Listeria monocytogenes cross-contamination of cheese: risk throughout the food supply chain.

Listeria monocytogenes has been the most common microbial cause of cheese-related recalls in both the United States and Canada in recent years. Since L. monocytogenes is inactivated by pasteurization, the majority of these cases have been linked to environmental and cross-contamination of fresh-soft, soft-ripened, and semi-soft cheeses. Cross-contamination of foods with L. monocytogenes is a continuous risk throughout the food supply chain and presents unique challenges for subsequent illness and outbreak investigations. Reports on outbreaks of listeriosis attributed to cross-contamination downstream from primary processing help highlight the critical role of epidemiological investigation coupled with coordinated molecular subtyping and surveillance in the recognition and investigation of complex foodborne outbreaks. Despite their complexity, environmental sampling throughout the supply chain coupled with improved genotyping approaches and concomitant analysis of foodborne illness epidemiological exposure data are needed to help resolve these and similar cases more rapidly and with greater confidence.

Microbiological quality of raw milk used for small-scale artisan cheese production in Vermont: effect of farm characteristics and practices.

This study 1) evaluated the overall milk quality and prevalence of 4 target pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and Escherichia coli O157:H7) in raw milk used for small-scale artisan cheesemaking and 2) examined specific farm characteristics and practices and their effect on bacterial and somatic cell counts (SCC). Raw milk samples were collected weekly from 21 artisan cheese operations (6 organic) in the state of Vermont that manufactured raw-milk cheese from cow (12), goat (5), or sheep (4) milk during the summer of 2008. Individual samples were examined for standard plate counts (SPC), coliform counts (CC), and SCC. Samples were also screened for target pathogens both quantitatively and qualitatively by direct plating and PCR. Overall, 86% of samples had SPC <10,000 cfu/mL, with 42% <1,000 cfu/mL. Additionally, 68% of samples tested were within pasteurized milk standards for coliform bacteria under the United States' Grade A Pasteurized Milk Ordinance at <10 cfu/mL. Log(10) SPC and CC did not differ significantly among species. Similarly, method of sample delivery (shipped or picked up), farm type (organic or conventional), and duration of milking (year-round or seasonal) did not have significant effects on farm aggregated mean log(10) SPC, CC, or SCC. Strong positive correlations were observed between herd size and mean log(10) SPC and between log(10) SPC and CC as well as SCC when data from all animal species were combined. Although SCC for cow milk were significantly lower than those for goat and sheep milk, 98, 71, and 92% of cow, sheep, and goat milk samples, respectively, were within the compliance limits of the United States' Grade A Pasteurized Milk Ordinance for SCC. Fourteen of the 21 farms (67%) were positive for Staph. aureus, detected in 38% of samples at an average level of 20 cfu/mL. Neither L. monocytogenes, E. coli O157:H7, or Salmonella spp. were detected or recovered from any of the 101 samples tested. Our results indicate that the majority of raw milk produced for small-scale artisan cheesemaking was of high microbiological quality with no detectable target pathogens despite the repeat sampling of farms. These data will help to inform risk assessments that evaluate the microbiological safety of artisan and farmstead cheeses, particularly those manufactured from raw milk.

Role of diabetologist in evaluating diabetic retinopathy.

OBJECTIVE: To evaluate the ability of diabetologists to screen diabetic patients for diabetic retinopathy. RESEARCH DESIGN AND METHODS: Comparison of eye examination performed by diabetologists with direct ophthalmoscopy through an undilated pupil and by ophthalmologists through a dilated pupil with seven-field stereoscopic fundus photography (gold standard). The study consisted of 67 insulin-dependent and non-insulin-dependent diabetic outpatients attending a diabetes clinic. RESULTS: On the basis of fundus photography, patients were classified as having no or insignificant (30%), minimal (31%), moderate (24%), or severe (15%) retinopathy. The diabetologists and ophthalmologists performed similarly in their ability to classify severity of diabetic retinopathy accurately. When no or insignificant retinopathy (isolated microaneurysms only) was detected by examination, clinically significant retinopathy detected by fundus photography was highly unlikely (less than 5%). On the other hand, if more than isolated microaneurysms were seen on examination, all examiners missed more severe lesions detected by fundus photography. Patients with corrected visual acuity worse than 20/30 had a high likelihood (100%) of moderate or severe retinopathy. CONCLUSIONS: Motivated well-trained diabetologists can screen for diabetic retinopathy. The absence of detectable lesions by direct ophthalmoscopy indicates that automatic referral to an ophthalmologist is not necessary. However, if any level of retinopathy is detected or corrected acuity is worse than 20/30, referral to an ophthalmologist is required. In this setting, fundus photography is advised because it is the most sensitive means of detecting clinically significant retinopathy. If other nonophthalmologists can be trained to achieve similar results, current recommendations for ophthalmologic referral that require annual ophthalmologic examinations for most diabetic patients may need to be reconsidered.

