Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We assessed the risk of progression from infection to disease as a function of DAT and rK39 serological titers.Leishmania donovani. In each cohort two serosurveys had been conducted. Based on results of initial surveys, subjects were classified as seronegative, moderately seropositive or strongly seropositive using both DAT and rK39. Based on the combination of first and second survey results we identified seroconvertors for both markers. Seroconvertors were subdivided in high and low titer convertors. Subjects were followed up for at least one year following the second survey. Incident VL cases were recorded and verified.We used available data on four cohorts from villages in India and Nepal that are highly endemic for We assessed a total of 32,529 enrolled subjects, for a total follow-up time of 72,169 person years. Altogether 235 incident VL cases were documented. The probability of progression to disease was strongly associated with initial serostatus and with seroconversion; this was particularly the case for those with high titers and most prominently among seroconvertors. For high titer DAT convertors the hazard ratio reached as high as 97.4 when compared to non-convertors. The strengths of the associations varied between cohorts and between markers but similar trends were observed between the four cohorts and the two markers.There is a strongly increased risk of progressing to disease among DAT and/or rK39 seropositives with high titers. The options for prophylactic treatment for this group merit further investigation, as it could be of clinical benefit if it prevents progression to disease. Prophylactic treatment might also have a public health benefit if it can be corroborated that these asymptomatically infected individuals are infectious for sand flies. L. donovani with DAT and rK39. After an interval of 6–12 months, a second round of surveys was conducted in which seroconvertors were identified, i.e. persons seronegative at baseline that had become seropositive in the interval between surveys. We subdivided the subjects according to serologic titers scored as negative, moderately positive or strongly positive. During follow-up we observed that the risk of progressing to disease was not very much different between seronegative and moderately seropositive subjects, but strongly increased for high titer seropositive subjects. This pattern was true for the initial serology, and even more pronounced among seroconvertors. Given the strongly increased risk of progression to disease among those with high DAT or rK39 titers, these data raise the question whether prophylactic treatment would be beneficial. The case for prophylactic treatment would be further strengthened if it could be shown that asymptomatically infected individuals are infectious to sand flies.To study the association between positive serology for VL among asymptomatic persons and progression to disease, we analyzed combined data from two different cohort studies in which over 32,000 subjects were enrolled. All subjects were screened for presence of antibodies to  Leishmania. In South America and the Mediterranean region, the disease is mainly caused by Leishmania infantum, which is a parasite of dogs opportunistically transmitted to humans. In East Africa and on the Indian subcontinent the disease is caused by L. donovani. L. donovani is essentially a parasite of humans, though recently there have been some reports of a possible animal reservoir L. infantum in a Brazilian study, and due to L. tropica in veterans of Operation Desert Storm L. infantum varied from 0.6 to 71% in endemic areas L. donovani or L. infantum to symptomatic clinical cases as 1∶2.4 in Sudan, 4∶1 in Kenya, 5.6∶1 in Ethiopia, 6∶1 to 18∶1 in Brazil, 4∶1 in Bangladesh and 8.9∶1 in India and Nepal Visceral leishmaniasis (VL) is a vector borne infectious disease that is endemic in parts of Asia, Africa, South America and Europe, and is caused by different species of the genus et al. 21 out of 91 polymerase chain reaction (PCR) and/or recombinant K39 (rK39) positive but asymptomatic individuals (23.1%) progressed to clinical VL over a follow-up period of one year et al. reported 38 VL cases among 55 asymptomatic seropositive subjects followed up for one year (69%) et al. followed up a cohort of 870 endemic subjects of whom 230 were Direct Agglutination Test (DAT) positive at onset and 120 were rK39 positive. Over a 2-year follow-up, 15 VL cases occurred among 583 persons negative on both tests (2.6%) versus 10 cases among 287 DAT and/or rK39 positive individuals (3.5%); the difference was not statistically significant et al. followed up a cohort of 9034 DAT negative subjects in high VL incidence villages in India and Nepal. The ratio of incident infection to incident disease was 8.9 to 1, the risk of disease was 11.5 times higher  in those that had converted from seronegative to seropositive during the first year when compared to the risk of those that remained seronegative at the start of follow-up Studies of the risk of progression from infection to disease have yielded contradictory results. In a study by Das et al. included a mixture of individuals with remote and recent seroconversion, Ostyn et al. were able to establish an association between positive serology and disease in individuals who were recently infected (recent seroconversion). Similarly, the asymptomatic subjects reported upon by Singh et al. were contacts of patients with VL, and as such they may have been recently infected and at higher risk of progression to disease. There has been recent controversy about the role of various antibody tests as markers of infection, and about the link between serologic status and disease risk in humans Whereas the antibody-positive cases reported by Gidwani We pooled prospectively obtained data from two population-based research projects focused on the epidemiology of VL in India and Nepal. The data were divided into four distinct cohorts. The first two cohorts belong to the KALANET research project which was concluded in India and Nepal in 2009; the two other cohorts belong to the NIH/TMRC research project that started in 2008 in India The main aim of the KALANET project was to investigate the effectiveness of insecticide-treated bed nets (ITN) as an intervention against VL. A paired-cluster randomized trial was conducted in 26 highly VL endemic villages in the Muzaffarpur district of Bihar, India (n = 16 clusters) and the Terai region of Nepal (n = 10 clusters). In the 13 intervention villages, ITN were distributed at the start of the trial, and the incidence rate of leishmania infection and VL in those villages was compared with that in 13 non-intervention villages over a two-year period. Incident infection, measured by seroconversion according to the DAT, was the primary outcome measure. Importantly, no differences in primary outcome were found between intervention and non-intervention villages, (seroconversion 5.4% vs. 5.5%), nor in the secondary outcome, incident VL (OR 0.99) In the framework of the NIH/TMRC research project, a population of approximately 85,000 persons living in 50 rural villages of Muzaffarpur district, a highly VL endemic district in Bihar India, have been under active surveillance since April 2008. From this population, hereafter called ‘TMRC - old area’, data are available for 13,134 subjects enrolled in the baseline serosurvey conducted in the villages with the highest VL incidence in the 3-year period preceding the survey (cohort 3). In 2010 the study population was expanded to an additional 10 villages named ‘TMRC-new area’. The additional villages were selected based on high reported VL incidence during the year preceding their entry. All subjects providing informed consent were included in a serosurvey. Data for 7,934 individuals from this ‘TMRC-new area’ (Cohort 4) were included in the present study.Altogether we thus have serology data available on 34,354 individuals enrolled in one of the four cohorts with a total of 76,113 person years follow-up. HIV prevalence in the area is assumed to be very low. In the TMRC study populations HIV testing was conducted among a subgroup of 1,787 persons of whom only 4 (0.2%) tested positive.Study procedures are extensively described in Picado et al (2010) and Hasker et al (2012) DAT and rK39 ELISA assays of serologic status were done from eluted filter paper blood as described elsewhere in the same laboratory at BHU India for the three Indian cohorts  and at BPKIHS Nepal for the Nepal samples (Cohort 2) For the KALANET cohort (Cohorts 1 and 2) results for rK39 were available from the baseline survey only. The cut-off for a seropositive test for rK39 ELISA was determined for the 4 cohorts separately, using the percentage point optical density  with the highest Youden index as a cut off with recent VL cases (diagnosed in the 24 months period preceding the survey) as reference. As a second cut off point, to distinguish moderately seropositive from highly seropositive, we used the mean OD plus two standard deviations among those who had never suffered VL at the time of the baseline surveys. A seroconvertor was defined as an individual who was rK39 negative in the baseline survey but rK39 positive in the follow-up survey with an increase in optical density of at least three percentage points. RK39 seroconvertors were also subdivided into moderate and high, using the cut offs described above for seropositivity.L. donovani has been confirmed; or a person with the combination of a clinical history typical for VL and a positive result of the rK39 test  and a good response to specific VL treatment. All cases reported were ascertained by study physicians.A case of VL was defined as a person with a clinical history typical for VL  and in whom the presence of Data were analyzed in Stata/IC V10.1 . For analytical purposes we considered the four cohorts pooled and separately. Kaplan-Meier curves of DAT or rK39 seropositive or seroconversion titer levels were made for the combined study population. Cox regression models were fitted to each cohort separately and the combined cohorts to test for (1) associations between initial DAT or rK39 sero-positivity and clinical VL, and for (2) associations between DAT or rK39 seroconversion and clinical VL. The latter was done only for the TMRC cohorts (cohort 3 and 4) since for the KALANET cohorts (cohort 1 and 2) rK39 results were only available at baseline. In the models for combined cohorts, the factor ‘cohort’ was included as a fixed effect. We tested for interactions, and if there were no interactions we presented a combined effect for the different study cohorts. During follow-up visits, information on all household members was obtained irrespective of whether they were present or absent. Deaths were censored on the reported date of decease.Both research projects (KALANET and TMRC) sought consent at three levels: community, household and individual. Communities were duly informed about the purpose of the studies and consent was sought from village leaders. For the KALANET trial ethical clearance was obtained from the ethical committees of BHU (India), the BPKIHS , the London School of Hygiene and Tropical Medicine (UK), and the University of Antwerp (Belgium). For the TMRC study ethical clearance was obtained from the review committee of the U.S. National Institutes of Health (NIH), as well as Institutional Review Boards of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, and the University of Iowa. All subjects provided written informed consent; in case of illiterate subjects, a thumb print plus a signature of an independent witness were used. For minors under the age of 18, written informed consent was obtained from a parent or guardian. Informed consent procedures for each of the studies were approved by the respective review boards.Among the total eligible study population of 34,354 subjects, 1,825 were excluded from further analysis because of a reported history of VL prior to the first serosurvey. Thus 32,529 subjects were included in the analysis, of whom 28,073 (86.3%) were also enrolled in a follow-up survey. Participants were divided over study populations and countries as indicated in Based on the criteria described in the ‘study procedures’ section, cut off values for rK39 ELISA expressed in percentage point optical density of a positive control (pp) varied between cohorts. The cut offs that distinguished seronegative from moderately seropositive individuals in the KALANET cohorts were 27 pp for Nepal and 34 pp for India. The cutoffs