Bronchoalveolar Lavage Fluid Samples Research Articles

Diagnostic Value of Metagenomic next-generation sequencing and X-pert in Bronchoalveolar lavage fluid for pneumonia in HIV-infected and HIV-uninfected patients

BackgroundThe pathogens causing unexplained pneumonia in both HIV-infected or HIV-unfected patients are likely to be complex. This retrospective study aimed to characterize the etiology of pneumonia in HIV-infected and HIV-uninfected patients using bronchoalveolar lavage fluid (BALF) analysis with metagenomic next-generation sequencing (mNGS) and X-pert MTB/RIF. MethodsBetween January 2022 and May2024, 141 HIV-infected and 104 HIV-uninfected patients admitted to Nanjing Second Hospital with pneumonia were included. BALF samples were collected and analyzed using mNGS to detect bacteria, fungi, viruses, tuberculosis (TB) and non-tuberculous mycobacteria (NTM), and X-pert for TB detection. Clinical data including CD4 T-cell counts, comorbidities, and ART status were collected and analyzed. ResultsHIV-uninfected patients were found to be older and exhibited a higher prevalence of comorbidities compared to HIV-infected patients. Despite higher median CD4 T-cell counts in HIV-uninfected individuals (412 cells/μL vs. 31 cells/μL in HIV-infected), TB detection rates using X-pert and mNGS were lower than anticipated, particularly in HIV-infected patients. Mixed-pathogen infections were significantly more prevalent in HIV-infected patients, especially those with lower CD4 T-cell counts. ART use showed variable impacts on pathogen diversity, with longer treatment durations associated with reduced infection complexity but persistent immunodeficiency in some cases.In patients with pneumonia, whether HIV-infected or HIV-uninfected, pathogens often exhibit complexity, underscoring the critical role of timely mNGS and X-pert analysis of BALF for early pathogen detection.

Nebulized Budesonide Prevents Airway Inflammation in Children with High Total IgE Levels After Open Heart Surgery with Cardiopulmonary Bypass: A Prospective Randomized Controlled Trial.

Cardiopulmonary bypass (CPB) is a crucial technique used to repair congenital heart defects (CHD); however, it may induce inflammatory response, leading to airway inflammation and need for prolonged mechanical ventilation. In this study, we aimed to evaluate the effect of budesonide nebulization in children with high serum total immunoglobulin E (tIgE) levels undergoing surgical repair of CHD via CPB. We conducted a randomized, single-center, controlled trial at a tertiary teaching hospital. One-hundred and one children with high tIgE were enrolled and randomized into the budesonide nebulization group (BUD group, n = 50) or the normal saline nebulization group (NS group, n = 51) between January 2020 and December 2020. Budesonide or normal saline was administered through a vibrating mesh nebulizer during mechanical ventilation every 8h. Blood and bronchoalveolar lavage fluid (BALF) samples were examined and data on airway mechanics and clinical outcomes were recorded. IL-6 and IL-8 levels in the blood and BALF samples significantly increased after CPB in both groups. Budesonide inhalation reduced IL-6 and IL-8 levels in the blood and BALF samples in children with high tIgE (P < 0.05). The mean airway pressure, PCO2, and oxygen index in the BUD group were significantly lower than those in the NS group after the first inhalation dose and persisted until almost 24h after surgery. The peak inspiratory pressure and drive pressure were lower in the BUD group than in the NS group at nearly 24h after surgery, with no significant difference at other time points. Additionally, the duration of mechanical ventilation, number of noninvasive ventilations after extubation, and number of patients using aerosol-inhaled bronchodilators after CICU in the BUD group were significantly lower than those in the NS group (P < 0.05). Children with high preoperative tIgE levels are at risk of airway inflammation after cardiopulmonary bypass. Inhaling budesonide during postoperative mechanical ventilation can reduce the intensity of inflammatory reactions, shorten the duration of mechanical ventilation, reduce airway pressure and the utilization of NIV after extubation.