Residual bodies in the retinal pigment epithelium induced by intravitreal netilmicin.

Intravitreal injection of aminoglycoside antibiotics is known to induce morphological changes in the retinal pigment epithelium (RPE) resembling a lipidosis. The RPE of netilmicin-treated rabbits displays a dose-related increase in autofluorescence compared to untreated controls. Netilmicin produces an accumulation of membrane-limited osmiophilic lamellated inclusions in the pigment epithelial cell. These inclusions measure from 1 to 3 microns in diameter, and have acid hydrolase activity demonstrated by cytidine monophosphate cytochemistry. These findings suggest that netilmicin-induced inclusions are residual bodies and that the accumulation of these residual bodies is responsible for the observed cellular lipidosis.

Elevation of vitreous leptin in diabetic retinopathy and retinal detachment.

PURPOSE: Leptin is a cytokine that regulates energy metabolism and is linked to diabetes mellitus through its metabolic actions. Leptin is angiogenic and promotes wound healing, and therefore this investigation was conducted to determine whether leptin is associated with neovascular and fibrotic complications of diabetes and other retinopathies. METHODS: Serum and vitreous samples were collected from patients classified by the presence and type of diabetic retinopathy or other ocular diseases. Leptin was measured in serum and vitreous by radioimmunoassay, and leptin and leptin receptor were localized in epiretinal membranes immunohistochemically. RESULTS: Leptin levels in serum and vitreous were higher in patients with diabetes than in those without, and vitreous leptin concentrations were especially elevated in patients with proliferative diabetic retinopathy or retinal detachment. Leptin and leptin receptor were detected in fibrovascular epiretinal membrane of patients with diabetes. CONCLUSIONS: Leptin in human vitreous is elevated in proliferative diabetic retinopathy, and retinal detachment and is present in fibrovascular epiretinal tissue. These data suggest an involvement of leptin in retinal disease.

Mass cultivation of bovine ocular pigment epithelial cells in microcarrier suspension culture.

Recent developments in tissue culture technology permit the mass cultivation of anchorage-dependent cells in a compact and easily maintained system by utilizing the large surface area of suspended microspheres for cellular attachment and proliferation. The present study describes the successful cultivation of pure bovine iris pigment epithelium and mixed bovine retinal pigment epithelium-choroid by microcarrier suspension culture. Ocular pigment epithelial cells were collected from enucleated calf eyes and cultured in conventional monolayer fashion on flat plates. Subsequently, cells were harvested by trypsinization and were inoculated into microcarrier culture. Confluence was obtained in 8 to 12 days and final cell densities were 1 x 10(6) cells/ml for iris pigment epithelium and 8 x 10(5) cells/ml for combined retinal pigment epithelium--choroid; the microcarrier density was 5 gm/L. Samples from ongoing microcarrier cultures were processed for light and electron microscopic examination. Culture of bovine ocular pigment epithelial cells by microcarrier suspension technique provides a simplified method of cultivation of large numbers of cells, with additional advantages in ongoing culture maintenance and accessibility.

Retinal toxicity of intravitreal gentamicin. An electron microscopic study.

Retinal ultrastructure was examined at various intervals following a single intravitreal injection of 100-4,000 micrograms of gentamicin in rabbit eyes. Three days after injections of 100-500 micrograms, numerous abnormal lamellar lysosomal inclusions were observed in the retinal pigment epithelium (RPE) and in macrophages in the subretinal space. These changes were typical of drug-induced lipid storage and were comparable to inclusions reported in kidney and other tissues as manifestations of gentamicin toxicity. One week after similar injections, focal areas of RPE necrosis and hyperplasia with disruption of outer segments appeared, but the inner segments and inner retina were intact. Doses of 800-4,000 micrograms produced a combined picture of RPE/macrophage lipidosis within the first 3 days, with increasing, superimposed, inner, retinal necrosis. This study provides the first evidence of lysosomal alterations in ocular tissues following the intravitreal injection of gentamicin and implicates the RPE as the primary site of observed toxicity.

