Joint analysis of the nPOD-Virus Group data: the association of enterovirus with type 1 diabetes is supported by multiple markers of infection in pancreas tissue

Abstract

Aims/hypothesis

Previous pathology studies have associated enterovirus infections with type 1 diabetes by examining the enterovirus capsid protein 1 (VP1) in autopsy pancreases obtained near diabetes diagnosis. The Network for Pancreatic Organ Donors with Diabetes (nPOD) has since obtained pancreases from organ donors with type 1 diabetes (with broad age and disease duration) and donors with disease-associated autoantibodies (AAbs), the latter representing preclinical disease. Two accompanying manuscripts from the nPOD-Virus Group report primary data from a coordinated analysis of multiple enterovirus indices. We aimed to comprehensively assess the association of multiple enterovirus markers with type 1 diabetes.

Methods

The nPOD-Virus Group examined pancreases from 197 donors, recovered between 2007 and 2019, classified into five groups: donors with type 1 diabetes, with residual insulin-containing islets (T1D-ICI group, n=41) or with only insulin-deficient islets (T1D-IDI, n=42); donors without diabetes who are AAb-negative (ND, n=83); and rare donors without diabetes expressing a single AAb (AAb⁺, n=22) or multiple AAbs (AAb⁺⁺, n=9). We assessed the overall association of multiple indicators of enterovirus infection, case-by-case and between donor groups, as well as assay agreement and reproducibility, using various statistical methods. We examined data from 645 assays performed across 197 nPOD donors.

Results

Detection of enterovirus indices by independent laboratories had high reproducibility, using both enterovirus-targeted and unbiased methods. T1D-ICI donors had significantly higher (p<0.001) proportions of positive assay outcomes (58.4%) vs T1D-IDI (10.3%), ND (17.8%) and AAb-positive donors (AAb⁺ 24.6%; AAb⁺⁺ 35.0%). Head-to-head comparisons revealed increased proportions of donors positive in two independent assays among T1D-ICI vs ND donors (VP1/HLA class I [HLA-I], p<0.0001; VP1/enterovirus-specific RT-PCR (EV-PCR), p=0.076; EV-PCR/HLA-I, p=0.016; proteomics/HLA-I, p<0.0001; VP1/proteomics, p=0.06). Among 110 donors examined for three markers (VP1, EV-PCR and HLA-I), 83.3% of T1D-ICI donors were positive in two or more assays vs 0% of ND (p<0.001), 26.7% of AAb⁺ (p=0.006), 28.6% of AAb⁺⁺ (p=0.023) and 0% of T1D-IDI (p<0.001) donors.

Conclusions/interpretation

The nPOD-Virus Group conducted, to date, the largest and most comprehensive analysis of multiple indices of pancreatic enterovirus infections in type 1 diabetes; these were more prevalent in T1D-ICI and AAb⁺⁺ donors than in other groups. Their preferential detection of these indices in donors with residual beta cells and autoimmunity implicates enterovirus infections across disease progression stages and supports a contribution to beta cell loss, directly or indirectly, even after diagnosis. The relatively small number of infected cells and the low amount of viral RNA support the existence of non-acute, low level, possibly persistent enterovirus infections in the pancreas.

Graphical Abstract