Quantitatively comparing weekly changes in retinal vascular characteristics of eyes eventually treated versus not treated for retinopathy of prematurity.

PURPOSE: To quantitatively compare retinal vascular characteristics over time in eyes eventually treated versus not treated for retinopathy of prematurity (ROP), using ROPtool analysis of narrow-field retinal images. METHODS: This longitudinal study used prospectively collected narrow-field retinal images of infants screened for ROP, prior to treatment, if needed. Images were analyzed using a methodology that combines quadrant-level measures from several images of the same eye. For the longitudinal analysis, one examination per postmenstrual age (PMA) was included per eye. We compared the following ROPtool indices and their change per week between eyes eventually treated versus not treated for ROP: tortuosity index (TI), dilation index (DI), sum of adjusted indices (SAI), and tortuosity-weighted plus (TWP). Analysis was performed on three levels: eye (mean value/eye), quadrant (highest quadrant value/eye), and blood vessel (highest blood vessel value/eye). RESULTS: Of 832 examinations (99 infants), 745 images (89.5%) had 3-4 quadrants analyzable by ROPtool. On the eye level, ROPtool indices differed between eyes eventually treated versus not treated at PMA of 33-35 and 37 weeks for TI, SAI, and TWP, and at PMA of 33-34 and 37 weeks for DI (P ≤ 0.0014), and change per week differed between eyes eventually treated versus not treated only for SAI at PMA of 32 weeks (P < 0.001). CONCLUSIONS: Quantitative analysis of retinal vascular characteristics using ROPtool can help predict eventual need for treatment for ROP as early as 32 weeks PMA. ROPtool index values were more useful than change in these indices to predict eyes that would eventually need treatment for ROP.

ROPtool analysis of plus and pre-plus disease in narrow-field images: a multi-image quadrant-level approach.

BACKGROUND: The presence of plus disease is important in determining when to treat retinopathy of prematurity (ROP), but the diagnosis of plus disease is subjective. Semiautomated computer programs (eg, ROPtool) can objectively measure retinal vascular characteristics in retinal images, but are limited by image quality. The purpose of this study was to evaluate whether ROPtool can accurately identify pre-plus and plus disease in narrow-field images of varying qualities using a new methodology that combines quadrant-level data from multiple images of a single retina. METHODS: This was a cross-sectional study of previously collected narrow-field retinal images of infants screened for ROP. Using one imaging session per infant, we evaluated the ability of ROPtool to analyze images using our new methodology and the accuracy of ROPtool indices (tortuosity index [TI], maximum tortuosity [Tmax], dilation index [DI], maximum dilation [Dmax], sum of adjusted indices [SAI], and tortuosity-weighted plus [TWP]) to identify pre-plus and plus disease in images compared to clinical examination findings. RESULTS: Of 198 eyes (from 99 infants) imaged, 769/792 quadrants (98%) were analyzable. Overall, 98% of eyes had 3-4 analyzable quadrants. For plus disease, area under the curves (AUCs) of receiver operating characteristic curves were: TWP (0.98) > TI (0.97) = Tmax (0.97) > SAI (0.96) > DI (0.88) > Dmax (0.84). For pre-plus or plus disease, AUCs were: TWP (0.95) > TI (0.94) = Tmax (0.94) = SAI (0.94) > DI (0.86) > Dmax (0.83). CONCLUSIONS: Using a novel methodology combining quadrant-level data, ROPtool can analyze narrow-field images of varying quality to identify pre-plus and plus disease with high accuracy.

Evaluating a Portable, Noncontact Fundus Camera for Retinopathy of Prematurity Screening by Nonophthalmologist Health Care Workers.

