Next-generation Sequencing Research Articles

Novel <i>FLNC</i> Gene Variant Associated with Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy, affecting approximately 1 in 500 people. It is the most common genetic cardiomyopathy inherited as a Mendelian trait in approximately 50% of patients, mainly due to pathogenic variants in genes encoding sarcomeric proteins. Mutations in the sarcomeric protein filamin C (<em>FLNC</em>) gene, with a cytogenetic localization on 7q32.1, have been linked to hypertrophic cardiomyopathy, as they have been determined to increase the risk of ventricular arrhythmia and sudden death. We present the case of a patient with HCM recognized by magnetic resonance imaging and echocardiography with a family history of cardiopathies. The molecular study in this patient was performed by next-generation sequencing on the Illumina MiniSeq instrument, comparing the results with international databases. In genetic studies, a novel mutation in the protein <em>FLNC</em> was detected. It is heterozygous, missence type. It is a variant where Cytosine is changed by timina at position 6305 of the <em>FLNC</em> gene. This produces the change of the amino acid proline by leucine at position 2102 of the Filamin C protein. The rare variant is located in Ig-like domain 19 within the ROD2 domain. This variant report suggests that there may indeed be a direct relationship between <em>FLNC</em> variants, mainly the ROD2 domain, and HCM. We think this new result should be considered for future genetic counseling of families affected by this type of cardiomyopathy.

Oral Microbiota Development in the First 60 Months: A Longitudinal Study.

Childhood is considered crucial in the establishment of future oral microbiota. However, the precise period of oral microbiota development remains unclear. This study aimed to identify the progression of oral microbiota formation in children. We longitudinally investigated the salivary microbiota of 54 children across 13 time points from 1 wk to 60 mo (5 y) old and their parents at 2 time points as a representative sample of the adult microbiota. Using next-generation sequencing, we obtained 10,000 gene sequences of the 16s rRNA V1-V2 region for each sample. The detection rate in children of 110 operational taxonomic units commonly detected in more than 85% of mothers and fathers, defined as the main constituent bacteria, was 25% at 1 wk old, increased to 80% between 6 and 18 mo old, and reached approximately 90% by 36 mo old. Early main constituent bacteria detected at 1 wk old were limited to Streptococcus, Rothia, and Gemella. At 6 to 18 mo old, the detection rates of various main constituent bacteria, including Neisseria, Haemophilus, and Fusobacterium, increased. UniFrac distance analysis showed that the oral microbiota of children approached that of adults at 6 to 18 mo old. In the weighted UniFrac distance index, unlike the unweighted index, there were no significant changes in children between 36 and 60 mo old from adults, and microbiota formation at 60 mo old was sufficiently advanced to be included within the range of adult individual differences. Our findings suggest that the initial 36 mo, particularly the period from 6 to 18 mo old, consists of a time window for oral microbiota maturation. In addition, the development of microbiota during this period may be critical for future oral disease prevention.

GVAF: generalized, flexible filtering software for annotated variant files.

In the rapidly advancing field of genomics, many tools have been developed to interpret genetic variants using next-generation sequencing (NGS) data. However, these tools often produce annotated variant files in different formats, which require specific software or programming skills to filter and analyze. To provide a filtering tool that can be used with diverse variant annotation tools without requiring specific software or programming skills. We developed Germline Variant Annotation and Filtering (GVAF), a command-line software tool that can handle annotated variant files in any table-shaped format. GVAF offers powerful filtering operations without the need for additional software or programming expertise. Built on the Java framework and bash scripts, it provides extensive features, including flexible filtering rules, recognition of genotype-related fields from variant call format (VCF) files, and customizable result generation. GVAF also integrates easily into existing data analysis pipelines. Compared to other tools, GVAF offers a broader range of functionalities, making it more flexible and intuitive for managing annotated variant files. This GVAF software and online manual is publicly available at https://www.sysbiolab.org/gvaf for academic users and is designed to streamline the variant interpretation process, aiding researchers in producing meaningful results.

