Needs assessment for direct ophthalmoscopy training in neurology residency.

BACKGROUND: Assessment of the ocular fundus, traditionally by direct ophthalmoscopy (DO), is essential to evaluate many neurologic diseases. However, the status of DO training in neurology residencies is unknown. We conducted a needs assessment to determine current attitudes, curricula, and gaps in DO training. METHODS: A survey was developed and administered to residents and program directors (PDs) at ACGME accredited neurology residencies in the United States. The survey assessed factors such as current DO curricula, perceived importance of DO, confidence of skills, and need for improvement. Data analysis was performed using the Mann Whitney U test and Fisher Exact Test. RESULTS: Nineteen PDs (11.6%) and 74 (41.1%) residents responded to the survey. 97.1% of residents and 100.0% of PDs believe DO is an important skill to learn. 29.4% of PDs expected graduating residents to have completed > 10 supervised DO exams, while 0.0% of graduating fourth year residents reported doing so (p = 0.03). 35.7% of graduating residents had never correctly identified an abnormal finding on DO. The number of times residents practiced DO unsupervised correlated with increasing confidence in all components of the DO exam (p < 0.05). Residents who felt their program emphasized DO were more likely to perform DO at least once a week compared to residents who did not perceive program emphasis (61.9% vs. 35.0%, p = 0.02) and were more confident in DO (p < 0.05). 66.7% of residents and 42.1% of PDs were not satisfied with current levels of DO training. 96.7% of residents and 78.9% of PDs felt it was important to improve curriculum for DO training. Supervised practice and practice skills sessions were identified as the most helpful interventions to improve DO training. CONCLUSIONS: The vast majority of neurology PDs and residents believe DO is an important skill to learn, are unsatisfied with the current level of DO training, and advocate for improvement in DO curricula. Current DO curricula have limited formal didactic training and supervised practice. The bulk of DO learning occurs through unsupervised practice, which is influenced by motivational factors such as perceived residency emphasis on DO learning.

Evaluation of an Instructional Video and Simulation Model for Teaching Slit Lamp Examination to Medical Students.

Purpose This article assesses the efficacy of an instructional video and model eye simulation for teaching slit lamp exam to medical students as compared to traditional preceptor teaching. Methods First through 4th year students from the University of California, San Francisco School of Medicine were recruited via email to participate in the study. Students were randomized into two groups. The experimental "model eye" group watched an instructional video on slit lamp exam, spent 10 minutes practicing on the model eye, then practiced for 25 minutes with a student partner. The control "preceptor teaching" group received 25 minutes of in-person preceptor teaching on slit lamp exam, then spent 25 minutes practicing with a student partner. Students were objectively assessed by a blinded grader who scored their examination skills with a 31-item checklist. Qualtrics surveys that measured student perceptions were distributed before and after the intervention. Results Seventeen medical students participated in the study. Students in the model eye group achieved higher mean objective assessment scores than students in the preceptor teaching group on skills relating to slit lamp set up (1.75, standard deviation [SD] = 0.50 and 1.50, SD = 0.80 out of 2 points, p = 0.03) and on the total score (1.69, SD = 0.6 and 1.48, SD = 0.8 out of 2 points, p < 0.01). Both groups reported a significant increase in their understanding of what a slit lamp is used for ( p < 0.01) and in their confidence using a slit lamp ( p < 0.01). All students felt their skills improved with the workshop, 94% found the workshop to be useful, and 88% enjoyed the workshop, with no intergroup differences on these metrics. Conclusion An instructional video combined with a simulation model is as effective as traditional preceptor teaching of the slit lamp exam. Such a teaching module may be considered as an adjunct to traditional methods.

Characterization of Infectious Keratitis in Opioid Users in a County Hospital Setting.

