Identifying Dormant Growth State of Mycobacteria by Orthogonal Analytical Approaches on a Single Cell and Ensemble Basis.

Tuberculosis is currently the single most deadly infectious disease in the world and a public health priority as defined by WHO. Although the disease is in general curable, treatment success is hampered by the necessity of a long and side effect prone treatment. Low treatment efficiency may be partly due to the special growth states that mycobacteria enter to avoid being killed by antibiotics and to persist longer within the host. Such growth states have been recently defined as dormant or persistent. We produced dormant model-organism cultures using an acidification model and characterized those by a multilayered approach using mass spectrometry (MALDI-TOF), microscopy (SEM, Raman), and microbiological techniques (CFU, OD600, ATP-levels). With a fast and 96-well-adapted extraction protocol, mycobacteria could be inactivated and extracted for MALDI-TOF analysis. For the first time, we demonstrate growth-state-dependent changes in the mass signatures of the culture, allowing for a reliable differentiation of dormant state and exponential growth. We also demonstrate resuscitation from dormant state back to exponential growth. Viable mycobacteria were immobilized, and single organisms were analyzed individually by Raman microscopy. For single-cell Raman microscopy, Mycobacterium smegmatis cultures were fixed using a new fast and gentle single-step immobilization technique on a hydrophobic glass slide. We were able to distinguish single viable bacteria in the dormant state from their rapidly growing, genetically identical counterparts, identifying the growth state of the culture based on single-organism spectra. This allows for the separation of heterogeneous cultures depending on their growth state using the destruction-free optical method of Raman microscopy.

Comparison of the bactericidal effect of cold atmospheric pressure plasma (CAPP), antimicrobial photodynamic therapy (aPDT), and polihexanide (PHX) in a novel wet surface model to mimic oral cavity application.

PURPOSE: Cold atmospheric pressure plasma (CAPP) is increasingly used for medical applications. The first devices are available from commercial manufactures, promising to improve wound healing and disinfection. The underlying antimicrobial mechanisms of CAPP are discussed, while the first results on its bactericidal efficiency against common bacterial species have already been published, with promising results. Most of the plasma sources used in these studies were built by the investigators themselves, and are not commercially available or licensed for clinical use. To evaluate the postulated bactericidal effects in clinical practice, we studied a commercially available, ready-to-use CAPP-device, which is also designed to be used in the field of dental, oral, and maxillofacial treatment. MATERIALS AND METHODS: Standardized bacterial cultures of two different pathogens (Acinetobacter baumannii and Staphylococcus aureus) were produced with defined colony-forming unit concentrations. Dilutions of these cultures were treated with a commercially available CAPP product according to the manufacturer's instructions in order to evaluate the antimicrobial activity of the technique. This in vitro study compared the CAPP treatment with established clinical therapies like polihexanide (PHX) and antimicrobial photodynamic therapy (aPDT). RESULTS: The bactericidal effect was evaluated in terms of reduction in colony-forming units after treatment of the bacterial samples with a defined dose of plasma, aPDT, or PHX. For CAPP, the bactericidal effect was found to be stronger in the Gram-negative isolate (A. baumannii) than in the Gram-positive S. aureus. A strong depth dependency was observed, especially with the Gram-negative isolate. Good bactericidal effects, with a reduction in bacterial load of more than 2 × log10, could only be observed in conditions of 0.3 mm of water-film thickness or less. Such a significant reduction in bactericidal effect depending on depth was not observed using aPDT or PHX in the studied depth range of 0.3-1.8 mm. CONCLUSION: CAPP treatment performed by the device (Plasma ONE) and configuration we used in this study seems to be ill suited for sufficiently killing Acinetobacter baumannii or Staphylococcus aureus in a moist infection site, as would be expected in the oral cavity. Established local antimicrobial therapies using PHX or aPDT showed better disinfectant properties. The clinical effect of improved wound healing, described by the manufacturer and some scientists, could not be investigated using this model. Given the results, however, it seems unlikely to be a direct consequence of bactericidal effects of CAPP in a wet environment. Further development of CAPP devices, or a different configuration (e.g. with a higher output, resulting in reactive nitrogen species-dominated, gas-phase chemistry), may enhance antibacterial effects in future, while tissue compatibility of such techniques remains to be elucidated further.

Cephalometric measurements and determination of general skull type of Cavalier King Charles Spaniels.

