Emanuel Schwarz

Paul C Guest

Hassan Rahmoune

Lan Wang

Yishai Levin

Erin Ingudomnukul

Liliana Ruta

Lindsey Kent

Michael Spain

Simon Baron-Cohen

Sabine Bahn

Sex-Specific Serum Biomarker Patterns in Adults with Asperger Syndrome

Keywords: Analysis of Variance, ANOVA, Autism-Spectrum Quotient, AQ, Asperger syndrome, AS, Brain Derived Neurotrophic Factor, BDNF, Diagnostic and Statistical Manual of Mental Disorders, DSM, Empathy Quotient, EQ, Extreme Male Brain, EMB, Fetal Testosterone, FT, PolyCystic Ovarian Syndrome, PCOS, Systemizing Quotient-revised, SQ-R, Asperger syndrome, serum, gender, sexual dimorphism, profiling, biomarker

Autism spectrum conditions have been hypothesized to be an exaggeration of normal male low-empathizing and high-systemizing behaviours. We tested this hypothesis at the molecular level by performing comprehensive multi-analyte profiling of blood serum from adult subjects with Asperger syndrome (AS) compared to controls. This led to identification of distinct sex-specific biomarker fingerprints for male and female subjects. Males with AS showed altered levels of 24 biomarkers including increased levels of cytokines and other inflammatory molecules. Multivariate statistical classification of males using this panel of 24 biomarkers revealed a marked separation between AS and controls with a sensitivity of 0.86 and specificity of 0.88. Testing this same panel in females did not result in a separation between the AS and control groups. In contrast, AS females showed altered levels of 17 biomarkers including growth factors and hormones such as androgens, growth hormone and insulin-related molecules. Classification of females using this biomarker panel resulted in a separation between AS and controls with sensitivities and specificities of 0.96 and 0.83 respectively, and testing this same panel in the male group did not result in a separation between the AS and control groups. The finding of elevated testosterone in AS females confirmed predictions from the "extreme male brain" and androgen theories of autism spectrum conditions. We conclude that to understand the aetiology and development of autism spectrum conditions, stratification by sex is essential.

Introduction

Autism spectrum conditions are diagnosed on the basis of difficulties in communication and social development, in addition to unusually narrow interests, repetitive behaviour and resistance to change (1). The two major subgroups are classic autism and Asperger syndrome (AS). AS differs from classic autism in that there is no history of language delay or learning difficulties [START_REF] Volkmar | Autism and autism spectrum disorders: diagnostic issues for the coming decade[END_REF]. The prevalence of autism spectrum conditions is 1% of the general population [START_REF] Baron-Cohen | Prevalence of autism-spectrum conditions: UK school-based population study[END_REF] and there is a marked sex difference, with males being 4-10 times more likely to be affected than females [START_REF] Wing | Asperger's syndrome: a clinical account[END_REF]. This suggests that sex-linked factors might play a role in the aetiology of the condition. In the general population, males on average perform at lower levels on tests of empathy and social sensitivity and show stronger interest in systems.

People with autism spectrum conditions show an exaggeration of this cognitive profile which has led to the "extreme male brain" (EMB) theory as an explanation of the psychological manifestations [START_REF] Baron-Cohen | The extreme male brain theory of autism[END_REF]. The EMB theory is supported by the finding of tomboyism and other masculine behaviors in females with autism spectrum conditions [START_REF] Knickmeyer | Sex-typical play: masculinization/defeminization in girls with an autism spectrum condition[END_REF]. The EMB theory has been extended recently to the neurological level. For example, in typical males the overall brain volume and specific regions such as the amygdala are larger. In autism spectrum conditions, these differences are even more extreme [START_REF] Baron-Cohen | Sex differences in the brain: implications for explaining autism[END_REF]. However, little is known about the molecular pathways underlying these effects.

The fetal androgen theory of autism spectrum conditions [START_REF] Baron-Cohen | Sex differences in the brain: implications for explaining autism[END_REF] suggests that increased levels of fetal testosterone (FT) leads to typical sex differences in brain structure and function. Individual differences in amniotic FT are positively-correlated with number of autistic traits [START_REF] Auyeung | Fetal testosterone and autistic traits[END_REF] and systemizing [START_REF] Auyeung | Foetal testosterone and the child systemizing quotient[END_REF], and negatively-correlated with language development, social development and empathy [START_REF] Chapman | Fetal testosterone and empathy: evidence from the empathy quotient (EQ) and the "reading the mind in the eyes" test[END_REF]. This theory is also supported by the observation of an increased rate of testosterone-related disorders such as polycystic ovary syndrome (PCOS), acne and hirsutism in women with autism spectrum conditions [START_REF] Ingudomnukul | Elevated rates of testosterone-related disorders in women with autism spectrum conditions[END_REF], a lower ratio of the 2 nd (index) and 4 th (ring) digit lengths (2D:4D) in both males and females with autism spectrum conditions [START_REF] Manning | The 2nd to 4th digit ratio and autism[END_REF], and an association of autism spectrum conditions with CAG repeats in the androgen receptor gene [START_REF] Henningsson | Possible association between the androgen receptor gene and autism spectrum disorder[END_REF], single nucleotide polymorphisms (SNPs) in the HOXD gene [START_REF] Sugie | Study of HOXD genes in autism particularly regarding the ratio of second to fourth digit length[END_REF], and an association between autistic traits and/or autism spectrum conditions with SNPs in sex-steroid related genes [START_REF] Chakrabarti | Genes related to sex steroids, neural growth, and social-emotional behavior are associated with autistic traits, empathy, and Asperger syndrome[END_REF].

Other molecular factors in the aetiology of autism spectrum conditions may not be sex-linked. Neurotrophins such as brain-derived neurotrophic factor (BDNF) are elevated in the blood of children with autism, which may be associated with early brain overgrowth [START_REF] Courchesne | Evidence of brain overgrowth in the first year of life in autism[END_REF] and SNPs in genes related to neural connectivity and neurotrophic pathways may also be involved [START_REF] Chakrabarti | Genes related to sex steroids, neural growth, and social-emotional behavior are associated with autistic traits, empathy, and Asperger syndrome[END_REF]. In addition, several studies have identified an over-activation of mast [START_REF] Castellani | Autism and immunity: revisited study[END_REF] and natural killer [START_REF] Vojdani | Low natural killer cell cytotoxic activity in autism: the role of glutathione, IL-2 and IL-15[END_REF] cells in autism spectrum conditions that may be associated with autoimmunity or adverse immune interactions during critical periods of brain development.

Autism spectrum conditions are also associated with familial autoimmune diseases and the presence of asthma, allergies and psoriasis during pregnancy [START_REF] Enstrom | Autoimmunity in autism[END_REF]. The interactions of these pathways in the nervous system are likely to be mediated by hormones and cytokines.

