Next-generation Sequencing Research Articles

Composite Hedges Nanopores codec system for rapid and portable DNA data readout with high INDEL-Correction

Reading digital information from highly dense but lightweight DNA medium nowadays relies on time-consuming next-generation sequencing. Nanopore sequencing holds the promise to overcome the efficiency problem, but high indel error rates lead to the requirement of large amount of high quality data for accurate readout. Here we introduce Composite Hedges Nanopores, capable of handling indel rates up to 15.9% and substitution rates up to 7.8%. The overall information density can be doubled from 0.59 to 1.17 by utilizing a degenerated eight-letter alphabet. We demonstrate that sequencing times of 20 and 120 minutes are sufficient for processing representative text and image files, respectively. Moreover, to achieve complete data recovery, it is estimated that text and image data require 4× and 8× physical redundancy of composite strands, respectively. Our codec system excels on both molecular design and equalized dictionary usage, laying a solid foundation approaching to real-time DNA data retrieval and encoding.

A phase 2, multicenter, clinical trial of CPX-351 in older patients with secondary or high-risk acute myeloid leukemia: PETHEMA-LAMVYX.

LAMVYX was a multicenter, single-arm, phase 2 trial designed to validate the safety and efficacy of CPX-351 in patients aged 60-75 years with newly diagnosed, secondary acute myeloid leukemia and to generate evidence on key issues not addressed in the preceding regulatory pivotal trial. The primary end point of the study was the complete remission (CR)/CR with incomplete hematologic recovery (CRi) rate after induction. Eligible patients were recommended to undergo allogeneic hematopoietic stem cell transplantation after the first consolidation cycle. Alternatively, patients could undergo up to six maintenance cycles with CPX-351. Twenty-nine patients (49%; 95% exact confidence interval [CI], 37%-62%) patients achieved a CR/CRi after one or two cycles of induction, with a measurable residual disease negativity rate of 67% as assessed by centralized, multiparameter flow cytometry. Among patients who had serial next-generation sequencing analyses available, clearance of somatic mutations that were present at diagnosis was achieved in 7 (35%). The median follow-up among survivors was 16.8 months (range, 8.7-24.3 months). The median event-free survival was 3.0 months (95% CI, 1.4-7.3 months), and the median overall survival was 7.4 months (95% CI, 3.7-12.7 months). In landmark analyses at day +100 from diagnosis, the 1-year overall and event-free survival rate among patients who underwent allogeneic hematopoietic stem cell transplantation was 70% (95% CI, 47%-100%) and 70% (95% CI, 47%-100%), respectively. The corresponding values were 89% (95% CI, 71%-100%) and 44% (95% CI, 21%-92%), respectively, for patients who entered the maintenance phase. No significant longitudinal changes were observed in severity index or quality-of-life visual analog scale scores. The current data provide novel insights that might inform the clinical positioning and optimal use of CPX-351, complementing previous results (ClinicalTrials.gov identifier NCT04230239).

Primary seminal vesicle diffuse large B-cell lymphoma: a case report and review of the literatures

Primary seminal vesicle lymphoma is a remarkably rare condition, predominantly manifesting as diffuse large B-cell lymphoma. Due to its rarity and nonspecific clinical presentations, it is often misdiagnosed or overlooked. Here, we report a case of a 68-year-old male diagnosed with primary seminal vesicle lymphoma, coinciding with prostate cancer. The diagnosis followed initial findings of elevated prostate-specific antigen levels and abnormal magnetic resonance imaging of the prostate and left seminal vesicle. Suspicion of prostate cancer led to a radical resection of both the prostate and seminal vesicle. Subsequent pathological examination and next-generation sequencing post-surgery confirmed the diagnosis of primary seminal vesicle diffuse large B-cell lymphoma, characterized by CD79B mutation type (MCD type). The patient was treated with six cycles of the R-CHOP regimen (rituximab, cyclophosphamide, vincristine, doxorubicin, prednisone), achieving complete metabolic remission as confirmed by positron emission tomography-computed tomography. Fifteen months post-treatment, the patient’s condition remains favorable. Through our literature review of additional six cases of primary seminal vesicle lymphoma, we aim to elucidate the typical clinical presentations, imaging features, pathological characteristics, genetic mutations, and therapeutic strategies, aiming to contribute to better detection and management of this rare malignancy. This case underscores the diagnostic challenges and emphasizes the necessity for heightened clinical suspicion and definitive pathological examination in the management of primary seminal vesicle lymphoma.

