The virome in early life and childhood and development of islet autoimmunity and type 1 diabetes: A systematic review and meta‐analysis of observational studies

Summary Viruses are postulated as primary candidate triggers of islet autoimmunity (IA) and type 1 diabetes (T1D), based on considerable epidemiological and experimental evidence. Recent studies have investigated the association between all viruses (the ‘virome’) and IA/T1D using metagenomic next‐generation sequencing (mNGS). Current associations between the early life virome and the development of IA/T1D were analysed in a systematic review and meta‐analysis of human observational studies from Medline and EMBASE (published 2000–June 2020), without language restriction. Inclusion criteria were as follows: cohort and case–control studies examining the virome using mNGS in clinical specimens of children ≤18 years who developed IA/T1D. The National Health and Medical Research Council level of evidence scale and Newcastle–Ottawa scale were used for study appraisal. Meta‐analysis for exposure to specific viruses was performed using random‐effects models, and the strength of association was measured using odds ratios (ORs) and 95% confidence intervals (CIs). Eligible studies (one case–control, nine nested case–control) included 1,425 participants (695 cases, 730 controls) and examined IA (n = 1,023) or T1D (n = 402). Meta‐analysis identified small but significant associations between IA and number of stool samples positive for all enteroviruses (OR 1.14, 95% CI 1.00–1.29, p = 0.05; heterogeneity χ 2 = 1.51, p = 0.68, I 2 = 0%), consecutive positivity for enteroviruses (1.55, 1.09–2.20, p = 0.01; χ 2 = 0.19, p = 0.91, I 2 = 0%) and number of stool samples positive specifically for enterovirus B (1.20, 1.01–1.42, p = 0.04; χ 2 = 0.03, p = 0.86, I 2 = 0%). Virome analyses to date have demonstrated associations between enteroviruses and IA that may be clinically significant. However, larger prospective mNGS studies with more frequent sampling and follow‐up from pregnancy are required to further elucidate associations between early virus exposure and IA/T1D.


Summary
Viruses are postulated as primary candidate triggers of islet autoimmunity (IA) and Newcastle-Ottawa scale were used for study appraisal. Meta-analysis for exposure to specific viruses was performed using random-effects models, and the strength of association was measured using odds ratios (ORs) and 95% confidence intervals (CIs).

| INTRODUCTION
Type 1 diabetes (T1D) is common, affecting more than 600,000 children aged <15 years worldwide. 1 T1D is preceded by islet autoimmunity (IA) lasting months to decades. 2 It is defined serologically as multiple autoantibodies against one or more T1D-associated autoantigens, including insulin (IAA), glutamic-acid decarboxylase (GADA), tyrosine phosphatase-like insulinoma antigen 2 (IA2A), islet cell cytoplasmic proteins (ICA) and β-cell-specific zinc transporter 8 (ZnT8A). 3 T1D pathogenesis results from a complex interplay of genetic predisposition 4,5 and environmental exposures. 6,7 Accumulating evidence supports the influence of environmental factors, particularly viruses. The increased incidence of T1D is too high to be attributed to genetics alone, 1,8 with data showing seasonal IA/T1D clustering, 9 geographical variation in incidence 10 and more frequent in utero and early-life infections in affected individuals. 11,12 Higher rates of enterovirus (EV) infection, detected by serological 13,14 or molecular methods, [15][16][17] have been observed in T1D patients at diagnosis versus unaffected controls, or prospectively in individuals who subsequently develop IA and/or T1D versus those who do not. Accordingly, our previous meta-analysis investigating EV using molecular methods demonstrated significant association between EV and IA (odds ratio [OR] 3.7, 95% confidence interval [CI] 2.1-6.8, p < 0.001) or T1D (9.8, 5.5-17.4, p < 0.001). 18 In addition, EV proteins and RNA have been isolated from pancreata of affected patients, with upregulated EV receptors selectively expressed in pancreatic islets. 19,20 However, inconsistencies in findings 21-23 make it difficult to establish a definitive causal association. Importantly, substantial investigation bias exists for EVs in previous studies. 22 In contrast, only a limited number of studies have reported on the potential associations of other viruses with T1D, including mumps, 24 cytomegalovirus, 25 rotavirus, 26 parechovirus, 27,28 Epstein-Barr virus, 29 rubella 12 and parvovirus. 30 In an effort to alleviate this bias towards EVs, a growing number of studies are applying high-throughput metagenomic next-generation sequencing (mNGS) to comprehensively characterise the population of all known human viruses (the 'virome'), simultaneously. Here, we report the first systematic review and meta-analysis of observational studies using mNGS to investigate vertebrate-infecting DNA and RNA viruses in children ≤18 years, and subsequent development of IA or T1D. Analysis of bacteriophage has been excluded from this review.
The unbiased viral mNGS in early life and childhood has potential to comprehensively identify diabetogenic viruses increasing the IA/T1D risk or viruses affording protection. This may present new opportunities to intervene through antiviral medications or vaccination.

