Mapping end-of-life and anticipatory medications in palliative care patients using a longitudinal general practice database.

OBJECTIVE: End-of-life and anticipatory medications (AMs) have been widely used in various health care settings for people approaching end-of-life. Lack of access to medications at times of need may result in unnecessary hospital admissions and increased patient and family distress in managing palliative care at home. The study aimed to map the use of end-of-life and AM in a cohort of palliative care patients through the use of the Population Level Analysis and Reporting Data Space and to discuss the results through stakeholder consultation of the relevant organizations. METHODS: A retrospective observational cohort study of 799 palliative care patients in 25 Australian general practice health records with a palliative care referral was undertaken over a period of 10 years. This was followed by stakeholders' consultation with palliative care nurse practitioners and general practitioners who have palliative care patients. RESULTS: End-of-life and AM prescribing have been increasing over the recent years. Only a small percentage (13.5%) of palliative care patients received medications through general practice. Stakeholders' consultation on AM prescribing showed that there is confusion about identifying patients needing medications for end-of-life and mixed knowledge about palliative care referral pathways. SIGNIFICANCE OF RESULTS: Improved knowledge and information around referral pathways enabling access to palliative care services for general practice patients and their caregivers are needed. Similarly, the increased utility of screening tools to identify patients with palliative care needs may be useful for health care practitioners to ensure timely care is provided.

Pharmacy DOTS Initiative (PDI): A case study on integrating pharmacies in the tuberculosis directly observed treatment-short course (TB DOTS) network in the Philippines

Background@#Tuberculosis (TB) is a disease that has continuously burdened Filipinos. Various programs have been launched by public and private sectors to decrease the incidence of TB and to scale up TB prevention and control in the country. In line with this, pharmacists have been contributing in the campaign against TB since 2004 through the implementation of the Pharmacy DOTS Initiative (PDI). Through the project Innovations and Multi-Sectorial Partnerships to Achieve Control of TB (IMPACT), PDI was relaunched in the country in 2014. @*Objectives@#This case study aims to evaluate the impact of PDI on TB prevention and control by assessing the effectiveness of the technical assistance package rolled out during program implementation. @*Methods@#A review of documents was done to evaluate the achievement of the specific targets of PDI. @*Results@#Among the targets, the percentage of actively referring pharmacies and the number of referrals made throughout the program failed to meet the target. The remaining program targets such as the establishment of a referral system, training of pharmacy personnel, adoption of a TB DOTS curriculum in pharmacy schools, and presence of national legislation, policies, and guidelines relevant to PDI were satisfactorily met. @*Conclusion@#PDI had a good response at the start of its implementation, but several issues resulted in the inability to sustain the interventions and achieve set targets.

Differential Identification of Mycobacterial Species Using High-Resolution Melting Analysis.

Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus-M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.

Pathogen Identification in Suspected Cases of Pyogenic Spondylodiscitis.

Pyogenic spinal infection continues to represent a worldwide problem. In approximately one-third of patients with pyogenic spondylodiscitis, the infectious agent is never identified. Of the cases that lead to organismal identification, bacteria are more commonly isolated from the spine rather than fungi and parasites. This study applied universal prokaryotic 16S rRNA PCR as a rapid diagnostic tool for the detection of bacterial agents in specimens from patients suspected of pyogenic spondylodiscitis. Gram and Ziehl-Neelsen staining were used as a preliminary screening measure for microbiologic evaluation of patient samples. PCR amplification targeting 16S rRNA gene was performed on DNA extracted from 57 cases including specimens from epidural abscesses, vertebral, and disc biopsies. Positive samples were directly sequenced. MRI findings demonstrated that disc destruction and inflammation were the major imaging features of suspected pyogenic spondylodiscitis cases, as 44 cases showed such features. The most common site of infection was the lumbar spine (66.7%), followed by thoracic spine (19%), the sacroiliac joint (9.5%), and lumbar-thoracic spine (4.8%) regions. A total of 21 samples amplified the 16S rRNA-PCR product. Sanger sequencing of the PCR products identified the following bacteriological agents: Mycobacterium tuberculosis (n = 9; 42.9%), Staphylococcus aureus (n = 6; 28.5%), Mycobacterium abscessus (n = 5; 23.8%), and Mycobacterium chelonae (n = 1; 4.8%). 36 samples displayed no visible 16S rRNA PCR signal, which suggested that non-bacterial infectious agents (e.g., fungi) or non-infectious processes (e.g., inflammatory, or neoplastic) may be responsible for some of these cases. The L3-L4 site (23.8%) was the most frequent site of infection. Single disc/vertebral infection were observed in 9 patients (42.85%), while 12 patients (57.15%) had 2 infected adjacent vertebrae. Elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) inflammatory markers were noted in majority of the patients. In conclusion, microbiological methods and MRI findings are vital components for the proper diagnosis of pyogenic spondylodiscitis. Our findings suggest that molecular methods such as clinical application of 16S rRNA PCR and sequencing may be useful as adjunctive diagnostic tools for pyogenic spondylodiscitis. The rapid turnaround time of 16S rRNA PCR and sequencing submission and results can potentially decrease the time to diagnosis and improve the therapeutic management and outcome of these infections. Although S. aureus and M. tuberculosis were the most common causes of pyogenic spinal infections in this study, other infectious agents and non-infectious etiologies should be considered. Based on study results, we advise that antibiotic therapy should be initiated after a definitive etiological diagnosis.

