Rafi Ahmad

• Mahar, Nirmal Singh; Branders, Sverre; Grabherr, Manfred; Gupta, Ishaan & Ahmad, Rafi (2026). K-MARVEL: K-Mer-based antimicrobial resistance virtual exploration lab. Nature Communications. doi: 10.1038/s41467-026-77438-8. Fulltekst i vitenarkiv Vis sammendrag

  Abstract The rapid spread of antimicrobial resistance (AMR) necessitates new computational surveillance tools. Current methods for analysis of next-generation sequencing data have trade-offs: assembly-based approaches are computationally intensive, and direct long-read mapping is hampered by high error rates that obscure resistance-conferring mutations. Here, we present K-MARVEL, an open-source method that captures antimicrobial resistance genes (ARGs) and resistance-conferring mutations from both short- and long-read datasets. Operating in protein k-mer space, K-MARVEL tolerates nucleotide-level sequencing errors. We benchmarked K-MARVEL on 209 long-read and 205 short-read datasets across 22 bacterial species and achieved F1-scores of 0.976 (short-read) and 0.958 (long-read), outperforming assembly-based methods in speed and memory usage. K-MARVEL had higher F1-scores than seven widely used short-read-based ARG classifiers (0.979) and two widely used long-read-based classifiers (0.961) for homology-model-based ARGs. K-MARVEL can accurately identify both homologous ARGs and structural genes containing resistance-conferring mutations, including multiple variants, directly from raw sequencing data.

• Branders, Sverre; Grabherr, Manfred & Ahmad, Rafi (2026). Mango: unearthing patterns in large-scale biological data through interactive correlation analysis | BMC Genomics | Springer Nature Link. BMC Genomics. doi: 10.1186/s12864-026-13249-x. Fulltekst i vitenarkiv Vis sammendrag

  Integrating different types of biological data is often challenging due to the presence of both numerical and categorical data. This complexity makes it harder to evaluate causal biological effects, especially when confounders like population structure, sampling methods, or multi-omics integration can lead to incorrect conclusions. We introduce Mango, an interactive correlation browser designed for visually exploring any tabular data type, regardless of their distribution. Our results on genomic and transcriptomic datasets demonstrate that these correlations can effectively distinguish between biases and causal relationships in large-scale data. Mango uses a novel algorithm for non-parametric correlation of numeric to categorical data called Median-Ranked Label Encoding and is implemented in C++/Java and supported on Linux. Source code is available under the GNU GPLv3 license from [1].

• Sverre, Branders,; Grabherr, Manfred & Ahmad, Rafi (2026). Voyager: rapid taxonomic characterization of long-read mixed-species sequencing samples in sorted motif distance space | Genome Biology | Springer Nature Link. Genome Biology. ISSN 1474-7596. doi: 10.1186/s13059-026-04235-3. Fulltekst i vitenarkiv Vis sammendrag

  Voyager is a novel algorithm that complements real-time sequencing by rapidly mapping long sequencing reads with insertion and deletion errors to a known database of organisms. It utilizes Sorted Motif Distance Space, distances between exact matches of short motifs sorted by rank, to represent sequence similarity in a highly compressed, computationally efficient manner while enabling strain-level discrimination. Voyager incorporates a deconvolution algorithm to discern closely related genomes. It achieves an average species-level accuracy of 99% across 15 datasets, outperforming all evaluated methods. Voyager maintains CPU runtimes comparable to the current fastest methods while using less than half the memory.

• Ali, Jawad; Bellankimath, Anurag Basavaraj; Hira, Jonathan; Chapagain, Crystal; Opgård, Silje Therese & Simonsen, Gunnar Skov [Vis alle 7 forfattere av denne artikkelen] (2026). Development and optimization of host DNA depletion in blood cultures using a saponin and salt-activated nuclease-based method. Frontiers in Microbiology. 17. doi: 10.3389/fmicb.2026.1784408. Fulltekst i vitenarkiv Vis sammendrag

  Early diagnosis of sepsis and bloodstream infections is essential to reduce mortality and combat antimicrobial resistance. However, current diagnostic methods are slow and laborious. Nanopore sequencing has the potential to be a rapid diagnostic tool for sepsis. Nonetheless, extracting bacterial DNA from blood samples remains challenging due to the abundance of host DNA and the presence of inhibitory substances. This study aimed to develop and optimize a method using saponin and salt-activated nucleases (SAN) for efficient host DNA depletion in blood cultures. Different saponin concentrations (2%–5%), HL-SAN and M-SAN nucleases, and various salt conditions (NaCl and MgCl2) were tested for their ability to reduce host DNA in Escherichia coli and Staphylococcus aureus blood cultures. The effects of bead-beating durations of 6 and 10 min on DNA fragment size distribution were also evaluated. The optimized method was validated using spiked and clinical blood cultures of the most prevalent BSI causing pathogens. Results showed that 4% of saponin treatment combined with 250 units (10 μL) of SAN nucleases achieved efficient host DNA depletion in spiked blood cultures. Compared to undepleted samples, HL-SAN achieved up to 38-fold reduction in host DNA for both E. coli and S. aureus. Likewise, M-SAN resulted in ~10-fold reduction in E. coli and ~4-fold reduction in S. aureus samples. Although SAN enzymes combined with higher salt concentrations showed slightly better host DNA removal, the difference was not statistically significant. Shortening the bead-beating time to 6 min improved the recovery of longer DNA fragments compared to 10 min. The optimized method validated using spiked and clinical-positive blood cultures, demonstrated efficient host depletion and bacterial DNA recovery.

• Wei, Qian; Hajjar, Ehsan; Cornillot‐Clément, Selma; Solli, Elise; Diaz, Nuria Garcia & Landskron, Johannes [Vis alle 9 forfattere av denne artikkelen] (2026). High-Throughput Phenotypic Screen to Identify FoxP3 Regulators in Primary T Cells. ACS Chemical Biology. ISSN 1554-8929. 21(4), s. 764–778. doi: 10.1021/acschembio.5c01019. Fulltekst i vitenarkiv Vis sammendrag

  FoxP3+ regulatory T cells (Tregs) maintain immune homeostasis by suppressing excessive antiself-immunity of effector T cells (Teffs) and thereby regulate autoimmunity and inhibit antitumor immune responses. Thus, Treg-targeting is a popular therapeutic strategy for both autoimmune diseases and cancer treatment. However, specific regulators of Tregs are needed to bypass adverse effects elicited by strategies that target molecules with overlapping expression between Tregs and other T cell subsets. We aim to identify small molecules that specifically target the key Treg lineage-defining transcription factor FoxP3. Here, we describe a high-throughput, flow-cytometry-based phenotypic screening assay, its data processing pipeline, and downstream methods for compound validation. We developed a set of algorithms to easily exclude compounds with high toxicity and to filter out autofluorescent compounds. Our screening assay is performed on human primary T cells by measuring FoxP3 levels in CD4+ T cells, using an automated liquid handling pipeline. Selected hit candidates from the screening were validated and assessed through functional analyses. From screening a library of approved drugs, we identified several candidates that regulate Treg functions through FoxP3. Seventeen chemical analogs of one hit compound were identified and characterized in cell-based assays, revealing structure–activity relationships and identifying compounds with more potent effects on the regulation of FoxP3 levels and Treg function. We conclude that this novel screening method could successfully identify FoxP3 regulators with an effect on Treg functions.

• Giacobbe, Daniele Roberto; Ahmad, Rafi; Akilli, Fatih Mehmet; Ascandari, Abdulaziz; Eyre, David W. & Gallardo-Pizarro, Antonio [Vis alle 17 forfattere av denne artikkelen] (2026). How ready are we to use artificial intelligence in our fight against Antimicrobial resistance? An ESGAID and EAAS perspective. Expert Review of Anti-Infective Therapy. ISSN 1478-7210. 24(1), s. 39–61. doi: 10.1080/14787210.2026.2625382. Fulltekst i vitenarkiv Vis sammendrag

  Introduction Antimicrobial resistance (AMR) remains one of the greatest threats to global health, requiring innovative approaches to antibiotic discovery, surveillance, diagnosis, and prescribing. In recent years, artificial intelligence (AI) has increasingly been applied across these domains, with the dual aim of accelerating research and strengthening antimicrobial stewardship. Areas covered This perspective summarizes current advances and challenges in applying AI for tackling AMR. We examine the role of AI in antibiotic discovery, laboratory surveillance, diagnosis of resistant infections, and clinical decision support systems. Finally, we address the ethical and regulatory landscape, data transparency, and liability concerns. Expert opinion AI offers unprecedented opportunities across the continuum of our efforts to counteract AMR, yet its adoption faces substantial hurdles. Some central challenges include the balance between model accuracy and explainability, the lack of widespread digital access, quality and transparency of training datasets, and usability for clinicians. Progress will depend on multidisciplinary collaboration, robust regulatory oversight, and the development of training programs equipping future healthcare professionals with AI-aware reasoning skills. Ultimately, AI should not replace but rather augment human reasoning in the fight against AMR, aligning innovation with ethical principles to ensure safer, more equitable AI-enhanced antibiotic prescribing and antimicrobial stewardship.