The general skull morphology of the head of the Cavalier King Charles Spaniel (CKCS) was examined and compared with cephalometric indices of brachycephalic, mesaticephalic, and dolichocephalic heads. Measurements were taken from computed tomography images. Defined landmarks for linear measurements of were identified using three-dimensional (3D) models. The calculated parameters of the CKCS were different from all parameters of mesaticephalic dogs but were the same as parameters from brachycephalic dogs. However, the CKCS had a wider braincase in relation to length than in other brachycephalic breeds. Studies of the etiology of the chiari-like malformation in the CKCS should therefore focus on brachycephalic control groups. As Chari-like malformation has only been reported in brachycephalic breeds, its etiology could be associated with a higher grade of brachycephaly, meaning a shorter longitudinal extension of the skull. This has been suggested for other breeds.

Efficacy of diclofenac sodium ophthalmic solution versus placebo in reducing inflammation following cataract extraction and posterior chamber lens implantation.

One hundred forty-eight patients were enrolled in a randomized, prospective, placebo-controlled clinical trial evaluating the efficacy of diclofenac sodium (Voltaren Ophthalmic) in reducing ocular inflammation following extracapsular cataract extraction with posterior chamber intraocular lens implantation. Eligible patients were enrolled and randomized (2:1 diclofenac:placebo) if the sum of anterior chamber cells plus flare one day postoperatively (baseline) was at least four. None of the patients received concomitant steroidal anti-inflammatory treatment. The 99 patients receiving diclofenac sodium had significantly greater improvement from baseline in summed flare plus cell score than the 49 placebo patients at two to five days and seven to nine days after baseline. Similarly, diclofenac sodium patients had significantly less post-baseline conjunctival erythema and ciliary flush than placebo patients. Significantly more diclofenac sodium patients than placebo patients showed moderate to marked improvement from baseline in overall assessment of inflammatory response. Forty-nine percent of placebo patients but only 17% of diclofenac patients were considered therapeutic failures (P < .001). By five to seven days, 82% of diclofenac sodium patients and 59% of placebo patients had corrected visual acuities of 20/40 or better (P < .001). There were no clinically important differences in mean intraocular pressure at any visit.

Improvement in visual acuity in chronic aphakic and pseudophakic cystoid macular edema after treatment with topical 0.5% ketorolac tromethamine.

Ketorolac tromethamine 0.5% ophthalmic solution treatment was compared to placebo treatment in 120 patients with chronic aphakic or pseudophakic cystoid macular edema (six-month or more duration of distance visual acuity of 20/40 or less and angiographic evidence of cystoid changes) during a four- to five-month double-masked, multicenter study in which patients were randomly assigned. A statistically significant improvement in distance visual acuity (two lines or more) was observed in the ketorolac-treated group as compared to the placebo-treated group after 30 days (P = .038), 60 days (P = .017), and 90 days (P = .008) of treatment. This improvement in visual acuity remained statistically significant one month after cessation of treatment (P = .001). Nine ketorolac-treated patients and two placebo-treated patients demonstrated a decrease in visual acuity one month after treatment was discontinued. Seven of the nine ketorolac-treated patients experienced an improvement in visual acuity after retreatment as compared to none of the placebo-treated patients. This study offers evidence for a more optimistic outlook in the medical treatment of chronic aphakic and pseudophakic cystoid macular edema.

Hyperopic thermokeratoplasty: clinical evaluation.

A new procedure, hyperopic thermokeratoplasty (HTK), developed in the Soviet Union for the correction of hyperopia, uses controlled thermal burns of the corneal stroma with a retractable probe tip preset to penetrate the cornea at 95% depth. The coagulations are applied in a radial pattern for spherical hyperopia. Only the peripheral cornea is treated and the effect is titrated by varying the optical zone and number of rays. The thermal effect flattens the peripheral cornea and steepens the central cornea. In this report, we prospectively evaluated the refractive results of a group of 61 HTK patients. Mean preoperative spherical equivalent was 3.9 diopters (D). Mean follow-up to date is 5.2 months, with 44% of cases evaluated at six months and 31% at one year. The initial effect of surgery (at one day) was a mean decrease in hyperopia of 6.0 D (standard error of the mean [S.E.] = 0.3 D), resulting in a mean spherical equivalent of -2.1 D (S.E. = 0.2 D). There was a steep regression of effect between one day and two months at which point average refraction was close to emmetropia. After two months, there was a gradual but continuing regression of effect, leveling off after six months. At five to six months, 63% of cases were undercorrected by at least a diopter; at 9 to 12 months, 83% of cases were undercorrected. The overall change in spherical equivalent at each time increased as optical zone size decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Endophthalmitis investigative protocol: a plan for source identification and patient protection.

