Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status.

Potential Mycobacterium tuberculosis (Mtb) transmission during different pulmonary tuberculosis (TB) disease states is poorly understood. We quantified viable aerosolized Mtb from TB clinic attendees following diagnosis and through six months' follow-up thereafter. Presumptive TB patients (n=102) were classified by laboratory, radiological, and clinical features into Group A: Sputum-Xpert Ultra-positive TB (n=52), Group B: Sputum-Xpert Ultra-negative TB (n=20), or Group C: TB undiagnosed (n=30). All groups were assessed for Mtb bioaerosol release at baseline, and subsequently at 2 wk, 2 mo, and 6 mo. Groups A and B were notified to the national TB program and received standard anti-TB chemotherapy; Mtb was isolated from 92% and 90% at presentation, 87% and 74% at 2 wk, 54% and 44% at 2 mo and 32% and 20% at 6 mo, respectively. Surprisingly, similar numbers were detected in Group C not initiating TB treatment: 93%, 70%, 48% and 22% at the same timepoints. A temporal association was observed between Mtb bioaerosol release and TB symptoms in all three groups. Persistence of Mtb bioaerosol positivity was observed in ~30% of participants irrespective of TB chemotherapy. Captured Mtb bacilli were predominantly acid-fast stain-negative and poorly culturable; however, three bioaerosol samples yielded sufficient biomass following culture for whole-genome sequencing, revealing two different Mtb lineages. Detection of viable aerosolized Mtb in clinic attendees, independent of TB diagnosis, suggests that unidentified Mtb transmitters might contribute a significant attributable proportion of community exposure. Additional longitudinal studies with sputum culture-positive and -negative control participants are required to investigate this possibility.

Improvement of a mouse infection model to capture Pseudomonas aeruginosa chronic physiology in cystic fibrosis.

Laboratory models are central to microbiology research, advancing the understanding of bacterial physiology by mimicking natural environments, from soil to the human microbiome. When studying host-bacteria interactions, animal models enable investigators to examine bacterial dynamics associated with a host, and in the case of human infections, animal models are necessary to translate basic research into clinical treatments. Efforts toward improving animal infection models are typically based on reproducing host genotypes/phenotypes and disease manifestations, leaving a gap in how well the physiology of microbes reflects their behavior in a human host. Understanding bacterial physiology is vital because it dictates host response and bacterial interactions with antimicrobials. Thus, our goal was to develop an animal model that accurately recapitulates bacterial physiology in human infection. The system we chose to model was a chronic Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF). To accomplish this goal, we leveraged a framework that we recently developed to evaluate model accuracy by calculating the percentage of bacterial genes that are expressed similarly in a model to how they are expressed in their infection environment. We combined two complementary models of P. aeruginosa infection-an in vitro synthetic CF sputum model (SCFM2) and a mouse acute pneumonia model. This combined model captured the chronic physiology of P. aeruginosa in CF better than the standard mouse infection model, showing the power of a data-driven approach to refining animal models. In addition, the results of this work challenge the assumption that a chronic infection model requires long-term colonization.

Virological assessment of hospitalized patients with COVID-2019.

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.

Genetic regulators of sputum mucin concentration and their associations with COPD phenotypes.

Hyper-secretion and/or hyper-concentration of mucus is a defining feature of multiple obstructive lung diseases, including chronic obstructive pulmonary disease (COPD). Mucus itself is composed of a mixture of water, ions, salt and proteins, of which the gel-forming mucins, MUC5AC and MUC5B, are the most abundant. Recent studies have linked the concentrations of these proteins in sputum to COPD phenotypes, including chronic bronchitis (CB) and acute exacerbations (AE). We sought to determine whether common genetic variants influence sputum mucin concentrations and whether these variants are also associated with COPD phenotypes, specifically CB and AE. We performed a GWAS to identify quantitative trait loci for sputum mucin protein concentration (pQTL) in the Sub-Populations and InteRmediate Outcome Measures in COPD Study (SPIROMICS, n = 708 for total mucin, n = 215 for MUC5AC, MUC5B). Subsequently, we tested for associations of mucin pQTL with CB and AE using regression modeling (n = 822-1300). Replication analysis was conducted using data from COPDGene (n = 5740) and by examining results from the UK Biobank. We identified one genome-wide significant pQTL for MUC5AC (rs75401036) and two for MUC5B (rs140324259, rs10001928). The strongest association for MUC5B, with rs140324259 on chromosome 11, explained 14% of variation in sputum MUC5B. Despite being associated with lower MUC5B, the C allele of rs140324259 conferred increased risk of CB (odds ratio (OR) = 1.42; 95% confidence interval (CI): 1.10-1.80) as well as AE ascertained over three years of follow up (OR = 1.41; 95% CI: 1.02-1.94). Associations between rs140324259 and CB or AE did not replicate in COPDGene. However, in the UK Biobank, rs140324259 was associated with phenotypes that define CB, namely chronic mucus production and cough, again with the C allele conferring increased risk. We conclude that sputum MUC5AC and MUC5B concentrations are associated with common genetic variants, and the top locus for MUC5B may influence COPD phenotypes, in particular CB.