Identification of Mycobacterium tuberculosis in Clinical Specimens of Patients Suspected of Having Extrapulmonary Tuberculosis by Application of Nested PCR on Five Different Genes.

Definitive and rapid diagnosis of extrapulmonary tuberculosis (EPTB) is challenging since conventional techniques have limitations due to the paucibacillary nature of the disease. To increase the sensitivity of detection of Mycobacterium tuberculosis (MTB) in EPTB specimens, we performed a nested PCR assay targeting several genes of MTB on EPTB specimens. A total of 100 clinical specimens from suspected cases of EPTB were processed. Standard staining for acid fast bacilli (AFB) was performed as the preliminary screening test. Extracted DNAs from specimens were subjected to Nested PCR technique for the detection of five different MTB target genes of IS6110, IS1081, hsp65kd, mbp64, and mtp40. On performing AFB staining, only 13% of specimens were positive, of which ascites fluid (33.3%), followed by pleural effusion (30.8%) showed the greatest AFB positivity rate. We demonstrated slight improvement in yields in lymph node which comprised the majority of specimens in this study, by employing PCR targeted to IS6110- and hsp65-genes in comparison to AFB staining. However, the yields in ascites fluid and pleural effusion were not substantially improved by PCR, but those from bone and wound were, as in nested PCR employing either gene, the same positivity rate were obtained for ascites fluid (33.3%), while for pleural effusion specimens only IS1081 based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employing IS1081 gene. Here, we report higher detection rate of EPTB in clinical specimens using five different targeted MTB genes. This nested PCR approach facilitates the comparison and the selection of the most frequently detected genes. Of course this study demonstrated the priority of IS1081 followed by mtp40 and IS6110, among the five tested genes and indicates the effectiveness of any of the three genes in the design of an efficient nested-PCR test that facilitates an early diagnosis of paucibacillary EPTB cases, which are difficult to diagnose with the available standard.

Isolation of Streptococcus pyogenes from children with pharyngitis and emm type analysis.

BACKGROUND: The group A streptococcus (GAS) M protein, encoded by the emm gene, acts as a major virulence factor. Emm-typing is the GAS gold standard molecular typing and is based on the DNA sequence of the nucleotides of the emm gene. The aim of the present study was to isolate GAS from patients and to detect the emm types of the isolates using emm typing. METHODS: A total of 1000 throat samples were collected from patients with pharyngitis referred to Aboozar Children's Hospital in Ahvaz, Iran. We performed antimicrobial susceptibility testing on all isolates using the Kirby-Bauer disk diffusion method. Additionally, amplification of the emm gene was performed using polymerase chain reaction using the standard primers and described protocol. RESULTS: From all throat samples screened, 25 isolates (2.5%) were identified as GAS. Antibiotic susceptibility testing revealed that all the GAS isolates were susceptible to penicillin and erythromycin, but 44% showed resistance to vancomycin. Based on polymerase chain reaction for the emm gene, the obtained emm types were: emm-3, observed in 20 isolates (80%); emm-1 observed in four isolates (16%); and emm-75 observed in one isolate (4%). CONCLUSION: The result of the present study showed that penicillin and erythromycin are still the most effective antibiotics against the organism. The emm typing revealed that emm type-3 was detected in most of the isolates from patients with purulent pharyngitis. On the basis of the findings of this study, we may conclude that emm typing provides new insights on the genetic diversity of the M proteins, and is of demonstrable value for molecular studies of GAS.

