Face mask sampling (FMS) for tuberculosis shows lower diagnostic sensitivity than sputum sampling in Guinea.

BACKGROUND: Pulmonary tuberculosis (PTB) diagnosis relies on sputum examination, a challenge in sputum-scarce patients. Alternative non-invasive sampling methods such as face mask sampling (FMS) have been proposed. OBJECTIVE: To evaluate the value of FMS for PTB diagnosis by assessing its agreement with sputum samples processed by GeneXpert MTB/RIF (Ultra)(Xpert) testing, and describe FMS sensitivity and specificity. METHODS: This was a prospective study conducted at the Carrière TB clinic in Guinea. Presumptive TB patients willing to participate were asked to wear a surgical mask containing a polyvinyl alcohol (PVA) strip for thirty minutes. Subsequently, two spot sputum samples were collected, of which one was processed by microscopy on site and the other by Xpert in Guinea's National Reference Laboratory of Mycobacteriology (LNRM). The first 30 FMS were processed at the Supranational Reference Laboratory in Antwerp, Belgium, and the following 118 FMS in the LNRM. RESULTS: One hundred fifty patients participated, of whom 148 had valid results for both mask and sputum. Sputum smear microscopy was positive for 47 (31.8%) patients while sputum-Xpert detected MTB in 54 (36.5%) patients. Among the 54 patients testing sputum-Xpert positive, 26 (48.1%) yielded a positive FMS-Xpert result, while four sputum-Xpert negative patients tested positive for FMS and 90 patients were Xpert-negative for both sputum and mask samples, suggesting a moderate level of agreement (k-value of 0.47). The overall mask sensitivity was 48.1%, with 95.7% specificity. CONCLUSION: In our setting, Xpert testing on FMS did not yield a high level of agreement to sputum sample.

Viral emissions into the air and environment after SARS-CoV-2 human challenge: a phase 1, open label, first-in-human study.

BACKGROUND: Effectively implementing strategies to curb SARS-CoV-2 transmission requires understanding who is contagious and when. Although viral load on upper respiratory swabs has commonly been used to infer contagiousness, measuring viral emissions might be more accurate to indicate the chance of onward transmission and identify likely routes. We aimed to correlate viral emissions, viral load in the upper respiratory tract, and symptoms, longitudinally, in participants who were experimentally infected with SARS-CoV-2. METHODS: In this phase 1, open label, first-in-human SARS-CoV-2 experimental infection study at quarantine unit at the Royal Free London NHS Foundation Trust, London, UK, healthy adults aged 18-30 years who were unvaccinated for SARS-CoV-2, not previously known to have been infected with SARS-CoV-2, and seronegative at screening were recruited. Participants were inoculated with 10 50% tissue culture infectious dose of pre-alpha wild-type SARS-CoV-2 (Asp614Gly) by intranasal drops and remained in individual negative pressure rooms for a minimum of 14 days. Nose and throat swabs were collected daily. Emissions were collected daily from the air (using a Coriolis µ air sampler and directly into facemasks) and the surrounding environment (via surface and hand swabs). All samples were collected by researchers, and tested by using PCR, plaque assay, or lateral flow antigen test. Symptom scores were collected using self-reported symptom diaries three times daily. The study is registered with ClinicalTrials.gov, NCT04865237. FINDINGS: Between March 6 and July 8, 2021, 36 participants (ten female and 26 male) were recruited and 18 (53%) of 34 participants became infected, resulting in protracted high viral loads in the nose and throat following a short incubation period, with mild-to-moderate symptoms. Two participants were excluded from the per-protocol analysis owing to seroconversion between screening and inoculation, identified post hoc. Viral RNA was detected in 63 (25%) of 252 Coriolis air samples from 16 participants, 109 (43%) of 252 mask samples from 17 participants, 67 (27%) of 252 hand swabs from 16 participants, and 371 (29%) of 1260 surface swabs from 18 participants. Viable SARS-CoV-2 was collected from breath captured in 16 masks and from 13 surfaces, including four small frequently touched surfaces and nine larger surfaces where airborne virus could deposit. Viral emissions correlated more strongly with viral load in nasal swabs than throat swabs. Two individuals emitted 86% of airborne virus, and the majority of airborne virus collected was released on 3 days. Individuals who reported the highest total symptom scores were not those who emitted most virus. Very few emissions occurred before the first reported symptom (7%) and hardly any before the first positive lateral flow antigen test (2%). INTERPRETATION: After controlled experimental inoculation, the timing, extent, and routes of viral emissions was heterogeneous. We observed that a minority of participants were high airborne virus emitters, giving support to the notion of superspreading individuals or events. Our data implicates the nose as the most important source of emissions. Frequent self-testing coupled with isolation upon awareness of first symptoms could reduce onward transmissions. FUNDING: UK Vaccine Taskforce of the Department for Business, Energy and Industrial Strategy of Her Majesty's Government.

