Sputum Gene Research Articles

Clinical profiles and related factors in tuberculosis patients with positive sputum smear mycobacterium tuberculosis tests

The aim of this study was to explore the related factors linked to the development and infectivity of tuberculosis. This was achieved by comparing the clinical characteristics of patients with pulmonary tuberculosis (TB) who tested positive in smear Mycobacterium tuberculosis tests with this who tested negative in smear mycobacterium tests but positive in sputum Gene Xpert tests. We gathered clinical data of 1612 recently hospitalized patients diagnosed with pulmonary tuberculosis who tested positive either in sputum Gene-Xpert test or sputum smear Mycobacterium tuberculosis tests. The data was collected from January 1, 2018 to August 5, 2023, at Sichuan Provincial People's Hospital. We conducted separately analyzes and comparisons of the clinical characteristics between the two groups of patients, aiming to discussed the related factors influencing the development and infectivity of tuberculosis. In comparison to the GeneXpert positive group, the sputum smear positive group exhibited a higher proportion of elderly patients (aged 75–89) and individuals classified as underweight (BMI < 18.5 kg/m2). Furthermore, this group was more prone to experiencing symptoms such as weight loss, coughing and sputum production, hemoptysis, shortness of breath, and difficulty breathing. Moreover, they are also more likely to develop extrapulmonary tuberculosis, such as tuberculous meningitis, tuberculous pleurisy, and tuberculous peritonitis. These clinical features, when present, not only increase the likelihood of a positive result in sputum smear tests but also suggest a high infectivity of pulmonary tuberculosis. Elderly individuals (aged 75 to 89) who are underweight (BMI < 18.5 kg/m2), display symptom of cough, expectoration, hemoptysis and dyspnea-particularly cough and expectoration-and those with extra pulmonary tuberculosis serve as indicators of highly infectious pulmonary tuberculosis patients. These patients may present with more severe condition, carrying a higher bacteria, and being more prone to bacterial elimination. Identification of these patients is crucial, and prompt actions such as timely and rapid isolation measures, cutting off transmission routes, and early empirical treatment of tuberculosis are essential to control the development of the disease.

Multi-drug resistant gene mutation analysis in Mycobacterium tuberculosis by molecular techniques.

Rifampicin (RIF) and isoniazid (INH), two most potent antibiotics, are prescribed to cure tuberculosis. Mycobacterium tuberculosis, the causative agent of multidrug-resistant tuberculosis (MDR-TB), is resistant to these first-line drugs. Here, two molecular techniques were demonstrated such as PCR sequencing-based and GeneXpert assay for rapidly identifying MDR-TB. Pulmonary samples (sputum) were collected from 55 MDR-TB suspected patients from the National Tuberculosis Reference Laboratory (NTRL), Dhaka where the research work was partially accomplished and continued in the department of Microbiology, University of Dhaka, Bangladesh. We strived for sequencing technique as well as GeneXpert assay to identify mutations in rpoB and katG genes in MTB strains and sputum directly. Culture-based drug susceptibility testing (DST) was performed to measure the efficacy of the molecular methods employed. When analyzed, rpoB gene mutations at codons 531 (54.54%), 526 (14.54%), and 516 (10.91%) were found by sequencing in 80% of the samples. Nucleotide substitution at katG315 (AGC→ACC) was spotted in 16 (76.19%) out of 21 samples. When comparing the sequencing results with DST, sensitivity and specificity were investigated to determine drug-resistance (rifampicin-resistance were 98 and 100% whereas isoniazid-resistance were 94 and 100% respectively). Additionally, as a point of comparison with DST, only 85.45% of RIF mono-resistant TB cases were accurately evaluated by the GeneXpert assay. This research supports the adoption of PCR sequencing approach as an efficient tool in detecting MDR-TB, counting the higher sensitivity and specificity as well as the short period to produce the results.

