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Results for “amplicon sequencing”

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PubMed2006

Co-circulation of genotype IA and new variant IB hepatitis A virus in outbreaks of acute hepatitis in Hungary--2003/2004.

Hepatitis A virus (HAV) is one of the most important causes of acute infectious hepatitis worldwide. In Hungary, the reported number of HAV infections has been decreasing in the last four decades, nevertheless, still, each year 500-800 new cases and multiple outbreaks occur, particularly in the northeast region of Hungary. In Hungary, serology is used routinely to establish the diagnosis of HAV infection without genetic analysis of HAV strains for molecular epidemiology. In this study, serum samples collected from symptomatic patients were tested by enzyme-immunoassay (anti-HAV-IgM ELISA) to establish the cause of three acute hepatitis A outbreaks (outbreak 1--from low prevalence region in Southwest Hungary in 2003 and outbreaks 2 and 3 from the endemic region in Northeast Hungary in 2004). Outbreak strains were characterized by reverse transcription-polymerase chain reaction (RT-PCR) amplification of a 360 bp viral VP1/2A region, amplicon sequencing and phylogenetic analysis. Four, seven, and three sera from outbreaks 1, 2, and 3, respectively, were investigated by RT-PCR for HAV genome and HAV RNA was detected in 4 (100%), 4 (57%), and 2 (67%) samples. All strains belonged to genotype I HAV. Outbreak 1 was caused by the new variant subtype IB and outbreaks 2 and 3 caused by genetically identical subtype IA strains. The Hungarian IA and IB hepatitis A viruses had the highest nucleotide identity, 98.4% and 99.0%, to IT-SCH-00 and IT-MAR-02 strains, respectively, detected in year 2000 and 2002 in Italy. Endemic subtype IA and probably imported new variant subtype IB HAV viruses was detected in outbreaks of hepatitis in Hungary that are closely related genetically to HAV strains in Italy.

Acute Disease↗

PubMed2025

Microbiological Investigations for Chikungunya Virus in Children With Acute Encephalitis Syndrome in a Non-Outbreak Setting in Southern India.

Chikungunya virus (CHIKV) is an emerging cause of acute encephalitis syndrome (AES) in India, with limited data on its role in childhood AES in southern India. We systematically evaluated children with AES in southern India during a non-epidemic period for CHIKV. Serum and cerebrospinal fluid (CSF) samples were tested for CHIKV using IgM ELISA and real-time reverse transcriptase PCR. Amplicon sequencing was performed on PCR-positive samples. Clinical and laboratory features were compared between children with and without CSF CHIKV positivity (PCR/IgM antibodies). Of 376 children with AES, 20 (5.3%) had positive CHIKV tests. Co-infections were common, particularly with scrub typhus. Children presented with diverse symptoms affecting various organ systems. Neurological manifestations included meningism, seizures, cerebellar signs, behavioral abnormalities, cranial nerve involvement, involuntary movements, and hemiparesis/hemiplegia. Children with CSF CHIKV positivity showed more focal neurological deficits and transaminitis, and less musculoskeletal symptoms. Sequencing confirmation of CHIKV was made in all patients with positive CHIKV PCR, revealing a close relationship with 2016 Kenyan and Indian strains, albeit in a different clade within the East/Central/South African genotype. Along with important mutations known to impact CHIKV infectivity, four novel amino acid substitutions were detected in envelope protein coding regions. Our findings underscore the importance of routine and comprehensive CHIKV testing for children with AES, irrespective of season/outbreak. The high rate of co-infections warrants further research. Continued genomic surveillance is essential to monitor emerging mutations with epidemic potential, increased severity and the risk of neurological disease.

Humans↗

PubMed2000

Diagnostic value of cytokine assays in cerebrospinal fluid in culture-negative, polymerase chain reaction-positive bacterial meningitis.

Analysis of bacterial DNA using a polymerase chain reaction performed with broad-range eubacterial 16S rDNA primers may yield a diagnosis of bacterial meningitis in cases where Gram staining of cerebrospinal fluid (CFS), antigen detection techniques or culture fail. Since this PCR technique occasionally gives false-positive results due to contamination of samples or laboratory reagents, a study was performed to establish the diagnostic value of assaying concentrations of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in 90 CSF samples. A high correlation was found between a positive PCR result and the concentrations of TNF-alpha and IL-10, indicating that cytokine assays may be used as a confirmatory test. The findings suggested that a combination of the PCR technique, amplicon sequencing and assay of TNF-alpha and IL-10 concentrations in CSF is a reliable and cost-effective procedure for diagnosis of culture-negative bacterial meningitis.

