DNA In Bronchoalveolar Lavage Fluid Research Articles

Mitochondrial DNA in bronchoalveolar lavage fluid is associated with the prognosis of idiopathic pulmonary fibrosis: a single cohort study

BackgroundExtracellular mitochondrial DNA (mtDNA) is released from damaged cells and increases in the serum and bronchoalveolar lavage fluid (BALF) of idiopathic pulmonary fibrosis (IPF) patients. While increased levels of serum mtDNA have been reported to be linked to disease progression and the future development of acute exacerbation (AE) of IPF (AE-IPF), the clinical significance of mtDNA in BALF (BALF-mtDNA) remains unclear. We investigated the relationships between BALF-mtDNA levels and other clinical variables and prognosis in IPF.MethodsExtracellular mtDNA levels in BALF samples collected from IPF patients were determined using droplet-digital PCR. Levels of extracellular nucleolar DNA in BALF (BALF-nucDNA) were also determined as a marker for simple cell collapse. Patient characteristics and survival information were retrospectively reviewed.ResultsmtDNA levels in serum and BALF did not correlate with each other. In 27 patients with paired BALF samples obtained in a stable state and at the time of AE diagnosis, BALF-mtDNA levels were significantly increased at the time of AE. Elevated BALF-mtDNA levels were associated with inflammation or disordered pulmonary function in a stable state (n = 90), while being associated with age and BALF-neutrophils at the time of AE (n = 38). BALF-mtDNA ≥ 4234.3 copies/µL in a stable state (median survival time (MST): 42.4 vs. 79.6 months, p < 0.001) and ≥ 11,194.3 copies/µL at the time of AE (MST: 2.6 vs. 20.0 months, p = 0.03) were associated with shorter survival after BALF collection, even after adjusting for other known prognostic factors. On the other hand, BALF-nucDNA showed different trends in correlation with other clinical variables and did not show any significant association with survival time.ConclusionsElevated BALF-mtDNA was associated with a poor prognosis in both IPF and AE-IPF. Of note, at the time of AE, it sharply distinguished survivors from non-survivors. Given the trends shown by analyses for BALF-nucDNA, the elevation of BALF-mtDNA might not simply reflect the impact of cell collapse. Further studies are required to explore the underlying mechanisms and clinical applications of BALF-mtDNA in IPF.

HHV-6B detection and host gene expression implicate HHV-6B as pulmonary pathogen after hematopoietic cell transplant

Limited understanding of the immunopathogenesis of human herpesvirus 6B (HHV-6B) has prevented its acceptance as a pulmonary pathogen after hematopoietic cell transplant (HCT). In this prospective multicenter study of patients undergoing bronchoalveolar lavage (BAL) for pneumonia after allogeneic HCT, we test blood and BAL fluid (BALF) for HHV-6B DNA and mRNA transcripts associated with lytic infection and perform RNA-seq on paired blood. Among 116 participants, HHV-6B DNA is detected in 37% of BALs, 49% of which also have HHV-6B mRNA detection. We establish HHV-6B DNA viral load thresholds in BALF that are highly predictive of HHV-6B mRNA detection and associated with increased risk for overall mortality and death from respiratory failure. Participants with HHV-6B DNA in BALF exhibit distinct host gene expression signatures, notable for enriched interferon signaling pathways in participants clinically diagnosed with idiopathic pneumonia. These data implicate HHV-6B as a pulmonary pathogen after allogeneic HCT.

94P Harnessing circulating tumor DNA in bronchoalveolar lavage fluid for precise molecular diagnosis of NSCLC

94P Harnessing circulating tumor DNA in bronchoalveolar lavage fluid for precise molecular diagnosis of NSCLC

Qingjin Huatan decoction attenuates lipopolysaccharide-induced acute lung injury in mice by controlling platelet-associated formation of neutrophil extracellular traps

