Anurag Basavaraj Bellankimath

• 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.

• 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.

• 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.

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• 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
• Bellankimath, Anurag Basavaraj (2026). Accelerating the Diagnosis and Management of Urinary Tract Infections. 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
• 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
• 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
• 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
• 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
• 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
• 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.

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