for strongly seropositive individuals were 44 and 42 pp respectively. The cut offs for individuals in the TMRC cohorts were lower, i.e., 15 and 21 pp for the ‘old area’, and 11 and 27 pp for the ‘new area’.Overall 235 VL cases occurred among the72,169 total person years observed, i.e. 3.3 per 1,000 person year at risk. As shown in the Kaplan-Meier graphs in Because the interactions between titer levels and study cohorts were statistically significant (p = 0.0001 both for DAT and for rK39), hazard ratios are presented for each of the populations separately . There wThe pattern of progression to VL according to the baseline rK39 status was similar to that according to DAT . IndividThe associations between seroconversion and progression to symptomatic VL were very strong for those with high titers, measured with either DAT or rK39 . The risThe overall hazard ratio of developing symptomatic VL when comparing non-convertors to convertors with moderate DAT titers was 6.9 (95% CI 3.4–14.3). Among seroconvertors with high titers the hazard ratio was 97.4 (95% CI 53.5–177.4), with 12% (15 out of 123) of subjects progressing to disease . There wUnlike the data for DAT titers, there was no significant association between moderate titer rK39 conversion and progression to symptomatic VL in the two TMRC cohorts. However there was a very strong and highly significant association with progression to symptomatic VL among those who converted with high rK39 titers. Hazard ratios ranged from 15.9 (95% CI 2.1–121.4) to 123.9 (95% CI 52.7–291.2) among the cohorts . There wL. donovani infection in India and Nepal. The data revealed strong associations between the magnitude of positive serology, measured by DAT and/or rK39, and the risk of progressing to symptomatic VL. A documented recent seroconversion was also associated with a risk of developing symptomatic VL. These associations were consistent and statistically significant among individuals with high titers of either serological marker. Among those seroconverting to high titers the risk of progressing to disease was particularly strong, reaching a hazard ratio of 97.4 among individuals with high DAT titers. The relatively long intervals between surveys (6–12 months) must have caused us to miss some seroconversions among later VL cases, which would lead to an underestimation of the true hazard ratio. Even though the strengths of the associations varied between cohorts and between markers, consistent patterns were observed across the four cohorts and the two serologic markers.This study analyzed data from four large cohorts of individuals living in regions endemic for With the exception of individuals who seroconverted according to DAT titer, variations between the strengths of associations precluded us from being able to calculate overall effects including all four cohorts. These variations were probably due to chance, since the numbers of subjects in each of the titer level groups considering all seropositive persons or all individuals who seroconverted was not very high. As a result, the absolute numbers of individuals developing VL were low especially in the groups with moderately positive titers. Overall fewer than 1 in 10 infected persons developed symptomatic disease. Another possible source of variation could be the positive predictive value of the tests for detecting infection, which varies with prevalence. However the variation observed could also be explained by the epidemiological status of individuals in each of the cohorts. VL tends to cause micro-epidemics that saturate an area and then “move on” to a neighboring area Leishmania species, our study populations exhibited a strong association between serological markers and progression to symptomatic VL among asymptomatic individuals et al. found no seropositive individuals among 100 Indian controls living in non-endemic regions, providing further evidence for the fact that positive serology is a result of infection with VL parasites and not due to cross reactivity Leishmania infection in epidemiological studies, and as tools to monitor the impact of interventions, especially when high titer cut offs are used.Contrary to what has been reported in some smaller cohort studies and among studies of other M. tuberculosis infection are not important as sources of transmission, whereas it is still a subject of debate whether individuals with asymptomatic infection constitute a reservoir impacting the incidence of VL The potential public health impact of asymptomatic VL infection remains undefined. There are substantial numbers of asymptomatic seropositive individuals in areas of Bihar and Nepal with endemic VL. According to the current study these individuals are at increased risk of progression to disease. The fact that 15.2% of individuals in the new TMRC area with high positive DAT titers progressed to symptomatic VL raises the question of whether prophylactic treatment should be considered. Prophylactic treatment for tuberculosis is recommended for persons with a positive tuberculin skin test, particularly if skin test conversion is recent. The lifetime risk of progression from tuberculosis infection to disease among non-immunodeficient hosts however is no more than 10%, half of which occurs in the first years following infection L.donovani as measured in DAT or rK39 are strongly associated with risk to disease progression. This relationship is even more pronounced among individuals who have recently seroconverted.At population level, specific antibodies against Checklist S1STROBE checklist.(DOC)Click here for additional data file.


Congenital hypothyroidism (CH), usually of the primary and permanent variety, is an eminently preventable cause of growth retardation and mental handicap whose outlook has been transformed by newborn screening, usually involving the measurement of capillary thyroid stimulating hormone (TSH). Severe primary CH, due for example to athyreosis, may result in subtle cognitive, behavioural and sensori-motor deficits, but the extent to which these can be offset by optimal postnatal diagnosis and management remains uncertain. This is because the available adult follow-up data reflect the outcome of previous management in the 1970’s and 1980’s, and also because the accurate neuro-psychological assessment of children is difficult, particularly in the preschool population. There is an urgent need to develop new consensus guidelines and to ensure that the children managed according to such guidelines are systematically and prospectively assessed so that good quality outcome data become available. In this review, key recommendations in the management of CH include: screening at day 3 so that severely affected infants can begin treatment within the first 10 days of life; setting the TSH referral cut-off at 8-10 mU/L; adopting a disciplined diagnostic algorithm to evaluate referred cases, with measurement of venous free thyroxine (T4), TSH and thyroglobulin combined with dual ultrasound and radioisotope imaging; initial treatment with a T4 dose of 50 μg daily in infants weighing ≥ 2.5 kg and 15 μg/kg/day in infants weighing < 2.5 kg followed by weekly review until thyroid function is normalised; and maintenance of free T4 levels between 15-26 pmol/L and TSH between 0.5-5 mU/L thereafter to avoid both under- and overtreatment. Conflict of interest:None declared. Newborn screening for congenital hypothyroidism (CH) was pioneered in the late 1970s and established in most Western European countries, North America and Australasia by the mid-1980’s. In countries that can afford newborn screening, treatment within the first 28 days of life - so-called ‘early treatment’ - has transformed the outlook for children with CH so that severe growth retardation with mental handicap (cretinism) is no longer seen. Although the prevalence of frank learning disability intelligence quotient (IQ <70) in children diagnosed clinically, prior to the introduction of newborn screening, is lower than previously thought at 8-25%  on newborn screening. Our recommendations are drawn with reference to current UK and Scottish guidelines; from our personal experience at the Royal Hospital for Sick Children in Glasgow; and from the European Society for Paediatric Endocrinology (ESPE) International Consensus Meeting on Congenital Hypothyroidism which was held in Rome in November 2011 and at which one of us (MD) was a participant. Definitions and PrevalenceAccording to the United States Rare Diseases Act of 2002, CH falls into the category of a rare disease  but is nevertheless the commonest paediatric endocrine disorder. It is described as primary when the gland itself is affected and central when the defect lies in the hypothalamo-pituitary axis; compensated when the hypothalamo-pituitary-thyroid axis is jeopardised but still manages to maintain normal thyroxine (T4) levels and decompensated when normal thyroid hormone levels cannot be maintained. The childhood and adult reference ranges for total and free T4 (fT4) are 80-160 nmol/L and 9-26 pmol/L, but during the first two weeks of life, T4 levels may be as high as 68 pmol/L, falling to 12-30 pmol/L thereafter . True thyroid hypoplasia is rare and its presence should prompt investigation for a mutation in the TSH receptor gene or in PAX8 ,10 When Apparent athyreosis may be due to an ectopic gland that is too small and poorly functioning to be seen on thyroid imaging and also to inactivating TSH receptor mutations . TransieOutcome in Early-treated Congenital HypothyroidismThe surprisingly good prognosis in severe primary CH can be explained by transplacental supply of maternal T4 . ProvideThe problems reported in early-treated subjects with CH can be broadly grouped into two categories as cognitive and behavioural, including deficits in IQ, memory and attention; and sensori-motor, including problems with fine motor, balance and auditory function. Cognitive and Behaviour ProblemsThe prevalence of frank learning disability in screened populations with CH is very low. Only 1.4% of Scottish patients diagnosed between 1979-1993 attended special schools (17), while Rovet and Ehrlich found that 4% of Canadian patients were in special education at Grade 6 level compared with 7% of controls . There are however data to show an IQ deficit in some children with early-treated CH. Tillotson et al  reportedSensori-motor ProblemsEarlier studies during childhood from the Netherlands and Toronto particularly have reported visual-motor impairment during childhood in CH ,25,26,27Hearing impairment in CH is of considerable interest. David Grant’s group in the UK reported mild hearing impairment in childhood , and abnInterpretation of the Available Outcome Data in Congenital HypothyroidismThere is overwhelming evidence that the severity of CH is a major factor predicting outcome, patients with athyreosis faring worse than those with other forms of thyroid dysgenesis such as sublingual ectopia ,26,27,28At present, the extent to which optimal postnatal treatment can offset intrauterine deficit from severe CH remains uncertain, and it is important to judge the information from existing studies in the context of the subjects concerned usually being born before or during the early 1980’s. The median age at treatment, for example 28 days in the cohorts of Salerno et al  and KempScreening, Diagnosis, Assessment, and Management of Congenital Hypothyroidism with Reference to European, UK and Scottish GuidelinesCapillary Screening Methods and Hormone Cut-OffsThe technique of capillary screening involves sampling from a heel-prick stab within a few days of birth and drying the blood onto circles on prepared filter paper which is then mailed to a regional laboratory. Assays are available for the measurement of total T4 , TSH and thyroid binding globulin (TBG). Despite a few notable exceptions in the USA and the Netherlands, most screening programmes measure TSH alone. An alternative is a two-tiered technique in which total T4 is tested initially, followed by TSH measurement when T4 is below an agreed cut-off, for example - 0.8 standard deviation (SD) or less than the mean score for the day of testing. This technique has the advantage of detecting central as well as primary hypothyroidism, but its sensitivity in diagnosing the former is limited  since toIf TSH screening alone is used, what should the cut-off be? In Greece, reducing the TSH cut-off from 20 mU/L to 10 mU/L led to the discovery of CH in 56 of 200 patients diagnosed between 2000 and 2002, with a 10-fold increase in recall rate (40). Cheetham’s group in Newcastle have shown that even a blood