Malignant Small Round Cell Tumor: A Rare Aggressive Malignancy-Case Report

Background: Malignant Small round Cell Tumor (MSRCT) is very rare group of tumors. Malignant cells have similar histologic appearance making it difficult to distinguish one from another, and poses a diagnostic challenge with needs for a multimodal approach. Immunohistochemistry and immunophenotyping by flow cytometry, reverse transcriptase polymer chain reaction (RT-PCR) and fluorescence in situ hybridization are needed to confirm. The appropriate and best treatment in early stages of MSRCT is early surgical excision with chemoradiotherapy based on patient specification. Inspite of all these, tumors have poor prognosis and chance of recurrence are high. Method: Case study of a 27-year-old male who presented with a non-productive cough for 4 weeks, with feverish feeling with weight loss and shortness of breath and a new onset precordial chest pain non radiating and worse on exertion. On examination his vitals were: BP 121/91 mm of hg, pulse rate 114bpm, temperature 36.3 degrees Celsius, respiratory rate 24/per min, SPO2 96%.On examination, Elevated Jugular Venous Pressure, hyperactive precordium, displaced apex beat to 6th intercostal space anterior axillary line, muffled heart sounds and bilateral lower limb edema. Respiratory exam was unremarkable. Sputum gene expert was negative for mycobacterium tuberculosis and covid 19 rapid test was negative. Chest X Ray showed cardiomegaly and a transthoracic echocardiography revealed moderate-large pericardial effusion, with mild collapse of right atrium in diastole. Pericardiocentesis done showed hemorrhagic effusion with lymphocytes cell component, patient was initiated on treatment for TB pericarditis empirically. Three months later a chest CT scan done revealed extensive mediastinal/pericardial mass with lung nodules. An ultrasound guided mediastinal/Pericardial biopsy was done, revealed a poorly differentiated small round cell carcinoma. Results: Patient expired 14 weeks from the initial onset of symptoms while awaiting immunohistochemistry studies for specific diagnosis of the tumor and oncology review. Discussion: This demonstrates the complexity of establishing differential diagnosis of MSRCT in a resource limited setting with subsequent delay in treatment initiation, heralds poor prognosis. This also confirms difficulties in establishing differential diagnosis in poorly differentiated tumor.

A compensatory RNase E variation increases Iron Piracy and Virulence in multidrug-resistant Pseudomonas aeruginosa during Macrophage infection.

During chronic cystic fibrosis (CF) infections, evolved Pseudomonas aeruginosa antibiotic resistance is linked to increased pulmonary exacerbations, decreased lung function, and hospitalizations. However, the virulence mechanisms underlying worse outcomes caused by antibiotic resistant infections are poorly understood. Here, we investigated evolved aztreonam resistant P. aeruginosa virulence mechanisms. Using a macrophage infection model combined with genomic and transcriptomic analyses, we show that a compensatory mutation in the rne gene, encoding RNase E, increased pyoverdine and pyochelin siderophore gene expression, causing macrophage ferroptosis and lysis. We show that iron-bound pyochelin was sufficient to cause macrophage ferroptosis and lysis, however, apo-pyochelin, iron-bound pyoverdine, or apo-pyoverdine were insufficient to kill macrophages. Macrophage killing could be eliminated by treatment with the iron mimetic gallium. RNase E variants were abundant in clinical isolates, and CF sputum gene expression data show that clinical isolates phenocopied RNase E variant functions during macrophage infection. Together these data show how P. aeruginosa RNase E variants can cause host damage via increased siderophore production and host cell ferroptosis but may also be targets for gallium precision therapy.

Severity of Lung Function Impairment Drives Transcriptional Phenotypes of COPD and Relates to Immune and Metabolic Processes.

This study sought to characterize transcriptional phenotypes of COPD through unsupervised clustering of sputum gene expression profiles, and further investigate mechanisms underlying the characteristics of these clusters. Induced sputum samples were collected from patients with stable COPD (n = 72) and healthy controls (n = 15). Induced sputum was collected for inflammatory cell counts, and RNA extracted. Transcriptional profiles were generated (Illumina Humanref-8 V2) and analyzed by GeneSpring GX14.9.1. Unsupervised hierarchical clustering and differential gene expression analysis were performed, and gene alterations validated in the ECLIPSE dataset (GSE22148). We identified 2 main clusters (Cluster 1 [n = 35] and Cluster 2 [n = 37]), which further divided into 4 sub-clusters (Sub-clusters 1.1 [n = 14], 1.2 [n = 21], 2.1 [n = 20] and 2.2 [n = 17]). Compared with Cluster 1, Cluster 2 was associated with significantly lower lung function (p = 0.014), more severe disease (p = 0.009) and breathlessness (p = 0.035), and increased sputum neutrophils (p = 0.031). Sub-cluster 1.1 had significantly higher proportion of people with comorbid cardiovascular disease compared to the other 3 sub-clusters (92.5% vs 57.1%, 50% and 52.9%, p < 0.013). Through supervised analysis we determined that degree of airflow limitation (GOLD stage) was the predominant factor driving gene expression differences in our transcriptional clusters. There were 452 genes (adjusted p < 0.05 and ≥2 fold) altered in GOLD stage 3 and 4 versus 1 and 2, of which 281 (62%) were also found to be significantly expressed between these GOLD stages in the ECLIPSE data set (GSE22148). Differentially expressed genes were largely downregulated in GOLD stages 3 and 4 and connected in 5 networks relating to lipoprotein and cholesterol metabolism; metabolic processes in oxidation/reduction and mitochondrial function; antigen processing and presentation; regulation of complement activation and innate immune responses; and immune and metabolic processes. Severity of lung function drives 2 distinct transcriptional phenotypes of COPD and relates to immune and metabolic processes.