A survey of randomly selected ophthalmic outpatient surgical facilities revealed a lack of established investigative protocols to follow when postoperative endophthalmitis occurs. The survey also provided information about the incidence of endophthalmitis, the microorganisms encountered, and the precautions used to prevent postoperative infections. We present an endophthalmitis investigative protocol which provides a task list, investigation forms, incidence log, and quality assurance measures to identify patients at risk and to minimize the risk of recurrence of this rare but serious complication of ophthalmic surgery. This standardized protocol will also assist in source identification when aseptic iritis occurs postoperatively. The purpose of this paper is to stimulate a dialogue which may result in an international standard for an endophthalmitis investigative protocol.

Effect of thermokeratoplasty on corneal curvature.

A cadaver eye model was used to evaluate and quantify the use of thermokeratoplasty for steepening the central cornea to correct hyperopia. Four groups of eye-bank eyes were treated with four separate surgical plans. Each plan involved the placement of controlled thermal burns (in the depths of the corneal stroma, using a cautery probe) applied in a radial pattern up to a premarked optical zone. The plans differed in the sequence of surgical steps. All plans progressively added radials and applications (to decrease optical zone) in various sequences. Corneal curvature was measured at baseline and at each surgical step. As more surgery was done within each plan, the corneas became progressively steeper. Total mean changes in corneal curvature ranged from 16.26 diopters to 19.76 diopters, depending on the plan. At each optical zone, as the number of radials increased, the effect increased. With progressively smaller optical zone size, the effect also increased.

Radial thermokeratoplasty for the correction of hyperopia.

Hyperopic thermokeratoplasty involves making controlled thermal burns in the corneal stroma in a radial pattern up to a premarked clear zone. It steepens the central cornea and reduces the hyperopia. A series of 117 radial thermokeratoplasty eyes done in the Soviet Union were analyzed. Mean preoperative hyperopia of +5.27 diopters (range = +0.50 to +17.00 D) was reduced at 12 months after surgery by a mean of -3.48 D, resulting in a mean postoperative spherical equivalent of +1.84 D. Average correction was 70.8%. Forty percent of eyes were corrected to less than 1.00 D residual refractive error; however, 58% were undercorrected by 1.00 D or more. The proportion of eyes seeing 20/40 or better unaided increased from 10% preoperatively to 52% by 3 months after surgery and remained at 53% at 12 months after surgery. Overall refractive stability was demonstrated during the 1st postoperative year. Stepwise regression showed that none of the preoperative or surgical factors significantly predicted outcome when all eyes were evaluated.

Delayed regression of effect in myopic epikeratophakia vs myopic keratomileusis for high myopia.

We compared the effectiveness and stability of our first six consecutive myopic epikeratophakia procedures (mean follow-up 18 months, range 14 to 26) with our first four consecutive cryolathe myopic keratomileusis procedures (mean follow-up 28 months, range 14 to 35). Myopic keratomileusis reduced preoperative myopia (range -8.9 to -12.1 diopters) by 77% to 94% in four patients. Refraction and visual acuity remained stable for over 1 to 3 years. In contrast, only three of the six myopic epikeratophakia procedures had stable results. The remaining three eyes which were within 0.38 D of emmetropia at the time of suture removal showed a loss of effect, resulting in myopia worse than their preoperative values in two eyes and a small correction in one eye. Only one of the six eyes achieved an uncorrected visual acuity better than 20/200. We conclude that myopic keratomileusis may be the procedure of choice for treating individuals with myopia of -8 to -15 D, because the incidence of late regression of effect after myopic epikeratophakia using the techniques in this study is unacceptable.

Advantages and limitations of current soft intraocular lenses.

