Potential resistant morphotypes of Acanthamoeba castellanii expressed in multipurpose contact lens disinfection systems.

OBJECTIVES: The free-living amoeba Acanthamoeba castellanii is a rare cause of contact lens-associated microbial keratitis. The cyst stage of this amoeba is noted for its resistance to disinfection by multipurpose contact lens solutions (MPS). This report examines and reviews the potential survival modes of A. castellanii in MPS. METHODS: Trophozoites of A. castellanii (ATCC 30234) at densities from 10 to near 10 were incubated in 3 different MPS in a laminar flow hood for 24 hours at ambient temperatures. The dried films of MPS and phosphate-buffered saline (PBS) controls were examined before and after the addition of a peptone-yeast extract-glucose recovery broth (PYG) for the presence of amoeboid trophozoites and resistance stages over at least 7 days. The parallel exposure of trophozoites to MPS without evaporation or addition of PYG was similarly examined. RESULTS: Amoeboid trophozoites were not recovered in PYG nor were cyst-like structures observed in any MPS with near 10 densities. Progressively with 10 to 10 trophozoites, varied survival modes, particularly aggregates of trophozoites associated with cyst-like structures and occasional amoeboid forms and double-walled cysts with ostioles, became more evident. These morphotypes were most prominent after evaporation and typically first observed in the PYG. CONCLUSIONS: Trophozoites of A. castellanii near 10 and progressively to 10 densities are capable of expressing a variety of "short-term" survival modes in MPS, notably with the added stress of evaporation. Expression of these alternate survival modes in MPS may relate, in part, to contamination of contact lens cases and difficulties in developing standardized MPS efficacy tests.

Differences among strains of the Fusarium oxysporum-F. solani complexes in their penetration of hydrogel contact lenses and subsequent susceptibility to multipurpose contact lens disinfection solutions.

PURPOSE: To examine in vitro conditions for attachment and penetration of silicone hydrogel (SH) lenses by clinical isolates of the Fusarium oxysporum-F. solani complexes and the relative susceptibilities of the fusaria in the lens matrices to multipurpose contact lens solutions (MPSs). METHODS: SH soft contact lenses were soaked in Sabouraud dextrose broth (SAB) for 2 hours and transferred to 3.0 mL of phosphate-buffered saline (PBS). The lenses were inoculated with representative isolates of both complexes and incubated on a shaker at ambient temperature. Lenses were examined daily by light microscopy before and after rinsing and rubbing in MPS. Selected lenses penetrated by fungi were rinsed and rubbed with MPS and held in MPS for 6 hours, transferred to PBS with 0.03% SAB, and examined daily. RESULTS: The degree and rate of lens penetration of contact lenses by isolates of the F. oxysporum-F. solani complexes varied with lens type and the strain. Isolates obtained from patients with Fusarium keratitis produced on and within lenses chlamydospores that seemed similar to those observed in lenses actually worn by patients when they developed Fusarium keratitis. Clinical isolates showed greater capacities than those of a standard test strain to penetrate lenses and to survive exposures to various MPSs. In general, isolates of F. solani were more readily removed from lenses by rubbing than were isolates of F. oxysporum. CONCLUSIONS: The ability of Fusarium spp. to attach to and penetrate SH lenses in vitro varies with the lens type and strain, and this ability may make infectious keratitis more likely. We recommend the incorporation of a rubbing step in the MPS disinfection of hydrogel lenses to reduce the risk of fungal keratitis.

Attachment to and penetration of conventional and silicone hydrogel contact lenses by Fusarium solani and Ulocladium sp. in vitro.

