Shangtong Zhang

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.

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.

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 (104 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.

Toxic Effects of Ag(I) and Hg(II) on Candida albicans and C. maltosa: a Flow Cytometric Evaluation

ABSTRACT The effects of Ag(I) and Hg(II) on membrane potential and integrity of cells of Candida albicans and C. maltosa were determined with a flow cytometric procedure that employed an anionic membrane potential-sensitive dye, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, and a membrane integrity indicator, propidium iodide. The membrane potentials of cells of both species were reduced rapidly within 15 min of exposure to Ag(I). No threshold dose for Hg(II) existed, and cells of both species lost membrane potential gradually in Hg(II) solutions. Cells of both species lost membrane integrity more rapidly in Ag(I) solutions than in Hg(II) solutions. In Ag(I) solutions, the decrease in the numbers of cells recoverable in culture occurred at a rate similar to the rate of cell depolarization and membrane permeabilization. In Hg(II) solutions, loss of cell recoverability preceded the loss of membrane potential and membrane integrity. C. albicans, in contrast toC. maltosa, showed no loss of membrane integrity after exposure to Hg(II) solutions for 1 h. Different rates of binding of Ag(I) and Hg(II) between the two species suggest that the two ions target different primary sites.

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.

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 [104 ml−1 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.

Viability of Amoebae, Fungal Conidia, and Yeasts

Conventional methods for the evaluation of antimicrobials and disinfecting solutions with microorganisms involve culture-based techniques, which are time-consuming and underestimate the number of viable organisms. Rapid detection and viability measurements of microorganisms in homogenous and heterogenous microbial populations have been greatly enhanced by recent advances in the use of fluorescent stains in flow cytometry (FCM). FCM has been applied to enumerate, differentiate, and identify microorganisms, determine protein and DNA content of cells, analyze the physiological state of individual cells, and analyze the interaction of drugs, antibiotics, and antimicrobials with microbial cells. Four physiological states of cells can be distinguished by FCM: (1) reproductively viable, (2) metabolically active, (3) intact, and (4) permeabilized.FCM permits a rapid and quantitative measurement of the optical characteristics of cells as they pass through, in a single file, a focused beam of light. As cells are carried within a fast-flowing fluid stream and through the focus of exciting light, three parameters are measured: forward angle light scatter, side angle light scatter, and fluorescence emitted by dyes that have specific interaction with intracellular components of individual cells. FCM data that are presented in histogram and dot plots can be generated to give information on a variety of properties of interest among cells in the population as a whole.FCM offers major advantages in multiparameter data acquisition and multivariate data analysis, high-speed analysis, and cell-sorting capabilities. Disadvantages may be associated with the cost, which is usually over 100,000 (US Dollars) for a typical laser-based flow cytometer with just analyzing capabilities. Another disadvantage is that skilled personnel are usually required to operate these complex instruments so as to get optimum performance. A schematic overview of flow cytometry is presented in Fig. 1.