• Hira, Jonathan; Mahbub, Nasib Bin; Ali, Jawad & Ahmad, Rafi (2025). Low-cost in-house re-formulated brain heart infusion medium for effective planktonic growth and early detection of bloodstream bacterial pathogens. Frontiers in Microbiology. 16. doi: 10.3389/fmicb.2025.1680006. Fulltekst i vitenarkiv Vis sammendrag

  Sepsis, a clinically defined life-threatening condition, is a global contributor to high morbidity and mortality rates in humans. It is caused by systemic bloodstream bacterial infections, primarily involving aerobic pathogens such as Escherichia coli , Staphylococcus aureus , and Klebsiella pneumoniae . Rapid and accurate identification of these pathogens is a high-demand task, as prolonged diagnosis may increase the mortality rate among sepsis patients. Globally, commercial blood culture systems like the BD BACTEC™ FX blood culture system, which utilizes BD BACTEC™ PLUS Aerobic/F culture bottles (used in this study), are commonly used to detect aerobic bloodstream infections. However, due to high costs (∼$10.00–$15.00/bottle), limited availability of culture media (especially in low- and middle-income countries, and war zones), and a lack of customization for antibiotic susceptibility assay and epidemiology research, there is a need for secondary alternatives to facilitate the growth and identification of bloodborne pathogens. Therefore, we developed a low-cost (∼$4–$5/bottle) in-house culture medium with a newly improved formulation of Brain Heart Infusion media that enhances bacterial growth from spiked human blood tested on a panel of bacteria ( Escherichia coli , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterococcus faecalis ). The growth dynamics of these microbes in in-house formulated BHI-Blood+ culture media coincide with those in BACTEC™ Plus Aerobic/F culture vials, which primarily suggests the compatibility of bloodborne pathogens with this media and can be flagged positive <8 h based on cellular growth rate. Additionally, conventional qPCR-based early detection (<24 h) and validation with the Oxford Nanopore MinION NGS platform highlight the value of this in-house culture media as an alternative to commercial culture media in terms of low-cost availability.

• Chapagain, Crystal; Khezri, Abdolrahman; Ali, Jawad; Smistad, Marit; Sølverød, Liv Synnøve & Ahmad, Rafi (2025). Optimizing sample preparation for culture-free nanopore sequencing to enable rapid pathogen and antimicrobial resistance profiling in bovine mastitis. Frontiers in Microbiology. 16. doi: 10.3389/fmicb.2025.1680165. Fulltekst i vitenarkiv Vis sammendrag

  Long-read metagenomic sequencing allows for the rapid, culture-independent, and accurate identification of causative pathogens and antimicrobial resistance (AMR) profiles, supporting precise antibiotic use and reducing the spread of resistance. However, its application to mastitis milk is challenging due to the complex milk matrix, low bacterial count, and high somatic cell content. This study primarily aimed to further optimize our previously developed culture-free nanopore sequencing protocol for milk samples from mastitis cases. Additional optimizations included combining centrifugation, gradient centrifugation, and fat fraction treatment with Tween 20 and citric acid. Subsequently, four DNA extraction kits (Blood and Tissue, Molysis Complete5, HostZero, and SPINeasy Host depletion) were evaluated for their ability to remove host DNA and enrich bacterial DNA for long-read sequencing with Oxford Nanopore technologies. qPCR was used to quantify bacterial and bovine DNA, allowing comparison of host depletion efficiency among the kits. Our results show that simple centrifugation effectively concentrates bacterial cells, removing the need for chemical treatments. The HostZero kit consistently produced higher DNA yields, improved DNA integrity, and more effective host DNA depletion. Using nanopore sequencing, both Gram-positive and Gram-negative mastitis pathogens, along with their AMR genes, were successfully detected. Overall, this study underscores the importance of an effective DNA extraction method for the direct sequencing of mastitis milk samples. Additionally, our findings support the potential of direct metagenomic sequencing as a rapid, culture-free approach for identifying mastitis pathogens and their resistance profiles.

• Bellankimath, Anurag Basavaraj; Branders, Sverre; Kegel, Isabell; Ali, Jawad; Asadikhoeini, Fatemeh & Johansen, Truls Erik Bjerklund [Vis alle 10 forfattere av denne artikkelen] (2025). Metagenomic sequencing enables accurate pathogen and antimicrobial susceptibility profiling in complicated UTIs in approximately four hours. Nature Communications. 17. doi: 10.1038/s41467-025-66865-8. Fulltekst i vitenarkiv Vis sammendrag

  Urinary tract infections (UTIs) affect 405 million people worldwide. Current diagnostics rely on cultures, which can take 2 to 4 days. This study evaluates eleven culture-independent methods for sample preparation from 78 complicated UTI patients, followed by real-time nanopore sequencing and data analysis. The metagenomic results are highly consistent with culture-based clinical routines (MALDI-TOF/VITEK-2). The optimized method demonstrated an accuracy score of 99% (100/101) for pathogen identification and 90% (589/653) for antimicrobial susceptibility profiling with 95% specificity. The method’s robustness is highlighted by its ability to accurately identify pathogens with as few as 32 bacterial cells/µL and a low bacterial-to-host cell ratio limit of 0.5. Additionally, mNGS identified 13 pathogens that routine diagnostics missed, which were subsequently confirmed by Vivalytic or PCR. This method is up to 30% more economical than published studies and commercial kits. DNA yield and flow cytometry can be used for pre-screening to reduce costs, which is crucial for clinical adoption. This research highlights the rapid diagnosis of clinical UTIs using a cost-effective and scalable method that requires around four hours from sample collection to informed decision-making. Furthermore, it aims to improve antimicrobial and diagnostic stewardship by reducing empirical treatment and ensuring more judicious antibiotic use.

• Khezri, Abdolrahman; Branders, Sverre; Bellankimath, Anurag Basavaraj; Ali, Jawad; Chapagain, Crystal & Asadikhoeini, Fatemeh [Vis alle 8 forfattere av denne artikkelen] (2025). MysteryMaster: scraping the bottom of the barrel of barcoded Oxford nanopore reads. BMC Bioinformatics. 26. doi: 10.1186/s12859-025-06266-2. Fulltekst i vitenarkiv Vis sammendrag

  Background The high error rate associated with Oxford Nanopore sequencing technology adversely affects demultiplexing. To improve demultiplexing and reduce unclassified reads from nanopore sequencing data, we developed MysteryMaster , a demultiplexer that utilizes the optimal sequence aligner, Cola. Results When compared to Oxford Nanopore´s Dorado and Guppy demultiplexing tools across three datasets of 37 diverse samples with established ground truth, we found that MysteryMaster accurately identifies a similar or greater percentage of reads among the different basecalling models: Fast, HAC, and SUP. MysteryMaster performs slightly better than the other tools on data that was basecalled using the Fast basecalled model, while its performance in HAC and SUP data is similar to Dorado’s. MysteryMaster has a false positive rate of just 0.41% with default settings. Conclusions While MysteryMaster can function as a standalone demultiplexer tool, the sequential application of Dorado and MysteryMaster produced the best overall performance.

• Wei, Qian; Foyn, Håvard; Landskron, Johannes; Wang, Shixiong; Rye, Inga Hansine & Skånland, Sigrid S [Vis alle 10 forfattere av denne artikkelen] (2025). Identification of a group of 9-amino-acridines that selectively downregulate regulatory T cell functions through FoxP3. iScience. 28(3). doi: 10.1016/j.isci.2025.111931. Fulltekst i vitenarkiv Vis sammendrag

  FoxP3+ regulatory T cells (Tregs) are responsible for immune homeostasis by suppressing excessive anti-self-immunity. Tregs facilitate tumor growth by inhibiting anti-tumor immunity. Here, we explored the targeting of FoxP3 as a basis for new immunotherapies. In a high-throughput phenotypic screening of a drug repurposing library using human primary T cells, we identified quinacrine as a FoxP3 downregulator. In silico searches based on the structure of quinacrine, testing of sub-libraries of analogs in vitro, and validation identified a subset of 9-amino-acridines that selectively abrogated Treg suppressive functions. Mechanistically, these acridines interfered with the DNA-binding activity of FoxP3 and inhibited FoxP3-regulated downstream gene regulation. Release from Treg suppression by 9-amino-acridines increased anti-tumor immune responses both in cancer patient samples and in mice in a syngeneic tumor model. Our study highlights the feasibility of screening for small molecular inhibitors of FoxP3 as an approach to pursuing Treg-based immunotherapy.

