Absence of urinary opioid peptides in children with autism

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1 Additional appendies are published online only at ad.bmj.om/ontent/vol93/ issue9 1 Great Ormond Street Hospital for Children, Great Ormond Street, London, UK; 2 Evelina Children s Hospital, Westminster Bridge Road, London, UK; 3 Moredun Researh Institute, Peniuik, Midlothian, Sotland, UK; 4 Biomathematis and Statistis Sotland, JCMB, The King s Buildings, Edinburgh, Sotland, UK; 5 Child Life and Health, College of Mediine and Veterinary Mediine, University of Edinburgh, Edinburgh, Sotland, UK; 6 Family and Child Psyhology Researh Centre, Psyhology Department, City University, London, UK Correspondene to: Dr Hilary Cass, Great Ormond Street Hospital for Children, Great Ormond Street, London, UK; assh@gosh.nhs.uk Aepted 11 February 2008 Published Online First 12 Marh 2008 Absene of urinary opioid peptides in hildren with autism H Cass, 1 P Gringras, 2 J Marh, 3 I MKendrik, 4 A E O Hare, 5 L Owen, 6 C Pollin 3 ABSTRACT Objetive: It has been laimed for a number of years that the urine of hildren with autism ontains exogenously derived opioid peptides. This finding is said to reflet a disturbane in the integrity of the gut epithelium, at as a diagnosti marker for autism and predit treatment response to a diet exluding gluten and asein. The aim of the present study was to determine whether exogenous or endogenous peptides were present in the urine of hildren with autism or of ontrol hildren. Design: Case-ontrol study Setting: Cases were reruited from two tertiary referral entres speialising in autisti spetrum disorders, while ontrols were reruited from mainstream primary and seondary shools in the same geographial area. Partiipants: 65 boys with autism, mean age 7.4 years (range 5 11) and 158 ontrol boys, mean age 7.8 years (range ). Investigations: Urine samples were examined by high pressure liquid hromatography (HPLC) and matrix assisted laser desorbtion ionisation-time of flight mass spetrometry (MALDI-TOF MS) for the presene of a number of putative opioid peptides. Outomes: There were no signifiant differenes between the HPLC urinary profiles of the hildren affeted by autism and the typially developing ontrols. In those ases where HPLC showed peaks in the loations at whih opioid peptides might be expeted to be found, MALDI-TOF established that these peaks did not, in fat, represent opioid peptides. Conlusions: Given the lak of evidene for any opioid peptiduria in hildren with autism, opioid peptides an neither serve as a biomedial marker for autism nor be employed to predit or monitor response to a asein- and gluten-free diet. Autism is a severe lifelong developmental disorder, defined on the basis of behavioural harateristis whih result from impairments in soial ommuniation and reiproal soial interation, repetitive and restritive behaviours, and imaginary thought. 1 It is one of the most ommon serious developmental disabilities of hildhood. 2 An underlying aetiology for their autism is identified for a minority of hildren, but this proportion rises in the learning disabled population. 3 For the majority of hildren for whom no underlying aetiology is identified, there are no evidene-based biomedial investigations to guide intervention. This has been identified as a key area for researh. 4 This an be a frustrating state of affairs for parents who are ontending with a diagnosis of autism for their hild. Most psyho-eduational interventions are onsidered helpful, although they too lak an evidene base. 5 In addition, many parents ontinue What is already known on this topi Claims that autisti hildren suffer from a ondition known as leaky gut, resulting in exogenously derived opioid peptides being present in their urine, have resulted in large numbers of parents seeking urinalysis in ommerial laboratories. Proponents of the leaky gut theory advoate the use of opioid peptiduria as a biomedial marker of the disorder and a pointer towards dietary intervention. What this study adds Original artile This study finds no evidene of opioid peptiduria in male hildren with autism or Asperger syndrome, nor evidene for any other differenes in urinary profiles between hildren with autism and Asperger syndrome and a mathed