Human Herpesvirus-6B Infection and Alterations of Gut Microbiome in Patients with Fibromyalgia: A Pilot Study

Fibromyalgia (FM) is a chronic disorder characterized by widespread musculoskeletal pain often accompanied by fatigue, sleep disturbances, memory issues, and mood disorders. The exact cause of FM remains unknown, and diagnosis is typically based on a history of persistent widespread pain, as there are no objective biomarkers usable in diagnosis of this disorder available. The aim of this study was to identify measurable indicators specific to FM with potential as biomarkers. This study included 17 individuals diagnosed with FM and 24 apparently healthy persons. Using real-time polymerase chain reaction (qPCR), we detected the presence of human herpesvirus (HHV)-6A and B genomic sequences in DNA isolated from peripheral blood mononuclear cells (PBMCs) and buccal swabs. HHV-6-specific IgG and IgM class antibodies, along with proinflammatory cytokine levels, were measured using enzyme-linked immunosorbent assay (ELISA) and bead-based multiplex assays. Additionally, the gut microbiome was analyzed through next-generation sequencing. HHV-6B was more frequently detected in the PBMCs of FM patients. FM patients with a body mass index (BMI) of 30 or higher exhibited elevated cytokine levels compared to the control group with the same BMI range. Gut microbiome analysis revealed significant differences in both α-diversity and β-diversity between the FM and control groups, indicating a shift in species abundance in the FM group.

PathVar: A Customisable NGS Variant Calling Algorithm Implicates Novel Candidate Genes and Pathways in Hemiplegic Migraine.

The exponential growth of next-generation sequencing (NGS) data requires innovative bioinformatics approaches to unravel the genetic underpinnings of diseases. Hemiplegic migraine (HM), a debilitating neurological disorder with a genetic basis, is one such condition that warrants further investigation. Notably, the genetic heterogeneity of HM is underscored by the fact that approximately two-thirds of patients lack pathogenic variants in the known causal ion channel genes. In this context, we have developed PathVar, a novel bioinformatics algorithm that harnesses publicly available tools and software for pathogenic variant discovery in NGS data. PathVar integrates a suite of tools, including HaplotypeCaller from the Genome Analysis Toolkit (GATK) for variant calling, Variant Effect Predictor (VEP) and ANNOVAR for variant annotation, and TAPES for assigning the American College of Medical Genetics and Genomics (ACMG) pathogenicity labels. Applying PathVar to whole exome sequencing data from 184 HM patients, we detected 648 variants that are probably pathogenic in multiple patients. Moreover, we have identified several candidate genes for HM, many of which cluster around the Rho GTPases pathway. Future research can leverage PathVar to generate high quality, candidate pathogenic variants, which may enhance our understanding of HM and other complex diseases.

Full-genome characterization of Turkish Rose Yellow Vein Virus (RYVV) Isolates

Rose yellow vein virus (RYVV) is a viral agent that has been identified in roses in recent years and is known to exist in a very limited area in the world. However, in studies conducted in our country, the presence of the agent has been detected in different regions and provinces. Since direct chemical control of viral diseases is not possible as in other biotic diseases, molecular characterization of the agent forms the basis for possible resistance or gene silencing studies to be conducted in the coming years. Thus, 44 RYVV isolates previously identified in our country were used for full genome studies. In full genome studies, overlapping PCR method was used with 7 different species-specific primer pairs. The full genomes of 3 of these isolates were obtained. The full genomes of the other isolates could not be obtained. It was determined that the genome size of the 3 RYVV isolates whose full genomes were obtained was 9314 nucleotides. It was determined that the only RYVV isolate found in the GenBank and the Türkiye isolates also showed a high level of nucleotide sequence similarity. However, the fact that the entire genome of the other isolates could not be obtained and the ones obtained were amplified with primer pairs designed according to the single isolate in the GenBank database may be due to the presence of different strains of RYVV isolates. For this reason, it is thought that next-generation sequencing studies should be prioritized to obtain complete genome information in future studies.