Clinicopathologic and Genomic Features of Invasive Stratified Mucin-producing Carcinoma of the Uterine Cervix Coexisting With High-grade Squamous Intraepithelial Lesion.

Invasive stratified mucin-producing carcinoma (ISMC) is a specific type of adenocarcinoma of the cervix, which is associated with human papillomavirus infection and often coexists with other types of carcinomas. However, given its rarity, understanding of this disease remains insufficient. We present a unique case of ISMC of the cervix coexisting with a high-grade squamous intraepithelial lesion (HSIL). In addition to histologic and immunohistochemical feature observation, genomic profiling of the 2 lesions was performed. Histologically, the ISMC and HSIL lesions were independent of each other. Aside from the typical morphology, various architectural features of ISMC were observed. Immunohistochemically, the ISMC and HSIL lesions were strongly and diffusely positive for p16 and exhibited high Ki-67 expression. The ISMC lesion was also positive for CK7, MUC5AC, and MUC6, while it was negative for PAX-8. The HSIL lesion was positive for CK5/6 and p40. The combined positive score of PD-L1 was 55. The other markers were all negative in both lesions, and the p53 was wild-type. Next-generation sequencing analysis revealed multiple gene mutations in the ISMC and HSIL lesions. A total of 88 gene mutations were identified in the ISMC lesion, while 20 gene mutations were identified in the HSIL lesion. Three mutations (ERBB2, histidine decarboxylase gene [HDC], and BSN) were detected in the ISMC and HSIL lesions. Both lesions had a low tumor mutation burden and microsatellite-stable status. No copy number-associated variants or structural variations were identified in either lesion. These results suggest that patients with ISMC may benefit from PD-L1 immunotherapy and targeted therapy.

A Beginner's Guide to Genomics in Complex Neurological Disorders

In view of the recent progress in genomics and next-generation sequencing technologies, we consider here their contribution toward neurological disorders. We describe the revolutionary impact that whole-genome and whole-exome sequencing have had on diagnostics and the identification of important genetic loci associated with a range of disorders. Neurological disorders affecting the central and peripheral nervous systems are complex, both in diagnosis and treatment. Herein we show that by applying multiomic tools to assist researchers in identifying new biomarkers and therapeutic approaches, the diagnosis, treatment, and prognosis of these disorders can be significantly improved.

Clinical validation of peripheral blood mononuclear cell DNA methylation markers for accurate early detection of hepatocellular carcinoma in Asian patients

BackgroundHepatocellular carcinoma (HCC), a leading cause of cancer-related deaths globally, poses significant challenges in early detection. Improved diagnostic accuracy can drastically influence patient outcomes, emphasizing the need for innovative, non-invasive biomarkers.Methods:This study utilized a cohort of 402 participants, including healthy controls, chronic hepatitis patients, and HCC patients from Bangladesh, to evaluate DNA methylation signatures in peripheral blood mononuclear cells (PBMC). We performed targeted next-generation sequencing on selected genes previously identified to assess their methylation dynamics. The development of M8 and M4 scores was based on these dynamics, using Receiver Operating Characteristic (ROC) analysis to determine their effectiveness in detecting early-stage HCC alongside existing markers such as epiLiver and alpha-fetoprotein (AFP).Results:Integration of M8 and M4 scores with epiLiver and AFP significantly enhances diagnostic sensitivity for early-stage HCC. The M4+epiLiver score achieves a sensitivity of 79.4% in Stage A HCC, while combining M4 with AFP increases sensitivity to 88.2–95.7% across all stages, indicating a superior diagnostic performance compared to each marker used alone.Conclusions:Our study confirms that combining gene methylation profiles with established diagnostic markers substantially improves the sensitivity of detecting early-stage HCC. This integrated diagnostic approach holds promise for advancing non-invasive cancer diagnostics, potentially leading to earlier treatment interventions and improved survival rates for high-risk patients.