spot cut-off of 10 mU/L  will miss mild cases of CH and have suggested that the increased detection with a level of 6 mU/L may justify the extra cost and higher recall rate . HoweverOptimal Timing of Capillary Blood Spot Sampling and NotificationIn high risk cases (e.g. when a sibling is affected), cord blood should be sampled for TSH, but as a rule, screening within the first 48 hours of birth should be avoided since the TSH surge occurs during this time . The recth-75th centile) age of 20 days (16-25 days) did not show age at starting treatment to be a factor in educational attainment. Similarly, Kempers et al  has been surprisingly difficult to prove. Boileau et al  found IQrs et al  were unars et al  reportedrs et al . Part ofMoreover, there are commonsense grounds for starting T4 treatment (and hence undertaking newborn screening) as soon after birth as possible. It is known that intrauterine hypothyroidism in babies with athyreosis adversely affects outcome and that diagnosis beyond one month leads to neurological impairment. It is logical, therefore, to minimise the gap between the abrupt discontinuation of maternal T4 supply at birth and the initiation of postnatal treatment in order to protect infants with severe disease. Moreover, severe hypothyroidism is known to cause unwanted symptoms during the postnatal period including feeding difficulties, prolonged jaundice and hypothermia, all of which the clinician wishes to avoid. There is, therefore, a case for carrying out newborn screening for CH on day 3 of life, once the logistics of screening for other disorders in the newborn period have been taken into consideration, so that treatment can be started within the first 8-10 days of life. Discussions are underway in the UK for this change to be implemented. Initial Visit: Clinical assessment, venous sampling, counselling, and initial treatment Clinical AssessmentInitial clinical assessment following notification should include a thorough history and examination as shown in Cardiac and hip examination should receive special attention given the reported increase in congenital heart disease [14/243 5.8%)] – and developmental dysplasia of the hip [8/243 (3.8%)] in one series (47), although the Scottish prevalence figures from 1979 – 2010 are lower at 8/525 (1.5%) and 6/525 (1.1%) . Dysmorphic features should be sought since syndromic disorders may be associated with either transient TSH elevation  of heparinised blood should be taken prior to treatment together with 1 mL of clotted blood for TG measurement. At the initial visit, additional blood may be taken for thyroid peroxidase and TSH-receptor antibodies from both mother and infant if there is a family history of thyroid disease, and DNA samples from both parents and infant with a family history of consanguinity. Otherwise, these investigations may be deferred until thyroid imaging has been carried out see . X-ray of KneeThis is practised in some centres since bone retardation on knee X-ray reflects the severity of intrauterine hypothyroidism and has been shown to correlate with IQ outcome .Initial CounsellingAt the first visit, the parents are often deeply shocked by the disclosure that their apparently perfect baby may have a lifelong thyroid disorder. It should also be remembered that the mother will usually be less than 2 weeks post partum and may be experiencing some of the common difficulties with sleep pattern and feeding that occur at this time. In our view, extensive counselling and explanation, and thyroid imaging, should not be carried out at this stage. Instead, we advise simply informing the family that the baby probably does have an underactive thyroid gland which will require lifelong treatment and that the outlook is excellent provided that the T4 replacement treatment is given faithfully, and ensuring that the family understand what medication to give, and how. Initial TreatmentUnless capillary TSH elevation is relatively mild (below 40 mU/L), we advise starting T4 treatment without waiting for the venous results. Given the evidence for better IQ outcome using higher, rather than lower doses of T4, we advocate an initial T4 dose of 50 μg daily in infants weighing ≥2.5 kg and 15 μg/kg/day in infants weighing <2.5 kg, consistent with previous European guidelines .There are few published data on the mode of T4 administration. A survey we conducted amongst British paediatricians showed that the majority recommended giving T4 in tablet form, crushed in 5 mL of milk or boiled water, either using teaspoon or syringe . Since tIf Evotrox is used, great care should be taken to ensure that the correct strength (25 or 50μg/5mL) is consistently prescribed so as to avoid confusion and consequent over- or under-dosage with T4. Whatever T4 preparation is used, the dispensing pharmacy must ensure that the parents fully understand how to prepare and give the medication. Parents should be encouraged to err on the side of repeating the medication in the event of minor vomiting after giving medication. There is a strong case for providing written instructions regarding T4 administration, something that is not currently advocated by UK or ESPE guidelines. Details on procedure  to be followed at the second visit are given in Diagnostic ImagingWe advise carrying out both thyroid ultrasound and isotope imaging in all babies referred with TSH elevation provided that they are fit for transfer to a specialist centre, in contrast to preterm and sick babies who are unable to leave the neonatal unit. T4 treatment will affect isotope uptake if TSH is suppressed to <5 mU/L, but this is unlikely if isotope scanning is carried out within 5 days of starting T4 treatment and, as indicated above, we recommend carrying out scanning at the second visit, 3-5 days after starting treatment. Whatever decision is taken, as to the timing of thyroid imaging, this should never be allowed to postpone T4 treatment. We recognise that not all centres, especially in the UK, either carry out or advocate diagnostic thyroid imaging in infants referred with TSH elevation. However, we strongly recommend this approach since:• Imaging aids genetic counselling. In the case of dyshormonogenesis, it ensures the appropriate targeting of further molecular genetic study and also early diagnosis (with a cord blood sample) in future siblings. • Providing the family with visual evidence that their baby (who is often asymptomatic) has a lifelong disorder is helpful.• Giving a firm diagnosis  not only resolves uncertainty and removes the need for further investigation in later childhood, but also reinforces the need for full compliance with lifelong medication.• Determining the precise diagnosis may inform subsequent T4 dosing.The potential pitfalls of both thyroid ultrasound and radioisotope scanning must be recognised. We and others have drawn attention to the diagnostic trap of misdiagnosing non-thyroidal tissue in thyroid fossa as thyroid dyplasia/hypoplasia ,52. HoweFurther InvestigationIf thyroid imaging shows an ectopic thyroid gland, or athyreosis with unrecordable TG, then further investigation is not usually required. The finding of a normal or hypoplastic gland in situ with either reduced or absent isotope uptake raises the possibility of allo-immune thyroid dysfunction, and further blood from both mother and child should be taken for thyroid peroxidase and TSH-receptor antibody testing. Transplacental passage of maternal antibodies may cause transient CH accounting for 2.7% of referrals in one series, some of whom were siblings  and, verA normal or large gland in situ, especially if radioisotope uptake is increased, suggests dyshormonogenesis due to an inherited enzyme defect. In this situation, blood from parents and infant should be taken for DNA extraction and mutational analysis . However, the pattern of large/normal gland in situ with avid isotope uptake is also seen in iodine deficiency. At present in the UK, the iodine status of infant and mother is not routinely assessed, but data from the North East of England indicate iodine deficiency in 3.5% of pregnant women with borderline deficiency in 40% . More reFurther CounsellingThe second visit, 4-5 days after initial assessment, is an opportune time to give the parents a more detailed verbal account of CH and to let them see the diagnostic images of their baby in order to reinforce the reality of the condition. We also give out a booklet, freely available from the Child Growth Foundation website (www. childgrowthfoundation.org/cgf_ booklets - Thyroid disorders) and obtain written informed consent for their child’s data to be entered onto our database. Subsequent Management and Follow-up Initial Dose TitrationFollowing the initial and second visit, we advise weekly assessment including thyroid function tests until the venous TSH has fallen to below 5 mU/L, aiming to normalise TSH within 14 days, as recommended by Selva et al . During Subsequent Dose SchedulingOnce the TSH has normalised, we recommend calculating surface area using the formula ? [length (cm) x weight (kg)/3600] and tailoring the replacement dose to the child’s needs, which will depend on the type and severity of CH. We consider it more logical to increase the dose pre-emptively rather than allowing the brain to ‘see’ too little T4, thus provoking a rise in TSH and prompting an increase in dosage. The full replacement dose (e.g. in athyreosis) is around 100 μg/m2/day (3-4 μg/kg/day), but infants with milder disease  will require less. A suggested dosage schedule, based on the requirement in the absence of thyroid tissue, is given in Most children do well when free T4 is kept between 9-26 pmol/L and TSH to between 0.5 and 5 mU/L, but some seem to be more sensitive to T4. Thus, parents need to be aware of the symptoms of T4 overtreatment, which include poor sleeping, heat intolerance, and irritability. Frequency and Structure of Clinic VisitsOur suggested schedule for each visit is outlined in We do not routinely carry out pubertal assessment or bone age estimation in our patients since we would not normally do this assessment either in normal children and adolescents, and the patients with CH can be regarded as normal individuals who simply need to be on replacement treatment. Given the concerning reports of hearing impairment in adults with CH , we are Re-evaluation of Patients with TSH Elevation of Uncertain CausePatients with clear evidence of thyroid dysgenesis on diagnostic imaging, or episode(s) of frank TSH elevation (> 15 mU/L) beyond infancy despite T4 treatment, do not normally require retesting. Children who do require re-evaluation can be broadly grouped into three categories; those who: • Were preterm and/or sick at the time of testing • Have congenital malformations and/or dysmorphic syndromes (including Down’s syndrome)• Show mild TSH elevation of uncertain cause +/- normal gland in situRe-evaluation should be deferred until 2-3 years of age, depending on the severity of TSH elevation at initial assessment. Treatment can be reduced and stopped over a 4- week period and thyroid function tested 2 weeks later. Diagnostic imaging can be carried out at this stage if no previous imaging has been performed. Transition to Adult CareThe age at transition from paediatric to adult care is at the discretion of the clinician and family. It is important for all patients to understand their condition and the long-term health implications of good compliance with T4 treatment. Girls with CH also need to be aware of the importance of preconceptual euthyroidism so that the foetus receives sufficient T4 during the first trimester. We therefore arrange for girls to be seen by an adult endocrinologist in our transition clinic, whereas we would normally aim to discharge boys to the care of their general practitioner at this age. ConclusionsThe outlook in primary CH is generally good, and it is likely that the advent of earlier and higher dose treatment over the past two decades will be shown to have improved outcome. The assessment of both the impact of disease severity and the degree to which this can be offset by postnatal management is difficult and requires long-term follow-up into adulthood, by which time practice is bound to have changed. Although much can be learned from the admirable French cohort studies of Léger et al ,34, therAcknowledgementsWe wish to thank Dr Tim Cheetham, Mrs Joan Mackenzie, Emma Jane Gault, and Denise Bannon for their help in preparing this review. We also thank our paediatric colleagues from all over Scotland for their support and input into our congenital hypothyroid database.