Screening and identification of key biomarkers associated with endometriosis using bioinformatics and next-generation sequencing data analysis

BackgroundEndometriosis is a common cause of endometrial-type mucosa outside the uterine cavity with symptoms such as painful periods, chronic pelvic pain, pain with intercourse and infertility. However, the early diagnosis of endometriosis is still restricted. The purpose of this investigation is to identify and validate the key biomarkers of endometriosis.MethodsNext-generation sequencing dataset GSE243039 was obtained from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) between endometriosis and normal control samples were identified. After screening of DEGs, gene ontology (GO) and REACTOME pathway enrichment analyses were performed. Furthermore, a protein–protein interaction (PPI) network was constructed and modules were analyzed using the Human Integrated Protein–Protein Interaction rEference database and Cytoscape software, and hub genes were identified. Subsequently, a network between miRNAs and hub genes, and network between TFs and hub genes were constructed using the miRNet and NetworkAnalyst tool, and possible key miRNAs and TFs were predicted. Finally, receiver operating characteristic curve analysis was used to validate the hub genes.ResultsA total of 958 DEGs, including 479 upregulated genes and 479 downregulated genes, were screened between endometriosis and normal control samples. GO and REACTOME pathway enrichment analyses of the 958 DEGs showed that they were mainly involved in multicellular organismal process, developmental process, signaling by GPCR and muscle contraction. Further analysis of the PPI network and modules identified 10 hub genes, including vcam1, snca, prkcb, adrb2, foxq1, mdfi, actbl2, prkd1, dapk1 and actc1. Possible target miRNAs, including hsa-mir-3143 and hsa-mir-2110, and target TFs, including tcf3 (transcription factor 3) and clock (clock circadian regulator), were predicted by constructing a miRNA-hub gene regulatory network and TF-hub gene regulatory network.ConclusionsThis investigation used bioinformatics techniques to explore the potential and novel biomarkers. These biomarkers might provide new ideas and methods for the early diagnosis, treatment and monitoring of endometriosis.

Clinical characteristics of 29 cases of pulmonary mucormycosis treated with and without surgery

Objective: To analyze the clinical characteristics of patients with pulmonary mucormycosis treated with and without surgery. Methods: This was a single-center, retrospective study. We retrieved "pulmonary mucormycosis" from the electronic medical records of China-Japan Friendship Hospital between 2016 and 2022. A total of 29 patients with pulmonary mucormycosis were collected. There were 19 males and 10 females with a median age of 49 (47, 67) years. Mann-Whitney U test, χ² test, Kaplan-Meier curve and log-rank test were used to compare the differences between groups. Results: The most common underlying disease was diabetes (19, 65.5%). The most frequent imaging findings were consolidation (25, 86.2%) and nodule or mass (21, 72.4%). Bronchial stenosis (16, 55.2%), obstruction by fungal plugs (18, 62.1%), pseudomembranous necrotizing bronchitis (19, 65.5%) were common. Treatment strategies were developed by the multi-disciplinary team (MDT). Among 16 patients who did not undergo surgery, 10 had bilateral multifocal lesions and 6 had unifocal lesions. All patients received antifungal therapies, and surgeries were performed in 13 (44.8%) patients. Patients who underwent surgery had numerically lower in-hospital mortality (15.4% vs. 31.3%, P=0.410). Involvement of unilateral multiple lesions was more common in patients who underwent surgeries (6/13 vs. 1/16, P=0.019). Patients who underwent surgery were more likely to have lobar and segmental bronchial involvement (13/13 vs. 9/16, P=0.007). A total of 15 patients underwent mNGS, 14 (93.3%) had positive results. Performing metagenomic next generation sequencing for diagnosis shortened the time from disease onset to diagnosis (log-rank P=0.014). Conclusion: Metagenomic next-generation sequencing aided early diagnosis. The patients who underwent surgery included unilateral multiple lesions and visualisation of endobronchial abnormalities on lobar or segmental bronchus in unilateral lung.