Intracranial tuberculoma in human immunodeficiency virus-infected patient: a case report

Intracranial tuberculoma is one of the most devastating forms of Mycobacterium tuberculosis infection, presenting in 1% of all tuberculosis cases. This condition is significantly associated with high morbidity and mortality and frequently occurs in immunocompromised patients such as patient with human immunodeficiency virus (HIV) infection. Symptoms of intracranial tuberculoma include headache, nausea, vomiting, ataxia, diplopia, hemiparesis, and seizure. We reported a 34-years-old male patient presented with progressive left side weakness for 1 month prior to admission. Patient also complained of intermittent fever, headache, productive cough, night sweats, and unintentional weight loss. On neurological examination, power of left upper and lower limb was 3. Patient had a positive test result for HIV serologic examination. Chest X-ray showed infiltrate in the apical segment of right lower lobe of the lung. Mycobacterium tuberculosis was found on sputum gene expert examination. Head computed tomography (CT) scan showed multiple hypodense lesions with contrast enhancement in right caudate nucleus and right lentiform nucleus, suggestive for tuberculoma. Patient was treated with fixed-dose combination of antituberculosis, corticosteroid, and other symptomatic medication and showed a significant clinical improvement.

Pseudomonas aeruginosa is oxygen-deprived during infection in cystic fibrosis lungs, reducing the effectiveness of antibiotics.

Pseudomonas aeruginosa infects the lungs of patients with cystic fibrosis. Sputum expectorated from the lungs of patients contains low levels of oxygen, indicating that P. aeruginosa may be oxygen-deprived during infection. During in vitro growth under oxygen-limiting conditions, a P. aeruginosa reference strain increases expression of a cytochrome oxidase with a high affinity for oxygen, and of nitrate and nitrite reductases that enable it to use nitrate instead of oxygen during respiration. Here, we quantified transcription of the genes encoding these three enzymes in sputum samples from 18 infected patients, and in bacteria isolated from the sputum samples and grown in aerobic and anaerobic culture. In culture, expression of all three genes was increased by averages of 20- to 500-fold in anaerobically grown bacteria compared with those grown aerobically, although expression levels varied greatly between isolates. Expression of the same genes in sputum was similar to that of the corresponding bacteria in anaerobic culture. The isolated bacteria were less susceptible to tobramycin and ciprofloxacin, two widely used anti-pseudomonal antibiotics, when grown anaerobically than when grown aerobically. Our findings show that P. aeruginosa experiences oxygen starvation during infection in cystic fibrosis, reducing the effectiveness of antibiotic treatment.

PS-R02-3: MASSIVE TUBERCULOUS PERICARDIAL EFFUSION WITHOUT CARDIAC TAMPONADE OR MEDIASTINAL LYMPHADENOPATHY IN AN 88 YEAR OLD MALE: A CASE REPORT

Tuberculosis, although endemic in Asia does not commonly affect the heart and would usually present as constrictive pericarditis. Pericardial involvement heralds an unfavorable prognosis and remains a challenge to clinicians. Cardiac tamponade is common in patients with massive pericardial effusions. In the Philippines, it is the second most common cause of cardiac tamponade as documented in the five-year CAPTIVE Heart Study. Mediastinal lymphadenopathy occurs in all cases and denotes lymphatic spread to the pericardium. We present a rare case of an elderly patient presenting with two month history of dyspnea without fever, weight loss, or chest pain. Echocardiography revealed thickened pericardium with fibrinous strands and massive pericardial effusion of 91 mm without tamponade physiology. CT scan revealed absence of mediastinal or hilar lymphadenopathy. Sputum gene xpert was positive. Anti-tuberculosis regimen was started. Patient also then underwent anterolateral thoracotomy, pleuropericardial window with biopsy. Histopathology revealed chronic inflammatory cells, peripherally located granulomas composed of epitheloid cells and Langhans giant cells confirming tuberculosis as the cause of the pericardial effusion. Large pericardial effusion of more than 20 mm is a predictor to the development of hemodynamic instability and subsequent tamponade. In comparison to 2D Echocardiography findings of the patient with a pericardial effusion measurement of 91 mm, still no tamponade physiology was noted even if the effusion existed for more than a month. Mortality is high even in the absence of cardiac tamponade. Mediastinal lymphadenopathy does not occur in all cases of tuberculous pericardial effusion but is still a possibility as seen in the case. The threshold of chronic massive pericardial effusion in developing into tamponade may be higher than previously noted as documented in the case.

