Enterovirus RNA in longitudinal blood samples and risk of islet autoimmunity in children with a high

ARTICLEEnterovirus RNA in longitudinal blood samples and risk of islet autoimmunity in children with a high genetic riskof type1diabetes:the MIDIA studyOndrej Cinek&Lars C.Stene&Lenka Kramna&German Tapia&Sami Oikarinen&Elisabet Witsø&Trond Rasmussen&Peter A.Torjesen&Heikki Hyöty&Kjersti S.RønningenReceived:26February2014/Accepted:18June2014#Springer-Verlag Berlin Heidelberg2014AbstractAims/hypothesis Only a few longitudinal molecular studies of enterovirus and islet autoimmunity have been reported,and positive results seem to be limited to Finland.We aimed to investigate an association between enterovirus RNA in blood and islet autoimmunity in the MIDIA study from Norway,a country which largely shares environmental and economic features with Finland.Methods We analysed serial blood samples collected at ages 3,6,and9months and then annually from45children who developed confirmed positivity for at least two autoantibodies (against insulin,GAD65and IA-2)and92matched con-trols,all from a cohort of children with a single high-risk HLA-DQ-DR genotype.Enterovirus was tested in RNA extracted from frozen blood cell pellets,using real-time RT-PCR with stringent performance control.Results Out of807blood samples,72(8.9%)were positive for enterovirus.There was no association between enterovirus RNA and islet autoimmunity in samples obtained strictly before(7.6%cases,10.0%controls,OR0.75[95%CI0.36, 1.57]),or strictly after the first detection of islet autoantibodies (10.5%case,5.8%controls,OR2.00[95%CI0.64,6.27]). However,there was a tendency towards a higher frequency of enterovirus detection in the first islet autoantibody-positive sample(15.8%)compared with the corresponding time point in matched controls(3.2%,OR8.7[95%CI0.97,77]). Neither of these results was changed by adjusting for potential confounders,restricting to various time intervals or employing various definitions of enterovirus positivity.Conclusions/interpretation Positivity for enterovirus RNA in blood did not predict the later induction of islet autoanti-bodies,but enterovirus tended to be detected more often at the islet autoantibody seroconversion stage.Keywords Autoimmunity.Enterovirus.Infancy. Longitudinal Study.RNA.RT-PCR.Virus-associated aetiologyAbbreviationDAISY Diabetes Autoimmunity Study in the YoungIntroductionEnterovirus infection has long been suspected to trigger or accelerate islet autoimmunity,which precedes type1diabetes Electronic supplementary material The online version of this article(doi:10.1007/s00125-014-3327-4)contains peer-reviewed but uneditedsupplementary material,which is available to authorised users.O.Cinek(*):L.KramnaDepartment of Paediatrics,2nd Faculty of Medicine,CharlesUniversity in Prague and University Hospital Motol,V Uvalu84,15006Prague5,Czech Republice-mail:Ondrej.Cinek@Lfmotol.cuni.czL.C.Stene:G.Tapia:E.Witsø:T.RasmussenDivision of Epidemiology,Norwegian Institute of Public Health,Oslo,NorwayS.Oikarinen:H.HyötyDepartment of Virology,University of Tampere,Tampere,FinlandP.A.TorjesenHormone Laboratory,Oslo University Hospital,Oslo,NorwayH.HyötyFimlab Laboratories,Pirkanmaa Hospital District,Tampere,FinlandK.S.RønningenDepartment of Pediatric Research,Oslo University Hospital,Oslo,NorwayDiabetologiaDOI10.1007/s00125-014-3327-4[1,2].Although several serological patient–control studies observed an association between enterovirus antibodies and type1diabetes,their validity has been disputed[3],mostly because of insufficient matching or unreliable antibody as-says.The advent of molecular testing opened new avenues of research into enteroviruses as potential causative agents for type1diabetes.Infections detected in the blood and its components seem to be more strongly associated with an autoimmune response compared with gut infections detected in faeces[1,4].Published prospective studies on islet autoimmunity that utilise molecular detection of enterovirus from the blood are surprisingly scarce.A recent review[5]and our literature search identified six publications utilising molecular detection from blood in case–control sets nested within prospective cohorts.Indications of an association between enterovirus infection and islet autoimmunity in these prospective studies came from Finland[6–9],whereas another prospective mo-lecular study,Diabetes Autoimmunity Study in the Young (DAISY),from the USA[10]did not support such an associ-ation.Moreover,significant heterogeneity exists in the defini-tions of islet autoimmunity,in sampling frequency and in enterovirus detection methods.The present study used the Norwegian MIDIA(Norwegian acronym for‘Environmental Triggers of Type1Diabetes’) birth cohort.Norway shares many characteristics with its neighbour Finland:among these are a very high incidence