Multilocus Sequence Typing Is a Reliable Alternative Method to DNA Fingerprinting for Discriminating among Strains of Candida albicans

Multilocus sequence typing (MLST) has emerged as a powerful new DNA-typing tool for the evaluation of intraspecies genetic relatedness. This method relies on DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. However, the results of the MLST scheme for Candida albicans have heretofore never been formally compared to those of other established typing techniques. To assess the value of MLST relative to those of other DNA fingerprinting tools for discriminating among strains of C. albicans, we applied it to a previously well-characterized set of 29 C. albicans isolates evaluated by the random amplified polymorphic DNA (RAPD), multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization probe techniques. MLST identified three clusters of genetically related isolates, with 82.3% direct concordance with MLEE, 82.7% with RAPD analysis, and 86.2% with the Ca3 Southern hybridization technique. When MLST was applied to a subset of 22 isolates of unrelated origins, it identified 21 independent diploid sequence types (DSTs), resulting in a discriminatory power of 99.6%. These DSTs were 96.9, 99.6, and 99.6% concordant with the genotypes identified by RAPD analysis, MLEE, and Ca3 Southern hybridization, respectively. These results demonstrate that MLST is a highly effective technique that performs at least comparably to other established DNA fingerprinting techniques

spa Typing Method for Discriminating among Staphylococcus aureus Isolates: Implications for Use of a Single Marker To Detect Genetic Micro- and Macrovariation

Strain typing of microbial pathogens has two major aims: (i) to index genetic microvariation for use in outbreak investigations and (ii) to index genetic macrovariation for use in phylogenetic and population-based analyses. Until now, there has been no clear indication that one genetic marker can efficiently be used for both purposes. Previously, we had shown that DNA sequence analysis of the protein A gene variable repeat region (spa typing) provides a rapid and accurate method to discriminate Staphylococcus aureus outbreak isolates from those deemed epidemiologically unrelated. Here, using the hypothesis that the genetic macrovariation within a low-level recombinogenic species would accurately be characterized by a single-locus marker, we tested whether spa typing could congruently index the extensive genetic variation detected by a whole-genome DNA microarray in a collection of 36 isolates, which was recovered from 10 countries on four continents over a period of four decades, that is representative of the breadth of diversity within S. aureus. Using spa and coa typing, pulsed-field gel electrophoresis (PFGE), and microarray and multilocus enzyme electrophoresis (MLEE) data in molecular epidemiologic and evolutionary analyses, we determined that S. aureus likely has a primarily clonal population structure and that spa typing can singly index genetic variation with 88% direct concordance with the microarray and can correctly assign isolates to phylogenetic lineages. spa typing performed better than MLEE, PFGE, and coa typing in discriminatory power and in the degree of agreement with the microarray at various phylogenetic depths. This study showed that genetic analysis of the repeat region of protein A comprehensively characterizes both micro- and macrovariation in the primarily clonal population structure of S. aureus

Use of a Multiplex Molecular Beacon Platform for Rapid Detection of Methicillin and Vancomycin Resistance in Staphylococcus aureus

Drug resistance, particularly vancomycin and methicillin resistance, in Staphylococcus aureus continues to emerge as a significant public health threat in both the hospital and community settings. In addition to the limited treatment options, S. aureus strains acquire and express numerous virulence factors that continue to increase its ability to cause a wide spectrum of human disease. As a result, empirical treatment decisions are confounded and there is a heightened need for a diagnostic test (or assay) to rapidly identify antibiotic resistance and specific virulence determinants and indicate the appropriate treatment. To that end we developed a platform using multiplex molecular beacon probes with real-time PCR for the rapid detection of drug resistance-determining genes and virulence factors in S. aureus. In this study, we demonstrate the specificity and sensitivity of our platform for detection of the genes conferring methicillin (mecA) and vancomycin (vanA) resistance as well as a gene encoding the virulence factor Panton-Valentine leucocidin (lukF) in S. aureus isolates

Dissociations of the Fluocinolone Acetonide Implant: The Multicenter Uveitis Steroid Treatment (MUST) Trial and Follow-up Study