• Khezri, Abdolrahman; Ali, Jawad & Ahmad, Rafi (2025). Clinical diagnostics of bacterial infections and their resistance to antibiotics-Current state and novel enabling technologies implementation perspectives. I Kaur, Punit & Sharma, Priyanka (Red.), Bacterial Enzymes as Targets for Drug Discovery: Meeting the Challenges of Antibiotic Resistance. Academic Press. ISSN 9780443222221. s. 41–50. doi: 10.1016/B978-0-443-22222-1.00018-0. Fulltekst i vitenarkiv Vis sammendrag

  Microbial infection can seriously threaten human life due to pathogen's ability to impose pathological changes and antibiotic resistance, where pathogens develop abilities to defeat antibiotics. Therefore, developing time-efficient and effective methods for pathogen and resistance identification is paramount. This chapter covers traditional culture-dependent and newly developed culture-free methods for pathogen detection and resistance identification. Most of the techniques used in microbiology have their own advantages and disadvantages. From a futuristic perspective, it is essential that the diagnostic methods emphasize early pathogen detection and resistance identification with more focus on multipathogen infection, low bacterial load infection, and, in the case of sequencing, an effective host depletion as well as developing user-friendly and effective bioinformatic tools. Furthermore, clinical effectiveness should be considered for the advanced and new point-of-care diagnostic tests.

• Diaz, Nuria Garcia; Solli, Elise; Hajjar, Ehsan; Cornillot‐Clément, Selma; Landskron, Johannes & Ahmad, Rafi [Vis alle 8 forfattere av denne artikkelen] (2025). MAPK and STAT3 Inhibitors Modulate FoxP3 Expression and Regulatory T Cell Function. European Journal of Immunology. ISSN 0014-2980. 55(2). doi: 10.1002/eji.202451225. Fulltekst i vitenarkiv Vis sammendrag

  Regulatory T cells (Tregs) are a subset of T cells defined by the expression of Forkhead box protein P3 (FoxP3) playing a crucial role in regulating effector T cell activity. Tregs accumulate in the tumor microenvironment facilitating tumor growth. Thus, targeting FoxP3+ Tregs could improve cancer immunotherapies. Here, we conducted a high-throughput, phenotypic screening of a drug repurposing library to identify compounds downregulating FoxP3 expression in human primary T cells. We identified the tyrosine kinase inhibitor bosutinib and the STAT3 inhibitor nifuroxazide effectively downregulating FoxP3 expression. To identify more potent compounds, structural analogs of these two compounds were searched and validated. These analogs were found to reduce FoxP3 expression in a similar- or more potent manner than the original hits. All compounds inhibited Treg suppressive functions and reduced the expression of Treg activation markers. Importantly, bosutinib disrupted FAK and CaMKII signaling more potently in Tregs, whilst nifuroxazide and its analog NA16 targeted STAT3 protein levels more effectively in Tregs. Additionally, bosutinib and NA16 targeted effector Tregs more effectively than other Treg subsets. In summary, bosutinib, nifuroxazide, and their analogs inhibited FoxP3 expression, Treg suppressive abilities, and Treg activation effectively, which could serve as tools for the improvement of current cancer immunotherapies.

• Karlsholm, Line Strand; Ahmad, Rafi; Taman, Hagar; Fenton, Christopher Graham & Paulssen, Ruth H (2025). Actively expressed microbiota in mucosal biopsies of treatment-naïve ulcerative colitis patients. Gut Microbes Reports. 2(1). doi: 10.1080/29933935.2025.2512763. Fulltekst i vitenarkiv Vis sammendrag

  The gut microbiota contributes to an aberrant immune dysfunction associated with inflammatory bowel disease (IBD) and is believed to contribute to the disease development of ulcerative colitis (UC). In this study, a whole-transcriptomic data set containing mucosal tissue biopsies obtained from treatment-naïve UC patients (n = 33) and control subjects (n = 15) were utilized to determine potential differences in microbial compositions located at the site of inflammation. Transcriptomic sequences that mapped to the human genome were removed from the dataset. The remaining were mapped to their respective Operational Taxonomic Units (OTUs) resulting in an overview of the microbial composition at the genus level. Results showed an overall decrease in microbial diversity in mucosal UC patient samples. However, an increase of the opportunistic pathogens Acinetobacter and Comamonas was observed in UC samples. Comamonas is an environmental pathogen. In addition to their role in mild but persistent infections, Comamonas species may play a role in active UC. The experimental approach described here provides new insights into the UC microbiota composition directly at the site of inflammation, which could contribute to a better understanding of UC pathogenesis. The obtained results might provide an opportunity to investigate host–microbe interactions in active UC.

• Ali, Jawad; Johansen, Wenche & Ahmad, Rafi (2024). Short turnaround time of seven to nine hours from sample collection until informed decision for sepsis treatment using nanopore sequencing. Scientific Reports. 14(1). doi: 10.1038/s41598-024-55635-z. Fulltekst i vitenarkiv Vis sammendrag

  Bloodstream infections (BSIs) and sepsis are major health problems, annually claiming millions of lives. Traditional blood culture techniques, employed to identify sepsis-causing pathogens and assess antibiotic susceptibility, usually take 2–4 days. Early and accurate antibiotic prescription is vital in sepsis to mitigate mortality and antibiotic resistance. This study aimed to reduce the wait time for sepsis diagnosis by employing shorter blood culture incubation times for BD BACTEC™ bottles using standard laboratory incubators, followed by real-time nanopore sequencing and data analysis. The method was tested on nine blood samples spiked with clinical isolates from the six most prevalent sepsis-causing pathogens. The results showed that pathogen identification was possible at as low as 102–104 CFU/mL, achieved after just 2 h of incubation and within 40 min of nanopore sequencing. Moreover, all the antimicrobial resistance genes were identified at 103–107 CFU/mL, achieved after incubation for 5 h and only 10 min to 3 h of sequencing. Therefore, the total turnaround time from sample collection to the information required for an informed decision on the right antibiotic treatment was between 7 and 9 h. These results hold significant promise for better clinical management of sepsis compared with current culture-based methods.

• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Vis alle 7 forfattere av denne artikkelen] (2024). Culture and amplification-free nanopore sequencing for rapid detection of pathogens and antimicrobial resistance genes from urine. European Journal of Clinical Microbiology and Infectious Diseases. ISSN 0934-9723. 43(11), s. 2177–2190. doi: 10.1007/s10096-024-04929-1. Fulltekst i vitenarkiv Vis sammendrag

  Purpose Urinary Tract Infections (UTIs) are among the most prevalent infections globally. Every year, approximately 150 million people are diagnosed with UTIs worldwide. The current state-of-the-art diagnostic methods are culture-based and have a turnaround time of 2–4 days for pathogen identification and susceptibility testing.<p> <p>Methods This study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Urine samples were spiked at clinically significant concentrations of 10³ -10⁵ CFU/ml. Three DNA extraction kits (BioStic, PowerFood, and Blood and Tissue) were investigated based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. After DNA extraction, the samples were sequenced using MinION and Flongle flow cells. <p>Results The Blood and Tissue kit outperformed the other kits based on the investigated parameters. Using nanopore sequencing, all the pathogens and corresponding genes were only identified at a spike concentration of 10<p>5</sup> CFU/ml, achieved after 10 min and 3 hours of sequencing, respectively. However, some pathogens and antibiotic-resistance genes (ARG) could be identified from spikes at 10³ colony formation units (CFU/mL). The overall turnaround time was five hours, from sample preparation to sequencing-based identification of pathogen ID and antimicrobial resistance genes. <p>Conclusion This study demonstrates excellent promise in reducing the time required for informed antibiotic administration from 48 to 72 h to five hours, thereby reducing the number of empirical doses and increasing the chance of saving lives.