TRANSFORMING HEALTHCARE DELIVERY THROUGH BIG DATA IN HOSPITAL MANAGEMENT SYSTEMS: A REVIEW OF RECENT LITERATURE TRENDS

This systematic review investigates the transformative role of big data technologies in biomedical research, analyzing 40 peer-reviewed articles published between 2010 and 2023. The review specifically explores advancements in next-generation sequencing (NGS), multi-omics approaches, machine learning, and artificial intelligence (AI), all of which have significantly enhanced the understanding of complex biological systems and diseases. NGS has emerged as a key tool in personalized medicine, enabling rapid and cost-effective genome sequencing that has facilitated the identification of genetic mutations and biomarkers associated with various diseases, particularly in oncology. Of the 40 studies reviewed, 12 focused on the integration of multi-omics data—genomics, transcriptomics, proteomics, and metabolomics—to provide a comprehensive view of biological processes. These multi-omics approaches have been instrumental in identifying biomarkers for disease progression and response to treatments, offering new avenues for drug development and precision medicine. Additionally, 15 studies highlighted the growing application of machine learning and AI algorithms in managing and analyzing vast biomedical datasets. These tools are now critical in uncovering hidden patterns within large datasets, predicting disease outcomes, and improving the accuracy of clinical decision-making. However, 10 studies emphasized ongoing challenges related to data storage, privacy concerns, and the lack of standardized data formats, which hinder effective data sharing across institutions. Despite these challenges, the integration of AI, IoT devices, and big data analytics is paving the way for more personalized, real-time healthcare monitoring and treatment solutions. This review concludes that while significant advancements have been made, further efforts are required to address the ethical and technical barriers that limit the full potential of big data technologies in biomedical research.

Structural alterations of the EGFR gene in glioblastoma samples as a prognostic factor and molecular target for therapy

Introduction. Epidermal growth factor receptor (EGFR) is a transmembrane protein of the receptor tyrosine kinase family that is activated in various cancers (non-small cell lung cancer, colorectal cancer, head and neck tumors). In glial brain tumors, increased EGFR expression levels are characteristic of the most aggressive subtype, glioblastoma. Frequent structural changes of EGFR in glioblastoma are amplification of the chromosome region where the EGFR gene is located, point mutations, as well as deletion of exons 2–7 of the EGFR gene leading to the formation of EGFRvIII transcript.Aim. To determine structural changes of the EGFR gene (point mutations and amplification of the EGFR gene, EGFRvIII transcript) in tumor samples using different methods and to evaluate their potential clinical significance.Materials and methods. The study included 75 patients with brain gliomas (70 of them glioblastoma) aged 34 to 78 years (mean age 56 years). DNA and RNA isolation was performed from fresh frozen tumor tissue, as well as from peripheral blood leukocytes. EGFR gene mutations were determined by next-generation sequencing (NGS), and β allele frequency (BAF) comparative analysis (normal-tumor) was performed to determine the copy number of chromosome 7 regions. Quantitative polymerase chain reaction was used to confirm the EGFR gene amplification in tumor samples, and reverse transcription-PCR was used to detect EGFRvIII variant.Results. The NGS method revealed 11/70 (16 %) mutations in coding regions of EGFR gene in glioblastoma samples, the EGFR gene amplification was detected in 26/70 (37 %) cases; no structural changes of the EGFR gene were detected in 5 glioma samples (astrocytoma, oligodendroglioma). All cases of EGFR gene amplification detected by NGS were confirmed by quantitative polymerase chain reaction. To search for EGFRvIII transcript, 31 tumor RNA samples were examined, of which EGFR amplification was present in 12 samples. EGFRvIII transcript was detected only in samples with EGFR gene amplification – 4/12 (33 %). To assess the clinical significance of structural gene alterations, the frequency of occurrence in primary and recurrent glioblastoma samples was compared.Conclusion. The NGS method allows to detect both point mutations and amplification of the EGFR gene. The EGFR gene amplification was associated with EGFRvIII mutation in 33 % of cases. No statistically significant differences in the frequency of structural changes in the EGFR gene between primary and relapsed glioblastomas were found.

Genomic features of newly diagnosed large B cell lymphoma with or without subsequent disease progression.

Genomic analysis has the potential to both risk-stratify and inform management of patients diagnosed with large B cell lymphomas (LBCL). We analyzed cases of newly diagnosed LBCL patients treated with standard immunochemotherapy from three publicly available cohorts of patients on which fluorescence in situ hybridization (FISH) and next generation sequencing (NGS) were performed to determine the frequency of genomic alterations based upon development of disease progression. Cases from 698 patients were analyzed, with 201 experiencing disease progression and 497 no disease progression by 24 months post-diagnosis. When analyzing for the presence of MYC rearrangement and MYC-BCL2 dual-rearrangement/double hit, as well as variants predicted to result in alteration of protein function in 15 genes common to NGS panels from all 3 cohorts, only MYC rearrangement and TP53 mutation were associated with significantly higher odds of disease progression on multivariate analysis. Additionally, cases from patients who experienced disease progression demonstrated a high frequency of specific genomic alterations when analyzed by cohort or cell of origin classification by immunohistochemistry when available. Individual genomic features of LBCL cases may predict for development of disease progression in newly diagnosed patients treated with standard therapies, as well occur at higher frequencies in cases of disease progression based upon geographic region and/or cell of origin status. These novel findings support efforts to evaluate genomic features as biomarkers for response to specific therapies in subsets of LBCL patients who experience disease progression, which may lead to discovery of more effective treatment options.

