Development of a single-tube, cell lysis-based, genus-specific PCR method for rapid identification of mycobacteria: optimization of cell lysis, PCR primers and conditions, and restriction pattern analysis

A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 h without conventional DNA isolation. The following three steps were optimized or developed: (i). a simple, 6-min direct cell lysis protocol as a PCR prestep for generation of DNA-template, (ii). an improved Mycobacterium-specific PCR amplification protocol with a broader species specificity using newly designed primers targeting a 228-bp region of the 65-kDa heat shock protein (hsp) gene and optimal PCR amplification conditions, and (iii). a genus-specific restriction analysis of the PCR product for conclusive identification of the unknown NTM isolates.     

Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.     

Outcomes and complications associated with perfluoro-n-octane and perfluoroperhydrophenanthrene in complex retinal detachment repair

OBJECTIVE: To compare rates of perfluorocarbon liquid (PFCL) intraocular retention, anatomic and visual acuity outcomes, and complications associated with intraoperative perfluoro-n-octane (Perfluoron) versus perfluoroperhydrophenanthrene (Vitreon) in retinal detachment repair. DESIGN: Retrospective noncomparative consecutive case series. PARTICIPANTS AND METHODS: Records of consecutive patients who underwent retinal detachment repair with intraoperative Perfluoron (n = 78) or Vitreon (n = 84) at the Bascom Palmer Eye Institute between November 1, 1991, and October 31, 1994, were retrospectively reviewed. MAIN OUTCOME MEASURES: Comparison of PFCL intraocular retention rates, retinal reattachment rates, visual acuity outcomes, and postoperative complication rates between the Perfluoron and Vitreon groups at postoperative day 1, week 1, month 1, month 3, and month 6. To assess the study's generalizability, the Perfluoron data obtained in the current study were compared with results of the Perfluoron Multicenter Clinical Study. RESULTS: Retained Perfluoron was noted less frequently (P < 0.03) than retained Vitreon at each postoperative visit assessed. At 6 months postoperatively, the cumulative rate of retained PFCL (noted at any of the study postoperative visits) was 7.8% of patients in the Perfluoron group and 38.3% in the Vitreon group (P < 0.001). No significant difference was found between the groups in retinal reattachment rates; at 6 months postoperatively, the retina was attached in 45 of 61 (74%) patients in the Perfluoron group and 36 of 57 (63%) patients in the Vitreon group (P = 0.2). There was a trend (P = 0.055) toward better 6-month visual acuity in the Perfluoron group compared with the Vitreon group. The incidence of corneal abnormality at any study visit was 46.7% in the Perfluoron group and 77.4% in the Vitreon group (P < 0.001). At 6 months postoperatively, the cumulative rate of elevated intraocular pressure (>25 mmHg) was 13 +/- 4% and 37 +/- 5%, respectively (P = 0.004). No significant difference was found between the groups in rates of postoperative hypotony. Results of this study are comparable with those observed in the Perfluoron Multicenter Clinical Study. CONCLUSIONS: In this study, Perfluoron was retained intraocularly less frequently than Vitreon. Although no significant difference was found between groups in retinal reattachment rates, Perfluoron is associated with slightly better 6-month visual acuity and lower rates of corneal abnormality and elevated intraocular pressure compared with Vitreon.     

Hemiretinal vein occlusion associated with membranous glomerulonephritis

PURPOSE: To report a patient in whom the finding of hemiretinal vein occlusion led to the diagnosis of membranous glomerulonephritis. DESIGN: Interventional case report. METHODS: A 44-year-old tennis instructor presented with a 1-week history of blurred vision in the left eye. Examination of the left eye demonstrated a best-corrected visual acuity of 20/40 and an inferior hemiretinal vein occlusion. RESULTS: Blood pressure was normal, and the patient was referred for a medical examination, which revealed membranous glomerulonephritis. The patient was treated with oral prednisone and cyclosporine. Four months after presentation, the left eye demonstrated resolution of the vascular abnormalities and had a best-corrected visual acuity of 20/20. CONCLUSION: Retinal vein occlusion may be associated with membranous glomerulonephritis. Treatment of the systemic disease may be associated with regression of the retinal vascular abnormalities.     

