Prof Mark Willcox

1
Prof Mark Willcox

• Institute for Eye Research, Vision Cooperative
  Research Centre and School of Optometry and
  Vision Science, University of New South Wales,
  Sydney, Australia

2
Outline of talk

• Key aspects to address
• Use of clinical isolates in testing
• Use of lens and lens case during disinfection
• New methodologies resulting from MoistureLoc and
  MoisturePlus failure analyses

3
Use of Clinical Isolates

• Using ISO Stand Alone test

4
Testing clinical isolates Stand Alone testing
MPS1
5
FungiMPS1
6
Gram positives MPS1
7
Gram negativesMPS1
8
FungiMPS1
9
Antifungal Efficacy of Individual MPDS on F.
solani after MRDTMPS1
10
Clinical Isolates

• Which clinical isolates?
• Isolates from those microbes that frequently
  cause contact lens associated MK?
• Isolates from Contact Lens Disinfection Cases?
• Should everyone use the same isolates?
• When does a clinical isolate become a laboratory
  isolate?

11
Recent Data from Epidemiological Studies in
Australia and UK

• AUSTRALIA
• 146 (62) cases were scraped
• 83 (49.4) culture proven
• 48 (58) Gram negative
• 37 P. aeruginosa or spp. (4 mixed cultures)
• 6 Serratia spp.
• 1 Klebsiella oxygenae
• 1 Other GNR
• 4 (5) Acanthamoeba
• 3 (4) Fungus/yeast
• 21 (25) Gram positive
• 5 CNS
• 4 S. aureus
• 2 Nocardia sp.
• 1 Strep. pneumoniae
• 1 Propionibacterium sp.

• UK
• Severe disease
• 7 Pseudomonas sp.
• 1 Staphylococcus sp.
• 1 Streptococcus viridans
• 1 Klebsiella
• 1 Acanthamoeba
• Moderate disease
• 20 Pseudomonas sp.
• 18 Staphylococcus sp.
• 3 Serratia marcescens
• 2 Corynebacterium sp.
• 2 Acanthamoeba
• 1 Streptococcus sp.
• 1 Acremonium sp.
• 1 Fusarium dimerum

Keay et al., 2007, Am J Ophthalmol Dart et al.,
2008, Ophthalmol
12
Types of microbes isolated from contact lens cases
Micro organism
times cultured
Median
Range
Propionibacterium sp
25.3
10
5-590
16
8
5-1,000
15.3
5
5-1,400
10.3
228,500
35-TNTC
6
470
5-TNTC
Streptococcus viridans
5.4
5
5-1,240
Bacillus spp
5.1
5
5-120
Micrococcus sp
5.1
5
5-365
3.8
13
5-30
Corynebacterium sp
3.7
23
5-2,500
3.4
155,000
5-TNTC
3.1
5
5-500
2.7
55
5-2,450
Burkholderia cepacia
2.5
25,500
10-TNTC
Achromobacter xylosoxidans
1.9
320
5-TNTC
Aeromonas hydrophila
1.8
33
5-60
Agrobacterium radiobacter
1.8
128,500
5-369,000
Klebsiella pneumoniae
1.8
33,000
330-189,500
Nocadia sp
1.4
5
5
Serraria liquifaciens
1.4
23,500
23,500-TNTC
Comamonas testosteroni
1.2
260,000
30-271,000
Enterobacter cloacae
1.2
2,433
15-TNTC
Klebsiella oxytoca
1.2
382,415
5-542,000
Moraxella sp
1.2
6,775
6,350-7,200
Morganella morganii
1.2
18
5-30
1.2
142,750
330-143,500
1.2
3,550
1,600-5,500
Staphylococcus lugdunensis
1
13
5-20
Acinetobacter sp
0.6
20
5-14.000
Flavobacterium odoratum
0.6
25
25
Non-haem streptococcus sp
0.6
5
5-15
Sphingomonas paucimobilis
0.6
35
35
Staphylococcus hyicus
0.6
80
5-80
Chryseomonas indologenes
0.5
10
10
13
Clinical Isolates

• Which clinical isolates?
• Isolates from those microbes that frequently
  cause contact lens associated MK?
• Pseudomonas aeruginosa, Staphylococcus aureus,
  Coagulase negative staphylococci, Serratia
  marcescens, Fusarium solanii, Acanthamoeba sp.,
• Isolates from Contact Lens Disinfection Cases?
• Stenotrophomonas maltophilia, Delftia
  acidovorans, Coagulase negative staphylococci,
  Propionibacterium sp.