Application of a quantitative framework to improve the accuracy of a bacterial infection model.

Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.

Endotypes of Exacerbation in Bronchiectasis: An Observational Cohort Study.

Rationale: Bronchiectasis is characterized by acute exacerbations, but the biological mechanisms underlying these events are poorly characterized. Objectives: To investigate the inflammatory and microbial characteristics of exacerbations of bronchiectasis. Methods: A total of 120 patients with bronchiectasis were enrolled and presented with acute exacerbations within 12 months. Spontaneous sputum samples were obtained during a period of clinical stability and again at exacerbation before receipt of antibiotic treatment. A validated rapid PCR assay for bacteria and viruses was used to classify exacerbations as bacterial, viral, or both. Sputum inflammatory assessments included label-free liquid chromatography-tandem mass spectrometry and measurement of sputum cytokines and neutrophil elastase activity. 16 s rRNA sequencing was used to characterize the microbiome. Measurements and Main Results: Bronchiectasis exacerbations showed profound molecular heterogeneity. At least one bacterium was identified in 103 samples (86%), and a high bacterial load (total bacterial load > 107 copies/g) was observed in 81 patients (68%). Respiratory viruses were identified in 55 (46%) patients, with rhinovirus being the most common virus (31%). PCR testing was more sensitive than culture. No consistent change in the microbiome was observed at exacerbation. Exacerbations were associated with increased neutrophil elastase, proteinase-3, IL-1ß, and CXCL8. These markers were particularly associated with bacterial and bacterial plus viral exacerbations. Distinct inflammatory and microbiome profiles were seen between different exacerbation subtypes, including bacterial, viral, and eosinophilic events in both hypothesis-led and hypothesis-free analysis using integrated microbiome and proteomics, demonstrating four subtypes of exacerbation. Conclusions: Bronchiectasis exacerbations are heterogeneous events with contributions from bacteria, viruses, and inflammatory dysregulation.

Accelerated Lung Function Decline and Mucus-Microbe Evolution in Chronic Obstructive Pulmonary Disease.

Rationale: Progressive lung function loss is recognized in chronic obstructive pulmonary disease (COPD); however, no study concurrently evaluates how accelerated lung function decline relates to mucus properties and the microbiome in COPD. Objectives: Longitudinal assessment of mucus and microbiome changes accompanying accelerated lung function decline in patients COPD. Methods: This was a prospective, longitudinal assessment of the London COPD cohort exhibiting the greatest FEV1 decline (n = 30; accelerated decline; 156 ml/yr FEV1 loss) and with no FEV1 decline (n = 28; nondecline; 49 ml/yr FEV1 gain) over time. Lung microbiomes from paired sputum (total 116 specimens) were assessed by shotgun metagenomics and corresponding mucus profiles evaluated for biochemical and biophysical properties. Measurements and Main Results: Biochemical and biophysical mucus properties are significantly altered in the accelerated decline group. Unsupervised principal component analysis showed clear separation, with mucus biochemistry associated with accelerated decline, whereas biophysical mucus characteristics contributed to interindividual variability. When mucus and microbes are considered together, an accelerated decline mucus-microbiome association emerges, characterized by increased mucin (MUC5AC [mucin 5AC] and MUC5B [mucin 5B]) concentration and the presence of Achromobacter and Klebsiella. As COPD progresses, mucus-microbiome shifts occur, initially characterized by low mucin concentration and transition from viscous to elastic dominance accompanied by the commensals Veillonella, Gemella, Rothia, and Prevotella (Global Initiative for Chronic Obstructive Lung Disease [GOLD] A and B) before transition to increased mucus viscosity, mucins, and DNA concentration together with the emergence of pathogenic microorganisms including Haemophilus, Moraxella, and Pseudomonas (GOLD E). Conclusions: Mucus-microbiome associations evolve over time with accelerated lung function decline, symptom progression, and exacerbations affording fresh therapeutic opportunities for early intervention.