Transmission of monkeypox/mpox virus: A narrative review of environmental, viral, host, and population factors in relation to the 2022 international outbreak.

Monkeypox virus (MPXV) has spread globally. Emerging studies have now provided evidence regarding MPXV transmission, that can inform rational evidence-based policies and reduce misinformation on this topic. We aimed to review the evidence on transmission of the virus. Real-world studies have isolated viable viruses from high-touch surfaces for as long as 15 days. Strong evidence suggests that the current circulating monkeypox (mpox) has evolved from previous outbreaks outside of Africa, but it is yet unknown whether these mutations may lead to an inherently increased infectivity of the virus. Strong evidence also suggests that the main route of current MPXV transmission is sexual; through either close contact or directly, with detection of culturable virus in saliva, nasopharynx, and sperm for prolonged periods and the presence of rashes mainly in genital areas. The milder clinical presentations and the potential presence of presymptomatic transmission in the current circulating variant compared to previous clades, as well as the dominance of spread amongst men who have sex with men (MSMs) suggests that mpox has a developed distinct clinical phenotype that has increased its transmissibility. Increased public awareness of MPXV transmission modalities may lead to earlier detection of the spillover of new cases into other groups.

Exhaled Mycobacterium tuberculosis Predicts Incident Infection in Household Contacts.

BACKGROUND: Halting transmission of Mycobacterium tuberculosis (Mtb) by identifying infectious individuals early is key to eradicating tuberculosis (TB). Here we evaluate face mask sampling as a tool for stratifying the infection risk of individuals with pulmonary TB (PTB) to their household contacts. METHODS: Forty-six sputum-positive PTB patients in The Gambia (August 2016-November 2017) consented to mask sampling prior to commencing treatment. Incident Mtb infection was defined in 181 of their 217 household contacts as QuantiFERON conversion or an increase in interferon-γ of ≥1 IU/mL, 6 months after index diagnosis. Multilevel mixed-effects logistical regression analysis with cluster adjustment by household was used to identify predictors of incident infection. RESULTS: Mtb was detected in 91% of PTB mask samples with high variation in IS6110 copies (5.3 × 102 to 1.2 × 107). A high mask Mtb level (≥20 000 IS6110 copies) was observed in 45% of cases and was independently associated with increased likelihood of incident Mtb infection in contacts (adjusted odds ratio, 3.20 [95% confidence interval, 1.26-8.12]; P = .01), compared with cases having low-positive/negative mask Mtb levels. Mask Mtb level was a better predictor of incident Mtb infection than sputum bacillary load, chest radiographic characteristics, or sleeping proximity. CONCLUSIONS: Mask sampling offers a sensitive and noninvasive tool to support the stratification of individuals who are most infectious in high-TB-burden settings. Our approach can provide better insight into community transmission in complex environments.

Exhaled SARS-CoV-2 RNA viral load kinetics measured by facemask sampling associates with household transmission.