of type1diabetes,latitude,shape of the territory,climatic con-ditions,a heterogeneous population density and socioeconom-ic conditions that include a high gross domestic product.To reduce differences in detection methods,this study used an assay very similar to the one used in the Finnish studies.In contrast to other studies,the participating children carried a single high-risk genotype,and whole blood rather than serum was used for enterovirus testing.Our aim was to investigate a putative association between the presence of enterovirus in the blood and islet auto-immunity in the Norwegian MIDIA study.MethodsEnterovirus frequency(i.e.members of Enterovirus A,B,C and D species)was compared between cases(participants with islet autoimmunity)and matched controls nested within a birth cohort of Norwegian children with the highest-risk HLA genotype.Study cohort After genetic screening of46,939newborns from the general Norwegian population during2001–2007, the MIDIA study identified1,047newborns with the HLA class II genotype conferring the highest risk of type1diabetes, DRB1*04:01-DQA1*03-DQB1*03:02/DRB1*03-DQA1*05-DQB1*02.Of these participants,911were recruited for follow-up.Three families later withdrew and their data were deleted.Of the remaining908children,blood samples and questionnaires were collected at ages3,6,9and12months, and annually thereafter,and plasma samples were tested for the presence of islet autoantibodies,indicating islet autoimmunity.Written parental consent was obtained.The study was approved by the Regional Committee for Medical Research Ethics(Office for Human Research Protections IRB name ‘Regional Med Resch Ethics Comm South IRB#2—South-East A’,IRB00001871)and the Norwegian Data Protection Authority.Nested case–control dataset By October2011,48out of908 children in the observed cohort[11,12]had developed islet autoimmunity(as defined below)and were included as cases. For each case,we randomly selected two controls from the cohort,matched for age(with a maximum tolerance of 1month wherever possible;this difference is narrow enough to account for the known enterovirus seasonality because sampling schedules of case and control participants were identical up to the first occurrence of islet autoantibodies) and county of residence(including the closest neighbouring county,if necessary).Children were ineligible as controls if they had one or more autoantibodies(as described below). Controls were followed at least until the time point of con-firmed islet autoimmunity in the corresponding case.To en-sure comparability,blood samples from cases that did not have a matching control sample were removed from the analysis.For three case and four control participants,there was not enough material for enterovirus testing,and they were therefore excluded from the analysis.The final case–control dataset included45cases and92controls:their characteristics are listed in Table1and generation of the case–control dataset is shown in Fig.1.Sample handling,islet autoantibody assays and case definition Capillary blood samples were collected into tubes containing EDTA and sent to the Norwegian Institute of Public Health by mail.Upon arrival,samples were centri-fuged:plasma was separated and tested for islet autoanti-bodies,while cell pellets containing residual plasma were stored at−80°C until further processing.All frozen samples were thawed once only.Autoantibodies to insulin,65kDa glutamic acid de-carboxylase(GAD-65)and tyrosine phosphatase-like protein IA-2were measured in duplicate by RIA in the hor-mone laboratory at Aker University Hospital,as previously described[12].We defined islet autoimmunity as detection of either(1)two or more islet autoantibodies in two or more consecutive samples,or(2)at least one islet autoantibody twice or two antibodies once followed by progression toDiabetologiatype 1diabetes during the current follow-up,or (3)at least one islet autoantibody in at least three consecutive samples,including the most recent sample.Nucleic acid extraction Frozen cell pellets containing red and white blood cells with a small amount of residual plasma were thawed at 4–8°C,and RNA was co-purified with DNA using the TRIzol Plus RNA Purification System (Invitrogen,Carls-bad,CA,USA)according to the manufacturer ’s protocol with minor modifications.Specifically,0.1ml of the blood cell pellet was added to 1ml Trizol containing 3.7μg carrier RNA (Qiagen,Hilden,Germany)and 0.5μl 1%(vol/vol in PCR-grade water)Armored RNA West Nile Virus (Asuragen Diagnostics,Austin,TX,USA)as an exogenous internal control.The extraction was then performed as indicated in the manufacturer ’s protocol until binding to PureLink columns and washing:these steps were not performed by centrifugation,but instead used a Qiagen vacuum manifold and vacuum pump set to −20to −30kPa,with columns connected to the