Impact of graphical user interface screen features on computer task accuracy and speed in a cohort of patients with age-related macular degeneration

PURPOSE: To investigate the impact of graphical user interface screen features on computer task performance in patients with age-related macular degeneration (AMD). DESIGN: Interventional case series. METHODS: Eighteen patients with visual impairment due to AMD were recruited from the Bascom Palmer Eye Institute Low Vision Clinic. Each patient underwent evaluation of visual acuity using the Early Treatment Diabetic Retinopathy Study protocol, contrast sensitivity using a Pelli-Robson chart, binocular simultaneous visual field using the Esterman program on an automated perimeter, and color vision using Farnsworth D-15. Each subject then completed computer icon identification tasks while the following screen features of the graphical user interface were varied: size of icons displayed, icon set size (number of icons displayed), and background color. Each patient performed all 125 computer tasks with each of five icon sizes (9.2 mm, 14.6 mm, 23.2 mm, 36.8 mm, 58.3 mm), each of five icon set sizes (2, 3, 4, 5, 6), and each of five different background colors (black, white, red, green, blue) in a randomly ordered fashion. Relationships between computer task performance (accuracy and speed) and graphical user interface screen features were studied. RESULTS: Icon size and icon set size are significantly associated with computer task accuracy (P <.001), whereas background color is not a significant predictor of task accuracy (P =.63). The impact of icon size on accuracy is nonlinear, with the data indicating that no additional improvement in accuracy is associated with increasing the icon size beyond 23.2 mm. The impact of icon set size on accuracy is linear, with a smaller icon set size significantly associated with greater computer accuracy. A larger icon size is significantly associated with a shorter time to task completion (P =.001); this relationship is largely linearly related to icon size. There was no significant impact of background color (P =.11) or set size (P =.37) on time to task completion. CONCLUSIONS: Modifications of graphical user interface design may permit improved computer task performance among patients with visual impairment due to AMD.     

Acute-onset postoperative endophthalmitis: review of incidence and visual outcomes (1995-2001)

BACKGROUND AND OBJECTIVE: To determine the incidence rate of acute-onset postoperative endophthalmitis and to assess visual acuity outcomes after treatment from the most recent 7 years (1995-2001) compared with the previous 11 years (1984-1994) among patients undergoing intraocular surgery at the same institution. PATIENTS AND METHODS: The medical records were reviewed of all patients undergoing intraocular surgery at the Bascom Palmer Eye Institute between January 1, 1995 and December 31, 2001. RESULTS: The 7-year incidence rate of acute-onset postoperative endophthalmitis was 0.05% (17 of 35,916 intraocular surgeries). The number of patients with endophthalmitis (incidence) and their median final visual acuity for each surgical category are as follows: cataract extraction: 8/21,972 (0.04%) - 20/100; glaucoma surgery: 4/1,970 (0.2%) - 20/70; penetrating keratoplasty: 2/2,362 (0.08%) - light perception; pars plana vitrectomy: 2/7,429 (0.03%) - hand movements; secondary intraocular lens placement: 1/485 (0.2%) - 20/40. Of the 8 cases of endophthalmitis after cataract surgery, 6 cases occurred after phacoemulsification and 2 of these cases had a dear corneal sutureless incision. CONCLUSION: The most recent 7-year incidence rate of acute-onset postoperative endophthalmitis is significantly lower than that of the previous 11 years (0.05% versus 0.09%; = 0.031) at the same institution. Visual acuity outcomes after treatment were generally better in cataract surgery, glaucoma surgery, and secondary intraocular lens categories compared to pars plana vitrectomy and penetrating keratoplasty categories.