Abstract Background Acute lung injury (ALI) is a severe and life-threatening lung inflammation with high morbidity and mortality, underscoring the importance to develop effective drugs. Qingjin Huatan decoction (QJHTD), as a classic ancient prescription, has been widely used for treating respiratory diseases. However, the role and mechanism of QJHTD against ALI remain unclear. Objective This study aimed to explore the therapeutic effect of QJHTD on lipopolysaccharide (LPS)-induced ALI in mice and uncover its mechanism. Methods The therapeutic effect of QJHTD on LPS-induced ALI in mice was evaluated by the histopathological changes in the lung tissue, the lung wet/dry weight ratio, and the levels of inflammatory cytokines and thrombin-antithrombin complexes. Transcriptomics was used to predict the mechanism of QJHTD in treating ALI. The expression levels of citrullinated histone 3 in the lung tissue, the content of cell-free DNA in the bronchoalveolar lavage fluid (BALF), and the platelet-associated formation of neutrophil extracellular traps (NETs) in vitro were determined. Results Qingjin Huatan decoction exerted protective effect against LPS-induced ALI by suppressing interstitial edema, maintaining the alveolar-capillary barrier, inhibiting the infiltration of neutrophils and platelets in the lung tissue, and lowering the levels of tumor necrosis factor α, interleukin 1β, interleukin 6, and thrombin-antithrombin complexes in BALF. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the formation of NETs was the main regulatory pathway for QJHTD against ALI. Qingjin Huatan decoction could treat ALI by inhibiting the release of NETs via reducing the content of citrullinated histone 3 in lung tissue and cell-free DNA in BALF in vivo, and suppressing the NETs formation induced by LPS-stimulated platelets under flow and static conditions in vitro. The formation of NETs was considered to bridge the interactions between neutrophils and platelets. Conclusions This research demonstrated the effects of QJHTD in treating ALI and provided new insights for clarifying the complex regulation of neutrophils, platelets, and NETs in ALI.

Poor accuracy of single serological IgM tests in children with suspected acute Mycoplasma pneumoniae infection in Guangzhou, China.

Introduction. Early and accurate diagnosis of Mycoplasma pneumoniae (MP) infection of children with pneumonia is at the core of treatment in clinical practice.Gap Statement. Serological immunoglobulin M (IgM) tests for MP infection of children in south China have been rarely described.Aim. To assess the diagnostic performance and clinical application of serodiagnosis of MP infection in paediatric pneumonia patients.Methodology. Serum samples from 144 children diagnosed with MP pneumonia were subjected to a particle agglutination (PA)-based IgM assay. Meanwhile, we used an established suspension array as the reference standard method for the detection of MP DNA in bronchoalveolar lavage fluid (BALF) from all patients to assess the reliability of serological assays.Results. When running immunological testing in single serum samples, 80.6 %(79/98) of cases were diagnosed with MP infection, whereas only 55 (56.1 %) cases were positive in MP DNA analysis. Furthermore, single serum tests for IgM during acute MP infection resulted in 85.5 % (47/55) sensitivity and 25.6 % (11/43) specificity. Nevertheless, immunological testing and MP DNA analysis yielded the same results when paired sera were available for MP IgM antibody testing.Conclusion. Paired serological IgM assays are necessary for the determination of an acute MP infection, whereas single serological IgM testing is unreliable. Moreover, even a short interval of two MP serological tests works well.

2094. A prospective multicenter study of HHV-6B genomic DNA and gene transcription in paired bronchoalveolar lavage fluid and blood from HCT recipients

