A Novel Form of Progressive Retinal Atrophy in Swedish Vallhund Dogs
2014

A E Cooper
S Ahonen
J S Rowlan
A Duncan
E H Seppälä
A Novel Form of Progressive Retinal Atrophy in Swedish Vallhund Dogs

PLoS ONE
991066102014Received April 18, 2014; Accepted July 30, 2014;Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: email@example.com (AMK)
Inherited retinal degenerations, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), represent leading causes of incurable blindness in humans. This is also true in dogs, where the term progressive retinal atrophy (PRA) is used to describe inherited photoreceptor degeneration resulting in progressive vision loss. Because of the similarities in ocular anatomy, including the presence of a cone photoreceptor-rich central retinal region, and the close genotypephenotype correlation, canine models contribute significantly to the understanding of retinal disease mechanisms and the development of new therapies. The screening of the pure-bred dog population for new forms of PRA represents an important strategy to establish new large animal models. By examining 324 dogs of the Swedish vallhund breed in seven countries and across three continents, we were able to describe a new and unique form of PRA characterized by the multifocal appearance of red and brown discoloration of the tapetal fundus followed over time by thinning of the retina. We propose three stages of the disease based on the appearance of the ocular fundus and associated visual deficits. Electroretinography revealed a gradual loss of both rod and cone photoreceptor-mediated function in Stages 2 and 3 of the disease. In the few dogs that suffered from pronounced vision loss, night-blindness occurred first in late Stage 2, followed by decreased day-vision in Stage 3. Histologic examinations confirmed the loss of photoreceptor cells at Stage 3, which was associated with the accumulation of autofluorescent material in the adjacent retinal pigment epithelium. Pedigree analysis was suggestive of an autosomal-recessive mode of inheritance. Mutations in six known canine retinal degeneration genes as well as hypovitaminosis E were excluded as causes of the disease. The observed variability in the age of disease onset and rate of progression suggest the presence of genetic and/or environmental disease modifiers.

Introduction

Inherited retinal diseases are among the leading causes for incurable vision loss in the human and canine populations [1][2][3][4][5]. In dogs, most of these conditions are classified as progressive retinal atrophy (PRA), and, similar to many forms of retinitis pigmentosa (RP) in human patients, primarily affect rod photoreceptors leading to initial night-vision loss followed by loss of cone photoreceptors and subsequent day-blindness [1,3,5]. Cone-rod dystrophies (crd) and primary cone degeneration (cd) have also been described in dogs with either combined initial day-and night-vision loss or exclusive day-blindness [1,5]. Inherited retinal disease can also result from a primary dysfunction of the retinal pigment epithelium (RPE) of which RPE65or BEST1-mutant dogs are examples [1,[5][6][7][8][9]. Because of the similarities in ocular anatomy, including the presence of a cone photoreceptor-rich central retinal region [10,11], and the frequently similar genotypephenotype correlation [1], canine retinal disease models contribute significantly to our understanding of retinal disease mechanisms and the development of new therapies for human patients [12][13][14][15][16][17][18][19][20]. Screening of the pure-bred dog population for new forms of PRA represents an important strategy to maintain breed health and to establish new large animal models.

Beginning in the late 1990s, Swedish and Finnish eye panelists recognized the emergence of a new retinal disease in Swedish vallhund dogs (Fig. 1). The morphologic retinal abnormalities seen by indirect ophthalmoscopy were different from any known forms of canine inherited retinopathy described to date (Finnish Kennel Club: http://jalostus.kennelliitto.fi/frmEtusivu.aspx; Swedish Kennel Club: http://kennet.skk.se/avelsdata/). With the gradual increase of the breed's international popularity, this Swedish vallhund retinopathy was also recognized in other parts of the world, including North America [2]. The purpose of this study was to characterize the phenotype and potential etiology of this condition in order to provide the basis for elimination of the disease in the canine population. Our findings are based on the examination of 324 Swedish vallhund dogs in seven countries and suggest that the Swedish vallhund retinopathy is a new form of PRA. An autosomal recessive mode of inheritance is most likely.

Materials and Methods

Ethics Statement

All of the described work was conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Approval from the local Institutional Animal Care and Use Committee was obtained when the clinical examination or sample collection was not part of the services requested by the dog owner or breeder (County Administrative Board of Southern Finland ESLH-2009-07827/Ym-23 and University of Pennsylvania IACUC protocols# 802604 and 803429).

Study Animals

Most of the results described in this study were obtained from Swedish vallhund dogs clinically examined by the authors (AMK and PV). The bulk of these dogs resided either in the United States or Finland, but additional dogs were also examined in Canada and Sweden. In addition, for pedigree and genetic analyses, Swedish vallhund dogs examined by other veterinary ophthalmologists or eye panelists were also included. These latter dogs resided in the United States, Canada, Finland, Sweden, the United Kingdom, the Netherlands, and Australia. Although other ocular and adnexal abnormalities such as persistent pupillary membranes and cataracts were noted, this study focused exclusively on retinal lesions.

Clinical Examination

All dogs were examined by hand-held slit-lamp biomicroscopy (either SL-14, Kowa Company, Tokyo, Japan; or HSO 10, Carl Zeiss Meditec AG, Jena, Germany) and binocular indirect ophthalmoscopy (Keeler All Pupil II; Keeler Instruments, Broomall, PA, USA) with a condensing lens (Pan Retinal 2.2D; Volk Optical, Mentor, OH, USA) under pharmacologic mydriasis with topical 1% tropicamide ophthalmic solution. All ocular abnormalities were documented and, for selected dogs, fundus photographs were taken with either a portable camera (RC-2, Kowa Company, Tokyo, Japan) or through a 20-diopter lens (Volk Optical, Mentor, OH, USA) with a digital video camera mounted on an indirect ophthalmoscope (Omega 2C, Heine Optotechnik, Herrsching, Germany). Images were visualized with Pinnacle studio plus software (Pinnacle, Mountain View, CA, USA) and individual frames extracted using Vegas Movie Studio HD Platinum 11.0 (Sony Creative Software, Inc, Middleton, WI, USA). Additionally, visual performance was assessed by distribution of a short questionnaire among 40 owners of PRA-affected Swedish vallhund dogs in Finland. Specifically, we inquired as to the owner's perception of the onset and severity of visual deficits observed in the dogs, such as the ability to see stationary or moving objects under different light conditions.

Electroretinography

Retinal function was recorded by electroretinography under general anesthesia in a total of 15 selected Swedish vallhund dogs in the United States, Sweden and Finland, using the same equipment and protocol (RETIport, Acrivet, Inc., Salt Lake City, UT, USA). Premedication by intramuscular injection occurred with acepromazine maleate (0.5 mg/kg) alone, or a combination of butorphanol tartrate (0.1-0.2 mg/kg) with either acepromazine maleate (0.01-0.02 mg/kg) or medetomidine HCl (20 mg/kg). While one dog was masked with an isoflurane/O 2 gas mixture, the remaining dogs were induced by intravenous injection of propofol (4-6 mg/kg to effect). All dogs were intubated and inhalation anesthesia maintained with an isoflurane/O 2 gas mixture on a semiclosed system. The dogs were placed in right lateral recumbency and left eyes were tested. Central position of the globe was achieved and maintained by retrobulbar injection of sterile saline caudal to the orbital ligament (volume to effect). In order to avoid muscle artifacts in the electroretinogram (ERG), an auriculopalpebral nerve block was applied by subcutaneous injection of 2% lidocaine HCl solution along the zygomatic arch. Two platinum subdermal needle electrodes (Grass Safelead Needle electrodes, Grass Technologies, West Warwick, RI, USA) were used: The reference electrode was placed subcutaneously approximately 10 mm from the lateral canthus, and the ground electrode was placed over the occipital protuberance [21]. A Kooijman/ Damhof ERG corneal contact lens with built-in white LED light source (Acrivet, Inc.) was used as the active electrode and applied with 2.5% hypromellose ophthalmic demulcent solution [22]. Following 20 minutes of dark adaptation, a single rod and maximal rod-cone responses were recorded with a flash intensity of 0.03 cd.s/m 2 (average of 20 sweeps at 0.2 Hz) or 3.0 cd.s/m 2 (average of 10 sweeps at 0.06 Hz) respectively. Subsequently, the eyes were light-adapted to a white uniform background light of 25 cd/m 2 from the Kooijman/Damhof ERG contact lens, and single cone (average of 10 sweeps at 0.5 Hz) and cone flicker (average of 30 sweeps at 28 Hz) responses were recorded with a 3.0 cd.s/m 2 flash intensity. For all recordings, the filters were set to allow a bandpass of 1 to 300 Hz. For each recording, amplitudes and implicit times were measured and compared between individual dogs. The small sample size precluded detailed statistical analyses.