Here we present the first systematic serum proteome profiling study of adults with the AS subtype of autism spectrum conditions. We had three major objectives: 1) to test the EMB theory at the molecular level using a multiplex analyte profiling approach, 2) to test the prediction from the fetal androgen theory that adult females with AS would have elevated testosterone levels and 3) to determine whether males and females with AS have distinct sex-specific serum biomarker fingerprints.

Methods

Sample collection

Protocols for recruitment, collection of clinical samples and the test methods were carried out in compliance with the Standards for Reporting of Diagnostic Accuracy (STARD) initiative [START_REF] Bossuyt | Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. Standards for Reporting of Diagnostic Accuracy[END_REF]. People with AS (n=45) were recruited via the Cambridge Autism Research Centre database of volunteers (www.autismresearchcentre.com), the National Autistic Society (UK) and local autism social groups in the United Kingdom. Controls (n=50) were recruited from the general population using leaflets and advertising and these individuals were matched to the AS group with respect to age, IQ, educational level, socioeconomic status, handedness, sex and body mass index (BMI) (Supplementary Table S2). AS was diagnosed by psychiatrists based on the Structured Clinical Interview for the Diagnostic and Statistical Manual-IV-Text Review Disorders and accepted international criteria (DSM-IV-TR). The Wechsler Abbreviated Scale of Intelligence was administered to all participants to measure Intelligence Quotient (IQ). All diagnoses and clinical tests were performed by psychiatrists under Good Clinical Practice compliance to minimize variability. Any patients whose clinical diagnosis required revision at a later stage were not included in the study.

Controls with a family history of serious mental illness or other medical conditions such as type II diabetes, hypertension, cardiovascular or autoimmune diseases were excluded.

Psychological measures

All participants were tested on the Autism-Spectrum Quotient (AQ) [START_REF] Baron-Cohen | The autism-spectrum quotient (AQ): evidence from Asperger syndrome/high-functioning autism, males and females, scientists and mathematicians[END_REF], as a measure of the number of autistic traits an individual possesses, the Empathy Quotient (EQ) and the Systemizing Quotient-revised (SQ-R) [START_REF] Wheelwright | Predicting Autism Spectrum Quotient (AQ) from the Systemizing Quotient-Revised (SQ-R) and Empathy Quotient (EQ)[END_REF] in order to test the EMB theory at the cognitive level. Normally, 80% of people with autism spectrum conditions score above a cut-off value of 32 (maximum = 50) on the AQ compared to only 2% for controls, a finding that has been confirmed cross-culturally [START_REF] Wakabayashi | The Autism-Spectrum Quotient (AQ) in Japan: A cross-cultural comparison[END_REF][START_REF] Hoekstra | Factor structure, reliability and criterion validity of the Autism-Spectrum Quotient (AQ): a study in Dutch population and patient groups[END_REF]. AQ predicts clinical diagnosis [START_REF] Woodbury-Smith | Screening adults for Asperger Syndrome using the AQ: a preliminary study of its diagnostic validity in clinical practice[END_REF] and has also been shown to be elevated among parents of children with autism spectrum conditions [START_REF] Bishop | Using self-report to identify the broad phenotype in parents of children with autistic spectrum disorders: a study using the Autism-Spectrum Quotient[END_REF], suggesting a heritability component.

Immunoassay profiling

We carried out HumanMAP® profiling of 147 analytes (see Table S1) in serum samples from the AS and control subjects in a Clinical Laboratory Improved Amendmentscertified laboratory at Rules Based Medicine (Austin, TX, USA). This technology has already been applied successfully in several clinical studies [START_REF] Bertenshaw | Multianalyte profiling of serum antigens and autoimmune and infectious disease molecules to identify biomarkers dysregulated in epithelial ovarian cancer[END_REF]. Analytes were measured in multiplexes in 25-50µL of serum samples. The assays were calibrated using standard curves and raw intensity measurements converted to protein concentrations using proprietary software. Analyses were conducted under blind conditions with respect to sample identities and the samples were analyzed randomly to avoid any sequential bias due to the presence or absence of diagnosis, participant age, or age of material.

Statistics

ANOVA was carried out on log-transformed data to investigate any interactions between sex and diagnosis for each molecular analyte and psychometric characteristic (AQ, EQ and SQ). To account for potential deviations from normality and homoscedasticity calculated interaction effects were compared against Aligned Rank Transform (ART) nonparametric analyses. For this purpose, Hodges-Lehmann estimates of the two main effects were subtracted from the raw scores, data ranked and analyzed using two-way ANOVA. P-values are stated without adjustment for multiple hypothesis correction as the necessity of this procedure remains to be controversial [START_REF] Rothman | No adjustments are needed for multiple comparisons[END_REF].

Sensitivity and specificity values were calculated based on a leave-one out cross validation procedure using the AdaBoost algorithm [START_REF] Hastie | The Elements of Statistical Learning[END_REF].

Results

Psychological measures

In this study, all participants were tested on the Autism-Spectrum Quotient (AQ) [START_REF] Baron-Cohen | The autism-spectrum quotient (AQ): evidence from Asperger syndrome/high-functioning autism, males and females, scientists and mathematicians[END_REF], the Empathy Quotient (EQ) and the Systemizing Quotient-revised (SQ-R) [START_REF] Wheelwright | Predicting Autism Spectrum Quotient (AQ) from the Systemizing Quotient-Revised (SQ-R) and Empathy Quotient (EQ)[END_REF]. The AS group had higher scores for AQ and SQ-R, and lower scores for EQ, replicating previous findings [START_REF] Baron-Cohen | The autism-spectrum quotient (AQ): evidence from Asperger syndrome/high-functioning autism, males and females, scientists and mathematicians[END_REF][START_REF] Wheelwright | Predicting Autism Spectrum Quotient (AQ) from the Systemizing Quotient-Revised (SQ-R) and Empathy Quotient (EQ)[END_REF]. Analysis of variance (ANOVA) testing showed a significant interaction between sex and diagnosis for all three measures (AQ, p=0.004; EQ, p=0.014; SQ, p=0.016) with females with AS having more "masculinized" scores compared to control females and compared to males with AS (Supplementary Table S2, Fig. 1). Interaction effects were comparable to results obtained form non-parametric analysis.

Immunoassay profiling

We carried out HumanMAP® profiling of 147 analytes in serum samples from the AS and control subjects. As a first step of the analysis, male and female subjects were analyzed separately due to the marked gender difference in prevalence rates. For this purpose, male and female subjects were randomized separately into training and blinded test sets to facilitate initial validation of gender specific biomarker signature. This is important since AS subjects are rare, and the prevalence is approximately 10 times lower for females than for males, making an independent replication of results difficult. Statistical analysis of the male training set identified a panel of 9 analytes which showed significant differences in expression between AS (n=12) and control (n=16) subjects (Supplementary Table S3).

Application of this panel to the male test set resulted in correct classification of 70% of AS (n=10) and 100% of control (n=10) subjects (P=0.003) (Fig. 2a). In contrast, application of the panel to the female test set could not distinguish AS subjects from controls (P=0.350).