FLNB gene, which is thought to have a role in cytoskeletal actin dynamics, is located within this chromosomal region and presents as a strong candidate for BMD regulation. We have previously identified significant associations between four SNPs in the FLNB gene and BMD in women. We have also previously identified associations between five SNPs located 5' of the transcription start site (TSS) and in intron 1 of the FLNB gene and expression of FLNB mRNA in osteoblasts in vitro. The latter five SNPs were genotyped in this study to test for association with BMD parameters in a family-based population of 769 Caucasian women.Low bone mineral density (BMD) is a primary risk factor for osteoporosis and is a highly heritable trait, but appears to be influenced by many genes. Genome-wide linkage studies have highlighted the chromosomal region 3p14-p22 as a quantitative trait locus for BMD (LOD 1.1 - 3.5). The P = 0.004 – 0.043). These three SNPs were also found to be significantly associated with total hip BMD Z-score (P = 0.014 – 0.026). We then combined the genotype data for these three SNPs with the four SNPs we previously identified as associated with BMD and performed a conditional analysis to determine whether they represent multiple independent associations with BMD. The results from this analysis suggested that these variants represent a single association signal.Using FBAT, significant associations were seen for femoral neck BMD Z-score with the SNPs rs11720285, rs11130605 and rs9809315 (FLNB gene and BMD in our data. FLNB is one of several genes located in 3p14-p22 that has been identified as significantly associated with BMD in Caucasian women.The SNPs identified in our studies as associated with BMD appear to be part of a single association signal between the  Postmenopausal osteoporosis is a systemic bone disease that is characterised by low bone mass and disturbed micro architecture of bone tissue, resulting in decreased bone strength and a corresponding increase in the risk of fracture . Bone maFilamin B (FLNB) gene, which is thought to have a role in cytoskeletal actin dynamics [FLNB gene have been implicated in a variety of genetic disorders characterised by skeletal malformation, some of which include: spondylocarpotarsal synostosis syndrome [FLNB and human stature variation in a genome-wide association study [The whole genome linkage scanning approach has identified multiple quantitative trait loci (QTL) for bone mineral density (BMD) , strongldynamics , is locasyndrome -19, Larssyndrome ,20, atelsyndrome ,21 and bsyndrome . Associaon study .FLNB gene and BMD in Caucasian women [FLNB mRNA in 96 human osteoblast cell lines [FLNB SNPs previously associated with BMD [FLNB gene that are independently associated with BMD.We have previously identified significant associations between four single nucleotide polymorphisms (SNPs) in the an women . Two of ll lines . Based oll lines  and examwith BMD  in a con2 test. LD analysis revealed that none of the 5 SNPs genotyped were in LD of r2 > 0.8 with each other . These three SNPs were also found to be significantly associated with total hip BMD Z-score . No significant associations were observed between any of the 5 SNPs examined and spine BMD Z-score. After correction of the data for testing 5 SNPs across 3 anatomical sites, the significant association between rs9809315 and femoral neck BMD Z-score was maintained (P = 0.028). Estimates of the additive genetic effect suggest that the minor alleles at rs11720285 (C), rs11130605 (T) and rs9809315 (T) are associated with an increased BMD Z-score at both the total hip and femoral neck sites .The genotype data for rs11720285, rs11130605 and rs9809315 was combined with that for the four other SNPs from the with BMD  for a coFLNB gene that were strongly associated with expression levels of FLNB mRNA [FLNB mRNA in our previous study [FLNB SNPs previously identified as associated with BMD and conditioned on the most significantly associated SNP, none of the associations remained significant. This suggests that these variants represent a single association signal between the FLNB gene and BMD in this study population. This is not the first time that common variation within a gene that has previously been identified as underlying a rare monogenetic form of osteoporosis and/or high bone mass has been implicated in BMD regulation, other examples including the SOST, CLCN7 and LRP5 genes [We previously identified 5 SNPs from the 5′ region of the LNB mRNA , 3 of whus study . HoweverP5 genes .FLNB mRNA expression levels in our previous study [SOX9 gene have been found to cause campomelic dysplasia [FLNB gene that have a minor allele frequency > 1% in Caucasians, both of which were genotyped in our previous study [Although rs2177153 demonstrated the most significant associations with BMD in the study population combined dataset , we cannus study . This cous study . The SNPus study ,30 and Uus study . Variatius study , while mysplasia , a diseaus study  and neitFLNB gene, including the SNPs rs9822918 and rs2177153 genotyped in our previous study [FLNB gene in osteoporosis, comparisons with this study and our previous study must be done with caution due to differences in study design as well as differences in the ethnicity of the study subjects.A recent study published by Li et al.  analysedus study , for assus study . AlthougFLNB gene in bone development [Flnb gene also has a role in bone. Flnb expression has been detected in vertebral bodies obtained from mouse embryos, and it has been suggested that the gene plays a role in vertebral segmentation, joint formation and endochondral ossification [Flnb gene and observed impaired development of the microvasculature and skeletal systems in Flnb-deficient embryos, few of which reached term. Those that were born were very small and had severe skeletal malformations including scoliotic and kyphotic spines, fusion of vertebral bodies, lack of intervertebral discs and reduced hyaline matrix in the extremities, thorax and vertebrae [Flnb-deficient mice presented with shortened distal limbs and small body size, abnormal spinal curvatures, dysmorphic facial/calvarial bones and develop fusion of the ribs and vetebrae, which appeared to be caused by a delay in chondrocyte development.In addition to the evidence suggesting a role for the human elopment -22, therfication . Zhou etfication  generateertebrae . Anotherertebrae  found thOver 70 binding partners have been identified to date for the filamin proteins ,38. TheyFLNB is the third gene from the 3p14-p22 region of the human genome that we have found to be associated with BMD in Caucasian women, the other two being ARHGEF3[RHOA[CTNNB1 gene located in 3p22.1, and 14 loci associated with risk of fracture at the genome-wide significance level [FLNB with BMD, the SNPs analysed here were not identified in this meta-analysis as associated with BMD at the genome-wide significance level (P < 5 × 10-8). The results of the meta-analysis must be treated with caution though as allele risk modelling suggested that only 5.8% of the total genetic variance in femoral neck BMD was accounted for by the genome-wide-significant SNPs reported in the study [g ARHGEF3 and RHOAGEF3[RHOA. InteresGEF3[RHOA,43,44. Ice level . Althoughe study . The rolFLNB gene and have been previously reported to be associated with FLNB mRNA expression in osteoblasts in vitro[Polymorphism at rs11720285, rs11130605 and rs9809315, all of which are located either 5′ of the TSS or in intron 1 of the  in vitro, has bee in vitro suggestsFLNB study [A total of 769 women from 335 families were recruited in Australia and the UK. This family-based population included siblings recruited in 1998 for a study of the genetics of osteoporosis  and is tNB study  with theAt a clinic visit data including age, height, weight, medical, gynaecological, and lifestyle data were recorded and a blood sample collected. Dual energy X-ray absorptiometry (DXA) BMD was assessed  at the lumbar spine L1-L4 and the total hip that includes an area from the femoral neck to just below the lesser trochanter. Within this area the femoral neck sub-region is widely used in clinical practice for prediction of fracture propensity and was therefore also included in the study. Due to the range of ages in this cohort, BMD data was adjusted for age prior to analysis by conversion to BMD Z-scores.Genomic DNA was extracted and purified from EDTA whole blood obtained from each subject . GenotypFLNB gene were selected for genotyping. These were selected based on previously published data suggesting that all five SNPs are significantly associated with expression of FLNB mRNA in 96 human osteoblast cell cultures [5 SNPs located either 5′ of the TSS or in intron 1 of the cultures .Data from the cohort was analysed using the FBAT (Family Based Association Tests) software to test for association within sib-pairs, a method based on the allelic transmission disequilibrium test . We usedP < 0.05 considered significant. LD between the different SNPs was evaluated using the JLIN software [UNPHASED v3.1.5 was used to estimate the genetic effect size in this cohort by generating an additive value as an estimate to how each allele influences the trait value relative to the most common allele . This prsoftware .in silico analysis of SNP targets was performed using the Functional Single Nucleotide Polymorphism (F-SNP) database [An database , which idatabase , a web-bThe authors declare they have no competing interests.BHM performed the genotyping, statistical analysis and drafted the manuscript. CM and RLP participated in the design and co-ordination of the study. TDS and SGW were involved in the recruitment of the study subjects and participated in study design and co-ordination. FD assisted in the statistical analysis of the data and study design. All authors read and approved the final manuscript.


