Sputum culture reversion in longer treatments with bedaquiline, delamanid, and repurposed drugs for drug-resistant tuberculosis.

Sputum culture reversion after conversion is an indicator of tuberculosis (TB) treatment failure. We analyze data from the endTB multi-country prospective observational cohort (NCT03259269) to estimate the frequency (primary endpoint) among individuals receiving a longer (18-to-20 month) regimen for multidrug- or rifampicin-resistant (MDR/RR) TB who experienced culture conversion. We also conduct Cox proportional hazard regression analyses to identify factors associated with reversion, including comorbidities, previous treatment, cavitary disease at conversion, low body mass index (BMI) at conversion, time to conversion, and number of likely-effective drugs. Of 1,286 patients, 54 (4.2%) experienced reversion, a median of 173 days (97-306) after conversion. Cavitary disease, BMI < 18.5, hepatitis C, prior treatment with second-line drugs, and longer time to initial culture conversion were positively associated with reversion. Reversion was uncommon. Those with cavitary disease, low BMI, hepatitis C, prior treatment with second-line drugs, and in whom culture conversion is delayed may benefit from close monitoring following conversion.

Implications of deduplication on the detection rates of multidrug-resistant organism (MDRO) in various specimens: insights from the hospital infection surveillance program.

BACKGROUND: Currently, different guidelines recommend using different methods to determine whether deduplication is necessary when determining the detection rates of multidrug-resistant organisms (MDROs). However, few studies have investigated the effect of deduplication on MDRO monitoring data. In this study, we aimed to investigate the influence of deduplication on the detection rates of MDROs in different specimens to assess its impact on infection surveillance outcomes. METHODS: Samples were collected from hospitalized patients admitted between January 2022 and December 2022; four types of specimens were collected from key monitored MDROs, including sputum samples, urine samples, blood samples, and bronchoalveolar lavage fluid (BALF) samples. In this study, we compared and analysed the detection rates of carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRECO), carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA) under two conditions: with and without deduplication. RESULTS: When all specimens were included, the detection rates of CRKP, CRAB, CRPA, and MRSA without deduplication (33.52%, 77.24%, 44.56%, and 56.58%, respectively) were significantly greater than those with deduplication (24.78%, 66.25%, 36.24%, and 50.83%, respectively) (all P < 0.05). The detection rates in sputum samples were significantly different between samples without duplication (28.39%, 76.19%, 46.95%, and 70.43%) and those with deduplication (19.99%, 63.00%, 38.05%, and 64.50%) (all P < 0.05). When deduplication was not performed, the rate of detection of CRKP in urine samples reached 30.05%, surpassing the rate observed with deduplication (21.56%) (P < 0.05). In BALF specimens, the detection rates of CRKP and CRPA without deduplication (39.78% and 53.23%, respectively) were greater than those with deduplication (31.62% and 42.20%, respectively) (P < 0.05). In blood samples, deduplication did not have a significant impact on the detection rates of MDROs. CONCLUSION: Deduplication had a significant effect on the detection rates of MDROs in sputum, urine, and BALF samples. Based on these data, we call for the Infection Prevention and Control Organization to align its analysis rules with those of the Bacterial Resistance Surveillance Organization when monitoring MDRO detection rates.

Monitoring COPD patients: systemic and bronchial eosinophilic inflammation in a 2-year follow-up.

