Factors affecting sedimentational separation of bacteria from blood.

Rapid diagnosis of blood infections requires fast and efficient separation of bacteria from blood. We have developed spinning hollow disks that separate bacteria from blood cells via the differences in sedimentation velocities of these particles. Factors affecting separation included the spinning speed and duration, and disk size. These factors were varied in dozens of experiments for which the volume of separated plasma, and the concentration of bacteria and red blood cells (RBCs) in separated plasma were measured. Data were correlated by a parameter of characteristic sedimentation length, which is the distance that an idealized RBC would travel during the entire spin. Results show that characteristic sedimentation length of 20 to 25 mm produces an optimal separation and collection of bacteria in plasma. This corresponds to spinning a 12-cm-diameter disk at 3,000 rpm for 13 s. Following the spin, a careful deceleration preserves the separation of cells from plasma and provides a bacterial recovery of about 61 ± 5%.

Rapid separation of very low concentrations of bacteria from blood.

A rapid and accurate diagnosis of the species and antibiotic resistance of bacteria in septic blood is vital to increase survival rates of patients with bloodstream infections, particularly those with carbapenem-resistant enterobacteriaceae (CRE) infections. The extremely low levels in blood (1 to 100CFU/ml) make rapid diagnosis difficult. In this study, very low concentrations of bacteria (6 to 200CFU/ml) were separated from 7ml of whole blood using rapid sedimentation in a spinning hollow disk that separated plasma from red and white cells, leaving most of the bacteria suspended in the plasma. Following less than a minute of spinning, the disk was slowed, the plasma was recovered, and the bacteria were isolated by vacuum filtration. The filters were grown on nutrient plates to determine the number of bacteria recovered from the blood. Experiments were done without red blood cell (RBC) lysis and with RBC lysis in the recovered plasma. While there was scatter in the data from blood with low bacterial concentrations, the mean average recovery was 69%. The gender of the blood donor made no statistical difference in bacterial recovery. These results show that this rapid technique recovers a significant amount of bacteria from blood containing clinically relevant low levels of bacteria, producing the bacteria in minutes. These bacteria could subsequently be identified by molecular techniques to quickly identify the infectious organism and its resistance profile, thus greatly reducing the time needed to correctly diagnose and treat a blood infection.

Rapid separation of bacteria from blood - Chemical aspects.

To rapidly diagnose infectious organisms causing blood sepsis, bacteria must be rapidly separated from blood, a very difficult process considering that concentrations of bacteria are many orders of magnitude lower than concentrations of blood cells. We have successfully separated bacteria from red and white blood cells using a sedimentation process in which the separation is driven by differences in density and size. Seven mL of whole human blood spiked with bacteria is placed in a 12-cm hollow disk and spun at 3000rpm for 1min. The red and white cells sediment more than 30-fold faster than bacteria, leaving much of the bacteria in the plasma. When the disk is slowly decelerated, the plasma flows to a collection site and the red and white cells are trapped in the disk. Analysis of the recovered plasma shows that about 36% of the bacteria is recovered in the plasma. The plasma is not perfectly clear of red blood cells, but about 94% have been removed. This paper describes the effects of various chemical aspects of this process, including the influence of anticoagulant chemistry on the separation efficiency and the use of wetting agents and platelet aggregators that may influence the bacterial recovery. In a clinical scenario, the recovered bacteria can be subsequently analyzed to determine their species and resistance to various antibiotics.

Macular microholes: pathogenesis and natural history.

AIMS: To study the natural history and evaluate optical coherence tomography (OCT) and the retinal thickness analyser (RTA) in patients with macular microholes. METHODS: The medical records of 22 patients with a well demarcated red intraretinal foveal or juxtafoveal defect were reviewed. Fluorescein angiography (FA), RTA, and OCT were performed. The main outcome measures were visual acuity (VA), and OCT and RTA characteristics of microholes. Long term follow up was available in 13 eyes of 12 patients. RESULTS: The patients had a mean age of 50 years and a mean refractive error of -0.93 dioptres. The presenting symptom was a central scotoma in 14 eyes and metamorphopsia in eight eyes. All patients had a corrected VA ranging from 20/16 to 20/125, with 20 out of 24 eyes (83%) having a VA > or =20/40. Symptoms remained stable or improved in 16 out of 22 patients (72%). OCT 2 findings were normal but an abnormality of the outer retina and/or a defect of the retinal pigment epithelium (RPE) were demonstrated on OCT 3 in 15 of 18 eyes (83%). The RTA topographic map demonstrated a defect at the site of the microhole in two out of 12 eyes. CONCLUSION: Although biomicroscopic examination suggested an inner foveal defect, the OCT 3 scans demonstrated a localised abnormality of the outer retina and/or RPE which could not be resolved using OCT 2. Macular microholes have a favourable long term prognosis with stable VA. Bilateral involvement is uncommon.

Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures.

We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV membranes. Furthermore, whereas SV2 is localized on the membranes of the LDCVs, chromogranin A, an acidic protein in secretory granules, is clearly in the core of the LDCVs. This is the first demonstration of these two antigens in such dose (approximately 20 nm) yet distinct compartments within a single organelle.

Watzke-Allen slit beam test in macular holes confirmed by optical coherence tomography.

OBJECTIVE: To examine the role, validity, and interpretation of Watzke-Allen slit beam testing in patients with idiopathic senile macular holes. METHODS: Thirty-seven consecutive patients with 40 full-thickness macular holes, confirmed on optical coherence tomography, were prospectively recruited. The Watzke-Allen slit beam test was used centrally and on the rim of the macular hole in both vertical and horizontal orientations. RESULTS: In 24 eyes, the beam was reported as thinned in both vertical and horizontal orientations when placed directly over the center of the macular hole. In 9 eyes, the Watzke-Allen slit was reported as broken in both vertical and horizontal orientations. In 6 eyes, the beam was reported as broken in one orientation and thinned in the other. In 1 eye, the beam was reported as kinked but not thinned or broken. When the beam was placed on the edge of the macular hole, all patients reported a displacement or bowing of the beam away from the center of the hole. CONCLUSIONS: These findings confirm tangential traction of photoreceptors from a central foveal dehiscence as the causative mechanism in the development of the majority of macular holes. Careful interpretation of the Watzke-Allen sign may offer a technique for preoperatively determining visual prognosis. Arch Ophthalmol. 2000;118:1059-1063

Expression of tyrosine hydroxylase in an immortalized human fetal astrocyte cell line; in vitro characterization and engraftment into the rodent striatum.

The use of primary human fetal tissue in the treatment of neurodegenerative disorders, while promising, faces several difficult technical and ethical issues. An alternative approach that would obviate these problems would be to use immortalized cell lines of human fetal central nervous system origin. An immortalized human fetal astrocyte cell line (SVG) has been established (45) and herein we describe the in vitro and in vivo characteristics of this cell line which suggest that it may be a useful vehicle for neural transplantation. The SVG cell line is vimentin, GFAP, Thy 1.1 and MHC class I positive, and negative for neurofilament and neuron specific enolase, consistent with its glial origin. To determine whether the cell line could be used as a drug delivery system, a cDNA expression vector for tyrosine hydroxylase was constructed (phTH/Neo) and stably expressed in the SVG cells for over 18 months as demonstrated by immunohistochemistry and Western blotting of the stable transfectants. HPLC analysis of the supernatant from these cells, termed SVG-TH, consistently found 4-6 pmol/ml/min of l-dopa produced with the addition of BH4 to the media. Furthermore, in cocultivation experiments with hNT neurons, PC-12 cells and primary rat fetal mesencephalic tissue, both the SVG and SVG-TH cells demonstrated neurotrophic potential, suggesting that they constituitively express factors with neuroregenerative potential. To determine the viability of these cells in vivo, SVG-TH cells were grafted into the striatum of Sprague-Dawley rats and followed over time. A panel of antibodies was used to unequivocally differentiate the engrafted cells from the host parenchyma, including antibodies to: SV40 large T antigen (expressed in the SVG-TH cells), human and rat MHC class 1, vimentin, GFAP, and tyrosine hydroxylase. While the graft was easily identified with the first week, over the course of a four week period of time the engrafted cells decreased in number. Concomittantly, rat CD4 and CD8 expression in the vicinity of the graft increased, consistent with xenograft rejection. When the SVG-TH cells were grafted to the lesioned striatum of a 6-hydroxydopamine lesioned rats, rotational behavior of the rat decreased as much as 80% initially, then slowly returned to baseline over the next four weeks, parallelling graft rejection. Thus, the SVG-TH cells can induce a functional recovery in an animal model of Parkinson's disease, however as a xenograft, the SVG cells are recognized by the immune system.

