Rapid diagnosis of Capnocytophaga canimorsus septic shock in an immunocompetent individual using real-time Nanopore sequencing: a case report.

BACKGROUND: Rapid diagnosis and appropriate treatment is imperative in bacterial sepsis due increasing risk of mortality with every hour without appropriate antibiotic therapy. Atypical infections with fastidious organisms may take more than 4 days to diagnose leading to calls for improved methods for rapidly diagnosing sepsis. Capnocytophaga canimorsus is a slow-growing, fastidious gram-negative bacillus which is a common commensal within the mouths of dogs, but rarely cause infections in humans. C. canimorsus sepsis risk factors include immunosuppression, alcoholism and elderly age. Here we report on the application of emerging nanopore sequencing methods to rapidly diagnose an atypical case of C. canimorsus septic shock. CASE PRESENTATION: A 62 year-old female patient was admitted to an intensive care unit with septic shock and multi-organ failure six days after a reported dog bite. Blood cultures were unable to detect a pathogen after 3 days despite observed intracellular bacilli on blood smears. Real-time nanopore sequencing was subsequently employed on whole blood to detect Capnocytophaga canimorsus in 19 h. The patient was not immunocompromised and did not have any other known risk factors. Whole-genome sequencing of clinical sample and of the offending dog's oral swabs showed near-identical C. canimorsus genomes. The patient responded to antibiotic treatment and was discharged from hospital 31 days after admission. CONCLUSIONS: Use of real-time nanopore sequencing reduced the time-to-diagnosis of Capnocytophaga canimorsus in this case from 6.25 days to 19 h. Capnocytophaga canimorsus should be considered in cases of suspected sepsis involving cat or dog contact, irrespective of the patient's known risk factors.

GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing.

BACKGROUND: Tandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations. We report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely - GtTR - which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation. RESULTS: We used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68 and 83% for capture sequence data and 200X WGS data respectively, improving to 87 and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25, 14, 12 and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results. CONCLUSIONS: The novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.

npInv: accurate detection and genotyping of inversions using long read sub-alignment.

BACKGROUND: Detection of genomic inversions remains challenging. Many existing methods primarily target inzversions with a non repetitive breakpoint, leaving inverted repeat (IR) mediated non-allelic homologous recombination (NAHR) inversions largely unexplored. RESULT: We present npInv, a novel tool specifically for detecting and genotyping NAHR inversion using long read sub-alignment of long read sequencing data. We benchmark npInv with other tools in both simulation and real data. We use npInv to generate a whole-genome inversion map for NA12878 consisting of 30 NAHR inversions (of which 15 are novel), including all previously known NAHR mediated inversions in NA12878 with flanking IR less than 7kb. Our genotyping accuracy on this dataset was 94%. We used PCR to confirm the presence of two of these novel inversions. We show that there is a near linear relationship between the length of flanking IR and the minimum inversion size, without inverted repeats. CONCLUSION: The application of npInv shows high accuracy in both simulation and real data. The results give deeper insight into understanding inversion.

Real-time demultiplexing Nanopore barcoded sequencing data with npBarcode.

MOTIVATION: The recent introduction of a barcoding protocol for Oxford Nanopore sequencing has increased the versatility of the technology. Several bioinformatics tools have been developed to demultiplex barcoded reads, but none of them supports streaming analysis. This limits the use of multiplexed sequencing in real-time applications, which is one of the main advantages of the technology. RESULTS: We introduced npBarcode, an open source and cross-platform tool for barcode demultiplexing in streaming fashion that can be used to pipe data to further real-time analyses. The tool also provides a friendly graphical user interface by integrating the module into npReader, making possible to monitor the progress concurrently when the sequencing is still in progress. We show that our algorithm achieves accuracies at least as good as competing tools. AVAILABILITY AND IMPLEMENTATION: npBarcode is bundled in Japsa-a Java tools kit for genome analysis, and is freely available at https://github.com/mdcao/japsa. CONTACT: s.nguyen@uq.edu.au or l.coin@imb.uq.edu.au. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetic variants in ICAM1, PPARGC1A and MTHFR are potentially associated with different phenotypes of diabetic retinopathy.