Separate analysis of the female training set resulted in identification of 15 analytes with significant differences in expression between AS subjects (n=13) and controls (n=14) (Supplementary Table S3). Application of this panel to the female test set resulted in correct classification of 90% of AS (n=10) and 90% of control (n=10) subjects (P=0.001) (Fig. 2b).

Application of the female-specific panel to the male test set could not discriminate AS subjects from controls (P>0.99), as above for the male-female cross-testing.

As a next step, serum samples from were compared between the total set of AS and control subjects. This led to identification 25 analytes which showed significant differences in expression between the groups (Supplementary Table S4). ANOVA found that 15 of these analytes also showed sex differences in expression. In addition, 14 separate analytes showed significant interactions between diagnosis and sex. Total testosterone showed no difference between AS and controls but free testosterone levels showed a significant interaction between diagnosis and sex as determined by calculating the free androgen index (FAI), defined as the ratio of total testosterone to sex hormone binding globulin (SHBG).

The significant gender-diagnosis interactions support the presence of gender-specific biomarker signatures. Given the biological reproducibility observed in independent training and test sets, we repeated the gender-specific analysis using all subjects to increase the statistical power. This approach resulted in identification of 24 analytes that were altered significantly in AS males (see Table 1a) and 17 that were altered significantly in AS females (see Table 1b). Only 4 analytes (IL-12p40, tissue factor, IL-1B, GOT1) were changed in the same direction in males and females, providing evidence for a minimal overlap of the molecular signatures (Fig. 3a). One protein (tenascin-C) showed the opposite regulation (increased in males and decreased in females).

Multivariate classification revealed that the combined panel of 24 differentially expressed analytes in males produced a separation between AS and control subjects with a sensitivity of 0.86 and a specificity of 0.88 (visualized using partial least squares discriminant analysis in Fig. 3b). Testing this same panel of 24 analytes in the female group did not result in a separation between AS and controls (Fig. 3b). Likewise, the combined panel of 17 differentially expressed analytes in females produced a separation between AS and control subjects with a sensitivity of 0.96 and a specificity of 0.83 and testing this same panel in the male group did not result in a separation between AS and controls (Fig. 3c).

Analytes that were altered in males with AS included several cytokines (IL-3, IL-4, IL-5, IL-10, IL-12p70, TNFα, ENA-78), fatty acid binding protein (FABP), the neuroendocrine secreted protein chromogranin A, and the cardiovascular and blood cell-associated proteins thrombopoietin (TPO) and erythropoietin (EPO).

The female-specific AS analytes showed a marked difference in nature and included growth factors, growth hormone, endothelin-1, brain derived neurotrophic factor (BDNF), luteinizing hormone and free testosterone.

Due to the association of growoth hormone deficiency in adult life and decreased insulin sensitivity [START_REF] Hana | The effects of GH replacement in adult GH-deficient patients: changes in body composition without concomitant changes in the adipokines and insulin resistance[END_REF], we also measured the levels of insulin in AS compared to controls as this was part of the HumanMAP® panel. There was no change for male AS subjects (p=0.469) although AS females showed a non-significant 1.57-fold increase compared to levels observed in control females (p=0.208) (data not shown). As insulin is co-released with residual levels of unprocessed proinsulin and the des31,32-proinsulin (31,32-PI) conversion intermediate, we also measured the levels these molecules specifically using sensitive twosite time resolved fluorometric (TRF) assays [START_REF] Guest | Increased levels of circulating insulin-related peptides in first onset, antipsychotic naïve schizophrenia patients[END_REF]. For insulin, the results of the TRF assay showed a high correlation (r 2 =0.98) with the multiplexed xMAP ® platform with an increase in AS females of 1.81-fold compared to control females although, again, this did not reach significance (Table 1c). The levels of proinsulin were not altered significantly although des31,32-proinsulin showed a significant 1.89-fold increase in females. The finding that glucose levels were relatively normal in the same subjects (1.11-fold; p=0.0670) suggested that the relatively high levels of insulin-related molecules may be due to insulin resistance.

Discussion

This is the first study demonstrating that males and females with AS have distinct biomarker fingerprints which can be detected in blood serum. For many of these molecules, this is the first description for their association with autism spectrum conditions. The remainder have been described previously thereby adding an extra layer of validity to the results. The finding that the biomarker fingerprint for AS males was comprised mostly of increased levels of cytokines is consistent with previous reports showing effects on immune function and autoimmune disorders in autism spectrum conditions [START_REF] Castellani | Autism and immunity: revisited study[END_REF][START_REF] Vojdani | Low natural killer cell cytotoxic activity in autism: the role of glutathione, IL-2 and IL-15[END_REF][START_REF] Enstrom | Autoimmunity in autism[END_REF]. The changes in testosterone and BDNF in females with AS are also consistent with the findings of other studies which have suggested that these molecules may be elevated in adults with autism spectrum conditions [START_REF] Ingudomnukul | Elevated rates of testosterone-related disorders in women with autism spectrum conditions[END_REF][START_REF] Courchesne | Evidence of brain overgrowth in the first year of life in autism[END_REF]. The observation of high testosterone levels and relatively low cytokine signature in females aligns well with previous studies which have suggested that testosterone may have protective effects on inflammatory disorders [START_REF] Kanda | Testosterone inhibits immunoglobulin production by human peripheral blood mononuclear cells[END_REF].

Our results showing that free testosterone levels were elevated in adults with AS lends further support to the androgen theory of autism spectrum conditions [START_REF] Baron-Cohen | Sex differences in the brain: implications for explaining autism[END_REF]. This effect was specific for females, which underlines the importance of recognizing sex as a key variable in autism research. Also, the finding of higher levels of biologically active testosterone in AS females provides molecular evidence of the EMB theory [START_REF] Baron-Cohen | The extreme male brain theory of autism[END_REF]. Females with AS showed an extreme of the male brain phenotype as seen by the AQ, EQ and SQ-R psychological measures suggesting that the masculinisation in cognition and androgens may be correlated. The increases in testosterone levels in AS females were paralleled by increases in the levels of luteinizing hormone in the same subjects. Previous studies have shown that luteinizing hormone pulsatility may predispose or cause hyperandrogenism in female adolescents [START_REF] Escobar | Acceleration of luteinizing hormone pulse frequency in adolescent girls with a history of central precocious puberty with versus without hyperandrogenism[END_REF] and has been associated with polycystic ovarian syndrome (PCOS) [START_REF] Barontini | Mechanisms of hypothalamic-pituitary-gonadal disruption in polycystic ovarian syndrome[END_REF]. This is consistent with our earlier work showing elevated rates of testosterone-related disorders including PCOS in women with AS [START_REF] Ingudomnukul | Elevated rates of testosterone-related disorders in women with autism spectrum conditions[END_REF].