Clinico-Epidemiological Profile and Diagnostic Procedures of Tuberculosis in Children: A Retrospective Study from OPD of Tertiary Care Center in Bangladesh

Background: Childhood Tuberculosis (TB) is an important cause of mortality and morbidity, which causes a significant TB burden in developing countries. Objectives: The aim of this study was to describe clinical profile and diagnostic procedures used in outpatient department (OPD) of a tertiary care hospital. Methods: It was a retrospective study conducted in OPD of Paediatrics Department, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh. Age range of studied populations, 3 months to 18 years who presented with clinical features of TB and were later diagnosed with tuberculosis according to national guidelines for the management of tuberculosis in children, 2021 were enrolled in this study. A total of 108 diagnosed cases of Tuberculosis between August 2021 and July 2022 were enrolled in this study. Data were collected from OPD records and transferred into MS excel sheet for data processing & analysis. Results: Among 834 suspected patients, 108 patients aged 3 months to 18 years were diagnosed with tuberculosis. The highest number of cases 47 (43.51%) were found in 11 - 18 years age group. Gender distribution of the patient showed 58 (53.70%) were male and 50 (46.29%) were female. Among 108 TB cases, 44 (40.74%) were pulmonary TB and 64 (59.25%) were extrapulmonary Tb. Among 64 extrapulmonary TB cases 38 (59.37%) cases were diagnosed as TB lymphadenitis. Fever 83 (76.85%), weight loss 62 (57.4%), cough 50 (46.29%), lump in neck and axilla 38 (35.18%) were found in most of the cases. A positive Mantoux test was found in 68 (62.96%) patients. Chest x-ray findings showed patchy opacity and consolidation in 46 (42.59%) cases. Suggestive FNAC from lymph node was observed among 35 (32.4%) cases. For bacteriological confirmation sputum gene Xpert and stool xpert ultra were positive among 10 (9.25%) and 23 (21.29%) cases. Conclusion: In this study, extrapulmonary TB (EPTB) cases were more than pulmonary TB (PTB) in children. Diagnosis of tuberculosis was based on clinical suspicion supported by various investigations especially bacteriological detection with stool X pert ultra in children.

Developing a Diagnostic Model to Predict the Risk of Asthma Based on Ten Macrophage-Related Gene Signatures.

Asthma (AS) is a chronic inflammatory disease of the airway, and macrophages contribute to AS remodeling. Our study aims at screening macrophage-related gene signatures to build a risk prediction model and explore its predictive abilities in AS diagnosis. Three microarray datasets were downloaded from the GEO database. The Limma package was used to screen differentially expressed genes (DEGs) between AS and controls. The ssGSEA algorithm was used to determine immune cell proportions. The Pearson correlation coefficient was computed to select the macrophage-related DEGs. The LASSO and RFE algorithms were implemented to filter the macrophage-related DEG signatures to establish a risk prediction model. Receiver operating characteristic (ROC) curves were used to assess the diagnostic ability of the prediction model. Finally, the qPCR was used to detect the expression of selected differential genes in sputum from healthy people and asthmatic patients. We obtained 1,189 DEGs between AS and controls from the combined datasets. By evaluating immune cell proportions, macrophages showed a significant difference between the two groups, and 439 DEGs were found to be associated with macrophages. These genes were mainly enriched in the gene ontology-biological process of immune and inflammatory responses, as well as in the KEGG pathways of cytokine-cytokine receptor interaction and biosynthesis of antibiotics. Finally, 10 macrophage-related DEG signatures (EARS2, ATP2A2, COLGALT1, GART, WNT5A, AK5, ZBTB16, CCL17, ADORA3, and CXCR4) were screened as an optimized gene set to predict AS diagnosis, and they showed diagnostic abilities with AUCs of 0.968 and 0.875 in ROC curves of combined and validation datasets, respectively. The mRNA expressions of EARS2, ATP2A2, COLGALT1, and GART in the control group were higher than in AS group, while the expressions of WNT5A, AK5, ZBTB16, CCL17, ADORA3, and CXCR4 in the control group were lower than that in the AS group. We proposed a diagnostic model based on 10 macrophage-related genes to predict AS risk.\.