© 2016 Elsevier Inc. Purpose: To describe fluocinolone acetonide implant dissociations in the Multicenter Uveitis Steroid Treatment (MUST) Trial. Design: Randomized clinical trial with extended follow-up. Methods: Review of data collected on the first implant in the eye(s) of participants. Dissociation was defined as the drug pellet no longer being affixed to the strut and categorized as spontaneous or surgically related. Results: A total of 250 eyes (146 patients) had at least 1 implant placed. Median follow-up time after implant placement was 6 years (range 0.5–9.2 years). Thirty-four dissociations were reported in 30 participants. There were 22 spontaneous events in 22 participants; 6-year cumulative risk of a spontaneous dissociation was 4.8% (95% confidence interval [CI]: 2.4%–9.1%). The earliest event occurred 4.8 years after placement. Nine of 22 eyes with data had a decline in visual acuity ≥5 letters temporally related to the dissociation. Thirty-nine implant removal surgeries were performed, 33 with replacement. Twelve dissociations were noted during implant removal surgeries in 10 participants (26%, 95% CI 15%–48%); 5 of these eyes had a decline in visual acuity ≥5 letters after surgery. The time from implant placement to removal surgery was longer for the surgeries at which dissociated implants were identified than for those without one (5.7 vs 3.7 years, P \u3c.001). Overall, visual acuity declined 15 or more letters from pre-implant values in 22% of affected eyes; declines were frequently associated with complications of uveitis or its treatment. Conclusion: There is an increasing risk of dissociation of Retisert implants during follow-up; the risk is greater with removal/exchange surgeries, but the risk of both spontaneous and surgically related events increases with longevity of the implants. In 22% of affected eyes visual acuity declined by 15 letters. In the context of eyes with moderate to severe uveitis for years, this rate is not unexpected

Factors Predicting Visual Acuity Outcome in Intermediate, Posterior, and Panuveitis: The Multicenter Uveitis Steroid Treatment (MUST) Trial

© 2015 Elsevier Inc. Purpose To identify factors associated with best-corrected visual acuity (BCVA) presentation and 2-year outcome in 479 intermediate, posterior, and panuveitic eyes. Design Cohort study using randomized controlled trial data. Methods Multicenter Uveitis Steroid Treatment (MUST) Trial masked BCVA measurements at baseline and at 2 years follow-up used gold-standard methods. Twenty-three clinical centers documented characteristics per protocol, which were evaluated as potential predictive factors for baseline BCVA and 2-year change in BCVA. Results Baseline factors significantly associated with reduced BCVA included age ≥50 vs \u3c50 years; posterior vs intermediate uveitis; uveitis duration \u3e10 vs \u3c6 years; anterior chamber (AC) flare \u3egrade 0; cataract; macular thickening; and exudative retinal detachment. Over 2 years, eyes better than 20/50 and 20/50 or worse at baseline improved, on average, by 1 letter (P =.52) and 10 letters (P \u3c.001), respectively. Both treatment groups and all sites of uveitis improved similarly. Factors associated with improved BCVA included resolution of active AC cells, resolution of macular thickening, and cataract surgery in an initially cataractous eye. Factors associated with worsening BCVA included longer duration of uveitis (6-10 or \u3e10 vs \u3c6 years), incident AC flare, cataract at both baseline and follow-up, pseudophakia at baseline, persistence or incidence of vitreous haze, and incidence of macular thickening. Conclusions Intermediate, posterior, and panuveitis have a similarly favorable prognosis with both systemic and fluocinolone acetonide implant treatment. Eyes with more prolonged/severe inflammatory damage and/or inflammatory findings initially or during follow-up have a worse visual acuity prognosis. The results indicate the value of implementing best practices in managing inflammation

Validity of the Prodromal Risk Syndrome for First Psychosis: Findings From the North American Prodrome Longitudinal Study

Treatment and prevention studies over the past decade have enrolled patients believed to be at risk for future psychosis. These patients were considered at risk for psychosis by virtue of meeting research criteria derived from retrospective accounts of the psychosis prodrome. This study evaluated the diagnostic validity of the prospective “prodromal risk syndrome” construct. Patients assessed by the Structured Interview for Prodromal Syndromes as meeting criteria of prodromal syndromes (n = 377) from the North American Prodrome Longitudinal Study were compared with normal comparison (NC, n = 196), help-seeking comparison (HSC, n = 198), familial high-risk (FHR, n = 40), and schizotypal personality disorder (SPD, n = 49) groups. Comparisons were made on variables from cross-sectional demographic, symptom, functional, comorbid diagnostic, and family history domains of assessment as well as on follow-up outcome. Prodromal risk syndrome patients as a group were robustly distinguished from NC subjects across all domains and distinguished from HSC subjects and from FHR subjects on most measures in many of these domains. Adolescent and young adult SPD patients, while distinct from prodromal patients on definitional grounds, were similar to prodromals on multiple measures, consistent with SPD in young patients possibly being an independent risk syndrome for psychosis. The strong evidence of diagnostic validity for the prodromal risk syndrome for first psychosis raises the question of its evaluation for inclusion in Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition)