Abstract Background We previously demonstrated frequent detection of HHV-6B DNA in bronchoalveolar lavage fluid (BALF) and its positive association with mortality in HCT recipients from 1992-2015 with lower respiratory tract disease (LRTD). Whether these findings remain pertinent in contemporary patients, the additive value of testing for viral gene transcription, and the correlation of HHV-6 detection in blood and BALF, are unknown. Methods We conducted a prospective study of allogeneic HCT recipients undergoing BAL for LRTD within 120 days of HCT at three cancer centers from 2015-2019. We collected and tested paired blood and BALF for HHV-6B DNA by qPCR and HHV-6B mRNA (U38 and U90 gene transcripts) among DNA positive samples using RT-qPCR. We described the detection of HHV-6B DNA and mRNA in blood and BALF, generated receiver operating characteristic (ROC) curves to determine the ability of BALF HHV-6B DNA detection to predict HHV-6B mRNA detection, and analyzed the association of HHV-6B DNA detection with mortality. Results We enrolled 116 allogeneic HCT recipients who underwent 125 BALs. HHV-6B DNA was detected in 45 of 122 BALF (37%) compared to 19 of 124 (15%) plasma samples. Among the 45 BALF samples with HHV-6B DNA detected, either HHV-6B mRNA transcript was detected in 22 (49%) (Figure 1). BALF HHV-6B DNA ≥ 218 copies/ml had an area under the curve of 0.93 for predicting detection of BALF viral mRNA (Figure 2). In turn, patients with BALF HHV-6B DNA ≥ 218 copies/mL had increased risk for mortality and death due to LRTD within 60 days after the BAL (Figure 3). This association remained after adjustment for age, oxygen use, and steroid use at the time of BAL in a multivariable Cox model (Figure 3). Conclusion HHV-6B was detected more frequently in BALF than plasma, suggesting compartment-specific reactivation. BALF HHV-6B DNA ≥ 218 copies/mL had high sensitivity and specificity for detection of viral gene transcription in BALF and was associated with increased mortality; this viral load is strikingly similar to the BALF viral load threshold of 251 copies/mL associated with mortality in our prior retrospective study. Together, these data suggest transcriptionally active HHV-6B is a clinically impactful pulmonary pathogen in contemporary HCT recipients. Disclosures Joshua A. Hill, MD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|Covance/CSL: Advisor/Consultant|CRISPR: Advisor/Consultant|Deverra: Grant/Research Support|Gilead: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|Merck: Grant/Research Support|Octapharma: Advisor/Consultant|OptumHealth: Advisor/Consultant|Oxford Immunotec: Grant/Research Support|Pfizer: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant Alpana waghmare, MD, Allovir: Grant/Research Support|Ansun BioPharma: Grant/Research Support|Kyorin Pharmaceutical: Advisor/Consultant|Pfizer: Grant/Research Support|Vir/GSK: Grant/Research Support Geoffrey Hill, M.D., FRACP, FRCPA, Applied Molecular Transport: Grant/Research Support|Compass Therapeutics: Grant/Research Support|Generon Corporation: Advisor/Consultant|Heat Biologics: Grant/Research Support|iTeos Therapeutics: Advisor/Consultant|iTeos Therapeutics: Grant/Research Support|Laevoroc Oncology: Grant/Research Support|NapaJen Pharma: Advisor/Consultant|Neoleukin Therapeutics: Advisor/Consultant|Serplus Technology: Grant/Research Support|Syndax Pharmaceuticals: Grant/Research Support Danielle M. Zerr, MD MPH, AlloVir: Advisor/Consultant Sanjeet S. Dadwal, MD, FACP, FIDSA, AlloVir: Advisor/Consultant|AlloVir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|Aseptiscope: Advisor/Consultant|Aseptiscope: Stocks/Bonds|Astellas: Speaker's Bureau|Cidara: Advisor/Consultant|Gilead: Grant/Research Support|Karius: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Speaker's Bureau|Takeda: Speaker's Bureau Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Amazon: Grant/Research Support|Ansun Biopharma: Grant/Research Support|EvrysBio: Advisor/Consultant|Gates Ventures: Grant/Research Support|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Helocyte: Advisor/Consultant|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Kyorin Pharmaceuticals: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Regeneron: Grant/Research Support|ReViral: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Grant/Research Support|Vir Biotechnology: Advisor/Consultant|Vir Biotechnology: Grant/Research Support.

Chronic Lung Allograft Dysfunction Is Associated with Increased Levels of Cell-Free Mitochondrial DNA in Bronchoalveolar Lavage Fluid of Lung Transplant Recipients.

Chronic Lung Allograft Dysfunction (CLAD) is a life-threatening complication that limits the long-term survival of lung transplantation patients. Early diagnosis remains the basis of efficient management of CLAD, making the need for distinctive biomarkers critical. This explorative study aimed to investigate the predictive power of mitochondrial DNA (mtDNA) derived from bronchoalveolar lavages (BAL) to detect CLAD. The study included 106 lung transplant recipients and analyzed 286 BAL samples for cell count, cell differentiation, and inflammatory and mitochondrial biomarkers, including mtDNA. A receiver operating curve analysis of mtDNA levels was used to assess its ability to detect CLAD. The results revealed a discriminatory pro-inflammatory cytokine profile in the BAL fluid of CLAD patients. The concentration of mtDNA increased in step with each CLAD stage, reaching its highest concentration in stage 4, and correlated significantly with decreasing FEV1. The receiver operating curve analysis of mtDNA in BAL revealed a moderate prediction of CLAD when all stages were grouped together (AUROC 0.75, p-value < 0.0001). This study has found the concentration mtDNA in BAL to be a potential predictor for the early detection of CLAD and the differentiation of different CLAD stages, independent of the underlying pathology.