PlaBacter: Determination of Microbial Diversity on Plastic Polymers in Topçu Pond

Climate change is one of the biggest problems of our world, and one of the biggest measures taken to solve the climate crisis is to reduce the consumption of oil and oil derivatives, which are considered among fossil resources. However, with the globalising world and increasing population, oil companies have turned to the plastic and packaging sector in order to meet the increasing demand for plastic consumption. This situation inevitably increases the environmental damage of plastic waste. In particular, the inability to use water resources correctly and the pollution of water by wastes left in the environment adversely affect the lives of living things on earth. Within the scope of the study, it was aimed to determine the microbial diversity living on polyethylene terephthalate (PET), which is known as the raw material of tools such as plastic bottles, food containers, and fishing line, which are left as waste in the environment, especially in rivers and lake shores. In this context, Topçu Pond, which is an important water source in the field of agricultural irrigation in Yozgat province, was selected as the study area. Genomic DNA (gDNA) was isolated from biofilm layers formed on water, sediment, and PET samples collected from the study area. The bacterial communities living in the samples were identified by analysing the 16S rDNA of the gDNAs with the next-generation sequencing method. Thus, we conducted the first microbial diversity study in Topçu Pond and defined the bacterial composition on PET samples in the pond.

Eosinophilic Meningitis in a Patient With Early HIV Infection, Latent Syphilis and HSV-2 Meningitis

Abstract Eosinophilic meningitis is an unusual condition, particularly in patients with HIV infection or syphilis. The detection of herpes simplex virus (HSV)-2 and HIV-1 in cerebrospinal fluid from a patient with acute eosinophilic meningitis has not been previously reported. Herein, we report a case of a 26-year-old cisgender man newly diagnosed with HIV-1 infection and latent syphilis, presenting with headache and low-grade fever. He was diagnosed with both acute eosinophilic meningitis and HSV-2 meningitis, and his condition improved after treatment with acyclovir and penicillin. The compelling case indicates that HSV-2 meningitis may manifest concurrently with acute eosinophilia meningitis in patients coinfected with HIV and Treponema pallidum. Metagenomics next-generation sequencing plays an important role in identifying and further confirming pathogens responsible for central nervous system infections.

Cutaneous Metastasis of Rectal Cancer as a Diagnostic Challenge: A Clinical Case and Literature Review

Background/Objectives: Metastatic colorectal cancer remains a fatal disease, with a 5-year survival rate lower than 15%. The most common metastatic sites are the lungs and the liver, while skin metastases are very rare and often indicate a poor prognosis with a lower survival rate. Methods. Herein, we present the clinical case of a 62-year-old female patient with rectal cancer metastases to the skin of the anogenital and abdominal regions, diagnosed 2 years after completion of treatment of the underlying disease. Results: Histological examination of the skin lesions revealed adenocarcinoma, and expression of the same immunohistochemical markers was also found in the primary tumor and in the cutaneous metastases. However, next-generation sequencing demonstrated differences in the mutational profiles of the primary tumor and metastasis to the skin. Somatic mutations in the APC, TP53, and PTPN11 genes were revealed in primary rectal adenocarcinoma, but another pathogenic TP53 mutation and a frameshift variant in the DYNC1I1 gene were found in cutaneous metastases. The patient underwent several courses of FOLFOX6 chemotherapy in combination with bevacizumab, but the treatment was unsuccessful. An analysis of 50 clinical cases from the literature concerning various manifestations of cutaneous metastases of rectal cancer showed a median survival of 8.5 months from the time of detection of the skin lesions. Conclusions: In this regard, careful skin examination of patients with rectal cancer and timely detection of cutaneous metastases are essential steps in the follow-up of patients who have undergone treatment of the primary tumor.