Bacteria↗

PubMed2026

Heavy-metal stress shapes habitat-specific microbial survival strategies in estuarine environments.

Estuarine ecosystems face increasing heavy metal pollution from rapid urbanization and industrialization, yet the microbial adaptive strategies to multiple metal stressors across different habitats remain poorly understood. This study investigated the diversity and composition of bacterial and fungal communities across free-living (FL), particle-attached (PA), and sediment (SE) fractions from three estuaries with varying heavy metal contamination, and further investigated functional adaptations of bacterial communities. High-throughput amplicon sequencing revealed habitat-specific communities, with SE hosting the highest alpha diversity and enrichment of metal-resistant genera such as Woeseia and Sva1033. Environmental filtering, particularly by Zn, was the dominant driver shaping bacterial assemblages across all habitats, whereas fungal communities displayed greater stochastic assembly patterns. Analysis of 44 high-quality bacterial metagenome-assembled genomes (MAGs) revealed diverse metal resistance genes (cusA, znuB, and zntA), along with enriched metabolic pathways for carbon, nitrogen, and sulfur cycling. Notably, both active efflux/oxidative stress defense and indirect immobilization mechanisms were observed across all habitats, but their relative importance differed: FL and PA communities exhibited a greater reliance on active metal efflux (czcAB) and oxidative stress defense (trxAB) to maintain intracellular homeostasis, whereas SE communities displayed a stronger genomic potential for sulfate reduction (dsrAB) that may contribute to metal immobilization through sulfide precipitation. This metabolic partitioning highlights the complementary roles of different habitats in mediating metal toxicity and biogeochemical cycling, providing new insights into microbial resilience in polluted estuaries and underscoring the urgency of addressing heavy-metal contamination in these critical ecosystems.

Estuaries↗

PubMed2026

Climate and soil shape Daqu wheat quality and seed microbiome via rhizosphere taxa and microbial assembly.

The grain quality and seed microbiome of Daqu wheat are fundamental determinants of Daqu fermentation performance; however, the mechanisms by which cultivation environments influence these traits via rhizosphere microbial communities remain unclear. Bacterial and fungal communities across the bulk soil-rhizosphere-seed continuum of three wheat cultivars grown in four ecoregions were characterized using absolute quantitative amplicon sequencing. The rhizosphere microbiome was treated as a central intermediary, while the response variables were seed microbial diversity and grain-quality traits, including starch content, protein content, and grain hardness. Twelve physicochemical properties of soil and 11 climatic factors were integrated into a multidimensional association framework. Environmental conditions exerted stronger influences on both seed quality traits and microbial diversity than cultivar identity. Distinct regional signatures were also evident in rhizosphere microbiomes, with environmental gradients explaining community variation more effectively than geographic distance. Bacterial communities exhibited greater sensitivity to environmental fluctuations than fungi. Mantel analyses identified available nitrogen, precipitation, and atmospheric pressure as significant drivers of core rhizosphere taxa (P < 0.05). iCAMP revealed that stochastic processes predominantly governed rhizosphere bacterial assembly, whereas stochastic and deterministic mechanisms jointly shaped fungal assembly. Partial least squares path modeling further uncovered a rhizosphere-mediated environment-seed cascade, wherein sunlight intensity and duration, atmospheric pressure, and soil nitrogen directly or indirectly affected seed wet gluten content, grain hardness, and seed microbial diversity through their influences on rhizosphere microbiota. Rhizosphere bacterial diversity was negatively associated with seed bacterial diversity (path coefficient = -0.118, P < 0.05), indicating that rhizosphere communities may shape seed endophytic bacterial assemblages via environmental filtering and competitive interactions. Collectively, these findings elucidate how environments shape the quality and seed microbiomes of Daqu wheat, providing scientific guidance for optimal site selection and the standardized production of high-quality brewing wheat for industrial Baijiu.

Triticum↗

PubMed2026

Depth-dependent microbial succession and interspecies hydrogen transfer drive pit mud maturation in Chinese strong-flavor baijiu fermentation.