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39-31.27% for rpoB-PCR, 36.44-60.83% for IS6110- PCR, 75.29-92.93% for nested-rpoB PCR, and 87.98-99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15-100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results.

Femtosecond dovetail penetrating keratoplasty: surgical technique and case report.

AIM: To report a dovetail configuration for femtosecond-enabled penetrating keratoplasty (PK) with the corresponding laser parameters and suturing technique. METHODS: A 40-year-old man, with a history of penetrating corneal injury as a child, underwent femtosecond-enabled dovetail keratoplasty, anterior vitrectomy and secondary intraocular lens suturing to repair his corneal scar and aphakia. A partial thickness dovetail pattern was performed in the recipient cornea using the femotsecond laser. The posterior side-cut was initiated approximately 100 microm anterior to the Descemet membrane and extended obliquely towards the outer edge of a ring lamellar cut, positioned at approximately 300 microm stromal depth. The anterior side-cut was extended from the internal edge of the ring lamellar cut to the corneal surface. Using an artificial chamber, the femtosecond laser was used to create a full-thickness 0.2 mm oversized femtosecond-enabled dovetail trephination with similar anterior lamellar depth (approximately 300 microm). Wound closure, using interrupted 10-0 nylon sutures, was guided by preplaced radial alignment laser microincisions and tongue-in-groove midstromal suture positioning. RESULTS AND DISCUSSION: Excellent alignment and stability of the donor and recipient tissue were observed immediately postoperatively and 5 months after surgery. The feasibility of the "dovetail" pattern of PK and the tongue-in-groove suture positioning is demonstrated.

Impediments to eye transplantation: ocular viability following optic-nerve transection or enucleation.

Maintenance of ocular viability is one of the major impediments to successful whole-eye transplantation. This review provides a comprehensive understanding of the current literature to help guide future studies in order to overcome this hurdle. A systematic multistage review of published literature was performed. Three specific questions were addressed: (1) Is recovery of visual function following eye transplantation greater in cold-blooded vertebrates when compared with mammals? (2) Is outer retina function following enucleation and reperfusion improved compared with enucleation alone? (3) Following optic-nerve transection, is there a correlation between retinal ganglion cell (RGC) survival and either time after transection or proximity of the transection to the globe? In a majority of the studies performed in the literature, recovery of visual function can occur after whole-eye transplantation in cold-blooded vertebrates. Following enucleation (and reperfusion), outer retinal function is maintained from 4 to 9 h. RGC survival following optic-nerve transection is inversely related to both the time since transection and the proximity of transection to the globe. Lastly, neurotrophins can increase RGC survival following optic-nerve transection. This review of the literature suggests that the use of a donor eye is feasible for whole-eye transplantation.

Echo Doppler parameters predict response to cardiac resynchronization therapy.

BACKGROUND: Cardiac resynchronization therapy (CRT) has been shown to reduce heart failure related morbidity and mortality. However, approximately 30% of patients do not respond to CRT. We investigated the usefulness of Echo Doppler parameters to predict reverse remodelling, functional improvement and mortality following CRT. MATERIALS AND METHODS: Our population consists of 200 consecutive heart failure patients evaluated for ventricular dyssynchrony by echocardiography between February 1999 and May 2007 who subsequently received CRT. Patients were reassessed for signs of reverse remodelling after a mean follow-up of 10 months. Information on vital status was obtained from local registration authorities. RESULTS: Three parameters significantly predicted reverse remodelling in the logistic regression analysis: the Q-to-E-wave-delay (QED) at a cutoff of 550 ms (odds ratio 4.5, P-value 0.001), the interventricular mechanical delay (IVMD) at a cutoff of 60 ms (odds ratio 2.4, P-value 0.02), and the aortic electromechanical delay (A-EMD) at a cutoff of 140 ms (odds ratio 2.9, P-value 0.004). Furthermore, the QED and the IVMD also predicted all-cause mortality (hazard ratio 0.36, P-value 0.02 and 0.21, P-value 0.004, respectively). Adjustment for confounders did not alter the results. CONCLUSIONS: The QED and IVMD predict reverse remodelling and survival following CRT. These parameters are easy to obtain, provide valuable prognostic information, and should thus be measured in CRT candidates evaluated by echocardiography.

Detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa strains isolated from burn patients in Ahwaz, Iran.

The antimicrobial activities of 6 common antimicrobial agents including carbapenems were tested against 100 clinical Pseudomonas aeruginosa isolates using a disk diffusion method. All imipenem (IPM)-resistant isolates were screened for metallo-beta-lactamase (MBL) production by Etest assay and were subsequently subjected to polymerase chain reaction (PCR) analysis with the bla(IMP) and bla(VIM) genes. Of 41 IPM-nonsusceptible isolates detected, 8 (19.51%) appeared to produce MBL, as determined by Etest. Using PCR assay, these isolates were positive for bla(VIM) genes, whereas none were positive for bla(IMP) genes.

[Ablation on the undersurface of a LASIK flap. Instrument and method for continuous eye tracking]. / Ablation auf der Unterseite eines LASIK-Flaps. Neues Instrument und Verfahren zum kontinuierlichen Eye-Tracking.

BACKGROUND: The risk of iatrogenic keratectasia after laser in situ keratomileusis (LASIK) increases with thinner posterior stromal beds. Ablations on the undersurface of a LASIK flap could only be performed without the guidance of an eye tracker, which may lead to decentration. A new method for laser ablation with flying spot lasers on the undersurface of a LASIK flap was developed that enables the use of an active eye tracker by utilizing a novel instrument. The first clinical results are reported. PATIENTS AND METHODS: Patients wishing an enhancement procedure were eligible for a modified repeat LASIK procedure if the flaps cut in the initial procedure were thick enough to perform the intended additional ablation on the undersurface leaving at least 90 microm of flap thickness behind. (1) The horizontal axis and the center of the entrance pupil were marked on the epithelial side of the flap using gentian violet dye. (2) The flap was reflected on a newly designed flap holder which had a donut-shaped black marking. (3) The eye tracker was centered on the mark visible in transparency on the flap. (4) Ablation with a flying spot Bausch & Lomb Technolas 217z laser was performed on the undersurface of the flap with a superior hinge taking into account that in astigmatic ablations the cylinder axis had to be mirrored according to the formula: axis on the undersurface=180 degrees -axis on the stromal bed. (5) The flap was repositioned. RESULTS: Detection of the marking on the modified flap holder and continuous tracking instead of the real pupil was possible in all of the 12 eyes treated with this technique. It may be necessary to cover the real pupil during ablation in order not to confuse the eye tracker. Ablation could be performed without decentration or loss of best spectacle-corrected visual acuity. Refractive results in minor corrections were good without nomogram adjustment. CONCLUSIONS: Using this novel flap holder with a marking that is tracked instead of the real pupil, centered ablations with a flying spot laser on the undersurface of a LASIK flap are feasible. Thus, the additional risk of iatrogenic keratectasia associated with stromal enhancement ablations is avoided.

[LASEK: results after 1 year. Retrospective analysis based on the dioptric power matrix for moderate myopic and astigmatic correction]. / Einjahresergebnisse nach LASEK. Retrospektive Analyse mit Hilfe der Refraktionsmatrix bei der Korrektur von mittlerer Myopie und myopem Astigmatismus.