Lower limb pain is an important determinant of locomotor disability. Recurrent episodes of pain may have a cumulative effect on functional decline in later life. Locomotor disability (LMD) is common at older ages, and can lead to other significant disability and mortality. Prevalent pain has been shown to be associated with LMD. This article aimed to assess the association between changes in lower limb pain status (ascertained from a manikin) and changes in the level of self-reported LMD in a sample of UK adults age ⩾50 years, over a 6-year period . There was an average increase in the level of LMD over 6 years. Reports of an onset of lower limb pain were associated with a relative increase in LMD, independently of sociodemographic factors and the onset of selected comorbid diseases. A dose-response relationship was observed between the onset of multiple lower limb joint involvement and more frequent or intense pain and larger increases in LMD. Becoming free from lower limb pain was associated with a relative decrease in LMD, but did not return LMD scores to the level of those who had remained pain-free throughout. This is consistent with a cumulative effect on LMD of recurrent episodes of pain. Lower limb pain may be a key target for prevention and rehabilitation to reduce years lived with disability in later life. First, LMD has typically been studied as a dichotomous phenomenon (disabled versus not disabled). Yet in reality, LMD is likely to be a continuum. Studying changes in LMD across a spectrum of disability may provide additional insights compared to those based on the proportion of individuals crossing an arbitrary threshold In this study, using a measure of LMD that captures its natural continuum 22.1Data are taken from the North Staffordshire Osteoarthritis Project (NorStOP) 2.2All participants provided written informed consent to take part in the study. Ethical approval was obtained from the North Staffordshire Local Research Ethics Committee .2.3Gender and age at baseline were ascertained from the general practice records and verified by self-report. Participants reported their socioeconomic status at baseline in 3 ways. Educational attainment was derived from the question, “Did you go on from school to full time education or university?”. Possible responses were yes (“further education”) and no (“school-age education only”). Occupational class was assessed at baseline using current job title (most recent job for those who were not working) and classified according to the Standard Occupational Classification 2.4LMD was measured using an interval-level score obtained from the walking and stair-climbing items of the Short Form-36 Physical Functioning subscale 2.5Lower limb pain was assessed using a screening item and a body manikin. The screening item asked, “In the past 4 weeks, have you had pain that has lasted for one day or longer in any part of your body?” with possible responses of yes and no. Front and back views of the body manikin were displayed, and respondents were asked to shade where they had pain. Standard transparent templates with the borders marked were used to assess in which area(s) of the body respondents had reported pain. This method has been shown to be repeatable  2.6The lower limb pain characteristics at 3-year follow-up were considered at 3 sites: hip, knee, and foot. At each site, data were collected on the intensity of pain  and the frequency of pain  in the previous 6 months 2.7At 3- and 6-year follow-ups, respondents were asked to report whether they had comorbidities from a prespecified list that included chest problems, eyesight problems (excluding the need for glasses), and leg pain on walking. Participants also completed the Hospital Anxiety and Depression Scale (HADS) 2.8Tobit regression analysis was used to estimate mean  level of LMD at baseline and at 3- and 6-year follow-ups, overall and across the 8 lower limb pain trajectory groups. This technique allows for the skewed nature of the LMD score at each time point As more detailed information regarding the presence of comorbidities and the characteristics of pain at specific lower limb sites was available at the 3- and 6-year follow-ups, further investigations into the potentially causal association between the onset of lower limb pain and changes in LMD were carried out in this time period. For this analysis, the onset (or not) of lower limb pain in the 2 time periods  are considered to be analogous due to the arbitrary nature of the timing of the data collection in relation to participants’ pain. Hence this analysis is carried out in the 3- to 6-year follow-up period, i.e., participants were free of lower limb pain at 3 years and either had or did not have an onset of lower limb pain at 6 years.First, adjustment was made for the concurrent onset of morbidities  previously shown to be cross-sectionally associated with LMD and lower limb pain Second, to establish whether there was an association between the characteristics of the new-onset lower limb pain with changes in LMD, the presence of pain at the 3 lower limb joint sites  at 6-year follow-up was considered. Initially, the presence of pain in any of the 3 lower limb joint sites or not (based on the manikin) was modelled  in those without lower limb pain at 3 years. Then, in those free of lower limb pain at 3-year follow-up, the 3 lower limb pain characteristics at 6-year follow-up  were each modelled separately as potential predictors of change in LMD between 3- and 6-year follow-ups. The number of pain sites and intensity of pain were modelled using appropriate fractional polynomial functions. Three models were derived for each of any pain at 6 years and the 3 pain characteristics: a) adjusted for LMD at 3-year follow-up; b) additional adjustment for age group, gender, baseline socioeconomic status; and c) additional adjustment for the onset of comorbidities from 3- to 6-year follow-up. All analyses were carried out in Stata 11.33.1A total of 5129 potentially eligible participants responded to the baseline survey in March 2002, completed measures of lower limb pain and LMD, and consented to further contact  3.2The most common trajectory was that of lower limb pain at all 3 time points . This gr3.3Mean LMD scores over the 6-year study period, derived from the Tobit regression, are presented for each of the 8 trajectories of lower limb pain. For reference, the 2 extreme trajectories (NNN and YYY) are shown in both figures and those without baseline lower limb pain are shown in When considering those free of lower limb pain at 3-year follow-up , those e44.1Our findings suggest an independent, specific, potentially reversible, dose-response relationship between lower limb pain and LMD in community-dwelling adults age ⩾50 years. Against the background of a gradual increase in mean LMD levels over 6 years, the onset of lower limb pain accelerated this deterioration in function, whereas recovery from lower limb pain was associated with relative deceleration. The more widespread, frequent, or intense the lower limb pain, the more marked the decline in locomotor function. This pattern of associations remained after adjusting for several potential confounders.4.2This study builds on previous work examining the association between lower limb pain and LMD e.g.,  but goesAssociations between changes in pain status, LMD, and death were not investigated in this report. Although interesting and important, exclusion of known deaths should not bias our estimates of the association between pain and LMD. Mortality analyses were not undertaken because we do not know the complete vital status of nonresponders.The analyses presented here considered the possible confounding effects of a number of morbidities on the association between the onset of lower limb pain and changes in LMD. These morbidities have previously been associated with LMD Although the new measure of LMD used in this study can be considered an improvement over previously used definitions of LMD, it still has limitations that deserve consideration. Because it is formed from only 5 items, taken from the SF-36 physical functioning subscale 4.3The majority of previous studies have considered the association between prevalent musculoskeletal conditions and the subsequent onset of LMD over some fixed time period Shah et al. Our study builds on these previous findings of an association between prevalent pain and the onset of LMD in two ways. First, we utilised a continuous measure of LMD, which permitted changes in the level of LMD over time to be detected across a wide spectrum of function in the community. Second, by examining 3 time points over the course of 6 years, this study is the first to assess fluctuations in lower limb pain status and their association with LMD.4.4The association between the onset of lower limb pain and increases in LMD provide further support for the direct role of lower limb pain in the progression of LMD. Although recovery from pain resulted, on average, in a decrease in LMD, this group did not start from or return to the same level of LMD reported in the equivalent group of people who never experienced this pain. We may be observing the cumulative effect of recurrent episodes of pain on the underlying rate of decline in LMD. The failure of those recovering from pain to return to the LMD levels seen in those who never experienced the pain suggests a need for the primary prevention of lower limb pain. Although the prevention of all lower limb pain may be an unachievable target, the results of this study do provide evidence in favour of the treatment of lower limb pain, as well as rehabilitation of function in those presenting with disabling pain. Furthermore, the results are suggestive of a policy of identifying and targeting those with pain in order to prevent a cumulative effect of incomplete LMD recovery over time leading to a substantially larger amount of disability than in the pain-free population.Our study does not provide information enabling us to examine in detail the many different mechanisms by which lower limb pain might increase the rate of deterioration in self-reported locomotor disability. These include conscious and unconscious avoidance and modification of activities in direct response to actual or anticipated pain and related symptoms on mobility, a pathway mediated by deficits in executive function attributed to pain, and reduced capacity relating to muscle weakness secondary to arthrogenic muscle inhibition and atrophy. In addition, conceptual overlap between pain on activities and the subjective rating of limitation and the effect of pain on negative appraisal of function may also contribute to the observed relationship.4.5Using long intervals between measurements will underestimate the true incidence of episodes of LMD and lower limb pain. Given the dynamic nature of both, future studies using shorter intervals  have the potential to reduce the number of years lived with disability in older populations.The authors have no conflicts of interest to declare.