BACKGROUND: High blood eosinophils seem to predict exacerbations and response to inhaled corticosteroids (ICS) treatment in patients with chronic obstructive pulmonary disease (COPD). The aim of our study was to prospectively evaluate for 2 years, blood and sputum eosinophils in COPD patients treated with bronchodilators only at recruitment. METHODS: COPD patients in stable condition treated with bronchodilators only underwent monitoring of lung function, blood and sputum eosinophils, exacerbations and comorbidities every 6 months for 2 years. ICS was added during follow-up when symptoms worsened. RESULTS: 63 COPD patients were enrolled: 53 were followed for 1 year, 41 for 2 years, 10 dropped-out. After 2 years, ICS was added in 12/41 patients (29%) without any statistically significant difference at time points considered. Blood and sputum eosinophils did not change during follow-up. Only FEV1/FVC at T0 was predictive of ICS addition during the 2 year-follow-up (OR:0.91; 95% CI: 0.83-0.99, p = 0.03). ICS addition did not impact on delta (T24-T0) FEV1, blood and sputum eosinophils and exacerbations. After 2 years, patients who received ICS had higher blood eosinophils than those in bronchodilator therapy (p = 0.042). Patients with history of ischemic heart disease increased blood eosinophils after 2 years [p = 0.03 for both percentage and counts]. CONCLUSIONS: Blood and sputum eosinophils remained stable during the 2 year follow-up and were not associated with worsened symptoms or exacerbations. Almost 30% of mild/moderate COPD patients in bronchodilator therapy at enrollment, received ICS for worsened symptoms in a 2 year-follow-up and only FEV1/FVC at T0 seems to predict this addition. History of ischemic heart disease seems to be associated with a progressive increase of blood eosinophils.

The Role of Efflux Pumps transporter in Multi-drug Resistant Tuberculosis: Mycobacterial memberane protein(MmpL5).

BACKGROUND: The overexpression of efflux pumps (Eps) was reported to contribute to multidrug resistant tuberculosis (MDR-TB). Increases in Eps that expel structurally unrelated drugs contribute to reduced susceptibility by decreasing the intracellular concentration of antibiotics. In the present study, an association of mycobacterial membrane protein (MmpS5-MmpL5) Ep and its gene regulator (Rv0678) was investigated in MDR-tuberculosis isolates. METHODS: MTB strains were isolated from patients at two different intervals, i.e., once when they had persistent symptoms despite 3-15 ≥ months of treatment and once when they had started new combination therapy ≥2-3 months. Sputum specimens were subjected to Xpert MTB/rifampicin test and then further susceptibility testing using proportional method and multiplex polymerase chain reaction (PCR) were performed on them. The isolates were characterized using both 16S-23S RNA and hsp65 genes spacer (PCR-restriction fragment length polymorphism). Whole-genome sequencing (WGS) was investigated on two isolates from culture-positive specimen per patient. The protein structure was simulated using the SWISS-MODEL. The input format used for this web server was FASTA (amino acid sequence). Protein structure was also analysis using Ramachandran plot. RESULTS: WGS documented deletion, insertion, and substitution in transmembrane transport protein MmpL5 (Rv0676) of Eps. Majority of the studied isolates (n = 12; 92.3%) showed a unique deletion mutation at three positions: (a) from amino acid number 771 (isoleucine) to 776 (valine), (b) from amino acid number 785 (valine) to 793 (histidine), and (c) from amino acid number 798 (leucine) to 806 (glycine)." One isolate (7.6%) had no deletion mutation. In all isolates (n = 13; 100%), a large insertion mutation consisting of 94 amino acid was observed "from amino acid number 846 (isoleucine) to amino acid number 939 (leucine)". Thirty-eight substitutions in Rv0676 were detected, of which 92.3% were identical in the studied isolates. WGS of mycobacterial membrane proteins (MmpS5; Rv0677) and its gene regulator (Rv0678) documented no deletion, insertion, and substitution. No differences were observed between MmpS5-MmpL5 and its gene regulator in isolates that were collected at different intervals. CONCLUSIONS: Significant genetic mutation like insertion, deletion, and substitution within transmembrane transport protein MmpL5 (Rv0676) can change the functional balance of Eps and cause a reduction in drug susceptibility. This is the first report documenting a unique amino acid mutation (insertion and deletion ≥4-94) in Rv0676 among drug-resistant MTB. We suggest the changes in Mmpl5 (Rv0676) might occurred due to in-vivo sub-therapeutic drug stress within the host cell. Changes in MmpL5 are stable and detected through subsequent culture-positive specimens.

Correlation of Cyclic Threshold Values Generated by GeneXpert Ultra MTB/RIF and Fluorescence Microscopy to Predict Mycobacterial Burden in Suspected Cases of Pulmonary Tuberculosis.