The discrepancy of somatic BRCA1/2 pathogenic variants from two different platforms in epithelial ovarian, fallopian tube, and peritoneal cancer

The somatic BRCA1 or BRCA2 Pathogenic Variant (PV)/Likely PV (LPV) from Next Generation Sequencing (NGS) is the most important biomarker for PARP inhibitor use and maintenance-targeted therapies. A discrepancy in the detection rates of BRCA1 and BRCA2 PV/LPV was identified among the NGS platforms. The objective of this study was to compare the somatic BRCA results from two distinct platforms using the same cohort and to identify the causes of these differences. Patients with epithelial ovarian cancer who concurrently underwent tumor NGS using two different platforms between January 2022 and June 2023 were included in this study. The two platforms used were in-house tumor NGS (Illumina NextSeq 550Dx, SureSelectXT library kit, and datasets from 1000 Genomes, ESP6500, ExAC, and ClinVar) and GreenPlan homologous recombination deficiency (HRD) test (Illumina NextSeq 550Dx, customized Twist Bioscience library kit, and datasets from COSMIC and OncoKB). The results of somatic mutations in BRCA1 and BRCA2 were compared between the two platforms. Of the 118 patients, 11.9% (n = 14) exhibited a discordant interpretation of BRCA1 or BRCA2 between the two platforms. Eleven patients (9.3%) exhibited negative results in the in-house platform but positive results (eight seven as PV of BRCA1, one as PV of BRCA2, one as LPV of BRCA1, and two as LPV of BRCA2) in the GreenPlan HRD test, while three patients (2.6%) had positive BRCA pathogenic variants (two as PV of BRCA1 [c.3340G > T, c.5152 + 3 A > C], one as LPV of BRCA2 [c.8174G > T], and one as LPV of BRCA1 [c.5017_5019delCAC]) in the in-house platform but a negative result in the GreenPlan HRD test. The discordance rate of somatic BRCA1 or BRCA2 mutations from different platforms was approximately 12%. In the case of the strong implication of BRCA PV/LPV with a negative result with one genetic test, different platforms could be considered in limited cases. Careful interpretation and further studies for the cross-validation of gene analysis platforms are needed.

Novel FLNC variants in pediatric cardiomyopathy: an insight into disease mechanisms

BackgroundFLNC gene variants have predominantly been reported in adult populations with cardiomyopathies, and early-onset cases are less common. The genotype–phenotype relationship indicates that dilated cardiomyopathy (DCM) is often associated with FLNC truncating variants.MethodsWe conducted a comprehensive genetic analysis using next generation sequencing (NGS) to identify FLNC variants in patients with cardiovascular conditions. Detailed phenotypic and variant analyses were performed to characterize the clinical features and genetic alterations. Minigene assays and structural modeling were used to investigate the pathogenicity caused by the identified variants.ResultsIn a cohort of 58 patients, novel heterozygous FLNC variants, c.3962A > T (p.Glu1321Val) and c.7543C > T (p.Leu2515Phe), were identified in patients presenting with dilated and mixed restrictive/hypertrophic cardiomyopathies, respectively. The c.3962A > T variant disrupted normal splicing, as demonstrated through the splicing prediction tool and minigene studies, further emphasizing its pathogenic potential.ConclusionFor missense variants of FLNC in patients with DCM, the splicing effect of the variant should be carefully checked. Early detection and intervention are crucial given the high risk of sudden cardiac death and severe cardiac complications.

Metagenomic Analysis of Microbial Diversity in the Urban Air of Sofia (Bulgaria) during Spring and Winter

The main scientific interest for decades remained focused primarily on physical and chemical air pollutants, which severely limited our knowledge of microbial diversity in the atmosphere. The ecology of airborne microorganisms is poorly understood, even though they are a constant element of the air we breathe. Their exploration is very important for understanding health and ecological impacts, mainly in the highly urbanized cities. The main purpose of this study is to explore the biodiversity of microorganisms in the atmosphere over the densely populated centre of Sofia (Bulgaria) during spring and winter by Next-generation sequencing. The results showed that the most prevailing bacteria in the urban bioaerosols were representatives of phyla Firmicutes, Proteobacteria, Bacteroides, Actinobacteria. Many of the identified species were soil and leaf surface dwellers. Most of the detected fungal sequences belong to the phylum Ascomycota. Some pathogenic bacterial and fungal taxa were also found. This study provides the first information about microbial diversity in the outdoor air above Sofia and contributes to better understanding of urban airborne microbiota.