OBJECTIVES: No studies have examined longitudinal patterns of naturally exhaled SARS-CoV-2 RNA viral load (VL) during acute infection. We report this using facemask sampling (FMS) and assessed the relationship between emitted RNA VL and household transmission. METHODS: Between December 2020 and February 2021, we recruited participants within 24 hours of a positive RT-qPCR on upper respiratory tract sampling (URTS) (day 0). Participants gave FMS (for 1 hour) and URTS (self-taken) on seven occasions up to day 21. Samples were analysed by RT-qPCR (from sampling matrix strips within the mask) and symptom diaries were recorded. Household transmission was assessed through reporting of positive URTS RT-qPCR in household contacts. RESULTS: Analysis of 203 FMS and 190 URTS from 34 participants showed that RNA VL peaked within the first 5 days following sampling. Concomitant URTS, FMS RNA VL, and symptom scores, however, were poorly correlated, but a higher severity of reported symptoms was associated with FMS positivity up to day 5. Of 28 participants who had household contacts, 12 (43%) reported transmission. Frequency of household transmission was associated with the highest (peak) FMS RNA VL obtained (negative genome copies/strip: 0% household transmission; 1 to 1000 copies/strip: 20%; 1001 to 10 000 copies/strip: 57%; >10 000 copies/strip: 75%; p = 0.048; age adjusted OR of household transmission per log increase in copies/strip: 4.97; 95% CI, 1.20-20.55; p = 0.02) but not observed with peak URTS RNA VL. DISCUSSION: Exhaled RNA VL measured by FMS is highest in early infection, can be positive in symptomatic patients with concomitantly negative URTS, and is strongly associated with household transmission.

Visualization of exhaled breath metabolites reveals distinct diagnostic signatures for acute cardiorespiratory breathlessness.

Acute cardiorespiratory breathlessness accounts for one in eight of all emergency hospitalizations. Early, noninvasive diagnostic testing is a clinical priority that allows rapid triage and treatment. Here, we sought to find and replicate diagnostic breath volatile organic compound (VOC) biomarkers of acute cardiorespiratory disease and understand breath metabolite network enrichment in acute disease, with a view to gaining mechanistic insight of breath biochemical derangements. We collected and analyzed exhaled breath samples from 277 participants presenting acute cardiorespiratory exacerbations and aged-matched healthy volunteers. Topological data analysis phenotypes differentiated acute disease from health and acute cardiorespiratory exacerbation subtypes (acute heart failure, acute asthma, acute chronic obstructive pulmonary disease, and community-acquired pneumonia). A multibiomarker score (101 breath biomarkers) demonstrated good diagnostic sensitivity and specificity (≥80%) in both discovery and replication sets and was associated with all-cause mortality at 2 years. In addition, VOC biomarker scores differentiated metabolic subgroups of cardiorespiratory exacerbation. Louvain clustering of VOCs coupled with metabolite enrichment and similarity assessment revealed highly specific enrichment patterns in all acute disease subgroups, for example, selective enrichment of correlated C5-7 hydrocarbons and C3-5 carbonyls in heart failure and selective depletion of correlated aldehydes in acute asthma. This study identified breath VOCs that differentiate acute cardiorespiratory exacerbations and associated subtypes and metabolic clusters of disease-associated VOCs.

A Persistent Tuberculosis Outbreak in the UK Is Characterized by Hydrophobic fadB4-Deficient Mycobacterium tuberculosis That Replicates Rapidly in Macrophages.

The genetic diversity of Mycobacterium tuberculosis can influence disease severity and transmissibility. To better understand how this diversity influences individuals and communities, we phenotyped M. tuberculosis that was causing a persistent outbreak in the East Midlands, United Kingdom. Compared to nonoutbreak isolates, bacilli had higher lipid contents and more hydrophobic cell surfaces. In macrophage infection models, the bacteria increased more rapidly, provoked the enhanced accumulation of macrophage lipid droplets and enhanced the secretion of IL-1ß. Natural deletions in fadB4, nrdB, and plcC distinguished the outbreak isolates from other lineage 3 isolates in the region. fadB4 is annotated with a putative role in cell envelope biosynthesis, so the loss of this gene has the potential to alter the interactions of bacteria with immune cells. Reintroduction of fadB4 to the outbreak strain led to a phenotype that more closely resembled those of nonoutbreak strains. The improved understanding of the microbiological characteristics and the corresponding genetic polymorphisms that associate with outbreaks have the potential to inform tuberculosis control. IMPORTANCE Tuberculosis (TB) killed 1.5 million people in 2020 and affects every country. The extent to which the natural genetic diversity of Mycobacterium tuberculosis influences disease manifestation at both the individual and epidemiological levels remains poorly understood. Insights into how pathogen polymorphisms affect patterns of TB have the potential to translate into clinical and public health practice. Two distinct lineage 3 strains isolated from local TB outbreaks, one of which (CH) was rapidly terminated and the other of which (Lro) persistently transmitted for over a decade, provided us with an opportunity to study these issues. We compared genome sequences, microbiological characteristics, and early immune responses that were evoked upon infection. Our results indicate that the natural lack of fadB4 in the Lro strain contributes to its unique features.