Purpose: To determine risk factors and clinical course of corneal ulcers in the setting of opioid use. Methods: A retrospective cohort study was performed of patients presenting with bacterial or fungal keratitis at a county hospital from 2010-2021. Subjects were separated into three groups: opioid drug users (heroin, methadone, fentanyl), non-opioid drug users, and non-drug users. 24 opioid users, 77 non-opioid drug users, and 38 non-drug users were included in the study. Chi-square and t-tests were used to compare hospitalization for corneal ulcer treatment; length of hospitalization; loss to follow-up; final best corrected visual acuity (BCVA); medication noncompliance; time to ulcer resolution; and visual disability (defined either by the legal limit for driving in California or the federal limit for blindness). Results: Opioid users had higher rates of unemployment (p=0.002), homelessness (p=0.018), and psychiatric conditions (p=0.024) compared with non-opioid and non-drug users. They had more severe presentations, with worse initial BCVA of the affected eye (p=0.003), larger ulcer size (p=0.023), and higher rates of individuals below the legal vision thresholds for driving (p=0.009) and blindness (p=0.033) at initial presentation. Opioid use was associated with increased rate of hospitalization (p<0.001), higher fortified antibiotic use (p=0.009), worse final BCVA of the affected eye (p=0.020), and increased rates of BCVA worse than the legal vision thresholds for driving (p=0.043) and blindness (p<0.001) on final presentation. Conclusions: Infectious keratitis associated with opioid use is associated with more severe presentations and poorer outcomes, including higher rates of visual disability.

Unmasking of subclinical keratoconus with Descemet membrane endothelial keratoplasty in Fuchs endothelial dystrophy.

We report a case of Fuchs endothelial corneal dystrophy (FECD) with concurrent forme fruste keratoconus (KCN) that was unmasked with Descemet membrane endothelial keratoplasty (DMEK) in the right eye, but not with Descemet-stripping automated endothelial keratoplasty (DSAEK) in the left eye. The patient was a 65-year-old female with FECD who underwent uncomplicated combination cataract surgery and DMEK in the right eye. She subsequently developed intractable monocular diplopia associated with inferior displacement of the thinnest point of the cornea and subtle steepening noted on posterior corneal curvature on Scheimpflug tomography. The patient was diagnosed with forme fruste KCN. Altering the surgical plan to combine cataract surgery and DSAEK in the left eye successfully circumvented the development of symptomatic visual distortion. This is the first case providing comparable data from contralateral eyes in the same patient regarding the outcome of DMEK versus DSAEK in eyes with concurrent forme fruste KCN. DMEK appeared to unmask posterior corneal irregularities and resulted in visual distortion, whereas DSAEK did not. The additional stromal tissue in DSAEK grafts appears to help normalize alterations of the posterior corneal curvature and may be the preferred endothelial keratoplasty for patients with concurrent mild KCN.

Descemet membrane endothelial keratoplasty in eyes with glaucoma.

Endothelial keratoplasty has become the standard for the treatment of endothelial dysfunction. In Descemet membrane endothelial keratoplasty (DMEK), only the endothelium and Descemet membrane are transplanted, providing superior outcomes compared to Descemet stripping endothelial keratoplasty (DSEK). A substantial subset of patients who require DMEK have comorbid glaucoma. Even in eyes with complex anterior segment such as eyes with previous trabeculectomy or tube shunts, DMEK can restore meaningful vision and outperforms DSEK in terms of visual recovery, decreased rejection rate, and the need for high dose of topical steroids. However, accelerated endothelial cell loss and secondary graft failure have been described in eyes with previous glaucoma surgery, namely trabeculectomy and drainage device. During DMEK and DSEK procedures, raised intraocular pressure is required to attach the graft, which could worsen preexisting glaucoma or cause de novo glaucoma. Mechanisms of postoperative ocular hypertension include delayed air clearance, pupillary block, steroid response, and damage to angle structures. Medically treated glaucoma has increased risk for postoperative ocular hypertension. By understanding these additional complications and making appropriate modifications in surgical techniques and postoperative management, DMEK can be performed successfully and achieve very good visual outcome in eyes with glaucoma. Such modifications include precisely controlled unfolding technique, iridectomies that can help avoid pupillary block, tube shunts that can be trimmed to facilitate graft unfolding, air fill tension that can be adjusted, and postoperative steroid regimens that can be modified to decrease the risk for steroid response. Long-term survival of the DMEK graft, however, is shorter in eyes with previous glaucoma surgery than those without, as observed after other types of keratoplasty.