The clinical application value of multi-site mNGS detection of patients with sepsis in intensive care units

BackgroundSepsis remains a leading cause of mortality in intensive care units, and rapid and accurate pathogen detection is crucial for effective treatment. This study evaluated the clinical application of multi-site metagenomic next-generation sequencing (mNGS) for the diagnosis of sepsis, comparing its performance against conventional methods.MethodsA retrospective analysis was conducted on 69 patients with sepsis consecutively admitted to the Department of Intensive Care Medicine, Meizhou People’s Hospital. Samples of peripheral blood and infection sites were collected for mNGS and conventional method tests to compare the positive rate of mNGS and traditional pathogen detection methods and the distribution of pathogens. The methods used in this study included a comprehensive analysis of pathogen consistency between peripheral blood and infection site samples. Additionally, the correlation between the pathogens detected and clinical outcomes was investigated.ResultsOf the patients with sepsis, 57.97% experienced dyspnea, and 65.2% had underlying diseases, with hypertension being the most common. mNGS demonstrated a significantly higher pathogen detection rate (88%) compared to the conventional method tests (26%). The pathogen consistency rate was 60% between plasma and bronchoalveolar lavage fluid samples, and that of plasma and local body fluid samples was 63%. The most frequently detected pathogens were gram-negative bacteria, and Klebsiella pneumonia. There were no significant differences in the clinical features between the pathogens.ConclusionmNGS is significantly superior to conventional methods in pathogen detection. There was a notable high pathogen consistency detection between blood and local body fluid samples, supporting the clinical relevance of mNGS. This study highlights the superiority of mNGS in detecting a broad spectrum of pathogens quickly and accurately.Trial registrationNot applicable.

Enhancing lower respiratory tract infection diagnosis: implementation and clinical assessment of multiplex PCR-based and hybrid capture-based targeted next-generation sequencing

Shotgun metagenomic next-generation sequencing (mNGS) is widely used to detect pathogens in bronchoalveolar lavage fluid (BALF). However, mNGS is complex and expensive. This study explored the feasibility of targeted next-generation sequencing (tNGS) in distinguishing lower respiratory tract infections in clinical practice. We used 229 retrospective BALF samples to establish thresholds and diagnostic values in a prospective cohort of 251 patients. After target pathogen selection, primer and probe design, optimization experiments, and bioinformatics analysis, multiplex PCR-based tNGS (mp-tNGS) and hybrid capture-based tNGS (hc-tNGS), targeting 198 and 3060 pathogens (DNA and RNA co-detection workflow) were established and performed. mp-tNGS and hc-tNGS took 10.3 and 16h, respectively, with low sequencing data sizes of 0.1M and 1M reads, and test costs reduced to a quarter and half of mNGS. The LoDs of mp-tNGS and hc-tNGS were 50-450CFU/mL. mp-tNGS and hc-tNGS were highly accurate, with 86.5% and 87.3% (vs. 85.5% for mNGS) sensitivities and 90.0% and 88.0% (vs. 92.1% for mNGS) specificities. tNGS detection rates for casual pathogens were 84.3% and 89.5% (vs. 88.5% for mNGS), significantly higher than conventional microbiological tests (P<0.001). In seven samples, tNGS detected Pneumocystis jirovecii, a fungus not detected by mNGS. Whereas mNGS detected six samples with filamentous fungi (Rhizopus oryzae, Aureobasidium pullulans, Aspergillus niger complex, etc.) which missed by tNGS. The anaerobic bacteria as pathogen in eight samples was failed to detect by mp-tNGS. tNGS may offer a new, broad-spectrum, rapid, accurate and cost-effective approach to diagnosing respiratory infections. National Natural Science Foundation of China (81625014 and 82202535).

The Omega-6 Lipid pathway shift is associated with neutrophil influx and structural lung damage in early cysticfibrosis lung disease.

In cystic fibrosis (CF), an imbalanced lipid metabolism is associated with lung inflammation. Little is known about the role that specific lipid mediators (LMs) exert in CF lung inflammation, and whether their levels change during early disease progression. Therefore, we measured airway LM profiles of young CF patients, correlating these with disease-associated parameters. Levels of omega (ω)-3/6 PUFAs and their LM derivatives were determined in bronchoalveolar lavage fluid (BALF) of children with CF ages 1-5 using a targeted high-performance liquid chromatography-tandem mass spectrometry approach. Hierarchical clustering analysis was performed on relative LM levels. Individual relative LM levels were correlated with neutrophilic inflammation (BALF %Neu) and structural lung damage (PRAGMA-CF %Disease). Significant correlations were included in a backward multivariate linear regression model to identify the LMs that are best related to disease progression. A total of 65 BALF samples were analysed for ω-3/6 lipid content. LM profiles clustered into an arachidonic acid (AA)-enriched and a linoleic acid (LA)-enriched sample cluster. AA derivatives like 17-OH-DH-HETE, 5-HETE, 5,15-diHETE, 15-HETE, 15-KETE, LTB4 and 6-trans-LTB4 positively correlated with BALF %Neu and/or PRAGMA %Dis. Contrastingly, 9-HoTrE and the LA derivatives 9-HoDE, 9(10)-EpOME, 9(10)-DiHOME, 13-HoDE, 13-oxoODE and 12(13)-EpOME negatively correlated with BALF %Neu and/or PRAGMA %Dis. 6-trans-LTB4 was the strongest predictor for BALF %Neu. 5-HETE and 15-KETE contributed most to PRAGMA %Dis prediction. Our data provide more insight into the lung lipidome of infants with CF, and show that a shift from LA derivatives to AA derivatives in BALF associates with early CF lung disease progression.