Microbial communities in fermentation pit mud play a key role in determining the quality of Chinese strong-flavor baijiu (CSFB). However, the ecological processes underlying pit mud maturation across spatial and temporal scales remain unclear. In this study, amplicon sequencing and metagenomic analyses were employed to investigate the taxonomic succession, community assembly, and metabolic functions of bacterial and archaeal communities during the transition from fresh pit mud (FPM) to new pit mud (NPM) and old pit mud (OPM). A pronounced depth-dependent succession pattern was observed, with 4 cm representing a critical ecological boundary separating distinct community structures and maturation trajectories. During surface-layer maturation, community assembly shifted from stochastic to deterministic processes, accompanied by homogeneous selection and increasing network complexity. In contrast, stochastic processes remained dominant throughout deep-layer maturation. Metagenomic analyses revealed a functional transition from lactate and acetate production, primarily associated with Lactobacillus in FPM and NPM, to butyrate and caproate production associated with Clostridium and Caproiciproducens in OPM. This functional transition was accompanied by enhanced amino acid metabolism, which was associated with the enrichment of Proteiniphilum and Aminobacterium. Notably, methanogen-mediated interspecies hydrogen transfer (IHT) emerged as a key ecological feature during pit mud maturation. In OPM, IHT networks primarily involving Methanobacterium and Methanosarcina linked methanogenesis with reverse β-oxidation through diverse hydrogen-transfer pathways, reinforcing metabolic interactions underlying caproate production. These findings provide new insights into the ecological mechanisms underlying pit mud maturation and offer a theoretical basis for the directed cultivation of high-quality pit mud in CSFB production.

Hydrogen↗

PubMed2026

Spatially resolved multi-omics analysis of indigenous Bacillus-fortified high-temperature Daqu.

Layer-dependent patterns associated with indigenous Bacillus fortification on high-temperature Daqu remain unclear. Here, six indigenous functional Bacillus strains were combined to fortify Daqu at three inoculation levels (QH4, QH5, QH6), with non-fortified as the control (CK). Upper, middle, and lower shelf-layer samples were profiled by physicochemical measurements, volatilomics, organic acid analysis, untargeted metabolomics, 16S/ITS amplicon sequencing, and metagenomics. PERMANOVA showed significant effects of treatment, spatial layer, and their interaction on physicochemical, volatile, bacterial, and fungal profiles (P = 0.001). Among the three inoculation levels, QH5 showed the most balanced performance: QH5_M exhibited the highest observed mean peak temperature (63.3 °C; +4.5 °C relative to CK_M), and its group-mean temperature remained ≥ 60 °C for seven consecutive days. Multi-omics analyses indicated coordinated, non-linear, and layer-dependent differences associated with indigenous Bacillus fortification, with QH5_M showing the most pronounced combined thermal, pyrazine, substrate, microbial, and predicted functional profile. These findings indicate that moderate indigenous Bacillus fortification was associated with distinct layer-dependent thermal and flavor profiles and coordinated microbial, metabolic, and predicted functional differences.

Bacillus↗

PubMed2003

Genomics of the human Y-chromosome. 1. Association with male infertility.

The human Y chromosome contains over 60 million nucleotides, but least number of genes compared to any other chromosome and acts as a genetic determinant of the male characteristic features. The male specific region, MSY, comprising 95% of the Y chromosome represents a mosaic of heterochromatic and three classes of euchromatic (X-transposed, X-degenerate and ampliconic) sequences. Thus far, 156 transcription units, 78 protein-coding genes and 27 distinct proteins of the Y chromosome have been identified. The MSY euchromatic sequences show frequent gene conversion. Of the eight massive palindromes identified on the human Y chromosome, six harbor vital testis specific genes. The human male infertility has been attributed to mutations in the genes on Y chromosome and autosomes and failures of several physical and physiological attributes including paracrine controls. In addition, deletion of any one or all the three azoospermia (AZFa, AZFb or AZFc) factor(s) and some still unidentified regulatory elements located elsewhere in the genome result in infertility. Characterization of palindromic complexes on the long arm of Y chromosome encompassing AZFb and AZFc regions and identification of HERV15 class of endogenous retroviruses close to AZFa region have facilitated our understanding on the organization of azoospermia factors. Considerable overlap of the AZFb and AZFc regions encompassing a number of genes and transcripts has been shown to exist. However, barring details on AZF, information on the exact number of genes or the types of mutations prevalent in the infertile male is not available. Similarly, roles of sizable body of repetitive DNA present in close association with transcribing sequences on the Y chromosome are yet not clear. In a clinical setting with known cases of infertility, systematic search for loss or gain of these repeat elements would help understand their biological role(s). We present a brief overview on the genetic complexity of the human Y chromosome in the context of human male infertility.

