A one-step multiplex PCR for acanthamoeba keratitis diagnosis and quality samples control.

PURPOSE: As the number of cases of Acanthamoeba spp. keratitis (AK) is constantly growing, new diagnostic tools are needed to confirm and guide ophthalmologists in this clinically problematic diagnosis. Molecular diagnosis is particularly well adapted, although only a few real-time PCR techniques have been described recently. The aim of this study was to develop a new PCR technique for the diagnosis of AK by combining the detection of Acanthamoeba DNA with human DNA, thus allowing an accurate interpretation of the PCR result. METHODS: Different DNA extraction procedures were compared to ensure an optimized amplification of one Acanthamoeba genome. The analytical parameters of this new multiplex Acanthamoeba beta-globin PCR (MAB-PCR) were evaluated. Fourteen eye drops were tested as potential PCR inhibitors. A prospective series of 28 corneal scrapings was subjected to MAB-PCR. RESULTS: The best extraction procedure associated thermal-shock pretreatment followed by a manual extraction procedure. The MAB-PCR parameters displayed excellent specificity and sensitivity, with a detection of 0.02 genome of Acanthamoeba. No eye drops were total PCR inhibitors. Of 28 corneal scrapings, 18 were considered true negatives. Seven could not be interpreted because of insufficient scraping material. Three were considered true positives: every patient progressed favorably on specific and reliable treatment. CONCLUSIONS: The MAB-PCR is a new tool to diagnose AK. It allows rapid diagnosis and prompt treatment of this probably underestimated etiology of infectious keratitis. This optimized real-time PCR outperforms the gold standard for Acanthamoeba keratitis diagnosis and it allows a concomitant evaluation of the quality of the corneal scraping, which is necessary for a precise interpretation of the results.

Choroidal blood flow regulation after posture change or isometric exercise in men with obstructive sleep apnea syndrome.

PURPOSE: Obstructive sleep apnea (OSA) syndrome generates hypertension, atherosclerosis, and endothelial and autonomic dysfunction, which may mutually interact with ocular vascular regulation. Exercise and posture changes can be used to manipulate blood pressure, ocular perfusion pressure (OPP), or both. It was hypothesized that choroidal vascular reactivity in response to isometric exercise and posture changes could be altered in OSA patients. METHODS: Healthy men were matched 1:1 for body mass index, sex, and age with patients with newly diagnosed OSA without cardiovascular comorbidities. All subjects underwent sleep studies and cardiovascular phenotyping (24-hour blood pressure monitoring, arterial stiffness measurements, and cardiac and carotid echography). Choroidal reactivity was assessed by laser Doppler flowmetry, which measured subfoveal choroidal blood flow. RESULTS: During exercise, blood pressure parameters increased significantly within the same range, with a similar profile over time in OSA patients and control subjects. A significant linear relationship (P = 0.0003) was noted between choroidal vascular resistance and the OPP changes during exercise in OSA patients and control subjects. From the sitting to the supine position, a significant decrease in mean arterial pressure occurred in both groups (10.9%-13.4%; P < 0.001). In both populations, no significant change in choroidal blood flow or vascular resistance was found during the posture change. Choroidal blood flow responses to exercise and posture changes were unchanged after 6 to 9 months of continuous positive airway pressure treatment. CONCLUSIONS: This study strongly suggests that the regulation of choroidal blood flow, which depends on the orthosympathetic and parasympathetic systems, is unaltered in men with OSA who have no comorbidities.

Choroidal blood-flow responses to hyperoxia and hypercapnia in men with obstructive sleep apnea.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) impacts on macrovasculature and autonomic function and may therefore interfere with ocular microvascular regulation. We hypothesized that choroidal vascular reactivity to hyperoxia and hypercapnia was altered in patients with OSA compared with matched control subjects and would improve after treatment with continuous positive airway pressure (CPAP). METHODS: Sixteen healthy men were matched 1:1 for body mass index, sex, and age with 16 men with newly diagnosed OSA without comorbidities. Subjects underwent sleep studies, 24-hour blood pressure monitoring, arterial stiffness measurements, and cardiac and carotid echography. Overall, patients were middle-aged, lean, and otherwise healthy except for having OSA with a limited amount of desaturation, with, at most, subclinical lesions of the cardiovascular system, stage 1 hypertension, or both. Choroidal laser Doppler flowmetry provides a unique opportunity to assess microvascular function by measuring velocity, (ChBVel), volume (ChBVol), and relative subfoveal choroidal blood flow (ChBF). Vascular choroidal reactivity was studied during hyperoxia and hypercapnia (8% CO2) challenges before and after treatment with nasal CPAP. RESULTS: Patients with OSA and control subjects exhibited similar choroidal reactivity during hyperoxia (stability of choroidal blood flow) and hypercapnia (significant increases in ChBVel of 13.5% and in ChBF of 16%). Choroidal vasoreactivity to CO2 was positively associated with arterial stiffness in patients with OSA. Gas choroidal vasoreactivity was unchanged after 6 to 9 months of CPAP treatment. CONCLUSION: This study showed unimpaired choroidal vascular reactivity in otherwise healthy men with OSA. This suggests that patients with OSA, without comorbidities, have long-term adaptive mechanisms active in ocular microcirculation.

Twenty-four hour (Nyctohemeral) rhythm of intraocular pressure and ocular perfusion pressure in normal-tension glaucoma.

PURPOSE: To characterize the nyctohemeral rhythm of intraocular pressure (IOP) and ocular perfusion pressure (OPP) in patients with newly diagnosed, untreated normal tension glaucoma (NTG). METHODS: Twenty-seven patients with suspected NTG were prospectively included and underwent 24-hour monitoring of IOP and blood pressure (BP), polysomnography, and nailfold capillaropathy. The nyctohemeral rhythms of IOP, BP, and OPP were modeled with a nonlinear least-squares, dual-harmonic regression procedure, studying the mean value, the acrophase, the nadir and the amplitude of each rhythm. Nonparametric tests were used to study the relationship between the rhythm of IOP and vascular, sleep, and visual field parameters. RESULTS: Five patients were excluded from the analysis after the 24-hour curve of IOP, (IOP > 21 mm Hg during nighttime [n = 1] or daytime [n = 4]). Twenty-two (81%) patients received a diagnosis of NTG (IOP < 22 mm Hg over 24 hours). They exhibited a diurnal acrophase (54.5%), or a nocturnal acrophase (36.4%) of IOP. The remaining patients (9.1%) with NTG had no nyctohemeral rhythm. A significantly higher proportion of patients with capillaropathy and a higher nyctohemeral fluctuation of IOP characterized the IOP group with diurnal acrophase. A rhythm of OPP was found in all patients, (diurnal [58%] or nocturnal [42%]) acrophase) equally distributed between the two groups of IOP. Amplitude of OPP was not significantly associated with the severity or progression of glaucoma. CONCLUSIONS: A nyctohemeral rhythm of IOP exists in most of the patients with NTG, either with a nocturnal acrophase or a diurnal acrophase. The rhythm of OPP did not correlate with the IOP rhythm.