- Email: [email protected]
Detection of pyrogens adsorbed to intraocular lenses
LABORATORY SCIENCE
Detection of pyrogens adsorbed to intraocular lenses Evaluation of limulus amoebocyte lysate and in vitro pyrogen tests Liliana Werner, MD, PhD, Manfred Tetz, MD, Khalid Mentak, PhD, Margaret Aldred, Walter Zwisler, PhD
PURPOSE: To determine the ability of the limulus amoebocyte lysate (LAL) assay and the in vitro pyrogen test (IPT) to detect pyrogens adsorbed to intraocular lenses (IOLs). SETTING: Berlin Eye Research Institute, Berlin, Germany. METHODS: Fifteen of each of the following IOLs were used: MicroSil MS 612 ASP, AcrySof SA60AT, Superflex, Sensar, XACT, and LS-106 IOLs. The challenge organism suspensions were 103 CFU/mL and 104 CFU/mL Escherichia coli, 103 CFU/mL and 104 CFU/mL Pseudomonas putida, and 105 CFU/ mL and 106 CFU/mL Staphylococcus epidermidis. Two IOLs of each model were incubated at room temperature for at least 2 days in 0.6 mL of 1 of the suspensions. They were then gamma sterilized. The extract of 1 IOL was tested with the LAL assay; the other IOL was tested with the IPT. RESULTS: The LAL was negative for all incubated IOLs. The IPT was positive for all IOLs incubated in E coli and P putida suspensions, with the MicroSil MS 612 ASP, AcrySof SA60AT, XACT, and LS106 IOLs showing a severe reaction. The Superflex and Sensar IOLs had a slight to moderate response for lower bacterial concentrations and a moderate to severe response for higher concentrations. For S epidermidis, all IOLs showed a slight IPT response except XACT IOLs, which showed a nonpyrogenic response. CONCLUSIONS: Results indicate that the LAL test may fail to detect pyrogens adsorbed to IOLs and the IPT reliably detects pyrogens with a dose-dependent response. This has relevance in the investigation of toxic anterior segment syndrome outbreaks. J Cataract Refract Surg 2009; 35:1273–1280 Q 2009 ASCRS and ESCRS
Toxic anterior segment syndrome (TASS) is characterized by a sterile postoperative inflammation caused by a noninfectious agent gaining access to the anterior segment, resulting in toxic damage to intraocular structures. It starts within 12 to 48 hours of anterior segment surgery, is limited to the anterior segment of the eye, is always gram stain and culture negative, and improves with steroidal treatment.1,2 Possible causes of TASS include instruments or devices containing residues of enzymatic detergents, oxidized metal deposits, polishing or sterilizing compounds, and bacterial endotoxin contamination of solutions and devices.3–9 In the past 3 years, TASS has been a significantly reported complication after cataract surgery with intraocular lens (IOL) implantation.2,9 Q 2009 ASCRS and ESCRS Published by Elsevier Inc.
The surface of medical devices, such as IOLs, may be contaminated with bacteria during various manufacturing steps. Even though the devices are routinely sterilized by different methods, heat-stable pyrogenic bacterial byproducts and other types of microbial and nonmicrobial pyrogens may remain adsorbed to their surfaces.10,11 Therefore, regulatory agencies worldwide require strict control of manufacturing to ensure that medical products are pyrogen-free. Pyrogen testing of medical devices is currently based on the limulus amoebocyte lysate (LAL) and rabbit pyrogen test (RPT) assays. The LAL assay uses the immune defense system of the horseshoe crab. It can be performed with liquid samples only and detects only lipopolysaccharides (LPS) with high sensitivity.12–15 The RPT can also detect other microbial and nonmicrobial 0886-3350/09/$dsee front matter doi:10.1016/j.jcrs.2009.03.012
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pyrogens. It basically involves intravenous injections of pyrogen solutions and measurements of rectal temperature.14,16–18 However, pyrogen adsorbed to medical devices, which cannot be removed by rinsing, would not be detected by the LAL or RPT assay. The more recently developed in vitro pyrogen test (IPT) can detect microbial and nonmicrobial pyrogens in human whole blood or cryopreserved blood that is brought in contact with solutions or materials. The monocytes and macrophages in the blood recognize the pyrogens by releasing cytokines. One of the first to be released is interleukin (IL)-1b, which can be measured by enzyme-linked immunosorbent assay (ELISA).18-25 This study was designed to compare the LAL assay and the IPT in terms of detection of pyrogens that remain adsorbed to IOL surfaces. MATERIALS AND METHODS Challenge organism suspensions in this study were prepared from 2 gram-negative bacteria (Escherichia coli, reference strain American Type Culture Collection [ATCC] 8739; Pseudomonas putida, reference strain ATCC 12633) and from 1 gram-positive bacterium (Staphylococcus epidermidis, reference strain ATCC 14990; non-slime producer). Overnight cultures of the strains were prepared by incubation in 10.0 mL peptone solution at 35 G 2.5 C for 16 G 8 hours. Fresh work suspensions (0.1 mL) of each strain were then inoculated in 10.0 mL soybean–casein digest medium and incubated at 35 C G 2.5 C for 16 G 8 hours. Challenge organism suspensions containing more than 108 colonyforming units (CFU)/mL of each bacteria (spectrophotometrically determined) were diluted and adjusted in saline for injection to result in a suspension of cell material equivalent Submitted: October 8, 2008. Final revision submitted: February 25, 2009. Accepted: March 3, 2009. From the Berlin Eye Research Institute (Werner, Tetz), Berlin, and Qualis Laboratorium GmbH (Zwisler), Konstanz, Germany; John A. Moran Eye Center (Werner), University of Utah, Salt Lake City, Utah; and Advanced Vision Science Inc. (Mentak, Aldred), Goleta, California, USA. Dr. Mentak is a consultant to and Ms. Margaret Aldred is an employee of Advanced Vision Science, Inc. Qualis Laboratorium GmbH is a service laboratory for in vitro pyrogen testing. No other author has a financial or proprietary interest in any material or method mentioned. Presented in part as a poster at the annual meeting of the American Academy of Ophthalmology, Atlanta, Georgia, USA, November 2008. Supported in part by a research grant from Advanced Vision Science, Inc. Corresponding author: Liliana Werner, MD, PhD, Berlin Eye Research Institute, Alt-Moabit 98/99, D-10559, Berlin, Germany. E-mail: [email protected].
