Profiling volatile organic compounds from human plasma using GC × GC-ToFMS.

Volatile organic compounds (VOCs) originating from human metabolic activities can be detected in, for example, breath, urine, feces, and blood. Thus, attention has been given to identifying VOCs from the above matrices. Studies identifying and measuring human blood VOCs are limited to those focusing on monitoring specific pollutants, or blood storage and/or decomposition. However, a comprehensive characterization of VOCs in human blood collected for routine diagnostic testing is lacking. In this pilot study, 72 blood-derived plasma samples were obtained from apparently healthy adult participants. VOCs were extracted from plasma using solid-phase microextraction and analyzed using comprehensive two-dimensional gas chromatography tandem time-of-flight mass spectrometry. Chromatographic data were aligned, and putative compound identities were assigned via spectral library comparison. All statistical analysis, including contaminant removal, data normalization, and transformation were performed usingR. We identified 401 features which we called the pan volatilome of human plasma. Of the 401 features, 34 were present in all the samples with less than 15% variance (core molecules), 210 were present in ⩾10% but <100% of the samples (accessory molecules), and 157 were present in less than 10% of the samples (rare molecules). The core molecules, consisting of aliphatic, aromatic, and carbonyl compounds were validated using 25 additional samples. The validation accuracy was 99.9%. Of the 34 core molecules, 2 molecules (octan-2-one and 4-methyl heptane) have been identified from the plasma samples for the first time. Overall, our pilot study establishes the methodology of profiling VOCs in human plasma and will serve as a resource for blood-derived VOCs that can complement future biomarker studies using different matrices with more heterogeneous cohorts.

Machine Learning for The Prediction of Ranked Applicants and Matriculants to an Internal Medicine Residency Program.

Phenomenon: Residency programs throughout the country each receive hundreds to thousands of applications every year. Holistic review of this many applications is challenging, and to-date, few tools exist to streamline or assist in the process for selecting candidates to interview and rank. Machine learning could assist programs in predicting which applicants are likely to be ranked, and among ranked applicants, which are likely to matriculate.Approach: In the present study, we used the machine learning algorithm Random Forest (RF) to differentiate between ranked and unranked applicants as well as matriculants and ranked non-matriculants to an internal medicine residency program in northern New England over a three-year period. In total, 5,067 ERAS applications were received during the 2016-17, 2017-18, and 2018-19 application cycles. Of these, 4,256 (84.0%) were unranked applicants, 754 (14.9%) were ranked non-matriculants, and 57 (1.12%) were ranked matriculants.Findings: For differentiating between ranked and unranked applicants, the RF algorithm achieved an area under the receiver operating characteristic (AUROC) curve of 0.925 (95% CI: 0.918-0.932) and area under the precision-recall curve (AUPRC) of 0.652 (0.611-0.685), while for differentiating between matriculants and ranked non-matriculants, the AUROC was 0.597 (95% CI: 0.516-0.680) and AUPRC was 0.114 (0.075-0.167). The ranks of matriculated applicants were significantly higher using the algorithmic rank list as compared with the actual rank list for the 2017-18 (median rank: 98 versus 204, p < .001) and 2018-19 cycles (74 versus 192, p = .006), but not the 2016-17 cycle (97 versus 144, p = .37).Insights: The RF algorithm predicted which applicants among the overall applicant pool were ranked with impressive accuracy and identified matriculants among ranked candidates with modest but better-than-random accuracy. This approach could assist residency programs with triaging applicants based on the likelihood of a candidate being ranked and/or matriculating.

Serial T-SPOT.TB responses in Tanzanian adolescents: Transient, persistent and irregular conversions.