• Wester, Astrid Louise; Solberg, Svein Øivind; Gopinathan, Unni; Ahmad, Rafi & Graham, David W. (2024). Antimicrobial Resistance in a One Health and One World Perspective – Mechanisms and Perspectives. I Quah, Stella R. (Red.), International Encyclopedia of Public Health. Academic Press. ISSN 9780323972802. s. 50–73. doi: 10.1016/B978-0-323-99967-0.00148-4. Fulltekst i vitenarkiv
• Ahmadi, Asal; Khezri, Abdolrahman; Nørstebø, Håvard & Ahmad, Rafi (2023). A culture-, amplification-independent, and rapid method for identification of pathogens and antibiotic resistance profile in bovine mastitis milk. Frontiers in Microbiology. 13. doi: 10.3389/fmicb.2022.1104701. Fulltekst i vitenarkiv Vis sammendrag

  Introduction: Rapid and accurate diagnosis of causative pathogens in mastitis would minimize the imprudent use of antibiotics and, therefore, reduce the spread of antimicrobial resistance. Whole genome sequencing offers a unique opportunity to study the microbial community and antimicrobial resistance (AMR) in mastitis. However, the complexity of milk samples and the presence of a high amount of host DNA in milk from infected udders often make this very challenging.<p> <p>Methods: Here, we tested 24 bovine milk samples (18 mastitis and six nonmastitis) using four different commercial kits (Qiagens’ DNeasy R PowerFood R Microbial, Norgens’ Milk Bacterial DNA Isolation, and Molzyms’ MolYsis^TM Plus and Complete5) in combination with filtration, low-speed centrifugation, nuclease, and 10% bile extract of male bovine (Ox bile). Isolated DNA was quantified, checked for the presence/absence of host and pathogen using PCR and sequenced using MinION nanopore sequencing. Bioinformatics analysis was performed for taxonomic classification and antimicrobial resistance gene detection. <p>Results: The results showed that kits designed explicitly for bacterial DNA isolation from food and dairy matrices could not deplete/minimize host DNA. Following using MolYsisTM Complete 5 + 10% Ox bile + micrococcal nuclease combination, on average, 17% and 66.5% of reads were classified as bovine and Staphylococcus aureus reads, respectively. This combination also effectively enriched other mastitis pathogens, including Escherichia coli and Streptococcus dysgalactiae. Furthermore, using this approach, we identified important AMR genes such as Tet (A), Tet (38), fosB-Saur, and blaZ. We showed that even 40 min of the MinION run was enough for bacterial identification and detecting the first AMR gene. <p>Conclusion: We implemented an effective method (sensitivity of 100% and specificity of 92.3%) for host DNA removal and bacterial DNA enrichment (both gram-negative and positive) directly from bovine mastitis milk. To the best of our knowledge, this is the first culture- and amplificationindependent study using nanopore-based metagenomic sequencing for realtime detection of the pathogen (within 5 hours) and the AMR profile (within 5–9 hours), in mastitis milk samples. These results provide a promising and potential future on-farm adaptable approach for better clinical management of mastitis.

• Das, Sathi; Saxena, Kanchan; Khezri, Abdolrahman; Dubey, Vishesh Kumar; Ahmad, Azeem & Tinguely, Jean-Claude [Vis alle 9 forfattere av denne artikkelen] (2023). Rapid identification of antimicrobial drug resistance strains of E-coli using SERS nanowire chip. Proceedings of SPIE, the International Society for Optical Engineering. ISSN 0277-786X. 12396. doi: 10.1117/12.2650578. Fulltekst i vitenarkiv Vis sammendrag

  We have investigated the enhanced Raman spectra of AMR bacteria strains of E. coli using silver coated silicon nanowires SERS assay. Three different E. coli strains, E. coli CCUG17620, NCTC 13441, and A239, were detected using two different excitation laser wavelengths. We found stable and enhanced SERS spectrum using 785 nm laser as opposed to 532 nm. Future development of SERS-chip could offer a reliable platform for direct identification of the pathogen in bio-fluid samples at strains level.

• Ahmad, Azeem; Hettiarachchi, Ramith; Khezri, Abdolrahman; Ahluwalia, Balpreet Singh; Wadduwage, Dushan & Ahmad, Rafi (2023). Highly sensitive quantitative phase microscopy and deep learning aided with whole genome sequencing for rapid detection of infection and antimicrobial resistance. Frontiers in Microbiology. 14. doi: 10.3389/fmicb.2023.1154620. Fulltekst i vitenarkiv Vis sammendrag

  Current state-of-the-art infection and antimicrobial resistance (AMR) diagnostics are based on culture-based methods with a detection time of 48–96 h. Therefore, it is essential to develop novel methods that can do real-time diagnoses. Here, we demonstrate that the complimentary use of label-free optical assay with whole-genome sequencing (WGS) can enable rapid diagnosis of infection and AMR. Our assay is based on microscopy methods exploiting label-free, highly sensitive quantitative phase microscopy (QPM) followed by deep convolutional neural networks-based classification. The workflow was benchmarked on 21 clinical isolates from four WHO priority pathogens that were antibiotic susceptibility tested, and their AMR profile was determined by WGS. The proposed optical assay was in good agreement with the WGS characterization. Accurate classification based on the gram staining (100% recall for gram-negative and 83.4% for gram-positive), species (98.6%), and resistant/susceptible type (96.4%), as well as at the individual strain level (100% sensitivity in predicting 19 out of the 21 strains, with an overall accuracy of 95.45%). The results from this initial proof-of-concept study demonstrate the potential of the QPM assay as a rapid and first-stage tool for species, strain-level classification, and the presence or absence of AMR, which WGS can follow up for confirmation. Overall, a combined workflow with QPM and WGS complemented with deep learning data analyses could, in the future, be transformative for detecting and identifying pathogens and characterization of the AMR profile and antibiotic susceptibility.

• Avershina, Ekaterina; Khezri, Abdolrahman & Ahmad, Rafi (2023). Clinical Diagnostics of Bacterial Infections and Their Resistance to Antibiotics—Current State and Whole Genome Sequencing Implementation Perspectives. Antibiotics. 12(4). doi: 10.3390/antibiotics12040781. Fulltekst i vitenarkiv Vis sammendrag

  Antimicrobial resistance (AMR), defined as the ability of microorganisms to withstand antimicrobial treatment, is responsible for millions of deaths annually. The rapid spread of AMR across continents warrants systematic changes in healthcare routines and protocols. One of the fundamental issues with AMR spread is the lack of rapid diagnostic tools for pathogen identification and AMR detection. Resistance profile identification often depends on pathogen culturing and thus may last up to several days. This contributes to the misuse of antibiotics for viral infection, the use of inappropriate antibiotics, the overuse of broad-spectrum antibiotics, or delayed infection treatment. Current DNA sequencing technologies offer the potential to develop rapid infection and AMR diagnostic tools that can provide information in a few hours rather than days. However, these techniques commonly require advanced bioinformatics knowledge and, at present, are not suited for routine lab use. In this review, we give an overview of the AMR burden on healthcare, describe current pathogen identification and AMR screening methods, and provide perspectives on how DNA sequencing may be used for rapid diagnostics. Additionally, we discuss the common steps used for DNA data analysis, currently available pipelines, and tools for analysis. Direct, culture-independent sequencing has the potential to complement current culture-based methods in routine clinical settings. However, there is a need for a minimum set of standards in terms of evaluating the results generated. Additionally, we discuss the use of machine learning algorithms regarding pathogen phenotype detection (resistance/susceptibility to an antibiotic).

• Ali, Jawad; Joshi, Mukund; Ahmadi, Asal; Strætkvern, Knut Olav & Ahmad, Rafi (2023). Increased growth temperature and vitamin B12 supplementation reduces the lag time for rapid pathogen identification in BHI agar and blood cultures. F1000 Research. 12(131). doi: 10.12688/f1000research.129668.2. Fulltekst i vitenarkiv Vis sammendrag

  Background: The rapid diagnostics of pathogens is essential to prescribe appropriate and early antibiotic therapy. The current methods for pathogen detection require the bacteria to grow in a culture medium, which is time-consuming. This increases the mortality rate and the global burden of antimicrobial resistance. Culture-free detection methods are still under development and are not used in the clinical routine. Therefore decreasing the culture time for accurate detection of infection and resistance is vital for diagnosis. Methods: In this study, we wanted to investigate easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation (e.g., vitamin B12 and trace metals), that can significantly reduce the lag time (tlag). These factors were arranged in simple two-level factorial designs using Gram-positive (Escherichia coli and Pseudomonas aeruginosa) and Gram-negative (Staphylococcus aureus and Bacillus subtilis) bacteria, including clinical isolates with known antimicrobial resistance profiles. Blood samples spiked with a clinical isolate of E. coli CCUG17620 were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures. Results: We observed that increased incubation temperature (42°C) along with vitamin B12 supplementation significantly reduced the tlag (10 – 115 minutes or 4% - 49%) in pure clinical isolates and blood samples spiked with E. coli CCUG17620. In the case of the blood sample, PCR results also detected bacterial DNA after only 3h of incubation and at three times the CFU/mL. Conclusions: Enrichment of bacterial culture media with growth supplements such as vitamin B12 and increased incubation temperature can be a cheap and rapid method for the early detection of pathogens. This is a proof-of-concept study restricted to a few bacterial strains and growth conditions. In the future, the effect of other growth conditions and difficult-to-culture bacteria should be explored to shorten the lag phase.