Dynamic sodium chemical shift imaging for the study of aqueous humor flow.

Ocular images were obtained using sodium chemical shift imaging (CSI) and 1,4,7,10-tetraazacyclododecane-N,N'N",N"'-tetramethylenephospho nate thulium (III) [Tm(DOTP)5-], a paramagnetic chemical shift reagent. After injecting the shift reagent into the anterior chamber of rabbits, serial imaging was done, monitoring the change in chemical shift with time. Sodium CSI produced images of the eye in three dimensions, quantitatively depicting the spatial and temporal changes in the concentration of a paramagnetic tracer substance. The Tm(DOTP)5- is eliminated from the anterior chamber by first-order kinetics with a half-life of 49 min. These data suggest that this substance is eliminated from the anterior chamber at the same rate as aqueous humor is replaced. Sodium CSI shows promise as a valuable technique for monitoring fluid dynamics in the living eye.

A feasibility study of 23Na magnetic resonance imaging of human and rabbit vitreal disorders.

PURPOSE: To assess the clinical feasibility of sodium magnetic resonance imaging for the visualization and characterization of intraocular tissues. METHODS: 23Na magnetic resonance images were obtained of enucleated human eyes and of rabbit eyes in vivo. The magnetic resonance imaging technique used in this study provided slices of < 2 mm thickness and in-plane resolution of < 2 x 2 mm. From each of these slices local values of spin-spin relaxation time (T2*) were calculated from pixel intensities in each of the eight echoes. RESULTS: The images clearly display normal anatomic details of the lens and vitreous humor, and important pathologic details such as intravitreal and subretinal hemorrhages, ocular melanoma, and retinal detachments. Intraocular tissue identifications based on relative spin-spin relaxation time values and pixel intensities correlate with those made by standard diagnostic techniques. CONCLUSIONS: 23Na magnetic resonance imaging may be used for the visualization and characterization of intraocular tissues. Differentiation among vitreous humor, lens, aqueous humor, subretinal fluid, or hemorrhage and tumor may be based on image intensity and/or spin-spin relaxation times.

Experimental ocular surgery with a high-repetition-rate erbium:YAG laser.

PURPOSE: To evaluate the performance in ocular surgery and the ocular tissue interactions resulting from increasing the maximum repetition rate of a pulsed-mode erbium:YAG laser system from 30 to 200 pulses per second. METHODS: An erbium:YAG laser was used that emitted at 2.94 microm with an output graduated from 0.2 mJ to 25 mJ and a repetition rate from 2 Hz to 200 Hz and that was equipped with a flexible optical fiber attached to various interchangeable 20-gauge endoprobes to perform ocular surgery in enucleated pig eyes. The specific maneuvers were performed in close contact in nontransmitting aqueous media and included inner retinal ablation, retinotomy, lens capsulotomy, lens ablation, iridotomy, and iridectomy. Selected tissue specimens were examined by light microscopy. RESULTS: Increasing the repetition rate to the 200-Hz range significantly improved the smoothness, continuity, and speed of all surgical maneuvers. Compared with the 30-Hz rate, substantially lower energies per pulse were efficient with the 200-Hz rate. The "sticking effect" between the tip of the probe and the target tissue at low-repetition rates, which resulted in discontinuation of the surgical maneuver, particularly during lens surgery, was eliminated with the use of high-repetition rates. Use of high-repetition rates produced a zone of residual thermal damage less than 30 microm in all ocular tissues. The histologic findings of tissue interactions were comparable to those obtained in published studies in which the same wavelength and low hertz rates were used. CONCLUSIONS: The high-repetition-rate erbium:YAG laser technology described is advantageous, compared with low-repetition-rate erbium:YAG lasers, and is applicable in a variety of ocular surgical procedures. Innovations in endoprobe design and further study will determine its role in contemporary ocular surgery.

