The genotypic characterisation of Acanthamoeba isolates from human ocular samples.

BACKGROUND: We characterised 37 amoebae cultured from corneal scrapings, contact lenses or lens case solutions of patients with suspected Acanthamoeba keratitis. METHODS: The isolates were identified by their morphology and by PCR targeting the Acanthamoeba nuclear small-subunit rRNA gene. Acanthamoeba isolates were genotyped by DNA sequence analysis. RESULTS: The 37 isolates comprised 35 Acanthamoeba, one Hartmannella and one Vahlkampfia. Ten Acanthamoeba isolates were shown to be responsible for keratitis. CONCLUSION: Genotype T4 was the only Acanthamoeba genotype responsible for keratitis in this study, and represented 79% of non-pathogenic isolates.

[Usefulness of voriconazole in treatment of Phoma glomerata after penetrating injury]. / Endophtalmie à Phoma glomerata après plaie du globe et efficacité du traitement par voriconazole en intravitréen.

We report the first case of endophthalmitis caused by Phoma glomerata. A 32-year-old man who underwent retinal detachment surgery consecutive to a penetrating globe injury presented with endophthalmitis 7 days after surgery. Anterior chamber tap and intravitreal injection of antibiotics (ceftazidime and vancomycin) were performed systematically. Fungus was observed at microscopic examination of the aqueous humor and treatment with intravitreal injection of amphotericin B was decided. The patient failed to improve with intravitreal amphotericin B but responded clinically to intravitreal voriconazole. The fungus was identified after culture as Phoma glomerata. The MIC for amphotericin B was 1microg/ml, for caspofungin was 2microg/ml, and for itraconazole was 8microg/ml or more. The MIC for voriconazole was up to 8microg/ml. The clinical response after intravitreal injection may be related to the high concentrations reached in the vitreous. Because of severity and ominous prognosis of intraocular fungal infections and posttraumatic Phoma ocular infections, aggressive management is required by intravitreal voriconazole administration.

Comparison of PCR, microscopic examination and culture for the early diagnosis and characterization of Acanthamoeba isolates from ocular infections.

In the study presented here, PCR, microscopic examination and culture of corneal samples were compared as methods of confirming the clinical diagnosis of Acanthamoeba keratitis, a serious ocular infection that is difficult to diagnose and threatens eyesight. The three methods were applied to isolates obtained from 513 patients with clinical signs or risk factors suggesting Acanthamoeba infection. Acanthamoeba keratitis was diagnosed in 12 of these patients. Combined PCR assays were more sensitive (94%) than either microscopic examination (33%) or culture (7%). The Acanthamoeba isolates were characterized using DNA sequence analysis of the nuclear small-subunit rRNA gene, and T4 was the predominant genotype found.

[Diagnosis of Acanthamoeba spp. keratitis with PCR]. / Diagnostic par PCR des kératites à Acanthamoeba spp.

AIM: Evaluation of a PCR assay as a diagnostic tool for detection of Acanthamoeba spp. in patients presenting infectious keratitis. METHODS: Between August 2001 and November 2002, 342 clinical specimens consisting in corneal scrapings from 334 patients were tested for Acanthamoeba using direct microscopy, culture, and PCR. A fragment of Acanthamoeba 18S rRNA gene was amplified using a set of primers referred to as Nelson's primers. RESULTS: A diagnosis of Acanthamoeba keratitis was considered for nine patients. Amoeba growth in culture was unfruitful for all of these cases. Eight patients had corneal scrapings that tested positive with PCR; in two cases direct microscopy observations confirmed PCR results. For one patient, a negative PCR result was obtained; however, a second corneal sample and cysts staining on May-Grünwald-Giemsa were positive. A false-positive PCR result was noted related to another amebic genus. A risk factor was found in all Acanthamoeba keratitis cases (contact lenses, trauma). Topical treatment was successful, and keratoplasty was necessary afterwards for optical rehabilitation in five patients. CONCLUSION: This study suggests that PCR is a sensitive diagnostic tool, superior to conventional techniques for detecting Acanthamoeba in corneal lesions.

[Proteomic analysis associating two-dimensional electrophoresis and mass spectrometry to identify lacrimal proteins: a case study]. / Apport de l'analyse protéomique associant électrophorèse bi-dimensionnelle et spectrométrie de masse en lacrymologie.

PURPOSE: Identification of a lacrimal protein by proteomic analysis, i.e., two-dimensional electrophoresis and mass spectrometry. MATERIAL AND METHODS: We studied the tears of a 25-year-old female with adrenal gland hyperplasia and hyperandrogenism complaining of chronic dryness and mild bilateral papillary hypertrophy. An allergologic workup was negative. Agarose electrophoresis of the tears showed a bilateral high level of rapid migrated proteins. RESULTS: Dodecyl sulfate polyacrylamide gel electrophoresis of the tears from both eyes showed a highly stained 15-kDa band after Coomassie colloidal blue coloration compared to controls. On two-dimensional electrophoresis, this band focused on a single spot at pI 7.0. After tryptic digestion in gel, peptide mass fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry provided clear identification of cystatin SN. It is known that mRNA regulated by androgens and encoding glycoproteins homologous to human cystatin exists in the rat lacrimal gland. CONCLUSION: We conclude that the hyperandrogenism of the patient may be cause for the hypersecretion of this cystatin SN, giving an explanation for the high level of rapid migrated proteins (lipocalins). This result provides a concrete example of the proteomic tool used to identify lacrimal proteins, still largely unknown.