Relationship between inflammatory status and microbial composition in severe asthma and during exacerbation.

BACKGROUND: In T2-mediated severe asthma, biologic therapies, such as mepolizumab, are increasingly used to control disease. Current biomarkers can indicate adequate suppression of T2 inflammation, but it is unclear whether they provide information about airway microbial composition. We investigated the relationships between current T2 biomarkers and microbial profiles, characteristics associated with a ProteobacteriaHIGH microbial profile and the effects of mepolizumab on airway ecology. METHODS: Microbiota sequencing was performed on sputum samples obtained at stable and exacerbation state from 140 subjects with severe asthma participating in two clinical trials. Inflammatory subgroups were compared on the basis of biomarkers, including FeNO and sputum and blood eosinophils. ProteobacteriaHIGH subjects were identified by Proteobacteria to Firmicutes ratio ≥0.485. Where paired sputum from stable visits was available, we compared microbial composition at baseline and following ≥12 weeks of mepolizumab. RESULTS: Microbial composition was not related to inflammatory subgroup based on sputum or blood eosinophils. FeNO ≥50 ppb when stable and at exacerbation indicated a group with less dispersed microbial profiles characterised by high alpha-diversity and low Proteobacteria. ProteobacteriaHIGH subjects were neutrophilic and had a longer time from asthma diagnosis than ProteobacteriaLOW subjects. In those studied, mepolizumab did not alter airway bacterial load or lead to increased Proteobacteria. CONCLUSION: High FeNO could indicate a subgroup of severe asthma less likely to benefit from antimicrobial strategies at exacerbation or in the context of poor control. Where FeNO is <50 ppb, biomarkers of microbial composition are required to identify those likely to respond to microbiome-directed strategies. We found no evidence that mepolizumab alters airway microbial composition.

Development of a colorimetric assay for the detection of SARS-CoV-2 3CLpro activity.

Diagnostic testing continues to be an integral component of the strategy to contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) global pandemic, the causative agent of Coronavirus Disease 2019 (COVID-19). The SARS-CoV-2 genome encodes the 3C-like protease (3CLpro) which is essential for coronavirus replication. This study adapts an in vitro colorimetric gold nanoparticle (AuNP) based protease assay to specifically detect the activity of SARS-CoV-2 3CLpro as a purified recombinant protein and as a cellular protein exogenously expressed in HEK293T human cells. We also demonstrate that the specific sensitivity of the assay for SARS-CoV-2 3CLpro can be improved by use of an optimised peptide substrate and through hybrid dimerisation with inactive 3CLpro mutant monomers. These findings highlight the potential for further development of the AuNP protease assay to detect SARS-CoV-2 3CLpro activity as a novel, accessible and cost-effective diagnostic test for SARS-CoV-2 infection at the point-of-care. Importantly, this versatile assay could also be easily adapted to detect specific protease activity associated with other viruses or diseases conditions.

COVID-19 symptoms are reduced by targeted hydration of the nose, larynx and trachea.

Dehydration of the upper airways increases risks of respiratory diseases from COVID-19 to asthma and COPD. We find in human volunteer studies involving 464 human subjects in Germany, the US, and India that respiratory droplet generation increases by up to 4 orders of magnitude in dehydration-associated states of advanced age (n = 357), elevated BMI-age (n = 148), strenuous exercise (n = 20) and SARS-CoV-2 infection (n = 87), and falls with hydration of the nose, larynx and trachea by calcium-rich hypertonic salts. We also find in a protocol of exercise-induced airway dehydration that hydration of the airways by calcium-rich salts increases oxygenation relative to a non-treatment control (P < 0.05). In a random control study of COVID-19 positive subjects (n = 40), thrice-a-day delivery of the calcium-rich hypertonic salts (active) suppressed respiratory droplet generation by 51% ± 11% and increased oxygen saturation over three days of treatment by 48.08% ± 9.61% (P < 0.001), while no changes were observed in the nasal-saline control group. Self-reported symptoms significantly declined in the active group and did not decline in the control group. Hydration of the upper airways appears promising as a non-drug approach for reducing risks of respiratory diseases such as COVID-19.