ontrol group. There is therefore no evidene that opioid peptiduria performs as a biomedial marker of autism, nor that it an be usefully employed to predit or monitor response to a asein and gluten exlusion diet. These findings are ounter to the putative mehanism of the leaky gut syndrome. to pursue experimental biomedial treatments. These inlude adopting a asein- and gluten-free exlusion diet for their affeted hildren, although a reent Cohrane Review established that this intervention laked an evidene base. 6 The leaky gut hypothesis proposes that there may be gluten sensitivity and a speifi enteroolitis in hildren with autism, resulting in an exogenously derived opioid peptiduria. These opioid peptides are assumed to ross the blood brain barrier and result in autisti symptomatology. 7 Anumberofresearhers have proposed that asein and gluten exlusion diets exert their effet by reduing irulating exogenously derived opioid peptides These investigators have also presented evidene that prior to ommening this exlusion diet, some hildren with autism have opioid peptiduria and that this an guide whether they are likely to respond to this type of dietary intervention. 12 There are many problems with this hypothesis, inluding major weaknesses in terms of biologial and linial plausibility. 13 Children with autism do Arh Dis Child 2008;93: doi: /ad

2 not have an inreased rate of oelia disease. 14 Neither are they more likely to present to their general pratitioner with hroni inflammation of the gastro-intestinal trat, food intolerane or reurrent gastro-intestinal symptoms. 15 Furthermore, the early tehniques for establishing the presene of opioid peptiduria employed either immunologial approahes 16 using antibodies raised against gluten or asein derivatives whih are subjet to diffiulties of ross-reativity and non-speifi binding, or high pressure liquid hromotography (HPLC). Thus they do not provide information onerning sequene length or the identity of the peptide(s). Other researhers employing HPLC have been unable to repliate the findings of urinary peptides. 17 A study with a small number of subjets was also unable to identify exogenously derived peptides employing mass spetrometry. 18 The aim of the present study was to determine whether exogenous or endogenous peptides were present in the urine of hildren with autism in physiologially signifiant quantities. We aimed to determine if differenes existed in the amount and nature of suh peptides when omparing urine samples from hildren with autism and age- and sex-mathed ontrol hildren. METHODS Subjets Cases onsisted of 68 male hildren with autism aged 4 11 years reruited from two tertiary diagnosti entres speialising in the assessment of hildren with neurodevelopmental disorders. Informed onsent and assent was obtained from parents and from hildren where possible. In addition to relevant loal ethis ommittee approval, the National Autisti Soiety researh ethis ommittee gave approval for the study. Diagnosis in all ases was based on ICD-10 lassifiation for autism. A total of 61 subjets reeived a diagnosis of autism, and seven a diagnosis of Asperger syndrome. Unlear ases or those that met partial riteria, for example atypial autism, were exluded. Diagnosis was ahieved through a multi-disiplinary, multi-ageny assessment as desribed in the National Autism Plan for Children, 19 and ases were only seleted for this study when there was omplete agreement between all team members on the basis of interview, history and observations. Diagnoses were further validated by formal Autism Diagnosti Interview- Revised (ADI-R) interviews 20 in 41 ases (60%) with 100% onordane between linial and ADI-R diagnosis. IQ was assessed in all ases: 16 of the autisti group (and by definition, all seven of the Asperger group) had IQs.70. Children with progressive neurologial disorders and unstable epilepsy were exluded, as were those taking regular mediations inluding stimulants and antionvulsants. Three ases were on gluten-freedietsandthreeaseswereongluten- andasein-freediets. A total of 202 male ontrols aged between 4 and 12 years were reruited from mainstream infant and primary shools, based on the same ethis ommittee approval as given for the ases with autism. After permission was obtained from head teahers, a standard information leaflet was irulated to parents inviting them to partiipate in the study. Informed onsent and assent was obtained from parents and, where possible, from ontrol hildren. The Strengths and Diffiulties Questionnaire (SDQ) was ompleted by the parents of the ontrols in order to sreen out hildren with possible neuropsyhiatri disorders. 