BACKGROUND: Prospective studies of interferon-gamma release assays (IGRA) on healthy subjects in tuberculosis-endemic regions have not examined the long-term variability of serial assays. This issue is relevant to the interpretation of tuberculosis (TB) vaccine trials based on prevention of infection. METHODS: T-SPOT.TB assays were performed manually on healthy adolescents during a tuberculosis vaccine trial in Tanzania at 5 intervals over 3 years. Assay results were defined as negative, positive, borderline or invalid. Subsequently, microtiter plates were analyzed by an automated reader to obtain quantitative counts of spot forming cells (SFCs) for the present analysis. RESULTS: 3387 T-SPOT.TB samples were analyzed from 928 adolescents; manual and automated assay results were 97% concordant. Based on the quantitative results 143 (15%) participants were prevalent IGRA-positives at baseline, were ineligible for further study. Among the remaining IGRA-negative participants, the annual rate of IGRA conversion was 2·9%. Among 43 IGRA converters with repeat assays 12 (28%) were persistent converters, 16 (37%) were transient converters, and 15 (35%) comprised a new category defined as irregular converters (≥2 different subsequent results). ESAT-6 and CFP-10 responses were higher in prevalent than incident positives: 53 vs 36 for CFP-10 (p < 0·007); 44 vs 34 for ESAT-6 (p = 0·12). CONCLUSIONS: Definitions of IGRA conversion, reversion, and persistence depend critically on the frequency of testing. Multiple shifts in categories among adolescents in a TB-endemic country may represent multiple infections, variable host responses in subclinical infection, or assay variation. These findings should to be considered in the design and interpretation of TB vaccine trials based on prevention of infection. Household contact studies could determine whether even transient IGRA conversion might represent exposure to an active case of M. tuberculosis disease.

Volatile organic compound profiling to explore primary graft dysfunction after lung transplantation.

Primary graft dysfunction (PGD) is a major determinant of morbidity and mortality following lung transplantation. Delineating basic mechanisms and molecular signatures of PGD remain a fundamental challenge. This pilot study examines if the pulmonary volatile organic compound (VOC) spectrum relate to PGD and postoperative outcomes. The VOC profiles of 58 bronchoalveolar lavage fluid (BALF) and blind bronchial aspirate samples from 35 transplant patients were extracted using solid-phase-microextraction and analyzed with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry. The support vector machine algorithm was used to identify VOCs that could differentiate patients with severe from lower grade PGD. Using 20 statistically significant VOCs from the sample headspace collected immediately after transplantation (< 6 h), severe PGD was differentiable from low PGD with an AUROC of 0.90 and an accuracy of 0.83 on test set samples. The model was somewhat effective for later time points with an AUROC of 0.80. Three major chemical classes in the model were dominated by alkylated hydrocarbons, linear hydrocarbons, and aldehydes in severe PGD samples. These VOCs may have important clinical and mechanistic implications, therefore large-scale study and potential translation to breath analysis is recommended.

CT for estimating adequacy of lymph node dissection in patients with squamous cell carcinoma of the head and neck.

BACKGROUND: Indices obtained from lymph node dissection specimens, specifically lymph node yield (LNY) and lymph node ratio (LNR), have prognostic significance in the setting of head and neck squamous cell carcinoma (HNSCCa). However, there are currently no validated tools to estimate adequacy of planned lymph node dissection using preoperative data. The present study sought to evaluate CT-derived estimates of lymphatic tissue volumes as a preoperative tool to guide cervical node dissection. METHODS: Fifteen cervical lymph node dissections were performed in 14 subjects with HNSCCa. Preoperative CT-derived estimates of lymphatic tissue volumes were compared with gross pathology tissue volume estimates and pathologically-determined LNY. RESULTS: Resected tissue volume (calculated using the triaxial ellipsoid method) correlates with CT-derived preoperative lymphatic volume estimates (r = 0.74, p = 0.003) while LNY does not(r = - 0.12, p = 0.67). When excluding pathologically enlarged lymph nodes ("refined" data), a negative correlation was observed between refined CT-derived volume estimates and refined LNY (r = - 0.65, p = 0.009). CONCLUSION: In the setting of cervical lymph node dissection, CT-derived lymphatic volume estimates correlate with resected tissue volume, but refined CT-derived volume estimates correlate negatively with refined LNY. TRIAL REGISTRATION: Retrospectively registered. LEVEL OF EVIDENCE: 4.

Characterization of Ciprofloxacin Resistance Levels: Implications for Ototopical Therapy.