| Search strategy and selection criteria
This review is registered on PROSPERO (23 July 2020), registration number CRD42020188737. Two reviewers (Clare L. Faulkner and Yi Xuan Luo) independently conducted a systematic search for observational studies investigating the association between virome composition and/or abundance, and IA or T1D. EMBASE and MED-LINE databases were searched (2000-1 June 2020) using the strategy in Appendix S1. The search was performed without geographical or language restrictions and limited to studies in humans. Restriction to studies published from year 2000 onwards was informed by emergence of mNGS and other viral sequencing technologies. 31,32 This search was supplemented by manual searching of references of identified papers, key journals, OpenGrey and ProQuest to identify additional articles potentially missed by online indexes. PROSPERO was interrogated to confirm no recent/ongoing systematic reviews.
Eligible studies were observational (cohort, case-control and nested case-control; including letters or abstracts), using mNGS to characterise the virome in any clinical specimen in children aged ≤18 years who developed IA and/or T1D. Age restriction was imposed because IA often develops in childhood, suggesting viruses exert influence early in life. IA was defined as persistence of one or more autoantibodies against T1D-associated autoantigens (IAA, GADA, IA2A, ICA and ZnT8A) in ≥2 time-separated consecutive samples.
Transplacental autoantibodies were excluded, defined as transient presence of the same autoantibody in a child <18 months and his/her mother. T1D was defined using American Diabetes Association criteria. 33 Eligible studies were categorised into two groups based on the outcome: IA or T1D. Data were extracted on vertebrate-infecting viruses only, excluding studies that only analysed bacteriophage.  We used both fixed-and random-effects models; only results from random-effects models are presented due to heterogeneity of study populations. Statistical heterogeneity was explored using Cochrane's Q Test (χ 2 ) and the I 2 statistic, which indicate the proportion of variance of the summary effect attributable to between-study heterogeneity. A p < 0.10 was considered a statistically significant heterogeneity, while I 2 ≤ 25% and ≥75% were deemed low and high heterogeneity, respectively. Subgroup analyses were performed for geographical location, stool versus plasma, consecutive virus shedding and studies using comparable detection thresholds, and pooled ORs were calculated. Sensitivity and influence analyses were conducted by the study size. Data analysis was completed in Review Manager, Version 5.4 (Cochrane Collaboration), 36   Our systematic review is reported using meta-analysis of observational studies in epidemiology and Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.

| RESULTS
The search returned 778 publications (51 duplicates), leaving 727 articles for review. Title and abstract screening identified 12 publications for full-text review. Four were excluded: three were repeat data sets [37][38][39] ; one used targeted polymerase chain reaction (PCR) rather than pre-specified mNGS. 40 Eight were included-one abstract, 41 Tables 1 and 2). One IA study 48 with insufficient data was excluded from meta-analysis.

| Study characteristics
Six studies defined IA as positivity for ≥1 T1D-associated autoantibody; two defined as ≥2 autoantibodies (

| Quality of evidence
The NOS scores were ≥8 (

| Islet autoimmunity
Seven studies investigated vertebrate-infecting viruses and IA. No

| Type 1 diabetes
Two studies 41

| Strengths and weaknesses
To minimise bias, we implemented pre-defined eligibility criteria, screening by independent reviewers, no language restrictions and sources beyond indexed databases. Random-effects models may have provided more conservative effect estimates by accounting for study population heterogeneity and generating wider confidence intervals. 64 Indeed in most viral mNGS datasets, more than 50% of sequences exhibit no detectable sequence similarity to known reference sequences, contributing to the viral 'dark matter'. 80 It is plausible that these may include highly divergent or completely novel viruses that have yet to be discovered. 81   development. 12