Distinctive evolution of alveolar T cell responses is associated with clinical outcomes in unvaccinated patients with SARS-CoV-2 pneumonia.

The evolution of T cell molecular signatures in the distal lung of patients with severe pneumonia is understudied. Here, we analyzed T cell subsets in longitudinal bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia, including unvaccinated patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or with respiratory failure not linked to pneumonia. In patients with SARS-CoV-2 pneumonia, activation of interferon signaling pathways, low activation of the NF-κB pathway and preferential targeting of spike and nucleocapsid proteins early after intubation were associated with favorable outcomes, whereas loss of interferon signaling, activation of NF-κB-driven programs and specificity for the ORF1ab complex late in disease were associated with mortality. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize individuals who recover, whereas responses against nonstructural proteins and activation of NF-κB are associated with poor outcomes.

Clinical characteristics and microbial signatures in the lower airways of diabetic and nondiabetic patients with pneumonia.

The microbial signatures in diabetes with pneumonia and the risk factors of severe pneumonia (SP) in diabetic patients are not clear. Our study explored microbial signatures and the association between clinical characteristics and SP then constructed a risk model to find effective biomarkers for predicting pneumonia severity. Our study was conducted among 273 patients with pneumonia diagnosed and treated in our hospital from January 2018 to May 2021. Bronchoalveolar lavage fluid (BALF) samples and clinical data were collected. Metagenomic sequencing was applied after extracting the DNA from samples. Appropriate statistical methods were used to compare the microbial signatures and clinical characteristics in patients with or without diabetes mellitus (DM). In total, sixty-one pneumonia patients with diabetes and 212 pneumonia patients without diabetes were included. Sixty-six differential microorganisms were found to be associated with SP in diabetic patients. Some microbes correlated with clinical indicators of SP. The prediction model for SP was established and the receiver operating characteristic (ROC) curve demonstrated its accuracy, with the sensitivity and specificity of 0.82 and 0.91, respectively. Some microorganisms affect the severity of pneumonia. We identified the microbial signatures in the lower airways and the association between clinical characteristics and SP. The predictive model was more accurate in predicting SP by combining microbiological indicators and clinical characteristics, which might be beneficial to the early identification and management of patients with SP.

Metagenomic versus targeted next-generation sequencing for detection of microorganisms in bronchoalveolar lavage fluid among renal transplantation recipients.

Metagenomic next-generation sequencing (mNGS), which provides untargeted and unbiased pathogens detection, has been extensively applied to improve diagnosis of pulmonary infection. This study aimed to compare the clinical performance between mNGS and targeted NGS (tNGS) for microbial detection and identification in bronchoalveolar lavage fluid (BALF) from kidney transplantation recipients (KTRs). BALF samples with microbiological results from mNGS and conventional microbiological test (CMT) were included. For tNGS, samples were extracted, amplified by polymerase chain reaction with pathogen-specific primers, and sequenced on an Illumina Nextseq. A total of 99 BALF from 99 KTRs, among which 93 were diagnosed as pulmonary infection, were analyzed. Compared with CMT, both mNGS and tNGS showed higher positive rate and sensitivity (p<0.001) for overall, bacterial and fungal detection. Although the positive rate for mNGS and tNGS was comparable, mNGS significantly outperformed tNGS in sensitivity (100% vs. 93.55%, p<0.05), particularly for bacteria and virus (p<0.001). Moreover, the true positive rate for detected microbes of mNGS was superior over that of tNGS (73.97% vs. 63.15%, p<0.05), and the difference was also significant when specific for bacteria (94.59% vs. 64.81%, p<0.001) and fungi (93.85% vs. 72.58%, p<0.01). Additionally, we found that, unlike most microbes such as SARS-CoV-2, Aspergillus, and EBV, which were predominantly detected from recipients who underwent surgery over 3 years, Torque teno virus (TTV) were principally detected from recipients within 1-year post-transplant, and as post-transplantation time increased, the percentage of TTV positivity declined. Although tNGS was inferior to mNGS owing to lower sensitivity and true positive rate in identifying respiratory pathogens among KTRs, both considerably outperformed CMT.