21 Fifteen of the ontrol group sored in the borderline range on the SDQ with a further 18 soring in the abnormal range. Seven parents eleted not to omplete the SDQ. All these individuals were exluded from further analysis leaving 162 ontrols. Urine olletion and sampling For all ases and ontrols, first morning urine was olleted on dry ie and immediately transferred to a 270uC freezer. Samples were bar oded to allow analysis to be arried out blind to diagnosti status. Urine samples for reatinine (see tehnial appendix 1 in supplementary data) and peptide measurement were suessfully aptured and analysed for 158 of the ontrols and 65 of the ases (58 hildren with autism and seven hildren with Asperger syndrome). HPLC analysis All HPLC analysis was arried out on an Amersham Biosienes AKTA Basi system. All hromatography was onduted using a reversed phase C18 olumn (ACE 5 C mm; at. no. ACE ) with a Phenomenex pre-olumn guard (AJO- 6073) (see tehnial appendix 2 in supplementary data). UV absorbane measurements (215 nm) were automatially taken every 0.8 s over the first 118 min of the run, generating an ASCII file listing approximately 8800 data points. Where HPLC analysis of a urine sample indiated the presene of high and well-defined peaks exhibiting the orret retention time for one or more of the suspet opioid peptides ited in previous studies, 8 12 the sample was lassified as a andidate for further analysis. Samples from all groups showed suh peaks, but only those from the autism and Asperger syndrome group were reanalysed, as being the most likely to present opioids given the assumptions of the leaky-gut hypothesis. Twenty five suh samples were re-frationated by HPLC, and individual frations were taken for further analysis by matrix assisted laser desorbtion ionisationtime of flight mass spetrometry (MALDI-TOF MS) to determine if any peaks exhibiting the expeted m/z ratio were observed. Power alulations Power alulations were arried out using NQuery Advisor software. The alulations show that the reatinine adjusted spetra generate statistis whih, given the number of hildren in the study, would have high statistial power to detet differenes between the ontrol and autisti spetrum disorder groups (see olumn 7 in table 1). Comparisons between the ontrol group and the autism group had similar properties. The low number of repliates in the Asperger syndrome group means that all omparisons to that group alone would have low power (not shown). Prior validation of biohemial tehniques Before embarking on the analysis desribed in this study, we arried out extensive validation of every aspet of our laboratory methodology and assay tehnique. This inluded HPLC and MALDI-TOF analysis of syntheti opioid peptides (see tehnial appendix 3 in supplementary data), alulation of the lower limit of detetion of peptides in urine for both tehniques (see tehnial appendix 4 in supplementary data), measurement of peptide stability in urine and then alulation of the oeffiient of variation for system reproduibility to guide subsequent statistial analysis (see tehnial appendix 5 in supplementary data). RESULTS Statistial analysis of total peak area The total peak area (ie, the area under the urve) for eah urine sample was alulated and the data were examined to determine if a statistially signifiant differene in means existed between the groups (table 1). Data were ompared either in raw unorreted form or normalised to the equivalent of 200 nm reatinine per sample to aount for differenes in urinary reatinine ontent between samples. Comparisons were made 746 Arh Dis Child 2008;93: doi: /ad