PURPOSE: To explore phenotype-genotype correlations that may contribute to a better understanding of diabetic retinopathy (DR). PROCEDURES: An exploratory association study was performed to identify genetic variants associated with non-proliferative DR (NPDR) in 307 type 2 diabetic patients who were previously stratified into 3 different phenotypes of NPDR progression. The 307 patients were genotyped for 174 single nucleotide polymorphisms of 11 candidate genes (ACE, AGER, AKR1B1, ICAM1, MTHFR, NOS1, NOS3, PPARGC1A, TGFB1, TNF and VEGFA). RESULTS: Significant associations were observed for PPARGC1A rs16874120 with phenotype A (odds ratio, OR = 0.60, 95% confidence interval, CI 0.36-0.99), ICAM1 rs1801714 with phenotype B (OR = 3.32, 95% CI 1.05-10.50) and both PPARGC1A rs10213440 (OR = 2.00, 95% CI 1.07-3.73) and MTHFR rs1801133 (OR = 1.84, 95% CI 1.08-3.11) with phenotype C. CONCLUSIONS: RESULTS indicate that specific gene variants in ICAM1, PPARGC1A and MTHFR are associated with different NPDR phenotypes, being likely candidates to explain different disease mechanisms underlying the different phenotypes. This is the first study to show correlations between specific gene variants and NPDR phenotypes, opening new perspectives on DR.

The Use of Herbal Medicines Among Cancer Patients.

Background and objective The use of herbal medicines has been increasing among cancer patients, as a way to control cancer and treatment-related symptoms; however, many patients are reluctant to disclose this use to their medical practitioners. The fact that oncological treatments have a narrow therapeutic margin, associated with the lack of control and clinical evidence concerning these supplements, makes medication-herbal interactions a reality. These interactions could lead to increased toxicity or a decreased effectiveness of oncological treatment. In light of this, we aimed to assess the prevalence of herbal medicine use in a patient population at a Portuguese central hospital: Centro Hospitalar Lisboa Ocidental. Materials and methods Patients with breast, prostate, or colorectal cancer diagnoses between August 2022 and July 2023 and undergoing oncological treatment were included. Data were collected through a survey during their first appointment, as well as by consulting the patients' clinical files. An interaction evaluation was carried out to assess potential medication-herbal interactions. Finally, a statistical analysis was performed to identify predictive factors for the use of herbal medicines. Results Among the 65 patients included in the study, 52% were females, and the median age of the cohort was 65 years. Breast cancer was the most prevalent diagnosis and the majority of the patients were undergoing palliative treatment. We found that 46% of patients used herbal medicines regularly: to strengthen the immune system, detoxification of the body, and treat insomnia and constipation. A medication-herbal interaction was found in 37% of the cases, the most frequent being doxorubicin-vitamin C, through an antioxidant mechanism. The univariable analysis failed to show any predictive factors associated with the use of herbal medicines. Conclusions This study sheds light on herbal medicine use among cancer patients and the reality of medication-herbal interactions. There is an urgent need for further research and evidence-based medical protocols regarding herbal medicine use, especially in complex cases such as cancer patients, to provide better and safer care.

Towards intellectual freedom in an AI Ethics Global Community.

The recent incidents involving Dr. Timnit Gebru, Dr. Margaret Mitchell, and Google have triggered an important discussion emblematic of issues arising from the practice of AI Ethics research. We offer this paper and its bibliography as a resource to the global community of AI Ethics Researchers who argue for the protection and freedom of this research community. Corporate, as well as academic research settings, involve responsibility, duties, dissent, and conflicts of interest. This article is meant to provide a reference point at the beginning of this decade regarding matters of consensus and disagreement on how to enact AI Ethics for the good of our institutions, society, and individuals. We have herein identified issues that arise at the intersection of information technology, socially encoded behaviors, and biases, and individual researchers' work and responsibilities. We revisit some of the most pressing problems with AI decision-making and examine the difficult relationships between corporate interests and the early years of AI Ethics research. We propose several possible actions we can take collectively to support researchers throughout the field of AI Ethics, especially those from marginalized groups who may experience even more barriers in speaking out and having their research amplified. We promote the global community of AI Ethics researchers and the evolution of standards accepted in our profession guiding a technological future that makes life better for all.