Factors Predicting Visual Acuity Outcome in Intermediate, Posterior, and Panuveitis: The Multicenter Uveitis Steroid Treatment (MUST) Trial

PurposeTo identify factors associated with best-corrected visual acuity (BCVA) presentation and 2-year outcome in 479 intermediate, posterior, and panuveitic eyes.DesignCohort study using randomized controlled trial data.MethodsMulticenter Uveitis Steroid Treatment (MUST) Trial masked BCVA measurements at baseline and at 2 years follow-up used gold-standard methods. Twenty-three clinical centers documented characteristics per protocol, which were evaluated as potential predictive factors for baseline BCVA and 2-year change in BCVA.ResultsBaseline factors significantly associated with reduced BCVA included age ≥50 vs <50 years; posterior vs intermediate uveitis; uveitis duration >10 vs <6 years; anterior chamber (AC) flare >grade 0; cataract; macular thickening; and exudative retinal detachment. Over 2 years, eyes better than 20/50 and 20/50 or worse at baseline improved, on average, by 1 letter (P = .52) and 10 letters (P < .001), respectively. Both treatment groups and all sites of uveitis improved similarly. Factors associated with improved BCVA included resolution of active AC cells, resolution of macular thickening, and cataract surgery in an initially cataractous eye. Factors associated with worsening BCVA included longer duration of uveitis (6-10 or >10 vs <6 years), incident AC flare, cataract at both baseline and follow-up, pseudophakia at baseline, persistence or incidence of vitreous haze, and incidence of macular thickening.ConclusionsIntermediate, posterior, and panuveitis have a similarly favorable prognosis with both systemic and fluocinolone acetonide implant treatment. Eyes with more prolonged/severe inflammatory damage and/or inflammatory findings initially or during follow-up have a worse visual acuity prognosis. The results indicate the value of implementing best practices in managing inflammation

Dissociations of the Fluocinolone Acetonide Implant: The Multicenter Uveitis Steroid Treatment (MUST) Trial and Follow-up Study

PurposeTo describe fluocinolone acetonide implant dissociations in the Multicenter Uveitis Steroid Treatment (MUST) Trial.DesignRandomized clinical trial with extended follow-up.MethodsReview of data collected on the first implant in the eye(s) of participants. Dissociation was defined as the drug pellet no longer being affixed to the strut and categorized as spontaneous or surgically related.ResultsA total of 250 eyes (146 patients) had at least 1 implant placed. Median follow-up time after implant placement was 6 years (range 0.5-9.2 years). Thirty-four dissociations were reported in 30 participants. There were 22 spontaneous events in 22 participants; 6-year cumulative risk of a spontaneous dissociation was 4.8% (95% confidence interval [CI]: 2.4%-9.1%). The earliest event occurred 4.8 years after placement. Nine of 22 eyes with data had a decline in visual acuity ≥5 letters temporally related to the dissociation. Thirty-nine implant removal surgeries were performed, 33 with replacement. Twelve dissociations were noted during implant removal surgeries in 10 participants (26%, 95% CI 15%-48%); 5 of these eyes had a decline in visual acuity ≥5 letters after surgery. The time from implant placement to removal surgery was longer for the surgeries at which dissociated implants were identified than for those without one (5.7 vs 3.7 years, P < .001). Overall, visual acuity declined 15 or more letters from pre-implant values in 22% of affected eyes; declines were frequently associated with complications of uveitis or its treatment.ConclusionThere is an increasing risk of dissociation of Retisert implants during follow-up; the risk is greater with removal/exchange surgeries, but the risk of both spontaneous and surgically related events increases with longevity of the implants. In 22% of affected eyes visual acuity declined by 15 letters. In the context of eyes with moderate to severe uveitis for years, this rate is not unexpected