3 Table 1 Comparison of mean total peak area from the omplete (0 78 min) and trunated spetra (40 78 min) between the ontrol and autisti groups, using unorreted and reatinine orreted data Mean values (SE) Total peak area Control (n = 158) Total ases (n = 65) between the ontrol (n = 158) and omplete autism and Asperger syndrome dataset (n = 65) (ie, two groups in total), and also between the ontrol and the two diagnosti subgroups (ie, three groups in total). In eah ase, analysis was arried out using one-way ANOVA, and p values were ontrolled to allow for multiple omparisons using both Fisher s and Dunnett s methods, where the individual and family error rates respetively were 5%, and the normal group was seleted as the baseline omparison for the Dunnett proedure. Although the ases as a group exhibited a larger average total peak area than the ontrol group for both the full and trunated spetra (the latter being the region where suspet opioid peptides would be expeted to elute if present), the relatively large variation between hildren within eah group ensured that these effets were not statistially signifiant (table 1, rows A and B, olumns 1, 2 and 5). When the peak area data were orreted for differenes in reatinine onentration, the effet was to inrease the apparent differene in average total peak areas between the two groups (rows C and D, olumns 1 and 2). However, no statistial signifiane was apparent when omparing the means of the two groups (olumn 5). When repeating these analyses for the autism subgroups, there was no statistially signifiant evidene for differenes in the urinary mean peak areas assoiated with different subgroups (olumns 3, 4 and 6), either with or without reatinine orretion. In all ases, the differenes in mean total peak areas are small ompared with the differenes seen between different hildren in the same groups. Autism (n = 58) Asperger syndrome (n = 7) p Value, ontrol/ ases p Value, all groups Size of differene detetable A. Complete spetrum* 1209 (18) 1229 (28) 1219 (30) 1311 (86) % B. Trunated spetrum* (8.0) (12.4) (13.2) (37.9) % C. Creatinine adjusted omplete spetrum{ (0.003) (0.005) (0.005) (0.014) % D. Creatinine adjusted trunated spetrum{ (0.003) (0.005) (0.006) (0.016) % E. Creatinine ontent* (0.62) 27.7 (0.96) (1.02) (2.93) % *Using square root transformed data; {using repeated log transformed data. A omparison of the reatinine onentration in the urine is also shown. The final olumn presents the minimum differene between the ontrol and autisti spetrum disorder groups detetable at 8% statistial power given the observed variability, as a perentage of the observed mean of the ontrol group. SE, standard error. Visual omparison of average urinary traes of ase and ontrol urines: whole spetra The average HPLC spetra for ase and ontrol urine samples were generated by alulating the average absorbane reading at every 0.8 s data point throughout the run for the entire dataset (fig 1). The overall amount of material under the urve was slightly larger for ases ompared to the ontrol group for most peaks throughout both traes and not just in the region min. The averaged autism/asperger trae and ontrol group trae were very similar in overall profile, with no peaks being ase or ontrol speifi and any differenes in peak height being small (10 15%). Average HPLC spetra for the autism and IQ subgroups (see tehnial appendix 6 in supplementary data) show no obvious peaks in any groups. MALDI-TOF MS for opioid peptides By HPLC analysis alone, 25 urine samples from the autism/ Asperger group were seleted as showing peaks in approximately the orret loations to be opioid peptides. For all 25, MALDI-TOF MS analysis of the relevant fration found no instanes of ions of m/z orresponding to opioid peptides. These results indiate that the peaks observed on the HPLC trae were not opioid peptides. Two examples of this are shown in figure 2. HPLC data suggested that met-enkephalin and dermorphin may have been present in sample 263; however, this was not substantiated by subsequent MALDI-TOF MS of the relevant frations. Similarly with autism sample 59, the HPLC data Figure 1 Creatinine-orreted (equivalent of 200 nm loaded) average urinary trae from ase and ontrol samples over the full 78-min run. Arh Dis Child 2008;93: doi: /ad