Exhaled SARS-CoV-2 quantified by face-mask sampling in hospitalised patients with COVID-19.

BACKGROUND: Human to human transmission of SARS-CoV-2 is driven by the respiratory route but little is known about the pattern and quantity of virus output from exhaled breath. We have previously shown that face-mask sampling (FMS) can detect exhaled tubercle bacilli and have adapted its use to quantify exhaled SARS-CoV-2 RNA in patients admitted to hospital with Coronavirus Disease-2019 (COVID-19). METHODS: Between May and December 2020, we took two concomitant FMS and nasopharyngeal samples (NPS) over two days, starting within 24 h of a routine virus positive NPS in patients hospitalised with COVID-19, at University Hospitals of Leicester NHS Trust, UK. Participants were asked to wear a modified duckbilled facemask for 30 min, followed by a nasopharyngeal swab. Demographic, clinical, and radiological data, as well as International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) mortality and deterioration scores were obtained. Exposed masks were processed by removal, dissolution and analysis of sampling matrix strips fixed within the mask by RT-qPCR. Viral genome copy numbers were determined and results classified as Negative; Low: ≤999 copies; Medium: 1000-99,999 copies and High ≥ 100,000 copies per strip for FMS or per 100 µl for NPS. RESULTS: 102 FMS and NPS were collected from 66 routinely positive patients; median age: 61 (IQR 49 - 77), of which FMS was positive in 38% of individuals and concomitant NPS was positive in 50%. Positive FMS viral loads varied over five orders of magnitude (<10-3.3 x 106 genome copies/strip); 21 (32%) patients were asymptomatic at the time of sampling. High FMS viral load was associated with respiratory symptoms at time of sampling and shorter interval between sampling and symptom onset (FMS High: median (IQR) 2 days (2-3) vs FMS Negative: 7 days (7-10), p = 0.002). On multivariable linear regression analysis, higher FMS viral loads were associated with higher ISARIC mortality (Medium FMS vs Negative FMS gave an adjusted coefficient of 15.7, 95% CI 3.7-27.7, p = 0.01) and deterioration scores (High FMS vs Negative FMS gave an adjusted coefficient of 37.6, 95% CI 14.0 to 61.3, p = 0.002), while NPS viral loads showed no significant association. CONCLUSION: We demonstrate a simple and effective method for detecting and quantifying exhaled SARS-CoV-2 in hospitalised patients with COVID-19. Higher FMS viral loads were more likely to be associated with developing severe disease compared to NPS viral loads. Similar to NPS, FMS viral load was highest in early disease and in those with active respiratory symptoms, highlighting the potential role of FMS in understanding infectivity.

A Virulence Associated Siderophore Importer Reduces Antimicrobial Susceptibility of Klebsiella pneumoniae.

The accessory genomes of many pathogenic bacteria include ABC transporters that scavenge metal by siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug efflux. There are mechanistic and recently recognized structural similarities between siderophore importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ on antimicrobial resistance of Klebsiella pneumoniae. YbtPQ is encoded in the yersiniabactin cluster in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp increased the susceptibility of K. pneumoniae to all antimicrobials tested. The mechanism was dependent on the yersiniabactin importer YbtPQ and may involve antimicrobial efflux, since it was affected by the inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. pneumoniae is recognized as a reservoir of genes for the emergence of hospital outbreak strains and for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, to E. coli decreased its susceptibility to a broad range of antimicrobials. Thus a confirmed siderophore importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have a secondary function exporting antimicrobials.

