Bronchial Alveolar Lavage Samples Research Articles

Th1/17 Cells Are Expanded in Bronchial Alveolar Lavage (BAL) Fluid from Leukemia Patients with Checkpoint Inhibitor-Induced Pneumonitis

A number of clinical trials are evaluating immune checkpoint inhibitors in patients with AML/MDS with encouraging results (Daver N et al, EHA 2017; Garcia-Manero et al, EHA 2017). Approximately 5-10% of patients with leukemia treated with checkpoint inhibitors had immune related adverse event (irAE) of pneumonitis, one of the life-threatening irAEs. Increased awareness, distinction from infectious pneumonia, and early intervention are critical in the management of pneumonitis-irAEs in patients with leukemia. Especially in the setting of acute leukemia the ability to distinguish immune mediated pneumonitis from infectious pneumonia is critical to determine whether or not the addition of high-dose steroids is warranted. The mechanisms of pneumonitis-irAE, the most important step for risk stratification and early detection remain elusive.MethodsWe collected bronchial alveolar lavage (BAL) fluid from seven leukemia patients (6 AML/MDs and 1 Richter transformation) who received checkpoint inhibitor based therapy on clinical trials, and subsequently developed respiratory symptoms and had a bronchoscopy. As a control, we collected BAL fluid from two AML/MDS patients who had never received checkpoint inhibitor therapy. We stained BAL CD4 T cell with lineage specific surface markers using the following antibodies; anti-CD4 BUV396, anti-CXCR3 BV 421, anti-CD45RA BV786, anti-CD3 PerCP, anti-CCR6 FITC, anti-CD25 PE, anti-PD-1 PE-Dazzle, anti-CXCR5 APC, and anti-CD127 APC 700. BAL CD4 T cell subsets, including Th1, 2, 17, follicular helper T, and regulatory T cells, were enumerated and the proportion and profile of the CD4 T cell subsets within the total lymphocytes were compared between pneumonitis group and control group.ResultPatient demographics are described in Figure 1A. Mean age of the patients with pneumonitis-irAE was 56. Most of them were on nivolumab in combination with chemotherapy/hypomethylator therapy. The patients developed pneumonitis 52.7 ± 77.8 days (mean ± DS) after the first exposure to a checkpoint inhibitor. Compared to the control group, lymphocytes were expanded in pneumonitis group (% lymphocytes, disease versus control: 32.3% vs. 19.0%). This observation led us to further characterize the lymphocytes in available BAL samples, especially CD4 T cells given their central role in immune efficacy and toxicity. Interestingly, Th1/17 hybrid cells (CXCR3hi CCR6hi), known to be pathogenic in autoimmune diseases, were expanded in the pneumonitis group compared to the control (8.24 % vs 3.19 %) (Figure 1B and C) These Th1/17 hybrid cells express high level of PD-1, suggesting their hyperactive status. Although premature, there was a trend that regulatory T cells were decreased in pneumonitis group (Data not shown). Surface staining of CD4 T cells in BAL fluid revealed that both disease and control had no follicular helper T cells, the CD4 T cell subset governing humoral immunity. Consistently, B cells were barely detected in BAL in both groups (Data not shown).ConclusionThese results suggest the possibility of increased Th1/17 hybrid cells with decreased Tregs as a biomarker of pneumonitis. The high PD-1 expression on Th1/17 hybrid cells suggests activation (or exhaustion) in the lungs in patients with irAEs. Further investigation of Th1/17 hybrid cells and correlation with peripheral blood may provide an insight into the mechanisms of pneumonitis-irAEs and biomarkers for early detection. In parallel, decreased Treg in the BAL may indicate an increased susceptibility to immune activation and inflammation seen in irAEs. [Display omitted] DisclosuresKantarjian:Novartis: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding. Sharma:Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Evelo: Consultancy, Other: stock; Astellas: Consultancy; EMD Serono: Consultancy; Consetellation: Other: stock; Amgen: Consultancy; Kite Pharma: Consultancy, Other: stock; Neon: Consultancy, Other: stock; BMS: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy. Daver:Otsuka America Pharmaceutical, Inc.: Consultancy; Karyopharm: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Kiromic: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Pfizer Inc.: Consultancy, Research Funding; Jazz: Consultancy; Incyte Corporation: Honoraria, Research Funding.

Utility of GeneXpert in analysis of bronchoalveolar lavage samples from patients with suspected tuberculosis in an intermediate-burden setting

To explore the clinical role of GeneXpert in managing pulmonary tuberculosis (TB) in an intermediate burden city. Sputum acid-fast-bacilli (AFB) smear negative patients underwent bronchoscopy for bronchial alveolar lavage (BAL). Fluids collected were examined for AFB smear, TB culture, TB polymerase chain reaction (PCR) (Cobas Taqman) and for GeneXpert. From October 2015 to February 2017, 227 BAL samples were collected. Cough and haemoptysis were the presenting symptoms in 70.0% and 37.4%, respectively. Apical shadows on chest X-rays (CXR) and apical cavitations on computed tomography (CT) were commoner in GeneXpert positive cases (p = 0.01 and 0.02, respectively). Sensitivity and specificity of GeneXpert for TB diagnosis was 80% and 98% respectively. Positive and negative predictive value of the test was 92.3 and 95.1%, respectively. There were 9 false negative GeneXpert samples (8 were Cobas Taqman TB PCR negative): 6 were diagnosed by BAL culture, 2 by biopsy and one by Cobas Taqman TB PCR. There were 3 false positive cases with negative culture; 2 were put on empirical treatment with favourable clinical responses, while one defaulted follow-p. GeneXpert in BAL samples has high sensitivity and specificity. It enabled timely initiation of anti-TB treatment in clinical suspicious cases.