Chromosomes, Human, Y↗

PubMed2026

Enrichment of Lysobacter in a long-term organically managed agricultural field with low soilborne disease incidence.

Disease-suppressive soils, in which soilborne pathogens are naturally suppressed, offer a promising model for sustainable crop protection, particularly in organic farming systems where chemical disease control options are limited. Although disease suppression in these soils is considered to rely on biological control, the underlying mechanisms remain poorly understood. In this study, we investigated soil from a long-term organically managed field in Shiga Prefecture, Japan, where soilborne disease incidence has remained consistently low, to identify bacterial community features potentially associated with this field. The 16S rRNA gene amplicon sequencing indicated that this soil harbored a bacterial community distinct from those of nearby agricultural soils. Following the application of organic compounds, the genus Lysobacter, a taxon with known antagonistic activity against plant pathogens, was markedly enriched in response to proteinaceous organic inputs. This enrichment was consistent across sampling times and specific to certain proteinaceous organic inputs, whereas minimal effects were observed on chitin, N-acetyl-d-glucosamine, or cysteine. Broader soil surveys indicated that Lysobacter enrichment was not strictly associated with whether soils had been managed under organic or conventional farming practices. Stepwise multiple regression analysis identified 10 co-occurring bacterial genera that were strongly associated with Lysobacter abundance. These findings highlight condition-dependent Lysobacter enrichment as a characteristic microbial response to proteinaceous organic amendments in this low-disease-incidence field and provide microbial insights that may inform microbiome-based strategies for sustainable soil management.

Lysobacter↗

PubMed2005

Norovirus genotypes causing gastroenteritis outbreaks in Finland 1998-2002.

BACKGROUND: Outbreak investigation methods for enteric viruses were improved in 1990s when gene amplification techniques were established in viral laboratories. OBJECTIVES: The objective of the study was to determine the causative agents for Finnish viral gastroenteritis outbreaks. Our aim was also to further characterise the norovirus strains, reveal the temporal occurrence of norovirus (NV) genotypes and to study some epidemiological aspects concerning the outbreaks. STUDY DESIGN: A total of 416 Finnish viral gastroenteritis outbreaks that occurred during 5 years (1998-2002), excluding those among hospitalised children, were investigated for enteric viruses. Stool samples were screened by electron microscopy as well as analyzed by specific noro- and astrovirus RT-PCR tests. Amplicon sequence analysis was used to find out norovirus genotypes. RESULTS: Noroviruses caused 252 (60.6%) of the outbreaks; other viruses, astro- or rotavirus, caused four epidemics. Norovirus epidemics occurred in all kinds of settings, most often in hospitals (30.6%) and in restaurants and canteens (14.3%). Both NV genogroups were found every year, but NV GGII outbreaks always outnumbered those of GGI. All but one outbreak at hospitals and nursing homes were of genotype GII. Polymerase sequence analysis revealed a variety of NV genotypes; six GI and at least eight GII genotypes. The GI.3 Birmingham-like and GII.4 Bristol-like genotype appeared every year, whereas the other types were circulating for shorter periods or sporadically. During the study period the genotypes GII.4 (Bristol), GII.1 (Hawaii), an emerging genotype GIIb, and a new variant of GII.4 predominated in that order. Indication for rapid genetic changes in the genotype GII.4 was also noticed. CONCLUSIONS: Noroviruses were the most prevalent causative agents in the outbreaks. Many NV genotypes were circulating, and a shift in the predominant genotypes was evident between epidemic seasons.

Caliciviridae Infections↗

PubMed2006

Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: a useful adjunct to conventional tissue-based diagnostic methods.