4 Arh Dis Child: first published as /ad on 12 Marh Downloaded from Figure 2 HPLC traes of ases 263 (top left) and 56 (top right) showing peak intensity (absorbane at 215 nm) plotted against retention time. HPLC trae is shown plotted with ontinuous lines, overlaid with peaks demonstrating the loation of pure opioid peptides shown with dotted lines. The frations marked below the x axis were taken for further analysis by MALDI-TOF MS, and the traes generated by eah individual fration are shown plotted underneath. The opioid peptides predited to be present by virtue of the orret retention time are shown marked on the HPLC hart, together with the m/z ratio whih should be present on the MALDI trae if the peptide was indeed present in the fration. on 21 July 2018 by guest. Proteted by opyright. 748 Arh Dis Child 2008;93: doi: /ad

5 suggested the presene of b-asomorphin 7 and neuropeptide FF. Again MALDI-TOF MS showed they were not opioid peptides. These negative observations were reported for all samples examined by a ombination of HPLC and MALDI-TOF MS. DISCUSSION This study finds no evidene of opioid peptiduria in male hildren with autism or Asperger syndrome, nor is there any evidene for any differenes in urinary profiles between hildren with autism and Asperger syndrome and a mathed ontrol group. There is therefore no evidene that opioid peptiduria performs as a biomedial marker of autism, nor that it an be usefully employed to predit or monitor response to a asein and gluten exlusion diet. These findings are ounter to the putative mehanism of the leaky gut syndrome. There are a number of strengths in this present study whih support these onlusions. Firstly, we report the findings from urine analysis of hildren with well-defined autism and Asperger syndrome aross a broad range of ognitive abilities. Seondly, while the initial stage of our study employed the same HPLC tehnique as desribed by the main researh groups reporting opioid peptiduria, we had the advantage of welldefined syntheti opioid peptides with whih we were able to ondut extensive benh-side laboratory tests. These onfirm that the onditions under whih we onduted our experiments were rigorous and appropriate and that methodologial errors annot explain our failure to repliate their apparent demonstration of opioid peptiduria. Thirdly, the present study employed mass spetrometry as an additional means of analysing urinary derived material. This tehnique provides an exat moleular weight for any moleules and is more sensitive to peptides than HPLC. HPLC only gives information regarding retention time, but thousands of highly dissimilar moleules may share a ommon retention time. HPLC allows no onlusions to be drawn regarding mass or struture and therefore annot be used to separate a omplex mixture of analytes with any degree of auray at these levels. The importane of being able to ompare HPLC and MALDI-TOF analyses was demonstrated, for example, in those ases where HPLC showed peaks in the loations at whih opioid peptides might be expeted to be found. In all suh ases, MALDI-TOF established that these peaks did not, in fat, represent opioid peptides. It is worth revisiting assumptions desribed in earlier work by Ek et al that illustrate the importane of this point: The known peptides were reognised by their points of elution and by hromatography with peak augmentation, using ommerially bought syntheti peptides. It is, of ourse, oneivable that a hromatographially similar peptide may have appeared and run with the known peaks However, it is reasonable to think that these peaks are aseomorphin 1-8-like peptides. 22 We have shown that suh interpretations are not, in fat, reasonable and have led to false assumptions about the presene of exogenously derived opioid peptides. One possible weakness of this study is the fat that ases were reruited from tertiary entres and hene may have been more linially severe or may have differed systematially from hildren presenting to ommunity servies. We would argue that our deision in this partiular study to deliberately only inlude full-blown ases of autism or Asperger syndrome serves to strengthen the findings. Another possible weakness is our lak of a ontrol group mathed for the very low IQ hildren with autism. This would have been a signifiant weakness if peptides had been identified in either group and shown an assoiation with IQ. However, peptides were not found in either ase. Although it may be argued that peptides should be lower or absent in the three hildren on gluten-free diets and three hildren on