Subjects and sample material

The clinical cohorts used in Reports II, III and IV of this thesis are based on three independent studies: The Diabetes Prediction and Prevention Study (DIPP), The Diabetes and Autoimmunity Study in the Young Study (DAISY) and Viruses in Diabetes Study (VirDiab). Samples collected in these cohorts cover various phases of the development of T1D. In the DIPP study, samples were collected from birth to the onset of T1D, while in the DAISY study children were followed from the time of autoantibody seroconversion to the development of T1D. In the VirDiab study samples were collected at the onset of T1D (Fig. 3). These study populations were recruited from a wide geographical area including children from Finland, Sweden, England, France, Greece and the USA (Fig. 4). Altogether these three studies included 2905 serum and 1242 rectal swab samples collected from 337 children who developed T1D, 90 children who were positive for T1D associated autoantibodies and 389 control children. Samples were collected over 15 years from 1993 to 2007. The methodological publication (Report I) was based on 108 stool samples selected randomly from 27 children participating in the DIPP study.

Figure 3. Schematic presentation of sample collection in relation to T1D process in the DIPP, DAISY and VirDiab studies. Arrows indicate the time periods of sample collections related to T1D disease process.

DIPP DAISY VirDiab

Birth AAb T1D

Figure 4. Study centers on the map. The black rectangle indicates the centers of the DIPP study in Oulu, Tampere and Turku. The white triangle refers to the VirDiab study centers in Tampere, Linköping, London, Lille and Athens. The DAISY study center is indicated with a white rectangle in Denver.

4.1.1 DIPP study

The Diabetes Prediction and Prevention Study (DIPP) is a prospective birth-cohort study in which children at high or moderate genetic risk of developing T1D are followed from birth till the onset of T1D or 15 years of age. All parents with newborn infants at the University Hospitals in Oulu, Tampere and Turku are offered the possibility for screening T1D associated HLA-DQB1 alleles from a cord blood sample. Children with increased risk (HLA-DQB1*02/*0302), the *0302/x genotype [x ≠ *02, *0301, or *0602], and also boys with the genotype DQB1*02/y-DQA1*05/z [y ≠ *0301, *0302, *0602, *0603; z ≠ *0201] are recruited to the study on the consent of the parents. The protocol used in the present study has been described previously [156]. Serum samples were collected from birth at intervals of 3 to 6 months till the onset of T1D or the age of 15 years. The diabetes-associated ICA autoantibody was tested continuously from every sample and if the child became ICA positive all follow-up samples were screened also for IAA, GADA and IA-2A. After autoantibody seroconversion, the children were invited to a clinical visit every 3 months. Stool samples were collected from the high risk group every

month from the age of 3 months till two years of age. In addition, the clinical symptoms were recorded at every clinical visit using a questionnaire.

In Report I, 108 stool samples were selected randomly from 27 DIPP children aged from 3 to 24 months in Tampere DIPP center. For this period of time parents recorded the age when all new foods were added to the child’s diet and this data was recorded by nurses at clinical visits at the age of 3, 6, 12, 18 and 24 months. Children were categorized in a group which was exclusively breastfed, and another group which received supplementary food.

In Report II, the frequencies of enterovirus infections were compared between case and control children at various time points during the development of T1D: 1) whole follow-up time period from birth to the onset of T1D, 2) time from birth to the time point of six months before seroconversion to autoantibody, 3) time window of 6 months before autoantibody seroconversion, 4) time period from autoantibody seroconversion to the diagnosis of T1D and 5) at the onset of T1D (Fig 5.). In this study, enterovirus analyses were carried out from all serum samples collected from case children who developed clinical T1D and from samples collected from one to six, non-diabetic and autoantibody negative control children matched for age, gender, HLA T1D associated DQ alleles, time of birth (± 1 month) and University Hospital district. A total of 333 serum samples from 38 case (boys 18) and 993 samples from 140 control (69 boys) children were analyzed.

