Proposed ANSI Standards for In-office Disinfection of Contact Lenses

METHODS FOR CONTACT LENS REPROCESSING:

MICROBIOLOGICAL REQUIREMENTS [10/12/99]

A Proposal by the ad hoc Committee on Disinfection of Trial Contact Lenses, of the Contact Lens Subcommittee, Z80 Committee on Ophthalmic Optics, of the American National Standards Institute:

William J. Benjamin
Chairman, representing the ANSI Contact Lens Subcommittee

Peter C. Donshik
Representing the American Association of Ophthalmology (AAO), & the Contact Lens Association of Ophthalmologists (CLAO)

Michael J. Miller
Representing the Contact Lens Institute (CLI)

Mary Mowry-McKee
Representing the Contact Lens Institute (CLI)

Richard Sanders
Representing the Optician's Association of America (OAA)

Christopher Snyder
Representing the American Optometric Association (AOA) & the Contact Lens Section of that organization

Barry Van Duzee
Representing the Contact Lens Manufacturers Association (CLMA)


Principle

Products for in-office disinfection and storage of trial contact lenses are intended to reduce the risk of microbial contamination that may be introduced during patient-to-patient use. Therefore, regimens for use in the office should meet a higher requirement than regimens used by patients. This is due to the increased potential for cross contamination between patients in the office over that existing for single-patient use.

Trial lens disinfection may be achieved by utilizing a lens care solution containing an antimicrobial agent or agents, exposure to heat or ultraviolet radiation, terminal sterilization (e.g., autoclaving), or other anti-microbial systems. In actual practice, it is highly recommended that a cleaning (e.g., rubbing) and rinsing stage be administered prior to exposure to the antimicrobial system; this assures that the trial lens is ade-quately prepared for disinfection.

A "Stand Alone Test" challenges a disinfecting product with a standard inoculum of a representative range of microorganisms and establishes the extent of their viability loss at pre-determined time intervals compar-able with those during which the product may be used. For the purposes of the "Stand Alone Test," the "rubbing and rinsing" stage is omitted to allow for a standardized evaluation of the inherent antimicrobial effectiveness of the product.

A "Regimen Test" is a procedure which challenges a multifunctional disinfecting regimen with a standard inoculum of a representative range of microorganisms and which measures the viability loss after the regi-men is followed in the manner and quantity recommended in product labeling and/or practitioner instruc-tions. This procedure may include the steps of cleaning, rinsing, and soaking.

The size of the microbial challenge chosen in this test is not intended to be representative of the likely chal-lenge in practice but to provide countable numbers from which estimation of the rate and extent of viability loss can be determined. In carrying out the test for antimicrobial activity the qualitative and quantitative compositions of the product have to be known at the time of testing by either analytical testing or extrapo-lation. Appropriate measures shall be taken to inactivate or remove residual antimicrobial agents during culturing and counting of challenge organism survivors and the effectiveness of these measures shall be validated. The action of this process during the test shall be demonstrated by the construction of suitable controls.

The proposed standard for reprocessing of contact lenses in the office elevates the microbiological requirements above those required for single-patient use. The active element in the reprocessing must pass the "stand alone test" and the entire procedure the "regimen test," whereas for single-patient use, the care regimen is allowed to pass either of these tests. The number of challenge microorganisms has been increased by the addition of 3 viruses: Poliovirus, Adenovirus, and Herpes simplex. The criterion for passage of the regimen test is "No recovery" of the challenge organisms. The committee felt that there was little evidence clinically or microbiologically to set an exact criterion for the stand alone test, and has left a range of criteria for consideration by the industry. The range noted in the proposal (see Table 1) is from "no recovery" to a "3 log reduction" for all of the challenge microorganisms. This in itself is a raising of the bar for the stand alone test but the committee felt that further definition of an exact criterion would need to await a more precise scrutiny by the industry.

Methods of management for contact lenses

Steam sterilization may be used to terminally sterilize trial contact lenses between patient fittings. The sterility assurance level (SAL) shall then be equal to 10-6 or less.

Trial contact lenses may also be subjected to high level disinfection using a hydrogen peroxide system which includes a two-hour soak in 3% hydrogen peroxide solution followed by neutralization and/or dilution of the hydrogen peroxide to 75 ppm or less. The final solution used for storage of a contact lens shall be preserved.

Heat disinfection and other methods may be used for trial contact lenses if they meet the criteria stated below.

Methodology and performance requirements for trial contact lenses that are disinfected by lens care solutions and systems


Stand alone test

[Note to reader: The criteria of numerical reduction for bacteria, fungi, and viruses, to be found below and in Table 1 for the "stand alone" test, each range from "No recovery" to 3 logs of reduction. The definitive reduction figures to be cited here and in Table 1 will require much discussion at the ANSI Z80 Contact Lens Subcommittee, and the criteria will require verification before a standard is issued with regards to the methodology presented. It was felt best to leave a range of reduction recommended at this time until this report is reviewed by the industry and those criterion levels of reduction are more critically addressed.]


