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.

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.

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.

Microbial variations in sputum cultures among hospitalized patients with community-acquired pneumonia: differences in sputum microbiota between asthma and COPD patients.

OBJECTIVE: To assess differences in the sputum microbiota of community-acquired pneumonia (CAP) patients with either COPD or asthma, specifically focusing on a patient population in Turkey. METHODS: This retrospective study included hospitalized patients > 18 years of age with a diagnosis of pneumonia between January of 2021 and January of 2023. Participants were recruited from two hospitals, and three patient groups were considered: CAP patients with asthma, CAP patients with COPD, and CAP patients without COPD or asthma. RESULTS: A total of 246 patients with CAP were included in the study, 184 (74.8%) and 62 (25.2%) being males and females, with a mean age of 66 ± 14 years. Among the participants, 52.9% had COPD, 14.2% had asthma, and 32.9% had CAP but no COPD or asthma. Upon analysis of sputum cultures, positive sputum culture growth was observed in 52.9% of patients. The most commonly isolated microorganisms were Pseudomonas aeruginosa (n = 40), Acinetobacter baumannii (n = 20), Klebsiella pneumoniae (n = 16), and Moraxella catarrhalis (n = 8). CAP patients with COPD were more likely to have a positive sputum culture (p = 0.038), a history of antibiotic use within the past three months (p = 0.03), utilization of long-term home oxygen therapy (p < 0.001), and use of noninvasive ventilation (p = 0.001) when compared with the other patient groups. Additionally, CAP patients with COPD had a higher CURB-65 score when compared with CAP patients with asthma (p = 0.004). CONCLUSIONS: This study demonstrates that CAP patients with COPD tend to have more severe presentations, while CAP patients with asthma show varied microbial profiles, underscoring the need for patient-specific management strategies in CAP.

Highly diverse sputum microbiota correlates with the disease severity in patients with community-acquired pneumonia: a longitudinal cohort study.

BACKGROUND: Community-acquired pneumonia (CAP) is a common and serious condition that can be caused by a variety of pathogens. However, much remains unknown about how these pathogens interact with the lower respiratory commensals, and whether any correlation exists between the dysbiosis of the lower respiratory microbiota and disease severity and prognosis. METHODS: We conducted a retrospective cohort study to investigate the composition and dynamics of sputum microbiota in patients diagnosed with CAP. In total, 917 sputum specimens were collected consecutively from 350 CAP inpatients enrolled in six hospitals following admission. The V3-V4 region of the 16 S rRNA gene was then sequenced. RESULTS: The sputum microbiota in 71% of the samples were predominately composed of respiratory commensals. Conversely, 15% of the samples demonstrated dominance by five opportunistic pathogens. Additionally, 5% of the samples exhibited sterility, resembling the composition of negative controls. Compared to non-severe CAP patients, severe cases exhibited a more disrupted sputum microbiota, characterized by the highly dominant presence of potential pathogens, greater deviation from a healthy state, more significant alterations during hospitalization, and sparser bacterial interactions. The sputum microbiota on admission demonstrated a moderate prediction of disease severity (AUC = 0.74). Furthermore, different pathogenic infections were associated with specific microbiota alterations. Acinetobacter and Pseudomonas were more abundant in influenza A infections, with Acinetobacter was also enriched in Klebsiella pneumoniae infections. CONCLUSION: Collectively, our study demonstrated that pneumonia may not consistently correlate with severe dysbiosis of the respiratory microbiota. Instead, the degree of microbiota dysbiosis was correlated with disease severity in CAP patients.

Nebulised interferon beta-1a (SNG001) in the treatment of viral exacerbations of COPD.