PURPOSE: To evaluate (1) the feasibility of non-ophthalmologist healthcare workers (HCWs) to obtain images of sufficient quality for retinopathy of prematurity (ROP) screening using a FDA-approved portable, non-contact, narrow-field fundus camera (i.e., Pictor™), and (2) the accuracy of grading these images to identify infants who developed treatment-warranted (type 1) ROP. DESIGN: Prospective cohort study. SUBJECTS: Infants undergoing routine ROP screening examinations (i.e. birth weight ≤1500 grams and/or gestational age ≤30 weeks or selected infants with a birth weight of 1500-2000g or gestational age >30 weeks and an unstable clinical course). METHODS: We prospectively recruited infants undergoing ROP screening examinations at a community hospital. On the same day an ophthalmologist examined them, a trained HCW imaged their retinas using the non-contact camera. Two masked ROP experts graded these images remotely. We calculated both the percentage of gradable images (i.e. having at least 3 quadrants with sufficient image quality), as well as the accuracy of identifying infants who developed type 1 ROP. MAIN OUTCOME MEASURES: Percentage of gradable images and the sensitivity and specificity of each grader for identifying infants with type 1 ROP by grading for the presence of pre-plus or plus disease. RESULTS: Ninety-nine infants were included. Overall, 92.4% and 94.2% of all infant imaging sessions were considered gradable by graders 1 and 2, respectively. Amongst gradable images, the sensitivity of both graders for identifying type 1 ROP by grading for the presence of pre-plus or plus disease was 100% (95% confidence interval (CI): 95-100%) and the specificity 91% (95% CI: 83-95%) for grader 1 and 93% (95% CI: 86-96%) for grader 2. CONCLUSIONS: It was highly feasible for trained HCWs to obtain digital retinal images of sufficient quality for ROP screening using a non-contact fundus camera. By grading for the presence of pre-plus or plus disease, graders identified infants who developed type 1 ROP with high sensitivity and specificity. The use of portable, non-contact retinal cameras by trained HCWs could increase our workforce in ROP screening and identify infants needing an indirect ophthalmoscopy examination by an ophthalmologist.

Non-contact retinal imaging compared to indirect ophthalmoscopy for retinopathy of prematurity screening: infant safety profile.

OBJECTIVE: Most retinopathy of prematurity screening involves an ophthalmologist performing indirect ophthalmoscopy, which can be stressful to infants. The purpose of this study is to evaluate the safety profile (using cardiopulmonary events as an indicator) of imaging infants with a non-contact retinal camera compared to examining them using indirect ophthalmoscopy. STUDY DESIGN: Prospective cohort study of 99 infants at a community hospital who were examined using indirect ophthalmoscopy and imaged using a non-contact retinal camera for retinopathy of prematurity. We evaluated the difference in the occurrence of safety events (i.e., clinically significant bradycardia, tachycardia, oxygen desaturation, or apnea) following the clinical examination versus retinal imaging. RESULT: Safety events occurred after 0.8% (n = 1) of imaging sessions and 5.8% (n = 18) of clinical examinations (mean difference = -0.055 (p = 0.015), favoring imaging). CONCLUSION: Retinal imaging with a non-contact camera was well tolerated and less stressful to infants compared to indirect ophthalmoscopy by an ophthalmologist.

Preference of Formosan subterranean termites for blue-stained southern yellow pine sapwood.

Little research has been conducted to investigate interactions between the invasive Formosan subterranean termite, Coptotermes formosanus Shiraki, and pine bark beetles native to the southeastern United States. Facilitative interactions between these organisms could alter stand dynamics and impact wood utilization strategies. American Wood Protection Association Standard E1-09 choice tests were carried out to determine the feeding preference of Formosan subterranean termites for blue-stained versus unstained southern yellow pine sapwood. Three separate colonies of Formosan subterranean termites consumed on average twice as much air-dried blue-stained southern yellow pine sapwood over unstained air-dried controls. Additionally, Formosan subterranean termites consumed over five-times more kiln-dried blue-stained sapwood than unstained kiln-dried control wafers. The implications of these results are particularly relevant to pine forest ecology, nutrient cycling, and the utilization of blue-stained southern pine building products in the southeastern United States, where Formosan subterranean termites have become established.

Ciclosporin modulates the responses of canine progenitor epidermal keratinocytes (CPEK) to toll-like receptor agonists.