to 103 CFU/mL and 104 CFU/mL of E coli and P putida and 105 CFU/mL and 106 CFU/mL of S epidermidis. Fifteen of each of the following IOLs were used: the silicone MicroSil MS 612 ASP (Dr. Schmidt Intraocularlinsen GmbH); the hydrophilic acrylic Superflex (Rayner Intraocular Lenses); and the hydrophobic acrylic AcrySof SA60AT (Alcon Laboratories), Sensar (Abbott Medical Optics, formerly Advanced Medical Optics), and XACT (Advanced Vision Science), the last of which has a 4% water content; and the LS-106 poly(methyl methacrylate) (Lenstec Inc.). Two IOLs of each type were incubated for at least 2 days at room temperature inside pyrogen-free plastic vials containing 0.6 mL of 1 of the 6 challenge organism suspensions. They were then gamma sterilized inside the same vials at 25 G 3 kGy. After sterilization, they were removed from the incubation vials and rinsed twice with fresh LAL water to remove unattached and loosely attached bacteria. For each suspension, 1 IOL was placed in a new reaction tube. Next, 1.0 mL of fresh LAL water was added, the IOL was incubated for 1 hour at 37 C, and the eluate was submitted to the LAL test (in 2 parallels). The other IOL was placed in a new reaction tube to be tested with the IPT. The IOLs incubated in sterile saline were used as negative controls. As a positive control for the IPT, 1 IOL (spiked sample) was incubated overnight in the saline–blood assay with a defined amount of an endotoxin standard (E coli strain O-111; 0.50 endotoxin units [EU]/mL). As positive control for the LAL, an aliquot of the eluate of each IOL was spiked with endotoxin standard (0.25 EU/mL) to confirm the suitability of the assay (spiked eluate). Therefore, 1 negative control IOL was tested with the LAL assay and 1 negative IOL and 1 positive control IOL were tested with the IPT. Table 1 shows the overall design of the study. Two groups of control solutions were also used. In the first group, saline, 103 CFU/mL and 104 CFU/mL of E coli and P putida and 105 CFU/mL and 106 CFU/mL of S epidermidis prepared in saline were incubated for at least 2 days at room temperature and tested by the LAL assay and IPT without previous sterilization. To evaluate the reduction in pyrogens after sterilization, the second group of control solutions, which had the same composition as the first control group, was incubated for at least 2 days at room temperature and tested by the LAL assay and IPT after gamma sterilization. Detailed procedures for the LAL assay and the IPT have been described. Briefly, for the LAL assay, the gel-clot method was used, with a sensitivity of 0.125 EU/mL (LAL consumables, Charles River Laboratories).12–15 The eluate resulting from the extraction of each IOL was reacted with an aqueous extract of the blood cells (amebocytes) of horseshoe crabs (undisturbed for 1 hour at 37 C). A positive test (indicating the presence of an amount of endotoxin in the studied sample that equaled or exceeded the reagent’s labeled sensitivity) was defined as the formation of a firm gel capable of maintaining its integrity when the test tube was inverted 180 degrees. A negative test was characterized by the absence of gel or by the formation of a viscous mass that did not hold when the tube was inverted. For the IPT (Endosafe-IPT Kit, Charles River Laboratories), human whole blood, together with saline and the IOLs (and pyrogens in controls), was incubated for 18 G 4 hours at 37 C G 1 C in pyrogen-free reaction tubes/containers.18–25 After incubation, triplicate aliquots of 100 mL of each blood incubation were taken for the ELISA procedure. The aliquots were distributed into wells of a microplate,
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Table 1. Study and control IOLs used and tests performed.
IOL/Test MicroSil MS 612 ASP LAL IPT AcrySof SA60AT LAL IPT Superflex LAL IPT Sensar LAL IPT XACT LAL IPT LS-106 LAL IPT
E Coli (CFU/ml)
IOLs (n)
P Putida (CFU/mL)
IOLs (n)
S Epidermidis (CFU/mL)
IOLs (n)
Negative Control (n)
Positive Control (n)
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
CFU Z colony-forming units; E Coli Z Escherichia coli; IOL Z intraocular lens; IPT Z in vitro pyrogen test; LAL Z limulus amoebocyte lysate; P Putida Z Pseudomonas putida; S Epidermidis Z Staphylococcus epidermidis
which were coated with monoclonal antibodies specific for IL-1b. An enzyme-conjugated polyclonal antibody against IL-1 b was added. During a 90-minute incubation period, a sandwich complex consisting of 2 antibodies and the IL1b was formed. A chromogenic substrate (tetramethylbenzidine) reactive with the enzyme produced color development whose optical density was read at 450 nm and was directly related to the IL-1b concentration. The reactivity in the ELISA was reported as the relative optical density in comparison with the optical density of triplicate aliquots of serial dilutions of an endotoxin standard (1.00, 0.50, and 0.25 EU/ mL) to provide an estimate of the degree of pyrogenicity of the samples as follows: none Z optical density of studied sample less than or equal to 3 times that of the blank; slight Z optical density less than or equal to 0.25 EU/mL standard endotoxin (limit of detection of the IPT test); mild Z between optical density 0.25 EU/mL and 0.50 EU/mL; moderate Z between optical density 0.5 EU/mL and 1.00 EU/mL; severe Z optical density greater than 1.00 EU/mL.
RESULTS The LAL test was negative for all incubated study IOLs regardless of the bacterial challenge used as well as for the negative IOL controls. All spiked positive product controls (spiked eluates) showed positive results with the LAL test, confirming its suitability. In contrast, the IPT detected pyrogens adsorbed to IOLs with a dose-dependent response. Figure 1 shows the optical density results of the IPT for the study and control IOLs. For reference, the negative LAL results are shown as optical density Z 0 in the graphs in Figure 1. Table 2 shows an estimate of the degree of
pyrogenicity of the IOLs in the IPT according to their relative optical density compared with the optical density of the endotoxin standard dilution series. The IPT was positive for all IOLs incubated in E coli and P putida suspensions (Figure 1 and Table 2), with the MicroSil MS 612 ASP, AcrySof SA60AT, XACT, and LS-106 IOLs having a severe pyrogenic reaction. The Superflex and Sensar IOLs generally showed a slight to moderate response for lower bacterial concentrations and moderate to severe for the higher concentrations. For S epidermidis, all IOLs generally showed a slight IPT response except for the XACT IOLs, which showed a nonpyrogenic response for both concentrations. Table 3 shows the results of the LAL test for the control solutions. Figure 2 shows the optical density results of the IPT performed on the control solutions and Table 4, an estimate of the degree of pyrogenicity of the same solutions in the IPT test according to their relative optical density compared with that of the endotoxin standard dilution series. The LAL test detected endotoxin of gram-negative bacteria in liquid samples but no pyrogens associated with gram-positive bacteria in the same type of samples (Table 3). There was a decreased response for 103 CFU/mL E coli after sterilization. The IPT test detected pyrogens associated with gram-negative and gram-positive bacteria in liquid samples (Figure 2 and Table 4). After sterilization, there was a decreased response for 103 CFU/mL E coli and for 105 CFU/mL and 106 CFU/mL S epidermidis.
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Figure 1. Optical density results of the IPT performed on study IOLs and control IOLs. Although the negative response of LAL gel-clot test is not based on optical density measurement, it is shown in the graphs as optical density Z 0 for reference only. An optical density of 3 represents an extremely strong reaction with overflow of the detection signal in the photometer. A: MicroSil MS 612 ASP. B: AcrySof SA60AT. C: Superflex. D: Sensar. E: XACT. F: LS-106 (* Z optical density read at 450 nm with a reference wavelength filter of 620 nm; OD Z optical density). J CATARACT REFRACT SURG - VOL 35, JULY 2009
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Table 2. Results of the estimation of the pyrogenicity of the study and control IOLs in the IPT. Challenge Solution (CFU per mL/Response) IOL MicroSil MS 612 ASP AcrySof SA60AT Superflex Sensar XACT LS-106
E Coli (CFU/mL)/Reaction
P Putida (CFU/mL)/Reaction
S Epidermidis (CFU/mL)/Reaction
Negative Control Response
Positive Control Response
103/severe 104/severe 103/severe 104/severe 103/slight 104/severe 103/severe 104/severe 103/severe 104/severe 103/severe 104/severe
105/none 106/slight 105/slight 106/slight 105/none 106/slight 105/slight 106/slight 105/none 106/none 105/none 106/slight
None
Moderate
None
Mild
None
Moderate
None
Mild
None
Moderate
None
Moderate
103/severe 104/severe 103/severe 104/severe 103/mild 104/moderate 103/slight 104/severe 103/severe 104/severe 103/severe 104/severe
CFU Z colony-forming units; E Coli Z Escherichia coli; IOL Z intraocular lens; P Putida Z Pseudomonas putida; S Epidermidis Z Staphylococcus epidermidis
DISCUSSION A pyrogen is a fever-inducing compound that can trigger an inflammatory reaction in the host. Pyrogens are usually divided into microbial and nonmicrobial. Various structural components of bacteria, yeasts, and molds have been isolated and characterized as pyrogenic. Examples of microbial pyrogens are cell-wall molecules from gram-negative bacteria LPS; lipoteichoic acid, which is the LPS equivalent in gram-positive bacteria; fungal spores; and viral pyrogens.24 Medical devices and pharmaceutical containers are routinely sterilized by methods such as irradiation, autoclaving, and gas sterilization. Although they may be sterile, heat-stable pyrogenic bacterial byproducts and even pyrogens of a chemical nature (nonmicrobial) may remain on their surface and induce significant inflammatory reactions.10,11 Regulatory agencies worldwide require strict control of manufacturing to ensure that medical products are pyrogen-free. Endotoxins (eg, LPS in the outer membrane of various gram-negative bacteria) are the
most typical and most powerful pyrogens in the natural world. According to the American National Standards Institute (ANSI) and International Organization for Standardization (ISO), the current endotoxin limit for IOLs and ophthalmic viscosurgical devices (OVDs) is 2.0 EU/IOL (0.4 ng LPS) (ISO 11979-8, ANSI Z80.7) and 0.5 EU/OVD mL (0.1 ng LPS) (ISO 15798), respectively. The most widely accepted and currently used pyrogen test for medical devices and pharmaceuticals is the LAL assay, which is restricted to LPS detection. The hemolymph of the horseshoe crab (Limulus polyphemus) coagulates when brought in contact with LPS. The device to be tested must be rinsed with purified water, with or without a detergent, because the assay can only be performed with liquid samples. The simplest form of LAL testing is the LAL gel-clot assay. Manufacturers have also developed 2 additional techniques: turbidimetric LAL and the chromogenic LAL. Turbidimetric LAL assay contains enough coagulogen to form turbidity when cleaved by the clotting
Table 3. Results of the LAL test on control solutions. Challenge Solution (CFU/mL and [Response]) Condition Incubation R2 days; no sterilization Incubation R2 days; sterilization
E Coli
P Putida
103 [C] 104 [C] 103 [ ] 104 [C]
103 [C] 104 [C] 103 [C] 104 [C]
S Epidermidis 105 [ 106 [ 105 [ 106 [
Negative Control [Response]
] ] ] ]
CFU Z colony-forming units; E Coli Z Escherichia coli; P Putida Z Pseudomonas putida; S Epidermidis Z Staphylococcus epidermidis
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Figure 2. Optical density results of the IPT performed on the control solutions. An optical density of 3 represents an extremely strong reaction with overflow of the detection signal in the photometer (* Z optical density read at 450 nm with a reference wavelength filter of 620 nm; OD Z optical density).