PURPOSE: The aim of this study was to assess astigmatism magnitude and axis changes based on the dioptric power matrix in low to moderate levels of myopia and astigmatism treated with LASEK. PATIENTS AND METHODS: This retrospective analysis included 54 myopic eyes treated with LASEK with a minimum follow-up of 12 months. An epithelial flap was created by 25-45 s of 20% alcohol exposure. The corneal surface was ablated using two different excimer lasers and nomogram adjustment. The flap was repositioned and a bandage applied to the contact lens. Main outcome measures were manifest refraction as calculated with the dioptric power matrix, UCVA, BSVCA, and retreatment rate. RESULTS: Mean manifest refraction is shown in table 2 (Tabelle 2). UCVAs of 20/20 or better were found in 33% of eyes at 1 week and in more than 53% at 3 months to 1 year. The safety index remained > or =0.98 after postoperative week 4. The efficacy index varied between 0.91 and 0.98 after 1 month. CONCLUSION: LASEK for correction of low to moderate myopia and astigmatism seems to be a safe, effective, and stable option.

Diffuse lamellar keratitis: incidence, associations, outcomes, and a new classification system.

PURPOSE: To evaluate the incidence, associations, and visual outcomes in patients with diffuse lamellar keratitis (DLK) after laser in situ keratomileusis (LASIK). SETTING: University-based refractive surgery center, Boston, Massachusetts, USA. METHODS: This retrospective review comprised 2711 eyes that had LASIK between September 1996 and September 1999. All eyes that developed DLK after LASIK were included. They were divided into type I DLK (center sparing) or type II DLK (center involved) and then subdivided into A (sporadic-DLK not diagnosed in other patients treated on the same day) or B (cluster-other patients identified with DLK). Type IA corresponded to center sparing, sporadic; type IB, center sparing, cluster; type IIA, center involved, sporadic; and type IIB, center involved, cluster. The main outcome measures were incidence of DLK after LASIK, time to diagnosis, time to resolution, and changes in best spectacle-corrected visual acuity (BSCVA). Unpaired t tests were used for statistical analyses. RESULTS: Thirty-six eyes (1.3%) developed DLK. Type I occurred in 58.3% of cases (type IA, n = 18; type IB, n = 3) and type II, in 41.7% (type IIA, n = 10; type IIB, n = 5). The mean time to diagnosis was not statistically significantly different between type I (1.8 days) and type II (1.1 days). Fourteen eyes (38.9%) developed DLK after an epithelial defect, representing an odds ratio of 13 times. The association with an epithelial defect was statistically significantly greater with type I (11/21 eyes, 52.4%) than with type II (3/15 eyes, 20.0%; P =.05). The mean time to resolution was 3.5 days in type I (type IA = 3.6 days; type IB = 2.7 days). This was significantly shorter than in type II, which had a mean time to resolution of 12.1 days (type IIA = 9.3 days; type IIB = 10.2 days) (P =.001). Loss of 2 or more lines of BSCVA occurred in 2 of 5 patients with type IIB and in no patients with types IA, IB, or IIA. CONCLUSIONS: Epithelial defects after LASIK increased the risk of DLK occurrence, especially type I. Type II DLK was associated with a prolonged time to resolution and carried a significantly higher risk of BSCVA loss than type I.

Diminished corneal angiogenesis in gelatinase A-deficient mice.

The goal of the present study was to define the role of gelatinase A in angiogenesis. We performed corneal micropocket assays in gelatinase A-deficient mice and their age-matched wild-type littermates. The corneal neovascular area in gelatinase A-deficient mice (0.15+/-0.14 mm(2)) was significantly less than that of wild-type littermates (0.53+/-0.35 mm(2); P<0.01). Similarly, aortic ring assays showed significant reduction of endothelial outgrowth in gelatinase A-deficient mice (0.26+/-0.14 mm(2)) as compared to wild-type littermates (0.44+/-0.06 mm(2); P<0.05). These results suggest that gelatinase A may play an important role in the regulation of corneal angiogenesis.

Transplantation of adult human corneal endothelium ex vivo: a morphologic study.