Bacteria

The number of each bacterial challenge organism recovered per ml shall be reduced by a mean value of not less than 99.9 % (3.0 to 5.0 logs) within the minimum recommended soaking period, or there shall be no recovery of bacterial challenge organisms (see Table 1).


Molds and yeasts

The number of each fungal challenge organism recovered per ml shall be reduced by a mean value of not less than 99.9 % (3.0 to 5.0 logs) within the minimum recommended soaking period, or there shall be no recovery of fungal challenge organisms (see Table 1). There shall be no increase in the number of micro-organisms recovered at not less than four times the minimum recommended soaking period (within an experimental error of ± 0.5 logs).


Viruses

The number of each viral challenge organism recovered per ml, by the method specified in Annex C, shall be reduced by a mean value of not less than 99.9 % (3.0 to 5.0 logs). The reduction of 3.0 to 6.0 logs shall be reached within the minimum recommended soaking period, or there shall be no recovery of viral chal-lenge organisms (see Table 1).


Regimen test

For each microbial species, there shall be no recovery of challenge organisms for all lots tested (See Table 1). "No recovery" in these testing procedures ensures that no more than 10 cfu for each lens type/storage solution combination existed before dilution.

NOTE: Data from more than one lens type may not be combined to calculate the average. When quali-fying a lens care product regimen for use with one lens type, the average count for each species requires averaging the data from the 24 inoculated and treated lenses of the one lens type. When qualifying a lens care product regimen for use with more than one lens type, the average count for each species by lens type would require averaging the data from the 12 inoculated and treated lenses for each lens type. See Table 4 for numbers of lenses to be used.

Table 1 — Summary of performance requirements criteria for contact lens disinfection procedures

Mean log reduction at soaking time

Microorganism

FungiA

BacteriaA

VirusesA

Stand Alone Test CriteriaB

No recovery to 3 log reduction

No recovery to 3 log reduction

No recovery to 3 log reduction

Regimen Test CriteriaB

No recovery

No recovery

No recovery


AFS = Fusarium solani ATCC 36031;

CA = Candida albicans ATCC 10231;
SM = Serratia marcescens ATCC 13880;

PA = Pseudomonas aeruginosa ATCC 9027;
SA = Staphylococcus aureus ATCC 6538;

AV = Adenovirus Type 7 ATCC VR-7 or Type 8 ATCC (to be determined);

HS = Herpes simplex Type 1 ATCC VR-539;

PV = Poliovirus Type 2 ATCC VR-1464.

B "No recovery" is equivalent to an average of not more than 10 cfu per lens type/storage solution combination, or a 4-5 log numerical reduction.

Test methods for bacteria, molds and yeast

NOTE: Test methods (i.e. materials and methods, test organisms, culture maintenance, etc.) for regimen testing using viruses are specified in Annex C.


Materials and reagents

NOTE: The materials and reagents (i.e. the test organisms, media and reagents, equipment and samples) are common to both the stand alone procedure for disinfecting products and the regimen procedure for contact lens disinfection.


Bacterial and fungal test organisms

The strains listed in Table 2 shall be used.


NOTE: Test organisms from other culture collections that may be used are listed in Annex A.

Table 2 — Test organisms

Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
Serratia marcescens ATCC 13880
Candida albicans ATCC 10231
Fusarium solani ATCC 36031

Culture media and reagents:

  • Potato Dextrose Agar (PDA)
  • Tryptone Soya Agar (TSA)
  • Sabouraud Dextrose Agar (SDA)
  • Dulbecco's Phosphate Buffered Saline without calcium chloride and magnesium chloride (DPBS): 200 mg/l KCl, 200 mg/l KH2PO4, 8000 mg/l NaC1, and 2160 mg/l Na2HPO4 · 7H20 or suitable diluent.
  • Dulbecco's Phosphate Buffered Saline plus 0.05% w/v polysorbate 80 (DPBST) or suitable diluent.
  • Validated neutralising agents/media as required, for example, Dey-Engley Neutralising Broth (DEB) and Letheen Broth


Test equipment:

The following common laboratory equipment is required: Sterile pipettes, swabs, tubes, petri dishes (90 mm to 100 mm x 20 mm), etc. and suitable instruments for spectrophotometric determination of cell density, for colony counting, and for centrifugation.


Test samples

The product to be tested shall be representative of the product to be marketed. Aliquots should be taken directly from the final product container immediately prior to testing.


Three lots of product shall be tested. Each lot of product shall be tested with a separate inoculum prep-aration for each challenge organism.


Culture maintenance

Maintain the test cultures as recommended by the curator of the appropriate culture collection. Cultures should be no greater than 5 passes removed from the depository stock (ATCC, NCIB, NCTC, NCPF or other recognized culture depository; see Annex A). Each pass is a subculture of the previous pass.


Preparation of microbial challenge (Inoculum)

NOTE: The preparation of the microbial challenge organisms (inoculum) is common to both the stand alone procedure for disinfecting products and the regimen procedure for contact lens disinfection.


NOTE: For the regimen procedure for contact lens disinfection, organic soil may be included as part of the inoculum. See Annex E for an example.