Metagenomic next-generation sequencing targeted and metagenomic next-generation sequencing for pulmonary infection in HIV-infected and non-HIV-infected individuals.

When individuals infected with human immunodeficiency virus (HIV) experience pulmonary infections, they often exhibit severe symptoms and face a grim prognosis. Consequently, early, rapid, and accurate pathogen diagnosis is vital for informing effective treatment strategies. This study aimed to use metagenomic next-generation sequencing (mNGS) and targeted mNGS (tNGS) to elucidate the characteristics of pulmonary infections in HIV and non-HIV individuals. This study enrolled 90 patients with pulmonary infection at the Department of Infectious Diseases of The First Hospital of Jilin University from June 2022 to May 2023, and they were divided into HIV (n=46) and non-HIV (n=44) infection groups. Their bronchoalveolar lavage fluid (BALF) was collected for mNGS analysis to evaluate the differences in pulmonary infection pathogens, and tNGS detection was performed on BALF samples from 15 HIV-infected patients. A total of 37 pathogens were identified in this study, including 21 bacteria, 5 fungi, 5 viruses, 5 mycobacteria, and 1 mycoplasma. The sensitivity of mNGS was 78.9% (71/90), which is significantly higher than that of conventional methods (CTM) (39/90, P=1.5E-8). The combination of mNGS with CTM can greatly enhance the sensitivity of pathogen detection. The prevalence of Pneumocystis jirovecii (82.6% vs. 9.1%), cytomegalovirus (CMV) (58.7% vs. 0%), and Epstein-Barr virus (EBV) (17.4% vs. 2.3%) was significantly higher in the HIV infection group than in the non-HIV infection group (P<0.05). Although no statistically significant difference was observed, the detection rate of Mycobacteria was higher in HIV-infected patients (17.4%) than in the non-HIV group (6.8%). Furthermore, the tNGS results of BALF from 15 HIV-infected patients were not entirely consistent with the mNGS results., and the concordance rate of tNGS for the detection of main pathogens reached 86.7% (13/15). Next-generation sequencing (NGS) can accurately detect pathogens in the BALF of patients with pulmonary infection. The sensitivity of tNGS is comparable to that of mNGS. Therefore, this technique should be promoted in the clinic for better patient outcomes.

Distribution characteristics of human herpes viruses in the lower respiratory tract and their impact on 30-day mortality in community-acquired pneumonia patients.

Human herpes viruses (HHVs) are commonly detected in community-acquired pneumonia (CAP) patients, particularly those with complex complications, attracting increased attention from clinical practitioners. However, the significance of detecting HHVs in bronchoalveolar lavage fluid (BALF) with CAP patients is still unclear. This study retrospectively analyzed BALF samples from 64 CAP patients at the Kunming Third People's Hospital between August 2021 and December 2023. Metagenomic next generation sequencing (mNGS) was conducted on BALF samples during CAP onset. Multivariate Cox regression models were used to identify independent risk factors for 30-day all-cause mortality in CAP. HHVs were found in 84.4% of CAP patients, which were the most common pathogens (45.1%), followed by bacteria (30.2%) and fungi (11.5%). Bacterial-viral co-infections were most common, occurring in 39 patients. Notably, there was no significant difference in HHV presence between severe and non-severe CAP patients (EBV: P = 0.431, CMV: P = 0.825), except for HHV-7 (P = 0.025). In addition, there was no significant difference in the 30-day mortality between HHV positive and HHV negative groups (P = 0.470), as well as between the HHV-7 positive and HHV-7 negative groups (P = 0.910). However, neither HHVs nor HHV-7 was independent risk factors for 30-day mortality in CAP patients (HHVs: HR 1.171, P = 0.888; HHV-7: HR 1.947, P = 0.382). In summary, among the prevalent presence of multiple HHVs, EBV and CMV were the most prevalent in CAP patients. Patients with sCAP were more susceptible to HHV-7 than those with non-sCAP. These results provide valuable insights for clinicians in guiding appropriate interventions for CAP treatment.

Advancing quantitative PCR with color cycle multiplex amplification.