BACKGROUND: Respiratory viral infections are major drivers of chronic obstructive pulmonary disease (COPD) exacerbations. Interferon-ß is naturally produced in response to viral infection, limiting replication. This exploratory study aimed to demonstrate proof-of-mechanism, and evaluate the efficacy and safety of inhaled recombinant interferon-ß1a (SNG001) in COPD. Part 1 assessed the effects of SNG001 on induced sputum antiviral interferon-stimulated gene expression, sputum differential cell count, and respiratory function. Part 2 compared SNG001 and placebo on clinical efficacy, sputum and serum biomarkers, and viral clearance. METHODS: In Part 1, patients (N = 13) with stable COPD were randomised 4:1 to SNG001 or placebo once-daily for three days. In Part 2, patients (N = 109) with worsening symptoms and a positive respiratory viral test were randomised 1:1 to SNG001 or placebo once-daily for 14 days in two Groups: A (no moderate exacerbation); B (moderate COPD exacerbation [i.e., acute worsening of respiratory symptoms treated with antibiotics and/or oral corticosteroids]). RESULTS: In Part 1, SNG001 upregulated sputum interferon gene expression. In Part 2, there were minimal SNG001-placebo differences in the efficacy endpoints; however, whereas gene expression was initially upregulated by viral infection, then declined on placebo, levels were maintained with SNG001. Furthermore, the proportion of patients with detectable rhinovirus (the most common virus) on Day 7 was lower with SNG001. In Group B, serum C-reactive protein and the proportion of patients with purulent sputum increased with placebo (suggesting bacterial infection), but not with SNG001. The overall adverse event incidence was similar with both treatments. CONCLUSIONS: Overall, SNG001 was well-tolerated in patients with COPD, and upregulated lung antiviral defences to accelerate viral clearance. These findings warrant further investigation in a larger study. TRIAL REGISTRATION: EU clinical trials register (2017-003679-75), 6 October 2017.

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.

Determinants of sputum culture conversion time in multidrug-resistant tuberculosis patients in ALERT comprehensive specialized hospital, Addis Ababa, Ethiopia: A retrospective cohort study.

INTRODUCTION: The treatment response of multi-drug resistance tuberculosis (MDR-Tuberculosis) patients is mainly dictated by the sputum culture conversion. An earlier culture conversion is a remarkable indicator of the improvement in the treatment response. In this study, we aimed to determine the time to culture conversion and its associated factors among MDR-Tuberculosis patients in All Africa Leprosy, Tuberculosis and Rehabilitation Training Center (ALERT) Hospital, Addis Ababa, Ethiopia. METHODS: A retrospective cohort study was conducted on 120 MDR-Tuberculosis patients attending ALERT Hospital from 2018-2022. Kaplan-Meier methods were used to determine the time to initial sputum culture conversion. All relevant laboratory, socio-demographic characteristics, and other clinical data were collected by chart abstraction using a structure data extraction form. The log-rank test was used to determine the survival rate. To identify the predictors of culture conversion, bivariate and multivariate Cox proportional hazard regression analysis was used. The hazard ratio (HR) with a 95% confidence interval was used to estimate the effect of each variable on the initial culture conversion. A test with a P value of < 0.05 was considered statistically significant. RESULTS: From the total of 120 study participants, 89.2% (107/120) have shown a successful culture conversion. The median age of the participants was 30 years (IQR = 12). The study participants were followed for 408.6 person-months (34.05 person-years). The median time to initial sputum culture conversion was 80 days. The median time to initial sputum culture conversion among HIV-positive and HIV-negative participants was 61 days (IQR = 58-63.5) and 88 days (IQR = 75-91), respectively. HIV-negative and patients with previous treatment history were shown to be the predictor for a prolonged time to initial sputum culture conversion, (aHR = 0.24 (95% CI: 0.1-0.4), P value <0.001) and (aHR = 0.47 (95% CI: 0.31-0.71), P value <0.001) respectively. CONCLUSION: The median time to sputum culture conversion for HIV positive was found to be 61 days in our study. Notably, patients with a history of previous anti-tuberculosis treatment, HIV-negative status, and higher bacillary load at baseline exhibited delayed culture conversion. These findings underscore the importance of considering such patient characteristics in the management of MDR-TB cases, as tailored interventions and close monitoring may lead to more favorable treatment outcomes. By identifying individuals with these risk factors early in the treatment process, healthcare providers can implement targeted strategies to optimize patient care and improve overall treatment success rates in MDR-TB management programs.