BACKGROUND: Smear microscopy for acid-fast bacilli visualization is important to assess the infectivity rate in patients with pulmonary tuberculosis (PTB), but it has limited sensitivity; hence, it is important to find an alternative strategy. The aim of our study was to compare the fluorescence microscopy grading by Auramine O phenol staining technique of respiratory samples with the cyclic threshold (Ct) values of GeneXpert Ultra (Mycobacterium tuberculosis/rifampicin [MTB/RIF]) and assess the diagnostic efficacy of GeneXpert Ultra (MTB/RIF) compared to microscopy in suspected cases of PTB. METHODS: The study was conducted in the Mycobacteriology Laboratory, Department of Microbiology, in Kasturba Hospital, Manipal. The study was a prospective, single-centered, cross-sectional study. Four hundred and fifty-two respiratory samples were included in the study. An optimal Ct cutoff value for ruling smear-positivity and smear-negativity and the mean Ct cutoff value were calculated. Clinical and radiological data from the requisition forms were assessed. IBM SPSS statistics software version 22 was used. The correlation between GeneXpert Ultra (MTB/RIF) Ct values and smear status was calculated by polychoric correlation. The extended McNemar's test was used to find the association between the variables. RESULTS: GeneXpert Ultra (MTB/RIF) yielded a higher positivity rate of 22.2% compared to smear microscopy 17.2%. Ct value and smear grading yielded a positive correlation (P = 0.8681; P < 0.05). GeneXpert Ultra (MTB/RIF) yielded nontuberculous mycobacteria in five undetected cases and speciated as Mycobacterium abscessus complex. CONCLUSIONS: Our study confirms the GeneXpert Ultra (MTB/RIF) Ct value levels as a predictor of smear positivity.

Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

BACKGROUND: Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). METHODS: EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. RESULTS: We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. CONCLUSION: From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Disseminated Mycobacterium avium Infection with Different Clinical Presentation in Two Human Immunodeficiency Virus-positive Patients.

ABSTRACT: Microorganisms belonging to the Mycobacterium avium complex (MAC) are ubiquitous in the environment, but only a minority of infected persons develop disease. An underlying lung disease or immune deficiency is a prerequisite for clinical manifestation. However, disseminated MAC disease primarily manifests in people living with human immunodeficiency virus (HIV) in the severe immunodeficiency stage with a whole host of clinical symptoms. We present two cases of disseminated M. avium infection in people living with HIV in the stage of severe immunodeficiency. Both patients exhibited distinct disease progression, with the absence of pulmonary symptoms being a common characteristic. The first patient predominantly experienced high fever, accompanied by diarrhea and severe anemia. The normothermia in the second patient was incongruent with the presence of marked cachexia, severe abdominal pain, and magnetic resonance imaging evidence of abdominal lymph node involvement. The causative agent was isolated from both sputum and stools. The patients underwent treatment that comprised aminoglycoside, macrolide, ethambutol, and rifampicin. Although both patients achieved optimal viral suppression of HIV, the immunologic response to antiretroviral therapy was suboptimal. The first patient died in the setting of severe immunodeficiency due to the development of decompensated liver cirrhosis, while the second patient demonstrated a slight reverse course of the disease.

Domiciliary monitoring of exhaled nitric oxide in the management of asthma: a pilot study.