gluten- and asein-free diets, this does not explain their absene in the other 62 ases. The hypothesis of the leaky gut has long been ontroversial, with proponents iting links between autism and gut abnormalities, and advoating the use of opioid peptiduria as a biomedial marker of the disorder and a pointer towards intervention. However, there is now a large body of work whih refutes the theory and annot repliate the findings. We suggest that this present study demonstrates that hildren with autism should not be subjeted to investigation of urinary opioid peptides or their parents to the expense of the assays whih are still widely advertised on the internet by ommerial laboratories around the world. There is still a need to sientifially establish for parents whether a asein- and gluten-free diet is an effetive intervention for autism, but we have shown that there is no evidene that exogenously derived opioid peptides are the mehanism by whih suh diets may exert their effet. Aknowledgements: We thank all the hildren, their shools and their parents, the multidisiplinary teams at Harper House Children s Servie and the Wolfson Centre, Great Ormond Street Hospital, and Dr Adrian Dawkes for his help in study design and initial sample olletion and analysis. Funding: This researh was funded by the R&D Fund, RHSC, Edinburgh and the Chief Sientist Offie, Edinburgh. Dr Luy Owen s post was funded by Ortho-Clinial Diagnostis, a UK based diagnostis arm of Johnson and Johnson. Competing interests: JM has previously ated as an expert witness for the litigants in the MMR litigation ase onduted by Alexander Harris against three MMR vaine manufaturers, whih involved urinary analysis of both litigants and non-litigant ontrols using MALDI-TOF mass spetrometry. He has an autisti hild who was not part of this legal ase. Ethis approval: Obtained from the loal ethis ommittee and the National Autisti Soiety researh ethis ommittee. Patient onsent: Obtained. REFERENCES 1. World Health Organization. The ICD-10 Classifiation of Mental and Behavioral Disorders - Diagnosti Criteria for Researh. Geneva: Switzerland, Baird G, Simonoff E, Pikles A, et al. Prevalene of disorders of the autism spetrum in a population ohort of hildren in South Thames: the Speial Needs and Autism Projet (SNAP). Lanet 2006;368: Gillberg C, Soderstrom H. Learning disability. Lanet 2003;362: MRC. Review of autism researh - epidemiology and auses. London: Medial Researh Counil, Available from: Doumentreord/index.htm?d = MRC (aessed 24 April 2008). 5. Diggle T, MConahie HR, Randle VR. Parent-mediated early intervention for young hildren with autism spetrum disorder. Cohrane Database Syst Rev 2003;(1):CD Millward C, Ferriter M, Calver S, et al. Gluten- and asein-free diets for autisti spetrum disorder. Cohrane Database Syst Rev 2004;(2):CD Wakefield AJ, Murh SH, Anthony A, et al. Ileal lymphoid nodular hyperplasia, nonspeifi olitis and pervasive developmental disorder in hildren. Lanet 1998;351: Reihelt KL, Knivsberg AM, Nodland M, et al. Nature and onsequene of hyperpeptiduria and bovine asomorphine found in autisti syndromes. Brain Dysfuntion 1994;7: Reihelt KL, Knivsberg AM, Lind G, et al. Probable etiology and possible treatment of hildhood autism. Brain Dysfuntion 1991;4: Whiteley P, Rodgers J, Savery D, et al. A gluten-free diet as an intervention for autism and assoiated spetrum disorders: preliminary findings. Autism 1999;3(1): Shattok P, Whiteley P. Biohemial aspets in autism spetrum disorders: updating the opioid-exess theory and presenting new opportunities for biomedial intervention. Expert Opin Ther Targets 2002;6(2): Knivsberg AM, Reihelt KL, Hoien T, et al. A randomised, ontrolled study of dietary intervention in autisti syndromes. Nutr Neurosi 2002;5(4): Gershon M. Autism and the MMR vaine. Revisiones Vaunas 2002;2: MCarthy DM, Coleman M. Response of intestinal muosa to gluten hallenge in autisti subjets. Lanet 1979;2(8148): Blak C, Kaye JA, Jik H. Relation of hildhood gastrointestinal disorders to autism: nested ase-ontrol study using data from the UK General Pratie Researh Database. BMJ 2002;325(7361): Reihelt KL, Hole K, Hamberger A, et al. Biologially ative peptide-ontaining frations in shizophrenia and hildhood autism. Adv Biohem Psyhopharmaol 1981;28: Arh Dis Child 2008;93: doi: /ad