enzyme, but not enough to form a clot. In the chromogenic technique, the coagulogen is partially or wholly replaced by a chromogenic substrate. Kinetic turbidimetric and chromogenic tests, although more accurate and faster than the gel-clot assay, cannot be used for fluids with inherent turbidity such as blood and yellow-tinted liquids (eg, urine), and their performance can be compromised by any precipitation from solution.12–15 The LAL was licensed by the U.S. Food and Drug Administration in the 1980s as an alternative to the RPT. This latter is a viable mammalian model to test for endotoxin and nonendotoxin pyrogens as well as other areas in which the LAL test is limited. It is approved for use in United States and European pharmacopeias and involves a baseline measurement of the rectal temperature, injection of the test solution into an ear vein, and temperature measurements at 30-minute intervals 1 to 3 hours after injection. The rabbit rectal temperature has to raise 0.5 C above the control temperature for the solution to be considered
pyrogenic. Although not restricted to endotoxin detection, the RPT has the disadvantages of being costly and laborious. Because it involves animal consumption, the results are prone to species variations; in addition, stress may affect the body temperature of the animal. Although small test samples can also be implanted in the RPT, the destruction of tissue during implantation may cause an inflammatory reaction that does not necessarily reflect the pyrogenicity of the material.14,16–18 Both LAL assay and the RPT have a different sensitivity than humans toward different microbial components. An outbreak of TASS in a surgical center is an environmental and toxin control issue that requires a complete analysis of all medications, fluids, and devices used during the surgery.1–9 However, as shown in studies of materials for orthopedic implants,26,27 pyrogens from endotoxin-contaminated surfaces are only extractable to a certain extent. Therefore, pyrogens that remain attached to the surface of medical devices and cannot be recovered in soluble contaminations would not be detected by the LAL assay or the RPT. When pyrogens enter the circulation, the stimulation of peripheral blood monocytes and macrophages determines the release of proinflammatory cytokines such as IL-1b, among others. These transmit signals to increase the thermostatic set point through prostaglandin E2. These signals are further transmitted to the brain, where complex thermoregulatory mechanisms are triggered to increase the body temperature. Development of the IPT was based on in vitro reactions that are relevant to the above fever-inducing mechanisms in humans because the release of IL-1b in human blood brought in direct contact with the solution or material to be tested is measured by ELISA. The IPT has the potential to complement the LAL test as it can detect other pyrogens originating from gram-negative or gram-positive bacteria as well as other microbial and nonmicrobial pyrogens. It also has the potential to replace the RPT.18–25 A 3-year study by the European Center for the Validation of Alternative Methods has validated the IPT as an alternative to the RPT, and the National Institutes of Health Interagency Coordinating Committee on the
Table 4. Results of the estimation of the pyrogenicity of the control solutions in the IPT. Challenge Solution (CFU per mL/Response) Condition Incubation R2 days; no sterilization Incubation R2 days; sterilization
E Coli
P Putida
S Epidermidis
Negative Control
103/Severe 104/Severe 103/Severe 104/Severe
103/Severe 104/Severe 103/None 104/Severe
105/Severe 106/Severe 105/None 106/Slight
None
CFU Z colony-forming units; E Coli Z Escherichia coli; P Putida Z Pseudomonas putida; S Epidermidis Z Staphylococcus epidermidis
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Validation of Alternative Methods is currently evaluating the test (http://iccvam.niehs.nih.gov/methods/ pyrogen/pyr_PeerPanel.htm. Accessed March 10, 2009). We designed this study to compare the LAL assay and the IPT in their detection of pyrogens attached/ adsorbed to IOLs. Although directly relevant for pyrogen control in IOL manufacturing, the results in our study can be applied to evaluation of medical devices in general. After incubation in the challenge organism suspensions for at least 2 days, the IOLs were gamma sterilized.28 This method was chosen because it allowed sterilization in the same vials used for incubation. Also, some currently available IOLs packaged in solutions are gamma sterilized (eg, XACT). The IOLs were then rinsed to remove unattached and loosely attached bacteria; therefore, results from both tests would only be relevant to pyrogens that remained attached or adsorbed to the IOLs. Bacterial adhesion to IOL surfaces occurs in 2 phases. The first depends on the physical characteristics of the bacteria and the biomaterial of the IOLs and involves reversible attraction caused by forces such as electrostatic and Van der Waals forces and hydrophobic bonding. This phase takes place immediately on exposure of the biomaterial to the bacteria. In the second phase of adhesion, molecular-specific reactions between bacterial surface structures and the substrate become predominant. In most cases, this is mediated by the bacterial production of a polysaccharide glycocalyx (slime) on the IOL surface 4 to 24 hours after incubation.29,30 Our study related to the second phase; however, we used concentrations that were significantly lower than those generally used in studies of bacterial adhesion to IOLs (108 CFU/mL) related to enterococci bacteria,31 Pseudomonas species,32 or strains of S epidermidis.33 Several bacteria, even when present in very small numbers, can contaminate the surface of a medical device during the various manufacturing steps. This may occur through airborne contamination, cross contamination, or washing steps that involve water or other solutions. Two gram-negative bacteria were used in our study: P putida, which is a rod-shaped saprotrophic soil bacterium, and E coli, which is a facultative anaerobic and non-sporulating bacterium that is commonly found in the lower intestine of warm-blooded animals. The gram-positive bacterium used (S epidermidis) is frequently found on the skin of humans and animals and in mucous membranes. Indeed, it is recognized as the most common organism isolated from cases of endophthalmitis after cataract surgery with IOL implantation.34,35 In a study comparing adhesion of slime and nonslime-producing S epidermidis on IOL surfaces, the