Quantitative PCR (qPCR) is the gold standard for detection and quantitation of known DNA targets, but the scarcity of spectrally distinct fluorophores and filter sets limits the number of detectable targets. Here, we introduce color cycle multiplex amplification (CCMA) to significantly increase the number of detectable DNA targets in a single qPCR reaction using standard instrumentation. In CCMA, presence of one DNA target species results in a pre-programmed pattern of fluorescence increases. This pattern is distinguished by cycle thresholds (Cts) through rationally designed delays in amplification. For example, we design an assay wherein Staphylococcus aureus sequentially induces FAM, then Cy5.5, then ROX fluorescence increases with more than 3 cycles between each signal. CCMA offers notably higher potential for multiplexing because it uses fluorescence permutation rather than combination. With 4 distinct fluorescence colors, CCMA theoretically allows the detection of up to 136 distinct DNA target sequences using fluorescence permutation. Experimentally, we demonstrated a single-tube qPCR assay screening 21 sepsis-related bacterial DNA targets in samples of blood, sputum, pleural effusion and bronchoalveolar lavage fluid, with 89% clinical sensitivity and 100% clinical specificity, showing its potential as a powerful tool for advanced quantitative screening in molecular diagnostics.

Application of metagenomic next-generation sequencing and targeted metagenomic next-generation sequencing in diagnosing pulmonary infections in immunocompetent and immunocompromised patients.

Metagenomic next-generation sequencing (mNGS) technology has been widely used to diagnose various infections. Based on the most common pathogen profiles, targeted mNGS (tNGS) using multiplex PCR has been developed to detect pathogens with predesigned primers in the panel, significantly improving sensitivity and reducing economic burden on patients. However, there are few studies on summarizing pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China on large scale. From January 2021 to December 2023, bronchoalveolar lavage fluid (BALF) or sputum samples from 546 immunocompetent and immunocompromised patients with suspected community-acquired pneumonia were collected. Pathogen profiles in those patients on whom mNGS was performed were summarized. Additionally, we also evaluated the performance of tNGS in diagnosing pulmonary infections. Combined with results of mNGS and culture, we found that the most common bacterial pathogens were Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii in both immunocompromised and immunocompetent patients with high detection rates of Staphylococcus aureus and Enterococcus faecium, respectively. For fungal pathogens, Pneumocystis jirovecii was commonly detected in patients, while fungal infections in immunocompetent patients were mainly caused by Candida albicans. Most of viral infections in patients were caused by Human betaherpesvirus 5 and Human gammaherpesvirus 4. It is worth noting that, compared with immunocompetent patients (34.9%, 76/218), more mixed infections were found in immunocompromised patients (37.8%, 14/37). Additionally, taking final comprehensive clinical diagnoses as reference standard, total coincidence rate of BALF tNGS (81.4%, 48/59) was much higher than that of BALF mNGS (40.0%, 112/280). Our findings supplemented and classified the pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China. Most importantly, our findings can accelerate the development and design of tNGS specifically used for regional pulmonary infections.

Commercial loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of Pneumocystis pneumonia: An alternative to immunofluorescence assays

A commercial loop-mediated isothermal amplification (LAMP) assay is available for the detection of Pneumocytis jirovecii (Eazyplex®, Amplex diagnostics, Germany). Few centers currently use this LAMP assay in France. Recently, the commercialization of reagents used to perform the P. jirovecii immunofluorescence assay (IFA) was stopped. This study aimed to assess the position of the commercial LAMP P. jirovecii assay in the diagnostic strategy for Pneumocystis pneumonia.Over 24 months (August 1, 2021, to September 1, 2023), all bronchoalveolar lavage fluid (BALF) samples with a request for P. jirovecii detection were analyzed with the commercial Eazyplex® LAMP assay, using a Genie 2® device (Amplex, diagnostics), in parallel with the techniques used for direct examination. Specific P. jirovecii quantitative real-time PCR (qPCR) was subsequently performed.In total, 346 BALF samples were analyzed by Diff-Quik coloration, IFA, and the commercial Eazyplex® LAMP assay for initial screening.Twenty-six cases of PCP were retained based on radiological, biological and clinical criteria.Among the 26 cases of PCP, 11 BALF samples were positive using the initial screening techniques: four with the three techniques, six by IFA and Eazyplex®, and one only by IFA. The eleven BALF samples were positive with the specific P. jirovecii qPCR assay, with a mean quantification cycle (Cq) of 27 [19–32].The commercial Eazyplex® LAMP assay is able to provide a result in 25 min and its sensitivity is similar to that of BALF direct examination techniques, such as IFA, which is a technique no longer available on the European market. The sensitivity of the commercial Eazyplex® LAMP assay is however clearly inferior to that of the specific P. jirovecii qPCR assay and, therefore, cannot replace the specific qPCR, but may have a place in the diagnostic strategy.