HYPOTHESIS: Ciprofloxacin-resistant pathogens are inhibited by high concentrations of ciprofloxacin found in commercially-available ototopical solutions. BACKGROUND: Ciprofloxacin-resistant pathogens in otitis media are currently treated with ototopical ciprofloxacin suspensions. This is done irrespective of laboratory-reported ciprofloxacin susceptibility, under the assumption that the high concentration of ciprofloxacin applied topically is sufficient to overcome antimicrobial resistance. METHODS: We evaluated 34 ciprofloxacin-resistant isolates consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Corynebacterium spp., and Turicella otitidis. Ciprofloxacin minimum inhibitory concentration (MIC) assays and clinical ototopical solution minimum bactericidal concentration (CMBC) assays were performed. RESULTS: Amongst the ciprofloxacin-resistant isolates, ciprofloxacin MICs ranged from 8 to 256 mcg/ml (mean: 87.1 mcg/ml) and CMBCs ranged from 23.4 to 1500 mcg/ml (mean: 237.0 mcg/ml). There were no significant differences with respect to MIC in comparing P. aeruginosa versus Corynebacterium spp. (mean: 53.3 versus 55.2, p = 0.86), S. aureus versus P. aeruginosa (mean: 128.0 versus 53.3, p = 0.34), and S. aureus versus Corynebacterium spp. (mean: 128.0 versus 55.2, p = 0.09). The correlation between ciprofloxacin MIC and CMBC was poor (Pearson's r = -0.08, p = 0.75). CONCLUSIONS: Ciprofloxacin-resistant pathogens commonly recovered from otitis media exhibit highly variable ciprofloxacin MIC and CMBC levels. Ciprofloxacin was able to inhibit growth in all isolates tested at MIC levels less than or equal to 256 mcg/ml; however, CMBC's up to 1500 mcg/ml were observed within that same group. The clinical relevance of these in vitro MICs is unclear due in part to higher bactericidal concentrations (CMBC) in several strains. Our results suggest that treatment failures may be due to a combination of factors rather than high-level resistance alone.

Tensor Veli Palatinopexy as a Novel Treatment for Eustachian Tube Dysfunction: A Cadaveric Feasibility Study.

OBJECTIVE: To demonstrate the feasibility of a novel procedure whereby a suture is placed transorally in the tensor veli palatini muscle to tighten it, thereby dilating the cartilaginous portion of the eustachian tube (ET). STUDY DESIGN: The study design was a prospective cadaveric experiment to examine the feasibility of a novel treatment for ET dysfunction. SETTING: Academic medical center in a research-oriented operating room with intraoperative computed tomography (CT) capabilities. METHODS: Seven fresh-frozen cadaver heads were obtained, each of which was thawed for 36 hours prior to use. The preprocedural volumes of the cartilaginous ET were measured by filling the ET with an iodine-containing radiocontrast agent via the nasopharynx and then obtaining CT images. Submucosal flaps in the soft palate were raised, and sutures were placed in the tensor veli palatini bilaterally to increase tension. After completion of the procedure, contrast placement and CT imaging were repeated. Three-dimensional models of the ETs were created, and the volumes were measured and compared. RESULTS: Of the 14 ETs evaluated, 13 showed an increase in postprocedure volume. On average, postprocedure volumes showed a 57% increase from preprocedure volumes (mean relative change, 57.1%; P = .013). CONCLUSION: Placement of a tension-holding suture in the tensor veli palatini muscle can reliably dilate the cartilaginous portion of the ET. Such a procedure may be useful in the treatment of ET dysfunction.

DAR-901 vaccine for the prevention of infection with Mycobacterium tuberculosis among BCG-immunized adolescents in Tanzania: A randomized controlled, double-blind phase 2b trial.