Toll-like receptor (TLR) 2 dependent pathways have an important role in the antimicrobial defense of human keratinocytes, and various factors and compounds have been shown to affect those pathways. Investigating Toll-like receptor function in canine keratinocytes and the potential for their modulation is of similar relevance in dogs due to the frequency of staphylococcal skin infections in this species, particularly in the context of canine atopic dermatitis. This pilot study hypothesized that ciclosporin would have a modulatory effect on the cytokine and TLR mRNA expression of canine progenitor epidermal keratinocytes in response to TLR2 agonists. No detectable up-regulation of TLR2, TLR4, IL-8 and TNF-α mRNA was detected following exposure to FSL-1, Pam3CSK4 and staphylococcal peptidoglycan (PGN). Ciclosporin alone did not alter the expression levels of these transcripts but in the presence of ciclosporin, TNF-α mRNA expression was upregulated in response to all three agonists and both TNF-α and IL-8 transcript abundance was increased in response to Pam3CSK4. The enhanced responsiveness of canine keratinocytes to TLR2 agonists in response to ciclosporin may imply that administration of this drug might enhance the innate immune barrier of skin.

Occurrence of Baylisascaris transfuga in wild populations of European brown bears (Ursus arctos) as identified by a new PCR method.

The European brown bear (Ursus arctos) is a species present in limited areas of Europe and several small populations are considered endangered. This species can be affected by a range of parasites. In particular, the genus Baylisascaris is an emerging parasite of wild animals which can cause severe larva migrans syndrome in aberrant hosts, which include 100 species of birds, mammals and also humans. Baylisascaris transfuga is the species reported from bears, and with the exception of a few laboratory trials, little is known about the capacity of this species to infect other animals. Furthermore, the identification of this species has traditionally been based on light microscopy, using either morphology of the adults at necropsy or detection of the eggs in faeces, which are methods limited by the experience and the efforts of the observer. The current work was aimed at developing a specific PCR to detect the parasite directly from faecal samples of naturally infected brown bears in the field, without the need for previous flotation. Using eggs and adults of B. transfuga collected in Croatia, we first developed a PCR to detect a portion of the second internal transcribed spacer region (ITS-2) of ribosomal DNA and then applied it to bear faecal samples spiked with a known number of B. transfuga eggs. We show here for the first time that this method allows the detection of a minimum of two Baylisascaris eggs in 25mg of faecal material, thus it demonstrates a high diagnostic capacity that could be applied to evaluate the prevalence of the parasite in faecal samples from wild populations of brown bears.

First Report of Laurel Wilt Caused by Raffaelea lauricola on Sassafras in Mississippi.

Laurel wilt is caused by the fungus Raffaelea lauricola T.C. Harrin., Aghayeva & Fraedrich and is lethal to redbay (Persea borbonia (L.) Spreng.), sassafras (Sassafras albidum (Nutt.) Nees), and other species in the Lauraceae (1). The fungus is carried by the redbay ambrosia beetle (Xyleborus glabratus Eichh.), which is native to Asia. After being discovered in Georgia in 2002 (1), X. glabratus and R. lauricola have spread rapidly, causing extensive redbay mortality in South Carolina, Georgia, Florida, and Mississippi (1,4). The disease has also been confirmed on sassafras in Florida, South Carolina (1), and Georgia. Questions remain as to whether laurel wilt will continue to spread on sassafras, which often occurs as scattered trees in the eastern United States. In June 2010, a homeowner reported that a sassafras tree north of Van Cleave, MS (30.668°N, 88.686°W) had begun wilting in late May. This landscape tree had three 10-m high stems (~20 cm in diameter at breast height). Dark staining in the xylem was observed around the entire circumference of all three stems and nearly all leaves were bronze colored and wilted. No ambrosia beetle tunnels were observed in the stems. No other symptomatic Lauraceae were encountered in the wooded area within 300 m. The nearest known location with laurel wilt on redbay was ~15 km away (4). A Lindgren funnel trap baited with manuka oil (2) was placed at the site in June and monitored biweekly until November, but no X. glabratus adults were captured. Chips from discolored xylem of the sassafras were surface sterilized, plated on cycloheximide-streptomycin malt agar, and R. lauricola was readily isolated (1). Identity of the fungus (isolate C2792 in collection of T. Harrington) was confirmed by using partial sequences of the 28S rDNA (3). The sassafras sequence was identical to that of all known sequences of R. lauricola in the United States, including GenBank No. EU123076 (the holotype isolate from redbay). To confirm pathogenicity, isolate C2792 was grown on malt extract agar and three redbay (average: 141 cm high and 12 mm in diameter at soil interface) and three sassafras (average: 170 cm high and 17 mm in diameter at soil interface) potted plants were wound inoculated with 0.2 ml of a spore suspension (4.9 × 106 conidia/ml) (1). Three control plants of each species were inoculated with sterile deionized water. After 8 weeks in a growth chamber at 26°C, all inoculated redbay and sassafras plants exhibited xylem discoloration above and below the inoculation point, two of the redbay and two of the sassafras had died, and the other plant of each species exhibited partial wilt (the main terminal or one or more branches). All control plants were asymptomatic. R. lauricola was reisolated from all inoculated symptomatic plants but not from controls. To our knowledge, this is the first report of laurel wilt on sassafras in Mississippi. Both redbay (4) and sassafras appear to be highly susceptible to the disease as it moves westward. Sassafras is less attractive than redbay to X. glabratus and it was thought that this might contribute to slowing the spread of laurel wilt once outside the range of redbay (2). Nonetheless, our observations confirm that sassafras can be infected where laurel wilt on redbay is not in the immediate vicinity. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. L. Hanula et al. J. Econ. Entomol. 101:1276, 2008. (3) T. C. Harrington et al. Mycotaxon 111:337, 2010. (4) J. J. Riggins et al. Plant Dis. 94:634, 2010.