We report our clinical experience with six soft intraocular lenses: (1) STAAR silicone, (2) CooperVision/Schlegel silicone, (3) IOLAB silicone, (4) Allergan Medical Optics (AMO) silicone, (5) Fyodorov silicone, and (6) Alcon hydrogel (IOGEL). We found that these soft intraocular lenses have several common design problems. The single-size design of five of the six soft lenses can lead to a windshield-wiper decentration effect in lenses too small for larger eyes. We observed several instances in which the one-piece lenses became decentered, subluxed, and vaulted anteriorly against the iris or posteriorly away from the iris as postoperative capsulozonular changes occurred. We found that the insertion methods for these soft lenses need refining to reduce the risk of intraocular damage and wound stretching if the lenses are to be folded and inserted through a small 3.0 mm to 3.5 mm incision. The surface and physical characteristics of these lenses may also require further study. We observed folding grooves, sebacium deposits, and rust from the insertion instrument on some lenses inside the postoperative eye and pigment dispersion associated with hydrogel lenses. We conclude that small diameter polymethylmethacrylate implants are the best choice for small incision cataract surgery at this time.

Enhanced accuracy is necessary for refractive surgery instrumentation.

New optical zone markers, diamond knives, and gauge blocks were ordered from a random selection of manufacturers and inspected by an independent metallurgical engineer. This study revealed workmanship flaws and poor instrument accuracy. None of the 26 optical zone markers examined came with instructions as to whether the incisions were to be carried to or from the inside diameter or the outside diameter of the corneal impression made by the marker. Two markers had a 100 microns or greater error in their centering devices and one had a centering device off center by 210 microns. Two of the seven diamond micrometer knives had broken blade tips and uneven footplates; two knives had misaligned blades and uneven footplates, one knife had extremely roughly finished footplates, and one knife had a 100 microns error in its micrometer. All the knives had a wobble in the blade shaft upon advancement and had rounded footplates, making blade extension verification difficult. Five of seven blade gauges were accurate to within 10 microns; two had errors of more than 60 microns which could lead to incisions too deep or too shallow and micro- or macroperforations of the cornea during refractive surgery. It is recommended that all refractive surgery instruments be carefully inspected under a high power micron-level microscope at the time of purchase and routinely thereafter.

Refractive evaluation of astigmatic keratotomy procedures.

We evaluated the efficacy of three transverse incision astigmatic keratotomy procedures using a vector analysis that enabled us to determine the magnitude of the effect and the axis in which it occurred. These procedures were used for low levels of astigmatism (0.5 diopter [D] to 3.75 D): (1) staggered, radial-touching transverse (T) incisions, (2) non-radial-touching T incisions, and (3) T incisions with interrupted radial. Most procedures were performed in conjunction with radial keratotomy for spherical correction of myopia. The non-radial-touching T procedure was the least effective method of reducing astigmatism. The staggered, radial-touching T and T with interrupted radial procedures were equally effective in reducing over 93% (on the average) of the preoperative astigmatism. Of these two procedures, the T with interrupted radial procedure was the most accurate, with the effect of the surgery deviating less than ten degrees from the desired axis in over 70% of the eyes.

Small incisions to control astigmatism during cataract surgery.

Astigmatic changes in three series of cataract surgical procedures were compared. Two series comprised eyes having phacoemulsification and implantation of a foldable silicone lens through a 3.0 mm to 4.0 mm incision or a 5.8 mm diameter polymethylmethacrylate (PMMA) lens through a 6.0 mm incision. The third series comprised eyes having a planned extracapsular cataract extraction (ECCE) procedure through a 10.0 mm incision and implantation of a PMMA posterior chamber lens. Surgically induced cylinder changes were compared by examining preoperative and postoperative dioptric cylinder power changes without regard to axis changes and by using vector analysis to compute induced cylinder for cases with axis changes. Both phacoemulsification series had similar mean induced cylinder levels, which were significantly less than mean induced cylinder in the ECCE group at both three and six months after surgery. Over 70% of the eyes in the two small incision phacoemulsification groups achieved an uncorrected visual acuity of 20/40 or better at three months, whereas only 28% of the ECCE group achieved that acuity. We concluded that the phacoemulsification procedure induced significantly less astigmatism and provided faster visual rehabilitation than the ECCE procedure. Furthermore, the use of small diameter PMMA IOLs inserted through small incisions minimized surgically induced cylinder in a way comparable to the use of foldable silicone implants, while maintaining good visual results with fewer postoperative complications.

Hexagonal keratotomy for correction of low hyperopia: preliminary results of a prospective study.

