient in both condensins (Δmks/Δsmc). Infections
were analyzed by bacterial quantitation, biomicroscopy, and histology
on days 1, 2, and 3 post-infection to assess bacterial growth and
inflammation. Values represent N ≥ 5 eyes/group.
Results: PAO1 grew in C57BL/6J corneas to approximately 107
CFU/eye by day 3 post-infection and caused significant corneal
inflammation over 3 days. Δsmc grew to approximately 107 CFU/eye
by day 1 post-infection, but the corneal burden significantly declined
on days 2 and 3, and no Δsmc were recovered from 3/5 eyes on day 3
post-infection. The Δmks corneal burden declined over 3 days, and no
Δmks were recovered from 3/9 eyes on day 2 post-infection or from
2/5 eyes on day 3 post-infection. The Δmks/Δsmc corneal burden also
declined over 3 days, and no Δmks/Δsmc were recovered from 3/9
eyes on day 1 post-infection, from 6/9 eyes on day 2 post-infection,
or from 5/5 eyes on day 3 post-infection. Infection with Δsmc caused
somewhat reduced corneal inflammation compared to that caused by
PAO1. Infection with Δmks and Δmks/Δsmc did not cause corneal
inflammation.
Conclusions: The results indicate that a deficiency in P. aeruginosa
condensins arrests corneal virulence. These findings suggest that
condensins may be a plausible target for therapeutic intervention.
The exploration of condensins as a novel drug target is innovative,
as broad-spectrum condensin inhibition would target a chromosome
organization pathway not yet examined as a drug target.
Commercial Relationships: Michelle Callegan, None;
Phillip S. Coburn; Hang Zhao, None; Valentin V. Rybenkov, None
Support: OCAST HR-14-042 (to VVR), NIH/NEI R01EY024140
(to MCC), Research to Prevent Blindness (unrestricted to DMEI),
P30EY027125 Core Grant for Vision Research (to RE Anderson)
In Vitro Antibiotic Susceptibility of Ocular Pathogens Collected
from the Aqueous and Vitreous Humor during the ARMOR
Surveillance Study
Penny A. Asbell1, Heleen H. DeCory2, Daniel F. Sahm3,
Christine M. Sanfilippo2. 1Ophthalmology, Icahn School of Medicine
at Mount Sinai, New York, NY; 2Medical Affairs, Bausch + Lomb,
Rochester, NY; 3IHMA, Inc, Schaumburg, IL.
Purpose: Topical antibiotics are a key part of strategies employed
to minimize the incidence of intraocular infections prior to and
following ocular surgery. Despite such efforts, bacterial resistance
is prevalent and can reduce the effectiveness of antibiotic therapy.
Here we examined resistance profiles of common bacterial pathogens
isolated from the aqueous and vitreous humor to antibiotics routinely
used in ophthalmic practice.
Methods: From 2009 through 2015, 172 aqueous and vitreous humor
isolates were collected through the ARMOR surveillance study
including 11 Haemophilus influenzae, 10 Pseudomonas aeruginosa,
21 Streptococcus pneumoniae, 30 Staphylococcus aureus, and 100
coagulase-negative staphylococci (CoNS). Minimum inhibitory
concentrations (MICs) were determined by broth microdilution
according to Clinical and Laboratory Standards Institute guidelines.
Where applicable, isolates were categorized as susceptible,
intermediate, or resistant based on systemic breakpoints.
Results: Antibiotic resistance was prevalent among staphylococci,
particularly CoNS, with high rates of resistance to azithromycin (5055%), ciprofloxacin (30-53%), and oxacillin/methicillin (33-50%).
At least 70% of methicillin-resistant staphylococci demonstrated
multidrug resistance (≥3 antibiotic classes). Fluoroquinolone
MIC90s (in µg/mL) for CoNS, the most frequently isolated organism
from the aqueous and vitreous humor, were 4, 32, 64, 64, and 256
for besifloxacin, moxifloxacin, gatifloxacin, ciprofloxacin, and
levofloxacin, respectively, while ciprofloxacin was most potent
against P. aeruginosa. With the exception of 1 ciprofloxacinintermediate P. aeruginosa isolate, all P. aeruginosa and H.
influenzae strains were susceptible to all agents tested. Among S.
pneumoniae isolates, resistance was highest for azithromycin (33%),
chloramphenicol (10%), and oral penicillin (43%).
Conclusions: Aqueous and vitreous humor staphylococcal isolates
showed high levels of resistance to commonly used topical
ophthalmic antibiotics. To achieve effective prophylaxis in the
perioperative setting, current antibiotic resistance trends should be
considered when choosing appropriate therapy.