Evaluation of mucosal bacteria and histopathology, clinical disease activity and expression of Toll-like receptors in German shepherd dogs with chronic enteropathies.

The pathogenesis of chronic enteropathies in dogs likely involves an interaction between the intestinal immune system and luminal intestinal bacteria. German shepherd dogs (GSD) are particularly predisposed to chronic enteropathies. The present study sought to evaluate expression patterns of certain pattern recognition receptors of the innate immunity (Toll-like receptors, TLR), clinical disease activity and histopathological severity in GSD with chronic enteropathies. Mucosal biopsies were collected from the duodenum, colon and ileum of 13 affected GSD and 10 healthy greyhounds as controls. Dogs were objectively assessed using published scoring systems for clinical and histological severity of disease. Diversity of the duodenal microbiota was assessed by construction of 16S rRNA gene libraries. Expression of TLR2, TLR4, TLR5 and TLR9 in biopsies of the duodenum, ileum and colon was assessed by quantitative real-time PCR. TLR4 expression was increased in all intestinal segments in GSD, however, TLR5 expression was very low compared to the healthy dogs. The microbiota in the duodenum of GSDs was significantly different to that of the greyhounds, with an over-representation of 16S rRNA gene sequences belonging to the classes of Bacilli, and Erysipelotrichi, and to the orders of Lactobacillales, Actinomycetales and Erysipelotrichales. These findings could point to a distinct pathogenesis of chronic enteropathies in GSD, with differentially high and low expression of TLR4 and TLR5, respectively, and increased proportions of specific members of the Lactobacillales potentially playing a role.

First Occurrence of Laurel Wilt Disease Caused by Raffaelea lauricola on Redbay Trees in Mississippi.