Risk Factors Predicting Loss to Follow-Up, Medication Noncompliance, and Poor Visual Outcomes Among Patients With Infectious Keratitis at a Public County Hospital.

PURPOSE: Infectious keratitis is a vision-threatening condition requiring close follow-up and disciplined eye drop administration to achieve resolution. Although patients presenting to county hospitals often have more severe presentations, there is a paucity of risk and outcomes data in this setting. This study investigates risk factors predicting loss to follow-up (LTFU), medication noncompliance, and poor outcomes for infectious keratitis in the county hospital setting. METHODS: This was a retrospective case-control study at Zuckerberg San Francisco General Hospital and Trauma Center. Inclusion criteria were patients who had corneal cultures for suspected infectious bacterial or fungal keratitis between 2010 and 2021. Exclusion criteria were patients with viral keratitis only. Multivariable logistic regression was used to analyze the relationship of social and medical risk factors with LTFU, medication noncompliance, worsened visual acuity (VA), and delayed resolution time. RESULTS: Of 174 patients with infectious keratitis in this analysis, 69 (40.0%) had LTFU. Unemployment was associated with increased risk of LTFU (odds ratio 2.58, P = 0.049) and worse final VA ( P = 0.001). Noncompliance trended toward an association with homelessness (odds ratio 3.48, P = 0.095). Increasing age correlated with longer resolution time, with each 1-year increase associated with delayed resolution by 0.549 days ( P = 0.042). CONCLUSIONS: Patients experiencing unemployment, homelessness, or increased age demonstrate higher risk for treatment barriers including loss to follow-up and medication noncompliance, resulting in worse VA and delayed time to resolution. These risk factors should be considered when determining the need for more deliberate follow-up measures in patients with infectious keratitis.

Characterization of Polymicrobial and Antibiotic-Resistant Infectious Keratitis in a County Hospital Setting.

Purpose: Infectious keratitis is a serious cause of visual impairment, particularly in low-income communities. This study examines the associations between social risk factors and polymicrobial keratitis, multidrug resistance, pathogen spectrum, and outcomes at a county hospital. Methods: We performed a retrospective study of Zuckerberg San Francisco General Hospital patients treated for infectious keratitis from 2010-2021. Multivariable regression was performed to analyze the relationships between social, medical, and psychiatric risk factors with polymicrobial growth, multidrug resistance, and clinical outcomes. Results: Of 174 patients with infectious keratitis, 44 (25%) had polymicrobial growth. Six patients (14%) with polymicrobial growth had multidrug-resistant organisms. Homeless patients were more likely to present with polymicrobial infection (OR 3.4, p = 0.023), and polymicrobial infections were associated with multidrug-resistant organisms (p = 0.018). Smoking, drug use, HIV positivity, prior corneal pathology, and contact lens use were not associated with an increased risk of polymicrobial infection. Eleven patients (6.3%) were started on topical antibiotics prior to presentation; of these, none developed polymicrobial infections or multidrug-resistant organisms. Polymicrobial infections increased the likelihood to initiation of fortified antibiotics (OR 2.9, p = 0.011) but did not impact ulcer size, final visual acuity, time to resolution, or likelihood of emergent procedures. Conclusions: Homelessness correlates with an increased risk of polymicrobial keratitis and subsequent multidrug resistance, supporting initiation of broad antibiotic coverage in this population. Prior topical antibiotics did not increase risk of polymicrobial infection. Polymicrobial infection did not significantly worsen clinical outcomes.

Corneal Pigment Opacity From Face-Down Positioning After Pars Plana Vitrectomy, Treated With Descemet Stripping Only Technique.

