Prospective Study of the Diagnostic Accuracy of the In Vivo Laser Scanning Confocal Microscope for Severe Microbial Keratitis.

To determine the diagnostic accuracy of in vivo confocal microscopy (IVCM) for moderate to severe microbial keratitis (MK). Double-masked prospective cohort study. Consecutive patients presenting to Aravind Eye Hospital, Madurai, India, between February 2012 and February 2013 with MK (diameter ≥3 mm, excluding descemetocele, perforation, or herpetic keratitis). Following examination, the corneal ulcer was scanned by IVCM (HRT3/RCM, Heidelberg Engineering, Heidelberg, Germany). Images were graded for the presence or absence of fungal hyphae or Acanthamoeba cysts by the confocal microscopist who performed the scan (masked to microbial diagnosis) and 4 other experienced confocal graders (masked to clinical features and microbiology). The regrading of the shuffled image set was performed by 3 graders, 3 weeks later. Corneal-scrape samples were collected for microscopy and culture. The main outcome measures were sensitivity, specificity, and positive and negative predictive values of IVCM compared with those of a reference standard of positive culture or light microscopy. Sensitivities and specificities for multiple graders were pooled and 95% confidence intervals calculated using a bivariate random-effects regression model. The study enrolled 239 patients with MK. Fungal infection was detected in 176 (74%) and Acanthamoeba in 17 (7%) by microbiological methods. IVCM had an overall pooled (5 graders) sensitivity of 85.7% (95% confidence interval [CI]: 82.2%-88.6%) and pooled specificity of 81.4% (95% CI: 76.0%-85.9%) for fungal filament detection. For Acanthamoeba, the pooled sensitivity was 88.2% (95% CI: 76.2%-94.6%) and pooled specificity was 98.2% (95% CI: 94.9%-99.3%). Intergrader agreement was good: κ was 0.88 for definite fungus; κ was 0.72 for definite Acanthamoeba. Intragrader repeatability was high for both definite fungus (κ: 0.88-0.95) and definite Acanthamoeba classification (κ: 0.63-0.90). IVCM images from 11 patients were considered by all 5 graders to have a specific organism present (10 fungus, 1 Acanthamoeba) but had negative results via culture and light microscopy. Laser scanning IVCM performed with experienced confocal graders has high sensitivity, specificity, and test reproducibility for detecting fungal filaments and Acanthamoeba cysts in moderate to large corneal ulcers in India. This imaging modality was particularly useful for detecting organisms in deep ulcers in which culture and light microscopy results were negative

Detection and quantification of human adenovirus genomes in Acanthamoeba isolated from swimming pools

ABSTRACT Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV) in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16) were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR). HAdVs were detected in 62.5% (10/16) of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water

In Vivo Corneal Confocal Microscopy and Histopathology of Keratitis Fugax Hereditaria from a Pathogenic Variant in NLRP3

Purpose To apply in vivo corneal confocal microscopy (IVCM) to study the pathogenesis of keratitis (keratoendotheliitis) fugax hereditaria, an autosomal dominant cryopyrin-associated periodic keratitis, associated with the c.61G>C pathogenic variant in the NLRP3 gene, in its acute and chronic phase, and to report histopathological findings after penetrating keratoplasty. Design Observational case series Methods Study population: Six patients during an acute attack, 18 patients in the chronic phase, and one patient who underwent penetrating keratoplasty.Intervention: Sanger sequencing for the NLRP3 variant c.61C>G. Clinical examination, corneal photography, IVCM, light microscopy and immunohistochemistry. Main Outcome Measures: IVCM and histopathological findings. Results During the acute attack, hyperreflective cellular structures consistent with inflammatory cells transiently occupied the anterior to middle layers of the corneal stroma. Other corneal layers were unremarkable. With recurring attacks, central oval stromal opacities accumulated. IVCM revealed that they contained long hyperreflective needle-shaped structures in extracellular matrix. By light microscopy, the anterior half of the stroma displayed thin and finely vacuolated lamellae, and keratocytes throughout the stroma were immunopositive for syndecan. Conclusions The acute attacks and chronic stromal deposits mainly involve the anterior to middle layers of the corneal stroma, and the disease is primarily a keratitis rather than a keratoendotheliitis. IVCM shows that inflammatory cells invade only the stroma during an acute attack. IVCM and light microscopic findings suggest that the central corneal opacities represent gradual deposition of extracellular lipids. The disease could make a good in vivo model to study activation of the NLRP3 inflammasome in cryopyrin-associated periodic syndromes.Peer reviewe

Effects of oleoresin capsicum pepper spray on human corneal morphology and sensitivity

To examine the potential harmful effects on corneal structure, innervation, and sensitivity of a spray containing the neurotoxin capsaicin (oleoresin capsicum, OC)