Histopathology and Immunohistochemistry

Ocular histopathologic examination was performed in eight Swedish vallhund dogs that were euthanized for unrelated reasons, and the eyes donated by the owners. Most of the eyes were fixed in formalin, and routinely processed for paraffin embedding and hematoxylin and eosin (H&E) staining. For specific examination of lipopigment accumulation (ceroid/lipofuscin), tissue sections were stained with acid-fast and counterstained with hematoxylin. One eye was fixed for 24 h in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at 4uC. Then the anterior segment and the vitreous were removed, and the tissue was placed sequentially in 15 and 30% sucrose for 24 h each prior to embedding in optimal cutting temperature (OCT) medium for cryosectioning. Paraffin sections of normal and PRA-affected eyes originating from other canine breeds were provided as control samples by the Anatomic Pathology/Biopsy Service of the University of Pennsylvania School of Veterinary Medicine and the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW).

To further examine the morphology of cells and the localization of protein expression within the retina, immunohistochemical staining of both paraffin and OCT retinal sections was performed with the following antibodies (Table S1): human cone arrestin (for cone photoreceptors), rhodopsin (for rod photoreceptors), RPE65 (for the retinal pigment epithelium, RPE), glial fibrillary acidic protein (GFAP, for astrocytes and Müller cells), glutamine synthetase (for Müller cells) and G 0 alpha (for ON bipolar cells). Alexa Fluor-labeled goat anti-rabbit immunoglobulin (IgG) or goat anti-mouse IgG (Molecular Probes Inc., Eugene, OR, USA) was used as secondary antibody. 49,6-Diamidino-2-phenylindole (DAPI) stained the cell nuclei.

Stained tissues were evaluated using light and fluorescent microscopy with a Zeiss Axioplan microscope (Carl Zeiss Meditec, Dublin, CA, USA). Images were digitally captured (Spot 4.0 camera; Diagnostic Instruments Inc., Sterling Heights, MI, USA), and imported into a graphics program (Photoshop; Adobe, Mountain View, CA, USA) for display. Selected tissues were imaged by laser scanning confocal microscopy (Olympus Fluo-View FV1000; Olympus America, Center Valley, PA, USA).

Pedigree and Candidate Gene Analysis

In an attempt to determine genealogical relationships between as many of the examined Swedish vallhund dogs as possible, a master pedigree was constructed using GenoPro genealogy software (http://www.genepro.com/). Genealogical data was either accessed in public canine registries such as the Finnish Kennel Club's Koiranet (http://jalostus.kennelliitto.fi/frmEtusivu. aspx) or from pedigree information provided by the dog owners.

When the owner's consent was obtained, either whole blood (in EDTA-containing tubes) or buccal swabs (Eurotubo Cytobrush, sterile, 200 mm, Danlab, Helsinki, Finland) were collected for genomic DNA extraction (QIAmp DNABlood Mini Kit, Qiagen, Valencia, CA, USA, or Chemagic Magnetic Separation Module I, Chemagen Biopolymer-Technologie AG, Baeswieler, Germany for whole blood; or QIAamp DNA Mini Kit, Qiagen for buccal swabs). DNA concentration was measured using Nanodrop ND-1000 UV/Vis Spectrophotometer (Nanodrop technologies, Wilmington, Delaware, USA); and the DNA was stored at -20uC. Genes associated with the following forms of inherited canine retinal diseases were tested for association using fragment analysis in 11 PRA-affected and 11 unaffected Swedish vallhund dogs: canine multifocal retinopathy (cmr; gene: BEST1) [8,9], rod-cone dysplasia type 1 (rcd1; PDE6B) and type 3 (rcd3; PDE6A) [23][24][25][26], progressive rod-cone degeneration (prcd; PRCD) [27], canine Leber congenital amaurosis (LCA; RPE65) [6,7], cone-rod dystrophy (crd SWHD , NPHP4) [28], and achromatopsia/cone degeneration (ACHM/cd; CNGB3) [29,30]. PRA-unaffected dogs were defined as Swedish Vallhund dogs over the age of six years who were examined by a board certified veterinary ophthalmologist and confirmed free of retinal disease.

Primers were designed to amplify two flanking microsatellites using Primer3 software (http://frodo.wi.mit.edu/primer3/) and the canine whole genome sequence assembly (CanFam2.0: http:// www.ensembl.org/Canis_familiaris/; see Table S2 for PCR amplification primers). An 18-bp extension sequence was added to the 59-end of the forward primers to allow amplification of a fluorescently labeled third primer Fam-TGACCGGCAG-CAAAATTG for visualization. Microsatellites were PCR-amplified using the pooling method: Two pools, one containing DNA from PRA-affected dogs, and one containing DNA from control dogs were made, using the exact same amount of DNA (Table  S3). GeneScan LIZ-500 dye size standard (Applied Biosystems by Life Technologies, Carlsbad, CA, USA) was added to the PCR products, combined with Hi-Di formamide, and placed onto an ABI3730xl DNA analyzer (Applied Biosystems). The data was analyzed using Peak Scanner Software v1.0 (Applied Biosystems) and compared between the two groups. Possible PCR saturation was inspected to ensure that none of the amplifications had gone to completion. As the exact same quantity of DNA was used from each individual, the fragment sizes were able to be directly compared between pools. If differences were observed between case and control pools, individual samples were amplified separately using the same protocol. Minor differences were observed between case and control pools for BEST1, CNGB3 and NPHP4 genes, therefore the cases and controls were further amplified separately for these genes ( Table S3). The individual microsatellite data was then analyzed for the presence of segregation under a recessive model.

Evaluation of Serum Vitamin E Concentrations and Other Potential Risk Factors

Because some aspects of the retinal disease phenotype were remotely reminiscent of vitamin E deficiency [31,32], serum vitamin E measurements were performed in a total of 19 dogs including normal and PRA-affected Swedish vallhund dogs as well as mixed breed colony dogs from the Retinal Disease Studies Facility at the University of Pennsylvania School of Veterinary Medicine. Following collection, serum samples were frozen until analysis at the Pennsylvania Animal Diagnostic Laboratory System Toxicology Laboratory at the University of Pennsylvania School of Veterinary Medicine. For sample preparation, vitamin E was extracted from the serum, filtered, and analyzed with a liquid chromatography system equipped with fluorescence detector (Shimadzu, Columbia, MD, USA). For quality control, serum with certified vitamin E concentration from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) was obtained and analyzed together with each batch of our canine serum samples. All vitamin E measurements were reported as the concentration of the most biologically active homologue, alpha tocopherol. A Wilcoxon rank sum test was performed to compare the values between groups.