Diagnostic Yield of Bronchoscopic Alveolar Lavage Gene Xpert MTB/Rif in Smear Negative Clinico-Radiologically Suspected Case of Pulmonary Tuberculosis

Background: Early diagnosis of pulmonary tuberculosis is difficult but dreaded complications like cavitations, abscess, disseminations and fibrosis of lung parenchyma can be avoided. Sputum microscopy is still the most available test for diagnosis of pulmonary tuberculosis but sustainable number of active cases remains undiagnosed via this method alone. In strong clinico-radiological pulmonary tuberculosis patients who are sputum scarce or sputum microscopy and sputum Xpert MTB/Rif negative broncho-alveolar lavage may provide superior sample for Gene Xpert MTB/Rif in diagnosis of Pulmonary Tuberculosis.&#x0D; Materials and Methods: Over 2 years period, patients with strong clinico-radiological suspicion of pulmonary tuberculosis who had sputum microscopy and sputum Xpert MTB/Rif negative were included in the study. Fibro-optic bronchoscopy was done for BAL Xpert MTB/Rif test. Base line demographic, clinical and radiological data were systematically analysedby SPSS software version 17.&#x0D; Results: 64 patients were included in the study with mean age 41 ± 10.52 years. The most common symptoms were cough, sputum production and weight loss with frequency of 58 (90.6%), 31 (48.4%) and 21 (32.11%) patients respectively. The most common CT scan findings were consolidation, cavitation and tree in bud pattern. BAL Xpert MTB/Rif was positive in 11 (17.18%) patients. According to the CT findings BAL Xpert MTB/Rif was most commonly present in patients presenting with cavitary lung disease.&#x0D; Conclusion: Eleven patients had BAL gene Xpert/Rif positive out of 64 patients who were sputum scarce or had both microscopy and sputum gene Xpert/Rif negative. Bronchoscopy BAL Xpert/Rif could provide an additional diagnostic test in these groups of patients.

Role of Gene Xpert in smear negative pulmonary tuberculosis

BackgroundTuberculosis is a major health problem contributing to significant morbidity and mortality. Early diagnosis and treatment is the key for TB control. Sputum microscopy is a rapid and inexpensive test but due to low and variable sensitivity, many cases can be missed. Culture is considered to be the gold standard but is time consuming. Gene Xpert is a novel and rapid cartridge based nucleic acid amplification test (CBNAAT) that can be used for prompt diagnosis. AimTo compare the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Gene Xpert with culture in diagnosing tuberculosis in sputum smear negative patients. MethodsThe study is a prospective observational study conducted from December 2017 to January 2019 on 189 patients, who were sputum smear negative but had signs and symptoms suggestive of tuberculosis. Their respiratory samples were taken (either sputum or bronchoalveolar lavage) and sent for Gene Xpert. The results were compared with culture, which was taken as the gold standard, and diagnostic accuracy was assessed. ResultA total of 189 patients were included in the study. In 25 patients sputum was taken and in 164 patients BAL was taken (which included 22 patients in whom sputum Gene Xpert was negative but there was high clinical suspicion of tuberculosis). The sensitivity, specificity, PPV and NPV of Gene Xpert in diagnosing smear negative pulmonary tuberculosis was found to be 96.3%, 81.3%, 87.5% and 94.2% respectively. ConclusionGene Xpert can be used as a rapid diagnostic tool in patients who are sputum smear negative but have clinical features highly suggestive of tuberculosis. It additionally helps in detecting rifampicin resistance. But every Gene Xpert positive case does not necessarily mean an active disease, therefore, past history of tuberculosis along with radiological signs of disease activity are to be considered. In case of negative Gene Xpert but high clinico-radiological suspicion of TB, patients should be followed up on regular intervals, while awaiting their culture.