Fifteen sighted eyes of 11 patients had hexagonal keratotomy for the correction of low hyperopia. Mean preoperative hyperopia of +3.21 diopters (range +1.75 to +5.50) was reduced a mean of 2.16 diopters (range 0 to -3.25) and keratometry was increased a mean of +2.22 diopters (range +0.75 to +3.71). Follow-up averaged 9.5 months (range 2.0 to 17.5). Ten of the 15 eyes (67%) had at least six months follow-up. No eye had better than 20/80 uncorrected visual acuity preoperatively. Fourteen eyes (93%) had 20/80 or better uncorrected acuity at the most recent postoperative visit. Nine of the 15 eyes (60%) had uncorrected acuity of 20/40 or better. Refractive spherical equivalent appeared to stabilize by three months for most patients. Astigmatism was increased a mean of only +0.02 diopter and no serious complications occurred. Further follow-up is required to assess the safety and efficacy of hexagonal keratotomy for reducing low levels of hyperopia.

Glare disability devices for cataractous eyes: a consumer's guide.

We evaluated five commercially available glare testers to determine how accurately each device predicted outdoor Snellen acuity in cataract patients. Predicted outdoor Snellen acuity was compared to actual Snellen outdoor acuity for the Miller-Nadler glare tester, the brightness acuity tester (BAT), the InnoMed true vision analyzer (TVA), the VisTech VCT 8000, and the EyeCon 5. All five devices were evaluated in a single series of eyes. We ranked the five glare testers as follows based upon their ability to predict outdoor Snellen acuity within one Snellen line: (1) BAT (73%), (2) TVA (69%), (3) VisTech VCT 8000 (56%), (4) Miller-Nadler (47%), and (5) EyeCon 5 (15%). All five devices were also evaluated for their capacity to provide false negative or false positive results and for testing time per eye, test difficulty level for the patient, versatility, upgrading capacity, examining room adaptability, and price. We concluded that glare testing of cataract patients is necessary to measure visual disability and that glare testing devices vary in their ability to predict outdoor vision.

The relationship between indoor and outdoor Snellen visual acuity in cataract patients.

One hundred six cataractous eyes of 78 patients were evaluated to examine the relationship between indoor and outdoor Snellen visual acuity. While 81 of all cataractous eyes (76.4%) had Snellen visual acuities of 20/40 or better when tested indoors, only 33 eyes (31.2%) had 20/40 or better Snellen visual acuities when tested outdoors facing the sun. When tested indoors, only three eyes (2.8%) had Snellen visual acuities worse than 20/80, while 31 eyes (29.2%) had outdoor Snellen visual acuities worse than 20/80. Ten eyes (9.4%) had outdoor Snellen visual acuities worse than 20/200. Seventy-four eyes (69.8%) had outdoor visual acuities that were at least two Snellen lines worse than those measured indoors and 23 eyes (21.7%) had outdoor visual acuities that were at least five Snellen lines worse. The median difference between indoor and outdoor visual acuity was three Snellen lines. The need for increased precision in the ability to assess outdoor "real world" vision while in the ophthalmologist's examination room is discussed. Clearly, indoor Snellen visual acuity alone is insufficient to evaluate functional visual impairment in cataract patients.

The relationship between cataract type and glare disability as measured by the Miller-Nadler glare tester.

Cataract patients were tested for glare disability using the Miller-Nadler glare tester. Predicted outdoor visual acuity was then compared with the actual outdoor visual acuity. The Miller-Nadler glare test scores predicted actual outdoor visual impairment to within one Snellen line in 46.7% of the eyes, underestimated actual outdoor visual impairment by more than one Snellen line in 31.5% of the eyes, and overestimated outdoor visual impairment by more than one Snellen line in 21.7% of the eyes. Overall, 64.1% of the eyes had outdoor vision which was more closely predicted by their glare scores than by their indoor Snellen acuity. When the cataractous eyes were divided into three categories, eyes with pure nuclear sclerosis, eyes with nuclear sclerosis and posterior subcapsular opacities, and eyes with all other cataractous combinations, predictability differences were observed. Although we found that actual outdoor visual acuities were not precisely predicted by disability glare scores in a substantial proportion of our subjects, the glare scores were considerably more predictive than indoor Snellen acuity. Further development and field testing of glare testing devices as predictors of outdoor visual impairment is necessary.