An NMR blood test for uveal melanoma?

In a recent article a simple nuclear magnetic resonance (NMR) blood test was suggested for the detection of the presence of cancer. The test's sensitivity to uveal melanoma of both pre- and posttreatment status has been investigated. Cases in this study were 95 patients with uveal melanoma, and controls were 70 participants in an ongoing case control study of retinal eye disease being conducted at the Massachusetts Eye and Ear Infirmary. Proton NMR evaluations at 4.7 T (200 MHz) were performed on plasma obtained from EDTA and citrated blood samples. The average line-width values were calculated from each spectrum. Statistical analysis revealed that mean proton NMR line widths were essentially equal for patients with treated (18.7 Hz) and untreated tumors (18.4 Hz) and for controls (18.5 Hz). Results based on this data set suggest that proton NMR spectroscopy has little predictive power in the detection of uveal melanoma or in the monitoring of therapy.

Evaluation of the retinal toxicity and pharmacokinetics of dexamethasone after intravitreal injection.

The intravitreal injection of steroids may be potentially useful in the treatment of endophthalmitis and other ocular inflammatory diseases. The retinal toxicity and intraocular turnover of aqueous solutions of dexamethasone sodium phosphate in doses ranging from 440 to 4000 micrograms were evaluated in the rabbit; evaluation was also performed for a 0.1-mL injection of a commercially available preparation (dexamethasone phosphate [Decadron] injection, 4 mg/mL). After the 440-micrograms dose, a transient increase in staining of the Müller cells was observed, which normalized after 2 days. Progressively higher doses resulted in an increasing spectrum of disorganization in Müller and other retinal cells. The half-life of the intravitreally injected drug was 3.48 hours. These findings suggest a primary interference in Müller cell function, possibly through dexamethasone-induced alterations in retinal glutamate or glucose metabolism.

Amiodarone keratopathy: drug-induced lipid storage disease.

Therapeutic administration of amiodarone, an antiarrhythmic drug, to eight patients resulted in the formation of vortex-like figures within the anterior cornea. Clinical examination disclosed no visual loss or other ocular abnormality attributable to the drug. In one patient, light and transmission electron microscopy of corneal epithelium, bulbar conjunctiva, and cataractous lens revealed complex lipid deposits within lysosome-like intracytoplasmic inclusions in corneal, conjunctival, and lens epithelium, conjunctival fibrocytes, and conjunctival vascular endothelium. Amiodarone keratopathy is compared clinically and morphologically with the corneal alterations seen in Fabry's disease and in chloroquine use as an example of a drug-induced lipid storage disorder.

Intravitreal amikacin in the treatment of bacterial endophthalmitis.

Intravitreal administration of broad-spectrum antibiotics is a widely accepted component of initial therapy for bacterial endophthalmitis. Four cases of culture-proved bacterial endophthalmitis are reported to demonstrate the safety of intravitreal administration of amikacin sulfate in conjunction with cephalosporins in patients. The benefits of using amikacin rather than gentamicin sulfate in intravitreal drug therapy are discussed, and a wider role for amikacin as the aminoglycoside of choice for initial intravitreal injection in presumed bacterial endophthalmitis is suggested.

Intravitreal antibiotic therapy with vancomycin and aminoglycoside. An experimental study of combination and repetitive injections.

We evaluated the retinal toxic reaction of combined intravitreal therapy with regimens of 1.0 mg of vancomycin hydrochloride and 400 micrograms of amikacin sulfate or 1.0 mg of vancomycin hydrochloride and 100 micrograms of gentamicin sulfate in the rabbit eye. The effects of reinjecting each combination a second or third time, separated by 48-hour intervals, were also examined by ophthalmoscopy and light and transmission electron microscopy. The initial administration of either regimen did not produce toxic reaction. After two injections, five of six eyes with vancomycin-gentamicin therapy and three of six eyes with vancomycin-amikacin therapy displayed focal areas of retinal toxic reaction on histologic study. After three injections, all eyes treated with either regimen showed histologic evidence of more advanced retinal toxic reaction primarily involving the outer retina and retinal pigment epithelium. The present study demonstrates the safety of an initial intravitreal injection of vancomycin and aminoglycoside but shows increasing retinal toxic reaction with repetitive combination therapy.