• Das, Sathi; Saxena, Kanchan; Tinguely, Jean-Claude; Pal, Arijit; Wickramasinghe, Nima L. & Khezri, Abdolrahman [Vis alle 13 forfattere av denne artikkelen] (2023). SERS Nanowire Chip and Machine Learning-Enabled Classification of Wild-Type and Antibiotic-Resistant Bacteria at Species and Strain Levels. ACS Applied Materials & Interfaces. ISSN 1944-8244. 15(20), s. 24047–24058. doi: 10.1021/acsami.3c00612. Fulltekst i vitenarkiv Vis sammendrag

  Antimicrobial resistance (AMR) is a major health threat worldwide and the culture-based bacterial detection methods are slow. Surface-enhanced Raman spectroscopy (SERS) can be used to identify target analytes in real time with sensitivity down to the single-molecule level, providing a promising solution for the culture-free bacterial detection. We report the fabrication of SERS substrates having tightly packed silver (Ag) nanoparticles loaded onto long silicon nanowires (Si NWs) grown by the metal-assisted chemical etching (MACE) method for the detection of bacteria. The optimized SERS chips exhibited sensitivity down to 10–12 M concentration of R6G molecules and detected reproducible Raman spectra of bacteria down to a concentration of 100 colony forming units (CFU)/mL, which is a thousand times lower than the clinical threshold of bacterial infections like UTI (105 CFU/mL). A Siamese neural network model was used to classify SERS spectra from bacteria specimens. The trained model identified 12 different bacterial species, including those which are causative agents for tuberculosis and urinary tract infection (UTI). Next, the SERS chips and another Siamese neural network model were used to differentiate AMR strains from susceptible strains of Escherichia coli (E. coli). The enhancement offered by SERS chip-enabled acquisitions of Raman spectra of bacteria directly in the synthetic urine by spiking the sample with only 103 CFU/mL E. coli. Thus, the present study lays the ground for the identification and quantification of bacteria on SERS chips, thereby offering a potential future use for rapid, reproducible, label-free, and low limit detection of clinical pathogens.

• Khan, Yusuf; Hammarström, Daniel; Ellefsen, Stian & Ahmad, Rafi (2022). Normalization of gene expression data revisited: the three viewpoints of the transcriptome in human skeletal muscle undergoing load-induced hypertrophy and why they matter. BMC Bioinformatics. 23(1). doi: 10.1186/s12859-022-04791-y. Fulltekst i vitenarkiv Vis sammendrag

  Background The biological relevance and accuracy of gene expression data depend on the adequacy of data normalization. This is both due to its role in resolving and accounting for technical variation and errors, and its defining role in shaping the viewpoint of biological interpretations. Still, the choice of the normalization method is often not explicitly motivated although this choice may be particularly decisive for conclusions in studies involving pronounced cellular plasticity. In this study, we highlight the consequences of using three fundamentally different modes of normalization for interpreting RNA-seq data from human skeletal muscle undergoing exercise-training-induced growth. Briefly, 25 participants conducted 12 weeks of high-load resistance training. Muscle biopsy specimens were sampled from m. vastus lateralis before, after two weeks of training (week 2) and after the intervention (week 12), and were subsequently analyzed using RNA-seq. Transcript counts were modeled as (1) per-library-size, (2) per-total-RNA, and (3) per-sample-size (per-mg-tissue). Result Initially, the three modes of transcript modeling led to the identification of three unique sets of stable genes, which displayed differential expression profiles. Specifically, genes showing stable expression across samples in the per-library-size dataset displayed training-associated increases in per-total-RNA and per-sample-size datasets. These gene sets were then used for normalization of the entire dataset, providing transcript abundance estimates corresponding to each of the three biological viewpoints (i.e., per-library-size, per-total-RNA, and per-sample-size). The different normalization modes led to different conclusions, measured as training-associated changes in transcript expression. Briefly, for 27% and 20% of the transcripts, training was associated with changes in expression in per-total-RNA and per-sample-size scenarios, but not in the per-library-size scenario. At week 2, this led to opposite conclusions for 4% of the transcripts between per-library-size and per-sample-size datasets (↑ vs. ↓, respectively). Conclusion Scientists should be explicit with their choice of normalization strategies and should interpret the results of gene expression analyses with caution. This is particularly important for data sets involving a limited number of genes or involving growing or differentiating cellular models, where the risk of biased conclusions is pronounced.

• Avershina, Ekaterina; Frye, Stephan Alfons; ali, jawad; Taxt, Arne Michael & Ahmad, Rafi (2022). Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing. Frontiers in Microbiology. 13. doi: 10.3389/fmicb.2022.822402. Fulltekst i vitenarkiv Vis sammendrag

  Rapid bacterial identification and antimicrobial resistance gene (ARG) detection are crucial for fast optimization of antibiotic treatment, especially for septic patients where each hour of delayed antibiotic prescription might have lethal consequences. This work investigates whether the Oxford Nanopore Technology’s (ONT) Flongle sequencing platform is suitable for real-time sequencing directly from blood cultures to identify bacteria and detect resistance-encoding genes. For the analysis, we used pure bacterial cultures of four clinical isolates of Escherichia coli and Klebsiella pneumoniae and two blood samples spiked with either E. coli or K. pneumoniae that had been cultured overnight. We sequenced both the whole genome and plasmids isolated from these bacteria using two different sequencing kits. Generally, Flongle data allow rapid bacterial ID and resistome detection based on the first 1,000–3,000 generated sequences (10 min to 3 h from the sequencing start), albeit ARG variant identification did not always correspond to ONT MinION and Illumina sequencing-based data. Flongle data are sufficient for 99.9% genome coverage within at most 20,000 (clinical isolates) or 50,000 (positive blood cultures) sequences generated. The SQK-LSK110 Ligation kit resulted in higher genome coverage and more accurate bacterial identification than the SQK-RBK004 Rapid Barcode kit.

• Hanrahan, Niall; Lister, Adam; Highmore, Callum; Rajith, Leena; Avershina, Ekaterina & Ali, Jawad [Vis alle 9 forfattere av denne artikkelen] (2022). Enhanced Raman techniques for infection diagnostics. Proceedings of SPIE, the International Society for Optical Engineering. ISSN 0277-786X. 12203, s. 6–17. doi: 10.1117/12.2635324. Fulltekst i vitenarkiv Vis sammendrag

  In this paper we describe our recent work in multi-excitation surface enhanced Raman spectroscopy (MX-SERS), and its application for robust strain-level bacteria identification. The development of MX-SERS follows directly from our previous work in rapid bacterial identification using multiexcitation Raman spectroscopy (MX-Raman), which enabled highly accurate (up to 99.75%) strainlevel distinction of bacteria, including antibiotic resistant strains of bacteria and from within complex media. In this work we use the strong wavelength dependence of both the Raman scattering crosssection and the surface plasmon to demonstrate a novel capability in bacteria identification. Compared to MX-Raman, MX-SERS has up to 8x faster data acquisition speed as well as up to 4000x lower laser power incident on the sample. Furthermore, we fabricate SERS-active substrates with a simple and low-cost fabrication method that can be adapted to fit a chosen wavelength regime. This combination of strain-level sensitivity and high-speed detection, combined with a low-cost SERS substrate, has strong potential applications in clinical diagnostics, and could be integrated within a real-world pathogen detection workflow. This presents new capabilities in label-free bacterial detection including novel culture-free investigation capabilities, and an improved methodology for sample handling with minimal preparation and processing.

• Modica, Maria Vittoria; Ahmad, Rafi; Ainsworth, Stuart; Anderluh, Gregor; Antunes, Agostinho & Beis, Dimitris [Vis alle 23 forfattere av denne artikkelen] (2021). The new COST Action European Venom Network (EUVEN)—synergy and future perspectives of modern venomics. GigaScience. 10(3), s. 1–5. doi: 10.1093/gigascience/giab019. Fulltekst i vitenarkiv Vis sammendrag

  Venom research is a highly multidisciplinary field that involves multiple subfields of biology, informatics, pharmacology, medicine, and other areas. These different research facets are often technologically challenging and pursued by different teams lacking connection with each other. This lack of coordination hampers the full development of venom investigation and applications. The COST Action CA19144–European Venom Network was recently launched to promote synergistic interactions among different stakeholders and foster venom research at the European level.

