SARS-CoV-2 genome annotation revealed the presence of 10 open reading frames (ORFs), of which the last one (ORF10) is positioned downstream of the N gene. It is a hypothetical gene, which was speculated to encode a 38 aa protein. This hypothetical protein does not share sequence similarity with any other known protein and cannot be associated with a function. While the role of this ORF10 was proposed, there is growing evidence showing that the ORF10 is not a coding region. Here, we identified SARS-CoV-2 variants in which the ORF10 gene was prematurely terminated. The disease was not attenuated, and the transmissibility between humans was maintained. Also, in vitro, the strains replicated similarly to the related viruses with the intact ORF10. Altogether, based on clinical observation and laboratory analyses, it appears that the ORF10 protein is not essential in humans. This observation further proves that the ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended.

Forensic microbiology is a subject of interest and research development across the forensic community. Studies of pathogen outbreaks, biocrime or bioterrorism attacks, and analyses of crime scenes fall within this field. Significant progress has been made in evolving DNA sequencing technologies in recent decades. These newly emerged transformative tools have become available to both biomedicine and forensics. Based on the published literature, this review provides an overview of the current trends and developments of NGS (next-generation sequencing) technologies applied to forensic microbiology. These new methods present numerous advantages over traditional sequencing technology and are useful in several practical applications within this forensic field. This article then examines the main challenges and limitations of this technology in forensics, providing a comprehensive review of NGS technology capabilities in expanding the precision and effectiveness of microbial forensic investigations—with the aim of inspiring scientists, forensic experts, lawyers, public health professionals, and policymakers alike to approach this newly powerful sequencing tool appropriately.

The endophytic Basidiomycete Sporobolomyces ruberrimus protects its host Arabidopsis arenosa against metal toxicity. Plants inoculated with the fungus yielded more biomass and exhibited significantly fewer stress symptoms in medium mimicking mine dump conditions (medium supplemented with excess of Fe, Zn and Cd). Aside from fine-tuning plant metal homeostasis, the fungus was capable of precipitating Fe in the medium, most likely limiting host exposure to metal toxicity. The precipitated residue was identified by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), X-Ray Diffraction (XRD) and electron microscopy (SEM/TEM) with energy dispersive X-Ray analysis (EDX/SAED) techniques. The performed analyses revealed that the fungus transforms iron into amorphous (oxy)hydroxides and phosphates and immobilizes them in the form of a precipitate changing Fe behaviour in the MSR medium. Moreover, the complexation of free Fe ions by fungi could be obtained by biomolecules such as lipids, proteins, or biosynthesized redox-active molecules.

Background/Objectives: Patients receiving maintenance hemodialysis (HD) commonly exhibit malnutrition, chronic low-grade inflammation, and metabolic disturbances. Gut dysbiosis may contribute to these abnormalities through the gut-kidney axis. This study aimed to characterize gut microbiota composition in HD patients and examine its associations with demographic, clinical, and body composition parameters. Methods: This cross-sectional study included 96 patients with end-stage kidney disease on maintenance HD. Clinical, laboratory, inflammatory, nutritional, and bioimpedance-derived body composition data were collected, including Malnutrition-Inflammation Score (MIS). Stool samples were analyzed for gut microbiota composition. Associations with host-related variables were assessed using alpha- and beta-diversity analyses, subgroup comparisons, and Mantel testing. Results: Gut microbiota showed marked inter-individual heterogeneity at the genus level, with dominant taxa including Blautia, Faecalibacterium, Streptococcus, Gemmiger, Ruminococcus, Escherichia-Shigella, and Enterococcus. Chao1 richness was higher in men than in women. Shannon entropy and Chao1 richness were positively associated with age and visceral adipose tissue (VAT), while Faith’s phylogenetic diversity increased with age. In contrast, the Gini index was negatively associated with age and VAT, indicating a more even microbial structure in older individuals and those with higher visceral adiposity. Beta-diversity differed between men and women and across categories within the female subgroup. Mantel testing showed a modest but significant correlation between microbiota and metadata distances. Conclusions: Gut microbiota in maintenance HD patients is highly heterogeneous and associated mainly with sex, age, visceral adiposity, and overall host phenotype. These findings suggest that microbiota variation in HD reflects multidimensional host-related factors rather than a single clinical feature.