PURPOSE: To investigate the feasibility of transplanting untransformed human corneal endothelial cells as a treatment strategy and possible alternative for penetrating keratoplasty by growing donor cells in culture and then transplanting them to denuded Descemet's membrane of recipient corneas. METHODS: Corneas from adult donors (50-80 years old) were obtained from eye banks. To grow corneal endothelial cells, Descemet's membrane with associated cells was dissected from the stroma. Endothelial cells were released by ethylenediaminetetraacetic acid treatment, grown in medium containing multiple growth factors, and identified as being of endothelial origin by morphology and by reverse-transcription polymerase chain reaction for keratin 12 and collagen type VIII. In transplantation experiments, cultured cells were seeded onto denuded Descemet's membrane of a second donor cornea at 5 x 10(5) cells/mL. The recipient cornea was incubated in organ culture for as long as 2 weeks. The morphology and ultrastructure of the endothelium were evaluated 7 and 14 days after transplantation by transmission electron microscopy, and by immunolocalization of zonula occludins-1 (ZO-1). Endothelial cell density was calculated in transplants by counting ZO-1-stained cells. RESULTS: Corneal endothelial cells cultured from adult donors consistently grew well in culture medium. Cells were identified as corneal endothelium by characteristic morphology and messenger RNA expression. Morphologic and ultrastructural studies of corneas containing transplanted endothelial cells demonstrated that with time there was an increase in endothelial cell-Descemet's membrane adhesion, in the extent of cell-cell contacts and lateral interdigitation, and in formation of a single cell layer. ZO-1 staining revealed tight junction formation similar to that of corneas in vivo. Mean endothelial cell density in transplanted corneas was 1,895 cells/mm(2) (range, 1,503-2,159 cells/mm(2) ). CONCLUSION: Untransformed adult human corneal endothelial cells can be efficiently and consistently cultured and transplanted onto denuded Descemet's membrane. Transplanted cells in organ culture exhibit morphologic characteristics and cell densities similar to corneal endothelial cells in vivo. These results provide evidence for the feasibility of developing methods for in vivo transplantation of untransformed corneal endothelial cells cultured from adult donor tissue.

Matrilysin cleavage of corneal collagen type XVIII NC1 domain and generation of a 28-kDa fragment.

PURPOSE: To localize endostatin and collagen type XVIII in human corneas and to characterize the enzymatic action of matrix metalloproteinases (MMPs) in the cleavage of collagen type XVIII and generation of endostatin in the cornea. METHODS: Anti-endostatin and anti-hinge antibodies were generated using peptide fragments corresponding to the endostatin region and the adjacent nonendostatin hinge region of collagen XVIII noncollagenous (NC)1 domain, respectively. Confocal immunostaining was performed to localize collagen XVIII in human corneas. SV40-immortalized corneal epithelial cells were immunoprecipitated and incubated with active MMP-1, -2, -3, -7, or -9, and Western blot analysis was performed to study collagen XVIII cleavage. Incubation with MMP-7 was performed at various concentrations (0, 2, 4, and 6 microg/ml) and time intervals (0, 1, 5, and 12 hours). Purified recombinant NC1 fragment of collagen XVIII was also digested with MMP-7, and the cleavage product was sequenced. RESULTS: Collagen XVIII was immunolocalized to the human corneal epithelium, epithelial basement membrane, and Descemet membrane. Western blot analysis demonstrated a 180- to 200-kDa band corresponding to collagen XVIII. MMP-7 (but not MMP-1, -2, -3, and -9) cleaved corneal epithelium-derived collagen XVIII to generate a 28-kDa endostatin-spanning fragment in a time- and concentration-dependent fashion. MMP-7 cleaved purified recombinant 34-kDa NC1 fragment of collagen XVIII in the hinge region to generate a 28-kDa fragment. CONCLUSIONS: Collagen XVIII is present in human cornea. MMP-7 cleaves the collagen XVIII NC1 domain to generate a 28-kDa fragment in the cornea.

LASIK complications: etiology, management, and prevention.

Laser in situ keratomileusis (LASIK) is a rapidly evolving ophthalmic surgical procedure. Several anatomic and refractive complications have been identified. Anatomic complications include corneal flap abnormalities, epithelial ingrowth, and corneal ectasia. Refractive complications include unexpected refractive outcomes, irregular astigmatism, decentration, visual aberrations, and loss of vision. Infectious keratitis, dry eyes, and diffuse lamellar keratitis may also occur following LASIK. By examining the etiology, management, and prevention of these complications, the refractive surgeon may be able to improve visual outcomes and prevent vision-threatening problems. Reporting outcomes and mishaps of LASIK surgery will help refine our approach to the management of emerging complications.