PURPOSE: The purpose of this study was to report a case of corneal opacity resulting from pigment deposition after face-down positioning, which was treated with Descemet stripping only (DSO) to enable concurrent pars plana vitrectomy (PPV) for retinal detachment repair. METHODS: A 79-year-old man with a history of Fuchs endothelial dystrophy and retinal detachment presented for the repair of recurrent retinal detachment and evaluation of a central corneal opacity. RESULTS: The patient was found to have significant corneal endothelial pigment deposition obscuring the view to the fundus. A repeat macula-involving retinal detachment was visualized on limited fundoscopic examination and confirmed using ultrasonography. The patient subsequently underwent combination scleral buckle, DSO, and PPV. DSO achieved corneal clarity for the entire duration of the PPV and allowed for the necessary postoperative face-down positioning. Immunohistochemistry of the corneal specimen revealed deposition of retinal pigment epithelium as the origin of the pigment opacity. The corneal edema cleared at postoperative month 4, and the retina remained attached, resulting in an improvement of visual acuity from counting fingers preoperatively to 20/70. DISCUSSION: This is, to the best of our knowledge, the first case describing the formation of a corneal endothelial opacity because of retinal pigment epithelium deposition associated with face-down positioning after PPV for retinal detachment. DSO is a minimally invasive, viable alternative to endothelial keratoplasty or temporary keratoprosthesis placement for the clearance of focal corneal endothelial opacities for PPV.

Antimicrobial Peptide Expression at the Ocular Surface and Their Therapeutic Use in the Treatment of Microbial Keratitis.

Microbial keratitis is a common cause of ocular pain and visual impairment worldwide. The ocular surface has a relatively paucicellular microbial community, mostly found in the conjunctiva, while the cornea would be considered relatively sterile. However, in patients with microbial keratitis, the cornea can be infected with multiple pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium sp. Treatment with topical antimicrobials serves as the standard of care for microbial keratitis, however, due to high rates of pathogen resistance to current antimicrobial medications, alternative therapeutic strategies must be developed. Multiple studies have characterized the expression and activity of antimicrobial peptides (AMPs), endogenous peptides with key antimicrobial and wound healing properties, on the ocular surface. Recent studies and clinical trials provide promise for the use of AMPs as therapeutic agents. This article reviews the repertoire of AMPs expressed at the ocular surface, how expression of these AMPs can be modulated, and the potential for harnessing the AMPs as potential therapeutics for patients with microbial keratitis.

Corneal ectasia associated with posterior lamellar opacification.

BACKGROUND: Concomitant corneal ectasia and posterior lamellar corneal opacification is rare, and the genetic relationship between these two conditions is unclear. We report the genetic and clinical characterization of this phenotype in three unrelated individuals. MATERIALS AND METHODS: One previously reported affected individual and two unreported, unrelated, affected individuals were recruited for the study. Subjects and unaffected relatives underwent slit lamp examination, refraction, and multi-modal imaging. Saliva samples were obtained from two of the three affected individuals, from which DNA was extracted. Sanger sequencing was performed to identify mutations in genes associated with posterior amorphous corneal dystrophy (PACD), brittle cornea syndrome (BCS), and posterior polymorphous corneal dystrophy (PPCD), while copy number variation (CNV) analysis was used to identify CNV in the PACD locus. RESULTS: Affected individuals demonstrated bilateral corneal steepening, stromal thinning and lamellar posterior corneal opacification. Corneal topography and tomography revealed conical or globular corneal steepening and decreased thickness. Anterior segment optical coherence tomography demonstrated hyperreflectivity of the posterior stroma in each of the affected individuals. Genetic testing did not detect a heterozygous deletion involving the PACD locus on chromosome 12 or a pathogenic mutation in the genes associated with BCS or PPCD. CONCLUSIONS: Corneal ectasia may be associated with posterior lamellar stromal opacification that appears consistent with PACD. However, genetic testing for PACD as well as BCS and PPCD in affected individuals fails to reveal pathogenic deletions or mutations, indicating that other genetic factors are involved.

Infectious Keratitis After Boston Type 1 Keratoprosthesis Implantation.

