Determination of protein profiles in vitreous of eyes with endophthalmitis with Surface Enhanced Laser desorption/ ionization time-of-flight (SELDI-tof) technology.

Endophthalmitis is a form of ocular inflammation which is clinically diagnosed.
The inflammation is primarily located in the vitreous and fluid of the anterior
chamber. The patient experiences serious visual loss, frequently combined with
intense ocular redness and pain. The inflammation is diagnosed with
biomicroscopy. Infection is confirmed by culturing of the vitreous.
Endophthalmitis is treated with wide-spectral antibiotics which are injected in
the vitreous.

Based on the outcome of the bacterial culture, treatment can be adapted to
match the diagnosed bacterium. In case of endophthalmitis, that develops within
6 weeks after cataract surgery, the bacterial species is identified in 70% of
vitreous samples. However, in 30% no bacterial growth is determined. (1) Until
today, it is not known whether negative cultures represent sterile
inflammations or infections in which the bacterial load is to low to yield a
positive culture. An additional disadvantage of regular culture methods is the
fact that they require at least 1-3 days. With new techniques the diagnostic
process of endophthalmitis could be optimised.

SELDI-tof analyses protein content in body fluids. This technique is extremely
suitable for microvolumia, since only 1 microlitre is required for analysis.
The proteins bind to microchips, are ionised by laser and subsequently
catapulted into a vacuum tube. The speed of migration of the proteins through
the tube (time of flight) defines the mass of the protein. The different masses
of diverse proteins result in a pattern of peaks. Inflammation-specific
biomarkers or bacterial-specific biomarkers can be identified by comparing
profiles of different patient groups (endophthalmitis versus controls).
SELDI-tof technology has proven to be very effective in detecting the cause of
intra-amnion infections, SARS and congenital CMV hepatitis. (2,3,4) The protein
profiles proved highly sensitive en specific for a diversity of bacteria and
viruses.

Currently no information is available about the utility of SELDI-tof technology
of research in ocular fluids. In this study, SELDI-tof technology is used as a
new diagnostic test in ocular inflammation based on infection. The high degree
of sensitivity and specificity of SELDI-tof technology (2,3,4) in combination
with a diagnostic process of only several hours could be a valuable addition to
current diagnostic methods.

Referencies:

1. Results of the Endophthalmitis Vitrectomy Study. A randomized trial of
immediate vitrectomy and of intravenous antibiotics for the treatment of
postoperative bacterial endophthalmitis. Endophthalmitis Vitrectomy Study
Group. Arch Ophthalmol 1995;113:1479-96.
2. Gravett MG, Novy MJ, Rosenfeld RG, et al. Diagnosis of intra-amniotic
infection
by proteomic profiling and identification of novel
biomarkers.
JAMA 2004;292:462-9.
3. Kang X, Xu Y, Wu X, et al. Proteomic fingerprints for potential application
to early
diagnosis of severe acute respiratory syndrome. Clin Chem
2005;51:56-64.
4. Ward DG, Suggett N, Cheng Y, et al. Identification of serum biomarkers for
colon
cancer by proteomic analysis. Br J Cancer 2006;%19;94:1898-905.

To evaluate the usefulness of SELDI-tof (surface enhanced laser desorption/
ionisation-technology) technology in determination of disease-specific protein
profiles of bacteria in vitreous of patients with endophthalmitis after
cataract surgery.
Protein profiles of vitreous samples from endophthalmitis patients with a
negative culture will be compared to those from patients in which the bacterial
cause is identified.

The patients with endophthalmitis will all have developed endophthalmitis
within 6 weeks after cataract surgery. Treatment of this disease exists of
retro bulbar anaesthesia followed by a vitreous biopsy (0.2-0.3 ml) with a
vitrectome. After biopsy broad spectral antibiotics are injected in the
vitreous cavity. The vitreous biopsy is sent for microbiological culture. For
the current research 0.1 ml vitreous will be kept and frozen at -80 degrees
Celsius. Additionally, of 10 patients a blood sample will be drawn. After
centrifuging the blood, serum will be aspirated and frozen at -80 degrees
Celsius. Both serum and vitreous will be coded before storage on the department
of Virology to protect the privacy of the patient.

The collection of blood samples will only take place after permission of the
patient through signing informed consent. The risk of taking the bloodsample
will be equal to the general risk of taking the bloodsamples as is done in eg.
out patients clinic.

Patients will not experience any additional discomfort f withdrawel of the
vitreous sample, because of all 3 patient groups a minimum of 0.2 ml vitreous
for culture or more (in case of pars plana vitrectomy) is extracted for
therapeutic purposes. The minimum required amount of vitreous for culture is
0.1 ml. Consequently, no extra vitreous needs to be collected for this study.
Both samples of vitreous and serum will be coded before arrival on the
department of Virology to protect patients privacy.