manifold through Qiagen V acConnectors.Before the final elution steps,columns were centrifuged for 2min at 12,000g to dry the membranes.Elution was performed into 120μl A VE buffer (Qiagen)and used for downstream applications after an over-night incubation at 4°C.Quantitative RT-PCR of enterovirus RNA Samples were test-ed for enterovirus,an exogenous internal control and human RNA content.Enterovirus RNA was assayed using the QuantiTect Probe RT-PCR Kit (Qiagen),using 900nM primers and 300nM probes (as reported in a previous Finnish study [13]).The combination of primers and probes reacts with an equal sensitivity to human Enterovirus A —D species (i.e.members of species Enterovirus A ,Enterovirus B ,En-terovirus C and Enterovirus D of the genus Enterovirus ,family Picornaviridae ,order Picornavirales ,according to the latest nomenclature of the International Committee on Taxonomy of Viruses,/,accessed 9May 2014).The assay does not cross-react with Rhinovirus A –C species.The analysis was carried out using ABI7300or ABI7700real-time thermocyclers and Sequence Detection Software (Applied Biosystems,Foster City,CA,USA).PCR conditions were 30min reverse transcription at 50°C,followed by 15min initial denaturation at 95°C,and 50cycles of 15s denaturation at 94°C and 1min combined annealing –synthesis at 60°C.Absolute quantification was performed using an eight-point standard curve spanning the range from 375,000to 1copy per microlitre Armored RNA West Nile Virus (quantitated spec-trophotometrically by the manufacturer).Samples were run in triplicate.The assay was sufficiently sensitive to consistently detect a single copy per microlitre source RNA;of note,very low virus quantities showed strong stochastic effects,with varying number of positive replicates.To ensure robustTable 1Characteristics of case and control participants in the present study CharacteristicParticipants Case (n =45)Control (n =92)Age at onset of islet autoimmunity (months)a 24.5(6.2–75)NA Female sex 28(62.2%)50(54.4%)Number of other children in the household b012(26.7%)30(32.6%)115(33.3%)45(48.9%)≥218(40.0%)17(18.5%)First-degree relatives with type 1diabetes c None 35(77.8%)87(94.5%)YesSiblings only 30Father only 32Mother only23Multiple family members 20Progression to type 1diabetes Yes20(44.4%)n.a.Age (years)at diabetes onset4.2(0.7–7.4)Blood samples in the matched analysis Total d258417Before the development of islet autoimmunity a,e144251Islet autoantibody seroconversion sample f3862After seroconversion g 76104Data are median (range),n (%)and n NA,not availableaAge at the time when the first islet autoantibody-positive sample was collectedbIncluding full siblings,half-siblings and step-siblings.The number of chil-dren was counted when the index child was 3months old.Case participants tend to have more children in the family compared with controls (p =0.018)cOnly full siblings were counted.Of the two cases with multiple affected first-degree relatives,the father,mother and one sibling were affected in one case,and two siblings and the father (but not the mother)were affected in the other case.First-degree relatives with diabetes were observed more often in the families of cases than controls,p =0.009dOf the 807samples tested for enterovirus and passing the quality control,675were included in the matched analysis.The remaining 132were removed because their matching sample was missingeThese include the samples before the first islet autoantibody-positive sample,and the corresponding samples from controls.Several initial blood samples taken in 2003and 2004were not available for enterovirus testing because of the lack of a cell pellet sample as the RNA source.Three cases and four controls were affected;thus,in total,45cases and 92controls were analysedfThe first islet autoantibody-positive sample and its matched control sample;several case samples could not be matched with a sufficiently similar control samplegFor association analysis with islet autoimmunity,samples without prop-erly age-matched controls (primarily after islet autoantibody seroconver-sion,owing to study design in which these participants were followed with more frequent blood samples)were removed from analysis,and not all cases and controls contributed samples to all three periods (before,during and after seroconversion)Diabetologiadetection,we defined samples as positive for enterovirus only if at least two out of three triplicate wells were positive. Negative controls were included in every extraction(at least one per every batch of15samples,at a random location within the batch)and independently in the PCR.Neither of these negative controls tested positive.In parallel with enterovirus and using an identical protocol, we also tested an exogenous internal control(a small amount of West Nile Virus fragment added to the first extraction step), and the