Complete Genome Sequences of Clinical Pandoraea fibrosis Isolates.

Pandoraea fibrosis is a newly identified Gram-negative bacterial species that was isolated from the respiratory tract of an Australian cystic fibrosis patient. The complete assembled genome sequences of two consecutive isolates (second isolate collected 11 months after antibiotic treatment) from the same individual are presented here.

Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing.

BACKGROUND: Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR). RESULTS: The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network-based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with ∼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, ß-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes. CONCLUSION: Overall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.

Retooling phage display with electrohydrodynamic nanomixing and nanopore sequencing.

Phage display methodologies offer a versatile platform for the isolation of single-chain Fv (scFv) molecules which may be rebuilt into monoclonal antibodies. Herein, we report on a complete workflow termed PhageXpress, for rapid selection of single-chain Fv sequences by leveraging electrohydrodynamic-manipulation of a solution containing phage library particles to enhance target binding whilst minimizing non-specific interactions. Our PhageXpress technique is combined with Oxford Nanopore Technologies' MinION sequencer and custom bioinformatics to achieve high-throughput screening of phage libraries. We performed 4 rounds of biopanning against Dengue virus (DENV) non-structural protein 1 (NS1) using traditional methods (4 week turnaround), which resulted in the isolation of 19 unique scFv clones. We validated the feasibility and efficiency of the PhageXpress method utilizing the same phage library and antigen target. Notably, we successfully mapped 14 of the 19 anti-NS1 scFv sequences (∼74%) with our new method, despite using ∼30-fold less particles during screening and conducting only a single round of biopanning. We believe this approach supersedes traditional methods for the discovery of bio-recognition molecules such as antibodies by speeding up the process for the development of therapeutic and diagnostic biologics.

Chiron: translating nanopore raw signal directly into nucleotide sequence using deep learning.

Sequencing by translocating DNA fragments through an array of nanopores is a rapidly maturing technology that offers faster and cheaper sequencing than other approaches. However, accurately deciphering the DNA sequence from the noisy and complex electrical signal is challenging. Here, we report Chiron, the first deep learning model to achieve end-to-end basecalling and directly translate the raw signal to DNA sequence without the error-prone segmentation step. Trained with only a small set of 4,000 reads, we show that our model provides state-of-the-art basecalling accuracy, even on previously unseen species. Chiron achieves basecalling speeds of more than 2,000 bases per second using desktop computer graphics processing units.

A complete high-quality MinION nanopore assembly of an extensively drug-resistant Mycobacterium tuberculosis Beijing lineage strain identifies novel variation in repetitive PE/PPE gene regions.

A better understanding of the genomic changes that facilitate the emergence and spread of drug-resistant Mycobacterium tuberculosis strains is currently required. Here, we report the use of the MinION nanopore sequencer (Oxford Nanopore Technologies) to sequence and assemble an extensively drug-resistant (XDR) isolate, which is part of a modern Beijing sub-lineage strain, prevalent in Western Province, Papua New Guinea. Using 238-fold coverage obtained from a single flow-cell, de novo assembly of nanopore reads resulted into one contiguous assembly with 99.92 % assembly accuracy. Incorporation of complementary short read sequences (Illumina) as part of consensus error correction resulted in a 4 404 064 bp genome with 99.98 % assembly accuracy. This assembly had an average nucleotide identity of 99.7 % relative to the reference genome, H37Rv. We assembled nearly all GC-rich repetitive PE/PPE family genes (166/168) and identified variants within these genes. With an estimated genotypic error rate of 5.3 % from MinION data, we demonstrated identification of variants to include the conventional drug resistance mutations, and those that contribute to the resistance phenotype (efflux pumps/transporter) and virulence. Reference-based alignment of the assembly allowed detection of deletions and insertions. MinION sequencing provided a fully annotated assembly of a transmissible XDR strain from an endemic setting and showed its utility to provide further understanding of genomic processes within Mycobacterium tuberculosis.