In an attempt to identify any environmental risk factors contributing to the development and progression of PRA in Swedish vallhund dogs, a short questionnaire was distributed to dog owners and breeders. Topics of inquiry included husbandry practices, as well as the previous occurence of any systemic or ocular disease.

Results

Clinical Phenotype

Between June 2005 and November 2011 we examined a total of 199 Swedish vallhund dogs. In addition, we evaluated eye examination data from certified veterinary ophthalmologists and eye panelists provided to us by the owners of an additional 125 Swedish vallhund dogs. In total, 113 of the 324 dogs examined exhibited a clinical phenotype consistent with Swedish vallhund retinopathy, resulting in an estimated disease prevalence of 34.9%. Based on all the available information, we identified a new retinal disease phenotype which appears to be breed specific since we are not aware of the presence of a similar disease phenotype in other dog breeds. By following individual affected dogs over time (Tables 1 and 2), we were able to define three distinct stages of the disease based on the severity of the clinical signs. The following phenotypic descriptions and numbers of affected dogs are based on the clinical examinations performed by the authors (AMK and PV) exclusively. Stage 1, the earliest stage with the mildest clinical signs, was characterized by a diffuse multifocal red or brown discoloration of the tapetal fundus (Fig. 2D-F). These abnormalities were seen in 34 dogs with a mean age (6 standard deviation) of 4.363.7 years, but were noted as early as 1.9 months and as late as 17.8 years of age. There were no visible lesions in the nontapetal fundus. Dogs in Stage 1 exhibited no clinical signs of vision loss. In Stage 2, the retina showed signs of degeneration, indicated by multifocal, geographic thinning, beginning in the periphery and spreading throughout the tapetal fundus (Fig. 2G-I). A total of 25 dogs were examined and found to have Stage 2 disease. The mean age at diagnosis was 6.263.1 years but changes were seen as early as 1.1 and as late as 12.6 years of age. While the majority of dogs with these degenerative changes did not appear to have vision problems initially, some owners reported that their dogs exhibited mild to moderate signs of night-blindness as the areas of retinal thinning expanded. Seven dogs with a mean age of 12.262.6 years were diagnosed with Stage 3 disease, characterized by more diffuse retinal thinning affecting most of the tapetal fundus ( Fig. 2J-L). Stage 3-changes were observed as early as 9.2 years and as late as 15.4 years. These dogs suffered from loss of night-vision and severely impaired day-vision; some dogs were assessed as completely blind by their owners.

In order to better understand the rate of disease progression, whenever possible, repeated examinations of specific dogs were performed over the 6.5-year period of the study. Although the precise duration that an animal spent in a particular disease stage could not be determined based on the voluntarily nature of the examinations and the widespread distribution of the study population across several continents, repeated examinations were used to provide a rough estimate of disease progression. When looking at dogs with multiple examinations, a total of five dogs were followed at Stage 1 for a minimum period of 0.9 years and a maximum period of 2.3 years (mean of 1.2 years) without any signs of progression to subsequent disease stages ( Table 2). One example of note is a dog that was repeatedly examined until the age of 17.8 years but never progressed beyond Stage 1. A total of four dogs were followed at Stage 2 for a minimum period of 1.8 years and a maximum period of 3.9 years (mean of 2.7 years) without any signs of progression to subsequent disease stages. The discrepancies noted with respect to rate of disease progression among Swedish vallhund dogs suggest that genetic or environmental disease modifiers are likely to play a role in the pathogenesis. During the study period, a total of ten dogs showed progression to subsequent disease stages with repeated examination ( Table 1). These dogs were followed for a minimum period of 1.1 years and a maximum period of 7.3 years (including other ophthalmologists' findings) with a mean of 4.0 years.

Evaluation of retinal function in 15 Swedish vallhund dogs (nine dogs at Stage 2, two dogs at Stage 3, and four normal control dogs) by electroretinography revealed a decrease of both rod and cone photoreceptor-mediated function in Stages 2 and 3 (Fig. 3). While the small sample size did not permit a detailed statistical analysis, our recordings indicate that relative cone function may be lost at a slower rate than rod function; this would be consistent with the dog owners' observations that night-blindness precedes loss of dayvision at Stage 3.

Unrelated to the herein described phenotype specific to Swedish vallhund retinopathy, we also observed focal and multifocal inactive chorioretinal scars in a number of dogs (n = 14). These lesions were characterized by focal areas of pigmentation that were often surrounded by a hyperreflective halo due to retinal thinning (Fig. S1). These 'bull's eye' lesions are most consistent with multifocal acquired chorioretinopathy (MAC), a disorder previously reported in herding and working dogs [33][34][35][36]. According to information provided by the dog owners, at least some of the Swedish vallhund dogs with MAC-like lesions suffered from severe gastrointestinal parasitism early in life which may have resulted in multifocal chorioretinitis. In three Swedish vallhund dogs we found these MAC lesions combined with the breed-specific retinopathy. Two Swedish vallhund dogs that were not examined by the authors were diagnosed with retinal folds (n = 2, 0.85% of all examinations), a mild form of retinal dysplasia.

Histologic Phenotype

Because our study involved only privately owned dogs and no purpose-bred research animals, the availability of tissue samples for histologic examination was limited to eight eyes generously donated by owners. As such, these samples were typically obtained from older dogs that died of causes unrelated to the retinopathy. The retinal disease status was not always known at the time of death and tissue collection. Despite these limitations, the available tissues still provided valuable information about changes in retinal architecture in advanced disease which may contribute to a better understanding of the etiopathogenesis. Most valuable was the tissue sample from a 15-year old Stage-3 retina with distinct regions of varying disease severity (Fig. 4B): Small islands of relatively normal appearing retina (Fig. 4B1) were surrounded by large areas of advanced retinal degeneration with loss of both rod and cone photoreceptors (Fig. 4B2). Regions with 'end-stage' disease showed severe retinal thinning with complete loss of normal architecture (Fig. 4B3). The transition between mildly and severely degenerated retinal regions was at times abrupt (Fig. 4C). The loss of photoreceptors was associated with the accumulation of autofluorescent material in the subretinal space and within the RPE (Fig. 4C-D). The presence of this material was more excessive than would be seen with other forms of PRA (e.g., prcd shown in Fig. 4E) or normal aging (Fig. 4F). We posit that this acid-fast positive, ceroid/lipofuscin-like material was likely the source of the red or brown discoloration clinically seen in the tapetal fundus and defining Stage 1 of the disease. However, based on the available data, the presence of autofluorescent material does not provide us with specific clues about the cellular and molecular disease mechanisms resulting in photoreceptor loss and retinal degeneration. We suspect that the disease-related increase in autofluorescence may be able to be visualized in the living animal when proper imaging tools are used. As such, this will be one of our future goals.

Pedigree and Candidate Gene Analysis

Pedigree analysis of nearly 300 Swedish vallhund dogs, including 125 affected animals, indicates a clear genetic contribution (Fig. 5). While variability in the age of disease onset and incomplete pedigree information preclude us from drawing any definitive conclusions about the mode of inheritance, the available data is most consistent with an autosomal recessive pattern. This notion is supported by the lack of a sex predisposition, and the observation that affected animals can originate from the breeding of non-affected dogs.

In order to perform candidate gene screening, we collected blood samples from normal and affected Swedish vallhund dogs.

Seven known canine PRA genes were tested for possible association: BEST1 [8,9], PDE6B and PDE6A [23][24][25][26], PRCD [27], RPE65 [6,7], NPHP4 [28], and CNGB3 [29,30]. Flanking microsatellite markers were selected for each locus and tested in case and control pools for possible allelic differences. No PCR completion was detected in any of the pools. The affected dogs did not share common alleles in any loci of six candidate genes, excluding them as causative for the disease ( Table S3). As both PRCD markers were monomorphic and uninformative, no conclusions could be drawn about a possible involvement of this gene in the disease process.