Trends and Disparities in Next-Generation Sequencing (NGS) Tumor Testing in Patients with Metastatic Prostate Cancer (mPC)

Trends and Disparities in Next-Generation Sequencing (NGS) Tumor Testing in Patients with Metastatic Prostate Cancer (mPC)

Association of Gut Microbiota Composition with Stunting Incidence in Children under Five in Jakarta Slums.

Stunting can be linked to various factors, one of which is dysbiosis. This study aims to analyze the microbiota composition and related contributing factors of stunted and non-stunted children in the slum areas of Jakarta. The subjects in this study included 21 stunted (HAZ ≤ -2SD) and 21 non-stunted children (-2SD ≤ HAZ ≤ 3SD) aged 2-5 years. Microbiota analysis was performed by extracting DNA from the subjects' feces and then via 16S rRNA sequencing using next-generation sequencing (NGS). The results of this study showed that in stunted children, the abundance of Mitsuokella (24,469 OTUs), Alloprevotella (23,952 OTUs), and Providencia alcalifaciens (861 OTUs) was higher, while in non-stunted children, that of Blautia (29,755 OTUs), Lachnospiraceae (6134 OTUs), Bilophila (12,417 OTUs), Monoglobus (484 OTUs), Akkermansia muciniphila (1116 OTUs), Odoribacter splanchnicus (42,993 OTUs), and Bacteroides clarus (8900 OTUs) was higher. Differences in microbiota composition in the two groups were influenced by nutrient intake, birth history, breastfeeding history, handwashing habits before eating, drinking water sources, and water sources for other activities. This study highlights that stunted children have a significantly different gut microbiota composition compared to non-stunted children, with higher levels of pathogenic bacteria and lower levels of beneficial bacteria. Future research should focus on interventions that can improve the gut microbiota composition to prevent stunting in children.

Plasma Cell-free DNA Metagenomic Next-Generation Sequencing in Immunocompromised Children

Abstract Background Plasma cell-free DNA metagenomic next-generation sequencing (mNGS) is a novel diagnostic tool for infectious diseases [1]. Data suggests that plasma mNGS may be useful in immunocompromised adults, changing management in up to 60% of the patients [2]. A prospective study evaluating utility of plasma mNGS for pneumonia in immunocompromised adults concluded that mNGS provided an additive diagnostic value of 12.1% [3]. A similar study in adults with febrile neutropenia reported a sensitivity and specificity of 85 and 100%, respectively [4]. However, there is limited data evaluating the utility of mNGS in immunocompromised children. Methods We retrospectively reviewed all pediatric immunocompromised patients who had plasma mNGS testing performed from December 2018 to July 2023 at St. Jude Children’s Research Hospital. Electronic medical records were reviewed to extract relevant clinical data and microbiologic data including results of mNGS, contemporaneous cultures, and related antimicrobial treatment. Descriptive statistics are presented as frequencies and medians (interquartile range, IQR). Comparisons were performed using Fisher’s exact test or Wilcoxon rank-sum test, as appropriate, using R version 3.6.3. Results A total of 18 immunocompromised children underwent plasma mNGS testing. Acute lymphoblastic leukemia was the most common malignancy (33.3%), followed by acute myeloid leukemia (22.2%). Two patients (11.1%) had undergone hematopoietic cell transplant, both allogeneic. At the time of testing, 16 patients (88.9%) had fever and 10 (55.6%) had an absolute neutrophil count of <500/mm3. All except one were on antimicrobial therapy at the time of testing, with a median duration of 8 days (IQR=12.25). Only 9 (50%) patients had a positive mNGS result. A positive mNGS resulted in broadening or extending antibiotics course in 4 patients (22.2%); negative mNGS results did not lead to changes in management in any of the cases. There were no significant differences in age, sex, days of fever, neutropenia, gastrointestinal (GI) symptoms, and days of fever before testing between those with positive versus negative results. However, a higher frequency of GI symptoms at the time of testing among those with positive results was observed. Clinical characteristics and impact on management in patients with positive and negative results are summarized in Table 1. Conclusion Plasma mNGS had a limited impact on the management of immunocompromised children at our institution. Negative results did not lead to de-escalation of therapy, and positive results led to additional exposure to antibiotics. Further research is needed to determine the role of mNGS in this population, and the interpretation of positive testing in the setting of gastrointestinal disease.