The serum levels of BDNF were also increased specifically in females with AS. This is interesting since previous studies have shown increased levels of this growth factor in children with autism spectrum conditions in association with atypical cerebral development [START_REF] Courchesne | Evidence of brain overgrowth in the first year of life in autism[END_REF]. Females with AS also showed an approximate two-fold decrease in growth hormone levels, supporting a previous studies that found a growth hormone deficit in autism [START_REF] Ragusa | Growth hormone deficit in autism[END_REF]. The findings on growth hormone in autism spectrum conditions have not been consistent, with some studies reporting elevated levels of growth-related hormones (36) whereas others have found low cerebrospinal fluid insulin-like growth factor-I concentrations in children with autism [START_REF] Vanhala | Low levels of insulin-like growth factor-I in cerebrospinal fluid in children with autism[END_REF]. However, it should be noted that all these previous studies were conducted using children during the early developmental stages and not on adults as in the case of our study.

Since growth hormone deficiency in adult life has been associated with decreased insulin sensitivity [START_REF] Hana | The effects of GH replacement in adult GH-deficient patients: changes in body composition without concomitant changes in the adipokines and insulin resistance[END_REF], we also measured serum levels of insulin-related peptides and glucose. This showed that insulin and the conversion intermediate, des31,32-proinsulin, were both elevated even though glucose levels were relatively normal. This provided evidence suggesting that at least some of the females with AS may have insulin resistance. This is intriguing because of the link between hyperinsulinemia and hyperandrogenism as described in previous studies of PCOS and post-menopausal women [START_REF] Kebapcilar | Comparison of four different treatment regimens on coagulation parameters, hormonal and metabolic changes in women with polycystic ovary syndrome[END_REF][START_REF] Golden | Glucose and insulin components of the metabolic syndrome are associated with hyperandrogenism in postmenopausal women: the atherosclerosis risk in communities study[END_REF]. Importantly, treatment with insulin sensitizing agents such as rosiglitazone has been shown to improve insulin sensitivity, leading to alleviation of hyperandrogenism and the associated symptoms [START_REF] Zheng | Effect of rosiglitazone on insulin resistance and hyperandrogenism in polycystic ovary syndrome[END_REF]. This highlights the importance of further studies exploring the relationships between androgen abnormalities and BDNF, growth hormone and insulin signalling in autism spectrum conditions.

In AS males, the specific serum biomarker fingerprint showed only a small overlap with that in females with AS. In particular, the predominant feature of this signature in males was increased levels of several cytokines and other inflammatory molecules, consistent with the proposed role of these pathways in autism spectrum conditions [START_REF] Van Gent | Autism and the immune system[END_REF][START_REF] Molloy | Elevated cytokine levels in children with autism spectrum disorder[END_REF]. In contrast, AS females showed altered levels of biomarkers including growth factors and hormones such as androgens, growth hormone and insulin-related molecules. The distinct nature of these sexspecific molecular fingerprints was exemplified by statistical classification analyses which showed that these were only capable of providing good separation of AS and control subjects within the respective male and female groups. The male-specific biomarkers yielded good separation only of male AS and control subjects and the female-specific panel was capable of separating only AS females from control females. Although the observed classification accuracy needs to be reproduced in an independent cohort, these differences underscore the importance of stratifying samples according to sex in biological studies of autism spectrum conditions. The sex-specific molecular profiles observed here lead to the suggestion that either different compensatory mechanisms occur in males and females with autism spectrum conditions or that these conditions may develop through distinct sexspecific molecular pathways.

Assessing the specificity of the present fingerprints for AS against other autism spectrum conditions in young, pubertal and adult subjects could lead to the development of muchneeded novel diagnostic strategies associated with a personalised medicine strategy. The analytes identified through the present study could also be assessed through comparison with the biomarker fingerprints obtained for other neuropsychiatric disorders. In addition, application of a convergent functional genomic strategy, such as that proposed by Le-Niculescu and colleagues (43) could be useful for improving the sensitivity and specificity of emerging biomarker panels. Further studies of these factors could provide greater insight into the molecular mechanisms involved in both the aetiology and development of autism spectrum conditions. an AS (red) or control (black) subject. The model built on males using 24 AS male specific analytes (left) does not yield a separation in prediction of AS in females (right). c) The model built on females with AS and female controls using 17 female specific analytes (left) does not yield a separation when predicting AS in males (right). This is a standard statistical technique for characterizing data variances. A simplified projection of multi-dimensional data is given such that the greatest variance lies on the first coordinate, the second greatest variance on the next coordinate, and so on.

Tables

Figure legends

Fig. 1 .

 1 Fig.1. AQ, EQ and SQ in males and females with AS compared to controls.

Fig. 2 .

 2 Fig. 2. Classification and prediction of AS and control (Cont) subjects using separate panels of a) 10 male-specific analytes and b) 15 female-specific analytes. The analytes were identified in separate training sets for male and female AS and control subjects. These were used for blinded prediction of male and female test subjects. The plus symbol indicate classification as AS and the minus symbol indicates classification as controls. P values indicate the significance of the classification and were calculated using Fisher's exact test. Fig. 3. a) Venn diagram showing distinct and common serum biomarker profiles for males and females with AS. Blue typeface and encircled area indicates hormones and growth factors. Red type face and encircled area indicates cytokines and inflammatory factors. Black typeface indicates

Table 1

 1

	.
	a) Comparison of AS males and control males
	Analyte	p-value	FC
	Interleukin-12p40 (IL-12p40)	0.0015	2.88
	Fatty acid binding protein	0.0010	2.02
	IL-3	0.0313	1.98
	Erythropoietin (EPO)	0.0093	1.92
	Tissue factor (TF)	0.0001	1.70
	IL-5	0.0011	1.59
	Granulocyte colony stimulating factor (G-CSF)	0.0042	1.47
	IL-1B	0.0034	1.44
	Chromogranin A (CGA)	0.0124	1.42
	Neuronal cell adhesion molecule (NrCAM)	0.0303	1.36
	Tenascin-C	0.0300	1.32
	Tumour necrosis factor alpha (TNFα)	0.0044	1.29
	ENA-78	0.0493	1.26
	IL-18	0.0186	1.24
	Factor-VII	0.0316	1.21
	Connective tissue growth factor (CTGF)	0.0090	1.19
	IL-4	0.0267	1.19
	Thrombopoietin (TPO)	0.0022	1.19
	Stem cell factor	0.0098	1.18
	Sortilin 1	0.0078	1.14
	IL-10	0.0333	1.12
	IL-12p70	0.0466	1.10
	Intercellular adhesion molecule 1 (ICAM-1)	0.0466	1.10
	Serum Glutamic Oxaloacetic Transaminase (GOT1)	0.0008	0.83
	ENA-78= epithelial-derived neutrophil-activating protein 78
	b) Comparison of AS females and control females
	Analyte	p-value	FC
	NARG1	0.0082	2.87
	IL-12p40	0.0011	2.74
	FAI	0.0001	2.01
	IL-1B	0.0073	1.75
	Luteinizing hormone	0.0471	1.56
	IL-7	0.0224	1.47
	TF	0.0328	1.42
	BDNF	0.0150	1.20
	GOT1	0.0238	0.84
	Apolipoprotein-CIII (Apo-CIII)	0.0438	0.83
	Immunoglobulin M (IGM )	0.0130	0.76
	sRAGE	0.0425	0.76

Acknowledgements

This work was funded by the Stanley Medical Research Institute, Psynova Neurotech Ltd. and the Three Guineas Project Grant, Cambridge Lifespan Asperger Syndrome Service. Insulin-related assays were performed by Keith Burling at the NIHR Cambridge Biomedical Research Centre, Core Biochemistry Assay Laboratory, Addenbrookes Hospital, Cambridge, UK.