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optical density of the biomass of bacteria was consistently greater with the slime-producing strain.33 Therefore, one might expect less bacterial attachment (as well as less pyrogen attachment after sterilization of the IOLs) using a non-slime-producing strain. The IPT was able to detect pyrogens adsorbed to IOLs with a dose-dependent response, including in IOLs incubated with the non-slime-producing strain of S epidermidis used (with the exception of the XACT IOLs). The LAL test was only able to detect endotoxins in the control solutions prepared from gram-negative bacteria. Another significant finding in our study was that gamma sterilization reduced the pyrogenicity of the bacteria only to some extent. Regarding bacterial attachment per se, the design of our study does not allow us to drawn conclusions related to preferential attachment to certain IOL materials. In summary, this study found that the LAL test was not able to detect pyrogens adsorbed to the surface of different IOL materials. The IPT, which involved direct contact of the IOLs with whole blood, detected pyrogens in a dose-dependent response through reactions that are relevant for the human response. The results have direct applicability to the investigation of TASS outbreaks as well as to the pyrogenicity of medical devices in general. REFERENCES 1. Monson MC, Mamalis N, Olson RJ. Toxic anterior segment inflammation following cataract surgery. J Cataract Refract Surg 1992; 18:184–189 2. Mamalis N, Edelhauser HF, Dawson DG, Chew J, LeBoyer RM, Werner L. Toxic anterior segment syndrome. J Cataract Refract Surg 2006; 32:324–333 3. Parikh C, Sippy BD, Martin DF, Edelhauser HF. Effects of enzymatic sterilization detergents on the corneal endothelium. Arch Ophthalmol 2002; 120:165–172 4. Jehan FS, Mamalis N, Spencer TS, Fry LL, Kerstine RS, Olson RJ. Postoperative sterile endophthalmitis (TASS) associated with the MemoryLens. J Cataract Refract Surg 2000; 26:1773–1777 5. Werner L, Sher JH, Taylor JR, Mamalis N, Nash WA, Csordas JE, Green G, Maziarz EP, Liu XM. Toxic anterior segment syndrome and possible association with ointment in the anterior chamber following cataract surgery. J Cataract Refract Surg 2006; 32:227–235 6. Mathys KC, Cohen KL, Bagnell CR. Identification of unknown intraocular material after cataract surgery: evaluation of a potential cause of toxic anterior segment syndrome. J Cataract Refract Surg 2008; 34:465–469 7. Kreisler KR, Martin SS, Young CW, Anderson CW, Mamalis N. Postoperative inflammation following cataract extraction caused by bacterial contamination of the cleaning bath detergent. J Cataract Refract Surg 1992; 18:106–110 8. Whitby JL, Hitchins VM. Endotoxin levels in steam and reservoirs of table-top steam sterilizers. J Refract Surg 2002; 18:51–57 9. Kutty PK, Forster TS, Wood-Koob C, Thayer N, Nelson RB, Berke SJ, Pontacolone L, Beardsley TL, Edelhauser HF,
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Arduino MJ, Mamalis N, Srinivasan A. Multistate outbreak of toxic anterior segment syndrome, 2005. J Cataract Refract Surg 2008; 34:585–590 Nakata T. Destruction of typical endotoxins by dry heat as determined using LAL assay and pyrogen assay. J Parenter Sci Technol 1993; 47:258–264 Moesby L, Hansen EW, Christensen JD, Høyer CH, Juhl GL, Olsen HB. Dry and moist heat sterilisation cannot inactivate pyrogenicity of Gram positive microorganisms. Eur J Pharm Sci 2005; 26:318–323 Cooper JF, Hochstein HD, Seligmann EB Jr. The limulus test for endotoxin (pyrogen) in radiopharmaceuticals and biologicals. Bull Parenteral Drug Assoc 1972; 26:153–162 Papageorgiou J, Linke TA, Kapralos C, Nicholson BC, Steffensen DA. Extraction of cyanobacterial endotoxin. Environ Toxicol 2004; 19:82–87 Park C-Y, Jung S-H, Bak J-P, Lee S-S, Rhee D-K. Comparison of the rabbit pyrogen test and limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide. Biologicals 2005; 33:145–151 Ong KG, Leland JM, Zeng K, Barrett G, Zourob M, Grimes CA. A rapid highly-sensitive endotoxin detection system. Biosens Bioelectron 2006; 21:2270–2274 Tschumi J. Comparison of temperature rise interpretations between European and United States Pharmacopeias’ pyrogen tests. PDA J Pharm Sci Technol 2003; 57:218–227; erratum, 387 Hoffmann S, Lu¨deritz-Puchel U, Montag T, Hartung T. Optimisation of pyrogen testing in parenterals according to different pharmacopoeias by probabilistic modelling. J Endotoxin Res 2005; 11:25–31 Nakagawa Y, Maeda H, Murai T. Evaluation of the in vitro pyrogen test system based on proinflammatory cytokine release from human monocytes: comparison with a human whole blood culture test system and with the rabbit pyrogen test. Clin Diagn Lab Immunol 2002; 9:588–597. Available at: http://cvi.asm. org/cgi/reprint/9/3/588. Accessed March 10, 2009 Schindler S, Hartung T. Comparison and validation of novel pyrogen tests based on the human fever reaction; the Human(e) Pyrogen Test Study Group. Dev Biol (Basel) 2002; 111:181–186 Schindler S, Asmus S, von Aulock S, Wendel A, Hartung T, Fennrich S. Cryopreservation of human whole blood for pyrogenicity testing. J Immunol Methods 2004; 294:89–100 Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S, Mistry Y, Montag-Lessing T, Spreitzer I, Lo¨schner B, van Aalderen M, Bos R, Gommer M, Nibbeling R, WernerFelmayer G, Loitzl P, Jungi T, Brcic M, Bru¨gger P, Frey E, Bowe G, Casado J, Coecke S, de Lange J, Mogster B, Næss LM, Aaberge IS, Wendel A, Hartung T. International validation of novel pyrogen tests based on human monocytoid cells. J Immunol Methods 2005; 298:161–173 Daneshian M, Guenther A, Wendel A, Hartung T, von Aulock S. In vitro pyrogen test for toxic or immunomodulatory drugs. J Immunol Methods 2006; 313:169–175 Schindler S, Spreitzer I, Lo¨schner B, Hoffmann S, Hennes K, Halder M, Bru¨gger P, Frey E, Hartung T, Montag T. International validation of pyrogen tests based on cryopreserved human primary blood cells. J Immunol Methods 2006; 316:42–51
24. Mazzotti F, Beuttler J, Zeller R, Fink U, Schindler S, Wendel A, Hartung T, von Aulock S. In vitro pyrogen test – a new test method for solid medical devices. J Biomed Mater Res 2007; 80A:276–282 25. Hasiwa M, Kullmann K, von Aulock S, Klein C, Hartung T. An in vitro pyrogen safety test for immune-stimulating components on surfaces. Biomaterials 2007; 28:1367–1375 26. Daniels AU, Barnes FH, Charlebois SJ, Smith RA. Macrophage cytokine response to particles and lipopolysaccharide in vitro. J Biomed Mater Res 2000; 49:469–478 27. Cho DR, Shanbhag AS, Hong C-Y, Baran GR, Goldring SR. The role of adsorbed endotoxin in particle-induced stimulation of cytokine release. J Orthop Res 2002; 20:704–713 28. Ludwig K, Scheiffarth OF, von Meyer L. Reducing the amount of monomers in intraocular lenses through sterilization by gamma radiation. Ophthalmic Res 1988; 20:304–307 29. Anderson JM, Marchant RE. Biomaterials: factors favoring colonization and infection. In: Waldvogel FA, Bisno AL, eds, Infections Associated with Indwelling Medical Devices 3rd ed. Washington, DC, ASM Press, 2000; 89–109 30. Kodjikian L, Burillon C, Roques C, Pellon G, Renaud FN, Hartmann D, Freney J. Intraocular lenses, bacterial adhesion and endophthalmitis prevention: a review. Biomed Mater Eng 2004; 14:395–409 31. Kobayakawa S, Jett BD, Gilmore MS. Biofilm formation by Enterococcus faecalis in intraocular lens material. Curr Eye Res 2005; 30:741–745 32. Mencucci R, Dei R, Danielli D, Susini M, Menchini U. Folding procedure for acrylic intraocular lenses; effect on optic surfaces and bacterial adhesion. J Cataract Refract Surg 2004; 30:457–463 33. Okajima Y, Kobayakawa S, Tsuji A, Tochikubo T. Biofilm formation by staphylococcus epidermidis on intraocular lens material. Invest Ophthalmol Vis Sci 2006; 47:2971–2975. Available at: http://www.iovs.org/cgi/reprint/47/7/2971. Accessed March 11, 2009 34. Moshirfar M, Feiz V, Vitale AT, Wegelin JA, Basavanthappa S, Wolsey DH. Endophthalmitis after uncomplicated cataract surgery with the use of fourth-generation fluoroquinolones; a retrospective observational case series. Ophthalmology 2007; 114:686–691 35. Chiquet C, Cornut P-L, Benito Y, Thuret G, Maurin M, Lafontaine P-0, Pechinot A, Palombi K, Lina G, Bron A, Denis P, Carricajo A, Creuzot C, Romanet J-P, Vandenesch F. Eubacterial PCR for bacterial detection and identification in 100 acute postcataract surgery endophthalmitis; for the French Institutional Endophthalmitis Study (FRIENDS) Group. Invest Ophthalmol Vis Sci 2008; 49:1971–1978
