Toxic anterior segment syndrome (TASS) and diffuse lamellar keratitis (DLK) are both anomalous postsurgical inflammatory syndromes that generally follow otherwise uncomplicated ocular surgery.1–3 The exaggerated inflammatory reaction is often caused by a toxic substance introduced during surgery and usually responds to intensive topical steroid treatment. Because laser in situ keratomileusis (LASIK) typically manifests no intrastromal corneal inflammation, DLK is usually obvious and alarming when it appears.4 In contrast, TASS may be unrecognized and underreported because mild or moderate cases may appear to be within the normal spectrum of postsurgical inflammation. Severe TASS may be misdiagnosed and treated as culture-negative endophthalmitis.
The DLK and TASS literature reports a long list of potential causative factors that have been associated with specific outbreaks. The risk factors can be subdivided into the following categories: (1) disposable surgical items, (2) reusable surgical items, (3) intraocular and extraocular solutions used during surgery, (4) modifications in surgical techniques, and (5) instrument contamination during sterilization and processing. Among more than 30 potential causes identified in the literature, autoclave reservoir biofilms have received relatively little attention as risk factors for DLK or TASS. However, the ability of biofilms to potentiate innate immunity through the system of pattern recognition receptors (PRRs) has become more widely recognized during the last 15 to 20 years. A widely held perception that endotoxin is the sole bacterial byproduct triggering innate immunity and inflammation is now considered inaccurate. Biofilms harbor a wide variety of other biotoxins that include bacterial and fungal cell wall proteins, bacterial flagellin, DNA and RNA fragments, and microbial exotoxins. All have the potential to produce severe inflammation mediated by the natural system of innate immunity.
We report a cluster of DLK cases most likely caused by biotoxins originating from a well-developed biofilm on the inner wall of a reservoir-based tabletop autoclave (STATIM 2000). We reviewed the literature on DLK and TASS, as well as the contemporary science of innate immunity, to develop and support this hypothesis. Finally, we report a decontamination and cleaning protocol designed to prevent the accumulation of autoclave reservoir biofilm, which has prevented further anomalous DLK at this center.
This study was conducted under a protocol approved by the Committee for the Protection of Human Subjects, University of California at Berkeley. Between January 2010 and May 2011, patients presenting for refractive surgery to the Refractive Surgery Center at the University of California, Berkeley, School of Optometry, underwent a standardized presurgery workup for refractive surgery. After their refractive surgery, all patients were examined on the first postoperative day by a single observer (K.T.). If any grade of DLK was detected, patients were then examined by the ophthalmic surgeon (A.L.S.). Uncomplicated patients were next examined on day 4 and then at 1 month, 3 months, and 6 months. If DLK was detected, follow-up visits were conducted every 1 to 3 days to monitor response to treatment. Throughout the period during which an elevated incidence of DLK was detected, various modifications in technique were used in an attempt to identify and eliminate the cause (Table 1).
Data are expressed as the ratio of DLK cases relative to the total number of LASIK cases. Chi-squared analysis is used to determine statistical significance of differences between groups. P values of less than .05 are considered statistically significant.
The MEDLINE database was searched using the following keywords: TASS, DLK, TASS, DLK, biofilm, and autoclave reservoir contamination. Relevant articles were reviewed to assess the manner in which autoclave reservoirs were analyzed or implicated.
During the 22 months between September 2, 2010, and June 11, 2012, the DLK incidence at the Berkeley center increased from 4 cases in 88 (4.6%) to 147 cases in 395 (37.2%). The clinical course of 7 eyes with grade 4 DLK (central toxic keratopathy) was prolonged and required topical antiinflammatory treatment, intraocular pressure–lowering medications, and, in several cases, oral prednisone (Figure 1). The final 1-year corrected distance visual acuity (CDVA) was reduced to 20/25 in 2 of these 7 eyes. All other eyes recovered a CDVA of 20/20 or 20/25 in the case of 1 amblyopic eye. Four of the 7 eyes required refractive surgical enhancement. In addition, 1 eye developed primary epithelial ingrowth requiring surgical debridement, and 2 eyes developed early cataracts, potentially related to steroid use.
Once the elevated incidence of DLK was recognized, multiple modifications in products, techniques, and instruments were initiated as listed in Table 1. Every modification ultimately failed to halt the excessive DLK rate until the STATIM 2000 (SciCan) autoclave was replaced on June 6, 2012, and a new reservoir sterilization and surveillance was initiated. During the next 31 months, the incidence of DLK dropped to a consistently low baseline rate of 2.2% (14/632 cases), P < .00001 (Table 2 and Figure 2).