PURPOSE: To identify the incidence, risk factors, and outcomes of infectious keratitis after Boston type 1 keratoprosthesis (kpro) implantation. METHODS: Retrospective case series of kpro procedures at the Stein Eye Institute and the Centre Hospitalier de l'Université de Montréal between May 1, 2004, and December 31, 2018. Data were collected regarding ocular history, operative details, postoperative management, microbiologic profile, treatment, and outcomes. Log-rank test and Cox proportional hazard ratio (HR) were used to evaluate for an association between risk factors and outcomes. RESULTS: A total of 349 kpro procedures were performed in 295 eyes of 268 patients. Fifty-seven cases of presumed infectious keratitis were identified after 53 procedures (15.2%) in 50 eyes (16.9%) of 49 patients (18.3%). The incidences of culture-positive bacterial and fungal keratitis were 0.014 and 0.004 per eye-year, respectively. Persistent corneal epithelial defect formation (P < 0.001) and cicatricial disease (HR: 1.98, 95% confidence interval, 1.02-3.83) were associated with a significantly higher incidence of infectious keratitis. For the 53 cases with a known outcome, medical therapy achieved resolution of infection in 34 cases (64.2%), whereas kpro explantation was required in 19 cases (35.8%). Infectious keratitis was associated with an increased risk for kpro explantation (HR: 3.09, 95% confidence interval, 1.92-4.79). CONCLUSIONS: Infectious keratitis develops in approximately 17% of eyes after kpro implantation, with a higher rate of culture-positive bacterial than fungal keratitis. The observed rate of microbial keratitis suggests the need for additional topical antimicrobial prophylaxis in eyes at higher risk, such as those with preexisting cicatricial disease or postoperative persistent corneal epithelial defect formation.

Immune Checkpoint Inhibitor-associated Uveitis: Review of Treatments and Outcomes.

Purpose: Immune checkpoint inhibitors (ICPIs), novel immunotherapy agents employed in the treatment of metastatic melanoma and other solid tumors, are associated with immune-related adverse events, including ocular inflammation. We review the current literature on immune checkpoint inhibitor-associated uveitis (ICIPU).Methods: A comprehensive literature review utilizing MEDLINE/PubMed, Cochrane, and Web of Science databases was conducted. One hundred and twenty-six cases of ICPIU reported in the literature prior to January 31, 2020 were identified and reviewed.Results: ICPIs were associated with 126 cases of anterior uveitis, intermediate uveitis, posterior uveitis, and panuveitis from 67 reports in the literature. Patients typically developed intraocular inflammation a median of 9 weeks after initiation of ICPI and 83.6% of the patients developed uveitis within 6 months. The vast majority of patients recovered to within one line of baseline vision in response to topical, local, and/or systemic steroid treatment as well as the cessation of medication.Conclusions: Prompt recognition and steroid treatment of ICPIU are critical to the care of patients receiving ICPIs.

Outcomes of Limbal Stem Cell Transplant: A Meta-analysis.

Importance: Limbal stem cell transplant (LSCT) can be categorized as direct autologous limbal transplant (AULT), direct allogenic limbal transplant (ALLT), cultivated autologous limbal stem cells transplant (cAULT), and cultivated allogenic limbal stem cells transplant (cALLT). To our knowledge, there is no study directly comparing the outcomes and complications of these procedures. Objective: To evaluate the outcomes of different LSCT procedures. Data Source: We searched PubMed, EMBASE, Web of Science, and Cochrane without language filter for peer-reviewed articles about LSCT. The latest search was performed on June 30, 2019. Study Selection: Clinical studies with the outcome of at least 20 eyes after LSCT were included. Animal studies and studies of other surgical interventions were excluded. Data Extraction and Synthesis: Two reviewers independently abstracted the data from each study. Heterogeneity was evaluated with the I2 statistic, and a meta-analysis was performed using the random-effects model. Main Outcomes and Measures: Outcome measures included the improvement of ocular surface, visual acuity (VA), and adverse events of recipient eyes and donor eyes. Results: Forty studies (2202 eyes) with a mean (SD) follow-up of 31.3 (20.9) months met the inclusion criteria. The mean (SD) age of study participants was 38.4 (13.1) years, and men accounted for 74%. The number of eyes that underwent AULT, ALLT, cAULT, and cALLT were 505, 742, 771, and 184, respectively. Improvement of the ocular surface was achieved in 74.5% of all eyes, 85.7% of eyes after AULT (95% CI, 79.5%-90.3%), 84.7% after cAULT (95% CI, 77.2%-90.0%), 57.8% after ALLT (95% CI, 49.0%-66.1%), and 63.2% after cALLT (95% CI, 49.3%-75.2%). Autologous limbal transplantation resulted in a greater VA improvement rate (76%) than did the other 3 procedures (cAULT: 56.4%; ALLT: 52.3%; cALLT: 43.3%; all P < .001). The most common adverse events in all recipient eyes were recurrent/persistent epithelial erosion (10.5%; 95% CI, 7.2%-23.3%) and elevated intraocular pressure (intraocular pressure, 1.7%; 95% CI, 0.5%-7.8%). Patients who underwent ALLT had the highest rate of recurrent epithelial erosion (27.8%; 95% CI, 17.1%-41.9%) and intraocular pressure elevation (6.3%; 95% CI, 1.8%-19.4%). Conclusions and Relevance: These findings suggest LSCT can improve or stabilize the corneal surface with a low rate of severe ocular complications and that autologous LSCT may have a higher success rate and fewer complications than allogenic LSCT.