Conflict of interest

The study has been funded by Psynova Neurotech Ltd. There are no conflicts of interest.


Ph.D Fotini Stripeli

Kyriakou P&a

Paidon

M.D.; Z Sakkou

M.D.; N Papadopoulos

M.D V Georgiou

I Gratsia

; M Christodoulou

Ph.D Tsolia

PERFORMANCE OF RAPID INFLUENZA TESTING IN HOSPITALIZED CHILDREN

Keywords: influenza, children, rapid test

PURPOSE:Influenza infection is associated with high hospitalization rates among young children.Rapid diagnosis of influenza infection is particularly useful in order to prevent nosocomial infection and allows for timely initiation of antiviral treatment.We evaluated the performance of a rapid influenza test in hospitalized children during the influenza season. METHODS:All children(6mos-14yrs)hospitalized with fever and/or respiratory symptoms,admitted during the 2005 influenza season,participated in the study.A multiplex RT-PCR,able to identify IFV-A H1N1,H3N2 and IFV-Bsubtypes was performed on nasopharyngeal aspirates.The nasal swab was tested with a lateral-flow immunoassay(QuickVue Influenza Test).The performance of the rapid test was compared with the results of PCR. RESULTS:Influenza infection was diagnosed by PCR in41/217(19%)patients.Infection with influenza A virus(H3N2) was diagnosed in all cases.The performance of the QuickVue Influenza Test was estimated as follows:sensitivity67.5%, specificity96%, positive predictive value79% and negative predictive value93%.The sensitivity of the test was higher in infants 6-12months,in those with short duration of symptoms and in the peak phase of the epidemic. CONCLUSION:The QuickVue Influenza Test is useful and reasonably accurate to detect influenza infection in hospitalized children during the influenza season. Infection with influenza virus is unlikely if the test is negative. A positive result suggests that infection is probable if influenza virus circulates in the community.

Please find enclosed for consideration the revised manuscript entitled "Performance of rapid influenza testing in hospitalized children" (Ref.: Ms. No. EJCMID-D-09-00502) The manuscript has been modified along the lines suggested in your letter and taking into consideration all the suggestions raised by the reviewer. We appreciate the constructive comments by the reviewer that have allowed us to improve the presentation of this study. The changes introduced are specified in the enclosed response to the comments of the reviewer. In addition, all changes have been highlighted in the text for ease of re-review. We believe that we have satisfactory answered all your queries as well as those of your reviewer. We would be pleased if you find that this revised manuscript is now suitable for publication in European Journal of Clinical Microbiology & Infectious Diseases Sincerely yours,

STRIPELI FOTINI MD, PhD Pediatrician

Point by point response to the reviewer We thank the reviewer for his/her most kind comments and for the attention paid to our manuscript. His/her constructive comments have allowed us to improve the presentation of this study Answers to his/her specific comments and suggestions follow:

1.

Abstract1: We acknowledge the reviewer for this specific comment. We added the study period to the methods section (page 2, line 3).

2.

Abstract2: As suggested, we added in the results that all cases had influenza A (page 2, line 9).

3.

Introduction: As suggested, we have deleted the last sentence of the introduction 4.

Methods1: We thank the reviewer for pointing out this point; we have made the appropriate correction (page 3, line 18). 5.

Methods2: As suggested, we added in the methods section how the vaccination status of each patient was determined (page 3, line 3). [START_REF] Zambon | Diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic or molecular detection of influenza[END_REF].

Results 1: We thank the reviewer for pointing out this point; by mistake the mean temperature was written to be 37.9±0.9 instead of 38.9±0.9. The appropriate correction has been made (page 6, line 4). [START_REF] Peltola | Accuracy of clinical diagnosis of influenza in outpatient children[END_REF].

Results 2: As suggested we reversed the order of 5th and 6th paragraph and also the tables 2 and 3. 8.

Results 3: We thank the reviewer for pointing out this point; we have made the appropriate correction to the tables. 9.

Discussion 1: We acknowledge the reviewer for this specific comment; we have made the appropriate correction (page 9, line 13).

Alex van Belkum

Editor-in-Chief

00502)

The manuscript has been modified along the lines suggested in your letter and taking into consideration all the suggestions raised by the reviewer. We appreciate the constructive comments by the reviewer that have allowed us to improve the presentation of this study. The changes introduced are specified in the enclosed response to the comments of the reviewer. In addition, all changes have been highlighted in the text for ease of re-review.

We believe that we have satisfactory answered all your queries as well as those of your reviewer. We would be pleased if you find that this revised manuscript is now suitable for publication in European Journal of Clinical Microbiology & Infectious Diseases Sincerely yours,

STRIPELI FOTINI MD, PhD

Pediatrician

Point by point response to the reviewer

We thank the reviewer for his/her most kind comments and for the attention paid to our manuscript. His/her constructive comments have allowed us to improve the presentation of this study Answers to his/her specific comments and suggestions follow:

1. Abstract1: We acknowledge the reviewer for this specific comment. We added the study period to the methods section (page 2, line 3).

2. Abstract2: As suggested, we added in the results that all cases had influenza A (page 2, line 9).

Introduction:

As suggested, we have deleted the last sentence of the introduction 4. Methods1: We thank the reviewer for pointing out this point; we have made the appropriate correction (page 3, line 18). 5. Methods2: As suggested, we added in the methods section how the vaccination status of each patient was determined (page 3, line 3).

Results 1:

We thank the reviewer for pointing out this point; by mistake the mean temperature was written to be 37.9±0.9 instead of 38.9±0.9. The appropriate correction has been made (page 6, line 4).

Results 2:

As suggested we reversed the order of 5 th and 6 th paragraph and also the tables 2 and 3.

Results 3:

We thank the reviewer for pointing out this point; we have made the appropriate correction to the tables. 9. Discussion 1: We acknowledge the reviewer for this specific comment; we have made the appropriate correction (page 9, line 13).