Loss of Airway Phylogenetic Diversity Is Associated with Clinical and Pathobiological Markers of Disease Development in Chronic Obstructive Pulmonary Disease.

Rationale: The airway microbiome has the potential to shape chronic obstructive pulmonary disease (COPD) pathogenesis, but its relationship to outcomes in milder disease is unestablished. Objectives: To identify sputum microbiome characteristics associated with markers of COPD in participants of the Subpopulations and Intermediate Outcome Measures of COPD Study (SPIROMICS). Methods: Sputum DNA from 877 participants was analyzed using 16S ribosomal RNA gene sequencing. Relationships between baseline airway microbiota composition and clinical, radiographic, and mucoinflammatory markers, including longitudinal lung function trajectory, were examined. Measurements and Main Results: Participant data represented predominantly milder disease (Global Initiative for Chronic Obstructive Lung Disease stage 0-2 obstruction in 732 of 877 participants). Phylogenetic diversity (i.e., range of different species within a sample) correlated positively with baseline lung function, decreased with higher Global Initiative for Chronic Obstructive Lung Disease stage, and correlated negatively with symptom burden, radiographic markers of airway disease, and total mucin concentrations (P < 0.001). In covariate-adjusted regression models, organisms robustly associated with better lung function included Alloprevotella, Oribacterium, and Veillonella species. Conversely, lower lung function, greater symptoms, and radiographic measures of small airway disease were associated with enrichment in members of Streptococcus, Actinobacillus, Actinomyces, and other genera. Baseline sputum microbiota features were also associated with lung function trajectory during SPIROMICS follow-up (stable/improved, decline, or rapid decline groups). The stable/improved group (slope of FEV1 regression ⩾66th percentile) had greater bacterial diversity at baseline associated with enrichment in Prevotella, Leptotrichia, and Neisseria species. In contrast, the rapid decline group (FEV1 slope ⩽33rd percentile) had significantly lower baseline diversity associated with enrichment in Streptococcus species. Conclusions: In SPIROMICS, baseline airway microbiota features demonstrate divergent associations with better or worse COPD-related outcomes.

Biomarker Predictors of Clinical Efficacy of the Anti-IgE Biologic Omalizumab in Severe Asthma in Adults: Results of the SoMOSA Study.

Background: The anti-IgE monoclonal antibody omalizumab is widely used for severe asthma. This study aimed to identify biomarkers that predict clinical improvement during 1 year of omalizumab treatment. Methods: One-year open-label Study of Mechanisms of action of Omalizumab in Severe Asthma (SoMOSA) involving 216 patients with severe (Global Initiative for Asthma step 4/5) uncontrolled atopic asthma (at least two severe exacerbations in the previous year) taking high-dose inhaled corticosteroids and long-acting ß-agonists with or without maintenance oral corticosteroids. It had two phases: 0-16 weeks, to assess early clinical improvement by Global Evaluation of Therapeutic Effectiveness (GETE); and 16-52 weeks, to assess late responses based on ⩾50% reduction in exacerbations or mOCS dose. All participants provided samples (exhaled breath, blood, sputum, urine) before and after 16 weeks of omalizumab treatment. Measurements and Main Results: A total of 191 patients completed phase 1; 63% had early improvement. Of 173 who completed phase 2, 69% had reduced exacerbations by ⩾50% and 57% (37 of 65) taking mOCSs had reduced their dose by ⩾50%. The primary outcomes 2,3-dinor-11-ß-PGF2α, GETE score, and standard clinical biomarkers (blood and sputum eosinophils, exhaled nitric oxide, serum IgE) did not predict either clinical response. Five volatile organic compounds and five plasma lipid biomarkers strongly predicted the ⩾50% reduction in exacerbations (receiver operating characteristic areas under the curve of 0.780 and 0.922, respectively) and early responses (areas under the curve of 0.835 and 0.949, respectively). In an independent cohort, gas chromatography/mass spectrometry biomarkers differentiated between severe and mild asthma. Conclusions: This is the first discovery of omics biomarkers that predict improvement in asthma with biologic agent treatment. Prospective validation and development for clinical use is justified.

