Identifying Clinically Relevant Bacteria Directly from Culture and Clinical Samples with a Handheld Mass Spectrometry Probe.

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.

Development and validation of a paper spray mass spectrometry method for the rapid quantitation of remdesivir and its active metabolite, GS-441524, in human plasma.

Introduction: Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several single-stranded RNA viruses, including the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2). It is currently the only FDA-approved antiviral agent for the treatment of individuals with COVID-19 caused by SARS-CoV-2. However, remdesivir pharmacokinetics/pharmacodynamics (PK/PD) and toxicity data in humans are extremely limited. It is imperative that precise analytical methods for the quantification of remdesivir and its active metabolite, GS-441524, are developed for use in further studies. We report, herein, the first validated anti-viral paper spray-mass spectrometry (PS-MS/MS) assay for the quantification of remdesivir and GS-441524 in human plasma. We seek to highlight the utility of PS-MS/MS technology and automation advancements for its potential future use in clinical research and the clinical laboratory setting. Methods: Calibration curves for remdesivir and GS-441524 were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Briefly, plasma samples were spotted on a paper substrate contained within pre-manufactured plastic cassette plates, and the spots were dried for 1 h. The samples were then analyzed directly for 1.2 min utilizing PS-MS/MS. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer utilizing automated technology. Results: The calibration ranges were 20 - 5000 and 100 - 25000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within ± 15%. Plasma calibrant stability was assessed and degradation for the 4 °C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 °C. No interference, matrix effects, or carryover was discovered during the validation process. Conclusions: PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir and GS-441524, which may be useful for clinical PK/PD, therapeutic drug monitoring (TDM), and toxicity assessment, particularly during the current COVID-19 pandemic and future viral outbreaks.

Cutting Instruments to Cut Costs: A Simple Initiative with Breast Surgical Operating Room Trays that Resulted in Substantial Savings.

BACKGROUND: A substantial expense in surgical care is incurred in the operating room (OR). We evaluated the financial impact of a systematic reduction in instrument tray contents on charges for breast surgery procedures. METHODS: A catalog of OR trays historically used for breast procedures (excisional biopsy, segmental and total mastectomy with or without axillary staging) was reviewed by four dedicated breast surgeons and downsized to a single tray accommodating all surgeon preferences. A matched-case comparison was performed pre- and post-downsizing. Cost analysis for salary and benefits (S&B) and unit supply cost (USC) pre- and post-downsizing were carried out. Instrument number, OR tray weights, set-up, and breakdown times were also compared. RESULTS: Post-downsizing, OR tray counts were reduced from 132 to 67 instruments (49%) and tray weight decreased from 30 to 20 pounds (33%). Scrub technician set-up and breakdown times were shorter by 22% and 25%, respectively. Comparing 449 matched cases (239 pre- and 210 post-downsizing), S&B and USC post-downsizing were decreased collectively for all procedures (p < 0.0001). With an average variance of S&B and USC (pre- to post-intervention) of $354, and an annualized case load of 813 operations, this could translate into S&B and USC savings of $287,802 per year. CONCLUSION: Simply downsizing OR breast trays resulted in decreased combined S&B and USC per procedure, leading to a substantial cost savings for the healthcare system. This measure aligns with a value and quality-based approach to patient care and could be easily replicated across institutions and specialties.

Study on the Gas-Phase Reactivity of Charged Pyridynes.

The reactivities of three isomeric, charged ortho-pyridynes, the 1,2-, 2,3-, and 3,4-didehydropyridinium cations, were examined in the gas phase using Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry. The structures of selected product ions were probed using collision-activated dissociation (CAD) experiments in a linear quadrupole ion trap (LQIT) mass spectrometer. Mechanisms based on quantum chemical calculations are proposed for the formation of all major products. The products of the reactions of the charged ortho-pyridynes in the gas phase were found to closely resemble those formed upon reactions of neutral ortho-arynes in solution, but the mechanisms of these reactions exhibit striking differences. Additionally, no radical reactions were observed for any of the charged ortho-pyridynes examined, in contrast to previous proposals that ortho-benzyne can occasionally react via radical mechanisms. Finally, the relative reactivities of those charged gaseous ortho-pyridynes that yielded similar product distributions were found to be affected mainly by the (calculated) vertical electron affinities of the dehydrocarbon sites, which suggests that the reactivity of these species is controlled by polar effects.