Figure 5. Outline of the analysis on role of enterovirus infections in different stages of T1D process in Report II. Birth-T1D = time from birth to onset of T1D. Birth - before 6 month period prior AAb = time from birth to 6 months before the seroconversion to positivity for the first autoantibody. 6 month period prior AAb = time window 6 months before autoantibody seroconversion, AAb-T1D = period from autoantibody seroconversion to diagnosis of T1D and T1D = at the onset of T1D.

Birth AAb T1D

Birth–before 6 month period prior AAb

6-month period

prior AAb AAb–T1D T1D Birth-T1D

4.1.2 DAISY study

The Diabetes and Autoimmunity Study in the Young (DAISY) study has followed two groups of children. One group was at an increased genetic risk of T1D without a diabetic relative, and in the other group of children, had a diabetic sibling or parent.

Altogether, 2365 children in both groups were screened longitudinally for three islet autoantibodies IAA, GADA and IA-A2 at ages 9, 15 and 24 months and annually thereafter. Children who tested positive for at least one autoantibody were followed with 3 to 6 months intervals (median 4 months) [157].

Report III is based on a cohort of 140 children who tested positive for at least one islet autoantibody in two or more consecutive samples and who provided at least one serum or rectal swab sample for enterovirus testing. Altogether, 1081 serum and 1242 rectal swab sample were collected in 1295 clinical visits for enterovirus analyses.

These samples were collected between 1993 and 2007 in Denver, United States of America.

4.1.3 VirDiab study

The Viruses in Diabetes Study (VirDiab) is a multicenter case-control study, which was carried out in five European countries during the years 2001-2005. Newly diagnosed T1D children were recruited in Tampere (Finland), Linköping (Sweden), London (England), Lille (France) and Athens (Greece). T1D children were matched pair-wise according to the time of sampling, gender, age and country. In Finland, the control children were recruited from DIPP study and they were matched with case children for the HLA-conferred risk for T1D. In other populations, control subjects were healthy school children or children from the local hospital coming for minor surgical operations. The diagnosis of T1D was based on the WHO criteria.

In Report IV, altogether 249 case-control pairs fulfilled these matching criteria and their samples were used for virus antibodies analyses. Antibodies against CBV1-6 serotypes were measured by a plaque reduction neutralization assay.

4.1.4 Study design for the analysis of PCR inhibition (I)

PCR inhibitors were tested in stool samples by spiking a standard amount of Semliki Forest Virus (SFV) RNA in nucleic acid fraction extracted from the stool sample before the RT-PCR reaction was performed. The amplification of SFV RNA in

spiked stool samples was compared to a positive control, which was a water sample spiked with the same amount of SFV RNA as the stool samples. The spiking of samples was done by mixing the SFV RNA in the master mix of RT-PCR reactions to minimize variation due to pipetting. All samples were tested with and without the addition of BSA to PCR mix to test if BSA inactivates possible inhibitors in the samples. In addition, the performances of two nucleic acid extraction kits were compared; one was based on silica columns and the other on magnetic glass particles.

The definition of inhibition was categorized in three classes: the sample was considered to be completely inhibited when no amplification was observed, partially inhibited when the sample gave a maximum 25% of the signal of the positive control and not inhibited when the signal was more than 25% of positive control (Fig. 5).

Figure 6. Schematic presentation of study design of RT-PCR inhibition test. Semliki forest virus RNA (SFV RNA) was used as a standard to estimate the amount of RT-PCR inhibition in the samples.

SFV RNA was quantified by specific RT-PCR method to estimate the amount of RT-PCR inhibition in the samples. (Adapted from Fig. 1 in Report I)

4.1.5 Virus strains

In Report I, Semliki forest virus, which was used as the control virus for monitoring PCR inhibitors was obtained from the University of Turku (a gift from the department of virology and Dr. Ari Hinkkanen).

In Report IV, CBV1-6 prototype strains were obtained from American Type Culture Collection (ATCC) and in addition, wild-type viruses CBV1 and CBV3 were isolated from Finland.