BACKGROUND: SRL172 prevented disease due to Mycobacterium tuberculosis in a Phase 3 trial. DAR-901 represents a scalable manufacturing process for SRL172. We sought to determine if DAR-901 would prevent infection with M. tuberculosis among BCG-primed adolescents age 13-15 years in Tanzania. METHODS: Adolescents with a negative T- SPOT.TBR interferon gamma release assay (IGRA) were randomized 1:1 to three intradermal injections of DAR-901 or saline placebo at 0, 2 and 4 months. Repeat IGRAs were performed at 2 months, and at 1, 2, and 3 years. The primary efficacy outcome was time to new TB infection (IGRA conversion to positive); the secondary outcome was time to persistent TB infection (IGRA conversion with repeat positive IGRA). RESULTS: Among 936 participants screened 667 were eligible and randomized to their first dose of vaccine or placebo (safety cohort). At 2 months, 625 participants remained IGRA-negative and were scheduled for the additional two doses (efficacy cohort). DAR-901 was safe and well-tolerated. One DAR-901 recipient developed a vaccine site abscess. Neither the primary nor secondary endpoints differed between the two treatment arms (p = 0.90 and p = 0.20, respectively). DAR-901 IGRA converters had median responses to ESAT-6 of 50.1 spot-forming cells (SFCs) vs. 19.6 SFCs in placebo IGRA converters (p = 0.03). CONCLUSIONS: A three-dose series of 1 mg DAR-901 was safe and well-tolerated but did not prevent initial or persistent IGRA conversion. DAR-901 recipients with IGRA conversion demonstrated enhanced immune responses to ESAT-6. Since protection against disease may require different immunologic responses than protection against infection a trial of DAR-901 to prevent TB disease is warranted. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov as NCT02712424.

Outcomes of a novel alloplastic technique for external auditory canal repair in tympanomastoidectomy.

OBJECTIVE: To analyze surgical outcomes of a novel alloplastic reconstruction technique for partial external auditory canal (EAC) defects in tympanomastoidectomy. METHODS: Retrospective study of 51 patients with cholesteatoma who underwent repair of partial EAC defects during tympanomastoidectomy at a tertiary referral center over 8 years. Nineteen patients were treated with a novel alloplastic graft technique using hydroxyapatite cement and bone pâté for EAC repair. Thirty-two patients treated with traditional cartilage repair of the EAC served as a control group. The primary outcomes measured were postoperative cholesteatoma recurrence rates, infection rates, and mean air-bone gap (ABG). RESULTS: Twenty of the 51 cases (39.2%) were revision surgeries for cholesteatoma recidivism, with a greater proportion of revision surgeries in the alloplastic group (57.9% vs 28.1%, P = .04). There was no significant difference in postoperative cholesteatoma recurrence (P = 1.00) or infection rates (P = .64) between the two techniques, with the alloplastic group experiencing slightly lower rates of recurrence (36.8%) and infection (5.3%) than cartilage repair (37.5% recurrence, 12.5% infection). Mean postoperative ABGs were comparable between the alloplastic (21.5 dB) and cartilage group (26.0 dB, P = .10). CONCLUSIONS: Composite alloplastic and bone pâté reconstruction is an effective technique to repair partial EAC defects in tympanomastoidectomy, with comparable postoperative hearing outcomes and no increased risk of cholesteatoma recurrence or infection compared to traditional cartilage repair. Recidivism rates were relatively high in both groups, likely due to the high rate of revision surgeries and aggressive nature of cholesteatoma within the cohort. LEVEL OF EVIDENCE: Level 3B.

The potential of CBC-derived ratios (monocyte-to-lymphocyte, neutrophil-to-lymphocyte, and platelet-to-lymphocyte) to predict or diagnose incident TB infection in Tanzanian adolescents.