Maximising the Synergy of Tumour Tissue and Liquid Biopsy Testing in Oncology Clinical Practice

The symposium took place during the 2024 European Society for Medical Oncology (ESMO) Congress in Barcelona, Spain, with the goal of highlighting the synergy between tissue and liquid biopsy testing in the diagnosis and treatment of solid tumours. Christian Rolfo, Director of the Division of Medical Oncology at The Ohio State University Comprehensive Caner Centre, Columbus, USA, set the stage with a discussion on the state-of-the-art liquid biopsy for solid tumour testing. He was followed by Guilhem Roubaud from the Bergonié Institute in Bordeaux, France, and Federico Cappuzzo from Regina Elena Institute, Rome, Italy, who delved into molecular testing challenges and applications in genitourinary and non-small cell lung cancer (NSCLC), respectively. Practical insights on next-generation sequencing (NGS) implementation was provided by Bence Sipos, TBAG of Molecular Pathology Baden-Württemberg, Germany, with real-world case studies presented by Sara Pilotto, University Hospital, Verona, Italy, and Roubaud. The faculty emphasised the importance of integrating liquid and tissue biopsies to perform tumour molecular profiling, which is crucial for accurate diagnosis and personalised treatment strategies. They also highlighted the critical role of rapid NGS in detecting a wide range of genetic alterations to enhance the precision of diagnosis and access to precision therapies.

Circ-CAMTA1 regulated by Ca2+ influx inhibited pyruvate carboxylase activity and modulate T cell function in patients with systemic lupus erythematosus

ObjectivesTo investigate the roles of Ca2+ influx-regulated circular RNAs (circRNAs) in T cells from patients with systemic lupus erythematosus (SLE).MethodsThe expression profile of circRNAs in Jurkat cells, co-cultured with and without ionomycin, was analyzed by next-generation sequencing and validated using real-time polymerase chain reaction. The identified Ca2+ influx-regulated circRNAs were further examined in T cells from 42 patients with SLE and 23 healthy controls. The biological function of specific circRNA was investigated using transfection and RNA pull-down assay.ResultsAfter validation, we confirmed that the expression levels of circ-ERCC4, circ-NFATC2, circ-MYH10, circ-CAMTA1, circ-ASH1L, circ-SOCS7, and circ-ASAP1 were consistently increased in Jurkat cells following Ca2+ influx. The expression levels of circ-CAMTA1, circ-ASH1L, and circ-ASAP1 were significantly lower in T cells from patients with SLE, with even lower levels observed in those with higher disease activity. Interferon (IFN)-α was found to suppress the expression of circ-CAMTA1. Circ-CAMTA1 bound to pyruvate carboxylase and inhibited its biological activity. Overexpression of circ-CAMTA1, but not its linear form, significantly decreased extracellular glucose levels. Furthermore, increased expression of circ-CAMTA1, but not its linear form, decreased miR-181c-5p expression, resulting increased IL-2 secretion.ConclusionThree Ca2+ influx-regulated circ-RNAs—circ-CAMTA1, circ-ASH1L, and circ-ASAP1 —were significantly reduced in T cells from patients with SLE and associated with disease activity. IFN-α suppressed the expression of circ-CAMTA1, which interacted with pyruvate carboxylase, inhibited its activity, affected glucose metabolism, and increased IL-2 secretion. These findings suggest that circ-CAMTA1 regulated by Ca²⁺ influx modulated T cell function in patients with SLE.