BACKGROUND: Whether asthma patients could benefit from home monitoring for fractional exhaled nitric oxide (flow of 50 mL/s, FeNO50) is unknown. We explore the application value of home monitoring FeNO50 in daily asthma management. METHODS: Twenty-two untreated, uncontrolled asthma patients were selected. Medical history, blood and sputum samples, pulmonary function, Asthma Control Test (ACT), and other clinical data of the subjects were collected. All subjects underwent daily monitoring for four weeks using a FeNO50 monitor and mobile spirometry (mSpirometry). The diurnal differences and dynamic changes were described. Compare the effect-acting time and the relative plateau of treatment between FeNO50 and mSpirometry monitoring. RESULTS: In the first two weeks, the morning median (IQR) level of FeNO50 was 44 (35, 56) ppb, which was significantly higher than the evening median level [41 (32, 53) ppb, P = 0.028]. The median (IQR) effect-acting time assessed by FeNO50 was 4 (3, 5) days, which was significantly earlier than each measure of mSpirometry (P < 0.05). FeNO50 reached the relative plateau significantly earlier than FEV1 (15 ± 2 days vs. 21 ± 3 days, P < 0.001). After treatment, the daily and weekly variation rates of FeNO50 showed a gradually decreasing trend (P < 0.05). The ACT score, sputum eosinophils, and blood eosinophils also significantly improved (P ≤ 0.01). CONCLUSIONS: The daily home monitoring of FeNO50 in asthmatic patients showed significant circadian rhythm, and the sensitivity of FeNO50 in evaluating the response to treatment was higher than mSpirometry. The daily and weekly variation rates of FeNO50 change dynamically with time, which may be used to assess the condition of asthma.

Comparison of the sputum microbiome between patients with stable nontuberculous mycobacterial pulmonary disease and patients requiring treatment.

BACKGROUND: We evaluated whether the sputum bacterial microbiome differs between nontuberculous mycobacteria pulmonary disease (NTM-PD) patients with stable disease not requiring antibiotic treatment and those requiring antibiotics. METHODS: We collected sputum samples from 21 clinically stable NTM-PD patients (stable group) and 14 NTM-PD patients needing antibiotic treatment (treatment group). We also obtained 13 follow-up samples from the stable group. We analyzed the 48 samples using 16S rRNA gene sequencing (V3-V4 region) and compared the groups. RESULTS: In the linear discriminant analysis effect size (LEfSe) analysis, the species Porphyromonas pasteri, Haemophilus parahaemolyticus, Prevotella nanceiensis, and Gemella haemolysans were significantly more prevalent in the sputum of the stable group compared to the treatment group. No taxa showed significant differences in alpha-/beta-diversity or LEfSe between the 21 baseline and 13 follow-up sputum samples in the stable group. In the stable group, the genus Bergeyella and species Prevotella oris were less common in patients who achieved spontaneous culture conversion (n = 9) compared to those with persistent NTM positivity (n = 12) (effect size 3.04, p = 0.039 for Bergeyella; effect size 3.64, p = 0.033 for P. oris). In the treatment group, H. parainfluenzae was more common in patients with treatment success (n = 7) than in treatment-refractory patients (n = 7) (effect size 4.74, p = 0.013). CONCLUSIONS: Our study identified distinct bacterial taxa in the sputum of NTM-PD patients based on disease status. These results suggest the presence of a microbial environment that helps maintain disease stability.

Comparative microbiome analysis in cystic fibrosis and non-cystic fibrosis bronchiectasis.

BACKGROUND: Bronchiectasis is a condition characterized by abnormal and irreversible bronchial dilation resulting from lung tissue damage and can be categorized into two main groups: cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). Both diseases are marked by recurrent infections, inflammatory exacerbations, and lung damage. Given that infections are the primary drivers of disease progression, characterization of the respiratory microbiome can shed light on compositional alterations and susceptibility to antimicrobial drugs in these cases compared to healthy individuals. METHODS: To assess the microbiota in the two studied diseases, 35 subjects were recruited, comprising 10 NCFB and 13 CF patients and 12 healthy individuals. Nasopharyngeal swabs and induced sputum were collected, and total DNA was extracted. The DNA was then sequenced by the shotgun method and evaluated using the SqueezeMeta pipeline and R. RESULTS: We observed reduced species diversity in both disease cohorts, along with distinct microbial compositions and profiles of antimicrobial resistance genes, compared to healthy individuals. The nasopharynx exhibited a consistent microbiota composition across all cohorts. Enrichment of members of the Burkholderiaceae family and an increased Firmicutes/Bacteroidetes ratio in the CF cohort emerged as key distinguishing factors compared to NCFB group. Staphylococcus aureus and Prevotella shahii also presented differential abundance in the CF and NCFB cohorts, respectively, in the lower respiratory tract. Considering antimicrobial resistance, a high number of genes related to antibiotic efflux were detected in both disease groups, which correlated with the patient's clinical data. CONCLUSIONS: Bronchiectasis is associated with reduced microbial diversity and a shift in microbial and resistome composition compared to healthy subjects. Despite some similarities, CF and NCFB present significant differences in microbiome composition and antimicrobial resistance profiles, suggesting the need for customized management strategies for each disease.