Sputum Gene Expression Reveals Dysregulation of Mast Cells and Basophils in Eosinophilic COPD

PurposeThe clinical and inflammatory associations of mast cells (MCs) and basophils in chronic obstructive pulmonary disease (COPD) are poorly understood. We previously developed and validated a qPCR-based MC/basophil gene signature in asthma to measure these cells in sputum samples. Here, we measured this gene signature in a COPD and control population to explore the relationship of sputum MCs/basophils to inflammatory and COPD clinical characteristics.Patients and MethodsMC/basophil signature genes (TPSAB1/TPSB2, CPA3, ENO2, GATA2, KIT, GPR56, HDC, SOCS2) were measured by qPCR in sputum from a COPD (n=96) and a non-respiratory control (n=17) population. Comparative analyses of gene expression between the COPD and the control population, and between eosinophilic COPD and non-eosinophilic COPD were tested. Logistic regression analysis and Spearman correlation were used to determine relationships of sputum MC/basophil genes to inflammatory (sputum eosinophil proportions, blood eosinophils) and clinical (age, body mass index, quality of life, lung function, past year exacerbations) characteristics of COPD.ResultsMC/basophil genes were increased in COPD versus control participants (CPA3, KIT, GATA2, HDC) and between eosinophilic-COPD and non-eosinophilic COPD (TPSB2, CPA3, HDC, SOCS2). We found all MC/basophil genes were positively intercorrelated. In COPD, MC/basophil genes were associated with eosinophilic airway inflammation (GATA2, TPSB2, CPA3, GPR56, HDC, SOCS2), blood eosinophilia (all genes) and decreased lung function (KIT, GATA2, GPR56, HDC).ConclusionWe demonstrate associations of MCs and basophils with eosinophilic inflammation and lower lung function in COPD. These findings are consistent with prior results in asthma and may represent a new tool for endotyping eosinophilic-COPD.

Pulmonary Tuberculosis mimicking a right Eventration on Radiography: The falsity of single view radiograph in the diagnosis of a precursor of pulmonary disease: A Case Report

Background: The diaphragm is one of the most important muscles of respiration in the body separating the abdomen from the thorax. Abnormalities of the diaphragm could be congenital or acquired, morphological or functional while pulmonary infection e.g. pulmonary tuberculosis, is implicated in its etiology. Case presentation: A 63-year- old man with six weeks history of cough productive of yellowish sputum. Chest X-ray showed a uniform well-circumscribed opacity in the right lower lobe abutting on or in continuum with the right diaphragm consistent with a diaphragmatic hump. Sputum Gene Xpert was positive for Mycobacterium tuberculosis. Chest CT scan revealed bilateral lymph node enlargement with hyperdense lesions in the anterior basal segment of the right lower lobe and medial bronchopulmonary segments of the right middle lobe. He was treated for 6 months with first-line anti-tuberculosis drugs. Discussion: The incidence of the diaphragmatic hump on chest radiograph worldwide and among Nigerians is unknown. The association of diaphragmatic hump with chest infection has been well document. The association of diaphragmatic hump with pulmonary tuberculosis is uncommon. Conclusion: A high index of suspicion is needed to diagnose pulmonary tuberculosis with atypical clinical and radiological presentations. Such prompt diagnosis will aid the treatment of the disease.

The sputum transcriptome better predicts COPD exacerbations after the withdrawal of inhaled corticosteroids than sputum eosinophils.

IntroductionContinuing inhaled corticosteroid (ICS) use does not benefit all patients with COPD, yet it is difficult to determine which patients may safely sustain ICS withdrawal. Although eosinophil levels can facilitate this decision, better biomarkers could improve personalised treatment decisions.MethodsWe performed transcriptional profiling of sputum to explore the molecular biology and compared the predictive value of an unbiased gene signature versus sputum eosinophils for exacerbations after ICS withdrawal in COPD patients. RNA-sequencing data of induced sputum samples from 43 COPD patients were associated with the time to exacerbation after ICS withdrawal. Expression profiles of differentially expressed genes were summarised to create gene signatures. In addition, we built a Bayesian network model to determine coregulatory networks related to the onset of COPD exacerbations after ICS withdrawal.ResultsIn multivariate analyses, we identified a gene signature (LGALS12, ALOX15, CLC, IL1RL1, CD24, EMR4P) associated with the time to first exacerbation after ICS withdrawal. The addition of this gene signature to a multiple Cox regression model explained more variance of time to exacerbations compared to a model using sputum eosinophils. The gene signature correlated with sputum eosinophil as well as macrophage cell counts. The Bayesian network model identified three coregulatory gene networks as well as sex to be related to an early versus late/nonexacerbation phenotype.ConclusionWe identified a sputum gene expression signature that exhibited a higher predictive value for predicting COPD exacerbations after ICS withdrawal than sputum eosinophilia. Future studies should investigate the utility of this signature, which might enhance personalised ICS treatment in COPD patients.