PURPOSE: To analyze the relative capacities of Fusarium solani and Ulocladium sp. to attach to and penetrate silicone hydrogel soft contact lenses. METHODS: Representative silicone hydrogel (SH, siloxy complexes) and conventional [hydroxyethylmethylacrylate (HEMA)] soft contact lenses were exposed to suspensions of F. solani and Ulocladium sp. in vitro (10 conidia/mL in phosphate-buffered saline). The lenses were incubated with shaking at ambient temperatures and examined after rinsing in a multipurpose contact lens solution (MPS) by light and scanning electron microscopy. RESULTS: Isolates of both genera firmly attached to and penetrated both lens types, but Ulocladium sp. did so in greater density and more rapidly than F. solani. The extent of firm attachment and time needed for penetration into the lenses varied with strain and substratum, particularly with the isolates of F. solani. Morphologic characteristics (eg, penetration pegs, microcycle conidiation, and chlamydospores) of F. solani in the SH and HEMA lenses were similar to those observed in several lenses from patients with ReNu with MoistureLoc (RML)-associated Fusarium keratitis. CONCLUSIONS: To our knowledge, this is the first report that F. solani produces coiled penetration pegs in the matrices of SH hydrophilic soft contact lenses similar in morphology to those found in HEMA lenses. F. solani attaches firmly to SH lenses and rarely penetrates the lens matrix, but viable fungal propagules may remain on the lens after vigorous rinsing with MPS. Failure to use a manual cleaning-disinfection procedure may help to explain the increased incidence of Fusarium keratitis associated with contact lens wear.

Growth and survival of Fusarium solani-F. oxysporum complex on stressed multipurpose contact lens care solution films on plastic surfaces in situ and in vitro.

PURPOSE: To analyze factors implicating the association of ReNu with MoistureLoc (ReNu ML) multipurpose contact lens solution (MPS) with the increased incidence of Fusarium keratitis. METHODS: Used contact lens cases with and without contact lenses and MPS containers were collected from patients with confirmed or possible Fusarium keratitis. Direct microscopy including transparent adhesive tape preparations and swab cultures were used to determine fungal colonization. Survival and growth of selected isolates of Fusarium spp. in drying MPS on plastic surfaces were determined by microscopy and recoverable colony counts on enriched agar. RESULTS: Discrete regions of fungal colonization, including occasional microcycle conidiation and chlamydospore formation, were observed on the surfaces of contact lens cases and, less often, on solution containers that had been used by patients with Fusarium keratitis associated with the use of ReNu ML. Isolates provisionally grouped with the F. solani-F. oxysporum complex were inhibited by fresh MPS in original solution containers and contact lens cases, but survived in stressed (drying) films of MPS, particularly ReNu ML. These in vitro test results were similar to the direct in situ observations of the materials from patients. CONCLUSIONS: Selective, rapid growth and survival of cells of the F. solani-F. oxysporum complex on plastic surfaces, particularly of contact lens cases with stressed ReNu ML films, may explain, in part, the recent Fusarium keratitis outbreak.

LuxS influences Escherichia coli biofilm formation through autoinducer-2-dependent and autoinducer-2-independent modalities.

Escherichia coli produces biofilms in response to the small molecule autoinducer-2 (AI-2), a product of the LuxS enzyme. LuxS is part of the activated methyl cycle and could also affect biofilm development by AI-2-independent effects on metabolism. A luxS deletion mutant of E. coli W3110 and an inducible plasmid-luxS-complemented strain were used to identify AI-2-independent phenotypes. Differential interference contrast microscopy revealed distinct surface colonization patterns. Confocal microscopy followed by quantitative image analysis determined differences in biofilm topography correlating with luxS expression; deletion mutant biofilms had a 'spreading' phenotype, whereas the complement had a 'climbing' phenotype. Addition of exogenous 4,5-dihydroxy-2,3-pentanedione (DPD), an AI-2 precursor, to the deletion mutant increased biofilm height and biomass, whereas addition of the methyl donor S-adenosyl methionine or aspartate prevented the luxS-complemented strain from producing a thick biofilm. The luxS-complemented strain autoaggregated, indicating that fimbriae production was inhibited, which was confirmed by transmission electron microscopy. DPD could not induce autoaggregation in the deletion mutant, demonstrating that fimbriation was an AI-2-independent phenotype. Carbon utilization was affected by LuxS, potentially contributing to the observed phenotypic differences. Overall, the work demonstrated that LuxS affected E. coli biofilm formation independently of AI-2 and could assist in adapting to diverse conditions.