Effects of mechanical insufflation-exsufflation on ventilator-free days in intensive care unit subjects with sputum retention; a randomized clinical trial.

BACKGROUND: Mechanical insufflation-exsufflation (MI-E) facilitates extubation. However, its potential to reduce the duration of ventilator use remains unclear. Therefore, the present study investigated whether the use of MI-E shortened the duration of mechanical ventilation in patients with high sputum retention. METHODS: A randomized open-label trial was conducted at a single intensive care unit (ICU) in Japan between November 2017 and June 2019. Ventilated subjects requiring suctioning at least once every hour due to sputum retention were randomly assigned to the MI-E group or conventional care group. The primary endpoint was the number of ventilator-free days on day 28. Secondary endpoints were ventilator days in surviving subjects, the length of ICU stay, and mortality and tracheostomy rates among survivors. RESULTS: Forty-eight subjects (81% males) with a median age of 72 years (interquartile range [IQR], 65-85 years) were enrolled. There were 27 subjects in the MI-E group and 21 in the control group. The median number of ventilator-free days was 21 (IQR, 13-24) and 18, respectively (IQR, 0-23) (P = .38). No significant differences were observed in the ICU length of stay (median, 10 days (IQR, 7-12) vs 12 days (IQR, 6-15); P = .31), mortality rate (19% vs 15%; odds ratio [OR], 1.36 [0.28-6.50]; P = .69), or tracheostomy rate among survivors (14% vs 28%; OR, 0.40 [0.08-1.91]; P = .25). CONCLUSION: In ventilated subjects in the ICU with high sputum retention, the use of MI-E did not significantly increase the number of ventilator-free days over that with conventional care.

Thoracic Society of Australia and New Zealand position statement: The safe clinical use of sputum induction for bio-sampling of the lower airways in children and adults.

Sputum induction is widely used in clinical settings for collection of biological samples from the lower airways. However, in recent years sputum induction has been associated with serious adverse events and even death. This position statement was commissioned by the Thoracic Society of Australia and New Zealand to address major adverse events of two deaths associated with sputum induction that have occurred in Australia in 2021, and outlines best practice for the safe use of sputum induction. The statement resulted from systematic literature searches by a multi-disciplinary group including respiratory physicians, nurses and physiotherapists (paediatric and adults focused). Consumers had input to an advanced draft of the position statement. The position statement covers indications for sputum induction, informed consent, scope of practice of personnel administering the procedure, infection control considerations, details about the sputum induction procedure, safety considerations and risk assessment in clinical settings.

Human genetic associations of the airway microbiome in chronic obstructive pulmonary disease.

Little is known about the relationships between human genetics and the airway microbiome. Deeply sequenced airway metagenomics, by simultaneously characterizing the microbiome and host genetics, provide a unique opportunity to assess the microbiome-host genetic associations. Here we performed a co-profiling of microbiome and host genetics with the identification of over 5 million single nucleotide polymorphisms (SNPs) through deep metagenomic sequencing in sputum of 99 chronic obstructive pulmonary disease (COPD) and 36 healthy individuals. Host genetic variation was the most significant factor associated with the microbiome except for geography and disease status, with its top 5 principal components accounting for 12.11% of the microbiome variability. Within COPD individuals, 113 SNPs mapped to candidate genes reported as genetically associated with COPD exhibited associations with 29 microbial species and 48 functional modules (P < 1 × 10-5), where Streptococcus salivarius exhibits the strongest association to SNP rs6917641 in TBC1D32 (P = 9.54 × 10-8). Integration of concurrent host transcriptomic data identified correlations between the expression of host genes and their genetically-linked microbiome features, including NUDT1, MAD1L1 and Veillonella parvula, TTLL9 and Stenotrophomonas maltophilia, and LTA4H and Haemophilus influenzae. Mendelian randomization analyses revealed a potential causal link between PARK7 expression and microbial type III secretion system, and a genetically-mediated association between COPD and increased relative abundance of airway Streptococcus intermedius. These results suggest a previously underappreciated role of host genetics in shaping the airway microbiome and provide fresh hypotheses for genetic-based host-microbiome interactions in COPD.

