Biology subjects

Bellankimath, A. B.

Publications and source records attributed to Bellankimath, A. B..

4 recordsLinked to original sources

bioRxiv2026

From culture to clarity in four hours: accelerating clinical management of bloodstream infections using metagenomics

BackgroundMetagenomic next-generation sequencing (mNGS) has the potential to transform clinical diagnostics for bloodstream infections (BSIs). However, its clinical utility is currently limited by several challenges, including the extraction of DNA from blood cultures. Our aim was to develop, evaluate, and optimize an in-house method for host depletion and bacterial DNA extraction from positive blood cultures to enable rapid mNGS-based pathogen detection and antimicrobial resistance profiling, informing clinical management of BSIs. Methods151 clinical blood cultures (115 positive and 36 negative) were processed for DNA extraction using an in-house-developed SEPSINN method for host depletion and bacterial DNA extraction. mNGS on the MinION was performed, and the results for pathogen identification and antimicrobial susceptibility predictions were compared with the routine clinical workflow. SEPSINN was also evaluated against a commercial DNA extraction method to assess its effectiveness in depleting host DNA and recovering bacterial DNA. ResultsThe SEPSINN method achieved up to 1000-fold depletion of host DNA and outperformed the commercial DNA extraction method. At the sample level, mNGS achieved 100% accuracy, specificity, and sensitivity, identifying at least one pathogen in all 115 positive blood cultures. At the pathogen level, mNGS showed 98% accuracy (120/123), specificity, and sensitivity. For antimicrobial susceptibility predictions, mNGS achieved an accuracy of 95% (1382/1451), a sensitivity of 88% (203/230), and a specificity of 97% (1179/1221). Moreover, the method also identified fungi, indicating a wider taxonomic range. mNGS resulted in an approximately 4-hour turnaround time for pathogen identification and resistance profiling. ConclusionsThe method can provide information on BSI clinical management within approximately 24 hours of receiving the sample, including the time required for culture positivity. This represents an important advancement in the clinical management of BSIs, with the potential to save lives and promote antibiotic stewardship.

microbiology↗

bioRxiv2026

Rapid clinical metagenomics enables early tailored therapy in complicated urinary tract infections and strengthens antimicrobial stewardship

Rapid and accurate diagnosis of UTIs remains difficult because culture-based methods are slow and less sensitive. This study evaluates URINN, a metagenomic workflow that detects uropathogens, antibiotic resistance genes, and virulence factors directly from patient urine samples. The optimized protocol was tested on a combined set of 349 clinical urine samples. URINN demonstrated 99% accuracy across all samples and 97% sensitivity for identifying 294 pathogens, including both bacteria and fungi. It predicted antibiotic susceptibility with 91% accuracy across 2099 antibiotics. The method detected pathogens at concentrations as low as 9.3 x 103 CFU/mL and provided results within approximately four hours. Flow cytometry and DNA yield analyses helped establish thresholds to differentiate culture-positive from culture-negative samples, with genome coverage linked to the accuracy of susceptibility predictions for certain species. Virulence profiling revealed that adherence and nutritional factors are crucial for colonization and persistence. Leukocyte counts were comparable between genders, but bacterial loads were higher in females. The catheterized group had significantly higher leukocyte counts, and their urine showed increased cephalosporin resistance. This approach could enhance clinical decision-making, support personalized treatment, and improve the management of complicated UTIs, thereby contributing to better UTI care and antibiotic stewardship.

genomics↗

bioRxiv2025

Development and optimization of the host DNA depletion in blood cultures using a saponin and SAN nucleases-based method

Bloodstream infections and sepsis are major health issues causing millions of deaths annually. Early and accurate diagnosis of sepsis is crucial for reducing mortality and combating antimicrobial resistance. However, current diagnostic methods are slow and time-consuming. Nanopore sequencing has the potential to serve as a rapid diagnostic method for sepsis. However, extracting bacterial DNA from blood samples is challenging due to the high content of host DNA. This study aimed to develop and optimize a method using saponin and SAN nucleases for effective host DNA depletion in blood cultures. Different concentrations of saponin, HL-SAN, and M-SAN nucleases, combined with various salt conditions (NaCl and MgCl2), were tested for their ability to deplete host DNA in blood cultures spiked with E. coli and S. aureus. The impact of different bead beating durations on DNA fragment sizes was also examined. The efficiency of host DNA depletion and bacterial DNA extraction was assessed using quantitative PCR (qPCR) and nanopore sequencing. Results indicated that 4% of saponin effectively lysed host cells, releasing DNA while preserving bacterial cells. The host DNA is then efficiently depleted using 250 units (10 L) of HL-SAN and M-SAN nucleases. Although SAN in combination with higher salt concentrations (2.5 M NaCl and 50 mM MgCl2) showed somewhat better host DNA removal, the difference was not statistically significant. Reducing bead beating time to 6 minutes improved the recovery of longer DNA fragments compared to 10 minutes. These results demonstrate an effective method for host DNA depletion using saponin and SAN nucleases, which is compatible with downstream processes, such as nanopore sequencing and qPCR.

microbiology↗

bioRxiv2024

Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing

PurposeUrinary Tract Infection (UTI) is a prevalent global health concern accounting for 1-3% of primary healthcare visits. The current methods for UTI diagnosis have a high turnaround time of 3-5 days for pathogen identification and susceptibility testing. This work is a proof-of-concept study aimed at determining the detection limit by establishing a culture and amplification-free DNA extraction methodology from spiked urine samples followed by real-time Nanopore sequencing and data analysis. MethodsThis study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Pathogens were spiked at two clinically significant concentrations of 103 and 105 CFU/ml. Three commercial DNA extraction kits were investigated based on the quantity of isolated DNA, average processing time, elution volume and the average cost incurred per extraction. The outperforming kit was used for direct DNA extraction and subsequent sequencing on MinION and Flongle flowcells. ResultsThe Blood and Tissue kit outperformed the other kits. All pathogens were identified at a concentration of 105 CFU/ml within ten minutes, and the corresponding AMR genes were detected within three hours of the sequencing start. The overall turnaround time including the DNA extraction and sequencing steps was five hours. Moreover, we also demonstrate that the identification of some pathogens and antibiotic-resistance genes was possible at a spike concentration of 103 CFU/mL. ConclusionThis study shows great promise toward reducing the time required for making an informed antibiotic administration from approximately 48 hours to five hours thereby reducing the number of empirical doses and saving lives.

microbiology↗