Clinical Features of Critical Coronavirus Disease 2019 in Children.

OBJECTIVES: We sought to describe the presentation, course, and outcomes of hospitalized pediatric coronavirus disease 2019 patients, with detailed description of those requiring mechanical ventilation, and comparisons between critically ill and noncritical hospitalized pediatric patients. DESIGN: Observational cohort study. SETTING: Riley Hospital for Children at Indiana University Health in Indianapolis in the early weeks of the coronavirus disease 2019 pandemic. PATIENTS: All hospitalized pediatric patients with confirmed coronavirus disease 2019 as of May 4, 2020, were included. INTERVENTIONS: Patients received therapies including hydroxychloroquine, remdesivir, tocilizumab, and convalescent serum and were managed according to an institutional algorithm based on evidence available at the time of presentation. MEASUREMENTS AND MAIN RESULTS: Of 407 children tested for severe acute respiratory syndrome-coronavirus 2 at our hospital, 24 were positive, and 19 required hospitalization. Seven (36.8%) were critically ill in ICU, and four (21%) required mechanical ventilation. Hospitalized children were predominantly male (14, 74%) and African-American or Hispanic (14, 74%), with a bimodal distribution of ages among young children less than or equal to 2 years old (8, 42%) and older adolescents ages 15-18 (6, 32%). Five of seven (71.4%) of critically ill patients were African-American (n = 3) or Hispanic (n = 2). Critical illness was associated with older age (p = 0.017), longer duration of symptoms (p = 0.036), and lower oxygen saturation on presentation (p = 0.016); with more thrombocytopenia (p = 0.015); higher C-reactive protein (p = 0.031); and lower WBC count (p = 0.039). Duration of mechanical ventilation averaged 14.1 days. One patient died. CONCLUSIONS: Severe, protracted coronavirus disease 2019 is seen in pediatric patients, including those without significant comorbidities. We observed a greater proportion of hospitalized children requiring mechanical ventilation than has been reported to date. Older children, African-American or Hispanic children, and males may be at risk for severe coronavirus disease 2019 requiring hospitalization. Hypoxia, thrombocytopenia, and elevated C-reactive protein may be useful markers of critical illness. Data regarding optimal management and therapies for pediatric coronavirus disease 2019 are urgently needed.

A statistical approach to optimizing paper spray mass spectrometry parameters.

RATIONALE: Paper spray mass spectrometry (PS-MS) was used to analyze and quantify ampicillin, a hydrophilic compound and frequently utilized antibiotic. Hydrophilic molecules are difficult to analyze via PS-MS due to their strong binding affinity to paper substrates and low ionization efficiency, among other reasons. METHODS: Solvent and paper parameters were optimized to increase the extraction of ampicillin from the paper substrate. After optimizing these key parameters, a Resolution IV 1/16 fractional factorial design with two center points was employed to screen eight different design parameters simultaneously. RESULTS: Pore size, sample volume, and solvent volume were the most significant factors affecting average peak area under the curve (AUC) and the signal-to-blank (S/B) ratio for the 1 µg/mL ampicillin calibrant. After optimizing the key parameters, a linear calibration curve with a range of 0.2 µg/mL to 100 µg/mL was generated (R2  = 0.98) and the limit of detection (LOD) and lower limit of quantification (LLOQ) were calculated to be 0.07 µg/mL and 0.25 µg/mL, respectively. CONCLUSIONS: The statistical optimization procedure undertaken here increased the mass spectral signal intensity by more than a factor of 40. This statistical method of screening followed by optimization experiments proved faster and more efficient, and produced more drastic improvements than typical one-factor-at-a-time experiments.

Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry.

Background 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 (lower limit of quantification [LLOQ]-upper limit of quantitation [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. Conclusions 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 the turnaround time and for use in clinical research.

