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Maternal-Fetal immune networks and viral signatures in the healthy amniotic cavity

The intrauterine environment has traditionally been viewed as a privileged site protected by the placental barrier. However, emerging evidence suggests that early in utero microbial exposure may prime the developing fetal immune system. Here, using target-enriched metagenomics and high-dimensional proteomics, we characterized the intra-amniotic viral landscape and immune networks in 114 healthy pregnancies including both normal and anomalous fetuses. We identify a sparse yet heterogeneous human viral signature in 26% of samples, predominantly composed of Herpesviridae, Polyomaviridae, and Picornaviridae. Although viral reads abundance was associated with fetal abnormalities, viral detection generally did not induce overt inflammatory activation, supporting a state of immune homeostasis within the amniotic cavity. Instead, viral presence was associated with subtle and selective immune modulation, including altered inducible antimicrobial peptide expression (HBD-2 and HBD-3), coupled with an attenuation of regulatory cytokines. Our results further reveal that the amniotic immune environment is primarily governed by gestational age, transitioning from a Th1-predominant "alert" phase to innate-readiness preceding parturition. These findings suggest that fragments of viral genetic material within the amniotic cavity may contribute to fetal immune instruction without triggering overt inflammation, providing a foundational framework for understanding how "silent" viral-exposure during gestation influences the developmental origins of neonatal immunity.

"Multiplex RT-PCR for SARS-CoV-2 variant surveillance in resource-limited settings: an in-house validation study in Cuba"

Background SARS-CoV-2 genomic surveillance is vital for public health, but whole-genome sequencing (WGS) remains costly and inaccessible in many resource-limited settings. We developed and validated a multiplex real-time RT-PCR assay for rapid, economical detection of key mutations associated with variants of interest (VOI) and concern (VOC). Methodology Two multiplex mixes (M1, M2) targeting eight mutations in the ORF1a and Spike genes were designed. Analytical validation included sensitivity, specificity, reproducibility, and limit of detection (LoD) using WHO international standards and a respiratory pathogen panel. In parallel, an in silico analysis evaluated oligonucleotide efficacy against 10.4 million SARS-CoV-2 genomes from GISAID/NCBI, assessing inclusivity, target-site secondary structure (RNAalifold), and hybridization energy (Primer3Plus). Results The assay demonstrated 100% clinical sensitivity among samples with valid RT-PCR results (41/42 samples yielded interpretable results, with one inhibited sample excluded from sensitivity calculation), a LoD of 5.7 log10 IU/mL, and 100% analytical specificity against 32 non-SARS-CoV-2 respiratory pathogens. Six out of eight oligonucleotide sets showed >96% inclusivity; two sets exhibited reduced inclusivity (94.03%, 90.14%) and structural features potentially affecting binding against emerging variants. The assay enables direct identification of major VOCs (Alpha, Beta, Gamma, Delta, Omicron) and indirect detection of multiple VOIs (P.2, Epsilon, Kappa, Eta, Iota, Lambda). Conclusion This standardized multiplex assay provides a rapid, sensitive, and low-cost alternative for SARS-CoV-2 variant surveillance in Cuba and similar settings. The integration of experimental and in silico validation offers a robust, adaptable framework to sustain diagnostic accuracy amid viral evolution, optimizing the allocation of scarce sequencing resources.

Accounting for allelic diversity and multicopy gene detection improves the accuracy of antibiotic resistance genotypic determination

Background Genomic prediction of antimicrobial resistance (AMR) relies on the accurate detection of resistance genes or allelic variants of core genes from raw or assembled genomes sequences. For several bacterial species and antibiotics, AMR genotype-phenotype discrepancies are common, indicating that important sources of error remain unresolved. For Enterococcus faecium, we focused on identifying the sources of discrepancies for tetracycline resistance, for which genotypic detection had shown particularly low accuracy. We investigated the effect of structural variation in antibiotic resistance genes (ARGs), including gene duplications, truncations, interruptions, and mixed configurations of complete and partial gene copies, as a source of genotype-phenotype discrepancies from short-read data. We conduct further extended investigations to other antibiotic families and into another bacterial species: Escherichia coli. Methods We analyzed collections of E. faecium and E. coli genomes, integrating high-quality complete assemblies, simulated Illumina short reads, and matched AMR phenotypic data. The integrity, copy number, and allelic diversity of ARGs were examined for multiple antibiotic classes, and their impact on ARG detection and accuracy of AMR determination was assessed using several commonly used bioinformatic tools (SRST2, ARIBA and AMRFinderPlus). Results For E. faecium, after ruling out the effect of specific tet allelic variants on tetracycline susceptibility, we found that the integrity and copy number of tet(M) had a major effect on detection accuracy. Duplicated and incomplete ARGs are also common in E. faecium genomes, particularly for macrolides (erm(B)) and aminoglycosides (ant(6)-Ia and aph(3')-IIIa). In E. coli, similar patterns were observed for tet(A), erm(B) and aminoglycoside-associated genes (aph(3')-IIIa and ant(6)-Ia). Across ARGs in both species, short-read mapping methods wrongly reported interrupted genes as complete in some instances, while assembly-based methods often failed to resolve complete copies of duplicated genes. Detection accuracy improved when tools were adapted to account for gene integrity and when extended AMR databases incorporating species-specific alleles were included. Conclusions Our findings reveal that bioinformatic limitations in dealing with ARG copy number and completeness, and in accounting for allelic variation, underly a substantial source of genotype-phenotype errors, highlighting the need for improved AMR databases and bioinformatic tools that consider these factors to achieve reliable genomic prediction of AMR.