Commercial Relationships: Penny A. Asbell, Bausch + Lomb (C);
Heleen H. DeCory, Bausch + Lomb; Daniel F. Sahm, IHMA, Inc;
Christine M. Sanfilippo, Bausch + Lomb
Evaluation of Surveillance Tools for Trachoma in Two Districts
in Nepal
Andrea I. Zambrano1, Beatriz E. Munoz1, Shekhar Sharma2,
Sailesh Mishra2, Laura Dize1, Katherine Crowley3, Lisa Rotondo3,
Sheila K. West1. 1Johns Hopkins Medicine, Baltimore, MD;
2
Nepal Netra Jyoti Sangh, Kathmandu, Nepal; 3RTI International,
Washington, DC.
Purpose: As more countries achieve elimination goals for active
trachoma, surveillance is necessary for verification of absence of
re-emergence. In Nepal, surveillance surveys in all formerly endemic
districts have started. During these pre-validation surveillance
surveys, we added a test of infection processed locally, and antibody
detection in young children, to the clinical grading of active trachoma
to evaluate the possible role of these tools in surveillance programs.
Methods: 15 randomly selected clusters within two districts were
chosen (30 clusters). In each cluster, 50 randomly selected children
ages 1-9 year olds and 100 adults ≥15 years old were examined for
TF±TI and TT respectively (1500 children and 3000 adults). Eye
swabs were taken from all children to test for C. trachomatis (CT)
using the Cepheid GeneXpert platform (Cepheid, Sunnyvale, CA).
Dried blood spots were collected from children 1-4 and 9 years (794
children) to determine antibody positivity to the C. trachomatis
antigen pgp3. Blood spots were processed on the Luminex-100
platform following standard procedures. Data were analyzed as
simple frequencies, and age stratified proportions.
Results: Districts were 2 and 4 years from last program activities.
In the sampled 1500 children, only 2 TF cases and one CT infection
were found. Overall antibody positivity was found in 2.4% of
samples with no increase in frequency by age. There was no evidence
for clustering of antibody positivity by community.
Conclusions: There is no evidence of re-emergence in those two
districts in Nepal. The absence of an increase in age seroprevalence
suggests interruption of transmission of C. trachomatis.
Commercial Relationships: Andrea I. Zambrano, None;
Beatriz E. Munoz, None; Shekhar Sharma, None; Sailesh Mishra,
None; Laura Dize, None; Katherine Crowley, None;
Lisa Rotondo, None; Sheila K. West, None
Support: Grant from the Bill and Melinda Gates Foundation to the
NTD Support Center; surveys were conducted with assistance from
RTI International with funding from the US Agency for International
Development and the ENVISION project. International Trachoma
Initiative supplied the Cepheid machine for use in Nepal.
Granulicatella Adiacens bleb-associated endophthalmitis
Samuel Yun, Muge Kesen. Ophthalmology, WVU Eye Institute,
Morgantown, WV.
Purpose: Granulicatella Adiacens is a type of nutritionally variant
Streptococci and part of normal flora in oral mucosa that requires
Vitamin B6 (Pyridoxine) in order to grow.1 Although well known
to cause treatment resistant-culture negative endocarditis, there is
paucity of its reports in ophthalmology literature.2 We report another
case of G. Adiacens and literature review to increase awareness of its
importance in culture negative endophthalmitis and its tolerance in
penicillin and vancomycin3.
Methods: A case report and review of literature
Results: 72 year old man presented with acute endophthalmitis in
the setting of history of glaucoma implant and scleral contact lens
wear for keratoconus. Although initially culture negative, subculture
result revealed G. Adiacens as a causative organism, which was
successfully treated with emergent pars plana vitrectomy (PPV), two
repeat intravitreal injections (IVI) of Vacomycin and Cefatazadime,
and systemic vancomycin and cefepime. In the literature review,
83 year old man with endophthalmitis from cataract surgery was
successfully treated with emergent PPV and IVI of Vancomycin and
Amikacin with complete recovery.4
Conclusions: One should suspect G. Adiacens in endophthalmitis
with pleomorphic gram stain and negative culture result.1 Given
tolerance to penicillin and vancomycin, one may consider
aminoglycoside in case of treatment resistant endophthalmitis.3
Commercial Relationships: Samuel Yun, None; Muge Kesen,
None
Chlamydia sp genotypes associated with inclusion conjunctivitis
in patients of an Ophthalmology Center in Mexico City
Ethel B. Guinto Arcos, Victor M. Bautista, Sergio U. Pliego-Nava,
Herlinda Mejia-Lopez. U968 Institut de la Vision, CONDE DE
VALENCIANA, Mexico City, Mexico.