Evaluation of Serum Vitamin E Concentrations and Other Potential Risk Factors

Because the fundic appearance in early stages of the Swedish vallhund retinopathy is reminiscent of vitamin E deficiency retinopathy [31,32,37], blood was collected and vitamin E (alpha tocopherol) levels were determined in three retinopathy-affected and six normal control Swedish vallhund dogs and compared to ten normal mixed-bred dogs housed in a well-controlled research colony. Overall, the serum vitamin E concentrations were lower in the Swedish vallhund dogs (18.8-44.5 mg/mL, median: 28 mg/ mL) compared to the normal mixed-bred colony dogs (36.2-48.8 mg/mL, median: 40.7 mg/mL; p = 0.01, Wilcoxon rank sum test), but well within the previously described normal range for this species [31,32,37]. Except for one individual, the measurements of the affected animals (18.8, 22.9, and 26.2 mg/mL) fell within the range of the normal Swedish vallhund dogs (19-44.5 mg/mL; median: 29 mg/mL), making vitamin E deficiency an unlikely etiology for Swedish vallhund retinopathy.

Discussion

Based on its inheritance patterns and the progressive nature of vision loss, the novel retinopathy observed in Swedish vallhund dogs appears to be a form of PRA. The phenotype of this disease is rather different from most known forms of PRA, with a multifocal rather than diffuse distribution of retinal degeneration. Furthermore, age of disease onset and rate of disease progression can vary considerably, even in closely related animals. Based on our observations gathered during ophthalmic examination of affected dogs, we propose three clinical stages of disease ranging from diffuse multifocal red/brown discoloration of the tapetal fundus without associated visual deficits (Stage 1), to geographic retinal thinning/degeneration with mild to moderate signs of nightblindness (Stage 2), to more diffuse retinal thinning/degeneration affecting most of the tapetal fundus and associated with nightvision loss and severely impaired day-vision (Stage 3). Paired with these clinical observations, functional and histologic examination of diseased retinas confirmed that this is a progressive retinopathy At the authors' discretion, selected examinations performed by other certified veterinary ophthalmologists were included in the data analysis in order to provide a more complete picture of disease progression. doi:10.1371/journal.pone.0106610.t001   affects both rod and cone photoreceptors as well as the RPE. One of the most unique features of this disease is the excessive accumulation of autofluorescent material within the RPE, which may be the source of the initial discoloration noted within the tapetal fundus. Our work to characterize this new canine retinopathy draws from well established collaborations between clinicians, geneticists, and dog breeders. Such an international effort was crucial considering that Swedish vallhund dogs are much fewer in numbers compared to other, more popular breeds. Our pedigree analysis included Swedish vallhund dogs examined in seven different countries, spanning three continents, and was highly suggestive of a genetic etiology and an autosomal recessive mode of inheritance. Because the affected dogs did not share common alleles, we excluded single, fully penetrant mutations in six known canine retinal disease genes: BEST1 [8,9], PDE6B and PDE6A [23][24][25][26], RPE65 [6,7], NPHP4 [28], and CNGB3 [29,30]. However, dominant or incompletely penetrant mutations in any of these genes cannot be formally ruled out. Overall, the candiate gene analysis suggests that the Swedish vallhund retinopathy is genetically distinct from other forms of PRA, and our ongoing genome-wide studies have identified a new, unreported PRA locus in this breed (Ahonen et al., unpublished data).

The most striking feature of Swedish vallhund PRA/retinopathy is the individual variability in disease progression, even among related dogs and those raised in similar environments. While some of the affected dogs progressed to subsequent stages of disease with repeated examination, others did not progress beyond early stages despite advanced age. This is reminiscent of canine cone-rod dystrophy 1 (cord1-PRA, suspected RPGRIP1 mutation) in the miniature long-haired dachshund, where genotype-phenotype discordance has been demonstrated within a genetically isolated population due to the presence of genetic disease modifiers [38]. Variability in disease phenotype caused by a particular mutation, either within one breed (e.g., X-linked PRA1) [39] or between multiple breeds (e.g., prcd) [40] has also been described in other types of PRA. Genetic disease modifiers that influence the severity of the disease phenotype also exist for other canine genetic disorders, such as Collie eye anomaly [41]. We suspect that genetic modifiers may be responsible for the variation in disease phenotype of PRA-affected Swedish vallhund dogs; as such, this will be a major focus of our future investigations.

Environmental disease modifiers may also play a role in determining onset and progression of PRA in the Swedish vallhund dogs. A classic example is the effect of light exposure on the progression of retinal degeneration in English mastiff dogs with autosomal dominant PRA [17]. Our attempt to identify environmental factors which may affect disease progression by questioning dog owners proved inconclusive. The observation of red/brown fundus changes at Stage 1 and the accumulation of autofluorescent lipofuscin-like material within the RPE was remotely reminiscent of vitamin E deficiency retinopathy [31,32,37]. However, normal serum vitamin E (alpha-tocopherol) concentrations in a few affected Swedish vallhund dogs failed to support such a connection.

We posit that the accumulation of autofluorescent, lipofuscinlike material corresponds to the red/brown discoloration clinically seen in the tapetal fundus, and may precede the loss of photoreceptors. Future investigations using specialized, highresolution in vivo imaging tools, such as optical coherence tomography and scanning laser ophthalmoscopy, will permit a much more detailed investigation into the source of the autofluorescence. Because this material appears to be more abundant in affected Swedish vallhund dogs compared to other forms of PRA with primary photoreceptor death or compared to the normal aging canine retinas, we suspect that the autofluorescent material is intimately associated with the disease-causing gene mutation. However, based on our available data, we are unable to conclude whether the Swedish vallhund disease primarily affects the RPE or the photoreceptors; both options are possible [42]. Pathologic accumulation of lipofuscin in the RPE is also a feature of several human diseases, including fundus flavimaculatus (Stargardt's disease) [43], but at this point it remains unclear which human condition is most closely modeled by Swedish vallhund PRA.

In summary, we describe a unique form of PRA in the Swedish vallhund dog. The novelty of this disease is not only based on the retinal disease phenotype, but also the variation in age of onset and rate of disease progression. An autosomal-recessive mode of inheritance appears most likely and mutations in several known retinal disease genes have been excluded. Genetic and/or environmental disease modifiers likely contribute to the disease  Tables 1 and 2 are highlighted in yellow and green, respectively. Squares, males; circles, females; white symbols, unaffected; black symbols, retinopathy affected. doi:10.1371/journal.pone.0106610.g005 phenotype, but hypovitaminosis E was excluded as a contributing factor. Figure S1 Multifocal acquired chorioretinopathy (MAC) in a Swedish vallhund dog. The multifocal black spots in the tapetal fundus represent post-inflammatory scars. With a classic 'bull's eye' lesion, the larger black pigmented spot is surrounded by a bright, yellow zone or halo, a sign of severe, focal retinal thinning/degeneration. (TIF)  Table S3 Association analysis of six known canine PRA genes in Swedish vallhund retinopathy. Eleven cases and eleven controls were pooled and genotyped for one to four microsatellite markers in each locus. The fragment sizes (bp) are marked in the table. The fact that the affected dogs do not share common alleles in any loci and same alleles were present in both cases and controls excludes these known PRA genes as candidates. For the CNGB3_29_35.82 and BEST1_18_57.49 markers, the pooled samples were individually genotyped to confirm the results. The NPHP4 markers were genotyped separately in a different set of dogs. (XLSX)