• Mølmen, Knut Sindre; Hammarström, Jens Konrad Daniel; Falch, Gunnar Slettaløkken; Grundtvig, Morten; Koll, Lise & Hanestadhaugen, Marita [Vis alle 14 forfattere av denne artikkelen] (2021). Chronic obstructive pulmonary disease does not impair responses to resistance training. Journal of Translational Medicine. 19(1). doi: 10.1186/s12967-021-02969-1. Fulltekst i vitenarkiv Vis sammendrag

  Background: Subjects with chronic obstructive pulmonary disease (COPD) are prone to accelerated decay of muscle strength and mass with advancing age. This is believed to be driven by disease-inherent systemic pathophysiologies, which are also assumed to drive muscle cells into a state of anabolic resistance, leading to impaired abilities to adapt to resistance exercise training. Currently, this phenomenon remains largely unstudied. In this study, we aimed to investigate the assumed negative effects of COPD for health- and muscle-related responsiveness to resistance training using a healthy control-based translational approach. Methods: Subjects with COPD (n = 20, GOLD II-III, FEV1predicted 57 ± 11%, age 69 ± 5) and healthy controls (Healthy, n = 58, FEV1predicted 112 ± 16%, age 67 ± 4) conducted identical whole-body resistance training interventions for 13 weeks, consisting of two weekly supervised training sessions. Leg exercises were performed unilaterally, with one leg conducting high-load training (10RM) and the contralateral leg conducting low-load training (30RM). Measurements included muscle strength (nvariables = 7), endurance performance (nvariables = 6), muscle mass (nvariables = 3), muscle quality, muscle biology (m. vastus lateralis; muscle fiber characteristics, RNA content including transcriptome) and health variables (body composition, blood). For core outcome domains, weighted combined factors were calculated from the range of singular assessments. Results: COPD displayed well-known pathophysiologies at baseline, including elevated levels of systemic low-grade inflammation ([c-reactive protein]serum), reduced muscle mass and functionality, and muscle biological aberrancies. Despite this, resistance training led to improved lower-limb muscle strength (15 ± 8%), muscle mass (7 ± 5%), muscle quality (8 ± 8%) and lower-limb/whole-body endurance performance (26 ± 12%/8 ± 9%) in COPD, resembling or exceeding responses in Healthy, measured in both relative and numeric change terms. Within the COPD cluster, lower FEV1predicted was associated with larger numeric and relative increases in muscle mass and superior relative improvements in maximal muscle strength. This was accompanied by similar changes in hallmarks of muscle biology such as rRNA-content↑, muscle fiber cross-sectional area↑, type IIX proportions↓, and changes in mRNA transcriptomics. Neither of the core outcome domains were differentially affected by resistance training load. Conclusions: COPD showed hitherto largely unrecognized responsiveness to resistance training, rejecting the notion of disease-related impairments and rather advocating such training as a potent measure to relieve pathophysiologies. Trial registration: ClinicalTrials.gov ID: NCT02598830. Registered November 6th 2015, https://clinicaltrials.gov/ct2/show/NCT02598830. Keywords: Anabolic resistance; COPD; Pathophysiology; Skeletal muscle; Strength training; Training load.

• Khezri, Abdolrahman; Avershina, Ekaterina & Ahmad, Rafi (2021). Hybrid Assembly Provides Improved Resolution of Plasmids, Antimicrobial Resistance Genes, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Clinical Isolates. Microorganisms. 9(12), s. 1–18. doi: 10.3390/microorganisms9122560. Fulltekst i vitenarkiv Vis sammendrag

  Emerging new sequencing technologies have provided researchers with a unique opportunity to study factors related to microbial pathogenicity, such as antimicrobial resistance (AMR) genes and virulence factors. However, the use of whole-genome sequence (WGS) data requires good knowledge of the bioinformatics involved, as well as the necessary techniques. In this study, a total of nine Escherichia coli and Klebsiella pneumoniae isolates from Norwegian clinical samples were sequenced using both MinION and Illumina platforms. Three out of nine samples were sequenced directly from blood culture, and one sample was sequenced from a mixed-blood culture. For genome assembly, several long-read, (Canu, Flye, Unicycler, and Miniasm), short-read (ABySS, Unicycler and SPAdes) and hybrid assemblers (Unicycler, hybridSPAdes, and MaSurCa) were tested. Assembled genomes from the best-performing assemblers (according to quality checks using QUAST and BUSCO) were subjected to downstream analyses. Flye and Unicycler assemblers performed best for the assembly of long and short reads, respectively. For hybrid assembly, Unicycler was the top-performing assembler and produced more circularized and complete genome assemblies. Hybrid assembled genomes performed substantially better in downstream analyses to predict putative plasmids, AMR genes and β-lactamase gene variants, compared to MinION and Illumina assemblies. Thus, hybrid assembly has the potential to reveal factors related to microbial pathogenicity in clinical and mixed samples.

• Mølmen, Knut Sindre; Hammarström, Jens Konrad Daniel; Pedersen, Karianne; Lie, Anne Cecilie Lian; Steile, Ragnvald B. & Nygård, Håvard [Vis alle 18 forfattere av denne artikkelen] (2021). Vitamin D3 supplementation does not enhance the effects of resistance training in older adults. Journal of Cachexia, Sarcopenia and Muscle. ISSN 2190-5991. 12(3), s. 599–628. doi: 10.1002/jcsm.12688. Fulltekst i vitenarkiv Vis sammendrag

  Background: Lifestyle therapy with resistance training is a potent measure to counteract age-related loss in muscle strength and mass. Unfortunately, many individuals fail to respond in the expected manner. This phenomenon is particularly common among older adults and those with chronic diseases (e.g. chronic obstructive pulmonary disease, COPD) and may involve endocrine variables such as vitamin D. At present, the effects of vitamin D supplementation on responses to resistance training remain largely unexplored. Methods: Ninety-five male and female participants (healthy, n = 71; COPD, n = 24; age 68 ± 5 years) were randomly assigned to receive either vitamin D3 or placebo supplementation for 28 weeks in a double-blinded manner (latitude 61°N, September-May). Seventy-eight participants completed the RCT, which was initiated by 12 weeks of supplementation-only (two weeks with 10 000 IU/day, followed by 2000 IU/day), followed by 13 weeks of combined supplementation (2000 IU/day) and supervised whole-body resistance training (twice weekly), interspersed with testing and measurements. Outcome measures included multiple assessments of muscle strength (nvariables = 7), endurance performance (n = 6), and muscle mass (n = 3, legs, primary), as well as muscle quality (legs), muscle biology (m. vastus lateralis; muscle fibre characteristics, transcriptome), and health-related variables (e.g. visceral fat mass and blood lipid profile). For main outcome domains such as muscle strength and muscle mass, weighted combined factors were calculated from the range of singular assessments. Results: Overall, 13 weeks of resistance training increased muscle strength (13% ± 8%), muscle mass (9% ± 8%), and endurance performance (one-legged, 23% ± 15%; whole-body, 8% ± 7%), assessed as weighted combined factors, and were associated with changes in health variables (e.g. visceral fat, -6% ± 21%; [LDL]serum , -4% ± 14%) and muscle tissue characteristics such as fibre type proportions (e.g. IIX, -3% points), myonuclei per fibre (30% ± 65%), total RNA/rRNA abundances (15%/6-19%), and transcriptome profiles (e.g. 312 differentially expressed genes). Vitamin D3 supplementation did not affect training-associated changes for any of the main outcome domains, despite robust increases in [25(OH)D]serum (∆49% vs. placebo). No conditional effects were observed for COPD vs. healthy or pre-RCT [25(OH)D]serum . In secondary analyses, vitamin D3 affected expression of gene sets involved in vascular functions in muscle tissue and strength gains in participants with high fat mass, which advocates further study. Conclusions: Vitamin D3 supplementation did not affect muscular responses to resistance training in older adults with or without COPD. Keywords: Cholecalciferol; Muscle plasticity; Strength training. © 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.