Primary rhegmatogenous retinal detachment: 20 years of change.

AIM: To compare characteristics, management, and outcome of two groups of patients with primary rhegmatogenous retinal detachment (RRD) presenting to the same vitreoretinal unit approximately 20 years apart. METHODS: 124 patients in 1979-80 and 126 cases in 1999 were compared. RESULTS: More cases were pseudophakic and fewer aphakic in 1999 than 1979-80. More cases of giant retinal tear and fewer dialyses were operated on in 1999. Vitrectomy was a primary procedure in 63% of cases in 1999 but only 1% in 1979-80. Anatomical success rates were statistically similar: 79.8% primary and 88.8% final success in 1979-80, and 84% primary and 93.6% final success in 1999. CONCLUSION: Surgical management of primary RRD has changed greatly in 20 years. Success rates have changed little, despite availability of differing surgical techniques.

Management of inferior retinal breaks during pars plana vitrectomy for retinal detachment.

AIMS: To determine whether it is necessary to support inferior retinal breaks with a scleral explant during pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment (RD). METHODS: A prospective study was carried out on nine eyes of nine consecutive patients undergoing PPV for primary RD with associated inferior retinal breaks and no significant proliferative vitreoretinopathy. RESULTS: Eight eyes were successfully reattached with a single operation. No cases presented with redetachment because of failed closure of the original inferior breaks. CONCLUSIONS: It is not necessary to support inferior retinal breaks with a scleral explant during PPV for primary RD repair in selected cases.

Optical coherence tomography of the vitreoretinal interface in macular hole formation.

AIMS: To image the vitreoretinal interface and provide further information on the pathogenesis of idiopathic macular hole formation. METHODS: Prospective recruitment of 80 eyes of 41 consecutive patients referred with a diagnosis of idiopathic full thickness macular hole (FTMH) to a teaching hospital retinal clinic. Both eyes of each patient underwent optical coherence tomography (OCT) imaging with vertical and horizontal scans centred on the fovea. RESULTS: A total of 30 eyes had stage 2 or 3 FTMHs and, of these, 21 had persistent vitreofoveal attachment and associated prefoveal opacities. 18 prefoveal opacities were identified by Goldmann contact lens examination and confirmed on OCT examination. Three prefoveal opacities were identified only on OCT examination. 10 eyes had stage 4 FTMHs and four cases were identified in whom the OCT appearance was consistent with impending, aborted, or lamellar macular holes. CONCLUSIONS: The wide range in OCT appearance of macular holes and associated prefoveal opacities suggests that, in at least some cases, a significant amount of retinal tissue is torn from the foveal area during macular hole formation. OCT imaging provides additional information on macular hole pathogenesis and is valuable in the planning of surgical intervention.

Acute posterior vitreous detachment: the predictive value of vitreous pigment and symptomatology.

AIM: To establish whether the presence of a retinal break can be predicted either by the presence of a positive Shafer's sign (pigment granules in the anterior vitreous) or symptomatology in patients presenting with an acute posterior vitreous detachment (PVD). METHODS: 200 eyes of 200 phakic patients with a symptomatic PVD of less than 1 month's duration underwent documentation of symptomatology and examination of the anterior vitreous for the presence of pigment granules. Indentation ophthalmoscopy was then carried out by an experienced vitreoretinal surgeon with no knowledge of the symptomatology or anterior vitreous gel examination findings. A second prospective group of 115 consecutive patients were assessed in a similar manner before primary rhegmatogenous retinal detachment repair. RESULTS: In 200 eyes presenting with an acute PVD, 25 were found to have an associated retinal break, 23 of which were also Shafer positive. In 115 eyes presenting for retinal detachment repair, 111 had an associated PVD and were found to be Shafer positive. Symptomatology was not predictive of an associated retinal break in the PVD group or in those presenting with a retinal detachment. CONCLUSION: The increased use of Shafer's sign is recommended as a valuable aid in determining which patients require urgent referral for an expert retinal examination. It is not possible to predict those patients with a retinal break secondary to PVD on the basis of symptomatology alone.