Lung microbiome composition and bronchial epithelial gene expression in patients with COPD versus healthy individuals: a bacterial 16S rRNA gene sequencing and host transcriptomic analysis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with airway inflammation and bacterial dysbiosis. The relationship between the airway microbiome and bronchial gene expression in COPD is poorly understood. We aimed to identify differences in the airway microbiome from bronchial brushings in patients with COPD and healthy individuals and to investigate whether any distinguishing bacteria are related to bronchial gene expression. METHODS: For this 16S rRNA gene sequencing and host transcriptomic analysis, individuals aged 45-75 years with mild-to-moderate COPD either receiving or not receiving inhaled corticosteroids and healthy individuals in the same age group were recruited as part of the Emphysema versus Airways Disease (EvA) consortium from nine centres in the UK, Germany, Italy, Poland, and Hungary. Individuals underwent clinical characterisation, spirometry, CT scans, and bronchoscopy. From bronchoscopic bronchial brush samples, we obtained the microbial profiles using 16S rRNA gene sequencing and gene expression using the RNA-Seq technique. We analysed bacterial genera relative abundance and the associations between genus abundance and clinical characteristics or between genus abundance and host lung transcriptional signals in patients with COPD versus healthy individuals, and in patients with COPD with versus without inhaled corticosteroids treatment. FINDINGS: Between February, 2009, and March, 2012, we obtained brush samples from 574 individuals. We used 546 of 574 samples for analysis, including 207 from healthy individuals and 339 from patients with COPD (192 with inhaled corticosteroids and 147 without). The bacterial genera that most strongly distinguished patients with COPD from healthy individuals were Prevotella (median relative abundance 33·5%, IQR 14·5-49·4, in patients with COPD vs 47·7%, 31·1-60·7, in healthy individuals; p<0·0001), Streptococcus (8·6%, 3·8-15·8, vs 5·3%, 3·0-10·1; p<0·0001), and Moraxella (0·05%, 0·02-0·14, vs 0·02%, 0-0·07; p<0·0001). Prevotella abundance was inversely related to COPD severity in terms of symptoms and positively related to lung function and exercise capacity. 446 samples had assessable RNA-seq data, 257 from patients with COPD (136 with inhaled corticosteroids and 121 without) and 189 from healthy individuals. No significant associations were observed between lung transcriptional signals from bronchial brushings and abundance of bacterial genera in patients with COPD without inhaled corticosteroids treatment and in healthy individuals. In patients with COPD treated with inhaled corticosteroids, Prevotella abundance was positively associated with expression of epithelial genes involved in tight junction promotion and Moraxella abundance was associated with expression of the IL-17 and TNF inflammatory pathways. INTERPRETATION: With increasing severity of COPD, the airway microbiome is associated with decreased abundance of Prevotella and increased abundance of Moraxella in concert with downregulation of genes promoting epithelial defence and upregulation of pro-inflammatory genes associated with inhaled corticosteroids use. Our work provides further insight in understanding the relationship between microbiome alteration and host inflammatory response, which might lead to novel therapeutic strategies for COPD. FUNDING: EU Seventh Framework Programme, National Institute for Health Research.

Inflammatory Endotype-associated Airway Microbiome in Chronic Obstructive Pulmonary Disease Clinical Stability and Exacerbations: A Multicohort Longitudinal Analysis.