B2M gene transcript,a commonly used control that is abundant in human blood[14].Threshold cycles were inspected to identify those strongly deviating from the mean (signifying a lack of cells or possible failure of extraction). Samples that exceeded two SDs of the threshold cycle from the average were marked as suspect of a low RNA content, and this fact was taken into account in downstream analyses. Forty-one samples(4.8%)had low RNA content;however, three of these were still enterovirus positive.A second set of nucleic acid extractions could not be performed because all blood sample material was used up in the first extraction. Therefore,we excluded these41suspect samples in the pri-mary analysis;in the sensitivity analysis,we re-ran all statis-tical analyses including these41samples,and results were essentially unchanged.Statistical analysis The association between enterovirus in-fections and islet autoimmunity was estimated using mixed effects logistic regression models with enterovirus positivity in a blood sample as the dependent variable,and case or control status(and other covariates)as independent variables. To account for the matched design and repeated measure-ments of enterovirus within individuals,random intercepts were specified for each matched set and for each individual within the matched set using the xtmelogit command with default settings in Stata,version12(StataCorp,College Sta-tion,TX,USA).The primary analysis used data from samples collected strictly before islet autoantibody seroconversion.Additional analyses included samples from the seroconver-sion period and after seroconversion.Case samples without a properly age-matched control(primarily after islet autoanti-body seroconversion,owing to the study design where these participants were followed with more frequent blood sam-ples),were removed from the analysis.We performed two sensitivity analyses:(1)we assessed the influence of also defining as enterovirus positive those sam-ples that tested positive in a single replicate,i.e.we extended the positivity definition to samples likely to be affected by the stochastic character of low copy number templates;(2)we performed a further sensitivity analysis including samples presumed to contain low levels of RNA,as indicated by assessment of control templates.ResultsEnterovirus in blood and islet autoimmunity A total of807 samples were tested,of which72(8.9%)were positive for enterovirus.Positivity for enterovirus was not associated withFig.1Generation of thecase–control set used in thepresent studyDiabetologiathe subsequent development of islet autoimmunity in samples taken prior to islet autoantibody seroconversion(Table2;OR 0.75[95%CI0.36,1.57]).There was a tendency towards an association with enterovirus detection in samples taken in the islet autoantibody seroconversion interval of case participants (6/38,15.8%),and the corresponding time point in controls (2/62,3.2%,OR8.7[95%CI0.97,77]).No significant association was observed after islet autoantibody seroconver-sion.The results are summarised in Table2,where unadjusted measures of association are presented along with results from models adjusted for potential confounders and modifiers(sex, first-degree relative with diabetes[yes or no]and number of other children in the household[coded as0,1and2or more]). The results were essentially unchanged upon adjustment.A graphical representation of enterovirus testing and islet autoimmunity data is provided in electronic supplementary material(ESM)Fig.1.The cumulative probability of a first enterovirus infection in blood was26%(95%CI19,34)by the second birthday and 38%(95%CI30,48)by3years of age(ESM Fig.2),with no significant difference between case and control participants in the timing of the first infection(data not shown).The estimated virus quantity in blood was generally low: only three samples had more than100copies per microlitre RNA extract,13had10–99copies per microlitre,and all remaining positive signals contained fewer than10copies per microlitre,where an accurate quantification is difficult.As our definition of enterovirus positivity required two or more positive replicates,we included a sensitivity analysis containing all low-quantity samples that were positive in only one of the three replicates(samples indicated by half-filled circles in ESM Fig.1;the total number of positive samples included in this relaxed definition of positivity was127/807, 15.7%).This analysis did not alter either of the above conclu-sions(data not shown).Enterovirus detection over the whole observation period The median number of samples tested for enterovirus per child was six(range1–14),and55participants(40.1%)had at least one positive sample during follow-up.Forty participants(29.2%) had one enterovirus RNA-positive blood sample during follow-up,13(9.5%)participants had two positive