The retired autoclave was stored with its reservoir drained until the DLK cluster had completely resolved. On April 4, 2013, the dry reservoir chamber sidewalls were cultured for bacterial and fungal contamination, revealing heavy growth of Pseudomonas aeruginosa and the Burkholderia cepacia complex. In the absence of any reservoir moisture, these bacteria presumably colonized the reservoir wall during the use of the autoclave and remained viable despite dry storage for 10 months.
Forty case reports, reviews, and other articles in the peer-reviewed literature between 1986 and 2019 relating to the topic of TASS5–44 (Table 3). Another 22 reports and articles on DLK1–3,45–63 (Table 4) were identified. Only 5 of these 62 total studies reported specifically investigating the potential role of autoclave reservoirs and their associated biofilms as the potential cause of TASS or DLK.3,13,35,46,52
Biofilms are polymicrobial cell populations that attach themselves to moist surfaces in a prolonged and durable manner and persist once the surface is dry. With sufficient time, virtually all moist surfaces are likely to develop a biofilm potentially comprising Gram-negative and positive bacterial and fungal elements. As the biofilm develops, it encloses itself in a matrix and becomes resistant to simple removal such as by rinsing with sterile water.64,65 Highly concentrated antibiotics, physical scrubbing, or exposure to boiling water may be necessary to remove the biofilm.66,67
Clinically, exposure to the constituents of biofilms can elicit severe immune responses resulting in damage to host tissues. Because of its small size and anterior chamber volume, the eye is especially sensitive. These immune responses involve the innate immune system generating a series of antimicrobial and inflammatory defenses in response to common microbial antigens. In contrast to acquired immunity, innate immune responses are considered to be nonspecific and lack immunological memory, that is, they target a broad range of microbes and are not boosted by previous exposure to antigen.
The scientific understanding of the system of innate immunity has expanded dramatically since 2000, when sterilizer reservoir biofilms were first implicated by Holland et al. in a cluster of 52 cases of DLK.50 Subsequently, the central role of PRRs in innate immunity was established. PRRs are present on and within inflammatory cells of many species where they detect the presence of pathogens by recognizing molecules unique to microorganisms. Initially thought to primarily recognize endotoxin, PRRs also mediate inflammatory responses to other bacterial and fungal cell wall proteins, bacterial flagellin, DNA and RNA fragments, and other microbial exotoxins. All of these byproducts of microbial cellular damage might be generated when microorganisms are exposed to high autoclave temperatures or steam.
If biological contaminants of a reservoir wall biofilm enter the autoclave chamber, they may be inoculated onto the surface of exposed ophthalmic instruments. The bacteria and fungi will be killed, and other byproducts will be broken down by heat and steam. However, fragments of these inactivated microbial byproducts may persist whereupon they could be introduced into the anterior chamber or corneal stroma in sufficient amounts to trigger PRR-mediated TASS or DLK.
We report a prolonged DLK cluster that continued despite multiple systematic changes in instrumentation and surgical protocols. This cluster of cases eventually ended following replacement of the entire tabletop autoclave. The absence of any further spikes in the DLK rate implicated the autoclave as the cause of our cluster. Therefore, the inner reservoir wall of the original autoclave was swabbed and cultured 10 months after it had been drained and retired. Despite regular draining and drying of the autoclave reservoir while in use, a polymicrobial population consistent with a well-developed biofilm was cultured from its walls. Both P aeruginosa and B cepacia bacteria cultured from the reservoir wall produce known ligands (eg, LPS, flagellin) to which PRRs can respond. We believe that these small molecular biotoxins, derived from the autoclave reservoir wall biofilm, were seeded onto the surgical instruments and then introduced into the cornea stroma. DLK was the innate immune response mediated by PRRs. Moreover, our ability to culture these microbes after an extended period of dry storage reflects the tenacity with which bacterial subpopulations are able to survive in a dormant state within a biofilm.68,69
A 2016 review of autoclave reservoir biofilms by Sorenson et al. surveyed regional outpatient surgery center tabletop autoclaves in association with a single-center cluster of TASS cases.35 Scanning electron micrographs were taken of a section of the autoclave reservoir wall obtained from the retired autoclave used to sterilize instruments implicated in 10 cases of TASS. These showed a well-developed biofilm where many bacterial elements appear to be in the predispersal phase (Figure 3). Calculations of reservoir wall bacterial density from these images suggest a surface population of approximately 10 billion cells, and a total population of many times that figure, given the multilayered nature of biofilms. Conceivably, their byproducts and dispersal elements could contaminate the fluid drawn into the reservoir to produce steam. In that study, 20 of the 25 autoclave reservoirs surveyed from regional ambulatory surgery centers demonstrated biofilm contamination, and 19 different bacterial or fungal species were identified.35Figure 4 shows the blood agar plates of 12 such autoclaves.