14
Clinical Isolates

• Should everyone use the same isolates?
• Choices can be either
• one or two clinical isolates for each nominated
  genera/species that can be used by all
  manufacturers
• OR
• isolates from each nominated species but not
  necessarily same between manufacturers
• Then adequate numbers of each genera/species
  should be used
• 5 strains of each species?

15
Clinical Isolates

• Enough isolates should be tested to ensure
  adequate coverage
• Advice for efficacy testing
• At least 2 log reduction for bacterial isolates
  and 1 log reduction for majority (50) of fungal
  isolates
• If using only 1-2 isolates then 1 log reduction
  for all isolates
• Stasis for Acanthamoebal isolates

16
Clinical isolates

• If clinical isolates are resistant to Stand Alone
  test then perform Regimen Test with current
  criteria plus additional SiHy lenses

17
Clinical Isolates

• When does a clinical isolate become a laboratory
  isolate?
• After too many passages in vitro
• Keep strains at -80oC and/or freeze-dried
• Resuscitate and passage only once to ensure
  purity
• Maintain good culturing/passaging records that
  can be audited
• Review and renew clinical isolates on 5 yearly
  basis

18
Depletion studies

• Stand alone testing in presence of contact lens

19
Methods

• The biocidal activity of solution in the presence
  of contact lens case and Balafilcon A lens
• Stand alone performed in lens case plus lens
• Bacterial load 1x106 cfu/ml
• Manufacturers recommended disinfection time
• Counts of bacteria in solution and on lenses
  (after vortexing to remove bacteria from lens
  surface)

20
Stand Alone Activity in Presence of Contact Lens
Depletion Study
Product was tested in a contact lens storage case
containing Balafilcon A lens for 6h disinfection
time
21
Stand Alone Activity in Presence of Contact Lens
Depletion Study
Product was tested in a contact lens storage case
containing Balafilcon A lens for 6h disinfection
time
22
Stand Alone Activity in Presence of Contact Lens
Depletion Study
Product was tested in a contact lens storage case
containing Balafilcon A lens for 6h disinfection
time
23
Depletion studies

• Which test
• Current stand alone or inoculate lens then
  perform test?
• Which lenses to use
• Group I and Group IV HEMA-based hydrogel
• One from each Silicone Hydrogel manufacturer
• Lotrafilcon B, Galyfilcon A, Balafilcon A,
  Comafilcon A
• Which lens case
• As supplied by manufacturer

24
Useful methods for evaluating new MPDS
25
Evaporation Methods

• 1. Levy et al., 2006
• MoistureLoc and ReNu MultiPlus were formulated,
  including all components, at 2 and 4
  concentrations. Biocidal efficacy against F.
  solani was assessed by comparing the concentrated
  formulations with a standard solution of
  MoistureLoc (designated 1)
• 2. Zhang et al., 2006
• MPDS allowed to air-dry for 3 to 4 hours
• Conidia (105/mL) added to the air-dried MPS film.
  
• The air-dried films of MPS on petri dish surfaces
  were inoculated with 0.1 mL SAB broth after 7
  days and examined microscopically at 24-hour
  intervals for fungal development.
• Or the films of MPS on plastic sections were
  examined directly by tape mount and light
  microscopy.
• 3. Current study
• Products were inoculated with F. solani then
  allowed to evaporate for 24h followed by top-up
  with fresh solutions and incubation of 6h (MRDT)

26
Simulated evaporation of solution - Levy
27
Evaporation onto plastic surfaces
28
Encystment of Acanthamoeba

• Kilvington et al., and Borazjani et al., 2008
• 3 mL of solution added to wells of a 12-place
  tissue culture plate.
• 3 to 5 x 105 trophozoites in lt30µL was added to
  each well
• Plates incubated at 24 to 28C for 24 hr.
• Then detergent sarkosyl was added to each well to
  a final concentration of 0.25 and mixed by
  vigorous pipetting.
• Hemocytometer counts were then made of the cyst
  numbers in the test and control wells.
• Microscopy also performed

29
Why was Complete MoisturePlus associated with
Acanthamoeba keratitis?
Kilvington et al. 2008 Eye Contact Lens. 34
133-139
30
Microscopy of Acanthamoeba
31
Role of excipients in MPDS
Kilvington et al. 2008 Eye Contact Lens. 34
133-139
32
Role of excipients in MPDS
Borazjani et al. 2008 ARVO poster
33
Suggested methods for future MPDS testing

• Encystment according to Kilvington et al., 2008
  and Borazjani et al., 2008
• Evaporation according to Zhang et al., 2006