Triclosan at environmentally relevant concentrations promotes horizontal transfer of multidrug resistance genes within and across bacterial genera.

BACKGROUND: Antibiotic resistance poses an increasing threat to public health. Horizontal gene transfer (HGT) promoted by antibiotics is recognized as a significant pathway to disseminate antibiotic resistance genes (ARGs). However, it is unclear whether non-antibiotic, anti-microbial (NAAM) chemicals can directly promote HGT of ARGs in the environment. OBJECTIVES: We aimed to investigate whether triclosan (TCS), a widely-used NAAM chemical in personal care products, is able to stimulate the conjugative transfer of antibiotic multi-resistance genes carried by plasmid within and across bacterial genera. METHODS: We established two model mating systems, to investigate intra-genera transfer and inter-genera transfer. Escherichia coli K-12 LE392 carrying IncP-α plasmid RP4 was used as the donor, and E. coli K-12 MG1655 or Pseudomonas putida KT2440 were the intra- and inter-genera recipients, respectively. The mechanisms of the HGT promoted by TCS were unveiled by detecting oxidative stress and cell membrane permeability, in combination with Nanopore sequencing, genome-wide RNA sequencing and proteomic analyses. RESULTS: Exposure of the bacteria to environmentally relevant concentrations of TCS (from 0.02 µg/L to 20 µg/L) significantly stimulated the conjugative transfer of plasmid-encoded multi-resistance genes within and across genera. The TCS exposure promoted ROS generation and damaged bacterial membrane, and caused increased expression of the SOS response regulatory genes umuC, dinB and dinD in the donor. In addition, higher expression levels of ATP synthesis encoding genes in E. coli and P. putida were found with increased TCS dosage. CONCLUSIONS: TCS could enhance the conjugative ARGs transfer between bacteria by triggering ROS overproduction at environmentally relevant concentrations. These findings improve our awareness of the hidden risks of NAAM chemicals on the spread of antibiotic resistance.

Insights into population structure of East African sweetpotato cultivars from hybrid assembly of chloroplast genomes.

Background: The chloroplast (cp) genome is an important resource for studying plant diversity and phylogeny. Assembly of the cp genomes from next-generation sequencing data is complicated by the presence of two large inverted repeats contained in the cp DNA. Methods: We constructed a complete circular cp genome assembly for the hexaploid sweetpotato using extremely low coverage (<1×) Oxford Nanopore whole-genome sequencing (WGS) data coupled with Illumina sequencing data for polishing. Results: The sweetpotato cp genome of 161,274 bp contains 152 genes, of which there are 96 protein coding genes, 8 rRNA genes and 48 tRNA genes. Using the cp genome assembly as a reference, we constructed complete cp genome assemblies for a further 17 sweetpotato cultivars from East Africa and an I. triloba line using Illumina WGS data. Analysis of the sweetpotato cp genomes demonstrated the presence of two distinct subpopulations in East Africa. Phylogenetic analysis of the cp genomes of the species from the Convolvulaceae Ipomoea section Batatas revealed that the most closely related diploid wild species of the hexaploid sweetpotato is I. trifida. Conclusions: Nanopore long reads are helpful in construction of cp genome assemblies, especially in solving the two long inverted repeats. We are generally able to extract cp sequences from WGS data of sufficiently high coverage for assembly of cp genomes. The cp genomes can be used to investigate the population structure and the phylogenetic relationship for the sweetpotato.

Follow up studies on the respiratory pattern and total cholinesterase activities in dichlorvos-poisoned rats.