Purpose: To identify the predominant genotypes of Chlamydia sp in
inclusion conjunctivits in Mexican population
Methods: 160 conjunctival samples from patients with clinical
diagnosis of inclusion conjunctivitis were evaluated. Molecular
diagnosis was performed by real time PCR for identification of
Chlamydia sp, using amplification of a segment of gene 16S
common in Chlamydia species. Genotype identification was made by
automated sequencing of OmpA gene using the Big Dye Terminator
Cycle Sequencing Ready kit (Applied Biosystems, Foster City,
CA). The obtained sequences were compared with those reported in
GenBank.
Results: The parcial sequences of ompA gene were analyzed
manually and genotypes were compared and aligned with the NCBI
database sequences via BLAST. The identification of the predominant
serotypes was achieved. The most frecuently found were D, E, F
and G servovars. In our study, all strains of E serovars were new
genotypicvariants of Chlamydia trachomatis similar to those found
in genital samples, recently reported in the GenBank by Indian
researchers.
Conclusions: Chlamydia sp. infections are the most common
bacterial sexually transmitted diseases in the world. The presence
of this disease in different areas throughout the globe and its
various ocular manifestations demonstrates the bacteria’s ability
to transcend race, class and age. Given the important impact of
Chlamydia on the world’s population in a variety of ways, clinicians
and epidemiologists must collaborate on the development of optimal
therapies and early diagnosis. The identification of Chlamydia sp.
serovars responsible for inclusion conjunctivitis will facilitate the
understanding and characterization of strains of epidemiological
importance in this population.
Commercial Relationships: Ethel B. Guinto Arcos, None;
Victor M. Bautista, None; Sergio U. Pliego-Nava, None;
Herlinda Mejia-Lopez, None
Carrageenan - a natural inhibitor of ocular chlamydial infection
in vitro and in vivo
Nadine Schuerer1, Elisabeth Stein1, Aleksandra Inic-Kanada1,
Sandra Belij1, Marijana Stojanovic2, Jacqueline Montanaro1,
Ehsan Ghasemian1, Emilija Marinkovic2, Ana Filipovic2,
Talin Barisani-Asenbauer1. 1OCUVAC - Laura Bassi, Center for
Pathophysiology, Infectiology and Immunology, Medical University
Vienna, Vienna, Austria; 2Department of Research and Development,
Institute of Virology, Vaccines and Sera – TORLAK, Belgrade,
Serbia.
Purpose: Ocular infection with Chlamydia trachomatis (Ct) is the
leading cause of infectious blindness. As Ct infects via extracellular
elementary bodies (EB), we suggest that carrageenan, a natural
extract from red seaweed that binds virus particles, might physically
bind EB and thereby prevent their attachment to epithelial cells. We
tested the hypothesis that carrageenan inhibits Ct infection in vitro
using an experimental ocular infection model and in vivo using our
guinea pig model.
Methods: Confluent monolayers of human conjunctival epithelial
(HCjE) cells were inoculated with Ct serovar B in the presence or
absence of carrageenan. Cells were cultured for 48 hours, then fixed
and stained with α-Chlamydia LPS antibody and visualized with
fluorescent microscopy. Hartley strain guinea pigs were treated either
with placebo or with 0.06 mg per eye of carrageenan for 2h before
infecting with 1x104 IFU of Chlamydia caviae (3 animals per group).
The palpebral and bulbar conjunctivae were evaluated for erythema,
edema, and exudation on days 4, 7, 14, and 21.
Results: HCjE cells treated with 1.2 mg/ml carrageenan showed
minimal infection (mean of 326±10.94 IFU), with a 7-fold reduction
compared to placebo treated cells (mean of 2403±89.47 IFU,
p=0.001). In the guinea pigs the pathology score was significantly
reduced in the group pre-treated with carrageenan at all time points
(p=0.05).
Conclusions: Carrageenan reduced the absolute number of infected
cells in vitro and the pathology in vivo, suggesting it should be
investigated further as treatment and/or prophylaxis for Ct infection.
Commercial Relationships: Nadine Schuerer, None;
Elisabeth Stein, None; Aleksandra Inic-Kanada,
None; Sandra Belij, None; Marijana Stojanovic, None;
Jacqueline Montanaro, None; Ehsan Ghasemian, None;
Emilija Marinkovic, None; Ana Filipovic, None; Talin BarisaniAsenbauer, None
Support: Laura Bassi FFG Project Number 822768
Clinical manifestations and diagnosis challenge of ocular
tuberculosis in Mexican population
Erick Rebolledo Enríquez1, Roberto Dalli2, Rosalva Bobadilla1,
Miguel Pedroza-Seres1, 2. 1Ophthalmology, Fundación Conde
de Valenciana, Mexico, Mexico; 2Uveitis, Clínica de Retina,
Guadalajara, Mexico.