Substituent Effects on the Reactivity of the 2,4,6-Tridehydropyridinium Cation, an Aromatic σ,σ,σ-Triradical.

2,4,6-Tridehydropyridinium cation (7) undergoes three consecutive atom or atom group abstractions from reagent molecules in the gas phase. By placing a π-electron-donating hydroxyl group between two radical sites, their reactivity can be quenched by enhancing their through-space coupling via a favorable resonance structure. Indeed, 3-hydroxy-2,4,6-tridehydropyridinium cation (8) abstracts only one atom or group of atoms from reagents. On the other hand, an electron-withdrawing cyano group between two of the radical sites (9) destabilizes the analogous resonance structure and diminishes through-space coupling between the radical sites, resulting in abstraction of three atoms, just like 7. However, the cyano-substituent also increases acidity to the point that 9 reacts pre-dominantly via proton transfer instead of undergoing radical reactions. Therefore, acidic triradicals may undergo nonradical, barrierless proton transfer reactions faster than radical reactions, which are usually accompanied by barriers. Examination of the analogous cyano-substituted mono-and biradicals revealed behavior similar to that of the corresponding unsubstituted species, with the exception of substantially greater reactivities due to their greater (calculated) vertical electron affinities. Finally, the 3-cyano-2,6-didehydropyridinium cation with a singlet ground state (S-T splitting: -11.9 kcal mol-1) was found to react exclusively from the lowest-energy triplet state by fast proton transfer reactions.

16S rRNA deep sequencing identifies Actinotignum schaalii as the major component of a polymicrobial intra-abdominal infection and implicates a urinary source.

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.

Experimental and computational studies on the formation of three para-benzyne analogues in the gas phase.

Experimental and computational studies on the formation of three gaseous, positively-charged para-benzyne analogues in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer are reported. The structures of the cations were examined by isolating them and allowing them to react with various neutral reagents whose reactions with aromatic carbon-centered σ-type mono- and biradicals are well understood. Cleavage of two iodine-carbon bonds in N-deuterated 1,4-diiodoisoquinolinium cation by collision-activated dissociation (CAD) produced a long-lived cation that showed nonradical reactivity, which was unexpected for a para-benzyne. However, the reactivity closely resembles that of an isomeric enediyne, N-deuterated 2-ethynylbenzonitrilium cation. A theoretical study on possible rearrangement reactions occurring during CAD revealed that the cation formed upon the first iodine atom loss undergoes ring-opening before the second iodine atom loss to form an enediyne instead of a para-benzyne. Similar results were obtained for the 5,8-didehydroisoquinolinium cation and the 2,5-didehydropyridinium cation. The findings for the 5,8-didehydroisoquinolinium cation are in contradiction with an earlier report on this cation. The cation described in the literature was regenerated by using the literature method and demonstrated to be the isomeric 5,7-didehydro-isoquinolinium cation and not the expected 5,8-isomer.

Does the 2,6-didehydropyridinium cation exist?

Reactive intermediates are key species involved in many chemical and biochemical processes. For example, carbon-centered aromatic σ,σ-biradicals formed in biological systems from naturally occurring enediyne antitumor antibiotics are responsible for the irreversible cleavage of double-stranded DNA caused by these prodrugs. However, because of their high reactivity, it is very difficult or impossible to isolate and investigate these biradicals. The aromatic σ,σ-biradical, 2,6-didehydropyridine, has been speculated for many years to be formed in certain organic reactions; however, no definitive proof of its generation has been obtained. We report here the successful generation of protonated 2,6-didehydropyridine and the examination of its chemical properties in the gas phase by using a Fourier transform ion cyclotron resonance mass spectrometer. The results suggest that a mixture of singlet (ground) state and triplet (excited) state 2,6-didehydropyridinium cations was generated. The two different states show qualitatively different reactivity, with the triplet state showing greater Brønsted acidity than that of the singlet state. The triplet state also shows much greater radical reactivity than that of the singlet state, as expected because of the coupling of the nonbonding electrons in the singlet state.