A human prospective study confirmed that the severity and time-course of organophosphate poisonings depend on the compound. Our purpose was to assess the ventilation at rest and cholinesterase activities from 5min to 72h in rats poisoned with dichlorvos at 40% of the MLD (5.12mg/kg). Ventilation at rest was recorded by whole body plethysmography and core temperature by infrared telemetry (DSI system). Results are expressed as mean±SEM. Statistical analyses used two-way ANOVA. Dichlorvos induced the onset of respiratory effects within 5min and hypothermia which peaked at 15min, both reversed within 90min post-injection. Dichlorvos significantly decreased respiratory frequency, resulting from an increase in expiratory time and associated with increased tidal volume. Tissues and whole blood cholinesterase activities were significantly decreased until the end of experiment. Our study showed that an inhibition of cholinesterase was correlated with an effect on respiratory functions at 15min and 60min. However, 24h post-poisoning, the increase in cholinesterase activity was not completed while ventilatory parameters were within the normal range. Respiratory effects were both qualitatively and quantitatively similar to those induced by diethylparaoxon. However the effects strongly differed between diethylparaoxon lasting hours while dichlorvos lasted tens of minutes.

Buprenorphine alters desmethylflunitrazepam disposition and flunitrazepam toxicity in rats.

High-dosage buprenorphine (BUP) consumed concomitantly with benzodiazepines (BZDs) including flunitrazepam (FZ) may cause life-threatening respiratory depression despite a BUP ceiling effect and BZDs' limited effects on ventilation. However, the mechanism of BUP/FZ interaction remains unknown. We hypothesized that BUP may alter the disposition of FZ active metabolites in vivo, contributing to respiratory toxicity. Plasma FZ, desmethylflunitrazepam (DMFZ), and 7-aminoflunitrazepam (7-AFZ) concentrations were measured using gas chromatography-mass spectrometry. Intravenous BUP 30 mg/kg pretreatment did not alter plasma FZ and 7-AFZ kinetics in Sprague-Dawley rats infused with 40 mg/kg FZ over 30 min, whereas resulting in a three-fold increase in the area under the curve (AUC) of DMFZ concentrations compared with control (p < 0.01). In contrast, BUP did not significantly modify plasma DMFZ concentrations after intravenous infusion of 7 mg/kg DMFZ, whereas resulting in a similar peak concentration to that generated from 40 mg/kg FZ administration. Regarding the effects on ventilation, BUP (30 mg/kg) as well as its combination with FZ (0.3 mg/kg) significantly increased PaCO(2), whereas only BUP/FZ combination decreased PaO(2) (p < 0.001). Interestingly, FZ (40 mg/kg) but not DMFZ (40 mg/kg) significantly increased PaCO(2) (p < 0.05), whereas DMFZ but not FZ decreased PaO(2) (p < 0.05). Thus, decrease in PaO(2) appears related to BUP-mediated effects on DMFZ disposition, although increases in PaCO(2) relate to direct BUP/FZ additive or synergistic dynamic interactions. We conclude that combined high-dosage BUP and FZ is responsible for increased respiratory toxicity in which BUP-mediated alteration in DMFZ disposition may play a significant role.

Enfrentando a mastectomia: análise dos relatos de mulheres mastectomizadas sobre questöes ligadas à sexualidade / Facing mastectomy: analysis of the discourse of mastectomized women about questions of sexuality

Este trabalho se propôs a investigar como seis mulheres mastectomizadas, com idades de 37 a 55 anos, e com tempo de cirurgia de 1 ano e meio a 8 anos, percebiam a sua sexualidade. Para exame dos relatos foi aplicada a técnica de análise de conteúdo, em que foram identificadas as seguintes categorias: informaçäo sobre a doença, reaçäo ao diagnóstico, relaçäo médico-paciente, eu e o meu corpo, eu e o olhar do outro, percepçäo de si mesma e relacionamentos amorosos.A análise dessas categorias demonstrou que, após a mastectomia, as mulheres apresentaram algumas limitaçöes e dificuldades em lidar com situaçöes que envolviam a exposiçäo do próprio corpo. No entanto, apesar dos temores, algumas mulheres produziram diversos modos de (re)significarem e expressarem a sua sexualidade de uma forma potencializadora para suas relaçöes cotidianas.