Browsing by Author "Kirkpatrick, Lindsey M."

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    16S rRNA deep sequencing identifies Actinotignum schaalii as the major component of a polymicrobial intra-abdominal infection and implicates a urinary source
    (Microbiology Society, 2017-05-03) Bryan, Andrew; Kirkpatrick, Lindsey M.; Manaloor, John J.; Salipante, Stephen J.; Pediatrics, School of Medicine
    Introduction. It can be difficult to catalogue the individual organisms comprising polymicrobial patient infections, both because conventional clinical microbiological culture does not facilitate the isolation and enumeration of all members of a complex microbial community, and because fastidious organisms may be mixed with organisms that grow rapidly in vitro. Empiric antimicrobial treatment is frequently employed based on the anatomical site and the suspected source of the infection, especially when an appropriately collected surgical specimen is not obtained., Case presentation. We present a case of an intra-abdominal infection in a patient with complex anatomy and recurrent urinary tract infections. Imaging did not reveal a clear source of infection, no growth was obtained from urine cultures and initial abdominal fluid cultures were also negative. In contrast, 16S rRNA deep sequencing of abdominal fluid samples revealed mixed bacterial populations with abundant anaerobes, including Actinotignum schaalii (Actinobaculum schaalii). Ultimately, only Enterobacter cloacae complex and meticillin-resistant Staphylococcus aureus, both of which were identified by sequencing, were recovered by culture., Conclusion. The clinical application of 16S rRNA deep sequencing can more comprehensively and accurately define the organisms present in an individual patient's polymicrobial infection than conventional microbiological culture, detecting species that are not recovered under standard culture conditions or that are otherwise unexpected. These results can facilitate effective antimicrobial stewardship and help elucidate the possible origins of infections.
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    Identifying Clinically Relevant Bacteria Directly from Culture and Clinical Samples with a Handheld Mass Spectrometry Probe
    (Oxford University Press, 2022) Povilaitis, Sydney C.; Chakraborty, Ashish; Kirkpatrick, Lindsey M.; Downey, Rachel D.; Hauger, Sarmistha B.; Eberlin, Livia S.; Pediatrics, School of Medicine
    Background: Rapid identification of bacteria is critical to prevent antimicrobial resistance and ensure positive patient outcomes. We have developed the MasSpec Pen, a handheld mass spectrometry-based device that enables rapid analysis of biological samples. Here, we evaluated the MasSpec Pen for identification of bacteria from culture and clinical samples. Methods: A total of 247 molecular profiles were obtained from 43 well-characterized strains of 8 bacteria species that are clinically relevant to osteoarticular infections, including Staphylococcus aureus, Group A and B Streptococcus, and Kingella kingae, using the MasSpec Pen coupled to a high-resolution mass spectrometer. The molecular profiles were used to generate statistical classifiers based on metabolites that were predictive of Gram stain category, genus, and species. Then, we directly analyzed samples from 4 patients, including surgical specimens and clinical isolates, and used the classifiers to predict the etiologic agent. Results: High accuracies were achieved for all levels of classification with a mean accuracy of 93.3% considering training and validation sets. Several biomolecules were detected at varied abundances between classes, many of which were selected as predictive features in the classifiers including glycerophospholipids and quorum-sensing molecules. The classifiers also enabled correct identification of Gram stain type and genus of the etiologic agent from 3 surgical specimens and all classification levels for clinical specimen isolates. Conclusions: The MasSpec Pen enables identification of several bacteria at different taxonomic levels in seconds from cultured samples and has potential for culture-independent identification of bacteria directly from clinical samples based on the detection of metabolic species.
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    P-2109. Culture-Free Identification of Microbes in Seconds Directly from Clinical Samples using the MasSpec Pen Technology
    (Oxford University Press, 2025-01-29) Downey, Rachel; Kumar, Manoj; Kirkpatrick, Lindsey M.; Hauger, Sarmistha Bhaduri; Johnson, Coreen; Dunn, James; Jackobs, Faith; Keating, Michael; Eberlin, Livia; Pediatrics, School of Medicine
    Background: Broad spectrum antibiotics are often used empirically in cases of suspected invasive infection requiring surgical intervention while awaiting results of conventional testing (cultures and molecular tests). Rapid and accurate diagnosis of the etiologic pathogens is critical to allow for selection of targeted antibiotics and improve outcomes for patients. We present current results of a large, multi-center clinical study using the MasSpec Pen (MSPen) technology to characterize the metabolic profiles of clinically relevant microbes in culture isolates and apply the technology to culture-independent identification of infectious agents directly in clinical samples. Methods: The MSPen allows direct sample analysis using a solvent droplet followed by immediate mass spectrometry analysis, with total acquisition time of ∼15 seconds (fig 1). In this study 785 samples (635 isolates and 150 clinical specimens) were analyzed using this technology to create distinct metabolic profiles to differentiate bacterial pathogens. 80% of data were used as a training set to develop such profiles; 20 percent of data were used as a test set to identify bacterial infection. Results: We identified over 400 bacterial metabolites and lipids in 10 different microbial species and compiled a unique metabolic profile for each that was used to directly identify specific microbes in cultures and clinical specimens. Among culture isolates, our statistical classifiers were able to achieve 99% accuracy for Gram-typing, and 99% accuracy among 10 bacteria in the test set (fig 2). Using species-specific metabolites and lipids, we were able to identify Pseudomonas aeruginosa directly from 3 infected specimens and Staphylococcus epidermidis directly from infected bone. Conclusion: Our results show the incredible promise that direct MSPen analysis has for rapid, culture-independent identification of bacteria from patient tissues. We are working to build classifiers to differentiate bacteria by specific m/z values to improve identification in tissue samples.
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    Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry
    (De Gruyter, 2020-01-13) Skaggs, Christine L.; Ren, Greta J.; Elgierari, El Taher M.; Sturmer, Lillian R.; Shi, Run Z.; Manicke, Nicholas E.; Kirkpatrick, Lindsey M.; Pediatrics, School of Medicine
    Introduction: Invasive fungal disease is a life-threatening condition that can be challenging to treat due to pathogen resistance, drug toxicity, and therapeutic failure secondary to suboptimal drug concentrations. Frequent therapeutic drug monitoring (TDM) is required for some anti-fungal agents to overcome these issues. Unfortunately, TDM at the institutional level is difficult, and samples are often sent to a commercial reference laboratory for analysis. To address this gap, the first paper spray-mass spectrometry assay for the simultaneous quantitation of five triazoles was developed. Methods: Calibration curves for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were created utilizing plasma-based calibrants and four stable isotopic internal standards. No sample preparation was needed. Plasma samples were spotted on a paper substrate in pre-manufactured plastic cartridges, and the dried plasma spots were analyzed directly utilizing paper spray-mass spectrometry (paper spray MS/MS). All experiments were performed on a Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer. Results: The calibration curves for the five anti-fungal agents showed good linearity (R2 = 0.98 – 1.00). The measured assay ranges (LLOQ – ULOQ) for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were 0.5 – 50 μg/mL, 0.1 – 10 μg/mL, 0.1 – 10 μg/mL, 0.1 – 10 μg/mL, and 0.1 – 10 μg/mL, respectively. The inter- and intra-day accuracy and precision were less than 25% over the respective ranges. Conclusion: We developed the first rapid paper spray MS/MS assay for simultaneous quantitation of five triazole anti-fungal agents in plasma. The method may be a powerful tool for near point-of-care TDM aimed at improving patient care by reducing turnaround time and for use in clinical research.