BACKGROUND: Ratios of different immune cell populations (i.e., monocyte-to-lymphocyte, neutrophil-to-lymphocyte, and platelet-to-lymphocyte ratios) have been studied as a means of predicting future tuberculosis (TB) disease risk or to assist in the diagnosis of incident TB disease. No studies to-date, however, have evaluated the potential of these ratios to predict or assist in the diagnosis of incident TB infection - the first step in the natural history of TB disease. METHODS: In this prospective study, we evaluated the complete blood count (CBC)-derived metrics of monocyte-to-lymphocyte ratio (MLR), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) as predictors of future TB infection risk or aids in the diagnosis of TB infection among 145 Tanzanian adolescents enrolled in the DAR-901 vaccine trial, using paired CBCs and interferon-gamma release assays (IGRAs) obtained at 0, 60 and 720 days after study enrollment. RESULTS: At baseline, there were no significant differences between study participants who remained persistently IGRA negative throughout the study period and those who subsequently converted to IGRA positive with respect to MLR (0.18 vs 0.17, p = 0.10), NLR (0.88 vs 1.02, p = 0.08), or PLR (115 vs 120, p = 0.28). Similarly, no significant differences were noted with respect to MLR, NLR, and PLR between IGRA converters and time-matched negative controls at the time of IGRA conversion. With respect to other blood cell measures, however, there were modest but significant differences between IGRA negatives and IGRA converters with respect to red blood cell count (4.8 vs 4.6 ×  106 cells/mcL, p = 0.008), hemoglobin (12.6 vs 12.3 g/dL, p = 0.01), and hematocrit (38.8 vs 37.8%, p = 0.005). CONCLUSIONS: In contrast to prior studies that have suggested that the ratios of different immune cell populations are associated with development of TB disease, our present findings do not demonstrate an association between these ratios and the development of TB infection. However, decreased red blood cell measures were associated with the subsequent development of TB infection, suggesting either that dysregulation of iron metabolism may play a role in TB pathogenesis or that following TB infection, iron dysregulation may precede IGRA positivity. TRIAL REGISTRATION: Clinicaltrials.gov NCT02712424 . Date of registration: March 14, 2016.

Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2.

Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins (Lsr2 and Lsr2-like, or LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 activity in model species and uncharacterized isolates resulted in the production of new metabolites not seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and provide global control over Streptomyces' arsenal of signaling and antagonistic compounds.

Natamycin and Voriconazole Exhibit Synergistic Interactions with Nonantifungal Ophthalmic Agents against Fusarium Species Ocular Isolates.

The in vitro activities of two antifungal drugs in combination with four nonantifungal ophthalmic agents were evaluated using a broth microdilution method and a collection of eight Fusarium ocular isolates that exhibited resistance to both natamycin (MICs, 14 to 32 µg/ml) and voriconazole (MICs, 4 to >128 µg/ml). Synergistic and indifferent interactions were observed for natamycin and 5-fluorouracil and natamycin with timolol dependent on the Fusarium isolate tested. Isolate-dependent synergistic and indifferent interactions were also observed for natamycin with EDTA and natamycin with dorzolamide. Synergistic or indifferent interactions were observed for voriconazole with timolol and voriconazole with 5-fluorouracil depending on Fusarium isolate. Taken together, these data suggest that commonly used ophthalmic agents enhance the in vitro activity of antifungal drugs against drug-recalcitrant ocular fungal pathogens.

Breath metabolome of mice infected with Pseudomonas aeruginosa.

INTRODUCTION: The measurement of specific volatile organic compounds in breath has been proposed as a potential diagnostic for a variety of diseases. The most well-studied bacterial lung infection in the breath field is that caused by Pseudomonas aeruginosa. OBJECTIVES: To determine a discriminatory core of molecules in the "breath-print" of mice during a lung infection with four strains of P. aeruginosa (PAO1, PA14, PAK, PA7). Furthermore, we attempted to extrapolate a strain-specific "breath-print" signature to investigate the possibility of recapitulating the genetic phylogenetic groups (Stewart et al. Pathog Dis 71(1), 20-25, 2014. https://doi.org/10.1111/2049-632X.12107 ). METHODS: Breath was collected into a Tedlar bag and shortly after drawn into a thermal desorption tube. The latter was then analyzed into a comprehensive multidimensional gas chromatography coupled with a time-of-flight mass spectrometer. Random forest algorithm was used for selecting the most discriminatory features and creating a prediction model. RESULTS: Three hundred and one molecules were significantly different between animals infected with P. aeruginosa, and those given a sham infection (PBS) or inoculated with UV-killed P. aeruginosa. Of those, nine metabolites could be used to discriminate between the three groups with an accuracy of 81%. Hierarchical clustering showed that the signature from breath was due to a specific response to live bacteria instead of a generic infection response. Furthermore, we identified ten additional volatile metabolites that could differentiate mice infected with different strains of P. aeruginosa. A phylogram generated from the ten metabolites showed that PAO1 and PA7 were the most distinct group, while PAK and PA14 were interspersed between the former two groups. CONCLUSIONS: To the best of our knowledge, this is the first study to report on a 'core' murine breath print, as well as, strain level differences between the compounds in breath. We provide identifications (by running commercially available analytical standards) to five breath compounds that are predictive of P. aeruginosa infection.