samples and two(1.5%)participants had three positive samples. Among the15participants for whom two or three enterovirus RNA-positive samples were observed during follow-up,five had consecutive samples positive for enterovirus RNA, whereas10had a second enterovirus RNA-positive sample after an intervening negative sample.If we assume that pos-itive consecutive samples formed part of a single infectious episode and the intermittent remaining positive samples rep-resented separate infection episodes,then there was a total of 67infectious episodes,of which5(7.5%)were‘prolonged’(group7,case;group11,case and the first control;group19, case;group36,case;ESM Fig.1).There was no detectableTable2Frequency of human enterovirus in blood,and islet autoimmunitySamples Participants OR(95%CI)aCase Control Unadjusted Adjusted bCollected before islet autoantibody seroconversion cEV−133226 1.0(reference) 1.0(reference)EV+11(7.6%)25(10.0%)0.75(0.36,1.57),p=0.440.60(0.27,1.32),p=0.20 Collected during seroconversion dEV−3260 1.0(reference) 1.0(reference)EV+6(15.8%)2(3.2%)8.7(0.97,77),p=0.0539.08(0.95,86),p=0.055 Collected after seroconversion eEV−6898 1.0(reference) 1.0(reference)EV+8(10.5%)6(5.8%) 2.00(0.64,6.27),p=0.23 2.76(0.87,8.77),p=0.086Data are n,n(%)and OR(95%CI)EV−,enterovirus negative samples;EV+,enterovirus positive samplesa Estimated from a mixed effects logistic regression model,with enterovirus positivity as the dependent variable and random intercepts for matched set and(within each matched set)individualsb Adjusted for sex,first-degree relative with type1diabetes(yes or no)and number of other children in the household(0,1or≥2).The final adjusted model does not include other tested covariates such as age,time of blood sampling and introduction of cow’s milk before3months of age since they did not influence the observed OR and did not improve the modelc Strictly excluding the sample first showing islet autoantibody positivityd Seroconversion time point defined as the first islet autoantibody-positive sample from case participants and corresponding matched controls.Note that for these samples we cannot know the temporal sequence of events,i.e.whether enterovirus RNA or islet autoantibody seroconversion occurred first e Seroconversion time point defined as first islet autoantibody-positive case sample and the control sample from the corresponding period Diabetologiaassociation between prolonged infections and seroconversion of islet autoantibodies.Modifiers of enterovirus positivity The highest frequency of enterovirus positivity was seen in samples obtained at2years of age(ESM Fig.3a),and the frequency decreased thereafter. There was an appreciable seasonal pattern,with a peak in late summer(p=0.019in the Walter and Elwood test[15];ESM Fig.3b).Enterovirus detection rates did not differ among calendar years.The frequency of enterovirus positivity was similar in children exposed to cow’s milk products before vs after 3months of age(OR0.68[95%CI0.36,1.24])and did not vary with sex(OR1.05[95%CI0.64,1.72]for males)nor with having first-degree relatives with diabetes(OR1.21 [95%CI0.61,2.39]).In contrast,enterovirus positivity was marginally increased when one or more other children were present in the household compared with none(OR 1.82[95%CI1.01,3.28]).We explored whether islet autoimmunity was associated with enterovirus RNA positivity in subgroups defined by the aforementioned factors(sex,children exposed before vs after 3months of age to cow’s milk products,first-degree relatives with diabetes,presence of other children in the household and calendar season of birth)using a stratified analysis and testing for interactions.None of these tests indicated any subgroup effects.DiscussionOur results do not support the hypothesis that enterovirus RNA in longitudinally collected blood samples predicts the subsequent development of islet autoimmunity,despite a high overall enterovirus frequency and inclusion of the highest number of cases among the prospective molecular studies published so far.Interestingly,in samples showing the first occurrence of islet autoantibodies we observed an almost significant increase in enterovirus RNA frequency.It is currently impos-sible to determine the sequence of events,i.e.whether entero-virus positivity occurred before islet autoimmunity or vice versa.If our enterovirus-positive samples reflected only acute viraemia(which is usually short),then only infections towards the end of the sample interval would be detected;thus,our data would support reverse causation(increased susceptibility to enterovirus in the blood as a result of islet autoimmunity). However,all but16positive samples contained only minute quantities of the virus,which is inconsistent with viraemic peaks.Our source of RNA(i.e.packed blood cells)may bear a certain‘memory’of past viral infections because peripheral white blood cells are permissive to enterovirus infection[16]and may be a better source of enterovirus RNA than plasma or serum[17,18].Thus,we speculate that the enterovirus posi-tivity we observed may reflect infection occurring over longer periods of time,leaving the direction of the causality unresolved.Study strengths Using a strict definition of positivity,we obtained a high enterovirus frequency of8.9%in blood sam-ples.This frequency is similar to that found in the DAISY study[10](although in that study a visit was deemed positive if serum,saliva or stool samples were positive)and is twofold higher than that of the Finnish DIPP(Diabetes Prediction and Prevention)study(4%and3%sera positivity in case and control participants)[7].Our high enterovirus frequency may thus also support the notion of the non-inferiority or superiority of whole blood compared with other sources of nucleic acid,as previously observed in testing for other viruses[19,20].Our study stems from the most homogeneous prospective cohort yet published,which reduces the possible variation in enterovirus frequency resulting from HLA genotypes[21].A strict matching protocol was employed,demonstrated by the clustering of infections across the matching groups(ESM Fig.1).Furthermore,factors modifying enterovirus positivity were either taken into consideration in the matched study design(age,place,season)or adjusted for in the analysis. Comparison with other studies The prospective studies pub-lished so far on enterovirus and islet autoimmunity show variations in several aspects,including sampling intervals and definitions of positivity[1].We specifically aimed to minimise methodological differences with prospective molec-ular studies from neighbouring Finland[6–9].In the Finnish studies,a significantly higher enterovirus RNA frequency was noted in sera taken in the6-month period before the first detection of islet autoantibodies,thus reflecting an excess of acute viraemic infections during that period [7,8],whereas no such association was observed in the present study.The differences in enterovirus frequency may partly explain this discrepancy.There is an unusually low enterovirus prevalence in Finland,which is several fold lower than in other populations[7,22–24].Assuming that a putative diabetogenic strain or serotype is hiding among a hundred or more other,innocuous strains,its effect may be easily overshadowed if these background strains are overly preva-lent.This may also be the case for the present Norwegian study,which shows the highest frequency so far of enterovirus RNA in the blood of prediabetic children.Study limitations One limitation shared by this study,as well as all other studies using pan-enterovirus RT-PCR analysis of blood or serum[7,8,10],is the lack of sequence information. Although detection using the5′-untranslated region is veryDiabetologiasensitive,the amplicon contains no information on serotype specificity.Amplification of the VP1(serotype-specific)re-gion is unfeasible in low positivity samples such as ours because degenerate PCR analysis requires relatively high template concentrations.Second,if a hit-and-run infection mechanism is involved in the pathogenesis of islet autoimmu-nity,then all currently available molecular studies—including ours—have an insufficient blood sampling frequency and would therefore detect only a small fraction of the total infections.In summary,we were not able to see a statistically signif-icant association between enterovirus RNA positivity in blood and the subsequent development of islet autoantibodies in children with a high genetic risk of type1diabetes.Although induction of islet autoantibodies tended to be associated with enterovirus detection in blood,we cannot conclude a causal relationship between the two events.Acknowledgements We thank the public healthcare nurses for their recruitment efforts for the MIDIA study and the follow-up of high-risk children,and the staff at the Biobank Department at the Norwegian Institute of Public Health.In particular,we would like to express our gratitude to all of the parents for their efforts in handling their children’s type1diabetes risk,for allowing samples to be taken from their children and for completing questionnaires.Funding This study and the MIDIA project were funded by the Norwegian Organization for Health and Rehabilitation(2008/0182),the Ministry of Health of the Czech Republic(IGA MZ11465–5),the Research Council of Norway(grants135893/330,155300/320,156477/730, 205086/F20and166515/V50),the Norwegian Diabetes Association,the Academy of Finland(grant to HH)and the Project for the Conceptual Development of Research Organisation00064203(University Hospital Motol,Prague,Czech Republic).OC’s sabbatical at HH’s laboratory in 2012was supported 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