In vitro interactions of Fusarium and Acanthamoeba with drying residues of multipurpose contact lens solutions.

PURPOSE: To examine in vitro effects of evaporation and drying of multipurpose contact lens solutions on survival of Fusarium and Acanthamoeba. METHODS: Conidia of representative Fusarium from the 2004-2006 keratitis outbreak and trophozoites of Acanthamoeba castellanii were inoculated into commercially available multipurpose contact lens care solutions. These solutions were inoculated with 10(2)-10(6) microbial propagules/mL and were evaporated for at least 24 hours. After drying, nutrient media for recovery of surviving organisms were added to the residues formed in the lids of 38 mm polystyrene Petri dishes. General morphologic patterns of the solution residuals and the distribution and morphologies of the microorganisms were recorded with microscopic imaging. RESULTS: Various multipurpose contact lens disinfection solutions formed distinctive dried residual patterns. Both Fusarium and Acanthamoeba at concentrations tested above 10(3) per mL of disinfection solution were recovered from dried films with replicate testing. Mature cysts of Acanthamoeba not evident in the inocula were observed in sparse numbers in all dried solutions except one (Complete Moisture Plus; Advanced Medical Optics) and control salines where precysts and mature cysts were common. Both fusaria and amoeba tended to be observed in discrete regions of the dried residues. CONCLUSIONS: Regions of drying films of multipurpose contact lens disinfection solutions on contact lens cases may induce and harbor dormant-resistant stages of Fusarium and Acanthamoeba. It is hypothesized that the evaporation and drying of multipurpose contact lens disinfection solutions may have been an added risk factor for case contamination among Fusarium and Acanthamoeba keratitis patients. The need for frequent replacement of contact lens cases is enforced.

Hyphal penetration of worn hydrogel contact lenses by Fusarium.

PURPOSE: To determine the relative susceptibility of worn and unworn hydrogel contact lenses to penetration by hyphae of the Fusarium solani-Fusarium oxysporum species complex. METHODS: Hydrogel contact lenses (lotrafilcon A, balafilcon A, senofilcon A, galyfilcon A, and etafilcon A) were removed from their original packages and placed directly on the eyes of 24 experienced contact lens wearers for 8-9 hours. Lenses were removed, each placed in 3.0 mL sterile phosphate buffered saline (PBS), and inoculated with 10 conidia of fusaria. Similar sets of unworn lenses were soaked in Sabouraud's dextrose broth for 2 hours prior to exposure to fusaria conidia in PBS. The entire surface of lenses was examined with microscopy for 14 days for the development of coiled hyphae in the lens matrix (ie, penetration pegs [PP]). RESULTS: A total of 21 of 54 worn hydrogel lenses representing 4 types of silicone hydrogel and one type of hydroxyethylmethacrylate lens were penetrated by the fusaria. Compared to unworn lenses, 9 of the 21 PP-positive worn lenses showed earlier and more extensive penetration than seen with the unworn lenses. Several worn lenses compared to their unworn counterparts showed negligible or delayed penetration. CONCLUSIONS: Worn hydrogel contact lenses without a history of exposure to disinfection solutions compared to unworn lenses of similar status may show enhanced or decreased susceptibilities to penetration by Fusarium. This suggests that tear characteristics are an additional factor in the invasive contamination of hydrogel contact lenses by Fusarium.

Relative in vitro rates of attachment and penetration of hydrogel soft contact lenses by haplotypes of fusarium.