Genotyping-by-sequencing shows high genetic diversity in Corylus avellana germplasm resistant to eastern filbert blight

European hazelnut (Corylus avellana) is an anemophilous, dichogamous, self-incompatible tree nut species. It is native to a large portion of Europe, Turkey, and the Caucasus region, across which a wealth of plant genetic resources is present. The objective of this study was to evaluate the genetic diversity of a core set of C. avellana representing the world’s germplasm using genotyping-by-sequencing derived single nucleotide polymorphism (SNP) markers and to classify novel eastern filbert blight (EFB) resistant or tolerant accessions. Two-hundred-twenty-two accessions underwent next-generation sequencing (NGS) to generate SNP markers. From this, 1,250 SNP markers were used to construct a neighbor-joining (NJ) dendrogram and perform a STRUCTURE and discriminant analysis of principal coordinates (DAPC) analyses. In the dendrogram, five major groups were established, which generally corresponded to geographic origins of the plant materials studied. In STRUCTURE, support was found for groupings at (K) = 3, (K) = 6, and (K) = 10 populations, with the greatest Δ-(K) value occurring at (K) = 10. Although the three different analyses indicated slightly different solutions, the overall results were generally consistent from the standpoint of identifying similar accession groupings. For many of the accessions, recorded origins tended to correspond with their genetic grouping, although there was also evidence of intermixing and likely movement of plant materials. Interestingly, in all three analyses, a vast majority of the new accessions from the Republic of Georgia formed their own distinct group, highlighting this geographic region as a unique pool of C. avellana genetic resources. Overall, EFB resistant/tolerant accessions were placed across a wide range of genetic backgrounds. Thus, our results indicate EFB resistance/tolerance is present across a wide spectrum of C. avellana genetic resources, with the Georgian accessions representing a new and relatively unique germplasm pool that can be incorporated into hazelnut breeding programs.

NF1 expression profiling in IDH-wildtype glioblastoma: genomic associations and survival outcomes

BackgroundNF1 inactivation is associated with sensitivity to MEK inhibitor targeted therapy in low-grade and some high-grade gliomas. NF1 loss may also be a harbinger of exploitable vulnerabilities in IDH-wildtype glioblastoma (GBM). Accurate and consistent detection of NF1 loss, however, is fraught given the large gene size, challenges with complete coverage and variant calling upon sequencing, and mechanisms of mRNA and protein regulation that result in early degradation in the absence of genomic alterations. Here, we seek to perform a composite analysis for NF1 loss accounting for genomic alterations and protein expression via immunohistochemistry. We also characterize the landscape of NF1 alterations in GBM.MethodsWe assembled a single-institution, retrospective cohort of 542 IDH-wildtype GBM with somatic next generation sequencing to investigate the frequency and nature of detected NF1 alterations. We selected 69 GBMs from which to build a tissue microarray (TMA) of 44 NF1-wildtype and 25 NF1-mutant cases. We performed NF1 immunohistochemistry using two different NF1 antibodies (NFC, Sigma-Aldrich; and iNF-07E, iNFixion Bioscience) and correlated results with clinical, genomic, and other immunohistochemical features.ResultsIn our retrospective cohort, we identified 88 IDH-wildtype GBM with NF1 alterations (16%). NF1 alterations were mutually exclusive with EGFR and MDM2 alterations (p-adj < 0.001, 0.05, respectively), but co-occurred with PIK3R1 alterations (Log2(OR) = − 1.6, p-adj = 0.03). Of the 63 scorable sporadic GBMs in the TMA, 14 harbored NF1 inactivating alterations and of those, 12 (86%) demonstrated minimal NF1 immunoreactivity by NFC antibody, compared to 8 (57%) by iNF-07E antibody. Among the 42 scorable NF1-wildtype GBM in the TMA, NF1 immunostaining was minimal in 18 (43%) by NFC antibody compared to 4 (10%) by iNF-07E antibody, potentially reflecting false positives or differential protein regulation. Minimal immunoreactivity by NFC antibody was associated with decreased median overall survival (8.5 vs. 16.4 months, p = 0.011). Cox proportional hazards model correcting for prognostic variables in this subset revealed HR 3.23 (95% CI 1.29–8.06, p = 0.01) associated with decreased NF1 expression by IHC.ConclusionNF1 immunostaining may serve as a sensitive surrogate marker of NF1 genomic inactivation and a valuable extension to next-generation sequencing for defining NF1 status. Minimal NF1 immunoreactivity is a poor prognostic marker, even in IDH-wildtype glioblastoma without apparent NF1 genomic alterations, but the underlying molecular mechanism requires further investigation.

