Jawad Ali

• Ali, Jawad; Bellankimath, Anurag Basavaraj; Hira, Jonathan; Chapagain, Crystal; Opgård, Silje Therese & Simonsen, Gunnar Skov [Show all 7 contributors for this article] (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. Full text in Research Archive Show summary

  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.

• 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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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 [Show all 10 contributors for this article] (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. Full text in Research Archive Show summary

  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 [Show all 8 contributors for this article] (2025). MysteryMaster: scraping the bottom of the barrel of barcoded Oxford nanopore reads. BMC Bioinformatics. 26. doi: 10.1186/s12859-025-06266-2. Full text in Research Archive Show summary

  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.

• 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. In Kaur, Punit & Sharma, Priyanka (Ed.), Bacterial Enzymes as Targets for Drug Discovery: Meeting the Challenges of Antibiotic Resistance. Academic Press. ISSN 9780443222221. p. 41–50. doi: 10.1016/B978-0-443-22222-1.00018-0. Full text in Research Archive Show summary

  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.

• 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. Full text in Research Archive Show summary

  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 [Show all 7 contributors for this article] (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), p. 2177–2190. doi: 10.1007/s10096-024-04929-1. Full text in Research Archive Show summary

  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. Full text in Research Archive
• Chapagain, Crystal; Khezri, Abdolrahman; Ali, Jawad; Smistad, Marit; Sølverød, Liv Synnøve & Ahmadi, Asal [Show all 7 contributors for this article] (2026). No culture, no amplification, just results: a five-hour breakthrough for bovine mastitis diagnostics. Full text in Research Archive
• Bellankimath, Anurag Basavaraj; Branders, Sverre; Kegel, Isabell; Ali, Jawad; Asadi, Fatemeh & Johansen, Truls Erik Bjerklund [Show all 10 contributors for this article] (2025). Accurate and rapid turnaround of four hours for the diagnosis of complicated UTIs using metagenomics. Full text in Research Archive
• Bellankimath, Anurag Basavaraj; Branders, Sverre; Kegel, Isabell; Ali, Jawad; Asadi, Fatemeh & Johansen, Truls E. Bjerklund [Show all 10 contributors for this article] (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. Full text in Research Archive
• Branders, Sverre; Kegel, Isabell; Ali, Jawad; Asadi, Fatemeh; Johansen, Truls E. Bjerklund & Imirzalioglu, Can [Show all 9 contributors for this article] (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. Full text in Research Archive
• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Show all 7 contributors for this article] (2025). Rapid Diagnosis of Urinary Tract Infection using Nanopore sequencing. Full text in Research Archive
• 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. Full text in Research Archive
• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Show all 7 contributors for this article] (2024). Rapid Detection of Clinically Relevant Uropathogens and Antimicrobial Resistance Genes in 5 Hours using Nanopore Sequencing. Full text in Research Archive
• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Show all 7 contributors for this article] (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. Full text in Research Archive
• Bellankimath, Anurag Basavaraj; Chapagain, Crystal; Branders, Sverre; Ali, Jawad; Wilson, Robert Charles & Johansen, Truls E. Bjerklund [Show all 7 contributors for this article] (2024). Rapid Detection of Clinically Relevant Uropathogens and Antimicrobial Resistance Genes in 5 Hours using Nanopore Sequencing. Full text in Research Archive
• Ali, Jawad & Ahmad, Rafi (2023). Rapid Diagnostics of Pathogens and AMR-encoding Genes in Blood Cultures Using Nanopore Sequencing . Full text in Research Archive
• 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. Full text in Research Archive
• Chapagain, Crystal; Ali, Jawad; Branders, Sverre & Ahmad, Rafi (2023). Rapid Detection of Pathogens and Antimicrobial Resistance in Urine Using Nanopore Sequencing. Full text in Research Archive
• Ali, Jawad & Ahmad, Rafi (2022). Rapid diagnostics of pathogens and AMR genes in blood samples using nanopore sequencing. Full text in Research Archive
• Ali, Jawad & Ahmad, Rafi (2022). Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing. Full text in Research Archive

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