Phacoemulsification and combined management of intraocular silicone oil.

We report two techniques in which cataract extraction is carried out by phacoemulsification through a corneal incision. If silicone oil is to be removed, a posterior continuous curvilinear capsulorhexis is performed, allowing aspiration of oil through the capsulorhexis followed by lens implantation into the remaining capsular bag. If silicone oil is to be left in situ, the remaining capsule is removed, converting the cataract extraction to an intracapsular-type procedure that retains the advantages of small incision surgery. These techniques offer good improvement in vision and retinal view with more rapid rehabilitation than conventional large incision surgery. The 20% retinal redetachment is comparable to that of other studies and we have not encountered any other significant complications with these techniques.

Electron microscopy study of explanted intraocular lenses from clinically noninfected eyes.

PURPOSE: To determine whether intraocular lenses (IOLs) in clinically noninfected eyes are coated with a significant, bacteria-containing biofilm. SETTING: The Oxford Eye Hospital, Oxford, United Kingdom. METHODS: Twenty-six IOLs, removed for reasons other than endophthalmitis from 26 patients attending the Oxford Eye Hospital over a 3 year period, were examined by electron microscopy. Immediately following explantation, the IOL was placed in glutaraldehyde 4% in 0.1 M phosphate buffer solution and processed for scanning electron microscopy (SEM). Areas of interest were reprocessed for transmission electron microscopy (TEM). RESULTS: There was no evidence of a bacterial biofilm on any IOL. In 5 IOLs, significant host cellular debris was seen at the tip of the haptic or at the optic-haptic junction. In 4 of them, clusters of coccoid-shaped structures were seen at the optic-haptic junction on SEM, but examination by TEM showed these structures to be melanosomes, not bacteria. CONCLUSION: We found no evidence to suggest that a significant number of IOLs are coated with a bacterial biofilm in clinically noninfected cases. We advocate the use of TEM to distinguish between coccoid bacteria and melanosomes.

Imaging beta cell regeneration and interactions with islet vasculature in transparent adult zebrafish.

Blood vessel networks provide nutrients and gaseous exchange that are essential for functions. Pancreatic islet capillaries deliver oxygen to endocrine cells while transporting hormones to organs and peripheral locations throughout the body. We have developed a zebrafish diabetes model in which adult islets can be followed in vivo during beta cell regeneration while calibrating changes in beta cell mass and fasting blood glucose levels. After genetic ablation, beta cells are initially dysfunctional or dying, and blood glucose levels increase fourfold. During a 2-week period, hyperglycemia eventually normalizes as beta cell mass regenerates. We show that mCherry-fluorescent, insulin-positive beta cells re-emerge in close contact with the vascular endothelium. Alterations in the dense vascular network of zebrafish islets were visualized by the expression of green fluorescent protein (GFP) in endothelial cells derived from the Fli transcription factor promoter. The rapid destruction and regeneration of beta cell mass was evaluated in the same animal over time, providing a functional model for investigating the interactions of islet cell types with vascular cells as well as the consequences of hyperglycemia on other tissues. Regenerating adult zebrafish can be utilized as vertebrate, metabolically active models for generating new insights into treatments for type 2 diabetes.

Trends in rates of retinal surgery in England from 1968 to 2004: studies of hospital statistics.

AIM: To describe trends over time and geographical variation in rates of vitreo-retinal surgery in England from 1968 to 2004. METHODS: Routinely collected hospital statistics were analysed for England, using the Hospital In-patient Enquiry and Hospital Episode Statistics from 1968 to 2004, and for the Oxford National Health Service Region using the Oxford Record Linkage Study from 1963 to 2004. RESULTS: Annual admission rates in England for surgery were about 5 episodes per 100 000 population in the 1960s, rising gradually to about 10 per 100 000 in the early 1990s, and then more sharply to 30 by 2004. The Oxford Record Linkage Study shows that multiple admissions per person were rare, which confirms that the observed increase represents a real increase in the number of people treated. Annual rates in England for buckle procedures declined to about 6 episodes per 100 000 population in 2004, whereas vitrectomy surgery rose to about 26 episodes per 100 000 population in 2004. CONCLUSION: Vitreo-retinal surgery has developed over the last 40 years in England, rapidly so over the past 15 years. Vitrectomy surgery has become much more common in England and buckle surgery has shown a steady decline. We demonstrate statistically significant geographical variation in the current annual rate of surgery between local authorities in England.