When faced with sensory stimuli, an organism may be required to detect very small differences in a physical parameter (discrimination), while in other situations it may have to generalize over many possible values of the same physical parameter. This decision may be based both on learned information and on sensory aspects of perception. In the present study we describe frequency processing in the behaving mouse using both discrimination and generalization as two key aspects of behaviour. We used a novel naturalistic behavioural apparatus designed for mice, the Audiobox, and paradigm contingencies that were identical for both auditory discrimination and generalization, the latter measured using latent inhibition. Mice learned to discriminate between frequencies that were an octave apart in a single trial. They showed significant discrimination between tone frequencies that were as close as 4–7%, and had d' of about 1 for ΔF of around 10%. In contrast, pre-exposure frequencies that were half an octave or less below the conditioned tone elicited latent inhibition, showing a generalization bandwidth of at least half an octave. Thus, in the same apparatus and using the same general memory paradigm, mice showed generalization gradients that were considerably wider than their discrimination threshold, indicating that environmental requirements and previous experience can determine whether the same two frequencies will be considered same or different. Remarkably, generalization gradients paralleled the typical bandwidths established in the auditory periphery and midbrain, suggesting that frequencies may be considered similar when falling within the same critical band. Two sensory stimuli may be highly discriminable, and yet sufficiently similar to be considered essentially the same. In natural speech, for example, two utterances of the same word by the same speaker, even with the same nominal pitch trajectory, would almost certainly be physically different and (with some effort) also discriminable from each other; nevertheless, as a rule, they would be judged ‘the same’. In natural contexts, both discrimination and generalization may have important roles. While discrimination is generally believed to be limited by peripheral sensory factors, generalization may be driven by context and task requirements Mice have good acoustic abilities. Audiograms, as well as thresholds for frequency or duration discrimination, are well described in the rodent literature using a variety of tasks Little is known about the capacity of mice to generalize across stimuli. We studied generalization using latent inhibition. Latent inhibition refers to the diminished capacity of a conditioned stimulus to be associated with reinforcement when that stimulus has been previously presented to the subject in a non-reinforced manner ad libitum. This paradigm is fully automatic, allowing both continuous recording of individual mouse behaviour in its living quarters, as well as the training and monitoring of several cohabiting mice. In consequence, we could train mice relatively quickly to discriminate between two frequencies and we could reliably measure generalization gradients using latent inhibition. The main result of this paper is the measurement, in the same apparatus and using the same technique, of small frequency discrimination thresholds (of few per cents) and much wider generalization gradients .Here we used the Audiobox, where training occurs automatically and the animals perform the task Ethics statement: Experiments were performed in The Hebrew University of Jerusalem and in the Max Planck Institute for Experimental Medicine in Göttingen. The joint ethics committee (IACUC) of the Hebrew University and Hadassah Medical Center approved the study protocol for animal welfare. The Hebrew University is an AAALAC International accredited institute. All animal experiments that took place in Göttingen were approved by and performed in accordance with the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, project license number 33.14-42502-04-10/0288.The main set of experiments was performed in Jerusalem. A replication of key subset of experiments was done in Göttingen, to check the reproducibility of data with this new apparatus. Some variations, such as roving of stimulus intensities, were introduced in Göttingen, and did not affect the results. We therefore report the results of the main experiments run in Jerusalem in the paper. The details of the replications in Göttingen are reported in the We used 135 C57BL/6JOlaHsd female mice obtained from a commercial supplier . The mice were 7–8 weeks (Israel) and 5–6 weeks (Germany) old at the time of arrival and were always kept in a light/dark 7am/7pm cycle. A few days after arrival, or 4 weeks after arrival in the group that began training with 9–10 weeks of age, each mouse was lightly anaesthetized with isoflurane vapour or avertin i.p. and a sterile transponder  was implanted subcutaneously in the upper back. In later replications, a stitch or histoacryl (Braun) was used to close the small hole left on the skin by the transponder injection. Once recovered from anaesthesia, the mice were placed in the Audiobox (see below).All behaviour was run in an Audiobox , a device developed for auditory research and based on the Intellicage . The model used in Jerusalem was a prototype, while the one used in Göttingen is commercially available (TSE). The Audiobox serves both as living quarters for the mice and as their testing arena. The mice are kept in groups of 8 to 10 animals. Each animal is individually identifiable through the use of the implanted transponder, and the behaviour of each mouse is automatically detected by two means: reading of the unique transponder carried by each mouse by an antenna at the entrance to the drinking corner (see below), and detection of specific behaviours (nose-poking and licking) through other sensors. In consequence, handling of the animals by the experimenter is reduced to the weekly cleaning of the cages and apparatus.ad libitum. Water is delivered in the second compartment of the Audiobox, the ‘corner’ , temperature regulated and kept in a 12 hr/12 hr dark/light cycle. The Audiobox consists of two compartments connected by a long corridor . One comWe used mainly tones between 6 and 15 kHz, and for some experiments between 3 and 19 kHz. This frequency range contains the area of highest sensitivity in a mouse audiogram. Natural background sounds, which the mouse might use for information and warning, tend to be within this frequency range. While some mouse vocalizations have frequencies above 45 kHz, other mouse communication calls also occupy this range: for example, wriggling calls emitted by pups contain mostly frequencies below 20 kHz Sounds were generated using Matlab (Mathworks) at a sampling rate of 96 or 48 kHz and written into computer files. Output was calibrated using either a Brüel and Kjaer (4939 ¼″ free field) or a GRAS (1/4″ 40BE) microphone. The microphone was placed at different positions within the corner, as well as outside the corner. Relevant sounds were played at the nominal intensities used in the study. Microphone signals were sampled at 96 kHz and analyzed in Matlab. Tones between 3 kHz and 19 kHz did not show any significant harmonic distortion. In the rare occasions when harmonics were present, they were at least 40 dB below the main signal. There was a linear correspondence between the nominal sound level and the sound level measured by the microphone.While sounds played inside the corner were significantly attenuated outside of the attenuated box (>20 dB), there was little attenuation between the corner and the corridor directly leading to it (about 10 dB). In consequence, mice in the corridor could hear the sound presented to the mouse inside the corner. This, however, did not seem to affect their behaviour. For example, animals that were pre-exposed to 13 kHz and then conditioned to the same frequency were run in several replications together with other mice that were pre-exposed to other frequencies. The amount of latent inhibition observed in the mice pre-exposed to 13 kHz was the same irrespective of the pre-exposure frequencies that cohabiting mice heard.Stimuli consisted of 30 ms pure tone pips, with 5 ms rise/fall linear slopes, repeated at a rate of 3 Hz. Tones were presented in the corner throughout the visit. All tone pips presented within a given visit had the same frequency. For the main experiments , sounds were played at a fixed intensity of 70 dB SPL. For the replications in Göttingen, we used a roving-intensity paradigm See .Throughout the duration of the experiment, one frequency (i.e. 6670 Hz) was always ‘safe’: when this frequency was presented during a visit, the mice could access the ports and drink water without an associated negative outcome. At some point within the training, a different tone frequency was associated with an air puff. The purpose of the training was to get the mice to learn this association.The aim of this experiment was to train mice to discriminate between tones of two different frequencies. Initially, these were two well separated tones (1 octave apart). We then explored the capacity of the mice to discriminate between progressively more similar tones, by moving the conditioned tone closer in frequency to the safe tone, in order to establish the just-noticeable difference (JND). We ran 3 replications of the discrimination task. The first one was run in Jerusalem using 9 mice, 7–8 weeks old at the time of testing, that heard a 6670 Hz as safe tone and 13340 Hz as conditioned tone. The other two were run in Goettingen See . The choHabituation phase (7 days): Immediately after the transponder implantation, mice were placed into the Audiobox. During this phase the doors giving access to the water within the corner remained constantly open and no sound was presented during the visits.Safe-only phase (4 days): Once the mice had learned to access the water and were drinking freely, the doors were closed and only opened when the mouse nose-poked into the port. At the same time, every visit to the corner was coupled, for the duration of the visit, with the presentation of the safe sound (tone pips).th visit out of every 20. Mice didn't seem to predict the appearance of a conditioned visit in Jerusalem, as reflected in the lack of change in the behaviour in the safe visits around conditioned visits. As in the safe-only phase, a train of tone pips was presented during the visit. The presence of the conditioned tone had a behavioural significance: a nose-poke during the presentation of the conditioned tone resulted in the delivery of an aversive air-puff, through a tube located on the ceiling of the corner, and the doors that gave access to the water did not open. The remaining visits were as during the safe-only phase.Introduction of a conditioned tone at 5% probability (3 days): The conditioned tone was now presented in 5% of the visits. These visits will be termed ‘conditioned visits’. They occurred on the 11th visit out of every 10.Conditioned tone at 10% probability (3–5 days): The conditioned tone was played in 10% of the visits. They occurred on the 6Conditioned tone at 17% probability (3 days): The conditioned tone was played in 17% of the visits (randomly once every 6 visits).Once discrimination at ΔF of 100% was stable with 17% conditioned visits, we began to lower the frequency of the conditioned tone. The safe tone remained constant in 83% of visits. The conditioned tone was reduced every two to three days from 13340 Hz , progressively down to 6803 (ΔF = 2%) in the first experiment run in Jerusalem , and safe-only phase (11 days), were run as described for the 2-tone discrimination. Following the safe-only phase, training consisted of the following stages see :Pre-exposure phase (5 days): The pre-exposed tone was now played in 17% of the visits (randomly once every 6 visits). This tone did not have behavioural significance and a nose-poke in these visits resulted, like in the safe visits, in the opening of the doors and access to water. The remaining 83% visits were associated with the safe tone as before.The pre-exposure tone had one of the following frequencies: 3335 Hz , 5609 , 7932 Hz , 9433 Hz , 11218 Hz , 13340 Hz , or 15864 Hz . Generally 2 or 3 groups of pre-exposure visits were run simultaneously, and often one of these groups was the group pre-exposed to the frequency that was conditioned later, 13340 Hz.Conditioned phase (5 days): This phase was identical for all mice, irrespective of their pre-exposure frequency. The pre-exposure visits were replaced by conditioned visits, also with a probability of 17%. For all mice, the conditioned tone had a frequency of 13340 Hz. During this phase, the conditioning tone had a behavioural significance: a nose-poke resulted in the delivery of an air-puff and the doors were not opened. The remaining 83% of the visits were safe visits. All the mice used in this report had at least 24 hours of conditioning.Discrimination performance was quantified by the standard measures from signal detection theory, the discriminability (d') and the Criterion (C) Negative values of C indicate that the mouse is more conservative  than the unbiased observer, while positive values indicate that it is less conservative.Since both d' and C cannot be calculated when either the hits or the false alarms reach levels of 100% or 0%, in the few cases where this happened we used 95% and 5% respectively for these calculations. This manipulation reduced d' slightly, and therefore our d' estimates are conservative.ad libitum, we first explored the capacity of C57BL/6JOlaHsd mice to discriminate frequencies of pure tones, characterizing their just noticeable differences (JNDs). Using the same general task contingencies, we then measured their ability to generalize across frequencies in an auditory latent-inhibition paradigm. One mouse died after anaesthesia and another was removed because it did not drink in the corner during the initial phases of training.Using a novel behavioural apparatus, the Audiobox (New Behavior and TSE), in which mice live for the duration of the experiment while performing the task vs. different judgements. This design made it possible to compare discrimination with generalization, which was also memory-based and included single-frequency trials. However, this task was also harder than standard frequency discrimination tests, which usually present the two frequencies during the same trial Frequency discrimination was tested in a memory-based design. In each trial, mice were presented with one frequency only, and had to decide whether to