Culture each test organism on agar slopes under the conditions given in Table 3.


Table 3 — Media and incubation conditions for growth of challenge organisms

Organism

Medium

Temperature
°C

Incubation time

P. aeruginosa

TSA

30 to 35

18 h to24 h

S. aureus

TSA

30 to 35

18 h to 24 h

S. marcescens

TSA

30 to 35

18 h to 24 h

C. albicans

SDA

20 to 25

42 h to 48 h

or C. albicans

SDA

30 to 35

18 h to 24 h

F. solani

PDA

20 to 25

10 d to 14 d

Use sterile DPBST or suitable diluent to harvest each culture; wash the surface growth, transfer it to a suitable vessel and vortex. Filter the spore suspensions through sterile glass wool, cheese cloth or gauze to remove hyphal fragments.


After harvesting, the cultured organisms may be washed using centrifugation. The bacterial suspensions may be filtered (e.g. 3 µm to 5 µm pore size) to produce a single cell dispersion. Then adjust all challenge cell suspensions with DPBST or other suitable diluent to a concentration of between 1 x 107 cfu/ml and 1 x 108 cfu/ml. Estimate the approximate cell concentration of each suspension by measuring the turbidity of the suspension or a dilution of the suspension using a spectrophotometer. The actual concentration of col-ony forming units per ml must be determined for each suspension, e.g. by the plate count method, at the time of the test.


NOTE: If centrifugation is used, each centrifugation should be conducted at 20°C to 25°C for no longer than the equivalent of 10 min at 4000 x g or less. Longer centrifugation times may be required at lower speeds.


NOTE: Use bacterial and yeast cell suspensions on the day of preparation. Spore suspensions may be used up to seven days following preparation if stored under refrigeration (2°C to 5°C).


Stand alone procedure


Inoculum challenge test procedure


Prepare one or more tubes (for each lot tested) containing a minimum of 10 ml of test solution per chal-lenge organism.

NOTE: Sample tubes are used rather than lens cases to allow effective technical execution of the test. Tubes of an appropriate material must be considered.

Inoculate the sample tube of the product to be tested with a suspension of test organisms sufficient to pro-vide a final count of between 1.0 x 105 and 1.0 x 106 cfu/ml. Ensure that the volume of inoculum does not exceed 1 % of the sample volume. Ensure complete dispersion of the inoculum by adequate mixing.

Store the inoculated product at 20°C to 25°C. The temperature must be monitored using a calibrated device and the temperature documented.


NOTE: If the product is sensitive to light it should be protected during the period of the test.

Take 1.0 ml aliquots of the inoculated product for determination of viable count at 25 %, 50 %, 75 % and 100 % of the minimum recommended disinfecting time for all organisms, and, in addition, not less than 400 % of the minimum recommended disinfecting time for yeast and mould. If overnight contact lens dis-infection is recommended, use a soaking time of 8 h.

Subject each of the 1.0 ml aliquots, removed at the specified time intervals, to a suitable series of decimal dilutions in validated neutralising media. Mix the suspension well by vortexing vigorously and let stand to allow neutralization to be completed.

If an antimicrobial agent in the formulation cannot be adequately inactivated or neutralized, eliminate it using a validated membrane filtration procedure (see Annex B).

Determine the viable count of organisms in appropriate dilutions by preparation of triplicate plates (unless otherwise justified) of a suitable recovery medium (e.g. TSA for bacteria and SDA for mould and yeast).

If membrane filtration has been employed to remove or neutralize antimicrobial agents, culture the mem-branes on these media as appropriate.

NOTE: If the pour plate method is utilised, keep the agar for pour plates below 50°C prior to pouring.

NOTE: The agar media used for determination of viable counts may also contain antimicrobial inactiva-tors or neutralizers, if required.

Incubate bacterial recovery plates at 30°C to 35°C. Incubate yeast recovery plates at 20°C to 25°C or 30°C to 35°C. Incubate mould recovery plates at 20°C to 25°C. Incubation times for optimal recovery of bacter-ia, yeast and moulds shall be determined. Minimum incubation times shall be based on recovery medium control testing (see 6.3.2). Record the number of cfu observed on countable plates.

NOTE: Plates should be observed periodically during incubation to prevent the occurrence of uncountable plates due to overgrowth.

Determine the average number of colony forming units on countable plates. Calculate the microbial reduc-tion at the specified time points.

NOTE: Countable plates refer to 30 cfu/plate to 300 cfu/plate for bacteria and yeast, and 8 cfu/plate to 80 cfu/plate for moulds, except when colonies are observed only for the 100 or 10-1 dilution plates.

No recovery of microorganisms shall be documented, e.g. by recording a „0" or „NR", when plates for all dilutions of a sample at a single time point have zero colonies.