Airway "Resistotypes" and Clinical Outcomes in Bronchiectasis.

Rationale: Chronic infection and inflammation shapes the airway microbiome in bronchiectasis. Utilizing whole-genome shotgun metagenomics to analyze the airway resistome provides insight into interplay between microbes, resistance genes, and clinical outcomes. Objectives: To apply whole-genome shotgun metagenomics to the airway microbiome in bronchiectasis to highlight a diverse pool of antimicrobial resistance genes: the "resistome," the clinical significance of which remains unclear. Methods: Individuals with bronchiectasis were prospectively recruited into cross-sectional and longitudinal cohorts (n = 280), including the international multicenter cross-sectional Cohort of Asian and Matched European Bronchiectasis 2 (CAMEB 2) study (n = 251) and two independent cohorts, one describing patients experiencing acute exacerbation and a further cohort of patients undergoing Pseudomonas aeruginosa eradication treatment. Sputum was subjected to metagenomic sequencing, and the bronchiectasis resistome was evaluated in association with clinical outcomes and underlying host microbiomes. Measurements and Main Results: The bronchiectasis resistome features a unique resistance gene profile and increased counts of aminoglycoside, bicyclomycin, phenicol, triclosan, and multidrug resistance genes. Longitudinally, it exhibits within-patient stability over time and during exacerbations despite between-patient heterogeneity. Proportional differences in baseline resistome profiles, including increased macrolide and multidrug resistance genes, associate with shorter intervals to the next exacerbation, whereas distinct resistome archetypes associate with frequent exacerbations, poorer lung function, geographic origin, and the host microbiome. Unsupervised analysis of resistome profiles identified two clinically relevant "resistotypes," RT1 and RT2, the latter characterized by poor clinical outcomes, increased multidrug resistance, and P. aeruginosa. Successful targeted eradication in P. aeruginosa-colonized individuals mediated reversion from RT2 to RT1, a more clinically favorable resistome profile demonstrating reduced resistance gene diversity. Conclusions: The bronchiectasis resistome associates with clinical outcomes, geographic origin, and the underlying host microbiome. Bronchiectasis resistotypes link to clinical disease and are modifiable through targeted antimicrobial therapy.

Activated sputum eosinophils associated with exacerbations in children on mepolizumab.

BACKGROUND: MUPPITS-2 was a randomized, placebo-controlled clinical trial that demonstrated mepolizumab (anti-IL-5) reduced exacerbations and blood and airway eosinophils in urban children with severe eosinophilic asthma. Despite this reduction in eosinophilia, exacerbation risk persisted in certain patients treated with mepolizumab. This raises the possibility that subpopulations of airway eosinophils exist that contribute to breakthrough exacerbations. OBJECTIVE: We aimed to determine the effect of mepolizumab on airway eosinophils in childhood asthma. METHODS: Sputum samples were obtained from 53 MUPPITS-2 participants. Airway eosinophils were characterized using mass cytometry and grouped into subpopulations using unsupervised clustering analyses of 38 surface and intracellular markers. Differences in frequency and immunophenotype of sputum eosinophil subpopulations were assessed based on treatment arm and frequency of exacerbations. RESULTS: Median sputum eosinophils were significantly lower among participants treated with mepolizumab compared with placebo (58% lower, 0.35% difference [95% CI 0.01, 0.74], P = .04). Clustering analysis identified 3 subpopulations of sputum eosinophils with varied expression of CD62L. CD62Lint and CD62Lhi eosinophils exhibited significantly elevated activation marker and eosinophil peroxidase expression, respectively. In mepolizumab-treated participants, CD62Lint and CD62Lhi eosinophils were more abundant in participants who experienced exacerbations than in those who did not (100% higher for CD62Lint, 0.04% difference [95% CI 0.0, 0.13], P = .04; 93% higher for CD62Lhi, 0.21% difference [95% CI 0.0, 0.77], P = .04). CONCLUSIONS: Children with eosinophilic asthma treated with mepolizumab had significantly lower sputum eosinophils. However, CD62Lint and CD62Lhi eosinophils were significantly elevated in children on mepolizumab who had exacerbations, suggesting that eosinophil subpopulations exist that contribute to exacerbations despite anti-IL-5 treatment.