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First author: Liliana Werner, MD, PhD Berlin Eye Research Institute, Berlin, Germany, and John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA
Detection of pyrogens adsorbed to intraocular lenses Evaluation of limulus amoebocyte lysate and in vitro pyrogen tests Liliana Werner, MD, PhD, Manfred Tetz, MD, Khalid Mentak, PhD, Margaret Aldred, Walter Zwisler, PhD
PURPOSE: To determine the ability of the limulus amoebocyte lysate (LAL) assay and the in vitro pyrogen test (IPT) to detect pyrogens adsorbed to intraocular lenses (IOLs). SETTING: Berlin Eye Research Institute, Berlin, Germany. METHODS: Fifteen of each of the following IOLs were used: MicroSil MS 612 ASP, AcrySof SA60AT, Superflex, Sensar, XACT, and LS-106 IOLs. The challenge organism suspensions were 103 CFU/mL and 104 CFU/mL Escherichia coli, 103 CFU/mL and 104 CFU/mL Pseudomonas putida, and 105 CFU/ mL and 106 CFU/mL Staphylococcus epidermidis. Two IOLs of each model were incubated at room temperature for at least 2 days in 0.6 mL of 1 of the suspensions. They were then gamma sterilized. The extract of 1 IOL was tested with the LAL assay; the other IOL was tested with the IPT. RESULTS: The LAL was negative for all incubated IOLs. The IPT was positive for all IOLs incubated in E coli and P putida suspensions, with the MicroSil MS 612 ASP, AcrySof SA60AT, XACT, and LS106 IOLs showing a severe reaction. The Superflex and Sensar IOLs had a slight to moderate response for lower bacterial concentrations and a moderate to severe response for higher concentrations. For S epidermidis, all IOLs showed a slight IPT response except XACT IOLs, which showed a nonpyrogenic response. CONCLUSIONS: Results indicate that the LAL test may fail to detect pyrogens adsorbed to IOLs and the IPT reliably detects pyrogens with a dose-dependent response. This has relevance in the investigation of toxic anterior segment syndrome outbreaks. J Cataract Refract Surg 2009; 35:1273–1280 Q 2009 ASCRS and ESCRS
Toxic anterior segment syndrome (TASS) is characterized by a sterile postoperative inflammation caused by a noninfectious agent gaining access to the anterior segment, resulting in toxic damage to intraocular structures. It starts within 12 to 48 hours of anterior segment surgery, is limited to the anterior segment of the eye, is always gram stain and culture negative, and improves with steroidal treatment.1,2 Possible causes of TASS include instruments or devices containing residues of enzymatic detergents, oxidized metal deposits, polishing or sterilizing compounds, and bacterial endotoxin contamination of solutions and devices.3–9 In the past 3 years, TASS has been a significantly reported complication after cataract surgery with intraocular lens (IOL) implantation.2,9 Q 2009 ASCRS and ESCRS Published by Elsevier Inc.
The surface of medical devices, such as IOLs, may be contaminated with bacteria during various manufacturing steps. Even though the devices are routinely sterilized by different methods, heat-stable pyrogenic bacterial byproducts and other types of microbial and nonmicrobial pyrogens may remain adsorbed to their surfaces.10,11 Therefore, regulatory agencies worldwide require strict control of manufacturing to ensure that medical products are pyrogen-free. Pyrogen testing of medical devices is currently based on the limulus amoebocyte lysate (LAL) and rabbit pyrogen test (RPT) assays. The LAL assay uses the immune defense system of the horseshoe crab. It can be performed with liquid samples only and detects only lipopolysaccharides (LPS) with high sensitivity.12–15 The RPT can also detect other microbial and nonmicrobial 0886-3350/09/$dsee front matter doi:10.1016/j.jcrs.2009.03.012
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pyrogens. It basically involves intravenous injections of pyrogen solutions and measurements of rectal temperature.14,16–18 However, pyrogen adsorbed to medical devices, which cannot be removed by rinsing, would not be detected by the LAL or RPT assay. The more recently developed in vitro pyrogen test (IPT) can detect microbial and nonmicrobial pyrogens in human whole blood or cryopreserved blood that is brought in contact with solutions or materials. The monocytes and macrophages in the blood recognize the pyrogens by releasing cytokines. One of the first to be released is interleukin (IL)-1b, which can be measured by enzyme-linked immunosorbent assay (ELISA).18-25 This study was designed to compare the LAL assay and the IPT in terms of detection of pyrogens that remain adsorbed to IOL surfaces. MATERIALS AND METHODS Challenge organism suspensions in this study were prepared from 2 gram-negative bacteria (Escherichia coli, reference strain American Type Culture Collection [ATCC] 8739; Pseudomonas putida, reference strain ATCC 12633) and from 1 gram-positive bacterium (Staphylococcus epidermidis, reference strain ATCC 14990; non-slime producer). Overnight cultures of the strains were prepared by incubation in 10.0 mL peptone solution at 35 G 2.5 C for 16 G 8 hours. Fresh work suspensions (0.1 mL) of each strain were then inoculated in 10.0 mL soybean–casein digest medium and incubated at 35 C G 2.5 C for 16 G 8 hours. Challenge organism suspensions containing more than 108 colonyforming units (CFU)/mL of each bacteria (spectrophotometrically determined) were diluted and adjusted in saline for injection to result in a suspension of cell material equivalent Submitted: October 8, 2008. Final revision submitted: February 25, 2009. Accepted: March 3, 2009. From the Berlin Eye Research Institute (Werner, Tetz), Berlin, and Qualis Laboratorium GmbH (Zwisler), Konstanz, Germany; John A. Moran Eye Center (Werner), University of Utah, Salt Lake City, Utah; and Advanced Vision Science Inc. (Mentak, Aldred), Goleta, California, USA. Dr. Mentak is a consultant to and Ms. Margaret Aldred is an employee of Advanced Vision Science, Inc. Qualis Laboratorium GmbH is a service laboratory for in vitro pyrogen testing. No other author has a financial or proprietary interest in any material or method mentioned. Presented in part as a poster at the annual meeting of the American Academy of Ophthalmology, Atlanta, Georgia, USA, November 2008. Supported in part by a research grant from Advanced Vision Science, Inc. Corresponding author: Liliana Werner, MD, PhD, Berlin Eye Research Institute, Alt-Moabit 98/99, D-10559, Berlin, Germany. E-mail: [email protected].