An endolaser probe with aspiration capability.

A new endolaser probe with aspiration capability can be used as a flute needle for drainage of subretinal fluid and for endolaser treatment of a retinotomy site after vitrectomy. This instrument allows removal of vitreous and subretinal fluid during air-fluid exchange and immediate endolaser photocoagulation without exchange of instruments.

Experimental transient exudative retinal detachment in the rat.

OBJECTIVE: To establish a new model of exudative retinal detachment in the rat. METHODS: Photothrombosis was produced in a single retinal vein using direct treatment with a dye laser operating in the yellow wavelength (577 nm). Control eyes received identical laser applications, but treatment was placed alongside the vessel and photothrombosis was not produced. Eyes were examined at intervals during the subsequent week with ophthalmoscopy, photography, fluorescein angiography, and light microscopy. RESULTS: In 13 (41%) of 32 eyes with photothrombosis, bullous retinal detachments developed 1 day after laser treatment, and continued occlusion of the vein was confirmed with fluorescein angiography. Detachments persisted for 2 to 4 days and spontaneously resolved; resolution coincided with restored venous patency at 5 to 7 days. None of the 20 control eyes developed bullous detachments (P < .005). CONCLUSIONS: Laser photothrombosis in the rat offers a simple and accessible model of transient exudative retinal detachment without the need for exogenous chromophores.

Effect of intravitreal dexamethasone in treatment of pneumococcal endophthalmitis in rabbits.

PURPOSE: To investigate whether corticosteroid therapy would decrease the inflammation and tissue damage associated with pneumococcal endophthalmitis. METHODS: Albino rabbits were injected intravitreally with 1000 live organisms of Streptococcus pneumoniae and randomized after 24 hours to treatment with intravitreal vancomycin hydrochloride alone (n = 10), combination intravitreal vancomycin and intravitreal dexamethasone (n = 10), or no treatment (n = 10). After 2 weeks, the eyes were examined clinically and enucleated for histopathologic examination. RESULTS: Eyes treated with vancomycin and dexamethasone had significantly less intraocular inflammation and more preservation of retinal tissue than untreated eyes or eyes treated with vancomycin alone (P < .05, Fisher's exact test). Untreated and vancomycin-treated eyes were indistinguishable on clinical and histologic examination. Marked anterior and posterior segment inflammation with total retinal necrosis was noted in eyes from both groups. CONCLUSION: Intravitreal corticosteroid therapy may play an important role in minimizing the inflammation and tissue damage associated with pneumococcal endophthalmitis.

The role of vitrectomy in the treatment of postoperative bacterial endophthalmitis. An experimental study.

While the diagnostic value of vitreous culture in the management of bacterial endophthalmitis is well established, the therapeutic value of vitrectomy in this condition is debated. The present experimental study uses an aphakic model of Staphylococcus aureus endophthalmitis in the rabbit. Animals were treated with the following: (1) intravitreal antibiotics alone; (2) intravitreal antibiotics with vitrectomy; (3) vitrectomy alone; and (4) no treatment. Eyes treated with antibiotics and vitrectomy displayed significantly clearer media at 14 days after therapy compared with eyes treated with antibiotics alone. There was also a greater tendency for eyes treated with antibiotics and vitrectomy to have negative cultures at 14 days, although this difference was not statistically significant. These findings are consistent with beneficial effects of therapeutic vitrectomy as an adjunct to intravitreal antibiotic therapy in an animal model of aphakic bacterial endophthalmitis.

Experimental drusen formation induced by intravitreal aminoglycoside injection.

A single intravitreal injection of aminoglycoside antibiotics in the rabbit induces changes in the retinal pigment epithelial layer that consist of disseminated yellow-white dots that are apparent six to ten months after injection. With fluorescein angiography, these dots have the characteristics of window defects or drusen. Histologic examination disclosed subepithelial amorphous material that stained positively with both periodic acid-Schiff and oil red O. Ultrastructural examination disclosed lipidic inclusions in the retinal pigment epithelial cells, basally directed cellular evagination, and basal accumulation of granular material, findings consistent with the reported morphologic features of hard drusen. These findings suggest that aminoglycoside-induced lesions may represent a model for retinal pigment epithelial degeneration and drusen formation.