Radiation exposure alters airway deformability and bony structure displacement during laryngoscopy.

BACKGROUND: Prior therapeutic radiation exposure in the setting of head and neck malignancies is associated with difficult airway instrumentation. We sought to characterize the anatomic changes that produce this phenotype. STUDY DESIGN: Retrospective review. METHODS: Five individuals with prior radiation therapy to the upper aerodigestive tract (previously irradiated) and 10 with no prior history of therapeutic radiation exposure (nonirradiated) were enrolled. Computed tomography images obtained before and during laryngoscope insertion ("uninstrumented" and "instrumented", respectively) were used to reconstruct three-dimensional representations of the pharyngeal airway, hyoid, and mandible. RESULTS: In the instrumented state, pharyngeal airway volumes were significantly greater in nonirradiated subjects relative to previously irradiated subjects (P = .01), and overall translation of both the hyoid and mandible was also greater in nonirradiated subjects (P = .01 and .04, respectively). CONCLUSION: Individuals with prior therapeutic radiation exposure to the upper aerodigestive tract differ from nonirradiated subjects with respect to airway deformation and bony structure translation during laryngoscopy. LEVEL OF EVIDENCE: 4.

Multi-modal Framework for Image-guided Trans-oral Surgery with Intraoperative Imaging and Deformation Modeling.

Treatment of throat cancers have improved due to minimally-invasive trans-oral approaches. Surgeons rely on preoperative imaging to guide their resection; however, large tissue deformations occur during trans-oral procedures due to placement of necessary retractors and laryngoscopes which hinders the surgeon's ability to accurately assess tumor extent and location of critical structures. We propose an image-guided framework utilizing intraoperative imaging and deformation modeling to improve surgeon accuracy and confidence. A CT-compatible laryngoscopy system previously developed was evaluated in this framework. Intraoperative images were acquired during laryngoscopy; force-sensing capabilities were enabled in the laryngoscope; and tracking of the scope and anatomic features was trialed. Tissue deformation and displacement were quantified and determined to be extensive, with values <; 4.6 cm in the tongue, <; 1.8 cm in bony structures, and <; 108.9 cm3 in airway volume change. Surgical navigation using intraoperative imaging and tracking was evaluated. Preliminary assessment of deformation modeling showed potential to supplement intraoperative imaging. Future work will involve streamlined integration of the components of this framework.

Exhaled human breath analysis in active pulmonary tuberculosis diagnostics by comprehensive gas chromatography-mass spectrometry and chemometric techniques.

Tuberculosis (TB) is the deadliest infectious disease, and yet accurate diagnostics for the disease are unavailable for many subpopulations. In this study, we investigate the possibility of using human breath for the diagnosis of active TB among TB suspect patients, considering also several risk factors for TB for smokers and those with human immunodeficiency virus (HIV). The analysis of exhaled breath, as an alternative to sputum-dependent tests, has the potential to provide a simple, fast, non-invasive, and readily available diagnostic service that could positively change TB detection. A total of 50 individuals from a clinic in South Africa were included in this pilot study. Human breath has been investigated in the setting of active TB using the thermal desorption-comprehensive two-dimensional gas chromatography-time of flight mass spectrometry methodology and chemometric techniques. From the entire spectrum of volatile metabolites in breath, three machine learning algorithms (support vector machines, partial least squares discriminant analysis, and random forest) to select discriminatory volatile molecules that could potentially be useful for active TB diagnosis were employed. Random forest showed the best overall performance, with sensitivities of 0.82 and 1.00 and specificities of 0.92 and 0.60 in the training and test data respectively. Unsupervised analysis of the compounds implicated by these algorithms suggests that they provide important information to cluster active TB from other patients. These results suggest that developing a non-invasive diagnostic for active TB using patient breath is a potentially rich avenue of research, including among patients with HIV comorbidities.

