High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.

A large variation in the severity of disease symptoms is one of the key open questions in coronavirus disease 2019 (COVID-19) pandemics. The fact that only a small subset of people infected with severe acute respiratory syndrome coronavirus 2 develops severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the immunoglobulin G (IgG) glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of interindividual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting N-acetylglucosamine in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing.

Research on post-vaccination antibody dynamics has become pivotal in estimating COVID-19 vaccine efficacy. We studied anti-SARS-CoV-2 Spike RBD IgG levels in 587 healthcare workers (2038 sera) who completed BNT162b2 vaccination. Average antibody titer 3 weeks after the first dose in COVID-19-naïve participants (median 873.5 AU/mL) was 18-fold higher than the test threshold, with a significant increase 1 month (median 9927.2 AU/mL) and an exponential decrease 3 (median 2976.7 AU/mL) and 6 (median 966.0 AU/mL) months after complete vaccination. Participants with a history of COVID-19 prior to vaccination showed significantly higher antibody levels, particularly after the first dose (median 14,280.2 AU/mL), with a slight decline 1 month (median 12,700.0 AU/mL) and an exponential decline in antibody titers 3 (median 4831.0 AU/mL) and 6 (median 1465.2 AU/mL) months after vaccination. Antibody levels of COVID-19-naïve subjects after the first dose were moderately correlated with age (r = −0.4). Multivariate analysis showed a strong independent correlation between IgG levels 6 months after vaccination and both IgG titers after the first dose and 1 month after vaccination (R² = 0.709). Regardless of pre-vaccination COVID-19 history, IgG levels 6 months after vaccination were comparable to antibody levels reached by COVID-19-naïve participants after the first vaccine dose.

Tick-borne diseases are a serious threat to both public and veterinary health. In this study, we used high-throughput sequencing to characterize the virome of three tick species implicated in the spread of vector-borne disease throughout Croatia. Ten viruses were identified, including seven potential novel species within the viral families Flaviviridae, Nyamiviridae, Rhabdoviridae, Peribunyaviridae, Phenuiviridae, and Nairoviridae.