Metagenomic next-generation sequencing in diagnosing perinephric abscess infection caused by Trichomonas vaginalis

Metagenomic next-generation sequencing in diagnosing perinephric abscess infection caused by Trichomonas vaginalis

Next generation sequencing identifies an increased diversity of microbes in post lavage specimens in infected TKA using a biofilm disrupting irrigant

BackgroundPeriprosthetic joint infection (PJI) is a devastating complication of joint arthroplasty. In chronic PJI, a biofilm envelops the surface of implants, which contains microbiota within an extra-microbial polymeric matrix (EMPM). Microbial identification is paramount for effective treatment. In this study, we use a multi-modal, EMPM disrupting, neoadjuvant irrigant and compare the microbiota detected pre-lavage to post-lavage by two techniques: culture and Next Generation Sequencing (NGS). We suspect more organisms to be identified after applying an EMPM disrupting irrigant. MethodsA multicenter, prospective study was conducted on 38 patients with known Total Knee Arthroplasty PJI. At initial arthrotomy, synovial fluid was obtained and analyzed for quantitative cultures and microbial NGS. Joint was then irrigated with Bactisure Lavage followed by Normal Saline. Post-lavage samples were similarly obtained and analyzed. ResultsIn pre-lavage samples for cultures, 55.3% of samples were positive, identifying 11 unique organisms. In post-lavage samples for cultures, 13.2% of samples were positive, identifying 5 unique organisms. In pre-lavage samples for NGS, 79% were DNA signal positive, identifying 126 unique organisms. In post-lavage samples for NGS, 74% of samples were DNA signal positive, identifying 177 unique organisms. Moreover, 135/177 of these organisms were not identified pre-lavage. ConclusionIn this pre-to-post irrigant study, culture showed a decrease in the number of identifiable organisms post-lavage. In contrast NGS revealed an increase in the number of identifiable organisms post-lavage. Furthermore, NGS identified 135 additional organisms, not detected pre-lavage. This suggests an increased diversity of microbes may exist within EMPM, which are not cultivable.

Sequence analysis and identification of a novel HLA-DPB1*02:01:69 allele by third-generation sequencing

To analyze the sequence of a novel HLA-DPB1 allele in an individual. A individual identified from the database of blood donors for matched platelet transfusion at the Blood Center of Zhejiang Province in May 2022 was selected as the study subject. HLA genotype of the individual was determined by next-generation sequencing (NGS) on an Ion Torrent S5 platform. The sequence of the HLA-DPB1 locus was also determined by NGS on an Illumina Miseq platform and third-generation sequencing using Oxford Nanopore MinION. This study was approved by the Blood Center of Zhejiang Province (Ethics No. 2021-001). A novel HLA-DPB1*02 allele was identified in the specimen, for which the closest genotype was HLA-DPB1*02:new,17:01:01G, with the variant located in exon 3. Meanwhile, the NGS also revealed a novel HLA-DPB1*17 allele, with the closest genotype being HLA-DPB1*02:01,17:new. Both the HLA-DPB1*17:01:01:01 and HLA-DPB1*02 alleles were identified by third-generation sequencing. Compared with the HLA-DPB1*02:01:02:01 allele, the novel allele had a G>A variation at position 369 in the exon 3, which however did not result in amino acid change. A novel HLA-DPB1 allele has been identified and validated by both NGS and TGS, which has been named as HLA-DPB1*02:01:69 by the World Health Organization Committee on Nomenclature of Factors of the HLA System.