Background/Objectives: Patients receiving maintenance hemodialysis (HD) commonly exhibit chronic low-grade inflammation, nutritional disturbances, altered body composition, and metabolic imbalance. Gut dysbiosis may contribute to these abnormalities through the gut–kidney axis; however, the relationship between the gut microbiota composition and host phenotype in HD patients remains incompletely characterized. This study aimed to characterize the gut microbiota composition in maintenance HD patients and assess its cross-sectional associations with demographic, inflammatory, nutritional, dialysis-related, and bioimpedance-derived body composition parameters. Methods: This single-center cross-sectional study included 96 patients with end-stage kidney disease undergoing maintenance HD. The primary objective was to characterize the gut microbiota composition in maintenance HD patients. Secondary objectives were to assess cross-sectional associations with demographic factors (sex, age) and bioimpedance-derived body composition (specifically VAT). Clinical and laboratory data, inflammatory markers, nutritional indicators, malnutrition–inflammation score (MIS), dialysis-related variables, and bioimpedance-derived body composition parameters were collected. Stool samples were analyzed using full-length 16S rRNA sequencing. The gut microbiota composition was assessed using taxonomic profiling, alpha-diversity and beta-diversity analyses, subgroup comparisons, and exploratory distance-based analyses. Associations were interpreted within a descriptive and hypothesis-generating framework. Results: The gut microbiota composition showed marked inter-individual heterogeneity at the genus level, with dominant taxa including Blautia, Faecalibacterium, Streptococcus, Gemmiger, Ruminococcus, Escherichia-Shigella, and Enterococcus. Chao1 richness was higher in men than in women. Shannon entropy and Chao1 richness were positively associated with age and visceral adipose tissue (VAT), while Faith’s phylogenetic diversity increased with age. In contrast, the Gini index was negatively associated with age and VAT, indicating a more even microbial community structure in older individuals and in those with higher visceral adiposity. Beta-diversity analyses suggested modest differences in microbial community structure according to sex and selected body composition-related categories, particularly in sex-stratified analyses. Exploratory distance-based analysis showed a modest association between overall microbiota dissimilarity and host phenotype dissimilarity, although this finding was limited by reduced sample overlap. Conclusions: The gut microbiota composition in maintenance HD patients was highly heterogeneous and showed cross-sectional associations, mainly with sex, age, visceral adiposity, and broader host phenotype. These findings suggest that microbiota variation in HD reflects multidimensional demographic, inflammatory, nutritional, metabolic, and body composition-related factors rather than a single clinical determinant. Larger longitudinal studies integrating standardized dietary, medication, metabolic, and clinical outcome data are needed to determine the prognostic relevance of these microbiota patterns.

Importance: SARS-CoV-2 genomic variants impacts the overall sensitivity of COVID-19 diagnosis, leading to false-negative diagnosis and the continued spread of the virus. Objective: To evaluate how nucleotide variability in target primer binding sites of the SARS-CoV-2 genomes may impact diagnosis using different recommended primer/probe sets, as well as to suggest the best primer/probes for diagnosis. Design: We downloaded 105,118 public SARS-CoV-2 genomes from GISAID (Sept, 25th, 2020), removed genomes of apparent worst quality (genome length 5% ambiguous bases) and missing metadata, and performed an analysis of complementarity for the 13 most used diagnostic primers/probe sets for RT-PCR detection. We calculated the N rate and % of genome recovery, with all primer/probe-sets considering viral origin and clade. Results: Our findings indicate that currently, the Paris_nCoV-IP2, -IP4 and WHO|E_Sarbeco primer/probe sets for COVID-19, to perform the best diagnostically worldwide, recovering >99.5% of the good quality SARS-CoV-2 genomes from GISAID, with no mismatches. The Chinese_CDC|2019-nCoV-NP primer/probe set, among the first to be designed during the pandemic, was the most susceptible to currently most abundant SARS-CoV-2 variants. Mismatches encompassing the binding sites for this set are more frequent in Clade-GR and are highly prevalent in over 30 countries globally, including Brazil and India, two of the hardest hit countries. Conclusions: Detection of SARS-CoV-2 in patients may be hampered by significant variability in parts of the viral genome that are targeted by some widely used primer sets. The geographic distribution of different viral clades indicates that continuous assessment of primer sets via sequencing-based surveillance and viral evolutionary analysis is critical to accurate diagnostics. This study highlights sequence variance in target regions that may reduce the efficiency of primer:target hybridization that in turn may lead to the undetected spread of the virus. As such, due to this variance, the Chinese_CDC|2019-nCoV-NP-set should be used with caution, or avoided, especially in countries with high prevalence of the GR clade. Key Points: Question: How variable are the binding-sites of primers/probes used for COVID-19 diagnosis? Findings: We investigated nucleotide variations in primer-binding sites used for COVID-19 diagnosis, in 93,143 SARS-CoV-2 genomes, and found primer sets targeting regions of increasingly nucleotide variance over time, such as the Chinese_CDC|2019-nCoV-NP. The frequency of these variations is higher in Clade-GR whose frequency is increasing worldwide. Paris_nCoV-IP2, IP4 and WHO|E_Sarbeco performed best. Meaning: We suggest the use of some sets to be halted and reinforce the importance of a continuous surveillance of SARS-CoV-2 variations to prompt the use of the best primers.