Mycoplasma Pneumoniae Infection and Persistent Wheezing in Young Children: A Retrospective Case-Control Study.

BackgroundTo investigate the clinical characteristics of children with persistent wheezing (PW) with Mycoplasma pneumoniae (MP) DNA in bronchoalveolar lavage fluid (BALF).MethodsThis retrospective case-control study included consecutive admitted children under 3 years of age who were diagnosed with PW and had MP DNA detected in BALF. Patients with mycoplasma pneumoniae pneumonia (MPP) and foreign-body aspiration (FBA) were enrolled as controls. The clinical characteristics of the groups were compared.ResultsDuring the study period, there were 89 patients diagnosed with PW without structural anomalies of the conductive airways, and 30 of these patients (33.7%, 30/89) with MP DNA detected in the BALF were selected as the study group. We included 44 patients with MPP and 44 patients with FBA as controls. Patients with MPP were older and had a higher occurrence of fever and C-reactive protein (CRP) than patients with PW (all P < 0.001). The median MP DNA copy number in patients with MPP was higher than that of patients with PW (P = 0.004). The median level of MP IgG in patients with PW was lower than that of patients with MPP and higher than that of patients with FBA (all P < 0.001). MP DNA copy number positively correlated with age (r = 0.392, P = 0.001) and CRP (r = 0.235, P = 0.048).ConclusionsOur study reveals that MP was highly detected in the BALF of PW patients. In addition, young patients with a low load of MP infection showed lower amounts of antibody, and a weak inflammatory response might be associated with PW.

MA25.07 Genomic Profiling of Lung Adenocarcinoma by Targeted NGS Using Extracellular Vesicle-Derived DNA in Bronchoalveolar Lavage Fluid

MA25.07 Genomic Profiling of Lung Adenocarcinoma by Targeted NGS Using Extracellular Vesicle-Derived DNA in Bronchoalveolar Lavage Fluid

P2.01-62 Extracellular Vesicle-Based Egfr Genotyping in Bronchoalveolar Lavage Fluid from Non-Small Cell Lung Cancer Patients

Liquid biopsies for EGFR genotyping using plasma cell-free DNA (cfDNA) have limited value due to low sensitivity. Extracellular vesicles (EV) have been proven to contain mutant EGFR DNA in bronchoalveolar lavage fluid (BALF) from non-small cell lung cancer (NSCLC) patients. As an alternative to plasma liquid biopsies using cfDNA, we investigated the feasibility of EV-based liquid biopsy for EGFR genotyping using BALF from NSCLC patients, prospectively. EV DNA was extracted from the BALF of NSCLC patients with confirmed tissue/cytology-based EGFR genotyping at initial diagnostic work-up stage. (N=137) The number of patients with stage I, II, III, and IV are 37 (27%), 6 (4.4%), 23 (16.8%), 71 (51.8%), respectively. The patients were selected by favorable demographic data for EGFR mutation. EGFR mutation testing was done by peptide nucleic acid (PNA) clamping-assisted fluorescence melting curve analysis. The overall sensitivity and specificity of EGFR genotyping using BALF EV DNA were 69.5% and 83.3% and the concordance rate was 77.4%. (Kappa=0.534, P=0.000) The sensitivity was significantly increased along with the stage (42.1% in stage I, II, 60.0% in stage III, and 100% in stage IV). Especially, in stage IV disease, BALF EV-based EGFR typing did not miss all of tissue-proven 30 EGFR mutant cases and detected 9 additional mutant cases. Detection of EGFR mutation becomes significantly more sensitive as T stage increases. (T1 = 43.8%, T2 = 75%, T3 and T4 = 100%) Turn-around time (TAT) for EGFR genotyping using BALF EV DNA was 1-2 days, while it takes usually 2 weeks for tissue typing. BALF EV-based EGFR genotyping is a novel and highly promising method with rapid TAT and incredible accuracy in stage IV NSCLC patients. Further research in early stage disease is necessary and the matter of over-detection should be clinically validated by evaluating EGFR-TKI drug response.