Objective sputum colour assessment and clinical outcomes in bronchiectasis: data from the European Bronchiectasis Registry (EMBARC).

BACKGROUND: A validated 4-point sputum colour chart can be used to objectively evaluate the levels of airway inflammation in bronchiectasis patients. In the European Bronchiectasis Registry (EMBARC), we tested whether sputum colour would be associated with disease severity and clinical outcomes. METHODS: We used a prospective, observational registry of adults with bronchiectasis conducted in 31 countries. Patients who did not produce spontaneous sputum were excluded from the analysis. The Murray sputum colour chart was used at baseline and at follow-up visits. Key outcomes were frequency of exacerbations, hospitalisations for severe exacerbations and mortality during up to 5-year follow-up. RESULTS: 13 484 patients were included in the analysis. More purulent sputum was associated with lower forced expiratory volume in 1 s (FEV1), worse quality of life, greater bacterial infection and a higher bronchiectasis severity index. Sputum colour was strongly associated with the risk of future exacerbations during follow-up. Compared to patients with mucoid sputum (reference group), patients with mucopurulent sputum experienced significantly more exacerbations (incident rate ratio (IRR) 1.29, 95% CI 1.22-1.38; p<0.0001), while the rates were even higher for patients with purulent (IRR 1.55, 95% CI 1.44-1.67; p<0.0001) and severely purulent sputum (IRR 1.91, 95% CI 1.52-2.39; p<0.0001). Hospitalisations for severe exacerbations were also associated with increasing sputum colour with rate ratios, compared to patients with mucoid sputum, of 1.41 (95% CI 1.29-1.56; p<0.0001), 1.98 (95% CI 1.77-2.21; p<0.0001) and 3.05 (95% CI 2.25-4.14; p<0.0001) for mucopurulent, purulent and severely purulent sputum, respectively. Mortality was significantly increased with increasing sputum purulence, hazard ratio 1.12 (95% CI 1.01-1.24; p=0.027), for each increment in sputum purulence. CONCLUSION: Sputum colour is a simple marker of disease severity and future risk of exacerbations, severe exacerbations and mortality in patients with bronchiectasis.

Bacterial interactome disturbance in chronic obstructive pulmonary disease clinical stability and exacerbations.

RATIONALE: Our understanding of airway dysbiosis in chronic obstructive pulmonary disease (COPD) remains incomplete, which may be improved by unraveling the complexity in microbial interactome. OBJECTIVES: To characterize reproducible features of airway bacterial interactome in COPD at clinical stability and during exacerbation, and evaluate their associations with disease phenotypes. METHODS: We performed weighted ensemble-based co-occurrence network analysis of 1742 sputum microbiomes from published and new microbiome datasets, comprising two case-control studies of stable COPD versus healthy control, two studies of COPD stability versus exacerbation, and one study with exacerbation-recovery time series data. RESULTS: Patients with COPD had reproducibly lower degree of negative bacterial interactions, i.e. total number of negative interactions as a proportion of total interactions, in their airway microbiome compared with healthy controls. Evaluation of the Haemophilus interactome showed that the antagonistic interaction networks of this established pathogen rather than its abundance consistently changed in COPD. Interactome dynamic analysis revealed reproducibly reduced antagonistic interactions but not diversity loss during COPD exacerbation, which recovered after treatment. In phenotypic analysis, unsupervised network clustering showed that loss of antagonistic interactions was associated with worse clinical symptoms (dyspnea), poorer lung function, exaggerated neutrophilic inflammation, and higher exacerbation risk. Furthermore, the frequent exacerbators (≥ 2 exacerbations per year) had significantly reduced antagonistic bacterial interactions while exhibiting subtle compositional changes in their airway microbiota. CONCLUSIONS: Bacterial interactome disturbance characterized by reduced antagonistic interactions, rather than change in pathogen abundance or diversity, is a reproducible feature of airway dysbiosis in COPD clinical stability and exacerbations, which suggests that we may target interactome rather than pathogen alone for disease treatment.