Controls


Inoculum Control


Prepare an inoculum count by dispersing an identical aliquot of the inoculum into the same volume of a suitable diluent (e.g., DPBST) as used in 6.3.1.1 to achieve a final concentration of 1.0 x 105 to 1.0 x 106 cfu/ml. Ensure that the volume of inoculum does not exceed 1% of the sample volume. Ensure dispersion of the inoculum by adequate mixing. Evaluate this control sample for cfu/ml at the beginning of the test in order to demonstrate the suitability of the medium used for growth of the test organism and provide an esti-mate of the initial inoculum concentration. Plate the appropriate aliquot from each tube onto the recovery agar plates in triplicate (unless otherwise justified).


Recovery medium control


Vortex a 1/10 dilution of the disinfecting product in the validated neutralising broth (1 ml into 9 ml). Let it stand to allow neutralization to be completed. Prepare a second control tube with 10 ml of a suitable diluent (e.g., DPBST). Inoculate the tubes with sufficient inoculum to result in 10-100 cfu of challenge organism per plate. Incubate for an appropriate period of time at ambient temperature. Plate the appropriate aliquot from each tube onto the recovery agar plates in triplicate (unless otherwise justified).

Incubate bacterial recovery plates at 30°C to 35°C. Incubate yeast recovery plates at 20°C to 25°C or 30°C to 35°C. Incubate mould recovery plates at 20°C to 25°C. Determine minimum incubation times for opti-mal recovery of bacteria, yeast and moulds.

Check that the recovery from the neutralizer broth is at least 50 % of the recovery in the second control tube. Perform this control for each challenge organism.

NOTE: If a dilution of greater than 1/10 is required for neutralization, then membrane filtration must be used.

Validate the neutralization of the product with each challenge organism initially and as appropriate.


Control Specification

If any control value is outside that specified, repeat the procedure as the associated test is invalid.


Regimen procedure

Lens inoculation

Carry out the test using lens types representative of those with which the regimen is intended to be used, e.g. Low water non-ionic, high water ionic, silicone acrylate, etc. New and unused lenses should be used for this test. When qualifying a lens care product regimen with a single lens type, inoculate each of eight (8) lenses for each microbial species per lot of test product; this results in testing a total of 24 lenses per formulation per species. When qualifying a lens care product regimen for use with all hydrophilic lenses, inoculate each of four (4) lenses from Group 1 (low water content non-ionic) and four (4) lenses from Group 4 (mid- and high water content ionic) for each microbial species per lot of test product; this results in testing a total of 12 lenses per lens type per formulation per species. Additional hydrophilic lens types may be tested; however, a minimum of four (4) lenses per lens type per species per lot of formulation must be used. In qualifying a lens care product regimen for use with all non-hydrophilic lenses, inoculate four (4) silicone acrylate lenses and four (4) fluorosilicone acrylate lenses per microbial species per lot of test pro-duct for a total of 12 lenses per lens type per formulation per species. Qualification of a lens care product regimen with all hydrophilic and all non-hydrophilic lenses requires testing with Group 1 and 4 hydrophilic lens types and silicone acrylate and fluorosilicone acrylate non-hydrophilic lenses.

Number of lenses per microbial species

Qualification for a single lens typeC

Qualification for all hydrophilic lensesB, D

Qualification for all
non-hydrophilic lensesB, D

Test sampleA

(e.g., Group 1)

Group 1

Group 4

Silicone
acrylate

Fluoro-silicone acrylate

Solution LOT 1

8

4

4

4

4

Solution LOT 2

8

4

4

4

4

Solution LOT 3

8

4

4

4

4

TotalD

24

12

12

12

12

A. Minimum of three (3) lots of lens care product to be tested.

B. If testing more than one lens type, a minimum of four (4) lenses per lens type per lot of lens care product per microbial species must be used.

C. If testing only one lens type, a minimum of eight (8) lenses per lens type per lot of lens care product per microbial species must be used.

D. Qualification of a lens care product regimen with all hydrophilic lenses and all non-hydrophilic lenses would require, at a minimum, testing the product with four lenses from each of the following lens types: Group 1 and Group 4 hydrophilic lenses and silicone acrylate and fluorosilicone acrylate non-hydrophlic lenses.



A. Minimum of three (3) lots of lens care product to be tested.

B. If testing more than one lens type, a minimum of four (4) lenses per lens type per lot of lens care product per microbial species must be used.

C. If testing only one lens type, a minimum of eight (8) lenses per lens type per lot of lens care product per microbial species must be used.

D. Qualification of a lens care product regimen with all hydrophilic lenses and all non-hydrophilic lenses would require, at a minimum, testing the product with four lenses from each of the following lens types: Group 1 and Group 4 hydrophilic lenses and silicone acrylate and fluorosilicone acrylate non-hydrophlic lenses.


Place test and control lenses, with concave surface uppermost, in a sterile petri dish. Inoculate each lens by placing 0.01 ml of inoculum on the underside of the lens at the point of contact between the petri dish and the lens. Also inoculate the upper surface by application of 0.01 ml of the same inoculum directly on to the concave surface of the lens.

Allow the inoculum to adsorb onto each lens for between 5 min and 10 min at 20°C to 25°C.