Supporting Information

Figure 1 .
1Appearance of Swedish vallhund dogs. The Swedish vallhund is a spitz-type herding breed, recognized since the age of the Vikings (700-1000 A.D.). The breed standard includes erect ears and a medium-length, hard haircoat. Coat color is a sable pattern in grey, red or combinations thereof. Tail may be natural (long, stub or bob) or docked. doi:10.1371/journal.pone.0106610.g001

Figure 3 .
3Standard electroretinograms (ERGs) recorded from normal and affected Swedish vallhund dogs. Compared to the ERG recordings of a normal dog (top row) both rod-and cone-mediated retinal functions are clearly reduced at Stage 2. ERG amplitudes are further decreased at Stage 3 (third row), especially rod-mediated responses, and may not even be recordable in some affected dogs (flat lines in fourth row). doi:10.1371/journal.pone.0106610.g003

Figure 2 .
2Clinical appearance of the ocular fundi of normal and affected Swedish vallhund dogs. (A-C) Normal appearance of the ocular fundus with the colored tapetum lucidum (responsible for eye shine), the well developed retinal vasculature, and the optic nerve head (arrows). It is not unusual to see a thin halo of altered reflectivity or conus papillaris around the optic nerve head as seen in B and C. (D-F) Stage 1 is characterized by multifocal red or brown discoloration of the tapetal fundus (arrows). (G-I) In Stage 2, the retina showed signs of degeneration, indicated by multifocal, geographic thinning (arrow heads), beginning in the periphery and spreading throughout the tapetal fundus. (J-L) Stage 3 is defined by a more diffuse retinal thinning recognized by the large, bright (hyperreflective) regions affecting most of the tapetal fundus (arrow heads). The conus papillaris also appears wider in Stage 3 (arrows). doi:10.1371/journal.pone.0106610.g002

Figure 4 .
4Histologic changes associated with Swedish vallhund retinopathy. (A) High quality section through a wildtype canine retina shows the normal retinal layers and architecture. (B) Different regions of the same 15-year old Swedish vallhund retina with Stage 3 disease. Small islands of relatively normal appearing retina (B1) are surrounded by severely degenerated retina characterized by the loss of photoreceptors and ONL thinning (B2). Retinal architecture is completely lost with end-stage disease and severe retinal degeneration (B3). (C) At the transition between relatively well preserved and completely absent (*) retinal architecture, loss of photoreceptors (cones are labeled red with hCAR antibody) is associated with accumulation of autofluorescent material (green) in the subretinal space and within the RPE (arrows). (D) Accumulation of autofluorescent material (green) within the RPE can also be seen in a well preserved region of a 13-year old Stage-2 affected retina (arrow), possibly indicating that increased autofluorescence may precede loss of photoreceptors. Accumulation of autofluorescent material within the RPE (arrows) can also be seen with other forms of PRA (E, prcd) or with normal aging (F, 11-year old normal canine retina) but to a much lesser extent than in affected Swedish vallhund dogs. Staining: H&E (A and B) and fluorescent immunolabeling (C-F). Cell nuclei are shown in blue with DAPI (C-F); hCAR, human cone arrestin; AF, autofluorescence. Calibration bars: 20 mm. Layers of the retina: RPE, retinal pigment epithelium; PR, photoreceptors; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer. doi:10.1371/journal.pone.0106610.g004

Figure 5 .
5Large Swedish vallhund pedigree. This pedigree is composed of nearly 300 dogs, including 125 animals affected with the breedspecific retinopathy. Affected dogs listed in

Table 1 .
1Swedish vallhund dogs that showed progression with repeated examination.Vallhund ID
Age at Stage 1 diagnosis (years) Age at Stage 2 diagnosis (years) Age at Stage 3 diagnosis (years)

CSC
2.2
3.4
_

GRP
3.9
4.9, 6.9, 9.1
_

LHU
0.6
2.0
_

PCC
1.5
3.7
_

GBB
3.9
4.9, 6.9, 9.1
_

FEE
_
9.6
13.4

FJJ
_
8.6, 8.7, 9.2
15.3

FY
_
4.6
8.3

JMM
_
11.4, 12.0
14.5

FL
_
5.3
12.6

Table 2 .
2Swedish vallhund dogs that showed no progression to subsequent disease stages with repeated examination.Vallhund ID
Age at Stage 1 diagnosis (years)
Age at Stage 2 diagnosis (years)
Age at Stage 3 diagnosis (years)

FHL
0.8, 1.8
_
_

JLG
16.9, 17.4, 17.8
_
_

JM
10.4, 11.4
_
_

KCY
5.2, 7.4
_
_

WAG
1.9, 2.9
_
_

CTG
_
8.7, 11.5, 12.6
_

HMAY
_
4.9, 7.1
_

KKB
_
1.1, 4.1
_

VM
_
2.2, 4.0
_

doi:10.1371/journal.pone.0106610.t002

Table S1
S1Primary antibodies used for immunohistochemistry. (DOCX)

Table S2
S2Primers used to amplify microsatellite markers. (XLSX)
PLOS ONE | www.plosone.org
September 2014 | Volume 9 | Issue 9 | e106610
AcknowledgmentsWe thank the breeders and owners of Swedish vallhund dogs worldwide for their permission to collect phenotype data as well as for the generous donation of blood and tissue samples. We also thank the following individuals for their technical support: Lisa Murphy and Daljit Vudathala
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Cosegregation of codon 807 mutation of the canine rod cGMP phosphodiesterase beta gene and rcd1. K Ray, V J Baldwin, G M Acland, S H Blanton, G D Aguirre, Invest Ophthalmol Vis Sci. 35Ray K, Baldwin VJ, Acland GM, Blanton SH, Aguirre GD (1994) Cosegregation of codon 807 mutation of the canine rod cGMP phosphodies- terase beta gene and rcd1. Invest Ophthalmol Vis Sci 35: 4291-4299.

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Accuracy of the "traffic light" clinical decision rule for serious bacterial infections in young children with fever: a retrospective cohort study OPEN ACCESS

Sukanya De Phd Student
Screening and Test Evaluation Program
School of Public Health
University of Sydney
2006SydneyAustralia

Gabrielle J Williams

School of Public Health and Community Medicine
University of New South Wales
2052Sydney

Department of Emergency Medicine
The Children's Hospital at Westmead
2145Sydney

Department of Infectious Diseases and Microbiology
The Children's Hospital at Westmead

Department of Nephrology
The Children's Hospital at Westmead

Accuracy of the "traffic light" clinical decision rule for serious bacterial infections in young children with fever: a retrospective cohort study OPEN ACCESS
10.1136/bmj.f8661 , Andrew Hayen associate professor of biostatistics 1 2 , Petra Macaskill professor of biostatistics 1 , Mary McCaskill medical director 3 , David Isaacs senior staff specialist 4 , Jonathan C Craig senior staff specialist 1 5
ObjectivesTo determine the accuracy of a clinical decision rule (the traffic light system developed by the National Institute for Health and Clinical Excellence (NICE)) for detecting three common serious bacterial infections (urinary tract infection, pneumonia, and bacteraemia) in young febrile children.Design Retrospective analysis of data from a two year prospective cohort study Setting A paediatric emergency department.Participants 15 781 cases of children under 5 years of age presenting with a febrile illness.Main outcome measuresClinical features were used to categorise each febrile episodes as low, intermediate, or high probability of serious bacterial infection (green, amber, and red zones of the traffic light system); these results were checked (using standard radiological and microbiological tests) for each of the infections of interest and for any serious bacterial infection.ResultsAfter combination of the intermediate and high risk categories, the NICE traffic light system had a test sensitivity of 85.8% (95% confidence interval 83.6% to 87.7%) and specificity of 28.5% (27.8% to 29.3%) for the detection of any serious bacterial infection. Of the 1140 cases of serious bacterial infection, 157 (13.8%) were test negative (in the green zone), and, of these, 108 (68.8%) were urinary tract infections. Adding urine analysis (leucocyte esterase or nitrite positive), reported in 3653 (23.1%) episodes, to the traffic light system improved the test performance: sensitivity 92.1% (89.3% to 94.1%), specificity 22.3% (20.9% to 23.8%), and relative positive likelihood ratio 1.