TB LAMP assay, a beneficial tool for the diagnosis of Tubercular meningitis in resource-limited settings.

INTRODUCTION: Tubercular meningitis (TBM) is a serious public health problem in developing countries as it leads to significant mortality and residual neurological sequelae. The estimated mortality due to TBM in India is 1.5 per 100,000 population. In resource-limited settings, only the Ziehl-Neelsen (ZN) stain, which has very little sensitivity, is available. The World Health Organization recommended the Loop Mediated Isothermal Amplification (TB LAMP) assay for pulmonary tuberculosis only. We evaluated this test for tubercular meningitis as well. METHODOLOGY: In a cross-sectional study of 2-year duration, we have taken 239 cerebrospinal fluid samples from suspected cases of tubercular meningitis patients. ZN staining along with Mycobacteria Growth Indicator Tube (MGIT) TB culture, Xpert MTB/RIF Ultra assay, and commercial TB LAMP assay were performed for each sample. RESULTS: Out of 239 samples, 40 samples (16.73%) were found TB LAMP assay positive, 48 samples (20.08%) were found Xpert ultra-assay positive, 12 samples (5.02%) were MGIT TB culture positive and acid-fast bacillus smear positive in ten samples (4.18 %). Out of 12 MGIT-positive samples, all samples (100%) were TB LAMP and Xpert ultra positive and one sample (8.33%) was ZN smear positive. In 199 negative samples from the TB LAMP assay, eight samples were positive by Xpert, none by MGIT TB culture and AFB smear. Sensitivity and specificity were found as 100% and 87.66%, respectively, for the TB LAMP assay. CONCLUSION: TB LAMP assay is a rapid, cost-effective, sensitive, and specific test for tubercular meningitis infection in resource-limited settings.

Increased tuberculosis case detection in Tanzanian children and adults using African giant pouched rats.

BACKGROUND: African giant pouched rats, trained by Anti-Persoonsmijnen Ontmijnende Product Ontwikkeling (APOPO), have demonstrated their ability to detect tuberculosis (TB) from sputum. We assessed rat-based case detection and compared the mycobacterium bacillary load (MTB-load) in children versus adults. METHODS: From January-December 2022, samples were collected prospectively from 69 Directly Observed Therapy (DOT) facilities' presumed TB patients. Using an average of five rats, APOPO re-evaluated patients with bacteriologically negative (sputum-smear microscopy or Xpert MTB/RIF) results. Rat-positive samples were tested using concentrated smear light-emitting diode microscopy to confirm TB detection before treatment initiation. The rats' identification of pulmonary TB is based on smelling TB-specific volatile organic compounds (VOCs) in sputum. Using STATA, Chi-square for odds ratio and confidence interval was calculated and evaluated: (1) the yield of rat-based TB detection compared to that of the health facilities; (2) rat-based TB detection in children versus adults; and (3) rats' ability to detect TB across MTB-loads and between children and adults. RESULTS: From 35,766 patients, 5.3% (1900/35,766) were smear-positive and 94.7% (33,866/35,766) were smear or Xpert-negatives at DOTS facility. Of those with negative results, 2029 TB cases were detected using rats, contributing to 52% (2029/3929 of total TB identified), which otherwise would have been missed. Compared to DOT facilities, rats were six-fold more likely to detect TB among Acid Fast Bacilli (AFB) 1+/scanty [90% (1829/2029) versus 60% (1139/1900), odds ratio, OR = 6.11, 95% confidence interval, CI: 5.14-7.26]; twice more likely to identify TB cases among children [71% (91/129) versus 51% (1795/3542), OR = 2.3, 95% CI: 1.59-3.42]; and twice more likely to identify TB cases among children with AFB 1+/scanty than adults with the same MTB-load [5% (86/1703) versus 3% (28/1067), OR = 2.0, 95% CI: 1.28-3.03]. CONCLUSIONS: Rats contributed over half of the TB cases identified in program settings, and children, especially those with a lower MTB-load, were more likely to be diagnosed with TB by rats. The chemical signatures, VOCs, were only available for adults, and further research describing the characteristics of VOCs in children versus adults may pave the way to enhance TB diagnosis in children.