Lens treatment


After adsorption of the inoculum, treat the lenses as described in the manufacturer's instructions to the practitioner for trial contact lens disinfection, including all steps of cleaning, rinsing and soaking as specified by the manufacturer. Test protocols should specify the parameters of the cleaning and rinsing procedures (e.g., rubbing and rinsing times and rinse volumes).

Recovery of surviving challenge organisms (see Annex B for a suggested method)

Dispense a suitable volume of a validated neutralizing medium into the filtration apparatus (B.1.2.1).

Transfer the entire contents of each test lens case (lens and solution) into the neutralizing medium in the filtration apparatus (B.1.2.2) having determined the neutralization exposure time prior to filtration (see Annex B).

Apply reduced pressure and filter the solution. Rinse the filter with a suitable volume of neutralizing medium.

Aseptically transfer the contact lens onto a bed of agar medium appropriate for recovery of the test organism. Pour some of the same agar medium (kept below 50°C) over the lens and allow to cool.

Apply the test filter to the surface of a plate of appropriate solid media (could be the same as used in 6.3.1.5).

Incubate bacterial recovery plates at 30°C to 35°C. Incubate yeast recovery plates at 20°C to 25°C or 30°C to 35°C. Incubate mould recovery plates at 20°C to 25°C. Incubation times for optimal recovery of bacter-ia, yeast and moulds shall be determined. Minimum incubation times shall be based on recovery medium control testing (see 6.4.4). Record the number of cfu observed on countable plates.


NOTE: Plates should be observed periodically during incubation to prevent the occurrence of uncountable plates due to overgrowth.


Controls

Lens inoculation control

For each microbial species tested, transfer three inoculated lenses to tubes of a suitable diluent (e.g., DPBST). Vortex for 30 s. Serially dilute and plate out appropriate dilutions in triplicate (unless otherwise justified) to permit a count of viable cells present.


NOTE: This count confirms that the number of organisms on the lens at the time of the regimen challenge is adequate. The mean of the counts should be not less than 2 x 105 cfu/lens and not greater than 2 x 106 cfu/lens.


Recovery medium control

Prepare filtration apparatus in triplicate (unless otherwise justified) as described in 6.4.3 with suitable vol-umes of the neutralizing medium and disinfecting product (see Annex B). Add 5 cfu to 100 cfu challenge organisms (one organism per filter), filter and cultivate as described in 6.4.3.

Confirm the inoculum on a suitable medium in triplicate (unless otherwise justified).

Ensure that the recovery on the filter from the neutralizer broth is at least 50 % of the inoculum.

Validate the neutralization of the product with each challenge organism initially and as appropriate.

Test reports for Stand Alone and Regimen Testing

The test report shall include:

  • the title of this Standard
  • the identification of the product
  • name of the product
  • batch number
  • expiration date
  • manufacturer
  • storage conditions
  • active substances(s) and its/their concentration(s) (optional)
  • the name/s of the operator/s
  • deviations from the protocol
  • date and time of incubation
  • storage time for inoculated product
  • results obtained

Annex A
(informative)

Test organisms from other culture collections

See tables A.1 and A.2.

NOTE: Cultures from various collections should be equivalent to ATCC strains.

Table A.1 — Test organisms from other culture collections

Pseudomonas aeruginosa

BUCSAV 278
IAM 10374

CCM 1961
IFO 13275

CIP 82.118
NCIMB 8626

DSM 1128
NRRL B-800

DSM 1385

Staphylococcus aureus

CIP 4.83

DSM 799

IFO 13276

NCIB 9518

NCTC 10788

Serratia marcescens

CCM 303
NCTC 10211

DSM 47

DSM 30121

CDC 813-60

NCIB 9155

Candida albicans

CBS 6431
NCPF 3179

CCY 29-3-106
NCYC 1363

CIP 48.72
VTT C-85161

DSM 1386

IFO 1594


Table A.2 — Culture collections and institutions

ATCC

American Type Culture Collection, Rockville, Md., USA

BUCSAV

Biologicky Ustav Ceskoslovenska Akademie Ved., Prague, Czechoslovakia

CBS

Centraalbureau voor Schimmelcultures, Baarn, The Netherlands

CCM

Czechoslovak Collection of Microorganisms, J.E. Purkyne University, Brno, Czechoslovakia

CCY

Czechoslovak Collection of Yeasts, Bratislava, Czechoslovakia

CDC

Centers for Disease Control, Atlanta, Georgia, USA

CIP

Collection de bactéries de l'Institute Pasteur, Paris, France

DSM

Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany

IAM

Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan

IFO

Institute for Fermentation, Osaka, Japan

NCIB

National Collection of Industrial Bacteria, Aberdeen, Scotland, U.K.

NCIMB

National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, U.K.

NCPF

National Collection of Pathgenic Fungi, Mycological Reference Laboratory, Central Public Health Laboratory, London, U.K.

NCTC

National Collection of Type Cultures, Central Public Health Laboratory, London, U.K.

NCYC

National Collection of Yeast Cultures, Nutfield, Surrey, U.K.