BACKGROUND: West Nile virus (WNV), a member of genus Flavivirus, causes febrile illness, encephalitis, meningitis, myelitis, and occasional deaths in humans. Although several reverse transcription-polymerase chain reaction (RT-PCR) assays have been developed for detection of WNV in serum, cerebrospinal fluid, and fresh tissues, the usefulness of WNV RT-PCR assays for RNA extracted from formalin-fixed human tissues has not previously been demonstrated. OBJECTIVE: The objective of this study was to evaluate the application of a RT-PCR technique for the detection of WNV in routinely processed, formalin-fixed, paraffin-embedded (FFPE) human tissues, and to compare it with conventional serology and immunohistochemistry (IHC). STUDY DESIGN: We performed two WNV-specific nested RT-PCR assays targeting the viral capsid, premembrane, and envelope genes in FFPE central nervous system tissue samples from 27 patients with fatal WNV encephalitis, as confirmed by serology or IHC, and compared the results. The presence of WNV in RT-PCR-positive samples was confirmed by amplicon sequencing. RESULTS: Twenty (74%) patients were WNV RT-PCR positive while 24 (89%) were seropositive. WNV IHC staining of neurons and neuronal processes was positive in fourteen (52%) patients. The concordance between IHC and serology was 41% (11/27) and between RT-PCR and serology was 63% (17/27). All 11 seropositive/IHC-positive patients and 6 (46%) of 13 seropositive/IHC-negative patients were RT-PCR positive while all 3 seronegatives were positive by both IHC and RT-PCR. CONCLUSIONS: In this study, RT-PCR was significantly more sensitive than IHC in detecting WNV infections and provided specific sequence information about the infecting virus. RT-PCR on FFPE tissues may be a particularly useful diagnostic tool in patients who die relatively soon after disease onset and for whom serology may be negative. Combined use of serology, IHC, and RT-PCR would be expected to have the best overall sensitivity and improve detection of fatal WNV infection.

Adult↗

PubMed2026

Restoration contexts shape the bacterial and fungal soil communities in desertification hotspots in the Brazilian semiarid region.

Desertification in the Brazilian semiarid has compromised ecosystem functionality, impacting soil microbial biodiversity. Thus, restoration strategies have been implemented, aiming to mitigate the negative impacts. However, little is known about their effects on soil microbial communities. In this study, we hypothesized that the two restoration contexts would promote distinct trajectories of soil microbial community recovery. We evaluated 36 soil samples collected from two desertification hotspots in the Brazilian semiarid, representing active (Gilbués) and passive (Irauçuba) restoration contexts. Soil DNA was extracted and subjected to 16S and ITS amplicon sequencing to characterize bacterial and fungal communities, respectively. Community differences were assessed using alpha-diversity metrics, redundancy analysis (RDA), and PERMANOVA. The results showed that within Gilbués (active restoration), bacterial and fungal community composition differed among soils under desertification and restoration. In Irauçuba (passive restoration), only native soils differed from both soils under desertification and restoration. Proteobacteria, Actinobacteriota, and Firmicutes (bacteria), and Ascomycota and Basidiomycota (fungi), were the dominant phyla in both hotspots. Bacterial and fungal communities showed distinct taxonomic patterns among native, degraded, and restored soils within each restoration context. Niche occupancy patterns also differed between restoration contexts. In conclusion, the two hotspots followed contrasting microbial recovery trajectories, demonstrating that restoration responses are context-dependent and vary according to the microbial groups, rather than supporting the universal superiority of one restoration strategy over the other.

Soil Microbiology↗

PubMed2026

Dynamics of antibiotic resistance genes co-occurrence with pathogenic and non-pathogenic bacteria throughout wastewater treatment processes.

Wastewater treatment plants (WWTPs) are recognized hotspots for antibiotic resistance genes (ARGs) and pathogenic bacteria. Despite advancements in treatment technologies, the persistence of ARGs and pathogenic bacteria remains a concern. In this study, we analyzed the dynamic changes in ARGs and bacterial communities throughout the treatment processes within an anaerobic-anoxic-oxic (AAO) WWTP over one week by using HT-qPCR coupled with 16S rRNA gene amplicon sequencing. The connectedness index, based on network analysis, showed that the dynamics of ARGs and mobile genetic elements (MGEs) were more strongly associated with potentially pathogenic bacteria than with non-pathogenic bacteria, suggesting that ARG immigration and dissemination in the WWTP were likely driven by potentially pathogenic taxa. The AAO treatment significantly reduced ARGs in final effluent (EF) (∼64 %) and residual sludge (RS) (∼81 %); however, potential hosts of ARGs such as Comamonas testosteroni and Clostridioides difficile persisted with minimal changes in relative abundance and remained detectable in EF and RS. Notably, the abundance of ARGs was lower in RS than in EF, and source tracking analysis identified influent as the primary source of ARGs and potentially pathogenic taxa in EF, underscoring the greater health risks associated with effluent discharge.