Bartonella infections are rare in blood-fed Ixodes scapularis and Ixodes pacificus ticks collected from rodents in the United States

BackgroundIxodes scapularis and Ixodes pacificus are important vectors of multiple pathogens in the United States. However, their role in transmission of Bartonella spp., which are commonly reported in rodents and fleas, has been debated. Our previous investigation on Bartonella spp. in host-seeking I. scapularis and I. pacificus showed Bartonella spp. were absent in the ticks, suggesting the two species are unlikely to contribute to Bartonella transmission. It is unclear whether the absence of Bartonella spp. in the host-seeking ticks was attributable to ticks not being exposed to Bartonella in nature or being exposed but unable to acquire or transstadially transmit the bacterium. To assess the likelihood of exposure and acquisition, we tested Ixodes spp. ticks collected from rodents for Bartonella infections.MethodsBlood-fed I. scapularis ticks (n = 792; consisting of 645 larvae and 147 nymphs), I. pacificus ticks (n = 45, all larvae), and Ixodes angustus ticks (n = 16, consisting of 11 larvae and 5 nymphs) collected from rodents from Minnesota and Washington were tested for Bartonella spp. using a quadruplex polymerase chain reaction (PCR) amplicon next-generation sequencing approach that targets Bartonella-specific fragments on gltA, ssrA, rpoB, and groEL. In parallel, rodents and fleas collected from the same field studies were investigated to compare the differences of Bartonella distribution among the ticks, fleas, and rodents.ResultsBartonella spp. were commonly detected in rodents and fleas, with prevalence of 25.6% in rodents and 36.8% in fleas from Minnesota; 27.9% in rodents and 45.2% in fleas from Washington. Of all tested ticks, Bartonella DNA was detected by gltA in only one larval I. scapularis tick from Minnesota.ConclusionsThe high prevalence of Bartonella spp. in rodents and fleas coupled with extremely low prevalence of Bartonella spp. in blood-fed ticks suggests that although Ixodes ticks commonly encounter Bartonella in rodents, they rarely acquire the infection through blood feeding. Notably, ticks were at various stages of feeding on rodents when they were collected. Laboratory transmission studies are needed to assess acquisition rates in fully blood-fed ticks and to assess transstadial transmission efficiency if ticks acquire Bartonella infections from feeding to repletion.Graphical

Oral supplementation of heat-killed Enterococcus faecalis strain EC-12 relieves gastrointestinal discomfort and alters the gut microecology in academically stressed students.

Stress significantly affects gastrointestinal and mental health, and the gut microbiota plays a pivotal role in this process. Enterococcus faecalis strain EC-12 (EC-12) is a lactic acid bacterium that has several health benefits. To investigate the impact of oral supplementation with heat-killed EC-12 on the discomfort caused by stress, a randomised, double-blind, placebo-controlled trial was conducted with students under academic stress taking EC-12 (n= 14) or a placebo (n= 13) daily for one week. Improvement in the students' symptoms was assessed using the visual analogue scale. Faecal microbiota was characterised by next-generation sequencing of 16S rRNA genes, and faecal metabolites and short-chain fatty acids were analysed using a GC-MS metabolomics approach. Significant improvements in abdominal pain and rumbling of the stomach were found in the EC-12 group compared to the placebo group, but no changes were observed in mental symptoms or salivary cortisol levels. The relative abundance of E. faecalis significantly increased in the EC-12 group after the trial; however, the composition and diversity of the gut microbiota did not change significantly. Functional analysis of the gut microbiota suggested that EC-12 intake alters specific metabolic pathways. Although the levels of faecal short-chain fatty acids did not change between the groups before and after the trial, EC-12 intake altered the composition of faecal metabolites, with a significant increase in tryptamine levels. The ratio of students with improved symptoms to those with increased tryptamine levels was calculated based on the number of students with elevated faecal tryptamine levels who showed symptomatic improvements. The ratio of improved rumbling stomach was higher than that of other types of digestive discomfort. These results suggest that oral supplementation with EC-12 has a potentially beneficial effect on stress-induced gastrointestinal discomfort, which may occur through alterations in gut microbiota composition and metabolism. This study was registered at the University Hospital Medical Information Network Center (UMIN) under the UMIN ID: UMIN000048184.