Evaluation of a Multiplex PCR Assay with Molecular Beacon Probes to Rapidly Detect Bacterial Pathogens Directly in Bronchial Alveolar Lavage (BAL) Samples from Patients with Hospital-Acquired Pneumonia (HAP)

BackgroundBacterial pneumonia is a common complication in hospitalized patients and it is associated with high morbidity and mortality. Standard culture-based methods may take 2–3 days to identify the etiologies of HAP, leading to delays in appropriate therapy. A rapid molecular assay that could diagnose the etiology of bacterial pneumonia directly from BAL samples within a few hours could facilitate faster and more directed administration of antimicrobial therapy.MethodsBAL samples were collected from hospitalized patients with suspected pneumonia, including ventilated patients, from December 2016 through April 2017. Genomic DNA was isolated from BAL samples using NucliSENS® easyMAG®. A panel of target-specific molecular beacon probes in a real time PCR assay (MB-PCR) was used to identify the following pathogens: universal bacterial Identification (16S rRNA), E. coli (uidA), K. pneumoniae (gapA), S. aureus (spa), P. aeruginosa (rpsL), A. baumannii (Ab-ITS) and the following resistance determinants: ESBLs (CTX-M, TEM and SHV), carbapenemases (NDM, VIM, IMP, OXA-48 and KPC) and mecA. The results of MB-PCR were then compared with quantitative culture results performed by the clinical microbiological lab.ResultsWe evaluated 53 BAL samples to identify the bacterial pathogen and key resistance determinants. Thirty-one samples yielded growth of ≥1 × 104 CFU/mL of bacteria by quantitative culture. The bacterial identification using MB-PCR for 16S rRNA correctly identified the presence of bacteria in all 31 samples (100% sensitivity). The MB-PCR identified P. aeruginosa (n = 5), S. aureus (n = 5), E. coli (n = 1), A. baumannii (n = 1), and K. pneumoniae (n = 1) in BAL samples that yielded ≥1 × 104 CFU/mL of the same pathogen by culture (100% sensitivity). The MB-PCR also identified blaTEM-harboring E. coli that grew ampicillin-resistant E. coli by culture. The specificity of the16S rRNA probe was 70%, as 7/53 BAL were false positive, whereas the specificity for the MB-PCR was 100% for P. aeruginosa, S. aureus, E. coli, and A. baumannii, and 98% for K. pneumoniae.ConclusionMultiplex MB-PCR assay is a rapid, sensitive and specific tool for detection of common bacterial causes of nosocomial pneumonia and important resistance determinants directly from BAL samples.Disclosures M. Satlin, Hardy Diagnostics: Investigator, Research support; S. G. Jenkins, Cormedix: Consultant, Consulting fee; Bayer: Consultant, Consulting fee Merck: Grant Investigator and Scientific Advisor, Research grant; T. J. Walsh, The Medicines Company: Consultant and Investigator, Consulting fee and Research grantAstellas: Consultant and Investigator, Consulting fee and Research grant Allergan: Consultant and Investigator, Consulting fee and Research grant Merck: Consultant and Investigator, Consulting fee and Research grant

Fulminant Invasive Pulmonary Aspergillosis After a Near-Drowning Accident in an Immunocompetent Patient.

To report on invasive aspergillosis infection in an immunocompetent adult after a near-drowning event, which allowed this pathogen to easily gain access to the human respiratory system and result in rapid, severe infection. A 51-year-old female developed severe pneumonia after a near-drowning accident. Two days after admission, a bronchial alveolar lavage (BAL) was performed and was positive for Aspergillus fumigatus. After a 30-day hospital course, multiple antifungals, and various routes of administration, the patient expired. Pneumonia is particularly common because of the aspiration of contaminated water. Whereas pneumococci, staphylococci, and Gram-negative bacteria are all common pathogens for this type of infection, fungi such as Aspergillus spp can also be involved and may be life threatening. Typically, these cases are reported in individuals with an immunodeficiency such as from receipt of myelosuppressive chemotherapy, bone marrow transplants, or lung transplants. Despite initiation of an appropriate empirical antifungal regimen, the rapid recovery of A fumigatus from pulmonary alveolar lavage and BAL samples as well as extremely elevated levels of galactomannan and (1→3)-β-D glucan may have indicated an invasive fungal infection (IFI). IFIs are uncommon in immunocompetent adults, but in the event of a near-drowning accident, environmental fungi can gain access to the human respiratory system and result in rapid, severe infection. Based on this case and the others described, it appears that near-drowning patients need an early initial evaluation for IFI.