Wastewater↗

PubMed2004

Quantitation of hepatitis B lamivudine resistant mutants by real-time amplification refractory mutation system PCR.

BACKGROUND/AIMS: Lamivudine is an antiviral drug that is used to treat hepatitis B virus (HBV) infection. Long-term therapy does not completely suppress viral replication, and resistant mutants emerge. Resistance is mediated by changes in the tyrosine-methionine-apartate-aspartate (YMDD) motif in the catalytic site of the HBV polymerase gene. We describe a method to detect and quantify mutant viral populations using amplification refractory mutation system (ARMS) PCR. METHODS: We developed a real-time ARMS-PCR to detect point mutations in the polymerase gene. Using real-time PCR (LightCycler) with a ResonSense probe, PCRs were performed using clones of the HBV polymerase gene containing the different YMDD mutations. Dilution series of the templates were made and tested against each of the primer pairs. This method was applied to quantify mutant virus in patient serum samples. RESULTS: As little as 0.01% mutant DNA in 10(5)-10(9) copies wild-type DNA were detected. The method is more sensitive than amplicon sequencing, which is the current method of mutant determination in the YMDD motif. CONCLUSIONS: This study demonstrates a rapid, highly sensitive and reproducible method of quantifying mutant HBV virus in lamivudine treated patients. It can be used to monitor patients before and during lamivudine therapy.

Amino Acid Sequence↗

PubMed2006

Analysis of the near full length genomes of HIV-1 subtypes B, F and BF recombinant from a cohort of 14 patients in São Paulo, Brazil.

The human immune deficiency virus (HIV) exhibits strikingly tremendous amount of genetic variability. Such feature is critically important for the virus to adapt to environmental changes by escaping the host immune system and by escaping candidate vaccine. Therefore, understanding of such diversity is fundamental for the design of successful drugs or vaccine, which is urgently needed to bring the HIV/AIDS epidemic under control. In this study, we investigated the magnitude of diversity of the HIV-1 near full-length genomes from patients previously assigned as infected with non-recombinant HIV-1 subtypes B and F1 variants based on small portion of viral genome. HIV-1 proviral DNA was extracted from 14 samples previously classified in our laboratory as six subtypes B and eight subtypes F on the basis of small amplicon sequencing. Reamplifications of DNA from these samples were carried out by an overlapping PCR followed by direct sequencing. The data were phylogenetically inferred. Sequence analysis revealed that two out of six partially identified subtype B and six out of eight partially identified subtype F were in fact BF recombinants throughout their full genomes. Two pairs BF recombinants had identical genomic recombination structure and distinct from the Argentinean CRF 12_BF strains, probably represents a novel circulating recombinant forms in Brazil. Our data provided new genetic material of some of the HIV-1 subtypes currently circulating in the country and points to the widespread of BF recombinants which are expected to change the epidemic nature by approaching the level of subtype B in Brazil.

Adult↗

PubMed2026

Molecular epidemiology and phylogeographic architecture of oncogenic intracellular bacteria in cervical cancer patients across Northern China.