COPD patients with chronic bronchitis and higher sputum eosinophil counts show increased type‐2 and PDE4 gene expression in sputum

Chronic obstructive pulmonary disease (COPD) patients with higher eosinophil counts are associated with increased clinical response to phosphodiesterase‐4‐inhibitors (PDE4i). However, the underlying inflammatory mechanisms associated with this increased response is not yet elucidated. This post hoc analysis focused on sputum gene expression in patients with chronic bronchitis who underwent 32‐day treatment with two doses of the inhaled PDE4i CHF6001 (tanimilast) or placebo on top of triple therapy. Biological characterization and treatment effects were assessed between patients with different sputum eosinophil levels (eosinophilhigh ≥ 3%; eosinophillow < 3%) at baseline (primary samples) or at the end of the treatment of the placebo arm (validation samples). Forty‐one genes were differentially expressed in primary samples (p‐adjusted for false discovery rate < 0.05); all up‐regulated in eosinophilhigh patients and functionally enriched for type‐2 and PDE4 inflammatory processes. Eleven out of nineteen genes having immune system biological processes annotations including IL5RA, ALOX15, IL1RL1, CLC, GATA1 and PDE4D were replicated using validation samples. The expression of a number of these inflammatory mediators was reduced by tanimilast treatment, with greater effects observed in eosinophilhigh patients. These findings suggest that type‐2 and PDE4 overexpression in COPD patients with higher sputum eosinophil counts contribute to the differential clinical response to PDE4i observed in previous clinical trials.

Multicentric evaluation of BioFire FilmArray Pneumonia Panel for rapid bacteriological documentation of pneumonia

ObjectivesTo evaluate performances of the rapid multiplex PCR assay BioFire FilmArray Pneumonia Panel (FA-PP) for detection of bacterial pathogens and antibiotic resistance genes in sputum, endotracheal aspirate (ETA) and bronchoalveolar lavage (BAL) specimens. MethodsThis prospective observational study was conducted in 11 French university hospitals (July to December 2018) and assessed performance of FA-PP by comparison with routine conventional methods. ResultsA total of 515 respiratory specimens were studied, including 58 sputa, 217 ETA and 240 BAL. The FA-PP detected at least one pathogen in 384 specimens, yielding an overall positivity rate of 74.6% (384/515). Of them, 353 (68.5%) specimens were positive for typical bacteria while eight atypical bacteria and 42 resistance genes were found. While identifying most bacterial pathogens isolated by culture (374/396, 94.4%), the FA-PP detected 294 additional species in 37.7% (194/515) of specimens. The FA-PP demonstrated positive percentage agreement and negative percentage agreement values of 94.4% (95% CI 91.7%–96.5%) and 96.0% (95% CI 95.5%–96.4%), respectively, when compared with culture. Of FA-PP false-negative results, 67.6% (46/68) corresponded to bacterial species not included in the panel. At the same semi-quantification level (in DNA copies/mL for FA-PP versus in CFU/mL for culture), the concordance rate was 43.4% (142/327) for culture-positive specimens with FA-PP reporting higher semi-quantification of ≥1 log10 in 48.6% (159/327) of cases. Interestingly, 90.1% of detected bacteria with ≥106 DNA copies/mL grew significantly in culture. ConclusionsFA-PP is a simple and rapid molecular test that could complement routine conventional methods for improvement of diagnosis accuracy of pneumonia.

Prevalence of culture positive Tuberculosis and utility of a clinical diagnostic tool for the diagnosis of Tuberculosis among HIV Infected Children attending HIV/AIDS Care and Treatment in Dodoma Municipality, Central Tanzania

ObjectiveTo evaluate the performance of the modified Keith Edwards TB clinical diagnostic tool among HIV infected children. MethodsCross sectional study of 252 HIV infected children < 15 years old suspected with TB at four HIV/AIDS Care Clinics in Dodoma, Tanzania from November 2018 - March 2019. The modified Keith Edwards TB clinical diagnostic tool was compared to gastric aspirates, lymphnode aspirates or sputum gene x-pert MTB/RIF and TB culture. Sensitivity, specificity, negative and positive predictive value of the clinical tool were determined. Data was analyzed using SPSS version 25. ResultsOut of 252 children evaluated, 13.5% (34/252) had TB using the clinical diagnostic tool and 5.2% (13/252) had culture positive TB. The sensitivity of the clinical tool was 76.9%, specificity of 90%. Culture positive TB predictors were lymphadenopathy (AOR 13.74, 95%CI (3.86 – 48.86) p value < 0.001), weight loss (AOR 3.19,95%CI (1.38 – 7.36) p value 0.007), and difficulty breathing (AOR 7.25, 95%CI (1.54 – 34.16) p value 0.012). ConclusionThe utility of the modified Keith Edwards clinical diagnostic tool for Tuberculosis diagnosis among HIV infected children is limited, calling for further validation. HIV infected children with lymphadenopathy, failure to thrive and difficulty in breathing are at high risk of Tuberculosis.