Preventive Effects of Carnosine on Lipopolysaccharide-induced Lung Injury

Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute lung injury, which involves neutrophilic inflammation and pulmonary cell death. Reactive oxygen species (ROS) play important roles in ARDS development. New compounds for inhibiting the onset and progression of ARDS are required. Carnosine (β-alanyl-L-histidine) is a small di-peptide with numerous activities, including antioxidant effects, metal chelation, proton buffering capacity and the inhibition of protein carbonylation and glycoxidation. We have examined the preventive effects of carnosine on tissue injury, oedema and inflammation in a murine model for ARDS. Oral administration of carnosine suppressed lipopolysaccharide (LPS)-induced vascular permeability, tissue injury and inflammation in the lung. In vivo imaging analysis revealed that LPS administration increased the level of ROS and that this increase was inhibited by carnosine administration. Carnosine also suppressed LPS-induced neutrophilic inflammation (evaluated by activation of myeloperoxidase in the lung and increased extracellular DNA in bronchoalveolar lavage fluid). Furthermore, carnosine administration suppressed the LPS-induced endoplasmic reticulum stress response in vivo. These results suggest that the oral administration of carnosine suppresses LPS-induced lung injury via carnosine’s ROS-reducing activity. Therefore, carnosine may be beneficial for suppressing the onset and progression of ARDS.

Mechanical ventilation induces neutrophil extracellular trap formation.

Mechanical ventilation can injure the lung and induce a proinflammatory state; such ventilator-induced lung injury (VILI) is associated with neutrophil influx. Neutrophils release DNA and granular proteins as cytotoxic neutrophil extracellular traps (NETs). The authors hypothesized that NETs were produced in a VILI model and may contribute to injury. In a two-hit lipopolysaccharide/VILI mouse model with and without intratracheal deoxyribonuclease (DNase) treatment or blockade of known inducers of NET formation (NETosis), the authors assessed compliance, bronchoalveolar lavage fluid protein, markers of NETs (citrullinated histone-3 and DNA), and markers of inflammation. Although lipopolysaccharide recruited neutrophils to airways, the addition of high tidal mechanical ventilation was required for significant induction of NETs markers (e.g., bronchoalveolar lavage fluid DNA: 0.4 ± 0.07 µg/ml [mean ± SEM], P < 0.05 vs. all others, n = 10 per group). High tidal volume mechanical ventilation increased airway high-mobility group box 1 protein (0.91 ± 0.138 vs. 0.60 ± 0.095) and interleukin-1β in lipopolysaccharide-treated mice (22.4 ± 0.87 vs. 17.0 ± 0.50 pg/ml, P < 0.001) and tended to increase monocyte chemoattractant protein-1 and interleukin-6. Intratracheal DNase treatment reduced NET markers (bronchoalveolar lavage fluid DNA: 0.23 ± 0.038 vs. 0.88 ± 0.135 µg/ml, P < 0.001; citrullinated histone-3: 443 ± 170 vs. 1,824 ± 403, P < 0.01, n = 8 to 10) and attenuated the loss of static compliance (0.9 ± 0.14 vs. 1.58 ± 0.17 ml/mmHg, P < 0.01, n = 19 to 20) without significantly impacting other measures of injury. Blockade of high-mobility group box 1 (with glycyrrhizin) or interleukin-1β (with anakinra) did not prevent NETosis or protect against injury. NETosis was induced in VILI, and DNase treatment eliminated NETs. In contrast to experimental transfusion-related acute lung injury, NETs do not play a major pathogenic role in the current model of VILI.

Extracellular DNA traps in bronchoalveolar fluid from a murine eosinophilic pulmonary response

Asthma is associated with a loss of the structural integrity of airway epithelium and dysfunction of the physical barrier, which protects airways from external harmful factors. Granulocyte activation causes the formation of extracellular traps, releasing web-like structures of DNA and proteins, being important to kill pathogens extracellularly. We investigated whether eosinophils infiltrating airways in an experimental model of asthma would induce eosinophil extracellular traps (EETs) in bronchoalveolar lavage fluid and lung tissue. We showed that an ovalbumin (OVA) asthma protocol presented a significant increase in eosinophil counts with increased extracellular DNA in bronchoalveolar lavage fluid as well as in lung tissue, confirming the presence of DNA traps colocalized with eosinophil peroxidase. EETs formation was reversed by DNase treatment. With these approaches, we demonstrated for the first time that OVA-challenged mice release extracellular DNA traps, which could aggravate pulmonary dysfunction.