BACKGROUND: Oncogenic intracellular bacteria, including Chlamydia trachomatis, Mycoplasma genitalium, and Fusobacterium nucleatum, have emerged as significant contributors to cervical carcinogenesis. Despite growing interest in microbial oncology, the molecular epidemiological landscape and phylogeographic distribution of these pathogens in Northern China remain poorly characterized. This study aimed to determine the prevalence, co-infection patterns, genotypic diversity, and spatial phylogeographic clustering of oncogenic intracellular bacteria among cervical cancer patients across five provinces of Northern China. METHODS: A cross-sectional, multi-center study was conducted between March 2022 and November 2024 across Shaanxi, Heilongjiang, Beijing, Shandong, and Inner Mongolia. Cervical swab specimens were collected from 1247 confirmed cervical cancer patients. Pathogen detection was performed using multiplex real-time polymerase chain reaction, 16S rRNA gene amplicon sequencing, and whole-genome sequencing. Phylogeographic analyses employed maximum likelihood and Bayesian evolutionary inference frameworks. Statistical analyses included multivariate logistic regression and geographic information system-based spatial clustering. RESULTS: The overall prevalence of at least one oncogenic intracellular bacterium was 68.3% (n = 852). Chlamydia trachomatis was the most prevalent pathogen detected in 41.2% of participants. Co-infection with two or more bacteria was identified in 29.7% of cases and was independently associated with advanced-stage cervical cancer (adjusted odds ratio = 2.87; 95% confidence interval: 1.94 to 4.23; p < 0.001). Phylogeographic analysis revealed three distinct molecular clades with evidence of bidirectional gene flow between Shaanxi and Heilongjiang. Whole-genome sequencing identified 14 novel virulence gene variants not previously characterized in Chinese clinical isolates. CONCLUSIONS: Oncogenic intracellular bacteria are highly prevalent and genotypically diverse among cervical cancer patients in Northern China. The identified phylogeographic clustering and novel virulence variants have direct implications for regional screening programs, targeted antimicrobial strategies, and the development of region-specific molecular diagnostic panels.

Cervical cancer↗

PubMed2026

Simultaneous quantitative detection of multiple low-frequency variants by high-dynamic-range capillary electrophoresis.

Sensitive and quantitative detection of low-frequency variants across multiple loci is critical for nucleic acid-based diagnostics, yet clinical implementation requires a balance among sensitivity, multiplexing capacity, cost, and operational simplicity. We previously developed a high-dynamic-range capillary electrophoresis system capable of detecting variants at allele frequencies below 1%; however, its application was limited to single-locus analysis. Here, we expanded this platform to multiplex detection by incorporating mobility-shift strategies into the assay design. This approach enabled simultaneous analysis of 15 hotspot variants across three clinically relevant loci: KRAS codons 12 and 13 and GNAS codon 201. Validation using synthetic oligonucleotides, formalin-fixed paraffin-embedded tissue, and liquid specimens demonstrated high quantitative accuracy over clinically relevant variant allele frequency ranges, with measured values closely matching expected values (R2 > 0.97). The assay showed high concordance with targeted amplicon sequencing and digital polymerase chain reaction for all variants at variant allele frequencies ≥1%, while also detecting selected variants below this threshold. Collectively, these results establish a multiplexed high-dynamic-range capillary electrophoresis assay for simultaneous, quantitative detection of low-frequency variants, offering a scalable and cost-effective approach for disease-focused gene panels in clinical laboratory settings.

HiDy↗

PubMed2026

Halolitoreus marinus gen. nov., sp. nov. and Halolitoreus rarus sp. nov., halophilic archaea isolated from diverse coastal tidal flats, and proposal of the novel family Halolitoreaceae fam. nov. in the order Halobacteriales within the class Halobacteria.

Coastal tidal flats represent dynamic saline environments that harbor largely unexplored haloarchaeal communities. In this study, amplicon sequencing, metagenomic analyses, and cultivation-based approaches revealed substantial haloarchaeal diversity in tidal flats from four provinces of eastern China despite their relatively low salinity. Five haloarchaeal strains, designated YSMS36T, DYSN1, QDMS2, CMSO5T, and ZSTT2, were isolated from diverse tidal flats. Theses strains shared 16S rRNA gene sequence similarities of 92.1-92.2% with their closest validly named relative, Salinilacihabitans rarus AD-4T. Phylogenetic analyses based on 16S rRNA and rpoB' gene sequences showed that the five strains formed a distinct and well-supported monophyletic lineage, separated from currently recognized members of the class Halobacteria. Average amino acid identity (AAI), average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) values between these five strains and the related Halobacteria representatives were 49.3-62.7%, 66.9-74.9%, and 16.2-29.5%, respectively, and well below the accepted thresholds for species and genus delineation. Phylogenomic analyses further supported their placement within a novel family of the order Halobacteriales. Based on phylogenetic, genomic, chemotaxonomic, and phenotypic analyses, these five strains represent two novel species of a novel genus within a novel family. The names, Halolitoreaceae fam. nov., Halolitoreus marinus gen. nov., sp. nov., and Halolitoreus rarus sp. nov. are herein proposed.

Coastal tidal flat↗