Laurel wilt is a lethal, nonnative vascular wilt disease of redbay (Persea borbonia), sassafras (Sassafras albidum), and other trees in the Lauraceae (1,4). It is caused by a fungus (Raffaelea lauricola) and transmitted by the redbay ambrosia beetle (Xyleborus glabratus), a nonnative insect first detected in Georgia in 2002 (1,2). Since introduction of the pathogen and vector (presumably from Asia), laurel wilt has caused extensive mortality to redbays in Georgia, Florida, and South Carolina (1). In June 2009, a landowner in Gautier, MS reported dead redbay trees. Signs and symptoms were identical to those reported for laurel wilt along the Atlantic Coast (wilted, bronze red foliage, and dark gray-to-black vascular discoloration) (1). Infected trees have subsequently been confirmed in and near the Pascagoula River Basin. Size of infected redbays ranged from 5 to 20 cm (diameter at breast height). No heavily decomposed or fallen redbays were noted. Many individual specimens exhibited extensive drying of stem wood and dry, wilted, light brown foliage. This indicates that introduction to the area may have occurred within the last 3 years. X. glabratus adults were collected (30°26'44.45″N, 88°39'41.83″W) in a Lindgren funnel trap baited with phoebe and manuka oil lures. Beetle identification was confirmed by USDA-APHIS, and voucher specimens were submitted to the Smithsonian National Museum of Natural History and the Mississippi Entomological Museum. Symptomatic redbay wood chips from the same location were surface sterilized and plated on cycloheximide-streptomycin malt agar and R. lauricola was isolated. A 1,026-bp portion of 18S rDNA (GenBank No. GQ996063) was amplified by PCR and sequenced using primers NS1 and NS4. BLASTn searches revealed perfect homology to R. lauricola isolate PL 697 (GQ329704). Two isolates of R. lauricola were recovered and prepared into separate spore suspensions (1 × 108 CFU/ml). Each isolate was inoculated into two healthy redbays. The inoculated redbays were placed in a growth chamber with two water-only controls. All inoculated plants, and none of the controls, exhibited wilt symptoms and died within 20 days. R. lauricola was recovered from the discolored sapwood of the inoculated plants, completing Koch's postulates. A model prediction for the natural dispersion of X. glabratus and R. lauricola estimated that these organisms may not reach Mississippi for 10 to 15 years (3). The current detection of laurel wilt in Mississippi is substantially ahead of this estimate. Currently, no records of laurel wilt have been reported from western Georgia, all of Alabama, or the panhandle of Florida. Confirmed locations in Mississippi are in Jackson County, along the Interstate 10 corridor and the Pascagoula River drainage. Due to the relatively large extent of the infestation (~64 km2, including hundreds of infected trees) eradication is not being attempted. Surveys, remote sensing, and phylogeographic analysis are underway to delineate the extent of infestation and discover the mode of introduction. The current outbreak of laurel wilt in Mississippi is likely the result of human transport of infested wood, either from Asia as a separate, new introduction or from previously infested areas in the southeastern United States. References: (1) S. W Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington et al. Mycotaxon 104:399, 2008. (3) F. Koch and W. Smith. Environ. Entomol. 37:442, 2008. (4) J. A. Smith et al. Plant Dis. 93:198, 2009.

Expression of Toll-like receptor 2 in duodenal biopsies from dogs with inflammatory bowel disease is associated with severity of disease.

There is growing evidence that aberrant innate immune responses towards the bacterial flora of the gut play a role in the pathogenesis of canine inflammatory bowel disease (IBD). Toll-like receptors (TLR) play an important role as primary sensors of invading pathogens and have gained significant attention in human IBD as differential expression and polymorphisms of certain TLR have been shown to occur in ulcerative colitis (UC) and Crohn's disease (CD). The aim of the current study was to evaluate the expression of two TLR important for recognition of commensals in the gut. TLR2 and TLR4 mRNA expression in duodenal biopsies from dogs with IBD was measured and correlated with clinical and histological disease severity. Endoscopic duodenal biopsies from 20 clinical cases and 7 healthy control dogs were used to extract mRNA. TLR2 and TLR4 mRNA expression was assessed using quantitative real-time PCR. TLR2 mRNA expression was significantly increased in the IBD dogs compared to controls, whereas TLR4 mRNA expression was similar in IBD and control cases. In addition, TLR2 mRNA expression was mildly correlated with clinical severity of disease, however, there was no correlation between TLR2 expression and histological severity of disease.

Mutagenicity of the malondialdehyde oligomerization products 2-(3'-oxo-1'-propenyl)-malondialdehyde and 2,4-dihydroxymethylene-3-(2,2-dimethoxyethyl)glutaraldehyde in Salmonella.

Malondialdehyde (MDA), a byproduct of non-enzymatic lipid peroxidation and prostaglandin biosynthesis, has been shown to be a weak frameshift mutagen in Salmonella mutagenicity assays. Because it is a dialdehyde, MDA can undergo self condensation to form polymeric products. It is possible that these condensation products are highly mutagenic and have contributed to previously reported estimates of MDA mutagenicity. We synthesized two major MDA polymerization products, (1) 2-(3'-oxo-1'-propenyl)-malondialdehyde [(MDA)2] and (2) 2,4-dihydroxymethylene-3-(2,2-dimethoxyethyl)glutaraldehyde [(MDA)3Me2] and tested their mutagenicity in the Salmonella frameshift tester strains hisD3052 and TA94 (hisD3052/pKM101). Analysis of the reversion rates revealed both (MDA)2 and (MDA)3Me2 to be weak mutagens, approximately equipotent to MDA. Although both (MDA)2 and (MDA)3Me2 are mutagenic, the fact that their formation is thermodynamically unfavorable under physiological conditions suggests they do not contribute significantly to the mutagenicity of MDA solutions.