INTRODUCTION

Seasonal influenza infection serves as a major cause of morbidity and mortality, particularly among high risk population groups. It has been associated with marked social and economic consequences, including high rates of academic and occupational absenteeism, as well as significant treatment and hospitalization costs. Influenza infection is associated with elevated hospitalization rates among young children, comparable to those observed among older adults or other high risk groups [START_REF] Neuzil | The effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children[END_REF][START_REF] Izurieta | Influenza and the rates of hospitalization for respiratory disease among infants and young children[END_REF][START_REF] Poehling | The underrecognized burden of influenza in young children[END_REF].

Influenza is also associated with an enhanced morbidity burden within both the outpatient and community settings [START_REF] Tsolia | Impact of influenza infection in healthy children examined as outpatients and their families[END_REF].

The diagnosis of influenza is usually based on patient's history, clinical sings and symptoms and on the knowledge of local epidemiological status. The combination of fever and cough was reported to increase the likelihood that respiratory disease was influenza, especially in adults [START_REF] Monto | Clinical signs and symptoms predicting influenza infection[END_REF][START_REF] Zambon | Diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic or molecular detection of influenza[END_REF] However, the clinical signs and symptoms of influenza infection among children frequently overlap with those caused by other viral infections present among the community [START_REF] Peltola | Accuracy of clinical diagnosis of influenza in outpatient children[END_REF]. Rapid diagnosis for influenza is useful in order to minimize nosocomial infections, thus allowing the timely institution of infection control measures [START_REF] Munoz | Influenza A virus outbreak in a neonatal intensive care unit[END_REF]. It also allows for the timely administration of antiviral treatment which is most effective if initiated within 48 hours since the onset of symptoms [START_REF] Harper | Expert Panel of the Infectious Diseases Society of America. Seasonal influenza in adults and children--diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the Infectious Diseases Society of America[END_REF][START_REF] Hayden | Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections. GG167 Influenza Study Group[END_REF][START_REF] Nicholson | Efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial. Neuraminidase Inhibitor Flu Treatment Investigator Group[END_REF]. Finally, tests that yield results rapidly can influence clinical management regarding antibiotic treatment decisions. As a result, the availability and utilization of influenza rapid diagnostic tests within both the laboratory and clinical settings have substantially increased during recent years. The influenza rapid diagnostic tests, providing results within 15 minutes, have also been applied as screening tests for influenza infection. More than ten types of rapid influenza diagnostic tests have been approved by the U.S. Food and Drug Administration (FDA).

The purpose of this study is to evaluate the performance of a rapid influenza test performed at the bedside of hospitalized children during one year's influenza season with the use of reverse transcription polymerase chain reaction (RT_PCR) as the standard diagnostic method. The performance of the rapid test was also compared to the clinical diagnosis of influenza in hospitalized children.

MATERIALS AND METHODS

A cross-sectional study design was conducted during the 2005 respiratoryinfection season in Athens, Greece. The study period was initiated on Jan. [START_REF] Fagan | Letter: Nomogram for Bayes theorem[END_REF] All study participants were assessed within 48 hours following hospital admission. A standardized questionnaire was completed by a study physician for all study participants. The questionnaire was applied in order to obtain detailed information about patients' demographics, previous medical history, the presence and duration of clinical symptoms and signs, physical findings, laboratory and radiological results. Information about the vaccination status against influenza was also obtained by vaccine records of the participants.

A nasopharyngeal aspirate (NPA) of 2 ml was obtained from each participant by a pediatrician through the application of a small flexible tube attached to a sterile mucus trap. If such secretions could not be obtained, 2 ml of sterile saline was introduced and re-aspirated. All collected specimens were stored at -70 o C until multiple xRT -PCR test was performed, within one month. A nasal swab of the turbinates was also obtained and tested right after collection for the presence of the influenza antigen, by the same researcher, through the application of the QuickVue Influenza Test.

Influenza virological diagnosis

The QuickVue Influenza Test (Quidel, San Diego, USA) was used for the rapid diagnosis of influenza infection. This test is based on a lateral-flow immunoassay and detects the presence of both influenza virus A and influenza virus B but does not differentiate between the two. The test was performed according to the manufacturer's instructions. The researcher was trained in how to perform the test and read the result. Specifically, the patient's specimen was placed in an extraction tube where the virus particles in the specimen were disrupted, thus exposing internal virus nucleoprotein. Following extraction, a test strip was placed within the tube, where nucleoproteins in the specimen reacted with both the lyophilized buffer and mouse monoclonal anti-influenza virus A and anti-influenza virus B antibodies contained in the test strip. A positive test result was obtained if a pink or red test line along with the blue control line appeared on the test strip, indicating that the extracted specimen contained influenza viral antigens. A negative test result was obtained if only a blue control line appeared, showing that no influenza viruses A or B antigens were present.

Reverse transcription of RNA material isolated from nasopharyngeal samples was performed as described previously [START_REF] Spyridaki | Comparison of nasal sampling methods for the detection of viral pathogens by RT-PCR -a GA²LEN project[END_REF]. RNA was extracted from 50μl of nasal aspirates using Trizol reagent (Invitrogen, CA, USA). Reverse transcription (cDNA synthesis) was performed in 20μl reactions using 8μl RNA, Superscript III reverse transcriptase (Invitrogen) and random hexamers.

Multiplex, nested (two rounds) PCRs for influenza virus (serotypes A/H1N1, A/H3N2, B) were conducted in 50-μl reactions consisting of 1x Buffer, 3mM Mg 2+ , 0.2mM dNTPs, 2U of Platinum Taq DNA polymerase (Invitrogen), and 0.2μM of each of previously published primers [START_REF] Zhang | Detection and identification of human influenza viruses by the polymerase chain reaction[END_REF]. Four microliters of cDNA were used in the first round PCR reaction mix, while 2μl of the PCR product of the first round were used in the amplification mixture of the second round [START_REF]Multiplex Reverse Trascription-PCR for Surveillance of Influenza A and B Viruses in England and Walles in 1995 and 1996[END_REF]. Samples were amplified in a PTC-200 DNA Engine thermocycler (MJ Research, MA, USA) with previously described thermocycling conditions [START_REF] Spyridaki | Comparison of nasal sampling methods for the detection of viral pathogens by RT-PCR -a GA²LEN project[END_REF].

Statistical analyses

The primary outcome of the study was assessed by comparing the performance of the QuickVue influenza test with that of the multiple xRT -PCR, the currently established criterion standard. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the QuickVue influenza test were evaluated both among the overall study population and according to the clinical symptoms of influenza. The sensitivity measures the proportion of actual positives which are correctly identified as such and the specificity measures the proportion of negatives which are correctly identified. However in practice, the PPV (the proportion of patients with confirmed influenza among patients with a positive rapid test) and NPV (the proportion of patients without influenza among patients who had negative rapid test) provide the most useful clinical information. Two other tools for the clinician which can help him to translate the test characteristics into clinically relevant terms are the positive and negative likelihood ratios (LRP and LRN respectively). The LRP (=sensitivity/(1-specificity)) and the LRN ((1-sensitivity)/specificity) are measures of the disciminating power of a test. LRPs of >10 and LRNs of <0.1 generate large changes from pretest probability to posttest probability. The translation of pre-test probabilities to post-test probabilities given the likelihood ratio is done with the use of Fagan TJ normograph [START_REF] Fagan | Letter: Nomogram for Bayes theorem[END_REF]. The chi-square analysis was applied in order to compare patients' characteristics. A p-value (p) of ≤0.05 was considered as the criterion for statistical significance for all analyses. Stata (Stata Corporation, Texas, USA) version 8 was used for all analyses.