6 17. Le Couter A, Trygstad O, Evered C, et al. Infantile autism and urinary exretion of peptides and protein-assoiated peptide omplexes. J Autism Dev Disord 1988;18(2): Hunter LC, O Hare A, Herron WJ, et al. Opioid peptides and dipeptidyl peptidase in autism (see omment). Dev Med Child Neurol 2003;45(2): National Initiative for Autism Sreening and Assessment. National autism plan for hildren (NAPC). London: The National Autisti Soiety, 2003:50. Images in paediatris Reurrent papular urtiaria in a 6-yearold girl CASE REPORT A 6-year-old female was referred for multiple pruriti lesions on her legs. No other members of the family had similar skin manifestations. The family reported having fleas at home previously, but the house had sine been fumigated. Examination revealed numerous 4 8 mm exoriated, erythematous papules in lusters on the legs (fig 1). The family was reassured that these lesions were probably seondary to flea bites. However, the patient ontinued to have intermittent rerudesenes. The family beame frustrated and septial beause no one else at home showed signs of inset bites. At the sixth-month follow-up, the patient s mother brought in some debris from the patient s bed. The material was examined mirosopially and identified to be a at flea (Ctenoephalides felis) larva (fig 2). The family at was treated for fleas and at follow up the patient was found to be lear. Children under 2 years typially do not have suffiient exposure to inset antigens to mount a robust hypersensitivity response. 2 Conversely, hildren over 10 years typially develop tolerane to the antigen and produe transient wheals instead of long-lasting papules. 24 This age-based suseptibility explains the ommon but ounterintuitive senario illustrated in this ase wherein only a single hild is affeted despite the whole family being exposed to the inset. Management of papular urtiaria prevention and eradiation is usually implemented without onfirmatory evidene of Figure 1 Clusters of exoriated erythematous papules and healing maules on the patient s lower extremities. 20. Lord C, Rutter M, Le Couter A. Autism Diagnosti Interview-Revised: a revised version of a diagnosti interview for aregivers of individuals with possible pervasive developmental disorders. J Autism Dev Disord 2002;24: Goodman R, Ford T, Simmons H, et al. Using the Strengths and Diffiulties Questionnaire (SDQ) to sreen for hild psyhiatri disorders in a ommunity sample. Int Rev Psyhiatry 2003;15(1 2): Ek J, Stensrud M, Reihelt KL. Gluten-free diet dereases urinary peptide levels in hildren with elia disease. J Pediatr Gastroenterol Nutr 1999;29(3): Figure 2 Photomirograph of the at flea larva (original magnifiation 106). the ulprit. 1 3 Unfortunately, preventative measures suh as home fumigation and treatment of pets are ostly and may seem exessive when families are septial of the diagnosis. 3 4 Identifying the ulprit helps motivate families to invest in prevention and an unover failures in eradiations. By verifying the persistene of the at flea in the patient s home despite fumigation, we pinpointed the at as the soure of re-infestation and potentially averted a frustrating yle of reurring eruptions for the patient. K P Thieu, 1 P A Lio 1,2,3 1 Harvard Medial Shool, 25 Shattuk Street, Boston, USA; 2 Department of Dermatology, Beth Israel Deaoness Medial Center, Boston, USA; 3 Children s Hospital Boston, Boston, USA Correspondene to: Peter A Lio, Beth Israel Deaoness Medial Center, Department of Dermatology, 330 Brookline Avenue, Boston, MA 02115; plio@bidm.harvard.edu Aknowledgements: We would like to thank Rihard J Pollak for his assistane in identifying the larva. Funding: None. Competing interests: None. Patient onsent: Parental/guardian onsent obtained. 2008;93:750. doi: /ad REFERENCES 1. Stibih AS, Shwartz RA. Papular urtiaria. Cutis. 2001;68: Hernandez RG, Cohen BA. Inset bite-indued hypersensitivity and the SCRATCH priniples: a new approah to papular urtiaria. Pediatris 2006;118:e Steen CJ, Carbonaro PA, Shwartz RA. Arthropods in dermatology. J Am Aad Dermatol 2004;50: Fitzpatrik TB, Johnson RA, Wollf K. Color Atlas and Synopsis of Clinial Dermatology. New York (NY): MGraw-Hill Companies, 2001: Arh Dis Child September 2008 Vol 93 No 9