Mixed Leptospira infections in domestic animals from a rural community with high leptospirosis endemicity.

Leptospirosis is one of the most common zoonoses in the world which is associated with a severe febrile disease in humans causing a variety of syndromes including meningitis, interstitial nephritis, hepatitis, and sometimes death. Leptospirosis is caused by different pathogenic Leptospira species divided into almost 30 serogroups and more than 300 serovars which are carried by some animal asymptomatic chronic infections. Humans can become infected through direct contact with animal urine or indirectly by coming into contact with fresh water or mud contaminated with urine. In this research, we looked for leptospiral DNA in urine samples from dogs living in a rural, low-income and highly endemic community in the coast of Ecuador. We used molecular biology and next-generation sequencing for the detection. Our results showed evidence of two Leptospira species, L interrogans and L. santarosai, genomes in three dogs. It has been widely known that animal carriers are typically infected with a single leptospiral strain. However, recent reports, including the present one, indicate that carrier animals may be coinfected with two or more leptospiral species.

High prevalence of cardiotropic viruses in endomyocardial biopsies from patients with inflammatory cardiomyopathy demonstrated by a novel targeted NGS approach

Abstract Background Viral infection of the heart muscle is a common cause of myocarditis and viral persistence can lead to chronic inflammatory cardiomyopathies (DCMi). A number of viruses including parvovirus B19 (B19V), adenovirus (AdV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes virus 6 (HHV-6) and enteroviruses (EV) are known to infect the myocardium, but their prevalence and role in persisting DCMi are not well understood. Virological etiologies can only be determined via endomyocardial biopsy (EMB), but the low tissue amount limits the number of conventional viral nucleic acid tests (virus-specific PCR) that can be performed in parallel. This study aimed to detect known and novel cardiotropic viruses in EMB of DCMi patients using a targeted next generation sequencing (NGS) approach. Methods A cohort of 33 patients with inflammatory cardiomyopathy (DCMi) (LVEF=29±12%) were retrospectively analyzed and compared to previous results of virus-specific PCR. DNA and RNA extracted from EMB were used for sequencing library preparation, and viral nucleic acids were enriched via a custom myBaits hybridization capture approach. Enriched libraries were sequenced on an Illumina MiSeq-platform in paired-end mode (500.000 reads/sample), and reads were taxonomically classified via Kraken2 and mapped against viral reference genomes using BWA-MEM2 or HiSat2, and subsequently deduplicated. Viral reads detected via both Kraken2 and mapping were classified as valid. Results Targeted NGS confirmed 19 out of 22 (86%) of the previous PCR-based viral detections. However, NGS was able to detect B19V DNA and RNA in an additional 11 and 15 patient samples respectively; CMV DNA/RNA in additional 5 and 4 samples respectively; EBV DNA/RNA in additional 5 and 1 samples respectively; and HHV-6 DNA/RNA in additional 3 and 1 samples respectively. Furthermore, viral reads of adenovirus type 2, adenovirus-associated virus type 2 (AAV-2), herpes viruses 7 and 8, and human parvovirus 4 were also found among the cohort. Thus, targeted NGS was able to identify 82 viral infections in a cohort of 33 DCMi patients, while only 22 were found using routine PCR tests. For routinely tested B19V, the use of the new NGS diagnostic approach improved sensitivity by 42% and specificity by 72%. Conclusions The presented NGS method represents a powerful new tool for the diagnosis of viral and inflammatory heart disease. The sensitivity is significantly higher than with conventional PCR approaches and the taxonomic classification is more specific. This enables the detection of previously false-negative routine viruses as well as potentially new cardiotropic pathogens from limited bioptic patient material – thus significantly increasing the diagnostic yield of EMB. In addition, our study demonstrates a high prevalence of non-enteroviral cardiotropic viruses in DCMi patients, shedding new light on the etiology of this heterogenous diseases and paving the way for more specific future treatments.