[Clinical management and prognosis in Acanthamoeba keratitis: a retrospective study of 25 cases]. / Prise en charge clinique et pronostic des kératites amibiennes. Etude rétrospective à propos de 25cas.

PURPOSE: To assess risk factors, clinical characteristics, diagnosis and outcome in patients with Acanthamoeba keratitis. METHODS: We reviewed 23 patients (25 eyes) treated for Acanthamoeba keratitis in the XV-XX Hospital between November 1994 and October 2000. Acanthamoeba were found either in corneal scrapings and/or contact lenses and/or storage solutions. RESULTS: A predisposing factor was found in 22/23 patients: most of them (20/22) were contact lens (CL) wearers, two patients had a recent history of corneal trauma, and 15/23 patients had been treated for presumed herpetic keratitis before examination in our hospital. The average delay between first symptoms and diagnosis was 2 months. All patients were treated with two or three topical antiparasitic eye drops for 2 to 6 months. Five patients received systemic treatment (ketoconazole or itraconazole). Nine eyes received surgical treatment: penetrating keratoplasty in eight cases, conjunctival flap in one case, enucleation in one case. Visual outcome was poor in ten eyes (less than 20/60) because of delayed diagnosis. CONCLUSION: Acanthamoeba keratitis is a serious vision-threatening disease. Early diagnosis and treatment are essential for improving visual outcome. Methods such as confocal microscopy and Acanthamoeba-PCR, allowing earlier diagnosis and treatment, will improve the management of Acanthamoeba keratitis.

Psychoneuroendocrine outcomes of short-term crowding stress.

One-week exposure to crowding stress has been reported to affect body weight and pituitary-adrenal axis activity. This study has first evaluated whether the aforementioned changes are associated with altered exploratory activity. One-week crowding (16 rats/cage) diminished body weight gains, compared to the control situation (four rats/cage): actually, this difference was mainly due to a marked loss in food intake and body weight following the first day of crowding. On the other hand, 1-week crowded rats were not different from their controls when placed for the first time in an open field, as shown by the analysis of their exploratory activities, their number of grooming episodes, and their defecation. Psychoneuroendocrine effects of 1-day crowding were then investigated: actually, open field behaviors were not modified but basal plasma glucose levels were decreased. Moreover, neither plasma ACTH and corticosterone levels nor 2-min cold swim-induced increases in corticosterone levels were affected. Besides, cold swim-induced increases in plasma ACTH levels were amplified in crowded rats, and these increases were associated with hyperglycemia in crowded, but not in control rats. It is suggested that 1-day exposure to crowding has differential consequences upon feeding and exploratory behaviors while potentiating the responsiveness of stress hormones.

Effects of cold stress on some 5-HT1A, 5-HT1C and 5-HT2 receptor-mediated responses.

The purpose of the present study was to analyze the influence of stress (24-h cold exposure) on presynaptic 5-HT1A receptors, and on postsynaptic 5-HT1A, 5-HT1C and 5-HT2 receptors. Cold exposure for 24 h affected neither pargyline-induced decreases in 5-hydroxyindoleacetic acid (5-HIAA) levels in midbrain and rest of brain, nor plasma glucose and corticosterone levels. Treatment with the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.5-1 mg/kg), 3-5 h after the end of cold exposure triggered less intense flat body posture and forepaw treading in cold-exposed rats than in controls. On the other hand, 15- and 30-min plasma glucose responses to 8-OH-DPAT (0.25-0.5 mg/kg, 3-5 h after cold) or to the alpha 2-adrenoceptor agonist, clonidine (0.025 mg/kg), were not affected by cold, while the 15-min, but not the 30 min, plasma corticosterone response to 8-OH-DPAT was slightly amplified in cold-exposed rats. Cold exposure affected neither the inhibitory effect of 8-OH-DPAT (0.25-0.5 mg/kg, 3-5 h after cold) on midbrain 5-HIAA levels, nor the hypothermic effect of 8-OH-DPAT (0.5-1 mg/kg, 3-5 h after cold). Lastly, the hypoactivity elicited by the 5-HT1C receptor agonist, m-chlorophenyl-piperazine (1.5-3 mg/kg, 3-5 h after cold), or head shakes elicited by the 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (1-2 mg/kg, 3-5 h after cold), were of similar intensities in control and in cold-exposed rats.

Oxidative stress in ethanol intoxicated rats.

The present work has investigated the correlation between short-term ethanol administration and the free radicals production in rat liver. The non-protein-SH and lipid peroxides contents, superoxide dismutase and glutathione peroxidase activities were investigated. Increased lipid peroxides concentration, decreased glutathione content, significant increase in superoxide dismutase activity and a decrease in glutathione peroxidase activity were observed.

Redox changes in insulin-dependent diabetes mellitus.

The present work evaluates some aspects of the redox potential in individuals with insulin-dependent diabetes mellitus (IDDM). The plasma lipid peroxides level and the concentration of glutathione (GSH) were measured. The data obtained revealed a rise in lipoperoxides concentration and a decrease in GSH content suggesting that oxygen derived free radicals may play a role in the pathogenesis of IDDM.