NRRL

Northern Regional Research Center, U.S. Department of Agriculture, Peoria, Illinois, USA

VTT

Technical Research Centre of Finland, VTT Collection of Industrial Microorganisms, Espoo, Finland


Annex B
(informative)

Example of a membrane filtration procedure

B.1 Materials and reagents

B.1.1 Culture media and reagents

B.1.1.1 Diluting fluid, with or without neutralizers

B.1.1.2 Tryptone Soya Agar (TSA)

B.1.1.3 Dulbecco's Phosphate Buffered Saline without calcium chloride and magnesium chloride (DPBS): 200 mg/l KCl, 200 mg/l KH2PO4, 8000 mg/l NaCl, and 2,160 mg/l NA2HPO4 · 7H20 or suitable diluent

B.1.1.4 Dulbecco's Phosphate Buffered Saline plus 0.05% w/v polysorbate 80 (DPBST) or suitable diluent.

B.1.1.5 Validated neutralising agents/media as required, for example, Dey-Engley Neutralizing Broth (DEB) and Letheen Broth

B.1.2 Test equipment

Usual laboratory equipment (such as sterile pipettes, petri dishes, containers) together with the following:

B.1.2.1 Suitable sterile apparatus for holding the sterile membrane filter and filtrate

B.1.2.2 Suitable equipment for creating a vacuum or pressure to cause the liquid phase of the inoculated test solution to pass through the membrane filter aseptically.

NOTE: The membrane filter should have a nominal pore size of not greater than 0.45 µm, a diameter of at least 47 mm and should be free of chemicals which could be toxic to microbial cells.

B.2 Test method and results

B.2.1 Moisten the sterile membrane filter (B.1.2.1) in a sterile filter assembly (B.1.2.2) with sterile DPBST (B.1.1.4), or suitable diluent

B.2.2 Aseptically transfer a measured volume of the inoculated test solution into sterile DPBST (B.1.1.4) or diluting fluid.

B.2.3 Transfer the diluted solution to the membrane and filter immediately with the aid of vacuum or pressure. Dilute the sample applied to the filter with 50 ml to 100 ml of dilution fluid and thoroughly mix to ensure uniform distribution of the sample over the entire area of the filter.

NOTE: This will decrease the probability of multiple colony forming units being placed on the filter at the same location.

B.2.4 Wash the membrane filter with several volumes of diluting fluid which may contain additional neutralising agents as needed.

NOTE: Three volumes of diluting fluid (100 ml each) are usually sufficient to remove and/or dilute the antimicrobial agent. The actual volume should be determined empirically for each formulation for each challenge organism.

B.2.5 Incubate the membrane filter with appropriate media to allow growth of colony forming units on the surface of the filter.

NOTE: This may be accomplished by aseptic removal of the membrane filter from the filter assembly unit and placement of the membrane on the surface of a sterile agar plate which does not have obvious liquid on the surface or the membrane may be enclosed in an agar sandwich. Alternatively, a sterile membrane filter unit may be used which requires addition of sterile media to the sealed filter and incubation of the membrane in situ. Media should be used which are appropriate for the type of challenge organism and the specific formulation under test. Time of the incubation should be established.

B.2.6 Determine the average number of colony forming units on the countable membrane filters (3 to 100 cfu/47 mm filter for bacteria and yeast and 3 to 10 cfu/47 mm filter for moulds). Calculate and document the cfu/ml of the inoculated solution.

B.3 Controls

Confirm neutralizer efficacy by transferring an aliquot of the uninoculated test solution into 50 ml to 100 ml of sterile diluting fluid using the same ratio of volume of test solution to volume of diluting fluid. Apply the entire volume to the membrane and filter using vacuum or pressure. Wash the filter with several volumes of the diluting fluid using the same volume as used for the test procedure. Transfer 5 to 100 cfu challenge organisms (one species per filter) into 100 ml of diluting fluid and apply to the membrane. Incubate the membrane filter in contact with media as described in the test procedure (see B.2.5).

Repeat the procedure using diluting fluid not exposed to the test solution. Compare counts with those derived by the same method but using a suitable diluent (e.g., DPBST), instead of the test solution. Confirm the inoculum on a suitable medium in triplicate (unless otherwise justified). Ensure that the recovery on the filter from the neutralizer broth is at least 50 % of the inoculum.

Annex C
(informative)

Regimen Procedure Using Viruses

PRINCIPLE: The procedure is designed to determine the virucidal activity of the disinfectant regimen for both short-term disinfection and long-term storage. The contact lenses and disinfectant solution will be assayed separately for increase sensitivity of the assay. Most disinfectants are toxic to the cell culture sys-tem. As virus is detected based on infection and cell death, the disinfectant must be diluted beyond the toxic level before virus can be detected. Testing the contact lens after removal from the disinfectant will allow for increased sensitivity as the contact lens will contain only small residuals of the disinfectant. The disinfectant will then be tested for viable virus after determining the dilution beyond the toxic level.

REFERENCES:

U.S. EPA "Efficacy Data Requirements: Virucide" (Proposed method prepared by Registration Division, Office of Pesticide Programs, EPA, 1976).