The characteristics of microbiome in the upper respiratory tract of COVID-19 patients.

BACKGROUND: Co-infection with other pathogens in coronavirus disease 2019 (COVID-19) patients exacerbates disease severity and impacts patient prognosis. Clarifying the exact pathogens co-infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is premise of the precise treatment for COVID-19 patients. METHODS: Sputum samples were collected from 17 patients in the COVID-19 positive group and 18 patients in the COVID-19 negative group. DNA extraction was performed to obtain the total DNA. Sequencing analysis using 16S and ITS rRNA gene was carried out to analyze the composition of bacterial and fungal communities. Meanwhile, all the samples were inoculated for culture. RESULTS: We did not observe significant differences in bacterial composition between the COVID-19 positive and negative groups. However, a significantly higher abundance of Candida albicans was observed in the upper respiratory tract samples from the COVID-19 positive group compared to the COVID-19 negative group. Moreover, the Candida albicans strains isolated from COVID-19 positive group exhibited impaired secretion of aspartyl proteinases. CONCLUSION: COVID-19 positive patients demonstrate a notable increase in the abundance of Candida albicans, along with a decrease in the levels of aspartyl proteinases, indicating the alteration of microbiota composition of upper respiratory tract.

Urinary lipoarabinomannan in individuals with sputum-negative pulmonary tuberculosis.

BACKGROUND OBJECTIVES: Tuberculosis (TB) is a major global cause of ill health. Sputum microscopy for confirmation of presumptive pulmonary TB (PTB) has a reportedly low sensitivity of 22-43 per cent for single smear and up to 60 per cent under optimal conditions. National TB Elimination Programme in India recommends the use of cartridge-based nucleic acid amplification test (CBNAAT) and culture for microbiological confirmation in presumptive PTB individuals with sputum smear negative test. The use of lateral flow urine lipoarabinomannan (LF-LAM) is usually recommended for the diagnosis of TB in HIV-positive individuals with low CD4 counts or those who are seriously ill. The objective of this study was to detect urinary LAM using cage nanotechnology that does not require a physiologic or immunologic consequence of HIV infection for LAM quantification in human urine in 50 HIV-seronegative sputum smear-negative PTB individuals. METHODS: To study the diagnostic value of urinary LAM in sputum smear negative PTB individuals, a cage based nanotechnology ELISA technique was used for urinary LAM in three different groups of participants. Fifty smears negative PTB clinically diagnosed, 15 smear positive PTB and 15 post TB sequel individuals. Sputum was tested by smear, CBNAAT, and culture along with urine LAM before treatment. The results were interpreted by ROC curve in comparison to the standard tests like CBNAAT and culture. RESULTS: The mean urinary LAM value was 0.84 ng/ml in 37 culture-positive [Mycobacterium tuberculosis (M.tb)] and 0.49 ng/ml in 13 culture-negative (M.tb) smear-negative individuals with PTB, respectively. In 47 smear-negative PTB cases with microbiologically confirmed TB by CBNAAT, the mean urinary LAM was 0.76 ng/ml. The mean urinary LAM in post-TB sequel individuals was 0.47 ng/ml. As per the receiver operating characteristic curve, cut-off value of urinary LAM in individuals with smear-negative PTB microbiologically confirmed by: (i) CBNAAT was 0.695 ng/ml and (ii) culture was 0.615 ng/ml. INTERPRETATION CONCLUSIONS: The findings of this study suggest that individuals with smear-negative PTB and a urinary LAM value of >0.615 ng/ml were most likely to have microbiological confirmed TB while those with a LAM value <0.615 ng/ml >0.478 ng/ml are less likely and those with a value <0.478 ng/ml are unlikely to have microbiological confirmed TB.