Rationale: Understanding the role of the airway microbiome in chronic obstructive pulmonary disease (COPD) inflammatory endotypes may help to develop microbiome-based diagnostic and therapeutic approaches. Objectives: To understand the association of the airway microbiome with neutrophilic and eosinophilic COPD at stability and during exacerbations. Methods: An integrative analysis was performed on 1,706 sputum samples collected longitudinally from 510 patients with COPD recruited at four UK sites of the BEAT-COPD (Biomarkers to Target Antibiotic and Systemic COPD), COPDMAP (Chronic Obstructive Pulmonary Disease Medical Research Council/Association of the British Pharmaceutical Industry), and AERIS (Acute Exacerbation and Respiratory Infections in COPD) cohorts. The microbiome was analyzed using COPDMAP and AERIS as a discovery data set and BEAT-COPD as a validation data set. Measurements and Main Results: The airway microbiome in neutrophilic COPD was heterogeneous, with two primary community types differentiated by the predominance of Haemophilus. The Haemophilus-predominant subgroup had elevated sputum IL-1ß and TNFα (tumor necrosis factor α) and was relatively stable over time. The other neutrophilic subgroup with a balanced microbiome profile had elevated sputum and serum IL-17A and was temporally dynamic. Patients in this state at stability were susceptible to the greatest microbiome shifts during exacerbations. This subgroup can temporally switch to both neutrophilic Haemophilus-predominant and eosinophilic states that were otherwise mutually exclusive. Time-series analysis on the microbiome showed that the temporal trajectories of Campylobacter and Granulicatella were indicative of intrapatient switches from neutrophilic to eosinophilic inflammation, in track with patient sputum eosinophilia over time. Network analysis revealed distinct host-microbiome interaction patterns among neutrophilic Haemophilus-predominant, neutrophilic balanced microbiome, and eosinophilic subgroups. Conclusions: The airway microbiome can stratify neutrophilic COPD into subgroups that justify different therapies. Neutrophilic and eosinophilic COPD are interchangeable in some patients. Monitoring temporal variability of the airway microbiome may track patient inflammatory status over time.

Difference in persistent tuberculosis bacteria between in vitro and sputum from patients: implications for translational predictions.

This study aimed to investigate the number of persistent bacteria in sputum from tuberculosis patients compared to in vitro and to suggest a model-based approach for accounting for the potential difference. Sputum smear positive patients (n = 25) provided sputum samples prior to onset of chemotherapy. The number of cells detected by conventional agar colony forming unit (CFU) and most probable number (MPN) with Rpf supplementation were quantified. Persistent bacteria was assumed to be the difference between MPNrpf and CFU. The difference in persistent bacteria between in vitro and human sputum prior to chemotherapy was quantified using different model-based approaches. The persistent bacteria in sputum was 17% of the in vitro levels, suggesting a difference in phenotypic resistance, whereas no difference was found for multiplying bacterial subpopulations. Clinical trial simulations showed that the predicted time to 2 log fall in MPNrpf in a Phase 2a setting using in vitro pre-clinical efficacy information, would be almost 3 days longer if drug response was predicted ignoring the difference in phenotypic resistance. The discovered phenotypic differences between in vitro and humans prior to chemotherapy could have implications on translational efforts but can be accounted for using a model-based approach for translating in vitro to human drug response.

3-D printed polyvinyl alcohol matrix for detection of airborne pathogens in respiratory bacterial infections.

Novel sampling matrices were manufactured using 3D printing for the detection of respiratory pathogens in expired air. A specific configuration of the matrices was designed using Computer-Aided Design software. Polyvinyl alcohol (PVA) was printed using fused deposition modelling to create a multilayer matrix to enhance the capture of bacteria. The performance of these matrices was compared with gelatine filters that have been used for this work to date. PVA matrices (60 mm diameter) were contaminated with bacteria either by direct inoculation, or by aerosol exposure using an Omron A3 nebuliser. Rough and smooth morphotypes of Mycobacterium abscessus, M. smegmatis and M. bovis BCG, were used in this study to contaminate the matrices. PVA matrices and gelatine sampling filters were contaminated to compare recovery rates for quantitative analyses. These were dissolved in water, bacteria pelleted and DNA extracted followed by a Mycobacterium-specific quantitative Polymerase Chain Reaction (qPCR).The results showed that 3D printed PVA matrices are very effective to capture the bacteria. 3D printed PVA matrix and gelatine filters yielded results of the same order of magnitude for mycobacterial analyses, however, PVA matrix offers several advantages over the latter material. 3D printed PVA is considered as an economic and time-effective matrix as it is cheaper than gelatine filters. PVA is sufficiently robust to be handled and loaded into the surgical masks for sampling, compared to the brittle gelatine filters that required supportive frames. PVA is a synthetic material and it is suitable for DNA-based analyses, whilst gelatine is derived from animal collagen, and carries a high bacterial DNA background that interferes with the target DNA analysis. Furthermore, PVA dissolves in distilled water without requiring chemicals or enzymes, such as the case for gelatine hydrolysis. To summarise, 3D printed PVA sampling matrix is considered a promising tool used for microbiological diagnostic purposes.