Prospective multicentre accuracy evaluation of the FUJIFILM SILVAMP TB LAM test for the diagnosis of tuberculosis in people living with HIV demonstrates lot-to-lot variability.

There is an urgent need for rapid, non-sputum point-of-care diagnostics to detect tuberculosis. This prospective trial in seven high tuberculosis burden countries evaluated the diagnostic accuracy of the point-of-care urine-based lipoarabinomannan assay FUJIFILM SILVAMP TB LAM (FujiLAM) among inpatients and outpatients living with HIV. Diagnostic performance of FujiLAM was assessed against a mycobacterial reference standard (sputum culture, blood culture, and Xpert Ultra from urine and sputum at enrollment, and additional sputum culture ≤7 days from enrollment), an extended mycobacterial reference standard (eMRS), and a composite reference standard including clinical evaluation. Of 1637 participants considered for the analysis, 296 (18%) were tuberculosis positive by eMRS. Median age was 40 years, median CD4 cell count was 369 cells/ul, and 52% were female. Overall FujiLAM sensitivity was 54·4% (95% CI: 48·7-60·0), overall specificity was 85·2% (83·2-87·0) against eMRS. Sensitivity and specificity estimates varied between sites, ranging from 26·5% (95% CI: 17·4%-38·0%) to 73·2% (60·4%-83·0%), and 75·0 (65·0%-82·9%) to 96·5 (92·1%-98·5%), respectively. Post-hoc exploratory analysis identified significant variability in the performance of the six FujiLAM lots used in this study. Lot variability limited interpretation of FujiLAM test performance. Although results with the current version of FujiLAM are too variable for clinical decision-making, the lipoarabinomannan biomarker still holds promise for tuberculosis diagnostics. The trial is registered at clinicaltrials.gov (NCT04089423).

[Design and application of a new type of switchable semi-closed sputum suction device for transfer].

Sputum suction is a necessary measure to remove respiratory secretions and collect sputum specimens, and the traditional sputum suction device includes open and closed methods. However, the open sputum suction device can easily cause sputum splashing during operation, polluting the operator and the surrounding environment. The closed sputum suction device is more expensive than the open sputum suction device. Although it can be used repeatedly, the friction between the protective sleeve and the wall of the suction tube increases after repeated uses, which in turn affects the operation. In addition, the two sputum suction methods need to be connected to a sputum collector when the sputum specimen is collected. This process can easily lead to leakage and contamination of specimens, and at the same time increase the infection risk for patients. In order to overcome the above problems, the medical staff from the department of critical care medicine of the First Affiliated Hospital of Dalian Medical University invented a new type of switchable semi-closed sputum suction device, and obtained the National Utility Model Patent of China (ZL 2020 2 1564712.3). The device is composed of a suction device connection port, a sliding negative pressure switch, an adapter interface, a pipe body, a transparent film protective sleeve, a clip-on connection joint, and a sputum collector. The sliding negative pressure switch at the connection port of the suction device can realize one-person one-handed operation. The sputum collector is connected with the sputum suction device to ensure that the specimen is not exposed. The transparent film protective sleeve covers the sputum suction tube body to ensure that the whole process of sputum suction operation is pollution-free. The clip-on connection joint is connected with the artificial airway interface to avoid sputum splashing during sputum suction, and it is clamped immediately after operation to reduce environmental pollution. Compared with the existing sputum suction device, the new switchable semi-closed sputum suction device has a more reasonable design and lower cost, which is worthy of clinical promotion.

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.

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.

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.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of mortality in patients with cystic fibrosis (pwCF), necessitating treatment with antibiotics. Pf is a filamentous bacteriophage produced by Pa and acts as a structural element in Pa biofilms. Pf presence has been associated with antibiotic resistance and poor outcomes in pwCF, although the underlying mechanisms are unclear. We have investigated how Pf and sputum biopolymers impede antibiotic diffusion using pwCF sputum and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf in sputum reduces the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

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.

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.