The potential role of lung microbiota and lauroylcarnitine in T-cell activation associated with checkpoint inhibitor pneumonitis

Checkpoint inhibitor pneumonitis (CIP) is a potentially fatal adverse event characterized by new pulmonary infiltrates in cancer patients receiving immune checkpoint inhibitor therapy. This study aims to explore the interplay between lung microbiota, dysregulated metabolites, and host immunity in CIP. We recruited thirteen hospitalized CIP patients, eleven idiopathic pulmonary fibrosis (IPF) patients, and ten new-onset non-small cell lung cancer patients. Bronchoalveolar lavage fluid samples were collected for 16S rRNA gene sequencing. The percentages of immune cells were determined using manual counting and flow cytometry. Interactions among microbiota, metabolites, and lymphocytes were analyzed using cultured mouse splenocytes and human T cells. Proteobacteria emerged as the dominant phylum, notably abundant in both the CIP and IPF groups. Vibrio, Halomonas, Mangrovibacter, and Salinivibrio were the predominant microbiota because of their discriminative abundance patterns. Vibrio (r=0.72, P-adj=0.007) and Halomonas (r=0.65, P-adj=0.023) demonstrated strong correlations with lymphocytes. Vibrio metschnikovii and Mangrovibacter plantisponsors were more abundant in the CIP group than in the IPF group. Lauroylcarnitine, a key intermediary metabolite co-occurring with the predominant microbiota, exhibited a potent effect on cytokine secretion by mouse and human T cells, notably enhancing IFN-γ and TNF-α production from CD4 and CD8 cells invitro. Lauroylcarnitine, co-occurring with the predominant lung microbiota in CIP, could activate T cells invitro. These findings suggest potential involvement of lung microbiota and acylcarnitine metabolism dysregulation in the pathogenesis of CIP. This work was supported by Peking University People's Hospital Scientific Research Development Funds (RDJ2022-15) and Provincial Key Clinical Specialty Capacity Building Project 2020 (Department of the Respiratory Medicine).

Predictive performance of Metagenomic Next Generation Sequencing in early detection of post-liver transplantation infections

ObjectiveTo evaluate the predictive performance of metagenomic next-generation sequencing (mNGS) in identifying and predicting pulmonary infections following liver transplantation and to investigate its association with patient outcomes within the initial four-week post-transplantation period. MethodsWe retrospectively analyzed 41 liver transplant patients with suspected pulmonary infections from August 2022 to May 2023. Bronchoalveolar lavage fluid (BALF) samples were collected on the first postoperative day for metagenomic next generation sequencing (mNGS) and culture. The predictive capability of mNGS for subsequent infections was assessed by monitoring inflammatory biomarkers and comparing the detection rates with culture methods. Real-time Polymerase Chain Reaction (Rt-PCR) was used to monitor Human betaherpesvirus 5 (CMV) and Human parvovirus B19 (B19) weekly during a four-week postoperative period. Inflammatory biomarkers and blood coagulation function were evaluated on specific days throughout the first, third, fifth, and during four weeks following surgery. The study was conducted until August 2023 to evaluate the patients' prognostic survival outcome, classifying them into groups based on the mortality and survival. ResultsThe analysis included a total of 41 patients, comprising 32 males and 9 females, with an average age of 52 (47, 63) years. Within one week after liver transplantation, there were 7 cases of bacterial infections, 5 cases of fungal infections, 19 cases of mixed infections, 8 cases without any infection, and 2 cases with unidentified pathogen-associated infections. mNGS successfully predicted 39 (72 %) strains of pathogens, while culture-based methods only detected 28 (52 %) strains. Among the 8 patients diagnosed as non-infected, culture methods identified positive results in 4 cases (50 %), whereas mNGS yielded positive results in 7 cases (87.5 %). The detection rates of CMV and B19 by Rt-PCR within 4 weeks after liver transplantation were 61 % and 17 %, respectively (25/41, 7/41) among the patients. During the study period, a total of 9 patients succumbed while 32 patients survived. The death group and the survival group exhibited significant differences in CRP, HGB, and INR levels at specific monitoring time points. The proportion of CMV detection in blood was significantly higher in the death group compared to the surviving group. Elevated CRP level was identified as a prognostic risk factor. ConclusionsDespite the presence of false positives, mNGS still presents a potential advantage in predicting pulmonary infection pathogens following liver transplantation. Furthermore, the levels of CRP and CMV carrier status within four weeks post-surgery exhibit significant associations with patient survival and prognosis.

Next-Generation Sequencing for Characterizing Respiratory Tract Virome and Improving Detection of Viral Pathogens in Children With Pneumonia.