nose-poke or not. Thus, they could not rely on within-trial same Contrary to our expectations, in the main experiment, mice learned to discriminate between the two tones (6670 Hz and 13340 Hz) quickly and reliably, as indicated by the high percentage of conditioned visits without nose-pokes, calculated as the mean value across mice in blocks of 24 hours . While tMice, in fact, learned the discrimination between their first and second conditioned visits. In their first conditioned visit, all the mice but one (8/9) nose-poked, getting in consequence an air-puff (11% no nose-pokes). Already from the second conditioned visit, however, almost all mice (8/9) refrained from nose-poking. This increase in performance from visit 1 to visit 2 was significant . Most mice continued to nose-poke occasionally during conditioned visits (suffering in consequence an air puff), although at a low rate. Their behaviour suggested that they noticed the different frequency: when mice nose-poked during a conditioned visit, the latency to nose-poke was usually longer than the latency to nose-poke in safe visits. Visits followed a clear circadian rhythm during all phases, with a peak in activity around midnight, as expected from nocturnal animals see . Visit lVisits without nose-pokes constituted about 30% of all safe visits. They were also distributed unevenly throughout the day . Given tThe structure of safe visits was unaffected by the introduction of conditioning visits . The avevs 13.2 s). Conditioning also had an effect on the latency to nose-poke in the few visits in which there was a nose-poke, as discussed above , it was significant  = 11.6, p<0.01). When we moved the conditioned frequency back up to 20% above the safe frequency, performance returned to normal, indicating that the reduction in discrimination performance was not due to a loss of stimulus control.In the main experiment, once the mice achieved a stable discrimination performance between tones that were one octave apart, we lowered the conditioned frequency every three days, bringing it progressively closer to the safe tone frequency. The goal was to find the smallest frequency difference at which the animals could perform the task . As the conditioned frequency was lowered , the meaThe development of frequency discrimination in time is studied in vs. conditioned) x ΔF) resulted in a significant effect of safe versus conditioned tone  = 40.3, p<0.01), no effect of ΔF  = 1.47, p = 0.17), and no interaction  = 0.55; p = 0.81). It is possible that the large and consistent difference in latency between safe and conditioned tones at all ΔFs except possibly 4% and 2% swamped the effects of ΔF on latency in this analysis.In conditioned visits in which the animal nose-poked, the mean latency to nose-poke across mice became shorter as the discrimination became more difficult . In the Correct discrimination between two cues takes place when there is both high avoidance of the conditioned stimulus (hits) and low avoidance of the unconditioned stimulus . The d' value (see Methods) takes into account the rates of both hits and false alarms. The threshold for successful discrimination is often considered to be the frequency difference at which d' equals 1. In our case, the average d'  was also affected by the change in ΔF, independently of whether it was measured with  or withoTwo replications of the discrimination experiment were conducted in Göttingen See . They reTo test generalization across frequencies we used the latent inhibition paradigm in a new group of naïve mice. To elicit latent inhibition, a pre-exposure phase was introduced between the safe-only phase and the conditioned phase. The pre-exposure phase consisted of 83% safe visits (6670 Hz) that did not differ from previous safe visits and 17% pre-exposure visits in which another tone frequency was used as an additional safe frequency that was not associated with any aversive outcome: the mouse could nose-poke and obtain water as in any other safe visit. This frequency varied between 3335 Hz and 15864 Hz in different experimental groups ; we will use the terms ‘pre-exposure frequency’ and ‘pre-exposure visits’ below to refer to visits in which this additional frequency was presented. Subsequent to the pre-exposure phase, all mice were conditioned at 13340 Hz, irrespective of their pre-exposure frequency.The introduction of the pre-exposure frequency alone had no effect on the behaviour , to 11218 Hz (n = 16), and to 13340 Hz n = 31; . The perWe showed above that in the simple conditioning task (without pre-exposure) mice learned the discrimination with a single exposure to the conditioning frequency. There are two reasons that might explain why in the generalization task learning was slower, even in the group pre-exposed to 3335 Hz. In the simple conditioning task used for measuring frequency discrimination, the conditioned tone was first presented in only 5% of visits, and not 17% as in the present task. We know (data not shown) that increasing this probability leads to a decrease in the number of hits as well as an increase in the number of false alarms, which might be the reason why in the current task the best group needed about 4 trials to learn the discrimination. Alternatively, since the mice have already been exposed to two safe tones they might generalize some of that learning to any new frequency, in which case the slower learning is really a component of the latent inhibition which is common to all pre-exposure frequencies.Latent inhibition was therefore quantified by the rate of visits without nose-pokes during the first day of conditioning. The frequency tuning curve of latent inhibition is displayed in th hour after the beginning of conditioning the performance was reduced only by 12.5% and 7.3%, in the two groups respectively relative to the group pre-exposed to 3.3 kHz.Latent inhibition was transient: the effect was very pronounced during the 6 hours following the first conditioned visit , black bThe effect that conditioning had on the safe visits  with and without short visits, similarly to the analysis for the frequency discrimination experiment. Both the LI and the generalization to the safe tone are readily apparent in nd day of conditioning, when the generalization to the safe tone had partially subsided. Removing visits shorter than 1 second  showed the largest change following the transition from pre-exposure to conditioning, and a more gradual change thereafter . During Using a novel behavioural paradigm, we measured both discrimination and generalization for pure tone frequency in C57BL/6J mice. Discrimination thresholds (d' = 1) were 4%-7%, although mice could discriminate significantly above chance frequencies as close as 2% apart. In spite of these small discrimination thresholds, mice generalized spontaneously over a range of at least 0.5 octave. Thus, mice generalize over frequencies that are easily discriminable.ad libitum. Since they represent a problem for estimating the true level of false alarm rates in these tasks, future strategies could be devised to reduce these visits for studies that require low levels of false alarms.The Audiobox allows training and testing in the animal living quarters, where animals live in groups and handling is minimized to the weekly cleaning. The mice were neither food- nor water-deprived: performing the task ensured their access to water. This setting makes it possible for each mouse to develop a spontaneous pattern of behaviour. We saw, for example, very clear circadian rhythms with their activity, peaking around midnight. Mice made numerous visits to the corner but did not nose-poke in about one third of the safe visits. This behaviour presumably reflects their natural curiosity and may be characteristic of each strain or genetic background. It most probably does not reflect lack of motivation: the visits without nose-pokes were short and often occurred after a period of inactivity but sometimes also occurred between visits with nose-pokes. These short visits are presumably a feature of tasks in which performance is C57BL\6J mice carry a mutation in the Ahr1 gene that leads to age-related hearing loss Mice learned to discriminate between two frequencies that were one octave apart between their first and second conditioned visits. They achieved discrimination thresholds that were comparable or only slightly larger than other thresholds obtained in rodents. In highly-trained mice, frequency discrimination thresholds are at 3% for frequencies around 8 kHz ad-libitum training in the home cage is at least partially responsible for the fast learning we observed. Indeed, it has been shown previously that differences in housing conditions and training paradigms can lead to differences in auditory thresholds Mice learned to discriminate frequencies very quickly by comparison with other operant auditory protocols: significant frequency discrimination was observed already on the first day of conditioning. Admittedly, conditioning occurred after 7 days in the Audiobox. In contrast, NMRI mice trained with a go/no-go paradigm required about 600 trials to reach asymptotic performance Mice spontaneously categorized all new tone frequencies as dangerous once they learned the paradigm. This is reflected in Despite their relatively small JND, mice generalized across a wide frequency band  of pre-exposure frequencies, as judged by the level of latent inhibition in the response to 13 kHz induced by the pre-exposure to frequencies ranging between 3 and 16 kHz for different groups of mice. A similar generalization has been shown before in the rabbit using the eyelid classical conditioning, and tones as predictive stimuli The behavioural tuning curve of latent inhibition was asymmetrical, narrower for frequencies above the conditioned tone. Although the asymmetrical shape, as well as the precise half-width of the behavioural tuning curve of latent inhibition, could result from the presence and position of the safe tone 1 octave below the conditioned tone, this is unlikely. Indeed, the generalization tuning-curves in the studies of Siegel Tuning curve widths of half an octave are not uncommon in C57Bl/6 mouse nerve fibres for similar frequency ranges and for lower intensities than those used here In addition to the latent inhibition, we also observed an increase in the number of ‘false alarms’ - safe visits without nose-pokes. This increase was particularly prominent in the group pre-exposed to the conditioned frequency itself. This generalization of the ‘cautiousness’ to the safe tone was, unlike latent-inhibition itself, long lasting, and was due to reduced discriminability (d') between the two tones, rather than a more conservative threshold for nose poking (C). Thus, the generalization to the safe tone could reflect, in part, the stress levels of the animals, who learned to avoid the conditioned tone but were left with the impression that apparently safe stimuli might turn nasty without warning: ‘He who was scalded with boiling water is cautious with cold’.We show that mice can be trained to perform auditory discriminations of different complexity quickly, reliably, and with minimum handling. This might prove useful in behaviourally characterizing the growing number of mice with auditory system-related mutations Figure S1Discrimination (Göttingen replications). A–D is for the mice that begun with 9 weeks of age. A. Mean daily performance expressed as the fraction of visits without nose-pokes for the habituation and safe visits (white and light grey) and the conditioned visits (black and dark grey) for the group that had 6670 Hz as safe and the group that had 13340 Hz as safe. B. Single trial performance analysis across all mice: # mice that avoid nose-poking (n = 18) during the 1st 7 conditioned visits starting with the first visit in which each mouse received an air-puff because it nose-poked (dark grey), the very 1st 7 safe visits after habituation (white), and the 1st 7 safe visits after the 1st punished conditioned visit light grey). C. Mean distribution of nose-pokes per visits in the habituation, safe visits during conditioning and conditioned visits. D. Mean distribution of visit duration for all visits (black) and visits without nose-pokes (white) in the habituation, safe visits during conditioning and conditioned visits. The x axis is linear up to visit length of 10 seconds and logarithmic thereafter. E–F is for the mice that begun with 5–6 weeks of age. E. Mean daily performance expressed as the fraction of visits without nose-pokes for the habituation and safe visits (white and light grey) and the conditioned visits (black and dark grey). B. Single trial performance analysis across all mice: # mice that avoid nose-poking (n = 6) during the 1st 7 conditioned visits starting with the first visit in which each mouse received an air-puff because it nose-poked (dark grey), the very 1st 7 safe visits after habituation (white), and the 1st 7 safe visits after the 1st punished conditioned visit light grey).(TIF)Click here for additional data file.Figure S2Individual JNDs . A. Individual psychometric curves for the 9 mice used in Jerusalem as a measure of d' values for each ΔF used. B. The mean psychometric curve (dark) is plotted over a background of individual psychometric curves (light gray). The horizontal and vertical dotted lines represent the level for a d' of 1 and a ΔF of 10%, respectively.(TIF)Click here for additional data file.Figure S3JNDs (Göttingen replications). A–C is for mice that begun with 9 weeks of age and D–F for mice that begun with 5–6 weeks of age. A and D. Mean daily performance in fraction of visits without nose-pokes for the safe (white) and conditioned visits (grey) across phase, starting with the last 3 days of ΔF of 100%. B and E. mean d' as a function of ΔF with (grey) and without (white) removal of visits of 3 seconds or less duration. C and F. percentage mice that avoid nose-poking in the first 20 conditioned visits and subsequent safe visits in each of the phases, where the 100% phase begins with the lasts 3 days of conditioning at ΔF of 100%. The first dark grey data point is the first conditioned trial in that phase, the second grey point is the mean performance in conditioned trials 2 to 10, and the third grey point is the mean performance in trials 11 to 20. The light grey points are the 4 safe visits that follow each conditioned trial.(TIF)Click here for additional data file.Table S1Sequence of conditioned frequencies used for JND assessment in the Göttingen replications.(DOCX)Click here for additional data file.Text S1Description of Göttingen discrimination and JND replications.(DOCX)Click here for additional data file.