Airway hyperresponsiveness correlates with airway TSLP in asthma independent of eosinophilic inflammation.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is released from the airway epithelium in response to various environmental triggers, inducing a type-2 inflammatory response, and is associated with airway inflammation, airway hyperresponsiveness (AHR), and exacerbations. TSLP may also induce AHR via a direct effect on airway smooth muscle and mast cells, independently of type-2 inflammation, although association between airway TSLP and AHR across asthma phenotypes has been described sparsely. OBJECTIVES: This study sought to investigate the association between AHR and levels of TSLP in serum, sputum, and bronchoalveolar lavage in patients with asthma with and without type-2 inflammation. METHODS: A novel ultrasensitive assay was used to measure levels of TSLP in patients with asthma (serum, n = 182; sputum, n = 81; bronchoalveolar lavage, n = 85) and healthy controls (serum, n = 47). The distribution and association among airway and systemic TSLP, measures of AHR, type-2 inflammation, and severity of disease were assessed. RESULTS: TSLP in sputum was associated with AHR independently of levels of eosinophils and fractional exhaled nitric oxide (ρ = 0.49, P = .005). Serum TSLP was higher in both eosinophil-high and eosinophil-low asthma compared to healthy controls: geometric mean: 1600 fg/mL (95% CI: 1468-1744 fg/mL) and 1294 fg/mL (95% CI: 1167-1435 fg/mL) versus 846 fg/mL (95% CI: 661-1082 fg/mL), but did not correlate with the level of AHR. Increasing age, male sex, and eosinophils in blood were associated with higher levels of TSLP in serum, whereas lung function, inhaled corticosteroid dose, and symptom score were not. CONCLUSIONS: The association between TSLP in sputum and AHR to mannitol irrespective of markers of type-2 inflammation further supports a role of TSLP in AHR that is partially independent of eosinophilic inflammation.

Cost-effectiveness of Low-complexity Screening Tests in Community-based Case-finding for Tuberculosis.

INTRODUCTION: In high-burden settings, low-complexity screening tests for tuberculosis (TB) could expand the reach of community-based case-finding efforts. The potential costs and cost-effectiveness of approaches incorporating these tests are poorly understood. METHODS: We developed a microsimulation model assessing 3 approaches to community-based case-finding in hypothetical populations (India-, South Africa-, The Philippines-, Uganda-, and Vietnam-like settings) with TB prevalence 4 times that of national estimates: (1) screening with a point-of-care C-reactive protein (CRP) test, (2) screening with a more sensitive "Hypothetical Screening test" (95% sensitive for Xpert Ultra-positive TB, 70% specificity; equipment/labor costs similar to Xpert Ultra, but using a $2 cartridge) followed by sputum Xpert Ultra if positive, or (3) testing all individuals with sputum Xpert Ultra. Costs are expressed in 2023 US dollars and include treatment costs. RESULTS: Universal Xpert Ultra was estimated to cost a mean $4.0 million (95% uncertainty range: $3.5 to $4.6 million) and avert 3200 (2600 to 3900) TB-related disability-adjusted life years (DALYs) per 100 000 people screened ($670 [The Philippines] to $2000 [Vietnam] per DALY averted). CRP was projected to cost $550 (The Philippines) to $1500 (Vietnam) per DALY averted but with 44% fewer DALYs averted. The Hypothetical Screening test showed minimal benefit compared to universal Xpert Ultra, but if specificity were improved to 95% and per-test cost to $4.5 (all-inclusive), this strategy could cost $390 (The Philippines) to $940 (Vietnam) per DALY averted. CONCLUSIONS: Screening tests can meaningfully improve the cost-effectiveness of community-based case-finding for TB but only if they are sensitive, specific, and inexpensive.

New Manual Quantitative Polymerase Chain Reaction Assay Validated on Tongue Swabs Collected and Processed in Uganda Shows Sensitivity That Rivals Sputum-based Molecular Tuberculosis Diagnostics.

BACKGROUND: Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. METHODS: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester). RESULTS: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently. CONCLUSIONS: Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

Evidence for Tuberculosis in Individuals With Xpert Ultra "Trace" Sputum During Screening of High-Burden Communities.