Feasibility of day-case vitreoretinal surgery.

PURPOSE: The indications for vitreoretinal (VR) surgery are increasing as equipment and techniques available improve. In order to decrease demand on limited health resources, day-case surgery would be beneficial in many cases. This study combines a retrospective and prospective arm to examine the feasibility and safety of routine day-case VR surgery. METHODS: One hundred consecutive patients (50 retrospective and 50 prospective) undergoing VR surgery within the Royal Berkshire NHS trust were included. The retrospective arm aimed to identify the frequency and type of acute ophthalmic or medical intervention during postop overnight stay and the results were used to alter management in the prospective group. The prospective group consisted of patients undergoing a mixture of overnight stay and day-case surgery. All patients in the prospective group had routine subtenon marcaine anaesthesia together with prophylactic pre-operative intravenous acetazolamide. Patients deemed fit postoperatively were offered overnight ward discharge, with obligatory next-day review. RESULTS: In the retrospective arm, 56% required oral nonsteroidal analgesia on the day of surgery and one patient required narcotic analgesia. Twenty-two per cent patients required intraocular pressure (IOP) control on the day of surgery and one patient required medical intervention in the form of urinary catheterisation. Nineteen patients required intervention on next-day review. In the prospective arm, 86% preferred day case and were suitable, 6% were suitable for day-case but preferred overnight stay and 8% were deemed not fit for discharge. No patient required narcotic analgesics. No patient discharged returned as a casualty overnight. Only one patient required topical beta-blocker for the control of IOP on next-day review. CONCLUSIONS: These data suggest that many patients who are hospitalised overnight for VR surgery could be safely treated as day cases. Such a shift in the pattern of care for VR surgery could provide a significant improvement in health-care efficiency and minimise patient inconvenience.

A comparative study of the efficacy of 2.5% phenylephrine and 10% phenylephrine in pre-operative mydriasis for routine cataract surgery.

It is common practice in many ophthalmic units to administer multiple applications of 10% phenylephrine in combination with an anti-cholinergic agent to ensure adequate pupil mydriasis prior to routine cataract surgery. Phenylephrine is a pure alpha-1 adrenoreceptor agonist known to produce marked systemic vasoconstriction and associated hypertension with occasional profound reflex bradycardia. Many reviews have suggested caution in the use of 10% phenylephrine in the elderly or hypertensive patient. In a prospective, randomised trial we have assessed pupil dilation comparing the efficacy of 10% phenylephrine (53 patients) versus 2.5% phenylephrine (62 patients). When administered in conjunction with 1% cyclopentolate four times over 1 hour pre-operatively, 2.5% phenylephrine was found to be as effective as 10% phenylephrine in the initiation and maintenance of mydriasis during both extracapsular and phacoemulsification cataract extraction.

Optimisation of outpatient resource utilisation in cataract management.

PURPOSE: We assessed whether patients referred with a diagnosis of cataract require outpatient assessment before listing for surgery or whether the general practitioner could have direct access to the waiting list. We also studied whether pre-assessment clinics made a significant difference to management even when waiting times were long. METHODS: Data about patients referred with a diagnosis of cataract to the Oxford Eye Hospital and associated hospitals were collected. Seventy-five patients were prospectively studied and 100 patient records were retrospectively analysed. RESULTS: Twenty-six per cent of patients had a misdiagnosis or additional problems affecting management. Optometrists provided more information than general practitioners, but their diagnostic accuracy was equal (73% optometrists, 75% general practitioners). At the pre-assessment clinic pupil dilatation changed management in only 4% of patients. CONCLUSION: Listing patients on the basis of referral letters would be inappropriate in 1 in 4 patients. Pre-assessment clinics rarely picked up a clinically relevant change. Thus if outpatient consultation included a decision on the exact surgical plan including implant power, then pre-assessment clinics may not be necessary.