Emergence and Rapid Diagnosis of Talaromyces marneffei Infections in Renal Transplant Recipients by Next-Generation Sequencing

In the last few years, next-generation sequencing (NGS) has emerged as a technology for laboratory diagnosis of many culture-negative infections and slow-growing microorganisms. In this study, we describe the use of metagenomic NGS (mNGS) for rapid diagnosis of T. marneffei infection in a 37-year-old renal transplant recipient who presented with chronic pneumonia syndrome. Bronchoalveolar lavage for mNGS was positive for T. marneffei sequence reads. Prolonged incubation of the bronchoalveolar lavage revealed T. marneffei colonies after 6 days of incubation. Analysis of 23 cases of T. marneffei infections in renal transplant recipients from the literature revealed that the number of cases ranged from 1 to 4 cases per five years from 1990 to 2020; but increased rapidly to 9 cases from 2021 to 2023, with 7 of them diagnosed by NGS. Twenty of the 23 cases were from T. marneffei-endemic areas [southern part of mainland China (n = 9); Hong Kong (n = 4); northeastern India (n = 2); Indonesia (n = 1) and Taiwan (n = 4)]. For the 3 patients from non-T. marneffei-endemic areas [United Kingdom (n = 2) and Australia (n = 1)], they had travel histories to China and Vietnam respectively. The time taken for diagnosis by mNGS [median 1 (range 1 to 2) day] was significantly shorter than that for fungal culture [median 6 (range 3 to 15) days] (P = 0.002). mNGS is useful for picking up more cases of T. marneffei infections in renal transplant recipients as well as providing a rapid diagnosis. Talaromycosis is an emerging fungal infection in renal transplant recipients.

Detection of genomic alterations in liquid biopsies from patients with non-small cell lung cancer using FoundationOne Liquid CDx: a cost-effectiveness analysis

Objective Liquid biopsy (LB) is a non-invasive technique to detect genetic alterations by next-generation sequencing (NGS) when tissue biopsy is not available. This study aims to estimate in the Spanish setting, the cost-effectiveness of using FoundationOne Liquid CDx (F1L CDx), a novel blood-derived LB test based on NGS, versus non-molecular diagnosis (non-mDx) in patients with advanced non-small cell lung cancer (NSCLC) in whom tissue sampling is not feasible. Methods A joint model was developed combining a decision-tree with partitioned survival models to calculate the costs and health outcomes over a lifetime horizon, comparing F1L CDx in LB versus non-mDx. Only direct costs (expressed in €of 2023) were included and a 3% discount rate for future costs and effects was considered. Health outcomes were expressed in Life Years (LYs) and Quality-Adjusted Life Years (QALYs). Utilities and treatment efficacy were obtained from the literature. An expert panel of 11 Spanish oncologists determined the treatment allocation and validated all model inputs and assumptions. Several sensitivity analyses were performed to assess the robustness of the results. Results In a hypothetical cohort of 1,000 patients, LB using F1L CDx would detect 386 alterations, so those patients could be treated with targeted therapies or enrolled in clinical trials. Cost-effectiveness results showed that F1L CDx provides greater effectiveness than non-mDx (+383.95 LYs and +305.94 QALYs), with an additional cost of €2,898,308. The incremental cost-utility ratio was €9,473/QALY gained. The probabilistic sensitivity analysis confirmed the robustness of the cost-effectiveness results. Limitations Various limitations inherent to cost-effectiveness analyses were described. Conclusion LB with F1L CDx test is a cost-effective strategy in Spain for patients with advanced NSCLC without tissue sample available for molecular diagnosis, improving the personalized treatment of these patients.

Insights into Porphyromonas somerae in Bladder Cancer Patients: Urinary Detection by ddPCR.

To date, the increased awareness of the impact of microbes on human health has promoted scientific interest in microbiome studies for diagnostic and therapeutic purposes, revealing correlations between specific taxa and cancer. In particular, numerous species of Porphyromonas have been associated with several types of tumors. Previously, we studied the urobiome using Next-Generation Sequencing (NGS), and found an increase in Porphyromonas somerae in first morning urine of subjects affected by bladder cancer (BCa). Here, we aimed to confirm the presence of P. somerae in BCa patients by using droplet digital Polymerase Chain Reaction (ddPCR), testing a cohort of 102 male subjects over 50 years. Our findings showed a significant increase in P. somerae in the urine of the BCa group within both ddPCR and NGS, and a correlation between the two methods was observed at a statistical level. Moreover, P. somerae's identification with ddPCR confirmed a significant association between this bacterium and the presence of BCa, highlighting its potential role as a biomarker. This allows us to propose the ddPCR as a suitable method for first-stage BCa screening and follow-up.