ASTM E 1052 - 85. Standard Test Method for Efficacy of Virucidal Agents Intended for Special Applications.

EQUIPMENT:

  • Incubator set at 37° C ± 1 C, 5% ± 1% CO2 atmospheric conditions.
  • Cell culture (inverted) microscope.
  • Cell culture hood.
  • Balance accurate to 0.01 g.
  • Automatic pipettor (optional).
  • Micropipettor accurate to 0.01 ml.
  • Incubator set at 20-25 C.
  • Vortex.
  • Timer.


SUPPLIES:

  • Sterile 1, 5, and 10 ml pipettes.
  • Micropipette tips.
  • 96-well cell culture plates.
  • 6-well cell culture plates.
  • Sterile test tubes.
  • 150 cm2 cell culture flasks.
  • Human epithelioid cervical carcinoma cells (HeLa).
  • Contact lens.
  • Contact lens storage containers.


REAGENTS:

  • Crystal violet.
  • 10% formalin.


MEDIA:

  • 1X Minimum Essential Medium (MEM) with 10% Calf Serum (CS).
  • 1X MEM with 2% CS.
  • Sterile PBS.
  • Trypsin-EDTA solution.


SAFETY:

Lab coat and safety glasses must be worn at all times during testing. Observe other safety precautions as noted by the sponsor regarding handling of test samples.

Handle disinfectants with care.

Do not wear contact lenses when working in horizontal HEPA hoods unless you also wear goggles or protective eye wear which prevents airflow across the cornea.

Properly dispose of infectious waste. Avoid creating aerosols when working with cultures.


STOCK VIRUS PREPARATION

Maintain 150 cm2 flasks of human epithelioid cervical carcinoma cells (HeLa) in MEM with 10% CS.

Inoculate a confluent 150 cm2 flask of HeLa cells with 5.0 ml of virus stock at a multiplicity of infection of 0.01. Incubate the flask at 37 + 1° C with 5 ± 1% CO2.

Allow the virus to absorb for 30-60 minutes, then add approximately 20 ml MEM with 2% CS to the flask. Incubate the flask at 37 + 1° C with 5 ± 1% CO2.

Allow the flask to reach 4 + CPE, freeze at <-60° C then rapidly thaw repeatedly. This causes mechanical rupture of the cell membranes and releases virus into the media. Store the virus stock at <-60° C.


TCID50 PROCEDURE

Seed 96-well cell culture plates with 0.1 ml/well of a suspension of HeLa cells at a concentration of approximately 105 cells/ml. Incubate the plates at 37 + 1 °C with 5 + 1% CO2 until confluent.

Make serial 1:10 dilutions (0.1 ml into 0.9 ml) of the virus in MEM with 2% CS.

Remove the media from the 96-well cell culture plates. Add 0.1 ml/well of the virus dilutions to the culture plates. All dilutions are plated in five wells. Add MEM with 2% CS to five wells as negative cell controls. Allow the virus to absorb for 30-60 minutes at 37 ± 1 °C with 5 + 1% CO2.

After absorption, add 0.1 ml of MEM with 2% CS to each well on the cell culture plates. Incubate the plates at 37° C ± 1 °C with 5 ± 1% CO2 for 3-5 days.

Inactivate the plates with 10% formalin for 1 hour at room temperature or for longer time periods at 2-8 C.

Wash the plates with tap water, then stain with 0.1% crystal violet solution for 30 minutes.

Wash the plates with tap water and examine the wells for viral CPE. Score each well with CPE or without CPE.

Calculate the TCID50 using the Reed-Muench method as shown in Annex D.


LENS INOCULATION

Adjust the titer of viral particles to 107+ PFU/ml.

Test 4 replicates of each type of lens per test lot of product, at each exposure time. Prepare 3 replicates of each type of lens as virus controls (no exposure to the disinfectant).

Place test and control lenses, with concave surface uppermost, in a sterile petri dish.

Inoculate each lens by placing 0.01 ml of virus inoculum on the underside of the lens at the point of contact between the petri dish and the lens. Also inoculate the upper surface by application of 0.01 ml of the same inoculum directly on to the concave surface of the lens.

Allow the virus to absorb on each lens for 5-10 minutes at 20-25 °C.


DISINFECTANT EXPOSURE

After adsorption of the inoculum, treat the lenses as described in the manufacturer's instructions to the consumer for contact lens disinfection, including all steps of cleaning, rinsing and soaking as specified by the manufacturer. Test protocols should specify the parameters of the cleaning and rinsing procedures (e.g., rubbing and rinsing times and rinse volumes).

Place the lenses in the lens storage containers and fill with disinfectant.

Place the corresponding virus control lenses in the lens storage containers and fill with MEM with 2% CS.

Store at 20-25 °C for the recommended minimum soak time (i.e. 4 hours). At the end of the soak period, remove the test exposure sample and controls for virus recovery testing. Store the remaining samples at 20-25 °C for the maximum storage period (i.e. 30 days).

After the designated maximum storage time, remove the test exposure sample and controls for virus recovery testing.