Introduction

Febrile illnesses are one of the most common reasons for young children to present to primary care practitioners and may account for up to a third of presentations to emergency departments. [1][2][3] Depending on the setting, about 5-25% of fever episodes in young children are due to serious bacterial infections. [4][5][6][7][8][9][10] If not detected and managed in a timely manner, such infections may lead to complications, long term disability, and even death. 11 12 Young children and infants with serious bacterial infection may manifest few, if any, localising signs of systemic infection. [13][14][15] A key challenge for physicians in the clinical evaluation of febrile young children is being able to correctly triage febrile illnesses, identifying those likely to be due to serious bacterial infections in a timely manner while at the same time avoiding over-investigation and overmedication of children, most of whom will have self limiting viral illnesses.

Several clinical criteria and decision tools have been developed to assist clinicians in identifying which febrile children have a serious illness. Unfortunately these have either not been externally validated in independent datasets, do not perform consistently, have insufficient accuracy, or apply only to a limited age range. 8 16-20 Recently the UK National Institute for Health and Clinical Excellence (NICE) published a guideline which provides a traffic light system for the initial assessment and management of young children with fever. 21 The NICE traffic light system was designed for young children under 5 years of age and was intended for a range of settings (general practice, paediatric specialists, or remote assistance by health professionals), and is a colour coded checklist of symptoms and signs (see online supplementary table 1 on bmj.com). Children whose clinical features fall within the green zone are considered to be at low risk of serious illness, while those in the amber and red zones are at intermediate and high risk respectively. The NICE guidance recommends that further investigations be directed according to the level of risk (see online supplementary table 2 on bmj.com). Although it has been widely promulgated, the accuracy of this system for the detection of serious bacterial infections has not been validated to date.

The aims of our study were to determine the test performance of the NICE traffic light system for the detection of three of the most common serious bacterial infections in young febrile children-urinary tract infection, pneumonia, and bacteraemia-and to assess whether the addition of urine analysis, a near patient test with good performance characteristics, 22 improves the performance of the NICE traffic light system. For this study we used data collected prospectively for the Febrile Evaluation of Children in the Emergency Room (FEVER) study. 23 The FEVER study (conducted between July 2004 and June 2006) preceded the NICE fever guideline (published in May 2007).

Methods

Study design and setting

Details of the main FEVER study are reported elsewhere. 23 We incorporated the Standard for Reporting of Diagnostic Accuracy (STARD) guidelines for study reporting. 24

Recruitment

Consecutive children under 5 years old who presented with a febrile illness to the emergency department of the Children's Hospital at Westmead between 1 July 2004 and 30 June 2006 were eligible. Febrile illness was defined as any illness that met one or more of the following criteria: a measured axillary temperature ≥38.0°C; parental report of a temperature ≥38.0°C measured at home within the previous 24 hours; parental report that the child "felt hot" in the previous 24 hours; and a presenting problem related to fever (10th revision of the international classification of diseases, Australian modification codes R50, R50.0, R50.1. R50.9 and R 56.0), as determined by a triage nurse.

The unit of analysis was an instance of febrile illness. In the case of multiple presentations with the same illness, history and clinical evaluation data from the first visit only were used. Case definition for "same illness" was if the child presented within 24 hours of a previous visit or if the fever had persisted between visits to the emergency department without a fever-free period of at least 24 hours.

Study exclusion criteria

Children transferred from another hospital, those with malignancy, and transplant recipients were excluded.

Data collection

Each child was triaged at presentation to the emergency department using the Australasian triage scale. This scale consists of five categories based on the level of clinical urgency, with cases assigned to category one being the most urgent and cases assigned to category five being the least urgent. 25 Clinical information for each illness episode was recorded by the examining physician (emergency department physician, paediatric trainee, or emergency medicine trainee) into a mandatory template within the hospital's electronic record keeping system. This ensured a standardised assessment with entry of 40 clinical features (symptoms and signs) and relevant background medical information of the children for each episode of febrile illness. The template was completed after the initial physician assessment but before test results were obtained. Any tests ordered (including urine analysis) were at the discretion of the treating physician. Test results, emergency department diagnoses, and admission details were electronically linked to the research database.

Outcome definitions

The primary outcomes of interest were the three most common serious bacterial infections in young children with fever-urinary tract infection, pneumonia, and bacteraemia. There were too few cases of other forms of serious bacterial infection, such as meningitis, osteomyelitis, and septic arthritis, to provide robust estimates of the performance of the traffic light system for these infections separately. We included these illnesses in a fourth category, "any serious bacterial infection," in combination with the three common infections. In cases where multiple infections clustered together-for example, when a child developed concomitant urinary tract infection and bacteraemia-they were included in all relevant outcomes. Reference standard test criteria for the diagnosis of serious bacterial infection included culture positivity (for urinary tract infection and bacteraemia) or radiological criteria (for pneumonia) and are detailed elsewhere. 23

Follow-up

All eligible children were followed up until they fulfilled the case definition for serious bacterial infection or until the fever had resolved for over 24 hours. Follow-up was undertaken at 10-14 days after the emergency department visit. Hospital records were reviewed, and parental reports (via telephone call follow-up) of resolution of fever, antibiotic use, and attendance at other healthcare facilities were obtained. Copies of test reports and chest x ray films from other healthcare facilities were obtained with parental consent.

Applying the NICE traffic light system

We matched the items comprising the NICE traffic light system to equivalent items within the FEVER study febrile template (see supplementary table 3 on bmj.com), which allowed us to apply the system retrospectively to each febrile episode in the FEVER study cohort. Accordingly, each episode was categorised as having a low, intermediate, or high probability of serious bacterial infection based on whether their clinical features fitted in the green, amber, or red zones of the NICE traffic light system. This was then compared with the final diagnosis as determined by reference standard test results and follow-up.

Urine analysis

Urine analysis was done at presentation to the emergency department by the attending doctor or nurse in some children. The urine collection method was in accordance with local clinical practice and was not specified for the study. The urine analysis result was reported semi-quantitatively based on the test strip reference chart. For the purpose of our study, the presence of any level of nitrite or leucocyte esterase was considered as test positive.

Statistical analysis

For each of the four infection categories (urinary tract infection, bacteraemia, pneumonia, and any serious bacterial infection), sensitivity and specificity were calculated for two thresholds for test positivity (presence of one or more red zone features versus one or more red or amber zone features). These cut points are displayed graphically with a receiver operating characteristics curve. For the subgroup of children who had a urine analysis reported, we reassessed the test performance of the traffic light system with and without the urine analysis results. We investigated four thresholds for positive urine analysis: leucocyte esterase positive, nitrite positive, either leucocyte esterase or nitrite positive, and both leucocyte esterase and nitrite positive.

We compared the incremental gain in performance of the NICE traffic lights with the combination of the NICE traffic lights and urine analysis (test positive defined as presence of any one or more of the following: amber traffic light features, red traffic light features, leucocyte esterase positive in urine, nitrite positive in urine). Because of the expected trade-off in test sensitivity and specificity when adding a test, we assessed the incremental gain through the relative positive likelihood ratio of the combination of the NICE traffic lights and urine analysis, to the NICE traffic lights on its own. 26 27 We obtained 95% confidence intervals for the relative positive likelihood ratios; if the relative positive likelihood ratio is greater than 1 and its confidence interval does not include 1, the combined test has superior performance than the NICE traffic light system on its own. 26 An increase in the positive likelihood ratio would result in an increase in the positive predictive value of the test-that is, the probability of infection given a positive test result.