Purpose: To describe clinical manifestations of patients diagnosed
with ocular tuberculosis in a reference Centre in Mexico City
Methods: We analyzed medical records from patients diagnosed with
ocular tuberculosis from January 2004 to December 2014 in Uveitis
and Immunology department including demographic data, clinical
presentation, treatment and complication
Results: 4493 patients were seen at the service; 1.24% (75 eyes)
had the diagnosis of ocular tuberculosis. The presentation among
the genders was 52% in women vs 48% in men, with a mean age of
46 years at diagnosis. We had a mean follow-up of 18.25 months.
The chief complain at the onset of symptoms were: decrease vision
(57%), red eye (21%) and ocular pain (11%). We observed a right
eye predilection (65%), and 33% of bilateral cases. The clinical
presentation was: panuveitis (39%), anterior uveitis (20%), posterior
uveitis (16%), scleritis (10%), and others (15%). BCVA was logMAR
0.6020 or higher in 41% at first visit. Main diagnostic tests used
were: PCR of aqueous humor in 11%, Quanti-FERON-TB Gold in
36% and PPD in 55 % of patients. 75% of positive PPD patients
had an induration between 10 and 30 mm, and > 30 mm in 25%.
According to clinical manifestations and auxiliary tests, presumptive
and confirmed cases were 89.3% and 10.7% respectively. Regarding
treatment, isoniazid monotherapy was the most used regimen (38%),
followed by rifampicin/isoniazid/pyrazinamide/ethambutol therapy
(36%), and a double or triple regimen in 26% of patients.
At the last visit, 60% of positive Quanti-FERON-TB Gold patients,
treated with antifimic drugs improved inflammatory activity. BCVA
was improved or stabilized in all cases, whereas all positive cases
who did not received antifimic treatment had 2 lines drop in their
BCVA. Patients with positive PPD test treated with antifimic drugs
improved inflammatory activity in 67%, and improved or stabilized
BCVA in 68% of cases. Complications were present in 55% of cases,
the most common include: cataract in 25 eyes, ocular hypertension in
12, and cystoid macular edema in 6 eyes.
Conclusions: Ocular tuberculosis is an uncommon cause of
morbidity. Lack of specific clinical manifestations, uniform
criteria and high specific tests in developing countries can lead to
misdiagnosis and delay in management. Cultural and economic
factors play an important role in ocular tuberculosis approach,
leading to a higher rate of complication
Commercial Relationships: Erick Rebolledo Enríquez, None;
Roberto Dalli; Rosalva Bobadilla, None; Miguel Pedroza-Seres,
None
Methicillin-Resistant Staphylococcus Keratitis in a Referral
Ophthalmology Center
Ismael Ávila-Lule1, Alfredo Terán-Tejada2,
Nallely Ramos Betancourt2, Josué D. Rodríguez-Pedraza2,
Francisco Beltrán-Díaz De La Vega2, Everardo Hernandez-Quintela2.
1
Asociación para Evitar la Ceguera en México. Hospital “Dr. Luis
Sánchez Bulnes”, Mexico City, Mexico; 2Cornea and Refractive
Surgery, Asociación para Evitar la Ceguera en México, Hospital “Dr.
Luis Sánchez Bulnes”, Mexico City, Mexico.
Purpose: To evaluate the frequency of Methicillin-Resistant
Staphylococcus (MRS) Keratitis in a referral Ophthalmology Center
in Mexico City.
Methods: A retrospective, observational study was performed.
Data was collected from medical charts of patients who attended
from February 2014 to February 2015 with diagnosis of infectious
keratitis and positive cuture for Staphylococcus spp. The data base
was subsequently used to obtain basic demographic information and
results of gram stains, bacterial cultures, sensitivity and, resistance to
antibiotics. MRS were identified by being resistant to oxacillin and/or
cefoxitin disk diffusion.
Results: Two hundred-ninety four Staphylococcus spp were isolated
from 294 patients with keratitis during the study period. A hundredtwenty six (42.9%) were methicillin resistant by oxacillin disk,
and 55 (18.7%) by cefoxitin disk. The MRS isolates included S.
epidermidis (n= 148), S. aureus (n=60), S. Haemolyticus (n=21), S.
Saprophyticus (n=19), S. hominis (n= 13), S. intermedius (n=8), and
others. The MRS corneal isolates displayed extensive antimicrobial
resistance.
Conclusions: The emergence of Methicillin-resistant Staphylococcus
strains is clinically relevant because their resistance to multiple
antibiotics limits their treatment options. Moreover, the antibiotic
susceptibility is decreasing, introducing new challenges regarding its
treatment and making constant antibiotic surveillance a priority
Commercial Relationships: Ismael Ávila-Lule, None;
Alfredo Terán-Tejada, None; Nallely Ramos Betancourt, None;
Josué D. Rodríguez-Pedraza; Francisco Beltrán-Díaz De La Vega,
None; Everardo Hernandez-Quintela, None

Session 272 Infections

Transcription

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