Se alle arbeider i NVA

• Ahmad, Rafi (2026). Artificial Intelligence in Health: India–Norway Opportunities for Research, Innovation and Better Care. Live Panel Discussion. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Superbugs vs. Super Tech: How AI and Genomics Are Reshaping the Battle Against Infection and Antimicrobial Resistance. doi: https:/www.ntnu.edu/ikb/india-collaboration. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Superbugs vs. Super Tech: How AI and Genomics Are Reshaping the Battle Against Infection and AMR. doi: https:/symposium2026.imweb.me/home. Fulltekst i vitenarkiv
• Alvseike, Ole; Asadikhoeini, Fatemeh; Khezri, Abdolrahman & Ahmad, Rafi (2026). OH-AMR-Diag: rask påvisning av antibiotikaresistens. Fulltekst i vitenarkiv
• Naqvi, Tania; Ahluwalia, Balpreet Singh & Ahmad, Rafi (2026). MBA/Ag-rGO SERS immunosensing Platform for Sensitive and Selective E.coli Detection in Complex Biological Medium. Fulltekst i vitenarkiv
• Asadikhoeini, Fatemeh & Ahmad, Rafi (2026). Project progress report. Fulltekst i vitenarkiv
• Ali, Jawad; Bellankimath, Anurag Basavaraj; Opgård, Silje Therese; Manivannan, Emil Varman; Simonsen, Gunnar Skov & Ahmad, Rafi (2026). From culture to clarity in four hours: accelerating clinical management of bloodstream infections using metagenomics. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Clinical metagenomics for rapid detection of infections & antimicrobial resistance. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Accurate and rapid turnaround of four hours for the diagnosis of complicated UTIs using metagenomics. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Bringing Metagenomics to the Bedside: Personalized Management of cUTIs for Patient Care. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). K-MARVEL: K-Mer-based antimicrobial resistance virtual exploration lab. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). From culture to clarity in four hours: accelerating clinical management of bloodstream infections. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). From sample to treatment in four hours: bringing metagenomics precision to complicated UTI diagnostics and management. Fulltekst i vitenarkiv
• Chapagain, Crystal; Khezri, Abdolrahman; Ali, Jawad; Smistad, Marit; Sølverød, Liv Synnøve & Ahmadi, Asal [Vis alle 7 forfattere av denne artikkelen] (2026). No culture, no amplification, just results: a five-hour breakthrough for bovine mastitis diagnostics. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Ny norskutviklet hurtigtest kan redde liv. [TV]. NRK. doi: https:/www.nrk.no/innlandet/forskningsprosjekt-om-hurtigtest-av-urinprover-far-gode-resultater-1.17723731. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Ny norskutviklet hurtigtest kan redde liv. [TV]. NRK TV og Radio. doi: https:/tv.nrk.no/serie/distriktsnyheter-oestnytt/sesong/202601/episode/DKIN98011426. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Denne norske oppfinnelsen kan brukes i kampen mot resistente bakterier. [Journal]. Forskning.no. doi: https:/www.forskning.no/antibiotikaresistens-bakterier-bioteknologi/denne-norske-oppfinnelsen-kan-brukes-i-kampen-mot-resistente-bakterier/2601839?fbclid=IwY2xjawPgRxhleHRuA2FlbQIxMABzcnRjBmFwcF9pZBAyMjIwMzkxNzg4MjAwODkyAAEe6AWFikEI8gATT6WSIYsHQ4ivBYj-o8ovmhSv53NpjAxWVcrveEWTVDNWdUg_aem_odTqAdKi6EbZ_WPwXXodaA. Fulltekst i vitenarkiv
• Ahmad, Rafi (2026). Kan spare opptil 1,6 millioner antibiotikadoser hvert år. [Journal]. Aftenposten. doi: https:/www.aftenposten.no/norge/i/xr371l/norsk-oppfinnelse-kan-bidra-i-kampen-mot-antibiotikaresistens?utm_source=iosapp&utm_medium=share%0A. Fulltekst i vitenarkiv
• Naqvi, Tania; Ahluwalia, Balpreet Singh & Ahmad, Rafi (2025). Fabrication of SERS substrates for pathogen detection. Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). Rapid detection of Campylobacter spp. and its co-occurrence with other intestine flora in Norwegian broiler flocks​ (Project introduction and overview). Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). UTI-Diag: Reducing the burden of antimicrobial resistance – urinary tract infection using innovative and scalable rapid diagnosis. Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). Superbugs vs. Super Tech: How AI and Genomics Are Reshaping the Battle Against Infection and AMR. Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). AMR-Educate: A multinational and interdisciplinary research-integrated education partnership for a sustainable future against infection and antimicrobial resistance threats. Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). Hva er antibiotikaresistens? Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). AI vs. Superbugs: How Artificial Intelligence IsTransforming Our Fight Against Infections and Antimicrobial Resistance. Fulltekst i vitenarkiv
• Moe, Erlend; Lagmandokk, Ole - Andre & Ahmad, Rafi (2025). Rafis kamp. [Internet]. inn.no. doi: https:/www.inn.no/forskning/forskningsnyheter/rafis-kamp/. Fulltekst i vitenarkiv Vis sammendrag

  Han brenner for å bekjempe et av vår tids store folkehelseproblemer, antibiotikaresistens. Drivkraften har han med seg fra barndommen i India.

• Ahmad, Rafi (2025). Four-hour turnaround for diagnosing complex UTIs using nanopore sequencing. Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). Innovative Technologies for Rapid Detection of Infections and Antimicrobial Resistance. Fulltekst i vitenarkiv
• Bellankimath, Anurag Basavaraj; Branders, Sverre; Kegel, Isabell; Ali, Jawad; Asadi, Fatemeh & Johansen, Truls Erik Bjerklund [Vis alle 10 forfattere av denne artikkelen] (2025). Accurate and rapid turnaround of four hours for the diagnosis of complicated UTIs using metagenomics. Fulltekst i vitenarkiv
• Branders, Sverre; Grabherr, Manfred G. & Ahmad, Rafi (2025). Voyager: Real-time Taxonomic Characterization of Longread Mixed-species Sequencing Samples in Sorted Motif Distance Space. Fulltekst i vitenarkiv
• Bellankimath, Anurag Basavaraj; Branders, Sverre; Kegel, Isabell; Ali, Jawad; Asadi, Fatemeh & Johansen, Truls E. Bjerklund [Vis alle 10 forfattere av denne artikkelen] (2025). Turnaround time of just four hours from sample collection to informed decision-making for complicated urinary tract infections in a urology ward, using culture-independent metagenomics. Fulltekst i vitenarkiv
• Branders, Sverre; Kegel, Isabell; Ali, Jawad; Asadi, Fatemeh; Johansen, Truls E. Bjerklund & Imirzalioglu, Can [Vis alle 9 forfattere av denne artikkelen] (2025). Turnaround time of just four hours from sample collection to informed decision-making for complicated urinary tract infections in a urology ward, using culture-independent metagenomics. Fulltekst i vitenarkiv
• Branders, Sverre; Grabherr, Manfred G. & Ahmad, Rafi (2025). Voyager: novel real-time taxonomic characterization of long-read sequence data. Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). Novel enabling technologies for diagnostics of infection and antimicrobial resistance. Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). Four-hour turnaround for diagnosing complex UTIs using nanopore sequencing. Fulltekst i vitenarkiv
• Ahmad, Rafi (2025). Rapid point of care UTI diagnostics using nanopore sequencing. Fulltekst i vitenarkiv
• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Vis alle 7 forfattere av denne artikkelen] (2025). Rapid Diagnosis of Urinary Tract Infection using Nanopore sequencing. Fulltekst i vitenarkiv
• Ali, Jawad; Johansen, Wenche & Ahmad, Rafi (2024). Short turnaround time of seven to nine hours from sample collection until informed decision for sepsis treatment using nanopore sequencing. Fulltekst i vitenarkiv
• Branders, Sverre; Grabherr, Manfred G. & Ahmad, Rafi (2024). Real-time Taxonomic Characterization of Long-read Mixed-species Sequencing Samples in Sorted Motif Distance Space: Voyager. BioRxiv. ISSN 0362-4331. doi: 10.1101/2024.04.13.589333. Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). Metagenomic analysis for rapid detection of bacterial infection and antimicrobial resistance. Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). Urindyrkning og resistensbestemmelse på 24t. Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). Fra idé til product, en reise i produktutvikling. Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). Metagenomic analysis for rapid detection of bacterial infection and antimicrobial resistance. Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). Novel technological solutions for rapid diagnostics of infection and antimicrobial resistance: A One Health approach. Fulltekst i vitenarkiv
• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Vis alle 7 forfattere av denne artikkelen] (2024). Rapid Detection of Clinically Relevant Uropathogens and Antimicrobial Resistance Genes in 5 Hours using Nanopore Sequencing. Fulltekst i vitenarkiv
• Branders, Sverre; Ahmad, Rafi & Grabherr, Manfred G. (2024). Real-time Taxonomic Characterization of Long-read Mixed-species Sequencing Samples in Distance-space. Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). How and when is rapid point of diagnostic tools to arrive and reshape management of urosepsis? Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). Point of diagnosis to direct treatment: Emerging technologies? Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). Two to five hours of incubation of blood culture bottles in a standard laboratory incubator followed by nanopore sequencing is enough for the identification of sepsis pathogens and their antimicrobial resistance . Fulltekst i vitenarkiv
• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Vis alle 7 forfattere av denne artikkelen] (2024). Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing. BioRxiv. ISSN 0362-4331. doi: 10.1101/2024.03.04.582689. Fulltekst i vitenarkiv
• Branders, Sverre; Ahmad, Rafi & Grabherr, Manfred G. (2024). Real-time Taxonomic Characterization of Long-read Mixed-species Sequencing Samples in Distance-space. Fulltekst i vitenarkiv
• Ahmad, Rafi (2024). Short Turnaround Time of Seven to Nine Hours from Sample Collection Until Informed Decision for Sepsis Treatment Using Nanopore Sequencing. Fulltekst i vitenarkiv
• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Vis alle 7 forfattere av denne artikkelen] (2024). Rapid Detection of Clinically Relevant Uropathogens and Antimicrobial Resistance Genes in 5 Hours using Nanopore Sequencing. Fulltekst i vitenarkiv
• Ali, Jawad & Ahmad, Rafi (2023). Rapid Diagnostics of Pathogens and AMR-encoding Genes in Blood Cultures Using Nanopore Sequencing . Fulltekst i vitenarkiv
• Ahmad, Rafi (2023). Novel technological solutions for rapid diagnostics of infection and antimicrobial resistance:A One Health approach. Fulltekst i vitenarkiv
• Ahmad, Rafi (2023). A culture-amplification-independent, and rapid method for identification of pathogens and antibiotic resistance profile in bovine mastitis milk. Fulltekst i vitenarkiv
• Ahmad, Rafi (2023). AMR-Educate: Current Status and plans forward. Fulltekst i vitenarkiv
• Ali, Jawad & Ahmad, Rafi (2023). Multi-excitation Raman spectroscopy complements whole genome sequencing for rapid detection of bacterial infection and resistance in WHO priority pathogens. Fulltekst i vitenarkiv
• Chapagain, Crystal; Ali, Jawad; Branders, Sverre & Ahmad, Rafi (2023). Rapid Detection of Pathogens and Antimicrobial Resistance in Urine Using Nanopore Sequencing. Fulltekst i vitenarkiv
• Ahmad, Rafi (2023). What is AMR-Educate? Fulltekst i vitenarkiv
• Ahmad, Rafi (2023). Novel technological solutions for rapid diagnostics of infection and antimicrobial resistance. Fulltekst i vitenarkiv
• Ahmad, Rafi (2023). Novel technological solutions for rapid diagnostics of infection and antimicrobial resistance:A One Health approach. Fulltekst i vitenarkiv
• Ahmad, Rafi (2023). Transforming Research, Education, and Innovation in Antimicrobial Resistance. Fulltekst i vitenarkiv
• Ali, Jawad & Ahmad, Rafi (2022). Rapid diagnostics of pathogens and AMR genes in blood samples using nanopore sequencing. Fulltekst i vitenarkiv
• Ahmad, Rafi (2022). Presentation of AMR-Educate project. Fulltekst i vitenarkiv
• Ahmad, Rafi (2022). Novel technological solutions for rapid diagnostics of Infection and Antimicrobial resistance: test cases from human and animal health. Fulltekst i vitenarkiv
• Ali, Jawad & Ahmad, Rafi (2022). Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing. Fulltekst i vitenarkiv
• Ahmad, Rafi (2021). Personalized Healthcare: Bioinformatics & Drug Discovery Perspective. Fulltekst i vitenarkiv
• Bellankimath, Anurag Basavaraj; Ahmad, Rafi; Wilson, Robert Charles; Ahluwalia, Balpreet Singh & Johansen, Truls Erik Bjerklund (2026). Transforming urinary tract infection diagnostics: Clinical metagenomics for rapid pathogen identification and antimicrobial resistance profiling. Universitetet i Innlandet. ISSN 9788283806229. Fulltekst i vitenarkiv Vis sammendrag