Results

During the study period there were 19 889 visits by febrile children under 5 years of age. Patient flow in the FEVER study is detailed in figure 1⇓. Overall, 15 781 eligible febrile illnesses were included in our analysis, 1120 (7.1%) due to serious bacterial infection, with 1166 infections identified; urinary tract infection in 543 (3.4% of the 15 781), pneumonia in 533 (3.4%), bacteraemia in 64 (0.4%), osteomyelitis in 12 (0.08%), meningitis in 8 (0.05%), and septic arthritis in 6 (0.04%). Multiple infections per illness occurred uncommonly, with 44 of the 1120 (3.9%) due to two or more serious bacterial infections (two infections in 42 illnesses, and three infections in two illnesses).

Characteristics of included children and illnesses (table 1 )

Over a quarter of the illness episodes were in children under 1 year old, and almost half were in children aged between 1 and 3 years. Hospitalisation was required in 1912 (12.1%) fever episodes. Four hundred (82.8%) of the 483 febrile illnesses that received a triage category of one or two were in the NICE red zone. Among those receiving a triage category of three, 2371/4894 (48.4%) were in the red zone, while 1042/6936 (15.0%) with a triage category of four and 253/3468 (7.3%) with a triage category of five were in the red zone. Viral infection, viral upper respiratory tract infection, and gastroenteritis were the most common clinical diagnoses.

A dipstick urine analysis was performed in 3653 (23.1%) of the 15781 febrile illnesses. Urine collection method was recorded for only 2262 samples sent for urine culture. Of these 2262 samples, the in-out catheter method was used in 1022 (45.2%), a clean catch specimen in 691 (30.5%), mid-stream sample in 519 (22.9%), supra-pubic aspirate in 10 (0.4%), and a bag specimen in 20 (0.9%). The proportion of children who had urine analysis is detailed by patient characteristics in supplementary table 4 on bmj.com. Children who were more unwell (triage categories one and two), who required hospitalisation, or had higher temperatures at presentation were more likely to have a urine analysis. A greater proportion of children with a provisional diagnosis of fever with no focus had a urine analysis done than children with a provisional diagnosis of asthma, croup, bronchiolitis, pneumonia, or a focal bacterial infection.

Concordance between NICE traffic light system and the FEVER data fields

Nearly three quarters of the 43 items listed in the traffic light system had comparable fields in the FEVER study. Thirteen (30%) of the 43 symptoms and signs had an exact match in FEVER, while 19 (44%) symptoms and signs were captured by similarly phrased terminology in the FEVER dataset (supplementary table 3 on bmj.com). Eleven (26%) of the 43 symptoms and signs listed in the traffic light system did not have an equivalent field in the fever dataset. These mostly included items such as "a new lump," "swelling of a limb or joint," "non-weight bearing limb," and "bile stained vomit." Given that these are relatively specific clinical indicators for hernia, focal bone or joint pathology, and bowel obstruction respectively, it is unlikely that the absence of data relating to these would alter the performance of the traffic light system for the detection of urinary tract infections, bacteraemia, or pneumonia, which are the outcomes of interest for this study.  2⇓), with a relative positive likelihood ratio of 1.17 (95% confidence interval 1.12 to 1.23). For the detection of any serious bacterial infection, the relative positive likelihood ratio was 1.10 (1.06 to 1.14), also indicating improved performance of the combined test over the NICE traffic light system.

Test performance of NICE traffic light system for detection of bacteraemia, urinary tract infection, and pneumonia

Discussion

We found that the NICE traffic light system has moderate sensitivity but low specificity for detection of the three most common serious bacterial infections in febrile young children, namely bacteraemia, urinary tract infections, and pneumonia. Importantly the traffic light system missed a sizeable proportion of urinary tract infections. This is a substantial deficiency in a screening tool for serious bacterial infections in febrile young children, given that, after the introduction of pneumococcal conjugate vaccine, the prevalence of occult bacteraemia in febrile children presenting to emergency departments has decreased substantially to somewhere between 0.4% and 0.7%, 28 whereas the prevalence of urinary tract infection in children with fever without a clinically obvious source remains greater than 7%. 29 Adding urine analysis, a simple and inexpensive near-patient test, to the traffic light system led to an appreciable increase in the proportion of urinary tract infections detected, and a concomitant increase in the overall proportion of serious bacterial infections detected.

The NICE fever guidelines advise routine testing of urine in all children with fever without apparent source (including those who are in the green zone of the traffic light system), thus helping to avoid missed cases of urinary tract infections, but they do not include this test in the traffic light system itself. 21 30 Based on our findings, we strongly support this recommendation but suggest that urine analysis be added to the traffic light criteria. We recommend that urine analysis should be done routinely in children with fever and suspected bacterial infection and only children with a negative result should be classified as belonging to the green (low infection risk) zone. However, although such an addition would improve the discriminatory ability of the NICE traffic light system, clinicians who use this approach should be aware of the appreciable number of misclassified children that remain. In our subset that had urine analysis (n=3653), the traffic light system with urine analysis included had a 78.0% (2466/3161) false positive rate (over three quarters of illness episodes that were not due to a serious bacterial infection were test positive) and 8.1% false negative rate (40/492 of illness episodes due to serious bacterial infections were test negative). Given its low test specificity, clinicians must still judge each case based on its individual features to avoid over-investigation and overtreatment.

Comparison with other studies

To our knowledge, only one other study has evaluated the test performance of the NICE traffic light system. This was done in 700 children aged between 3 months and 16 years with suspected acute infection. 31 In that study the traffic light system had a reported test sensitivity of 85% and specificity of 29% for detection of illnesses (bacterial or non-bacterial in origin) that were considered to be serious or of intermediate severity (uncomplicated urinary tract infections were classified as being mild in severity). However, the number of children with an infection that was reported as serious or of intermediate severity was relatively low (313 in total, with only 67 cases of pneumonia with radiological confirmation, five cases of bacteraemia, and 30 urinary tract infections with systemic symptoms), and so test performance metrics were relatively imprecise. The study also included children up to 16 years old (median age 3 years), although the NICE traffic light system was designed for children up to 5 years old. Finally, the validity of these findings is uncertain because the reference standard test for illness severity was the unverified final diagnoses made by the treating physicians and not standard, microbiologically based definitions. The authors of that study acknowledge that they did not have data on all the "red" and "amber" features.

Strengths and limitations of study

Ours is the first study that has evaluated the test accuracy of the NICE traffic light system for the detection of serious bacterial infections in febrile children under 5 years old, for whom this test was intended, but it does have limitations. The data were not collected for this purpose, and so there was incomplete overlap between the clinical criteria in the NICE traffic light system and FEVER data fields. However, most of the clinical criteria required for the traffic light system-especially all the common criteria relevant to the evaluation of possible urinary tract infection, bacteraemia, and pneumonia-were captured by the corresponding FEVER data fields. We believe that the lack of fields in the FEVER data corresponding to NICE fields such as "a new lump," "swelling of a limb or joint," "not weight bearing," and "bile stained vomit" is unlikely to have affected the detection of the serious bacterial infections of interest to any significant extent, although these could well be of relevance when assessing for other serious infections or illnesses such as septic arthritis, osteomyelitis, or bowel obstruction. A second limitation was that the FEVER study had small numbers of serious bacterial infections other than urinary tract infection, bacteraemia, and pneumonia. As such, the validity of the NICE traffic light system for detection of other serious bacterial infections (such as meningitis, osteomyelitis, and septic arthritis) could not be assessed with confidence. However, the small number of cases of these infections in our study reflects the low prevalence of these infections in a setting of a developed country where immunisation is almost universal.