RESULTS

Among the eligible study population (N=311), 217 (69.8%) were tested by both PCR and QuickVue Influenza Test for the presence of influenza infection. The ninety-four (30.2%) eligible study participants were excluded from the study due to either denied informed consent for study participation or limited hospital stay duration (less than 48 hours).

Children who participated to the study were almost equally males (108/217) and females (109/217). Fifty two per cent of the participants were between the ages of 12 months and 5 years (Table 1). One hundred forty five children out of 217 (67%) were Greeks while 47 (22%) were Albanian, and 25 (11%) were of other ethnicities.

Fourteen study children (7%) were born at gestational age less than 37 weeks. Of the study population, 54 (26%) had predisposing conditions for influenza infection, including asthma (20%), seizures (4%) and congenital heart disease (2%).

Among the study population, 176 (86%) presented with fever (mean temperature: 38.9 ± 0.9 o C) and 29 children (14%) with respiratory symptoms only.

The subgroup of febrile children consisted of 149 patients (85%) with respiratory symptoms; 9 (5%) had vomiting and / or diarrhea, 2 (1%) had abdominal pain, 1 had myalgias (0.6%) and 6 (3.5%) seizures. The remaining 9 febrile children (5%) were admitted with the diagnosis of a febrile illness without any localizing signs. The main respiratory symptoms were rhinitis (96%) and cough (89%).

According to the PCR conducted, 18% of the study population had influenza infection, all of which was influenza A (H3N2). The characteristics of patients with and without influenza infection are shown in (Table 1).

QuickVue Influenza Test detected influenza virus in 27 samples (68%) found to be positive by PCR but did not detect virus in 13 samples positive by PCR. Seven samples (4%) were positive by QuickVue Influenza Test but negative by PCR. Based on these, the performance of the test was estimated as follows: sensitivity 67.5%, specificity 96%, positive predictive value 79% and negative predictive value 93% (Table 2). As it shown in the table the LRPs were high, 6.3 to 23, with most between 12 and 18 and the LRNs varied between 0.08 and 0.5 implying conclusive changes from pretest probability to posttest probability.

The overall sensitivity of the clinical symptoms of influenza (fever and cough) was 76%, with a positive predictive value of 18%; specificity and negative predictive value were 20% and 79%, respectively. With regard to the age of participants, the sensitivity and positive predictive value of clinical symptoms of influenza were higher among children aged between 6 and 12 months as compared with those children aged greater than 12 months (80% and 13% to 76% and 19% respectively). Similar results were found for specificity and negative predictive value (30% and 92% to 18% and 74% respectively). The positive likelihood ratios and the negative likelihood ratios varied between 0.9 and 1.1 and between 0.7 to 1.4, respectively (Table 3)

During the 4-week peak period of influenza activity which accounted for 70% of all instances of influenza virus identification, the sensitivity increased to 76% and the positive predictive value to 86%. Specificity and negative predictive values remained almost the same, 95% and 91% respectively. Sensitivity and positive predictive values were lower during the first and third phase of the epidemic (sensitivity 57% and 50% and positive predictive value 67% and 66% respectively). Specificity changed to 91% and 98% while negative predictive values changed to 87% and 96% respectively (Table 3).

The mean duration of symptoms at the time of admission was 4.57±2.43 days.

Stratification by reported disease duration, showed that sensitivity and specificity of the QuickVue Influenza Test among hospitalized children with disease duration <2 days were 76% and 100%, respectively. When QuickVue Influenza Test was performed in children with disease duration longer than 2 days an estimated sensitivity and specificity of 65% and 94%, respectively, was calculated. Rapid test had higher PPVs with samples from children with shorter disease duration (100 to 71%) but the NPV was almost the same for both groups (94 to 93%) (Table 3).

The sensitivities of QuickVue Influenza Test were high in samples collected from children aged ≤ 12 months (80%), but moderate with samples collected from children aged > 12 months (66%). The test was highly specific with samples from both age categories (95 to 96%). PPV was higher with samples from children >12 months (82 vs 67%) while NPV was approximately identical (92 to 97%) (Table 3).

DISCUSSION

Rapid identification of influenza cases is essential, particularly among hospitalized patients, for the initiation of specific antiviral treatment administration and consequent diminishment of the length of hospitalization. Moreover, rapid identification of influenza cases may contribute to the limitation of unnecessary antibiotic use and prevention of nosocomial transmission of the virus to high risk hospitalized population groups [START_REF] Sharma | Effect of rapid diagnosis of influenza virus type a on the emergency department management of febrile infants and toddlers[END_REF][START_REF] Bonner | Impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial[END_REF][START_REF] Abanses | Impact of rapid influenza testing at triage on management of febrile infants and young children[END_REF].

Previous studies have shown that during a local outbreak, a clinical presentation including both fever and cough was most indicative of influenza infection among the study population. Specifically, the detection of influenza infection presenting with both fever and cough was observed to have sensitivities ranging between 64 and 86%, specificities between 55 and 67%, and PPVs ranging between 48 and 87% [START_REF] Monto | Clinical signs and symptoms predicting influenza infection[END_REF][START_REF] Zambon | Diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic or molecular detection of influenza[END_REF][START_REF] Boivin | Predicting influenza infection during epidemics with use of clinical case definition[END_REF][START_REF] Ruest | Comparison of the Directigen flu A+B test, the QuickVue influenza test, and clinical case definition to viral culture and reverse transcription-PCR for rapid diagnosis of influenza virus infection[END_REF]. This contrasts other studies which reported that no clinical signs or symptoms are specific for influenza virus infection [START_REF] Carrat | Evaluation of clinical case definitions of influenza: detailed investigation of patients during the 1995-1996 epidemic in France[END_REF][START_REF] Nicholson | Acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden[END_REF]. The present study indicates that the clinical diagnosis of influenza, based on the combined presence of fever and cough, is difficult among children in everyday clinical practice.

The observed limited PPV indicates that only 18% of the true cases of influenza were identified as such based on patients' symptoms, while the specificity of 20% precludes the use of fever and cough for predicting influenza virus infections. The decreased LRPs values and the elevated LRNs values make the influenza infection unlikely with the use of clinical symptoms alone. The symptoms were least accurate among children aged less than one year. This finding is in accordance with the clinical experience of diagnosis of respiratory infections among infants, although the burden of influenza is greater in this age group [START_REF] Neuzil | Burden of interpandemic influenza in children younger than 5 years: a 25-year prospective study[END_REF][START_REF] Heikkinen | Burden of influenza in children in the community[END_REF].