  Urinary tract infections (UTIs) are among the most common bacterial infections world wide, impacting 405 million people and resulting in significant morbidity and healthcare costs. The routine diagnostic test, urine culture, takes 2-4 days to identify the pathogen and determine an appropriate antibiotic. This approach can lead to suboptimal clinical management, high antibiotic prescription rates, and improper use, which in turn foster antimicrobial resistance (AMR). Clinical metagenomics, which involves DNA sequencing of patient samples, offers a promising opportunity to revolutionize UTI diagnosis and treatment. Using Oxford Nanopore Technology (ONT), sequencing and data analysis can be performed in real-time. However, a crucial step for clinical application is the depletion of abundant human DNA, which dominates microbial DNA, as well as the removal of impurities and inhibitors present in the urine sample. Existing DNA extraction methods are not suitable for real-time sequencing because of their lengthy turnaround times, high costs, and challenges in managing clinical samples. The thesis aimed to develop, evaluate, and optimize DNA extraction methods for rapid pathogen identification and antibiotic susceptibility profiling in UTI diagnostics. We developed a culture-independent DNA extraction method that selectively removes human DNA and enriches microbial DNA. This method is accurate, fast, and cost-effective for clinical samples. In a proof-of-concept (PoC) study, urine samples from 78 patients with complicated UTI (cUTI) were evaluated using 11 different DNA extraction methods. The methods encompassed combinations of HL-SAN and M-SAN endonucleases, enzymatic and mechanical lysis strategies, and column and magnetic bead based DNA extraction techniques. Compared with routine culture-based diagnostic assays, the optimized method yielded the best pathogen identification performance (accuracy 99%, sensitivity 99%, specificity 100%) and antibiotic-susceptibility predictions (accuracy 90%, sensitivity 68%, specificity 95%). The method is also rapid, with a turnaround time of approximately 4 hours and a per sample cost of $36. Following modifications, a prospective validation study was carried out on an additional 296 cUTI samples, confirming pathogen identification performance consistent with the PoC study and slightly improved antibiotic susceptibility predictions. The limit of detection was 9.3 × 103 CFU/mL. The modifications also enabled the detection of Candida species, indicating the potential for comprehensive pathogen identification. The developed method’s usefulness extends beyond cUTI samples, as shown by the PoC study on blood cultures for diagnosing bloodstream infections. In this study, key parameters like saponin, endonucleases, and salt concentrations were fine tuned for the blood cultures. The optimized approach successfully depleted host material in blood cultures. In conclusion, the culture-independent sample preparation methods tackle the crucial challenge of DNA extraction from clinical samples. They provide a comprehensive and practical strategy to improve the diagnosis and management of UTIs and bloodstream infections, and to promote prudent antibiotic use and AMR stewardship. While additional validation is necessary, these findings show strong potential for broad clinical application and the potential to improve patient care.

• Branders, Sverre; Ahmad, Rafi; Grabherr, Manfred & Mahajan, Sumeet (2026). Machine Learning Solutions for Rapid Diagnostics of Infection and Antimicrobial Resistance. Universitet i Innlandet / University of Inland Norway. ISSN 9788283806175. Fulltekst i vitenarkiv Vis sammendrag

  Antimikrobiell resistens (AMR), mikrobers evne til å tåle legemidler somantibiotika og antivirale midler, truer global helse, utvikling og bærekraft. Overforbrukogfeilbrukav antimikrobielle midler er hovedårsakene til den økende forekomsten. Dagens kliniske diagnostikk bidrar til uhensiktsmessig antibiotikabruk, ettersom lang svartid ofte gjør omfattende empirisk behandling nødvendig. Nye, nøyaktige, kostnadseffektive og raske diagnostiske metoder for infeksjonssykdommer kan styrke antimikrobiell for valtning og sikre mer rasjonell bruk av antibiotika. Metagenomisk neste-generasjons sekvensering (mNGS) er en lovende metode for rask og agnostisk diagnostikk, men flere utfordringer begrenser fortsatt bruken i kliniske laboratorier. Målet med denne avhandlingen var å utvikle nye, brukervennlige algoritmiske og maskinlæringsbaserte (ML) løsninger for å muliggjøre rask metagenomikkbasert diagnostikk av infeksjoner og AMR. Fire programvareverktøy ble utviklet med fokus på brukervennlighet. Disse imple menterernyealgoritmerogML-metoderforåbidratilidentifikasjonogkarakterisering av patogener samt bestemmelse av antibiotikasensitivitet. Verktøyene har intuitive vi sualiseringer og grafiske brukergrensesnitt for å muliggjøre klinisk implementering. MysteryMaster utfører demultipleksering for Oxford Nanopore Technologies og kan gjenvinne opptil 33 % av tidligere uklassifiserte reads, noe som effektivt reduserer datatap. Voyager adresserer den sentrale utfordringen med rask og effektiv mapping av lange reads med innsettings- og delesjonsfeil mot referansegenomer ved å imple mentere en ny algoritme for mapping og redusere falske positive funn gjennom en dekonvolusjonsmetode. I en omfattende benchmark på tvers av 15 metagenomiske datasett med lange sekvenser overgikk Voyager eksisterende verktøy når det gjaldt nøyaktighet og minnebruk, samtidig som det var like raskt som dagens metoder. K MARVELidentifiserer nøyaktig antibiotikaresistensgener og punktmutasjoner direkte fra rå sekvenseringsdata ved hjelp av en ny protein-k-mer-basert algoritme. Denne metoden gir en betydelig hastighetsøkning sammenlignet med assembly-avhengige metoder, med tilsvarende nøyaktighet. Mango er en visuell korrelasjonssøker som integrerer tabulardata ved hjelp av en ny koderingsalgoritme. Resultatene viste at Mango raskt kan skille mellom kausale effekter og batch-effekter i storskala tabular data. Til slutt ble en mNGS-basert metode for patogenidentifikasjon og prediksjon av antibiotikasensitivitet optimalisert og evaluert i en klinisk pasientkohort med komplis erte urinveisinfeksjoner. Den optimaliserte metoden gir svar på omtrent fire timer til en kostnad som er lavere enn for kommersielt tilgjengelige metoder. I evalueringen oppnådde metoden 99 % samsvar i patogenidentifikasjon og 90 % samsvar i sensi tivitetsprediksjon sammenlignet med rutinemessige kliniske metoder. Funnenesompresenteres i denneavhandlingen adresserer sentrale algoritmiske og translasjonelle barrierer for klinisk implementering av mNGS-baserte hurtigdiagnos tiske metoder for infeksjon og AMR. Merutstrakt bruk av denneteknologien kan bidra til å oppdageutbrudd, styrkeinfeksjonsforebygging og-kontroll, diagnostikk og AMR forvaltning, forbedre pasientutfall og bidra til å bekjempe den globale AMR-krisen.

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