to 103 CFU/mL and 104 CFU/mL of E coli and P putida and 105 CFU/mL and 106 CFU/mL of S epidermidis. Fifteen of each of the following IOLs were used: the silicone MicroSil MS 612 ASP (Dr. Schmidt Intraocularlinsen GmbH); the hydrophilic acrylic Superflex (Rayner Intraocular Lenses); and the hydrophobic acrylic AcrySof SA60AT (Alcon Laboratories), Sensar (Abbott Medical Optics, formerly Advanced Medical Optics), and XACT (Advanced Vision Science), the last of which has a 4% water content; and the LS-106 poly(methyl methacrylate) (Lenstec Inc.). Two IOLs of each type were incubated for at least 2 days at room temperature inside pyrogen-free plastic vials containing 0.6 mL of 1 of the 6 challenge organism suspensions. They were then gamma sterilized inside the same vials at 25 G 3 kGy. After sterilization, they were removed from the incubation vials and rinsed twice with fresh LAL water to remove unattached and loosely attached bacteria. For each suspension, 1 IOL was placed in a new reaction tube. Next, 1.0 mL of fresh LAL water was added, the IOL was incubated for 1 hour at 37 C, and the eluate was submitted to the LAL test (in 2 parallels). The other IOL was placed in a new reaction tube to be tested with the IPT. The IOLs incubated in sterile saline were used as negative controls. As a positive control for the IPT, 1 IOL (spiked sample) was incubated overnight in the saline–blood assay with a defined amount of an endotoxin standard (E coli strain O-111; 0.50 endotoxin units [EU]/mL). As positive control for the LAL, an aliquot of the eluate of each IOL was spiked with endotoxin standard (0.25 EU/mL) to confirm the suitability of the assay (spiked eluate). Therefore, 1 negative control IOL was tested with the LAL assay and 1 negative IOL and 1 positive control IOL were tested with the IPT. Table 1 shows the overall design of the study. Two groups of control solutions were also used. In the first group, saline, 103 CFU/mL and 104 CFU/mL of E coli and P putida and 105 CFU/mL and 106 CFU/mL of S epidermidis prepared in saline were incubated for at least 2 days at room temperature and tested by the LAL assay and IPT without previous sterilization. To evaluate the reduction in pyrogens after sterilization, the second group of control solutions, which had the same composition as the first control group, was incubated for at least 2 days at room temperature and tested by the LAL assay and IPT after gamma sterilization. Detailed procedures for the LAL assay and the IPT have been described. Briefly, for the LAL assay, the gel-clot method was used, with a sensitivity of 0.125 EU/mL (LAL consumables, Charles River Laboratories).12–15 The eluate resulting from the extraction of each IOL was reacted with an aqueous extract of the blood cells (amebocytes) of horseshoe crabs (undisturbed for 1 hour at 37 C). A positive test (indicating the presence of an amount of endotoxin in the studied sample that equaled or exceeded the reagent’s labeled sensitivity) was defined as the formation of a firm gel capable of maintaining its integrity when the test tube was inverted 180 degrees. A negative test was characterized by the absence of gel or by the formation of a viscous mass that did not hold when the tube was inverted. For the IPT (Endosafe-IPT Kit, Charles River Laboratories), human whole blood, together with saline and the IOLs (and pyrogens in controls), was incubated for 18 G 4 hours at 37 C G 1 C in pyrogen-free reaction tubes/containers.18–25 After incubation, triplicate aliquots of 100 mL of each blood incubation were taken for the ELISA procedure. The aliquots were distributed into wells of a microplate,
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Table 1. Study and control IOLs used and tests performed.
IOL/Test MicroSil MS 612 ASP LAL IPT AcrySof SA60AT LAL IPT Superflex LAL IPT Sensar LAL IPT XACT LAL IPT LS-106 LAL IPT
E Coli (CFU/ml)
IOLs (n)
P Putida (CFU/mL)
IOLs (n)
S Epidermidis (CFU/mL)
IOLs (n)
Negative Control (n)
Positive Control (n)
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
103, 104 103, 104
2 2
103, 104 103, 104
2 2
105, 106 105, 106
2 2
1 1
Spiked eluate 1
CFU Z colony-forming units; E Coli Z Escherichia coli; IOL Z intraocular lens; IPT Z in vitro pyrogen test; LAL Z limulus amoebocyte lysate; P Putida Z Pseudomonas putida; S Epidermidis Z Staphylococcus epidermidis
which were coated with monoclonal antibodies specific for IL-1b. An enzyme-conjugated polyclonal antibody against IL-1 b was added. During a 90-minute incubation period, a sandwich complex consisting of 2 antibodies and the IL1b was formed. A chromogenic substrate (tetramethylbenzidine) reactive with the enzyme produced color development whose optical density was read at 450 nm and was directly related to the IL-1b concentration. The reactivity in the ELISA was reported as the relative optical density in comparison with the optical density of triplicate aliquots of serial dilutions of an endotoxin standard (1.00, 0.50, and 0.25 EU/ mL) to provide an estimate of the degree of pyrogenicity of the samples as follows: none Z optical density of studied sample less than or equal to 3 times that of the blank; slight Z optical density less than or equal to 0.25 EU/mL standard endotoxin (limit of detection of the IPT test); mild Z between optical density 0.25 EU/mL and 0.50 EU/mL; moderate Z between optical density 0.5 EU/mL and 1.00 EU/mL; severe Z optical density greater than 1.00 EU/mL.
RESULTS The LAL test was negative for all incubated study IOLs regardless of the bacterial challenge used as well as for the negative IOL controls. All spiked positive product controls (spiked eluates) showed positive results with the LAL test, confirming its suitability. In contrast, the IPT detected pyrogens adsorbed to IOLs with a dose-dependent response. Figure 1 shows the optical density results of the IPT for the study and control IOLs. For reference, the negative LAL results are shown as optical density Z 0 in the graphs in Figure 1. Table 2 shows an estimate of the degree of
pyrogenicity of the IOLs in the IPT according to their relative optical density compared with the optical density of the endotoxin standard dilution series. The IPT was positive for all IOLs incubated in E coli and P putida suspensions (Figure 1 and Table 2), with the MicroSil MS 612 ASP, AcrySof SA60AT, XACT, and LS-106 IOLs having a severe pyrogenic reaction. The Superflex and Sensar IOLs generally showed a slight to moderate response for lower bacterial concentrations and moderate to severe for the higher concentrations. For S epidermidis, all IOLs generally showed a slight IPT response except for the XACT IOLs, which showed a nonpyrogenic response for both concentrations. Table 3 shows the results of the LAL test for the control solutions. Figure 2 shows the optical density results of the IPT performed on the control solutions and Table 4, an estimate of the degree of pyrogenicity of the same solutions in the IPT test according to their relative optical density compared with that of the endotoxin standard dilution series. The LAL test detected endotoxin of gram-negative bacteria in liquid samples but no pyrogens associated with gram-positive bacteria in the same type of samples (Table 3). There was a decreased response for 103 CFU/mL E coli after sterilization. The IPT test detected pyrogens associated with gram-negative and gram-positive bacteria in liquid samples (Figure 2 and Table 4). After sterilization, there was a decreased response for 103 CFU/mL E coli and for 105 CFU/mL and 106 CFU/mL S epidermidis.