Histochemical and cellular changes accompanying the appearance of lung fibrosis in an experimental mouse model for Hermansky Pudlak syndrome

Hermansky Pudlak syndrome (HPS) is a heterogeneous recessive genetic disease with a tendency to develop lung fibrosis with aging. A mouse strain with two mutant HPS genes affecting separate vesicle trafficking pathways, C57BL/6-Hps1 ep-Ap3b1 pe, exhibits severe lung abnormalities at young ages, including enlarged alveolar type II (ATII) cells with giant lamellar bodies and foamy alveolar macrophages (AMs), which are readily identified histologically. In this study, the appearance of lung fibrosis in older animals was studied using classical histological and biochemical methods. The HPS double mutant mice, but not Chediak Higashi syndrome (C57BL/6-Lyst bg-J-J, CHS) or C57BL/6J black control (WT) mice, were found to develop lung fibrosis at about 17 months of age using Masson trichrome staining, which was confirmed by hydroxyproline analysis. TGF β1 levels were elevated in bronchial alveolar lavage samples at all ages tested in the double mutant, but not WT or CHS mice, indicative of a prefibrotic condition in this experimental strain; and AMs were highly positive for this cytokine using immunohistochemistry staining. Prosurfactant protein C staining for ATII cells showed redistribution and dysmorphism of these cells with aging, but there was no evidence for epithelial-mesenchymal transition of ATII cells by dual staining for prosurfactant C protein and α-smooth muscle actin. This investigation showed that the HPS double mutant mouse strain develops interstitial pneumonia (HPSIP) past 1 year of age, which may be initiated by abnormal ATII cells and exacerbated by AM activation. With prominent prefibrotic abnormalities, this double mutant may serve as a model for interventive therapy in HPS.

Pneumonia Caused byShigella sonneiin Man Returned from India

Pneumonia Caused by<i>Shigella sonnei</i>in Man Returned from India

Transcriptome Changes in Mycoplasma hyopneumoniae during Infection

Mycoplasma hyopneumoniae causes swine pneumonia and contributes significantly to the porcine respiratory disease complex. The mechanisms of pathogenesis are difficult to address, since there is a lack of genetic tools, but microarrays are available and can be used to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Mycoplasmas were collected from bronchial alveolar lavage samples and compared to broth-grown cells using microarrays. Bronchial alveolar lavage was performed on pigs 28 days postinfection, and mycoplasmas were isolated by differential centrifugation. Mycoplasma RNA-enriched preparations were then obtained from total RNA by subtracting eucaryotic ribosomal and messenger RNAs. Labeled cDNAs were generated with mycoplasma open reading frame-specific primers. Nine biological replicates were analyzed. During lung infection, our analysis indicated that 79 M. hyopneumoniae genes were differentially expressed (P < 0.01), at a false-discovery rate of <2.7%. Of the down-regulated genes, 28 of 46 (61%) lacked an assigned function, in comparison to 21 of 33 (63%) of up-regulated genes. Four down-regulated genes and two up-regulated genes encoded putative lipoproteins. secA (mhp295) (P = 0.003) and two glycerol transport permease genes (potA [mhp380; P = 0.006] and ugpA [mhp381; P = 0.003]) were up-regulated in vivo. Elongation factor EF-G (fusA [mhp083]) (P = 0.002), RNA polymerase beta chain (rpoC [mhp635]) (P = 0.003), adenylate kinase (adk [mhp208]) (P = 0.001), prolyl aminoacyl tRNA synthetase (proS [mhp397]) (P = 0.009), and cysteinyl-tRNA synthetase (cysS [mhp661]) (P < 0.001) were down-regulated in vivo.

Inhalation efficacy of RFI-641 in an African green monkey model of RSV infection.

Human respiratory syncytial virus (RSV) is a major cause of acute upper and lower respiratory tract infections. RFI-641 is a novel RSV fusion inhibitor with potent in vitro activity. In vivo efficacy of RFI was determined in an African green monkey model of RSV infection involving prophylactic and therapeutic administration by inhalation exposure. Inhalation was with an RFI-641 nebulizer reservoir concentration of 15 mg/ml for 15 minutes (short exposure) or 2 hours (long exposure). Efficacy and RFI-641 exposure was determined by collection of throat swabs, nasal washes and bronchial alveolar lavage (BAL) on selected days. The short-exposure group (15 minutes) exhibited no effect on the nasal, throat or BAL samples. The throat and nasal samples for the long-exposure group failed to show a consistent reduction in viral titers. RFI-641 2 hours exposure-treated monkeys showed a statistically significantly log reduction for BAL samples of 0.73-1.34 PFU/ml (P-value 0.003) over all the sampling days. Analysis indicates that the long-exposure group titer was lower than the control titer on day 7 and when averaged across days. The results of this study demonstrate the ability of RFI-641 to reduce the viral load of RSV after inhalation exposure in the primate model of respiratory infection.