BACKGROUND: "Trace" results on Xpert MTB/RIF Ultra ("Ultra"; Cepheid) -a molecular diagnostic test for tuberculosis (TB)-are often interpreted as an indication for TB treatment, but may also represent detection of nonviable bacilli or analytical error. In community-screening settings where individual TB risk is low, there is limited guidance on how to interpret Ultra-trace results. METHODS: We conducted systematic Ultra TB screening of adults and adolescents (≥15 years) in Kampala, Uganda, through door-to-door and event-based sputum collection. We enrolled individuals with trace-positive sputum for detailed clinical, radiographic, and microbiological (including 2 sputum cultures, repeat Ultra, and for people with HIV, urine lipoarabinomannan) evaluation, and compared those findings with similar evaluations in controls with Ultra-negative and Ultra-positive (non-trace) sputum. RESULTS: Of 21 957 people screened with Ultra, 211 (1.0%) tested positive, including 96 (46% of positives) with trace results. Of 92 people enrolled with trace-positive sputum; 12% (11/92) were HIV-positive and 14% (13/92) had prior TB. The prevalence of TB among participants with trace-positive sputum results was 14% (13/92) by culture, 24% (22/92) using broader microbiological criteria, and 26% (24/92) after accounting for clinical diagnosis. The prevalence of cough and of abnormal chest computed tomography (CT) findings were 32% and 26%, respectively, if Ultra-negative; 34% and 54% if trace-positive/non-microbiologically confirmed; 72% and 95% if trace-positive/microbiologically confirmed; and 71% and 93% if Ultra-positive (more than trace). CONCLUSIONS: Most individuals with trace-positive sputum in Ugandan communities did not have microbiologically confirmed TB but had more symptoms and chest CT abnormalities than people with Ultra-negative sputum.

Polymerase Chain Reaction on Respiratory Tract Specimens of Immunocompromised Patients to Diagnose Pneumocystis Pneumonia: A Systematic Review and Meta-analysis.

BACKGROUND: This meta-analysis examines the comparative diagnostic performance of polymerase chain reaction (PCR) for the diagnosis of Pneumocystis pneumonia (PCP) on different respiratory tract samples, in both human immunodeficiency virus (HIV) and non-HIV populations. METHODS: A total of 55 articles met inclusion criteria, including 11 434 PCR assays on respiratory specimens from 7835 patients at risk of PCP. QUADAS-2 tool indicated low risk of bias across all studies. Using a bivariate and random-effects meta-regression analysis, the diagnostic performance of PCR against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group definition of proven PCP was examined. RESULTS: Quantitative PCR (qPCR) on bronchoalveolar lavage fluid provided the highest pooled sensitivity of 98.7% (95% confidence interval [CI], 96.8%-99.5%), adequate specificity of 89.3% (95% CI, 84.4%-92.7%), negative likelihood ratio (LR-) of 0.014, and positive likelihood ratio (LR+) of 9.19. qPCR on induced sputum provided similarly high sensitivity of 99.0% (95% CI, 94.4%-99.3%) but a reduced specificity of 81.5% (95% CI, 72.1%-88.3%), LR- of 0.024, and LR+ of 5.30. qPCR on upper respiratory tract samples provided lower sensitivity of 89.2% (95% CI, 71.0%-96.5%), high specificity of 90.5% (95% CI, 80.9%-95.5%), LR- of 0.120, and LR+ of 9.34. There was no significant difference in sensitivity and specificity of PCR according to HIV status of patients. CONCLUSIONS: On deeper respiratory tract specimens, PCR negativity can be used to confidently exclude PCP, but PCR positivity will likely require clinical interpretation to distinguish between colonization and active infection, partially dependent on the strength of the PCR signal (indicative of fungal burden), the specimen type, and patient population tested.

A novel DNase assay reveals low DNase activity in severe asthma.

Secreted deoxyribonucleases (DNases), such as DNase-I and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 (n = 439) and from healthy controls (n = 89). We found that DNase activity was lower than normal in asthma [78.7 relative fluorescence units (RFU)/min vs. 120.4 RFU/min, P < 0.0001]. Compared to patients with asthma with sputum DNase activity in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post-translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway that is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation.NEW & NOTEWORTHY We developed a new DNase assay and used it to show that DNase activity is impaired in asthma airways.

The presence of cystic fibrosis-related diabetes modifies the sputum microbiome in cystic fibrosis disease.