Identification of fungal DNA in BALF from patients with home-related hypersensitivity pneumonitis

In Japan, a major type of home-related hypersensitivity pneumonitis (HP) is summer-type HP, which is caused by Trichosporon asahii (T. asahii) or Trichosporon mucoides. Some patients with home-related HP test negative for antibodies against Trichosporon; yet, a causative mold antigen cannot be identified. We analyzed 19 patients with home-related HP, 8 healthy volunteers, and 35 patients with other diseases. We extracted DNA from cell pellets of bronchoalveolar lavage fluid (BALF), amplified the DNA by PCR using Trichosporon-specific primers or other fungus-specific primers, and cloned as well as sequenced the PCR amplicon. Other primers used were specific for Acremonium chrysogenum, Aspergillus fumigatus, Aspergillus niger, Fusarium napiforme, Humicola fuscoatra, Penicillium corylophilum, and Pezizia domiciliana. We detected Trichosporon DNA (n = 17) and F. napiforme DNA (n = 2) by PCR in 19 patients with home-related HP; however, these species were not identified in healthy volunteers. After sequencing of the PCR amplicon for Trichosporon species, we identified T. asahii (n = 11), Trichosporon japonicum (n = 1), and Cryptococcus uzbekistanesis (n = 4). We could detect fungal DNA in BALF cell pellets from patients with home-related HP. These data suggest that this method might be useful to detect antigens responsible for home-related HP.

Detection of Human Cytomegalovirus in Bronchoalveolar Lavage Fluid of Lung Transplant Recipients Reflects Local Virus Replication and Not Contamination from the Throat

Whether bronchoalveolar lavage (BAL) fluid may be contaminated with oropharyngeal cytomegalovirus (CMV) has never been investigated. In an analysis of CMV DNA loads in 76 simultaneously obtained BAL fluid and throat wash samples from lung transplant recipients, we show that such contamination is unlikely and that detection of CMV DNA in BAL fluid reflects virus replication in the lung.

Seasonal variation in mortality of Pneumocystis jirovecii pneumonia in HIV-infected patients

A seasonal variation in the presentation of Pneumocystis jirovecii pneumonia (PCP) has been reported and a previous study from this centre noted a seasonal variation in mortality rates. This study examined seasonal influences (including climatic factors) within-host factors (clinical and laboratory-derived variables), the infectious burden of P. jirovecii in bronchoalveolar lavage (BAL) fluid, the presence of dihydropteroate synthase (DHPS) mutations in P. jirovecii, variations in knowledge and skills of junior medical staff, and mortality in 547 episodes of PCP occurring in 494 HIV-infected patients. The overall mortality rate was 13.5%. There was a seasonal variation in mortality: highest in autumn (21.2%) and lowest in spring (9.7%), P = 0.047. After adjustment was made for prognostic factors previously identified as being associated with mortality (increasing patient age, second/third episode of PCP, low haemoglobin, low PaO(2), presence of medical co-morbidity and pulmonary Kaposi sarcoma), there was no seasonal association with mortality, P = 0.249. The quantity of P. jirovecii DNA in BAL fluid showed no evidence of seasonal variation, P = 0.67; DHPS mutations were identified with equal frequency in each season and the mortality rate for February and August (when junior medical staff arrive in new posts) was 16.7%, only slightly greater than for other months (13.0%).

Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid

BackgroundThe diagnosis of invasive pulmonary aspergillosis (IPA) remains challenging. Culture and histopathological examination of bronchoalveolar lavage (BAL) fluid are useful but have suboptimal sensitivity and in the case of culture may require several days for fungal growth to be evident. Detection of Aspergillus DNA in BAL fluid by quantitative PCR (qPCR) offers the potential for earlier diagnosis and higher sensitivity. It is important to adopt quality control measures in PCR assays to address false positives and negatives which can hinder accurate evaluation of diagnostic performance.MethodsBAL fluid from 94 episodes of pneumonia in 81 patients was analyzed. Thirteen episodes were categorized as proven or probable IPA using Mycoses Study Group criteria. The pellet and the supernatant fractions of the BAL were separately assayed. A successful extraction was confirmed with a human 18S rRNA gene qPCR. Inhibition in each qPCR was measured using an exogenous DNA based internal amplification control (IAC). The presence of DNA from pathogens in the Aspergillus genus was detected using qPCR targeting fungal 18S rRNA gene.ResultsHuman 18S rRNA gene qPCR confirmed successful DNA extraction of all samples. IAC detected some degree of initial inhibition in 11 samples. When culture was used to diagnose IPA, the sensitivity and specificity were 84.5% and 100% respectively. Receiver-operating characteristic analysis of qPCR showed that a cutoff of 13 fg of Aspergillus genomic DNA generated a sensitivity, specificity, positive and negative predictive value of 77%, 88%, 50%, 96% respectively. BAL pellet and supernatant analyzed together resulted in sensitivity and specificity similar to BAL pellet alone. Some patients did not meet standard criteria for IPA, but had consistently high levels of Aspergillus DNA in BAL fluid by qPCR.ConclusionThe Aspergillus qPCR assay detected Aspergillus DNA in 76.9% of subjects with proven or probable IPA when the concentrated BAL fluid pellet fraction was used for diagnosis. There was no benefit from analyzing the BAL supernatant fraction. Use of both extraction and amplification controls provided optimal quality control for interpreting qPCR results and therefore may increase our understanding of the true potential of qPCR for the diagnosis of IPA.

Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients

Background: Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory tract samples using cytochemical stains. Molecular diagnosis of PCP is potentially more sensitive. Methods: A study...

Quantitative detection of herpes simplex virus DNA in the lower respiratory tract

Quantitation of herpes simplex virus (HSV) DNA in bronchoalveolar lavage specimens could indicate an infectious role in the lower respiratory tract. The aim of this study was to compare quantitative HSV DNA results from adult bronchoalveolar lavage specimens to clinical outcome. Quantitative real-time PCR assays targeting HSV and other herpes viruses were performed on adult bronchoalveolar lavage specimens obtained from a largely immunocompromised population during a 1-year period. The results were compared to patient characteristics and outcome. HSV DNA was detected in 11 (19%) of 57 bronchoalveolar lavage specimens with a mean viral level of 5.6 log genome equivalents/ml (range, 2.9-8.1 log). A threshold of HSV DNA levels equal or higher than 5.0 log (n = 7) was associated with mortality within 28 days following hospital admission (odds ratio [OR], 6.8; 95% confidence interval [CI], 1.2-39.2). A threshold level of 5.5 log was associated with mortality within 28 days of sampling (OR 8.5; 95% CI 1.2-62.1), only after excluding patients receiving specific antiviral medication. Patients with HSV DNA levels equal or higher than 7.5 log had severe respiratory failure. Viral pneumonia was histologically proven in one patient with 8.0 log at autopsy. No patient with HSV DNA levels below 5.5 log (n = 5) or DNA levels higher than 5.0 log of cytomegalovirus (CMV) (n = 3), Epstein-Barr virus (EBV) (n = 9), varicella-zoster virus (VZV) (n = 1), or human herpesvirus 6 (HHV-6) (n = 0) died within 28 days of hospital admission. We conclude that quantitative detection of HSV DNA in bronchoalveolar lavage fluid is a potential diagnostic tool for detection of relevant viral infection of the lower respiratory tract.

Human Herpesvirus 6 in Bronchalveolar Lavage Fluid after Lung Transplantation: A Risk Factor for Bronchiolitis Obliterans Syndrome?

Bronchiolitis obliterans syndrome (BOS) is the limiting factor to long-term survival after lung transplantation. Previous studies suggested respiratory viral tract infections are associated with the development of BOS. To identify the impact of virus detection in bronchoalveolar lavage (BAL) fluid, we analyzed BAL samples from 87 consecutive lung transplant recipients for human herpesvirus (HHV)-6, Epstein-Barr virus, Herpes simplex virus 1/2, Cytomegalovirus, respiratory syncytical virus and adenovirus by PCR. Acute rejection, BOS and death were recorded for a mean follow-up time of 3.27 +/- 0.47 years. Results of PCR analysis and other potential risk factors were entered into a Cox regression analysis of BOS predictors and death. Only acute rejection was a distinct risk factor for BOS of all stages, death and death from BOS. HHV-6 was detected in 20 patients. Univariate and multivariate analysis revealed that HHV-6 was associated with an increased risk to develop BOS > orb = stage 1 and death, separate from the risk attributable to acute rejection. Identification of HHV-6 DNA in BAL fluid is a potential risk factor for BOS. Our results warrant further studies to elucidate a possible causal link between HHV-6 and BOS.