The volatile molecular profiles of seven Streptococcus pneumoniae serotypes.

In this study, the volatile molecule profile of Streptococcus pneumoniae serotypes was evaluated using solid phase microextraction (SPME) and two dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS). Here, seven serotypes (6B, 14, 15, 18C, 19F, 9V, and 23F) were analyzed in an isogenic background. We identified 13 core molecules associated with all seven serotypes, and seven molecules that were differentially produced between serotypes. Serotype 14 was found to have the most distinct volatile profile, and could be discriminated from the other six serotypes in aggregate with an area under the curve (AUC) of 89%. This study suggests that molecules from S. pneumoniae culture headspace show potential for rapid serotype identification.

Detection of high-risk carbapenem-resistant Klebsiella pneumoniae and Enterobacter cloacae isolates using volatile molecular profiles.

Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are alarming in the clinical setting, as CRE isolates often exhibit resistance to most clinically-available antibiotics. Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenemase carried by CRE in North America and Europe, frequently detected in isolates of K. pneumoniae, Escherichia coli, and Enterobacter cloacae. Notably, KPC-expressing strains often arise from clonal lineages, with sequence type 258 (ST258) representing the dominant lineage in K. pneumoniae, ST131 in E. coli, and ST78 and ST171 in E. cloacae. Prior studies have demonstrated that carbapenem-resistant K. pneumoniae differs from carbapenem-susceptible K. pneumoniae at both the transcriptomic and soluble metabolomic levels. In the present study, we sought to determine whether carbapenem-resistant and carbapenem-susceptible isolates of K. pneumoniae, E. coli, and E. cloacae produce distinct volatile metabolic profiles. We were able to identify a volatile metabolic fingerprint that could discriminate between CRE and non-CRE with an area under the receiver operating characteristic curve (AUROC) as high as 0.912. Species-specific AUROCs were as high as 0.988 for K. pneumoniae and 1.000 for E. cloacae. Paradoxically, curing of KPC-expressing plasmids from a subset of K. pneumoniae isolates further accentuated the metabolic differences observed between ST258 and non-ST258.

Volatile fingerprinting of Pseudomonas aeruginosa and respiratory syncytial virus infection in an in vitro cystic fibrosis co-infection model.

Volatile molecules in exhaled breath represent potential biomarkers in the setting of infectious diseases, particularly those affecting the respiratory tract. In particular, Pseudomonas aeruginosa is a critically important respiratory pathogen in specific subsets of the population, such as those with cystic fibrosis (CF). Infections caused by P. aeruginosa can be particularly problematic when co-infection with respiratory syncytial virus (RSV) occurs, as this is correlated with the establishment of chronic P. aeruginosa infection. In the present study, we evaluate the volatile metabolites produced by P. aeruginosa (PAO1)-infected, RSV-infected, co-infected, or uninfected CF bronchial epithelial (CFBE) cells, in vitro. We identified a volatile metabolic signature that could discriminate between P. aeruginosa-infected and non-P. aeruginosa-infected CFBE with an area under the receiver operating characteristic curve (AUROC) of 0.850, using the machine learning algorithm random forest (RF). Although we could not discriminate between RSV-infected and non-RSV-infected CFBE (AUROC = 0.431), we note that sample classification probabilities for RSV-infected cell, generated using RF, were between those of uninfected CFBE and P. aeruginosa-infected CFBE, suggesting that RSV infection may result in a volatile metabolic profile that shares attributes with both of these groups. To more precisely elucidate the biological origins of the volatile metabolites that were discriminatory between P. aeruginosa-infected and non-P. aeruginosa-infected CFBE, we measured the volatile metabolites produced by P. aeruginosa grown in the absence of CFBE. Our findings suggest that the discriminatory metabolites produced likely result from the interaction of P. aeruginosa with the CFBE cells, rather than the metabolism of media components by the bacterium. Taken together, our findings support the notion that P. aeruginosa interacting with CFBE yields a particular volatile metabolic signature. Such a signature may have clinical utility in the monitoring of individuals with CF.