Pneumonia is typically caused by a variety of pathogenic microorganisms. Traditional research often focuses on the infection of a few microorganisms, whereas metagenomic studies focus on the impact of the bacteriome and mycobiome on respiratory diseases. Reports on the virome characteristics of pediatric pneumonia remain relatively scarce. We employed de novo assembly and combined homology- and feature-based methods to characterize the respiratory virome in whole-genome DNA sequencing samples from oropharynx (OP) swabs, nasopharynx (NP) swabs, and bronchoalveolar lavage fluids (BALF) of children with pneumonia. Significant differences were observed in the alpha and beta diversity indexes, as well as in the composition of the oropharyngeal virome, between pneumonia cases and controls. We identified 1137 viral operational taxonomic units (vOTUs) with significant differences, indicating a preference of pneumonia-reduced vOTUs for infecting Prevotella, Neisseria, and Veillonella, whereas pneumonia-enriched vOTUs included polyomavirus, human adenovirus, and phages targeting Staphylococcus, Streptococcus, Granulicatella, and Actinomyces. Comparative analysis revealed higher relative abundances and prevalence rates of pneumonia-enriched OP vOTUs in NP and BALF samples compared to pneumonia-reduced vOTUs. Additionally, virome analysis identified six pediatric patients with severe human adenovirus or polyomavirus infections, five of whom might have been undetected by targeted polymerase chain reaction (PCR)-based testing. This study offers insights into pediatric pneumonia respiratory viromes, highlighting frequent transmission of potentially pathogenic viruses and demonstrating virome analysis as a valuable adjunct for pathogen detection.

DNA methylation analysis in plasma for early diagnosis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) represents the most prevalent type of lung cancer. SHOX2 and RASSF1A methylation have been identified as important biomarkers for diagnosis and prognosis of lung cancer. Bronchoalveolar lavage fluid (BALF) exhibits good specificity and sensitivity in diagnosing pulmonary diseases, but its acquisition is challenging and may cause discomfort to patients. In clinical, plasma samples are more convenient to obtain than BALF; however, there is little research on the concurrent detection of SHOX2 and RASSF1A methylation in plasma. This study aims to assess the diagnostic value of a combined promoter methylation assay for SHOX2 and RASSF1A in early-stage LUAD using plasma samples. BALF and blood samples were obtained from 36 early-stage LUAD patients, with a control group of nineteen non-tumor individuals. The promoter methylation levels of SHOX2 and RASSF1A in all subjects were assessed using the human SHOX2 and RASSF1A gene methylation kit. The methylation detection rate of SHOX2 and RASSF1A in plasma was 61.11%, slightly lower than that in BALF (66.7%). The Chi-square test revealed no significant difference in the methylation rate between BALF and plasma (P > 0.05). The area under the receiver operating characteristic (ROC) curve analysis for blood was 0.806 (95% CI, 0.677 to 0.900), while for BALF it was 0.781 (95% CI, 0.649 to 0.881). Additionally, we conducted an analysis on the correlation between SHOX2 and RASSF1A methylation levels in plasma with gender, age, tumor differentiation, pathologic classification, and other clinicopathological variables; however, no significant correlations were observed. Measurement of SHOX2 and RASSF1A methylation for early diagnosis of LUAD can be achieved with high sensitivity and specificity by using plasma as a substitute for BALF samples.

Clinical, Laboratory, and Imaging Characteristics of Tropheryma Whipplei Detection in Bronchoalveolar Lavage Fluid Using Next-Generation Sequencing: A Case-Control Study.

The aim of this study was to assess the prevalence of Tropheryma whipplei (TW) infection in the population and to investigate the clinical symptoms, as well as the laboratory and imaging characteristics of patients testing positive for TW using next-generation sequencing (NGS). A retrospective review was conducted on 1346 bronchoalveolar lavage fluid (BALF) samples collected between January 2021 and September 2023. The case group comprised patients with TW detected using NGS while the control group included 65 randomly chosen Gram-positive bacterial infection patients without TW. Comparative analyses were carried out on the basic demographics, laboratory parameters, and imaging findings between the two groups. Additionally, the case group underwent an in-depth examination of underlying diseases, pathogens, final diagnoses, treatment strategies. The case group comprised of 51 patients with TW, constituting 3.8% of the total. There was no significant difference in gender and age between the case and control groups (P = 0.84, P = 0.07). Symptoms such as coughing, expectoration, wheezing, fever, and hemoptysis are less commonly detected in the case group with a higher incidence of chest pain when compared to the control group (P >0.05). The case group exhibited decreased albumin levels and increased C-reactive protein and D-dimer levels compared to normal levels. Imaging findings in the case group commonly included nodules, patchy images, and interstitial changes, the most common underlying disease is cardiovascular disease, and the most frequently co-occurring pathogen is the human herpesvirus. Among the case group, 27 patients received a final diagnosis of pneumonia, and 3 patients clinically diagnosed with Whipple's disease demonstrated improvement in both symptoms and imaging after treatment. NGS revealed a relatively low overall detection rate of TW-positive patients using BALF. TW was more prevalent in middle-aged and elderly male patients characterized by symptoms such as cough, expectoration, shortness of breath, and fever. Chest imaging in these cases typically showed nodules and interstitial changes.