Deteksi gen pneumolysin (ply) Streptococcus pneumoniae pada Sampel Klinis Usia Lanjut secara Kultur dan PCR

Latar Belakang : Bakteri Streptococcus pneumoniae, merupakan bakteri gram positif flora normal namun dapat menjadi bakteri pathogen penyebab pneumonia apabila sistem kekebalan tubuh menurun. sekitar 180 pneumonia komuniti dengan angka kematian antara 20 - 35 %. Pneumonia komuniti menduduki peringkat keempat dan sepuluh penyakit terbanyak yang dirawat per tahun. Populasi yang rentan terserang pneumonia adalah anak-anak usia kurang dari 2 tahun, usia lanjut. Tujuan: mendeteksi gen ply pada sputum penderita suspek pneumonia usia lanjut sebagai faktor virulensi dari S.pneumoniae secara kultur dan PCR. Metode: Metode penelitian ini merupakan penelitian deskriptif untuk mendeteksi adanya faktor virulensi gen ply S.pneumoniae pada sputum penderita suspek pneumonia pada lanjut usia dengan metode kultur dan PCR. Hasil Penelitian : secara konvensional (metode kultur) diperoleh 1 sampel dari 56 sampel yang positif bakteri gram positif S. pneumoniae pada metode molekuler (PCR) terdapat 7 sampel positif bakteri S.pneumoniae dari 56 sampel.. Kesimpulan : Dalam mendeteksi gen ply pada sputum penderita lansia suspek pneumonia sebagai faktor virulensi dari bakteri S.pneumoniae secara kultur terdeteksi 1 sampel positif dari 56 sampel dan dengan metode PCR terdeteksi 7 sampel positif dari 56 sampel. Dalam mendeteksi gen ply dengan menggunakan kultur dan PCR dapat dikatakan bahwa metode PCR lebih akurat, cepat dan tepat dalam mendeteksi gen ply S.pneumonia pada sampel sputum penderita suspek pneumonia usia lanjut.&#x0D;

Identification of novel epigenetic abnormalities as sputum biomarkers for lung cancer risk among smokers and COPD patients

ObjectivesSmoking is a common risk factor for chronic obstructive pulmonary disease (COPD) and lung cancer. Although COPD patients have higher risk of lung cancer compared to non-COPD smokers, the molecular links between these diseases are not well-defined. This study aims to identify genes that are downregulated by cigarette smoke and commonly repressed in COPD and lung cancer. Materials and methodsPrimary human airway epithelial cells (HAEC) were exposed to cigarette-smoke-extract (CSE) for 10-weeks and significantly suppressed genes were identified by transcriptome array. Epigenetic abnormalities of these genes in lung adenocarcinoma (LUAD) from patients with or without COPD were determined using genome-wide and gene-specific assays and by in vitro treatment of cell lines with trichostatin-A or 5-aza-2-deoxycytidine. ResultsThe ten most commonly downregulated genes following chronic CSE exposure of HAEC and show promoter hypermethylation in LUAD were selected. Among these, expression of CCNA1, SNCA, and ZNF549 was significantly reduced in lung tissues from COPD compared with non-COPD cases while expression of CCNA1 and SNCA was further downregulated in tumors with COPD. The promoter regions of all three genes were hypermethylated in LUAD but not normal or COPD lungs. The reduced expression and aberrant promoter hypermethylation of these genes in LUAD were independently validated using data from the Cancer Genome Atlas project. Importantly, SNCA and ZNF549 methylation detected in sputum DNA from LUAD (52% and 38%) cases were more prevalent compared to cancer-free smokers (26% and 15%), respectively (p < 0.02). ConclusionsOur data show that suppression of CCNA1, SNCA, and ZNF549 in lung cancer and COPD occurs with or without promoter hypermethylation, respectively. Detecting methylation of these and previously identified genes in sputum of cancer-free smokers may serve as non-invasive biomarkers for early detection of lung cancer among high risk smokers including COPD patients.