In the present study, influenza infection was identified in 18% of children aged 6 months to 14 years who were hospitalized with fever and/or respiratory symptoms. Influenza infections were detected with positive PCR for influenza A and B. PCR was used as the standard criterion because it detects more influenza cases than viral culture and has a greater accuracy [START_REF] Abanses | Impact of rapid influenza testing at triage on management of febrile infants and young children[END_REF][START_REF] Boivin | Predicting influenza infection during epidemics with use of clinical case definition[END_REF][START_REF] Schweiger | Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples[END_REF][START_REF] Pregliasco | Comparison of RT-PCR with other diagnostic assays for rapid detection of influenza viruses[END_REF]. Similarly, our study showed that the QuickVue Influenza Test has a high diagnostic yield for the detection of influenza infection among hospitalised children during the influenza season. The observed performance of the QuickVue Influenza Test is similar to that from previous studies with reported sensitivities and specificities varying between 74 and 93% and between 76 and 98% respectively [START_REF] Boivin | Predicting influenza infection during epidemics with use of clinical case definition[END_REF][START_REF] Poehling | Bedside diagnosis of influenzavirus infections in hospitalized children[END_REF][START_REF] Grijalva | Accuracy and interpretation of rapid influenza tests in children[END_REF][START_REF] Uyeki | Influenza diagnosis and treatment in children: a review of studies on clinically useful tests and antiviral treatment for influenza[END_REF]. The observed PPV (79%) indicates that the majority of patients with positive rapid test truly have influenza infection, while the even greater NPV ( 93%) indicates that influenza infection is unlikely if the test is negative. Moreover the LRPs were very high indicating that a positive result impacts the probability for influenza more than a negative result. For example if a child has 50% chance of influenza after history and physical, this chance increases to 90-95% (LRP 12-18) with a positive test and decreases to 25-30% (LRN 0.2-0.5) with a negative test. Notice also must be given to the rapid test negative / PCR positive subgroup. It is currently unknown whether these patients are truly able to transmit the infection to others due to low viral load that is detected only by the highly sensitive PCR; consequently it is a matter of further investigation whether these patients should be isolated to prevent transmission to others. Viral shedding is observed to be elevated and more prolonged in children, especially the younger ones, compared to adults. Moreover, viral shedding is enhanced during the first days of infection [START_REF] Glezen | Interpandemic influenza in the Houston area, 1974-76[END_REF][START_REF] Frank | Patterns of shedding of myxoviruses and paramyxoviruses in children[END_REF]. As a result, infected young children having the test performed during the first days of the disease are more likely to have a positive test as compared to older children with extended disease duration.

To this effect, the present study findings indicated that the sensitivity of the rapid test increased to 80% among children aged 6 to 12 months and to 75% among children with symptom onset less than 2 days.

With regard to the performance of the QuickVue Influenza Test according to the time phase of an influenza epidemic, the study findings indicated that during the peak of the influenza epidemic, a positive rapid test result is highly indicative (86%) of influenza infection, while a negative test result highly suggests the exclusion of infection (91%). In contrast, during the troughs of the influenza epidemic, the study findings indicated that only half of the positive rapid tests were confirmed influenza cases (PPV 50%). As a result, during such periods a confirmatory test, such as PCR or viral culture, for detecting influenza cases is necessary. Additionally, it should be noted that even during periods of low influenza activity a negative rapid test virtually excludes the diagnosis of influenza (NPV 96%).

Several points however must be interpreted with some caution. First, only influenza A was identified in the study samples, so the rapid test characteristics for influenza B infections could not be assessed. Second, the study was conducted in the inpatient setting and the conclusions from the QuickVue Influenza Test cannot be extrapolated to the ambulatory setting where some differences in disease characteristics may exist, such as viral titters and shorter duration of illness before hospital admission,.

Nowadays, under the threat of the ongoing novel influenza A (H1N1) pandemic it must be added that the use of point-of-care rapid influenza antigen detection tests may also improve the timeliness and appropriateness of decisions regarding the management of hospitalized or ambulatory children. Decisions regarding initiation of antiviral treatment and application of patient isolation measures may be greatly facilitated if reliable rapid diagnosis is available. Existing rapid antigen detection tests are generally targeted against preserved viral antigens and they have comparable analytical and clinical sensitivity for seasonal influenza A virus and for the new influenza A (H1N1) [START_REF] Chan | Analytical sensitivity of rapid influenza antigen detection tests for swine-origin influenza virus (H1N1)[END_REF][START_REF] Faix | Rapid-test sensitivity for novel swine-origin influenza A (H1N1) virus in humans[END_REF]. Further studies are required to examine the performance characteristics of the antigen detection tests for the diagnosis of influenza caused by this new virus.

In conclusion, our study suggests that the clinical case definition of influenza based on the presence of fever and cough is inaccurate for prediction of influenza virus infection in children. The QuickVue Influenza Test on the other hand is useful and reasonably accurate for the detection of influenza infection in hospitalised children during the influenza season. Infection with influenza virus is less likely if the test is negative. A positive result suggests that infection is probable if influenza virus circulates in the community. On the other hand, in accordance with the WHO statement for the use of rapid testing for influenza diagnosis (July 2005) during periods of low influenza activity, if rapid tests are used, positive results must be interpreted with caution and confirmed by a definitive influenza test. The sensitivity of the test is higher in younger children and in patients with shorter duration of symptoms. However, the results of this study cannot be extrapolated to patients presenting during different seasons.

  European Journal of Clinical Microbiology & Infectious Diseases Ref.: Ms. No. EJCMID-D-09-00502 "PERFORMANCE OF RAPID INFLUENZA TESTING IN HOSPITALIZED CHILDREN" Dear Editor, Please find enclosed for consideration the revised manuscript entitled "Performance of rapid influenza testing in hospitalized children" (Ref.: Ms. No. EJCMID-D-09-

  , 2005, following the detection of two consecutive pediatric patients with confirmed influenza virus infection, and was concluded on May 15, 2005, following the conclusion of the influenza season within the region. The study was approved by the Ethical Committee of the "P. & A. Kyriakou" Children's Hospital in Athens, Greece. Informed consent for study participation was required from the legal guardians of all eligible participants prior to study participation. All children, aged 6 months to 14 years, who presented with fever (37.8 o C, axillary) and/or respiratory symptoms and were consequently hospitalized at the "P. & A. Kyriakou" Children's Hospital during the period Jan. 15, 2005 through May 15, 2005, were eligible (N=311). No exclusion criteria, regarding demographic and/or socio-economic characteristics, for study participation were applied.

Characteristics Influenza (-)

N=177 1

Influenza (+)