Cystic fibrosis-related diabetes (CFRD) affects 40%-50% of adults with CF and is associated with a decline in respiratory health. The microbial flora of the lung is known to change with the development of CF disease, but how CFRD affects the microbiome has not been described. We analyzed the microbiome in sputa from 14 people with CF, 14 with CFRD, and two who were classed as pre-CFRD by extracting DNA and amplifying the variable V3-V4 region of the microbial 16S ribosomal RNA gene by PCR. Sequences were analyzed and sources were identified to genus level. We found that the α-diversity of the microbiome using Shannon's diversity index was increased in CFRD compared with CF. Bray Curtis dissimilarity analysis showed that there was separation of the microbiomes in CF and CFRD sputa. The most abundant phyla identified in the sputum samples were Firmicutes and Proteobacteria, Actinobacteriota and Bacteroidota, and the ratio of Firmicutes/Bacteroidota was reduced in CFRD compared with CF. Pseudomonas, Azhorizophilus, Porphyromonas, and Actinobacillus were more abundant in CFRD compared with CF, whereas Staphylococcus was less abundant. The relative abundance of these genera did not correlate with age; some correlated with a decline in FEV1/FVC but all correlated with hemoglobin A1C (HbA1c) indicating that development of CFRD mediates further changes to the respiratory microbiome in CF.NEW & NOTEWORTHY Cystic fibrosis-related diabetes (CFRD) is associated with a decline in respiratory health. We show for the first time that there was a change in the sputum microbiome of people with CFRD compared with CF that correlated with markers of raised blood glucose.

Sputum interleukin-6 level as a marker of severity during acute chest syndrome in children with sickle cell disease.

Acute chest syndrome (ACS) is a leading cause of morbimortality in sickle cell disease (SCD). In this prospective observational study, we investigated sputum interleukin-6 (IL-6) level as an ACS severity marker during 30 ACS episodes in 26 SCD children. Sputum IL-6 levels measured within the first 72 h of hospitalisation for ACS were significantly higher in patients with oxygen requirement ≥2 L/min, ventilation (invasive and/or non-invasive) length ≥5 days, bilateral and/or extensive opacities on chest X-ray or erythrocytapheresis requirement. Sputum IL-6 could serve as an ACS severity marker to help identify patients requiring targeted anti-inflammatory treatments such as tocilizumab.

Association of indicators of extensive disease and rifampin-resistant tuberculosis treatment outcomes: an individual participant data meta-analysis.

BACKGROUND: Indicators of extensive disease-acid fast bacilli (AFB) smear positivity and lung cavitation-have been inconsistently associated with clinical rifampin-resistant/multidrug-resistant tuberculosis (RR/MDR-TB) outcomes. We evaluated the association of these indicators with end-of-treatment outcomes. METHODS: We did an individual participant data meta-analysis of people treated for RR/MDR-TB with longer regimens with documented AFB smear and chest radiography findings. We compared people AFB smear-negative without cavities to people: (1) smear-negative with lung cavities; (2) smear-positive without lung cavities and (3) AFB smear-positive with lung cavities. Using multivariable logistic regression accounting for demographic, treatment and clinical factors, we calculated adjusted ORs (aOR) for any unfavourable outcome (death, lost to follow-up, failure/recurrence), and mortality and treatment failure/recurrence alone. RESULTS: We included 5596 participants; included participants significantly differed from excluded participants. Overall, 774 (13.8%) were AFB smear-negative without cavities, 647 (11.6%) only had cavities, 1424 (25.4%) were AFB smear-positive alone and 2751 (49.2%) were AFB smear-positive with cavities. The median age was 37 years (IQR: 28-47), 3580 (64%) were male and 686 (12.5%) had HIV. Compared with participants AFB smear-negative without cavities, aOR (95% CI) for any unfavourable outcome was 1.0 (0.8 to 1.4) for participants smear-negative with lung cavities, 1.2 (0.9 to 1.5) if smear-positive without cavities and 1.6 (1.3 to 2.0) if AFB smear-positive with lung cavities. Odds were only significantly increased for mortality (1.5, 95% CI 1.1 to 2.1) and failure/recurrence (2.2, 95% CI 1.5 to 3.3) among participants AFB smear-positive with lung cavities. CONCLUSION: Only the combination of AFB smear-positivity and lung cavitation was associated with unfavourable outcomes, suggesting they may benefit from stronger regimens.