NOTE: There may be additional processing steps (i.e. rubbing and/or rinsing) after the lenses are removed.

Assay the lens and disinfectant for viable virus.


CONTACT LENS VIRUS RECOVERY

Seed 6 well cell culture plates with 2.0 ml/well of a suspension of HeLa cells at a concentration of approx-imately 105 cells/ml. Incubate the plates at 37 + 1 °C with 5 + 1% CO2 until confluent.

Place the contact lens into a sterile test tube containing 2 ml of MEM with 2% CS. Vortex the mixture for 1 minute to extract any viable virus.

Remove the media from the 6 well test plate. Add the entire extract (2 ml) to one well. For each set of titrations, include 3 wells receiving 2 ml of MEM with 2% CS for negative cell controls.

Incubate the plates at 37 ± 1 ° C with 5 ± 1% CO2 for 3-5 days.

Inactivate the plates with 10% formalin for 1 hour at room temperature or for longer time periods at 2-8 C.

Wash the plates with tap water then stain with 0.1% crystal violet solution for 30 minutes.

Wash the plates with tap water and examine the wells for viral CPE. Score each well with CPE or without CPE.


DISINFECTANT VIRUS RECOVERY

Seed 96 well cell culture plates with 0.1 ml/well of a suspension of HeLa cells at a concentration of approximately 105 cells/ml. Incubate the plates at 37 + 1 ° C with 5 + 1% CO2 until confluent.

After the disinfectant exposure time, perform the TCID50 procedure on the disinfectant used to store the contact lens

Examine the wells for viral CPE. Score each well with CPE or without CPE. Calculate the TCID50 using the Reed and Muench method.


TOXICITY AND NEUTRALIZATION CONTROLS

Toxicity Controls (TCLD50): Most disinfectants are toxic at some level to the cell cultures used in the assay for the virus. Toxicity controls are performed to show the level of disinfectant that will cause CPE and thereby determine a detection limit for the assay.

Perform the contact lens disinfectant exposure and virus recovery procedure with 3 uninoculated lenses of each lens type. Plate the lens extracts in 6 well plates and the disinfectant dilutions in 96 well plates. Include a negative cell controls of MEM with 2% CS.

Score each well as with toxicity or without toxicity. Calculate the TCLD50 using the Reed and Muench method.

Neutralization Controls: Neutralization controls are an extension of the toxicity controls to show that low levels of virus can be recovered at the dilution where the disinfectant does not show toxicity to the cells.

Perform the contact lens disinfectant exposure and virus recovery procedure with 3 uninoculated lenses of each lens type. Before plating the lens extractions and dilutions of the disinfectant, spike each with approximately 10 virus. Include controls of MEM with 2% CS spiked with the same level of virus.

Score each well for virus CPE. Determine the dilution at which the virus CPE is equal to the virus CPE of the control.

Extraction Control: This control is included to confirm the detection limit of contact lens based on the extraction method used in the assay.

Inoculate 3 lenses as outlined in the lens inoculation procedure with 10 virus per lens. The dilution of the virus used is determined by the initial TCID50 titration of the stock virus.

Assay the lenses for viable virus as indicated in the contact lens virus recovery procedure. Include a control with the same amount of virus added to 2 ml of extraction media.

Score each well for virus CPE.

Annex D.

TCID50 CALCULATION

REED-MUENCH METHOD

DILUTION

TESTED

VIRUS

VIRAL CPE

SAMPLE

+/-

3

+

+

+

+

4

+

+

+

+

5

+

+

+

-

6

-

-

+

-

7

-

-

-

-


TCID50 CALCULATIONS

DILUTION

TOTAL +

TOTAL -

RATIO

PERCENT

3

12

0

12/12

100

4

8

0

8/8

100

5

4

1

4/5

80

6

1

4

1/5

20

7

0

8

0/8

0


TOTAL + = The total number of wells with CPE as counted coming up from the bottom of the chart.

TOTAL - = The total number of wells with CPE as counted coming down from the top of the chart.

RATIO = The number of wells with CPE over the total number of wells.

Pd (Proportionate distance) Calculation:

Annex E.

(informative)


SELECTED REFERENCES

Smith CA, Pepose JS (1999) Disinfection of tonometers and contact lenses in the office set-ting: are current techniques adequate? American Journal of Ophthalmology 127(1):77-84.

Slade JS, Pike EB, Eglin RP, Colbourne JS, Kurtz JB (1989) The survival of human immu-nodeficiency virus in water, sewage and sea water. Water Science and Technology 21:55-59.

ISO/DIS 14729 (1999) Ophthalmic optics – Contact lenses - Microbiological requirements for products and regimens for hygienic management of contact lenses. International Organ-ization for Standards, Geneva, Switzerland.

Premarket Notification (510(k)) Guidance Document for Contact Lens Care Products. United States Department of Health and Human Services, Food and Drug Administration, Center for Devices and Radiological Health, 9200 Corporate Blvd. (HFZ-460), Rockville, Maryland, USA; May 1, 1997.