The Utility of Real-Time PCR, Metagenomic Next-Generation Sequencing, and Culture in Bronchoalveolar Lavage Fluid for Diagnosis of Pulmonary Aspergillosis

Timely detection of Aspergillus infection is crucial given the high mortality rate of pulmonary aspergillosis (PA). Here, the diagnostic performances for PA of mycological culture, Aspergillus real-time Polymerase Chain Reaction (RT-PCR), and metagenomic next-generation sequencing (mNGS) assay from bronchoalveolar lavage fluid (BALF), were evaluated. Totally 139 patients with suspected fungal pneumonia were enrolled between December 2021 and July 2023, collecting 139 BALF samples for RT-PCR and culture, with 87 undergoing mNGS assay. The sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve (AUC) with 95% confidence intervals of these assays for PA were as follows: 35.3% (14.2–61.7%), 100.0% (94.0–100.0%), 100.0% (54.1–100.0%), 84.5% (79.3–88.6%), and 0.676 (0.560–0.779) for culture; 82.4% (56.6–96.2%), 98.3% (91.1–100.0%), 93.3% (66.4–99.0%), 95.2% (87.6–98.2%) and 0.903 (0.815–0.959) for same diagnostic performance of RT-PCR and mNGS; and 94.1% (71.3–99.9%), 96.7% (88.5–99.6%), 88.9% (67.1–96.9%), 98.3% (89.6–99.7%), 0.954 (0.880–0.989) for RT-PCR combining mNGS; RT-PCR, mNGS, and their combination significantly improved in AUC values over culture (p <0.001), but RT-PCR testing and mNGS had no significant difference with each other and their combination. Overall, the performance of culture was limited by low sensitivity, both RT-PCR and mNGS assays as single diagnostic tests are promising compared to culture and combined tests.

The diagnostic value of bronchoalveolar lavage fluid metagenomic next-generation sequencing in critically ill patients with respiratory tract infections.

Metagenomic next-generation sequencing (mNGS) is an unbiased and rapid method for detecting pathogens. This study enrolled 145 suspected severe pneumonia patients who were admitted to the Affiliated Hospital of Jining Medical University. This study primarily aimed to determine the diagnostic performance of mNGS and conventional microbiological tests (CMTs) using bronchoalveolar lavage fluid samples for detecting pathogens. Our findings indicated that mNGS performed significantly higher sensitivity (97.54% vs 28.68%, P < 0.001), coincidence (90.34% vs 35.17%, P < 0.001), and negative predictive value (80.00% vs 13.21%, P < 0.001) but performed lower specificity than CMTs (52.17% vs 87.5%, P < 0.001). Streptococcus pneumoniae as the most common bacterial pathogen had the largest proportion (22.90%, 30/131) in this study. In addition to bacteria, fungi, and virus, mNGS can detect a variety of atypical pathogens such as Mycobacterium tuberculosis and non-tuberculous. Mixed infections were common in patients with severe pneumonia, and bacterial-fungal-viral-atypical pathogens were the most complicated infection. After adjustments of antibiotics based on mNGS and CMTs, the clinical manifestation improved in 139 (95.86%, 139/145) patients. Our data demonstrated that mNGS had significant advantage in diagnosing respiratory tract infections, especially atypical pathogens and fungal infections. Pathogens were detected timely and comprehensively, contributing to the adjustments of antibiotic treatments timely and accurately, improving patient prognosis and decreasing mortality potentially.IMPORTANCEMetagenomic next-generation sequencing using bronchoalveolar lavage fluid can provide more comprehensive and accurate pathogens for respiratory tract infections, especially when considering the previous usage of empirical antibiotics before admission or complicated clinical presentation. This technology is expected to play an important role in the precise application of antimicrobial drugs in the future.