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Figure 1. Optical density results of the IPT performed on study IOLs and control IOLs. Although the negative response of LAL gel-clot test is not based on optical density measurement, it is shown in the graphs as optical density Z 0 for reference only. An optical density of 3 represents an extremely strong reaction with overflow of the detection signal in the photometer. A: MicroSil MS 612 ASP. B: AcrySof SA60AT. C: Superflex. D: Sensar. E: XACT. F: LS-106 (* Z optical density read at 450 nm with a reference wavelength filter of 620 nm; OD Z optical density). J CATARACT REFRACT SURG - VOL 35, JULY 2009
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Table 2. Results of the estimation of the pyrogenicity of the study and control IOLs in the IPT. Challenge Solution (CFU per mL/Response) IOL MicroSil MS 612 ASP AcrySof SA60AT Superflex Sensar XACT LS-106
E Coli (CFU/mL)/Reaction
P Putida (CFU/mL)/Reaction
S Epidermidis (CFU/mL)/Reaction
Negative Control Response
Positive Control Response
103/severe 104/severe 103/severe 104/severe 103/slight 104/severe 103/severe 104/severe 103/severe 104/severe 103/severe 104/severe
105/none 106/slight 105/slight 106/slight 105/none 106/slight 105/slight 106/slight 105/none 106/none 105/none 106/slight
None
Moderate
None
Mild
None
Moderate
None
Mild
None
Moderate
None
Moderate
103/severe 104/severe 103/severe 104/severe 103/mild 104/moderate 103/slight 104/severe 103/severe 104/severe 103/severe 104/severe
CFU Z colony-forming units; E Coli Z Escherichia coli; IOL Z intraocular lens; P Putida Z Pseudomonas putida; S Epidermidis Z Staphylococcus epidermidis
DISCUSSION A pyrogen is a fever-inducing compound that can trigger an inflammatory reaction in the host. Pyrogens are usually divided into microbial and nonmicrobial. Various structural components of bacteria, yeasts, and molds have been isolated and characterized as pyrogenic. Examples of microbial pyrogens are cell-wall molecules from gram-negative bacteria LPS; lipoteichoic acid, which is the LPS equivalent in gram-positive bacteria; fungal spores; and viral pyrogens.24 Medical devices and pharmaceutical containers are routinely sterilized by methods such as irradiation, autoclaving, and gas sterilization. Although they may be sterile, heat-stable pyrogenic bacterial byproducts and even pyrogens of a chemical nature (nonmicrobial) may remain on their surface and induce significant inflammatory reactions.10,11 Regulatory agencies worldwide require strict control of manufacturing to ensure that medical products are pyrogen-free. Endotoxins (eg, LPS in the outer membrane of various gram-negative bacteria) are the
most typical and most powerful pyrogens in the natural world. According to the American National Standards Institute (ANSI) and International Organization for Standardization (ISO), the current endotoxin limit for IOLs and ophthalmic viscosurgical devices (OVDs) is 2.0 EU/IOL (0.4 ng LPS) (ISO 11979-8, ANSI Z80.7) and 0.5 EU/OVD mL (0.1 ng LPS) (ISO 15798), respectively. The most widely accepted and currently used pyrogen test for medical devices and pharmaceuticals is the LAL assay, which is restricted to LPS detection. The hemolymph of the horseshoe crab (Limulus polyphemus) coagulates when brought in contact with LPS. The device to be tested must be rinsed with purified water, with or without a detergent, because the assay can only be performed with liquid samples. The simplest form of LAL testing is the LAL gel-clot assay. Manufacturers have also developed 2 additional techniques: turbidimetric LAL and the chromogenic LAL. Turbidimetric LAL assay contains enough coagulogen to form turbidity when cleaved by the clotting
Table 3. Results of the LAL test on control solutions. Challenge Solution (CFU/mL and [Response]) Condition Incubation R2 days; no sterilization Incubation R2 days; sterilization
E Coli
P Putida
103 [C] 104 [C] 103 [ ] 104 [C]
103 [C] 104 [C] 103 [C] 104 [C]
S Epidermidis 105 [ 106 [ 105 [ 106 [
Negative Control [Response]
] ] ] ]
CFU Z colony-forming units; E Coli Z Escherichia coli; P Putida Z Pseudomonas putida; S Epidermidis Z Staphylococcus epidermidis
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Figure 2. Optical density results of the IPT performed on the control solutions. An optical density of 3 represents an extremely strong reaction with overflow of the detection signal in the photometer (* Z optical density read at 450 nm with a reference wavelength filter of 620 nm; OD Z optical density).
enzyme, but not enough to form a clot. In the chromogenic technique, the coagulogen is partially or wholly replaced by a chromogenic substrate. Kinetic turbidimetric and chromogenic tests, although more accurate and faster than the gel-clot assay, cannot be used for fluids with inherent turbidity such as blood and yellow-tinted liquids (eg, urine), and their performance can be compromised by any precipitation from solution.12–15 The LAL was licensed by the U.S. Food and Drug Administration in the 1980s as an alternative to the RPT. This latter is a viable mammalian model to test for endotoxin and nonendotoxin pyrogens as well as other areas in which the LAL test is limited. It is approved for use in United States and European pharmacopeias and involves a baseline measurement of the rectal temperature, injection of the test solution into an ear vein, and temperature measurements at 30-minute intervals 1 to 3 hours after injection. The rabbit rectal temperature has to raise 0.5 C above the control temperature for the solution to be considered
pyrogenic. Although not restricted to endotoxin detection, the RPT has the disadvantages of being costly and laborious. Because it involves animal consumption, the results are prone to species variations; in addition, stress may affect the body temperature of the animal. Although small test samples can also be implanted in the RPT, the destruction of tissue during implantation may cause an inflammatory reaction that does not necessarily reflect the pyrogenicity of the material.14,16–18 Both LAL assay and the RPT have a different sensitivity than humans toward different microbial components. An outbreak of TASS in a surgical center is an environmental and toxin control issue that requires a complete analysis of all medications, fluids, and devices used during the surgery.1–9 However, as shown in studies of materials for orthopedic implants,26,27 pyrogens from endotoxin-contaminated surfaces are only extractable to a certain extent. Therefore, pyrogens that remain attached to the surface of medical devices and cannot be recovered in soluble contaminations would not be detected by the LAL assay or the RPT. When pyrogens enter the circulation, the stimulation of peripheral blood monocytes and macrophages determines the release of proinflammatory cytokines such as IL-1b, among others. These transmit signals to increase the thermostatic set point through prostaglandin E2. These signals are further transmitted to the brain, where complex thermoregulatory mechanisms are triggered to increase the body temperature. Development of the IPT was based on in vitro reactions that are relevant to the above fever-inducing mechanisms in humans because the release of IL-1b in human blood brought in direct contact with the solution or material to be tested is measured by ELISA. The IPT has the potential to complement the LAL test as it can detect other pyrogens originating from gram-negative or gram-positive bacteria as well as other microbial and nonmicrobial pyrogens. It also has the potential to replace the RPT.18–25 A 3-year study by the European Center for the Validation of Alternative Methods has validated the IPT as an alternative to the RPT, and the National Institutes of Health Interagency Coordinating Committee on the
Table 4. Results of the estimation of the pyrogenicity of the control solutions in the IPT. Challenge Solution (CFU per mL/Response) Condition Incubation R2 days; no sterilization Incubation R2 days; sterilization
E Coli
P Putida
S Epidermidis
Negative Control
103/Severe 104/Severe 103/Severe 104/Severe
103/Severe 104/Severe 103/None 104/Severe
105/Severe 106/Severe 105/None 106/Slight
None
CFU Z colony-forming units; E Coli Z Escherichia coli; P Putida Z Pseudomonas putida; S Epidermidis Z Staphylococcus epidermidis