Between 1986 and 2019, 40 articles on TASS were published in the peer-reviewed literature. Another 22 papers were published on DLK. Most of these preceded the description of PRRs and their role in innate immunity, which now implicates a more expansive list of microbial byproducts besides heat-stable endotoxin. This may have led the authors to overlook or underestimate the potential for inactivated biofilm components from the autoclave reservoir to cause TASS and DLK. For example, only 5 of these 62 articles report investigating autoclave reservoirs as a potential etiology (Tables 3 and 4). As a result, we believe that the ophthalmic surgical community remains largely unaware of the potential risks of biofilm formation and accumulation on the reservoir walls of steam autoclaves.
To reduce biofilm formation, the autoclave reservoir should be regularly drained and air dried, such as at the conclusion of each week's usage. However, this alone may not prevent biofilm formation. For example, a newly purchased autoclave reservoir developed culturable biofilm after only 20 days of use despite being drained and dried after each day of surgery (Figure 5). This finding was consistent with Holland's 1999 study demonstrating biofilm contamination of glass beads after 11 days of exposure to distilled water within a previously decontaminated autoclave reservoir. If the autoclave reservoir design precludes scrubbing or physical removal of biofilm, thermal destruction with boiling water is probably the best method to prevent and remove biofilm formation from the heat-stable plastic wall. Indeed, exposure to boiling water effectively eliminated biofilm viability in all 3 treated reservoirs from our 2016 data (Figure 6 shows one such culture set). Accordingly, we adopted this boiling water cleaning protocol for the reservoir of the STATIM autoclave, which replaced the older unit at the Berkeley ASC. Monthly cultures of this autoclave reservoir were negative for 12 consecutive months from August 2012 to August 2013, and similar surveillance cultures might be considered by other centers who adopt this protocol to establish its utility. We have continued the following maintenance protocol at the Berkeley center on a weekly basis since the resolution of the DLK cluster (Table 5):
- Step 1. Turn the unit's power switch to the “OFF” position.
- Step 2. Remove the reservoir cap and, if present, the course mesh filter.
- Step 3. Completely drain the reservoir by means of the drain tube.
- Step 4. Completely fill the reservoir (≈4.0 L) with boiling distilled water (do not use tap water).
- Step 5. Allow boiling water to remain in the reservoir for 2 to 3 minutes.
- Step 6. Drain the autoclave reservoir completely (using the drain tube) until next use.
- Step 7. Before next use, fill the reservoir with distilled water (room temperature).
- Step 8. Turn the power switch to the “ON” position, use as per the operator's manual.
Note that the inserted cassette required for the cycle contains no instruments, and that the near-capacity filling allows boiling water to contact the dependent and vertical surfaces of the autoclave reservoir wall.
We concluded that inactivated contaminants from the autoclave reservoir biofilm caused the cluster of DLK at the Berkeley center. Since the initiation of this cleaning protocol, the incidence of DLK has remained below 2.2% at this same center. Although this is an anecdotal observation from a single center, when considered together with 2 previously published clusters caused by reservoir contamination, we believe that reservoir wall biofilms are occult sources of steam contamination and pose an underappreciated risk for triggering DLK or TASS.35,50 After consulting with and reviewing this protocol with the manufacturer (SciCan), we endorse regular reservoir cleansing with boiling water in STATIM cassette autoclaves in an effort to mitigate this risk.
WHAT WAS KNOWN
- Toxic anterior segment syndrome (TASS) and diffuse lamellar keratitis (DLK) are uncommon inflammatory outcomes after uncomplicated ocular surgery. Many suspected causes have been suggested.
- Biofilms are endemic to nearly all moist surfaces and undergo a transformation rendering their microenvironment resistant to simple elimination.
- Biofilms harbor a wide variety of other biotoxins that include bacterial and fungal cell wall proteins, flagellin, DNA and RNA fragments, and microbial exotoxins, all of which stimulate the system of innate immunity via pattern recognition receptors (PRRs).
WHAT THIS PAPER ADDS
- The literature discussing TASS and DLK may have overlooked the role of autoclave reservoir biofilms because the understanding of PRRs and their role in innate immunity has only come to light in recent years.
- Fluid reservoirs of tabletop steam autoclaves can readily develop polymicrobial biofilms harboring microbial pathogens, whose inert molecular byproducts can cause DLK and TASS when introduced to the eye by surgical instruments.
- Stringent reservoir cleaning and maintenance may significantly reduce this risk by preventing and removing these biofilms. Ophthalmic surgical centers should consider following the reservoir sterilization protocol presented herein.
Stephanie McGovern, RN, and Linda Lee, RN assisted with data acquisition and patient coordination.
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