Our evaluation of the test performance of the traffic light system with the addition of urine analysis was potentially limited by the fact that only about a quarter of the FEVER cohort had a urine analysis. The decision to perform a urine analysis was at the treating clinician's discretion. As expected, the group of children who had urine analysis was at higher risk of urinary tract infection, and were different than those who did not have a urine analysis performed. This reflects standard clinical practice, where it is not routine to subject a young child with an obvious focus of infection as the fever source and who is at low risk of urinary tract infection to a potentially invasive test. Mandating a urine culture in all eligible children was not ethically justifiable. However, this may lead to verification bias, where only those who are positive for the index test (in this case the traffic light system, with or without the urine analysis), have the reference standard test performed (urine culture), leading to overestimates of test sensitivity. Although possible in our study, this bias may have been ameliorated because the traffic light system was not designed when the FEVER study was conducted, and so could not have been used to determine which children should have had a urine culture performed. Further, the design of our study required that all children were followed until the febrile illness resolved (two step verification process), which was achieved in 93% of all illnesses. Finally, even with a potentially optimistic estimate of sensitivity for the traffic light system, our study shows that improvements in performance are required, such as the inclusion of the urine analysis result.

It could be argued that the NICE guidance was designed to detect all serious illnesses, not just infections. The definition of serious illness in the NICE guidance was "an illness with fever that could cause death or disability if there was a delay in diagnosis or treatment." While this definition would be inclusive of serious bacterial infections, it also implies detection of serious illnesses that are not of bacterial origin such as Kawasaki disease, viral gastroenteritis with moderate or severe dehydration, metabolic conditions presenting with acid-base or electrolyte imbalance, or severe viral lower respiratory tract infections requiring supportive management. We assessed the accuracy of the traffic light system only for detecting serious bacterial infections and thus did not explore the full potential of this clinical tool. We also did not attempt to assess its clinical effectiveness or ease of application, nor clinicians' willingness to use it.

Conclusions

The NICE traffic light system has moderate test sensitivity but low specificity for the detection of the three most common serious bacterial infections in young febrile children. The system missed a substantial proportion of urinary tract infections, but the addition of urine analysis improved performance significantly, as reflected in the relative positive likelihood ratio.

With the addition of this relatively simple, non-invasive, and inexpensive near patient test, the traffic light system may be a useful triage tool for healthcare professionals for the initial evaluation of likelihood of serious bacterial infections in young febrile children. Clinical effectiveness and acceptability of the system for the detection of serious illnesses need to be assessed through randomised control trials.

What is already known on this topic

Diagnosis of serious bacterial infections in young children with febrile illnesses is challenging

The NICE guidelines on febrile illnesses in children provide a simple clinical tool (a traffic light system) to guide healthcare professionals' initial assessment of febrile children, but the accuracy of the tool for detecting serious bacterial infections in young children has not been tested

What this study adds

The NICE traffic light system has a moderate sensitivity and low specificity for detection of serious bacterial infections, and urinary tract infections in particular tend to go undetected with the system Adding urine analysis to the traffic light system enhances its sensitivity substantially and increases its positive predictive value, making it a potentially useful tool for the initial evaluation of febrile infants and young children.

The low test specificity of the traffic light system makes individual case based clinical judgement critical for clinicians to avoid overinvestigation and unnecessary hospitalisation or treatment

2| Performance of the NICE traffic light system with and without urine analysis for detection of serious bacterial infections. Values are percentages (95% confidence intervals)

Fig 2
2Receiver operating characteristics curve for performance of the NICE traffic light system, with or without the addition of urine analysis, for detection of serious bacterial infections

Combining the intermediate and high risk traffic light categories (presence of features that were in the amber or red zones) gave a test sensitivity of 85.8% (95% confidence interval 83.6% to 87.7%) and specificity of 28.5% (27.8% to 29.3%) for the detection of any serious bacterial infection. The high risk category (presence of one or more feature in the red zone) had a sensitivity of 47.9% (44.9% to 50.8%) and specificity of 75.9% (75.2% to 76.6%). Among the serious bacterial infections, 40/533 (7.5%) cases of pneumonia, 9/64 (14.1%) cases of bacteraemia, and 108/543 (19.9%) cases of urinary tract infections were in the green zone and would be missed using the NICE traffic light system. Urine analysis had been reported in 3653 (23.1%) of the 15 781 febrile episodes. In this subset, 492 febrile episodes had a final diagnosis of one or more serious bacterial infection (362 urinary tract infections, 118 cases of pneumonia, and 27 cases of bacteraemia). Threshold specific sensitivities and specificities for the combination of the traffic light system with urine analysis are detailed in table 2⇓. For the detection of urine tract No commercial reuse: See rights and reprints http://www.bmj.com/permissions Subscribe: http://www.bmj.com/subscribe infections, the combined test had improved performance over the NICE traffic light system on its own (fig

No commercial reuse: See rights and reprints http://www.bmj.com/permissions Subscribe: http://www.bmj.com/subscribe Tables Table 1| Characteristics of the study population *Triage categories are as per the Australasian triage scale. †Lymphadenitis, tonsillitis, otitis media, or cellulitis. No commercial reuse: See rights and reprints http://www.bmj.com/permissions Subscribe: http://www.bmj.com/subscribeBMJ 2013;346:f866 doi: 10.1136/bmj.f866 (Published 13 February 2013)
Page 6 of 9
No (%) of illnesses (n=15 781)
Characteristic

Age (months):

748 (4.7)
<3

925 (5.9)
3-5

2995 (19.0)
6-11

4865 (30.8)
12-23

2855 (18.1)
24-35

1971 (12.5)
36-47

1422 (9.0)
48-60

Sex:

6967 (44.1)
Male

8814 (55.9)
Female

Highest recorded temperature (°C):

3446 (21.8)
≤37.9

5624 (35.6)
38-38.9

4994 (31.6)
39-39.9

1717 (10.9)
≥40

Triage category*:

30 (0.2)
1

453 (2.9)
2

4894 (31.0)
3

6936 (43.9)
4

3468 (21.9)
5

NICE category:

4066 (25.8)
Red

7373 (46.7)
Amber

4342 (27.5)
Green

Provisional diagnoses:

5940 (37.6)
Viral infections

2186 (13.9)
Gastroenteritis

2125 (13.5)
Asthma, croup, bronchiolitis

1586 (10.1)
Focal infections †

1285 (8.1)
Fever no focus

641 (4.1)
Pneumonia

420 (2.7)
Febrile convulsion

295 (1.9)
Urinary tract infection

244 (1.5)
Allergy

45 (0.3)
Sepsis

22 (0.1)
Meningitis

13 (0.1)
Kawasaki disease

5 (0.03)
Osteomyelitis

974 (6.2)
All other diagnoses

Table

Competing interests: All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.".
Approval was obtained through the University of Sydney Human Research Ethics Committee (ID 2405) and the Royal Alexandra Hospital for Children Ethics Committee. Data sharing: No additional data availableapproval: Approval was obtained through the University of Sydney Human Research Ethics Committee (ID 2405) and the Royal Alexandra Hospital for Children Ethics Committee (ID 99023, 2004/113). Data sharing: No additional data available.

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†Test positive is presence of amber or red traffic light feature, or positive urine analysis, or both. No commercial reuse: See rights and reprints. †Test positive is presence of amber or red traffic light feature, or positive urine analysis, or both. No commercial reuse: See rights and reprints http://www.bmj.com/permissions Subscribe: http://www.bmj.com/subscribe