Stefan A. Boers

The majority of SARS-CoV-2 genomes obtained during the pandemic were derived by amplifying overlapping windows of the genome (‘tiled amplicons’), reconstructing their sequences and fitting them together. This leads to systematic errors in genomes unless the software is both aware of the amplicon scheme and of the error modes of amplicon sequencing. Additionally, over time, amplicon schemes need to be updated as new mutations in the virus interfere with the primer binding sites at the end of amplicons. Thus, waves of variants swept the world during the pandemic and were followed by waves of systematic errors in the genomes, which had significant impacts on the inferred phylogenetic tree. Here we reconstruct the genomes from all public data as of June 2024 using an assembly tool called Viridian ( https://github.com/iqbal-lab-org/viridian ), developed to rigorously process amplicon sequence data. With these high-quality consensus sequences we provide a global phylogenetic tree of 4,471,579 samples, viewable at https://viridian.taxonium.org . We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny.

This study investigates a diagnostic challenge in detecting Streptococcus pneumoniae using a Spn9802-targeted PCR assay. Four S. pneumoniae isolates tested lytA-positive but Spn9802-negative due to an insertion sequence (IS1515) within Spn9802, which was confirmed by whole-genome sequencing. This insertion may lead to false-negative results, posing concerns for PCR-based diagnostics. The study emphasizes the need for dual-target PCR and regular updates to diagnostic primers, underscoring the importance of continuous assay evaluation and post-market surveillance to ensure reliable pathogen detection.

The detection and identification of bacterial species in clinical samples are crucial for patient management and antibiotic treatment. When culture-based identification methods fail, 16 S rRNA gene next-generation sequencing (NGS) serves as a valuable alternative. However, its clinical utility is often limited by prolonged time to results (TtR) and limited species-level resolution. This study aimed to develop and validate a faster, more discriminative 16 S rRNA gene NGS workflow. Our current 16 S rRNA gene NGS protocol uses micelle-based PCR (micPCR) targeting the V4 region, followed by Illumina sequencing. This method ensures accurate quantification of 16 S rRNA gene copies in (low biomass) clinical samples by reducing PCR artefacts and correcting for background DNA contamination. To shorten the TtR and improve species-level determination, the micPCR protocol was adapted to amplify full-length 16s rRNA genes, followed by nanopore sequencing using the Flongle Flow Cell with automated data analysis using the Genome Detective platform. Testing with a synthetic microbial community and six clinical samples showed that the 16 S rRNA gene micPCR/nanopore sequencing protocol maintains good accuracy and sensitivity, reducing TtR to 24 h and enhancing species-level resolution. This optimized workflow improves clinical diagnostics, making it a valuable tool for guiding patient treatment decisions.

In 2022, a cluster of severe childhood hepatitis was detected primarily in Europe and North America, leading to a global alert by the World Health Organization. An association with adeno-associated virus 2 (AAV2) in conjunction with human adenoviruses was found. Five percent of the cases progressed to acute liver failure, necessitating transplantation. The mechanism of disease that accounts for fulminant liver failure in these patients remains incompletely described. An upsurge was observed of in the five total cases of acute liver failure that presented to the Dutch national referral center for pediatric liver transplantation in the spring of 2022. An in-depth molecular analysis of the mechanism of pediatric acute liver failure was performed using targeted transcriptomics and metagenomics to identify any virus present in the cases, immune profile haplotypes, and differentially expressed gene groups. Explanted liver tissue and plasma samples ( n = 15) were subjected to viral metagenomic and human transcriptomic profiling, targeting >600 inflammatory genes. Liver transcriptomic signatures of transplanted cases were compared with those of pediatric controls from a liver biobank ( n = 6). AAV2, adenoviruses, and herpesviruses were detected in liver explant tissue and plasma samples of the cases. Epstein-Barr virus and varicella zoster virus infection with pathognomonic clinical symptomatology preceded liver failure in two respective cases. AAV2 was detected in one-third of control livers. Excessive activation of monocyte pathways was detected in liver explants from cases compared with controls. Remarkably, this signature was comparable for AAV2, adenoviruses, and/or herpesviruses-positive transplant cases. Our multi-omic findings suggest a common transcriptomic profile, with an upregulation of monocyte pathways in the presented transplanted cases, which had similar severe clinical outcomes. In the cohort presented, AAV2 was not exclusively associated with acute liver failure, suggesting that other processes may have contributed to a uniform cascade of irreversible pathology. IMPORTANCE: Since the appearance of the cluster of pediatric hepatitis of unknown origin in 2022, several groups have reported an association of adenoviruses and AAV2 in a high number of cases in contrast to controls. The adenoviruses detected were heterogeneous in both species—adenovirus C and F—and sequences. The mechanisms of disease that accounts for fulminant liver failure, occurring in 5% of pediatric hepatitis cases, remain incompletely described. The current study adds to previous data by including pediatric acute liver failure cases during the upsurge, enabling the analyses of inflammation expression profiles in cases with different viruses in relation to pediatric controls. This led to the discovery of transcriptome upregulation of monocyte pathways in liver explants from the cases. This inflammatory transcriptomic signature was comparable for AAV2, adenoviruses, and/or herpesviruses-positive transplant cases.

Viral enrichment by probe hybridization has been reported to significantly increase the sensitivity of viral metagenomics. This study compares the analytical performance of two targeted metagenomic virus capture probe-based methods: (i) SeqCap EZ HyperCap by Roche (ViroCap) and (ii) Twist Comprehensive Viral Research Panel workflow, for diagnostic use. Sensitivity, specificity, and limit of detection were analyzed using 25 synthetic viral sequences spiked in increasing proportions of human background DNA, eight clinical samples, and American Type Culture Collection (ATCC) Virome Virus Mix. Sensitivity and specificity were 95% and higher for both methods using the synthetic and reference controls as gold standard. Combining thresholds for viral sequence read counts and genome coverage [respectively 500 reads per million (RPM) and 10% coverage] resulted in optimal prediction of true positive results. Limits of detection were approximately 50–500 copies/mL for both methods as determined by ddPCR. Increasing proportions of spike-in cell-free human background sequences up to 99.999% (50 ng/mL) did not negatively affect viral detection, suggesting effective capture of viral sequences. These data show analytical performances in ranges applicable to clinical samples, for both probe hybridization metagenomic approaches. This study supports further steps toward more widespread use of viral metagenomics for pathogen detection, in clinical and surveillance settings using low biomass samples. IMPORTANCE: Viral metagenomics has been gradually applied for broad-spectrum pathogen detection of infectious diseases, surveillance of emerging diseases, and pathogen discovery. Viral enrichment by probe hybridization methods has been reported to significantly increase the sensitivity of viral metagenomics. During the past years, a specific hybridization panel distributed by Roche has been adopted in a broad range of different clinical and zoonotic settings. Recently, Twist Bioscience has released a new hybridization panel targeting human and animal viruses. This is the first report comparing the performance of viral metagenomic hybridization panels.