PURPOSE: To investigate the relative abilities of different haplotypes of the Fusarium solani (FSSC)-Fusarium oxysporum (FOSC) complexes to attach to and invade hydrogel contact lenses. METHODS: Silicone hydrogel and traditional hydroxyethylmethacrylate soft contact lenses were exposed to conidia [10 ml in phosphate-buffered saline (PBS)] of different haplotypes of fusaria associated with the Fusarium keratitis outbreak of 2004-2006. Select lenses and fungi were examined under conditions of organic enrichment. The lenses were incubated with shaking at ambient temperatures, then examined microscopically for the presence of penetration pegs (PPs). RESULTS: Attachment to and penetration of balafilcon A lenses in PBS within 96 hours were observed with representative isolates of FSSC 1-a, 1-b, and 2-d. Densities and coiled morphology of the PPs were similar. Eight of 8 FOSC failed to attach and form PP in PBS without prior sorption of organics by the lens. Generally, FSSC 1 isolates showed more rapid development of PP. Representatives of all haplotypes, including FSSC 2-c (ATCC 36031, a standard challenge strain), showed at least sparse attachment and penetration of the balafilcon A lens and, to a lesser degree, the lotrafilcon A lens. The development of PP in etafilcon A and galyfilcon A lenses required extended incubations (>21 days) relative to balafilcon A lenses. CONCLUSIONS: Attachment to and penetration of unworn hydrogel soft contact lenses by Fusarium varied with the strain and lens type rather than with the clinical, environmental, or geographic source of the isolates. Without organic enrichment of the lenses, penetration was more rapid and extensive by representatives of FSSC 1. Penetration was slow and less extensive under these conditions with FOSC and FSSC 2-c and 3. Organic enrichment of the lenses typically favored development of PP by the FOSC. Attachment and penetration of lenses occurred sooner and to a greater extent with surface-treated silicone hydrogel lenses than with the hydroxyethylmethacrylate lens.

Fusarium keratitis and contact lens wear: facts and speculations.

Over the past several decades mycotic keratitis has been considered a rare sequel to hydrogel contact lens wear. In 2005--2006 an upswing in the incidence of Fusarium keratitis was associated with a disproportionate use of one multipurpose contact lens solution (MPS, ReNu with MoistureLoc, Bausch & Lomb, Rochester, NY). The MPS, as manufactured and marketed, was sterile and met regulatory guidelines for antimicrobial activity. A multivariant interaction of poor hygienic practices and the contact lens paraphernalia were associated with a mostly selective contamination in or on the lens storage case by members of the F. solani/F. oxysporum species complexes from the environment of the user. A decline of the anti-fusaria properties of the MPS in the lens case appeared related to its dissociation from drying, or dilution and the potential for sorption of antimicrobial solution components (e.g., alexidine) to various hydrogel lenses. These factors and capacities of the fusaria for rapid amplification by microcycle conidiation, production of dormant resistant cells, and potential for attachment and penetration of hydrogel lenses, were linked to the occasional selective fungal survival and growth during storage of the lens in MPS. Lack of a manual rubbing-cleaning step in the MPS disinfection process was considered a risk factor for keratitis.

Mold colonization during use of preservative-treated and untreated air filters, including HEPA filters from hospitals and commercial locations over an 8-year period (1996-2003).

High efficiency particulate arrestance (HEPA; 99.97% efficient at 0.3 microm) filters, filters with ASHRAE particulate arrestance rating of 90-95% at 1 mum (90-95% filters), and lower efficiency cellulosic-polyester filters from air conditioning systems in hospitals and commercial buildings were removed from the systems and examined microscopically for mold colonization. Cellulosic-type filters from systems with water entrainment problems typically were colonized, or became colonized upon incubation in moisture chambers. Species of Acremonium, Aspergillus, and Cladosporium were most common. With air filters of all types, treatment of filter media with an antimicrobial preservative tended to reduce or delay colonization. Mold colonization of HEPA and 90-95% filters was observed most often on the load surfaces, but two untreated HEPA filters were permeated with fungi, one with Aspergillus flavus, the other with Cladosporium sp. Air filters in heating, ventilating, and air conditioning (HVAC) systems, particularly those with chronic or periodic exposure to moisture, may serve as point sources for indoor molds.