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Validation of Alternative Methods is currently evaluating the test (http://iccvam.niehs.nih.gov/methods/ pyrogen/pyr_PeerPanel.htm. Accessed March 10, 2009). We designed this study to compare the LAL assay and the IPT in their detection of pyrogens attached/ adsorbed to IOLs. Although directly relevant for pyrogen control in IOL manufacturing, the results in our study can be applied to evaluation of medical devices in general. After incubation in the challenge organism suspensions for at least 2 days, the IOLs were gamma sterilized.28 This method was chosen because it allowed sterilization in the same vials used for incubation. Also, some currently available IOLs packaged in solutions are gamma sterilized (eg, XACT). The IOLs were then rinsed to remove unattached and loosely attached bacteria; therefore, results from both tests would only be relevant to pyrogens that remained attached or adsorbed to the IOLs. Bacterial adhesion to IOL surfaces occurs in 2 phases. The first depends on the physical characteristics of the bacteria and the biomaterial of the IOLs and involves reversible attraction caused by forces such as electrostatic and Van der Waals forces and hydrophobic bonding. This phase takes place immediately on exposure of the biomaterial to the bacteria. In the second phase of adhesion, molecular-specific reactions between bacterial surface structures and the substrate become predominant. In most cases, this is mediated by the bacterial production of a polysaccharide glycocalyx (slime) on the IOL surface 4 to 24 hours after incubation.29,30 Our study related to the second phase; however, we used concentrations that were significantly lower than those generally used in studies of bacterial adhesion to IOLs (108 CFU/mL) related to enterococci bacteria,31 Pseudomonas species,32 or strains of S epidermidis.33 Several bacteria, even when present in very small numbers, can contaminate the surface of a medical device during the various manufacturing steps. This may occur through airborne contamination, cross contamination, or washing steps that involve water or other solutions. Two gram-negative bacteria were used in our study: P putida, which is a rod-shaped saprotrophic soil bacterium, and E coli, which is a facultative anaerobic and non-sporulating bacterium that is commonly found in the lower intestine of warm-blooded animals. The gram-positive bacterium used (S epidermidis) is frequently found on the skin of humans and animals and in mucous membranes. Indeed, it is recognized as the most common organism isolated from cases of endophthalmitis after cataract surgery with IOL implantation.34,35 In a study comparing adhesion of slime and nonslime-producing S epidermidis on IOL surfaces, the
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optical density of the biomass of bacteria was consistently greater with the slime-producing strain.33 Therefore, one might expect less bacterial attachment (as well as less pyrogen attachment after sterilization of the IOLs) using a non-slime-producing strain. The IPT was able to detect pyrogens adsorbed to IOLs with a dose-dependent response, including in IOLs incubated with the non-slime-producing strain of S epidermidis used (with the exception of the XACT IOLs). The LAL test was only able to detect endotoxins in the control solutions prepared from gram-negative bacteria. Another significant finding in our study was that gamma sterilization reduced the pyrogenicity of the bacteria only to some extent. Regarding bacterial attachment per se, the design of our study does not allow us to drawn conclusions related to preferential attachment to certain IOL materials. In summary, this study found that the LAL test was not able to detect pyrogens adsorbed to the surface of different IOL materials. The IPT, which involved direct contact of the IOLs with whole blood, detected pyrogens in a dose-dependent response through reactions that are relevant for the human response. The results have direct applicability to the investigation of TASS outbreaks as well as to the pyrogenicity of medical devices in general. REFERENCES 1. Monson MC, Mamalis N, Olson RJ. Toxic anterior segment inflammation following cataract surgery. J Cataract Refract Surg 1992; 18:184–189 2. Mamalis N, Edelhauser HF, Dawson DG, Chew J, LeBoyer RM, Werner L. Toxic anterior segment syndrome. J Cataract Refract Surg 2006; 32:324–333 3. Parikh C, Sippy BD, Martin DF, Edelhauser HF. Effects of enzymatic sterilization detergents on the corneal endothelium. Arch Ophthalmol 2002; 120:165–172 4. Jehan FS, Mamalis N, Spencer TS, Fry LL, Kerstine RS, Olson RJ. Postoperative sterile endophthalmitis (TASS) associated with the MemoryLens. J Cataract Refract Surg 2000; 26:1773–1777 5. Werner L, Sher JH, Taylor JR, Mamalis N, Nash WA, Csordas JE, Green G, Maziarz EP, Liu XM. Toxic anterior segment syndrome and possible association with ointment in the anterior chamber following cataract surgery. J Cataract Refract Surg 2006; 32:227–235 6. Mathys KC, Cohen KL, Bagnell CR. Identification of unknown intraocular material after cataract surgery: evaluation of a potential cause of toxic anterior segment syndrome. J Cataract Refract Surg 2008; 34:465–469 7. Kreisler KR, Martin SS, Young CW, Anderson CW, Mamalis N. Postoperative inflammation following cataract extraction caused by bacterial contamination of the cleaning bath detergent. J Cataract Refract Surg 1992; 18:106–110 8. Whitby JL, Hitchins VM. Endotoxin levels in steam and reservoirs of table-top steam sterilizers. J Refract Surg 2002; 18:51–57 9. Kutty PK, Forster TS, Wood-Koob C, Thayer N, Nelson RB, Berke SJ, Pontacolone L, Beardsley TL, Edelhauser HF,
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24. Mazzotti F, Beuttler J, Zeller R, Fink U, Schindler S, Wendel A, Hartung T, von Aulock S. In vitro pyrogen test – a new test method for solid medical devices. J Biomed Mater Res 2007; 80A:276–282 25. Hasiwa M, Kullmann K, von Aulock S, Klein C, Hartung T. An in vitro pyrogen safety test for immune-stimulating components on surfaces. Biomaterials 2007; 28:1367–1375 26. Daniels AU, Barnes FH, Charlebois SJ, Smith RA. Macrophage cytokine response to particles and lipopolysaccharide in vitro. J Biomed Mater Res 2000; 49:469–478 27. Cho DR, Shanbhag AS, Hong C-Y, Baran GR, Goldring SR. The role of adsorbed endotoxin in particle-induced stimulation of cytokine release. J Orthop Res 2002; 20:704–713 28. Ludwig K, Scheiffarth OF, von Meyer L. Reducing the amount of monomers in intraocular lenses through sterilization by gamma radiation. Ophthalmic Res 1988; 20:304–307 29. Anderson JM, Marchant RE. Biomaterials: factors favoring colonization and infection. In: Waldvogel FA, Bisno AL, eds, Infections Associated with Indwelling Medical Devices 3rd ed. Washington, DC, ASM Press, 2000; 89–109 30. Kodjikian L, Burillon C, Roques C, Pellon G, Renaud FN, Hartmann D, Freney J. Intraocular lenses, bacterial adhesion and endophthalmitis prevention: a review. Biomed Mater Eng 2004; 14:395–409 31. Kobayakawa S, Jett BD, Gilmore MS. Biofilm formation by Enterococcus faecalis in intraocular lens material. Curr Eye Res 2005; 30:741–745 32. Mencucci R, Dei R, Danielli D, Susini M, Menchini U. Folding procedure for acrylic intraocular lenses; effect on optic surfaces and bacterial adhesion. J Cataract Refract Surg 2004; 30:457–463 33. Okajima Y, Kobayakawa S, Tsuji A, Tochikubo T. Biofilm formation by staphylococcus epidermidis on intraocular lens material. Invest Ophthalmol Vis Sci 2006; 47:2971–2975. Available at: http://www.iovs.org/cgi/reprint/47/7/2971. Accessed March 11, 2009 34. Moshirfar M, Feiz V, Vitale AT, Wegelin JA, Basavanthappa S, Wolsey DH. Endophthalmitis after uncomplicated cataract surgery with the use of fourth-generation fluoroquinolones; a retrospective observational case series. Ophthalmology 2007; 114:686–691 35. Chiquet C, Cornut P-L, Benito Y, Thuret G, Maurin M, Lafontaine P-0, Pechinot A, Palombi K, Lina G, Bron A, Denis P, Carricajo A, Creuzot C, Romanet J-P, Vandenesch F. Eubacterial PCR for bacterial detection and identification in 100 acute postcataract surgery endophthalmitis; for the French Institutional Endophthalmitis Study (FRIENDS) Group. Invest Ophthalmol Vis Sci 2008; 49:1971–1978
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First author: Liliana Werner, MD, PhD Berlin Eye Research Institute, Berlin, Germany, and John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA