Increased Prevalence of Enteroviral RNA in Blood Spots From Newborn Children Who Later Developed Type 1 Diabetes
A population-based case-control study
- Gisela G. Dahlquist, MD, PHD1,
- Jenny Forsberg, MS1,
- Lars Hagenfeldt, MD, PHD2†,
- Jens Boman, MD3 and
- Per Juto, MD, PHD3
- 1Department of Clinical Sciences-Paediatrics, Umeå University, Umeå, Sweden
- 2Centre of Inherited Metabolic Diseases, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden
- 3Department of Virology, Umeå University, Umeå, Sweden
- Address correspondence to Professor Gisela Dahlquist, Umeå University, Department of Clinical Sciences-Paediatrics, SE-901 87 Umeå, Sweden. E-mail:
Virus infections during fetal life may lead to persistent infections due to unresponsiveness of the immature immune system and by different mechanisms inducing autoimmunity in the β-cell (1). In a previous study, we found group-reacting antibodies to enterovirus more frequently increased in serum at delivery in a cohort of mothers whose children later developed diabetes than in control subjects (2). It has been argued that mothers of later diabetic children might have increased immune responses to certain viruses (3). Therefore, detection of the virus nucleic acid would be important to confirm.
Enteroviral infections typically occur as epidemics with a short viremic phase; therefore, they are detectable for only a short time in peripheral blood (4). Consequently, large series of blood or serum samples would be necessary. Since 1973, blood spots routinely taken on days 2–4 of life for analysis of inherited metabolic diseases in all newborns in Sweden are stored in a biobank. From this biobank, we collected blood spots from 600 children in the Swedish childhood diabetes register who were born during the years 1986–1995 and who had diabetes onset before 1996. For each case, a control sample was included from a child born on the same date and not found in the Swedish childhood diabetes register, and the control sample was stored adjacent to the case filter.
Nested enterovirus PCR was performed essentially according to Puig et al. (5). To exclude the possibility of false-positivity due to contamination, we excluded all case-control pairs with double positivity and each step of RNA extraction and analysis included positive and negative controls. All case-control pairs were analyzed in the same run. As references, representatives of the most common congenital viral infections were chosen for analysis: CMV (6) and parvo B19 virus (7). A total of 542 pairs of samples were valid and analyzed for enterovirus RNA.
Twenty-seven diabetic cases were RNA positive, as compared with 14 control subjects (odds ratio 1.98 [95% CI 1.04−3.77], two-tailed P = 0.04). Due to limited material, typing of enteroviral RNA by sequencing was not possible . For CMV and parvo B19 viruses, no differences were shown between cases and references in samples of 208 and 180 pairs, respectively.
The findings support the hypothesis that early enteroviral infections may play a role in the pathogenesis of type 1 diabetes. In our previous study of maternal sera, the etiological fraction of enteroviral infection in pregnancy (4) was calculated at 27%. Thus, early fetal or neonatal infections may explain a fraction of childhood diabetes cases.
This study was supported by grants from the Swedish Research Council (Medicine) (project no. 07531), the Swedish Diabetes Association, and the Västerbotten County Council.