Limbal Stem Cell Deficiency After Glaucoma Surgery.

PURPOSE: To characterize the clinical presentation of limbal stem cell deficiency (LSCD) associated with glaucoma surgeries. METHODS: This is a retrospective cross-sectional study of patients with LSCD and glaucoma who presented to the Stein Eye Institute at the University of California, Los Angeles, between 2009 and 2018. Patients who underwent trabeculectomy and/or aqueous shunt surgery were included. The severity of LSCD was staged using global consensus guidelines and a clinical scoring system, and basal epithelial cell density was measured by in vivo confocal microscopy. Anatomic locations of glaucoma and non-glaucoma surgeries, locations of LSCD, and severity of LSCD were compared. RESULTS: Fifty-one eyes of 41 patients with LSCD associated with glaucoma surgery were included in this study. LSCD in these patients uniquely featured sectoral replacement of corneal epithelium by conjunctival epithelium, without corneal neovascularization or pannus. The sites of glaucoma surgery strongly correlated with the locations of LSCD (P = 0.002). There was a trend toward increased severity of LSCD in eyes with 2 or more glaucoma surgeries as compared to eyes with 1 glaucoma surgery, although the difference did not reach statistical significance (P = 0.3). Use of topical glaucoma medications correlated with LSCD severity, while the impact of antimetabolites did not reach statistical significance. The location of glaucoma drainage surgery is correlated with the location of LSCD. CONCLUSIONS: LSCD associated with glaucoma surgery has clinical features distinct from LSCD resulting from other etiologies. Further study is required to delineate the full impact of glaucoma surgery on limbal stem cell function and survival.

ISCHEMIC CENTRAL RETINAL VEIN OCCLUSION IN THE ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR ERA.

PURPOSE: Anti-vascular endothelial growth factor therapy has improved the prognosis for patients with central retinal vein occlusion (CRVO). However, most studies published to date exclude ischemic CRVO. The purpose of this study was to describe the outcome in eyes with ischemic CRVO treated with anti-vascular endothelial growth factor therapy. METHODS: Thirty-seven patients with ischemic CRVO from 3 centers were followed for at least 6 months. Data on patient demographic, vision status, and anti-vascular endothelial growth factor treatments were collected. RESULTS: Average number of injections during the study period was 5. Younger age was associated with improved vision (P = 0.006). Patients with improved visual outcomes tended to have macular edema as the primary indication for treatment, whereas patients with worse outcomes tended to have neovascularization as the primary indication for treatment. CONCLUSION: This study highlights significant variability in the use of anti-vascular endothelial growth factor therapy for ischemic CRVO and underscores that eyes with neovascularization tend to have worse visual outcomes.

Clinical applications of fundus autofluorescence in retinal disease.

Fundus autofluorescence (FAF) is a non-invasive retinal imaging modality used in clinical practice to provide a density map of lipofuscin, the predominant ocular fluorophore, in the retinal pigment epithelium. Multiple commercially available imaging systems, including the fundus camera, the confocal scanning laser ophthalmoscope, and the ultra-widefield imaging device, are available to the clinician. Each offers unique advantages for evaluating various retinal diseases. The clinical applications of FAF continue to expand. It is now an essential tool for evaluating age related macular degeneration, macular dystrophies, retinitis pigmentosa, white dot syndromes, retinal drug toxicities, and various other retinal disorders. FAF may detect abnormalities beyond those detected on funduscopic exam, fluorescein angiography, or optical coherence tomography, and can be used to elucidate disease pathogenesis, form genotype-phenotype correlations, diagnose and monitor disease, and evaluate novel therapies. Given its ease of use, non-invasive nature, and value in characterizing retinal disease, FAF enjoys increasing clinical relevance. This review summarizes common ocular fluorophores, imaging modalities, and FAF findings for a wide spectrum of retinal disorders.