Adults as well as infants have the capacity to discriminate languages based on visual speech alone. Here, we investigated whether adults' ability to discriminate languages based on visual speech cues is influenced by the age of language acquisition. Adult participants who had all learned English (as a first or second language) but did not speak French were shown faces of bilingual (French/English) speakers silently reciting sentences in either language. Using only visual speech information, adults who had learned English from birth or as a second language before the age of 6 could discriminate between French and English significantly better than chance. However, adults who had learned English as a second language after age 6 failed to discriminate these two languages, suggesting that early childhood exposure is crucial for using relevant visual speech information to separate languages visually. These findings raise the possibility that lowered sensitivity to non-native visual speech cues may contribute to the difficulties encountered when learning a new language in adulthood. From the first days of life, language perception involves both auditory and visual speech information. The visual information available in talking faces contains linguistic cues often correlated with and complementary to the acoustic signal . One of the best known examples is the difficulty Japanese learners often have in discriminating the English /r/ vs. /l/ contrast Goto, . It is eSecond language learners also show differences with regard to prosodic or supra-segmental language contrasts  of the second language suggest that the auditory phonemic system appears to start losing plasticity in early childhood. For example, among children who acquired a second language after age 7, auditory phonetic perception and production of accent-free speech are less precise than among children who acquired their second language before age 7  and supra-segmental auditory speech perception, we hypothesized that visual language discrimination would also be influenced by the age at which the second language was learned. We therefore tested adults who had learned English at different ages. We divided the adults into three groups. The first group (Infant Exposure) was comprised of adults who had acquired English in infancy (by 2 years)—either as a single language or in a dual language-learning environment. Because an effect has been found for visual language discrimination between 6- and 8-months  conferred any short-term familiarity benefits, we would expect improved performance among the speakers in the blocked condition.In accordance with the Behavioral Research Ethics Board at the University of British Columbia, all participants gave informed consent before participating. There were 120 adult participants  represented native-like and (7) represented beginner. We switched to a more detailed questionnaire . The sentences from each language were then individually digitized with the sound removed, to create 8–13 s silent video clips. There were no significant differences between sentence lengths for the English [average 37.24 (SD = 6.00) syllables] and the French [average 33.24 (SD = 5.88) syllables] video clips.The faces of three balanced bilingual (French/English) speakers were recorded while they recited sentences in both English and French. The French and English sentences were taken from the French and English versions of the book “The Little Prince,” and were selected to overlap in content (same sentence translations) and to be roughly equivalent in length (see Participants were tested in a sound-attenuated room and sat at eye level with the monitor (17″) of a Pentium 4 PC. From a distance of ~75 cm, the participants watched 24 pairs of sentences, and each pair was played consecutively. For each pair of sentences, a white fixation point would first appear in the center of the black screen for 500 ms. Following this, a red frame with the speaker silently reciting one of the sentences would appear and was followed by a 1 s interval of black screen before the second sentence in the pair was played inside a green frame. Participants were asked to press the right mouse button (marked with an S) if they thought both clips were in the same language and the left mouse button (marked with as D) if they thought that they were from different languages. During the second sentence (green frame) participants had been instructed to respond as soon as they were sure of their judgment. If a response was not made during the second sentence, a white question mark appeared in the center of the black screen and was displayed until a response was made or 2000 ms elapsed. The language for each sentence clip was chosen pseudorandomly by the computer for each participant. The order and total number of sentences was set to be equiprobable, with each sentence appearing only once.The two sentences in a given trial were spoken by the same person and were different in content. In the random condition, the clips used in a given trial were selected randomly from one of the three speakers. In the blocked condition, eight clip pairs from each individual speaker were presented consecutively before moving on to the eight pairs from the next speaker. This allowed for a test of potential improvement across exposure to each speaker. The order of the speakers was counterbalanced for each condition and the speaker order for the blocks was counterbalanced across participants.t-tests revealed that across all ages of acquisition, both the English L1   and English L2   discriminated the languages significantly better than chance, and did so in both the Random  and Blocked  speaker blocks. A univariate analysis of variance (ANOVA) including sex, language background (English as L1 or English as L2), and speaker order (blocked or random) yielded only a significant main effect for language background .Using group mean averages, a series of one-sample F = 5.55, p < 0.05]. Planned comparisons focusing on the multilingual participant groups revealed that the Infant and Early multilingual age groups did not perform significantly different from each other , but did perform better than adults who acquired English in late childhood (6–15 years) . In fact, performance was significantly better than chance for multilingual learners who acquired English in M = 56%, t(19) = 2.69, p < 0.02] and learners who acquired English in early childhood , but not for participants who acquired English in late childhood . These results are graphically illustrated in Figure To probe whether age of acquisition of English had an effect on visual speech discrimination, we ran additional analyses. An ANOVA analyzing the effect of age of English acquisition  yielded a significant effect r(70) = 0.09, p = 0.48]r(79) = 0.02, p = 0.84] also failed to reach significance. However, there were significant group differences between the means for proficiency scores, 1.16 , 1.48 , and 1.95 ,  as well as the group means for years of experience, 20.1 , 16.5 , and 12.4 , .We performed several follow-up analyses with the multilingual groups in order to explore whether proficiency or number of years of experience, rather than age of acquisition  = 1.47, p = 0.16].To further probe the possibility that self-rated proficiency or years of experience with English may have contributed to our findings, we equated the Early and Late Multilingual groups by selecting subsets with equivalent proficiency scores or years of experience. We selected a subset of Late multilinguals who scored between 1 and 3 on the proficiency scale [with a mean score = 1.48(0.67) that was equivalent to the Early multilinguals = 1.53(0.60)]. The results from the full sample concerning the influence of AoA were replicated in the restricted Late multilingual sample as the late learning multilinguals again failed to perform significantly better than chance  while the late learning bilinguals did not .Similarly, we also tested the effect of AoA by selecting a subset of English L2 speakers who had an equivalent amount of experience in total number of years (12–19 years), and then within this group, compared the effects of early and late AoA. This resulted in 2 groups: 20 early bilinguals with a mean = 15.3(1.26) years of experience and 16 late bilinguals with a mean = 14.06(2.17) years of experience, wherein the mean years of exposure were not significantly different. The results from the full sample concerning the influence of AoA were replicated in this restricted sample: early bilinguals performed significantly better than chance [The age at which a language is learned  during childhood influences the ability to visually discriminate this language from others in adulthood. Interestingly, this effect of AoA could be examined separately from the influence of years of exposure or proficiency (self-rated). When tested on a visual language discrimination task, most participants who had learned English as a second language in late childhood (after 6 years) failed to discriminate English from French, whereas most participants who had learned English earlier, as infants (0–2 years old) or in early childhood (2–6 years old), succeeded. Allowing the participants to view the speakers in a blocked vs. random speaker order did not seem to have an influence on discrimination performance.According to prior research, infants who are familiar with both languages (French and English since early infancy) retain the capacity to continue discriminating the languages visually at 8 months, while their monolingual counterparts fail (Weikum et al., Sensitive periods have been previously identified for phonemic segment discrimination in auditory spoken languages (for a review see Werker and Tees, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.