Characterization of Otostrongylus circumlitus from Pacific harbor and northern elephant seals.

Otostrongylus circumlitus (Railliet, 1899) from Pacific harbor seals (Phoca vitulina richardsi) and northern elephant seals (Mirounga angustirostris) were examined using morphological and molecular methods to determine whether northern elephant seals along the central California coast are infected by the same species of Otostrongylus as are Pacific harbor seals in the same area. Fixed nematodes were examined and measured using light microscopy. The polymerase chain reaction (PCR) was used to amplify and sequence the second internal transcribed spacer (ITS-2) and D3 expansion (26S) regions of ribosomal DNA of O. circumlitus from Pacific harbor and northern elephant seals. The ITS-2 region was also amplified from Parafilaroides sp. from the Pacific harbor seal, northern elephant seal, and California sea lion (Zalophus californianus) and used for restriction fragment length polymorphism (RFLP) analysis. Morphologically, it was not possible to distinguish O. circumlitus from Pacific harbor and northern elephant seals, and over a consensus length of 443 base pairs (bp) for ITS-2 and 321 bp for D3 the sequences of O. circumlitus from both hosts were identical. With the PCR-RFLP assay, it was possible to distinguish O. circumlitus from Parafilaroides sp. The results suggest that O. circumlitus is the same species in Pacific harbor and northern elephant seals, and molecular methods make it possible to distinguish this nematode from related nematodes.

Why academic divisions of hematology/oncology are in trouble and some suggestions for resolution.

PURPOSE: Academic divisions of hematology/oncology seem to have difficulty recruiting and retaining excellent productive clinicians. A major reason for this is that salaries do not compete with the private sector for similar work. METHODS: We reviewed divisional finances productivity, and experiences from faculty members leaving. RESULTS: The academic salaries are approximately one third of practice because the chemotherapy concession has been given to the academic hospital. In addition, there may be substantial problems in under-billing, lack of attention to detail in billing, and poor collection practices. CONCLUSION: Academic practice still has much to offer, including opportunities for research and multidisciplinary team management, although the differences may narrow over the coming years. Attention to detail in the billing, collection for work performed, and increasing academic salaries to levels nearer to private practice are necessary components of the solution to recruit and retain quality productive clinicians.

Reactivity and mutagenicity of endogenous DNA oxopropenylating agents: base propenals, malondialdehyde, and N(epsilon)-oxopropenyllysine.

Malondialdehyde (MDA), a mutagenic product of lipid peroxidation, reacts with DNA to form the premutagenic lesion, pyrimido[1, 2-a]purin-10(3H)-one (M(1)G). M(1)G is present in normal human tissues, but the contribution of other endogenously produced MDA analogues is poorly understood. Oxidation of the DNA backbone can cause strand breaks and release base propenals, and MDA condensation with proteins yields N(epsilon)-oxopropenyllysine. Here we compare the M(1)G-forming ability and Salmonella typhimurium mutagenicity of MDA with adenine, thymine, and cytosine propenals and N(alpha)-acetyl-N(epsilon)-oxopropenyllysine methyl ester. Base propenals are 30-150 times more potent than MDA in M(1)G formation and are 30-60 times more mutagenic than MDA. In addition, the Fe-bleomycin complex, which generates base propenals, induced M(1)G, but gamma-radiation, which generates mostly MDA, did not. M(1)G formation by MDA and base propenals was concentration-dependent, time-dependent, and enhanced by acidic conditions. N(alpha)-Acetyl-N(epsilon)-oxopropenyllysine methyl ester was less reactive and less mutagenic than MDA. These differences in potency are consistent with differences in leaving group ability. This work supports a role for other MDA analogues, especially base propenals, in the formation of endogenous M(1)G adducts.

Evaluation of the neodymium-YAG laser for treatment of ocular opacities.

The Neodymium:YAG Laser was used to find damage thresholds for glass and PMMA IOLs. In addition, optimal cone angles of the laser in regard to laser safety was determined along with consideration of suitability of other laser wavelengths. Lastly, investigation of substitute solutions for optical breakdown compared to vitreous was evaluated.