Mutational Consequences of Ciprofloxacin in Escherichia coli.

We examined the mutagenic specificity of the widely used antibiotic ciprofloxacin (CPR), which displays weak to moderate mutagenic activity in several bacteria and generates short in-frame deletions in rpoB in Staphylococcus aureus To determine the spectrum of mutations in a system where any gene knockout would result in a recovered mutant, including frameshifts and both short and long deletions, we examined CPR-induced mutations in the thymidylate synthase-encoding thyA gene. Here, any mutation resulting in loss of thymidylate synthase activity generates trimethoprim (Trm) resistance. We found that deletions and insertions in all three reading frames predominated in the spectrum. They tend to be short deletions and cluster in two regions, one being a GC-rich region with potential extensive secondary structures. We also exploited the well-characterized rpoB-Rif(r) system in Escherichia coli to determine that cells grown in the presence of sublethal doses of CPR not only induced short in-frame deletions in rpoB, but also generated base substitution mutations resulting from induction of the SOS system. Some of the specific point mutations prominent in the spectrum of a strain that overproduces the dinB-encoded Pol IV were also present after growth in CPR. However, these mutations disappeared in CPR-treated dinB mutants, whereas the deletions remained. Moreover, CPR-induced deletions also occurred in a strain lacking all three SOS-induced polymerases. We discuss the implications of these findings for the consequences of overuse of CPR and other antibiotics.

KIR and HLA Genotypes Implicated in Reduced Killer Lymphocytes Immunity Are Associated with Vogt-Koyanagi-Harada Disease.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are killer lymphocytes that provide defense against viral infections and tumor transformation. Analogous to that of CTL, interactions of killer-cell immunoglobulin-like receptors (KIR) with specific human leukocyte antigen (HLA) class I ligands calibrate NK cell education and response. Gene families encoding KIRs and HLA ligands are located on different chromosomes, and feature variation in the number and type of genes. The independent segregation of KIR and HLA genes results in variable KIR-HLA interactions in individuals, which may impact disease susceptibility. We tested whether KIR-HLA combinations are associated with Vogt-Koyanagi-Harada (VKH) disease, a bilateral granulomatous panuveitis that has strong association with HLA-DR4. We present a case control study of 196 VKH patients and 209 controls from a highly homogeneous native population of Japan. KIR and HLA class I genes were typed using oligonucleotide hybridization method and analyzed using two-tailed Fisher's exact probabilities. The incidence of Bx-KIR genotypes was decreased in VKH patients (odds ratio [OR] 0.58, P = 0.007), due primarily to a decrease in centromeric B-KIR motif and its associated KIRs 2DS2, 2DL2, 2DS3, and 2DL5B. HLA-B22, implicated in poor immune response, was increased in VKH (OR = 4.25, P = 0.0001). HLA-Bw4, the ligand for KIR3DL1, was decreased in VKH (OR = 0.59, P = 0.01). The KIR-HLA combinations 2DL2+C1/C2 and 3DL1+Bw4, which function in NK education, were also decreased in VKH (OR = 0.49, P = 0.012; OR = 0.59, P = 0.013). Genotypes missing these two inhibitory KIR-HLA combinations in addition to missing activating KIRs 2DS2 and 2DS3 were more common in VKH (OR = 1.90, P = 0.002). These results suggest that synergistic hyporesponsiveness of NK cells (due to poor NK education along with missing of activating KIRs) and CTL (due to HLA-B22 restriction) fail to mount an effective immune response against viral-infection that may trigger VKH pathogenesis in genetically susceptible individuals, such as HLA-DR4 carriers.

Mutagen Synergy: Hypermutability Generated by Specific Pairs of Base Analogs.

UNLABELLED: We tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 × 10(-8)) in the rpoB gene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T→G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools. IMPORTANCE: Although mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent.