Pathogens In Bronchoalveolar Lavage Fluid Research Articles

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).

Bronchial lavage tNGS in the diagnosis of pulmonary tuberculosis.

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis, remains a significant global health concern. Targeted Next-Generation Sequencing (tNGS) has emerged as a rapid and comprehensive diagnostic tool for tuberculosis, offering advantages over traditional methods and serving as an effective alternative for drug susceptibility testing and the detection of drug-resistant tuberculosis. This study aimed to retrospectively analyze the clinical characteristics of pulmonary tuberculosis patients. After explore the application value of targeted next-generation sequencing technology in this patient population, providing valuable insights for clinical diagnosis and treatment. In this retrospective study, we analyzed data from 65 patients with laboratory-confirmed tuberculosis admitted to Tianjin Baodi Hospital from November 14, 2020, to February 1, 2023. Patients underwent bronchoalveolar lavage fluid (BALF) testing, including acid-fast staining, culture, and tNGS. Biopsies and histopathological examinations were performed on some patients, along with comprehensive radiological assessments for all. Among the 65 pulmonary tuberculosis patients, targeted next-generation sequencing detected pathogens in bronchoalveolar lavage fluid with a positivity rate of 93.8%, significantly higher than traditional methods such as acid-fast staining, culture, and pathology. Compared to bronchoalveolar lavage fluid smear, targeted next-generation sequencing demonstrated significantly higher diagnostic sensitivity (98.46% vs. 26.15%) and accuracy (98.46% vs. 26.15%). Targeted next-generation sequencing, with its high sensitivity and specificity compared to traditional methods, provides unique advantages in detecting pathogens among these patients, highlighting its importance in disease management.

Metagenomic Next-Generation Sequencing for Pathogens in Bronchoalveolar Lavage Fluid Improves the Survival of Patients with Pulmonary Complications After Allogeneic Hematopoietic Stem Cell Transplantation.

Unbiased metagenomic next-generation sequencing (mNGS) has been used for infection diagnosis. In this study, we explored the clinical diagnosis value of mNGS for pulmonary complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). From August 2019 to June 2021, a prospective study was performed to comparatively analyze the pathogenic results of mNGS and conventional tests for bronchoalveolar lavage fluid (BALF) from 134 cases involving 101 patients with pulmonary complications after allo-HSCT. More pathogens were identified by mNGS than with conventional tests (226 vs 120). For bacteria, the diagnostic sensitivity (P = 0.144) and specificity (P = 0.687) were similar between the two methods. For fungus except Pneumocystis jirovecii (PJ), conventional tests had a significantly higher sensitivity (P = 0.013) with a similarly high specificity (P = 0.109). The sensitivities for bacteria and fungi could be increased with the combination of the two methods. As for PJ, both the sensitivity (100%) and specificity (99.12%) of mNGS were very high. For viruses, the sensitivity of mNGS was significantly higher (P = 0.021) and the negative predictive value (NPV) was 95.74% (84.27-99.26%). Pulmonary infection complications accounted for 90.30% and bacterium was the most common pathogen whether in single infection (63.43%) or mixed infection (81.08%). The 6-month overall survival (OS) of 88.89% in the early group (mNGS ≤ 7days) was significantly higher than that of 65.52% (HR 0.287, 95% CI 0.101-0.819, P = 0.006) in the late group (mNGS > 7days). mNGS for BALF could facilitate accurate and fast diagnosis for pulmonary complications. Early mNGS could improve the prognosis of patients with pulmonary complications after allo-HSCT. ClinicalTrials.gov identifier, NCT04051372.

Clinical Evaluation of Metagenomic Next-Generation Sequencing and Identification of Risk Factors in Patients with Severe Community-Acquired Pneumonia.

Severe community-acquired pneumonia (SCAP) is the leading cause of death among patients with infectious diseases worldwide. This study aimed to evaluate the effectiveness of metagenomic next-generation sequencing (mNGS) through detecting pathogens in bronchoalveolar lavage fluid (BALF) and identifying risk factors for recovery in SCAP patients. This prospective study recruited 158 SCAP patients admitted to respiratory intensive care unit that were randomly divided into control and study groups, with receiving conventional tests and the same conventional tests plus mNGS, respectively. The diagnostic efficiency of mNGS was evaluated by comparing with conventional tests. Furthermore, univariate and multivariate logistic regression analyses were performed to determine the independent risk factors for recovery in SCAP patients, and a nomogram prediction model was established based on these factors. Within the study group, the pathogen detection rate was significantly higher with mNGS than that with conventional tests (84.81% vs 45.57%, P < 0.001), with a positive coincidence rate of 94.44%. Acinetobacter baumannii (21.52%, 17/79), Candida albicans (17.72%, 14/79), and Klebsiella pneumonia (15.19%, 12/79) were the top three common pathogens detected by mNGS. Of note, the improvement rate of patients in the study group was significantly higher than that in the control group. The further analysis revealed that the increased levels of interleukin-6, blood urea nitrogen, procalcitonin, the longer length of hospital stay, and bacterial infection were independent risk factors for recovery of SCAP patients, while mNGS detection status was a protective factor. The predictive model showed a good performance for the modeling and validation sets. Early mNGS exhibited a superior diagnostic efficiency to conventional tests in SCAP patients, which can reduce the risk of death in SCAP patients. Moreover, the clinical factors could also be used for the management and prognosis prediction of SCAP patients.

Utility of nanopore sequencing for detecting pathogens in bronchoalveolar lavage fluid from pediatric patients with respiratory failure

RNA viruses are the most frequent pathogens responsible for respiratory infections, particularly in pediatric patients. Next-generation sequencing, represented by Illumina sequencing, is one of the most comprehensive methods for identifying pathogens. Nanopore sequencing has been used to identify and analyze pathogens with a shorter sequencing time. In this study, we evaluated the utility of nanopore sequencing for the detection of RNA viruses in bronchoalveolar lavage fluid (BALF) of pediatric patients with respiratory failure. Using the seven BALF samples, we first compared the nanopore and Illumina sequencing results. The nanopore sequencing detected the same RNA viruses as the Illumina sequencing. Subsequently, BALF samples from 24 additional pediatric patients with respiratory failure were analyzed by nanopore sequencing, and RNA viral pathogens were detected in 10 out of 24 patients. Among these 10 patients, nanopore sequencing identified the same viral pathogens as detected by the PCR and viral antigen tests in five patients. Furthermore, additional RNA viral pathogens were detected by nanopore sequencing with high genome coverage in five patients that were not detected by PCR and viral antigen tests. In conclusion, nanopore sequencing could comprehensively detect RNA viral pathogens in BALF samples with equivalent sensitivity and genome coverage as Illumina sequencing. This rapid sequencing platform may be more beneficial for detecting RNA viruses in clinical settings.

Application of metagenomic next-generation sequencing in the diagnosis and resistome analysis of community-acquired pneumonia pathogens from bronchoalveolar lavage samples.

To perform a prospective diagnostic study exploring the clinical utility of metagenomic next-generation sequencing (mNGS) in diagnosing community-acquired pneumonia (CAP), and revealing resistome differences in bronchoalveolar lavage fluid (BALF) from CAP patients with varying severity of admission base on Pneumonia Patient Outcomes Research Team (PORT) risk classes. We compared the diagnostic performances of mNGS and conventional testing for the detection of pathogens in BALF from 59 CAP patients, and performed resistome differences analysis of metagenomic data from 59 BALF samples, namely, 25 from CAP patients with PORT score I (I group), 14 from CAP patients with PORT score II (II group), 12 from CAP patients with PORT score III (III group), and 8 from CAP patients with PORT score IV (IV group). The diagnostic sensitivities of mNGS and conventional testing for the detection of pathogens in BALF in patients with CAP were 96.6% (57/59) and 30.5% (18/59), respectively. There was a significant difference in the overall relative abundance of resistance genes between the four groups (P=0.014). The results of principal coordinate analysis based on Bray-Curtis dissimilarities showed that there were significant differences in the composition of resistance genes among the I, II, III, and IV groups (P=0.007). A large number of antibiotic resistance genes, such as those affiliated with multidrug, tetracycline, aminoglycoside, and fosfomycin resistance, were enriched in the IV group. In conclusion, mNGS has a high diagnostic value in CAP. There were significant differences present in microbiota resistance to antibiotics in BALF from CAP patients in different PORT risk classes, which should attract enough attention.

Methodological comparison of bronchoalveolar lavage fluid-based detection of respiratory pathogens in diagnosis of bacterium/fungus-associated pneumonia in critically ill patients.

Bacterium/fungus-associated pneumonia (BAP/FAP) is the prominent cause of high mortality and morbidity with important clinical impacts globally. Effective diagnostic methods and proper specimen types hopefully facilitate early diagnosis of pneumonia and prevent spread of drug-resistant bacteria/fungi among critically ill patients. In the present study, 342 bronchoalveolar lavage fluid (BALF) samples were collected from critically ill patients with pulmonary infections between November 2020 and March 2021. The BALF materials were comparatively employed to screen BAP/FAP through microscopy, culture, antigenic marker and PCR-based methods. The limit of detection (LOD) of cultures and PCR for bacteria/fungi was determined by serial dilution assays. Specimen slides were prepared with Gram staining for microscopic examinations. Microbial cultures and identifications underwent routine clinical protocols with the aid of mass spectrometry. (1,3)-β-D-glucan and galactomannan tests with BALF were carried out accordingly. Direct detection of pathogens in BALF was achieved through PCR, followed by sequencing and BLAST in GenBank database for pathogenic identification. The subjects' demographic and clinical characteristics were well evaluated. BAP/FAP was identified in approximately 47% of the subjects by the BALF-based PCR. The PCR-based diagnostic methods showed improved detection performance for fungi with good LOD, but performed similarly for bacteria, when compared to the cultures. There was poor agreement among traditional microscopy, culture and PCR assays for bacterial detections (kappa value, 0.184 to 0.277). For overall bacterial/fungal detections, the microscopy showed the lowest detecting rate, followed by the cultures, which displayed a slightly higher sensitivity than the microscopy did. The sensitivity of PCR was much higher than that of the other means of interest. However, the traditional cultures rather than antigenic marker-based approaches were moderately consistent with the PCR-based methods in fungal species identification, particularly for Candida and Aspergillus spp. Our findings further revealed that the age, length of hospital stay, invasive procedures and cerebral diseases were likely considered as main risk factors for BAP/FAP. Screening for BALF in critically ill patients with suspected pneumonia pertaining high risk factors using combined PCR-based molecular detection strategies would hopefully contribute to early diagnosis of BAP/FAP and improved prognosis of the patients.

The Impact of mNGS Technology in the Etiological Diagnosis of Severe Pneumonia in Children During the Epidemic of COVID-19.

Metagenomic next-generation sequencing (mNGS) is an emerging technique for pathogen detection. However, most literature on the clinical application of pediatrics generally comprises case reports or small-scale cohort studies. A total of 101 children with community-acquired severe pneumonia admitted to Tianjin Children's Hospital from November 2021 to February 2022 were included. Pathogens in bronchoalveolar lavage fluid (BALF) specimens were detected using mNGS. The performances of mNGS and conventional tests on pulmonary infection diagnosis and pathogen identification were compared. According to our data, mNGS had a broader spectrum for pathogen detection. The mNGS results of BALF showed that the number of children with severe pneumonia hospitalized for mycoplasma pneumoniae infection was more than that for other bacterial infections during the COVID-19 epidemic. In addition, 43 cases (42.6%) had been identified with mixed infection, including 36 cases (35.6%) of Mycoplasma pneumoniae mixed with other pathogenic bacteria. Analytically, the mNGS exhibited significantly enhanced detection in the BALF as compared with the conventional laboratory pathogenic detection approaches (P < 0.05). The Pearson correlation analysis revealed positive correlation between the time of fever during hospitalization and the number of mycoplasma sequences (P < 0.05). Compared with traditional methods, mNGS has a higher etiological detection rate and can comprehensively detect various pathogens of severe pneumonia. Therefore, mNGS of bronchoalveolar lavage fluid should be performed in children with severe pneumonia, which is of great significance for guiding treatment.

Clinical Evaluation of Metagenomic Next-Generation Sequencing for the detection of pathogens in BALF in severe community acquired pneumonia

BackgroundRapid and accurate identification of pathogens is very important for the treatment of Severe community-acquired pneumonia (SCAP) in children. Metagenomic Next-generation sequencing (mNGS) has been applied in the detection of pathogenic bacteria in recent years, while the overall evaluation the application of SCAP in children is lacking.MethodsIn our study, 84 cases of SCAP were enrolled. Bronchoalveolar lavage fluid (BALF) samples were analysed using mNGS; and sputum, blood, and BALF samples were analysed using conventional technology (CT).ResultsAmong the 84 children, 41 were boys, and 43 were girls, with an average age ranging from 2 months to 14 years. The pathogen detection rate of mNGS was higher than that of CT (83.3% [70/84] vs. 63.1% [53/84], P = 0.003). The mNGS was much greater than that of the CT in detecting Streptococcus pneumoniae (89.2% [25/29] vs. 44.8% [13/29], P = 0.001) and Haemophilus influenzae (91.7% [11/12] vs. 33.3% [4/12], P < 0.005). The mNGS also showed superior fungal detection performance compared with that of the CT (81.8% [9/11] vs. 18.2% [2/11], P = 0.004). The mNGS test can detect viruses, such as bocavirus, rhinovirus, and human metapneumovirus, which are not frequently recognised using CT. However, the mNGS detection rate was lower than that of the CT (52.4% [11/21] vs. 95.2% [20/21], P = 0.004) for Mycoplasma pneumoniae (MP). The detection rate of mNGS for mixed infection was greater than that of the CT, although statistical significance was not observed (26.3% [20/39] vs. 21.1% [16/39], P > 0.005). Treatment for 26 (31.0%) children was changed based on mNGS results, and their symptoms were reduced; nine patients had their antibiotic modified, five had antibiotics added, nine had their antifungal medication, and seven had their antiviral medication.ConclusionmNGS has unique advantages in the detection of SCAP pathogens in children, especially S. pneumoniae, H. influenzae, and fungi. However, the detection rate of MP using mNGS was lower than that of the CT. Additionally, mNGS can detect pathogens that are not generally covered by CT, which is extremely important for the modification of the treatment strategy.

Advancing Microbe Detection for Lower Respiratory Tract Infection Diagnosis and Management with Metagenomic Next-Generation Sequencing.

Due to limitations of traditional microbiological methods and the presence of the oropharyngeal normal flora, there are still many pathogens that cause lower respiratory tract infections (LRTIs) cannot be detected. Metagenomic next-generation sequencing (mNGS) has the potential capacity to solve this problem. This retrospective study successively reviewed 77 patients with LRTI and 29 patients without LRTI admitted to Tianjin Medical University General Hospital, China from August 2020 to June 2021. Pathogens in bronchoalveolar lavage fluid (BALF) specimens were detected adopting mNGS and traditional microbiological assays. The diagnostic performance of pathogens was compared between mNGS and BALF culture. The value of mNGS for aetiological and clinical impact investigation in LRTI was also evaluated. Among 77 patients with LRTI, 22.1%, 40.3%, and 65.0% of cases were detected as definite or probable pathogens by culture, all conventional microbiological tests, and mNGS, respectively. Using the final diagnosis as a gold standard, mNGS exhibited a sensitivity of 76.6% (95% confidence interval [CI], 65.6-85.5%), which was considerably superior to that of BALF culture (76.6% vs 18.2%; P < 0.01); specificity of 79.3% (95% CI, 60.3-92.0%), which was similar (79.3% vs 89.7%; P = 0.38); positive-predictive value of 90.8% (95% CI, 81.0-96.5%), and negative-predictive value of 56.1% (95% CI, 39.7-71.5%). According to our data, mNGS identified potential microorganisms in 66.7% (42/63) of culture-negative samples. Among 59 patients with pathogens identified by mNGS, conventional microbiological methods confirmed pathogenic infections in less than half (28/59) cases. Within the 77 patients, 34 (44.2%) patients received pathogen-directed therapy, 7 (9.1%) patients underwent antibiotic adjustment, and 3 (3.9%) patients stopped using antibiotics due to mNGS results. mNGS exhibits high accuracy in diagnosing LRTI, and combine with traditional microbiological tests, causative pathogens can be detected in approximately 70.0% of cases, thus yields a positive effect on antibiotic application.

Metagenomic Next-Generation Sequencing for Pathogens in Bronchoalveolar Lavage Fluid Improve the Survival of Patients with Pulmonary Complications after Allogeneic Hematopoietic Stem Cell Transplantation

Metagenomic Next-Generation Sequencing for Pathogens in Bronchoalveolar Lavage Fluid Improve the Survival of Patients with Pulmonary Complications after Allogeneic Hematopoietic Stem Cell Transplantation

Clinical value of metagenomic next-generation sequencing by Illumina and Nanopore for the detection of pathogens in bronchoalveolar lavage fluid in suspected community-acquired pneumonia patients.

At present, metagenomic next-generation sequencing (mNGS) based on Illumina platform has been widely reported for pathogen detection. There are few studies on the diagnosis of major pathogens and treatment regulation using mNGS based on Illumina versus Nanopore. We aim to evaluate the clinical value of metagenomic next-generation sequencing (mNGS) by Illumina and Nanopore for the detection of pathogens in bronchoalveolar lavage fluid (BALF) in suspected community-acquired pneumonia (CAP) patients. BALF samples collected from 66 suspected CAP patients within 48 hours of hospitalization were divided into two parts, one for conventional culture and the other for mNGS by two platforms (Illumina and Nanopore). The clinical value based on infection diagnosis, diagnostic performance for main pathogens and treatment guidance were assessed. More types of species were detected by Nanopore than Illumina, especially in viruses, fungus and mycobacterium. Illumina and Nanopore showed similar detectability in bacterium except for mycobacterium tuberculosis complex/nontuberculosis mycobacteria. Pathogenic infection was established or excluded in 53 of 66 patients. There was little difference in the coincidence rate between Illumina and Nanopore with the clinical diagnosis, but both were superior to the culture (57.81%, 59.38%, 25%, respectively). Compared with Illumina, the diagnostic area under the curve of Nanopore was higher in fungi, but lower in bacteria and Chlamydia psittaci. There was no statistically significant difference between Illumina and Nanopore in guiding drug treatment (56.1% vs. 50%, p=0.43), but both were superior to the culture (56.1% vs. 28.8%, p=0.01; 50% vs. 28.8%, p=0.01). Single inflammatory indicators could not be used to determine whether the patients with culture-negative BALF were established or excluded from infection. The species detected at 1 h and 4 h by Nanopore were consistent to some extent, and its turn-around time (TAT) was significantly shorter than Illumina (p<0.01). Illumina and Nanopore both have its own advantages in pathogenic diagnosis and play similar roles in infection diagnosis and guiding clinical treatment. Nanopore has a relatively short TAT, which may be promising in rapid etiological diagnosis of acute and critically ill patients.

Application of metagenomic next-generation sequencing in the detection of pathogens in bronchoalveolar lavage fluid of infants with severe pneumonia after congenital heart surgery.

BackgroundMetagenomic next-generation sequencing (mNGS) has become a valuable diagnostic tool in clinical etiology detection due to its rapidity, accuracy, and high throughput. However, the role of this technology in the diagnosis and treatment of infants with severe pneumonia after congenital heart surgery is still unclear.MethodsWe conducted a retrospective cohort study of infants with severe pneumonia after congenital heart surgery. Samples were collected from infants in the hospital’s cardiac intensive care unit between January 2010 and January 2022. The conventional microbiological test (CMT) group consisted of patients who underwent routine microbiological examination, and the infants’ bronchoalveolar lavage fluid was examined. The mNGS group consisted of patients who underwent mNGS and routine microbiological examinations.ResultsThe overall positive rate of mNGS was significantly higher than that of CMT (88.4 vs. 62.5%, P = 0.009). After receipt of the microbiological results, 30/43 (70%) patients in the mNGS group had a change in antibiotic use compared with 14/40 (35%) in the CMT group (P = 0.002). Subsequently, after adjusting the treatment plan according to the microbiological test results, the number of people with improved pulmonary infection in the mNGS group was significantly higher than that in the CMT group (63 vs. 28%, P < 0.05). In addition, the duration of invasive ventilation, length of CICU stay and total hospital length of stay in the mNGS group were significantly lower than those in the CMT group (P < 0.05).ConclusionmNGS is a valuable tool to determine the etiology of infants with severe pneumonia after congenital heart disease surgery. It can significantly improve the sensitivity of pathogen detection, which can help determine appropriate antimicrobial drugs, improve the diagnostic accuracy of the disease, and improve outcomes.

Clinical evaluation of metagenomic next-generation sequencing for detecting pathogens in bronchoalveolar lavage fluid collected from children with community-acquired pneumonia.

This study is to evaluate the usefulness of pathogen detection using metagenomic next-generation sequencing (mNGS) on bronchoalveolar lavage fluid (BALF) specimens from children with community-acquired pneumonia (CAP). We retrospectively collected BALF specimens from 121 children with CAP at Tianjin Children's Hospital from February 2021 to December 2021. The diagnostic performances of mNGS and conventional tests (CT) (culture and targeted polymerase chain reaction tests) were compared, using composite diagnosis as the reference standard. The results of mNGS and CT were compared based on pathogenic and non-pathogenic organisms. Pathogen profiles and co-infections between the mild CAP and severe CAP groups were also analyzed. The overall positive coincidence rate was 86.78% (105/121) for mNGS and 66.94% (81/121) for CT. The proportion of patients diagnosed using mNGS plus CT increased to 99.18%. Among the patients, 17.36% were confirmed only by mNGS; Streptococcus pneumoniae accounted for 52.38% and 23.8% of the patients were co-infected. Moreover, Bordetella pertussis and Human bocavirus (HBoV) were detected only using mNGS. Mycoplasma pneumoniae, which was identified in 89 (73.55%) of 121 children with CAP, was the most frequent pathogen detected using mNGS. The infection rate of M. pneumoniae in the severe CAP group was significantly higher than that in the mild CAP group (P = 0.007). The symptoms of single bacterial infections (except for mycoplasma) were milder than those of mycoplasma infections. mNGS identified more bacterial infections when compared to the CT methods and was able to identify co-infections which were initially missed on CT. Additionally, it was able to identify pathogens that were beyond the scope of the CT methods. The mNGS method is a powerful supplement to clinical diagnostic tools in respiratory infections, as it can increase the precision of diagnosis and guide the use of antibiotics.

The clinical application of metagenomic next-generation sequencing for detecting pathogens in bronchoalveolar lavage fluid: case reports and literature review

ABSTRACT Introduction Clinical metagenomic next-generation sequencing (mNGS) allows a comprehensive genetic analysis of microbial materials. Different from other traditional target-driven molecular diagnostic tests, such as PCR, mNGS is a hypothesis-free diagnostic approach that allows a comprehensive genetic analysis of the clinical specimens that cover nearly any common, rare, and new pathogens ranging broadly from viruses, bacteria, fungi to parasites. Areas covered In this article, we discussed the clinical application of the mNGS using two clinical cases as examples and described the use of mNGS to assist the diagnosis of parasitic pulmonary infection. The advantages and challenges in implementing mNGS in clinical microbiology are also discussed. Expert opinion mNGS is a promising technology that allows quick diagnosis of infectious diseases. Currently, a plethora of sequencing and analysis methods exists for mNGS, each with individual merits and pitfalls. While standards and best practices were proposed by various metagenomics working groups, they are yet to be widely adopted in the community. The development of a consensus set of guidelines is necessary to guide the usage of this new technology and the interpretation of NGS results before clinical adoption of mNGS testing.

Application of metagenomic next-generation sequencing for bronchoalveolar lavage diagnostics in patients with lower respiratory tract infections.

ObjectiveMetagenomic next-generation sequencing (mNGS) has the potential to overcome the shortcomings of traditional culture methods. This study aimed to assess the diagnostic value of mNGS in patients with lower respiratory tract infections (LRTIs).MethodsThis retrospective observational study sequentially enrolled 47 patients with LRTIs admitted to Shenzhen Hospital of Southern Medical University between February 2019 and November 2020. Pathogens in bronchoalveolar lavage fluid (BALF) samples were investigated to compare diagnoses by mNGS with culture methods.ResultsCompared with culture methods, mNGS had a diagnostic sensitivity of 80% and a specificity of 35.13% with an agreement rate of 44.68% between these two methods. mNGS significantly increased the pathogen detection rate.ConclusionsmNGS may show some advantages in identifying a wide range of LRTI pathogens, improving the sensitivity for viruses and atypical pathogens. The clinical application of NGS technology is worth looking forward to.

Diagnostic Significance of Metagenomic Next-Generation Sequencing for Community-Acquired Pneumonia in Southern China.

BackgroundThe morbidity and mortality of community-acquired pneumonia are relatively high, but many pneumonia pathogens cannot be identified accurately. As a new pathogen detection technology, metagenomic next-generation sequencing (mNGS) has been applied more and more clinically. We aimed to evaluate the diagnostic significance of mNGS for community-acquired pneumonia (CAP) in the south of China.MethodsOur study selected CAP patients who visited the 3rd Xiangya Hospital from May 2019 to April 2021. Pathogens in bronchoalveolar lavage fluid (BALF) specimens were detected using mNGS and traditional microbiological culture. mNGS group: detected by both mNGS and BALF culture; control group: detected only by BALF or sputum culture. The diagnostic performance of pathogens and the antibiotic adjustments were compared within mNGS group.ResultsThe incidence of acute respiratory distress syndrome (ARDS) was 28.3% in the mNGS group and 17.3% in the control group. Within the mNGS group, the positive rate of pathogens detected by mNGS was 64%, thus by BALF culture was only 28%. Pathogens detected by mNGS were consisted of bacteria (55%), fungi (18%), special pathogens (18%), and viruses (9%). The most detected pathogen by mNGS was Chlamydia psittaci. Among the pathogen-positive cases, 26% was not pathogen-covered by empirical antibiotics, so most of which were made an antibiotic adjustment.ConclusionsmNGS can detect pathogens in a more timely and accurate manner and assist clinicians to adjust antibiotics in time. Therefore, we recommend mNGS as the complementary diagnosis of severe pneumonia or complicated infections.

Diagnostic value of detection of pathogens in bronchoalveolar lavage fluid by metagenomics next-generation sequencing in organ transplant patients with pulmonary infection

To evaluate the diagnostic value of metagenomics next-generation sequencing (mNGS) in detecting pathogens in bronchoalveolar lavage fluid (BALF) for pulmonary infection in solid organ transplant patients in intensive care unit (ICU). A retrospective study was conducted, the BALF samples from 46 patients with post organ transplant pneumonia/suspected pneumonia admitted to the Department of Critical Care Medicine of the First Affiliated Hospital of University of Science and Technology of China from August 2018 to August 2021 were collected, all tested by simultaneous mNGS and conventional comprehensive microbial test (CMT), and the results of CMT were used as the reference standard to compare the differences in the diagnostic value of mNGS and CMT for pulmonary infections in solid organ transplant patients, and to analyze the diagnostic value of mNGS for mixed infections. (1) Pneumonia pathogens: a total of 31 pathogens were detected in 35 patients, including bacteria (16 species), fungi (9 species) and viruses (6 species). Among them, 25 pathogens were detected by mNGS and CMT, and only 19 pathogens were detected by mNGS. Among the microorganisms isolated by mNGS method, the detection rates of Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae were higher [51.4% (18/35), 42.9% (15/35), 31.4% (11/35), respectively]; Candida albicans, Aspergillus and Pneumocystis carinii were the most commonly detected fungi [31.4% (11/35), 22.9% (8/35), 22.9% (8/35), respectively]; 20 patients were positive for the virus, and the most commonly detected viruses were cytomegalovirus, herpesvirus and EB virus [28.6% (10/35), 20.0% (7/35), 17.1% (6/35), respectively]. In addition, one case of Brucella was detected by mNGS. (2) Diagnostic efficiency: as far as bacterial detection is concerned, 20 cases of negative results were obtained by CMT detection of 35 samples included in the study, and a total of 10 cases of positive results were obtained by mNGS detection of negative samples; the percentage of mNGS positive samples was significantly higher than that of CMT positive samples [odds ratio (OR) = 5.5, 95% confidence interval (95%CI) = 1.2-24.8, P = 0.02]. When compared with CMT, the sensitivity and specificity of mNGS were 93.3% and 50.0%, and the positive predictive value (PPV) and negative predictive value (NPV) were 58.3%, 91.1%. As far as fungal detection was concerned, there was no significant difference in the percentage of positive samples between the two methods (OR = 1.5, 95%CI = 0.5-4.2, P = 0.60); the sensitivity and specificity of mNGS were 72.2% and 64.7%, and the PPV and NPV were 68.4%, 68.8%; CMT test of the 35 included samples produced 17 negative results, and mNGS test of the negative samples produced 6 positive results. A total of 20 patients tested positive for the virus by mNGS. In addition, 23 patients (65.7%) were diagnosed with pulmonary mixed infection. The use of mNGS to detect pathogens in BALF can improve the sensitivity and specificity of bacterial identification of pulmonary infection in critically ill organ transplant patients, and mNGS has obvious advantages in detecting virus and identifying mixed infections.

The Application of Metagenomic Next-Generation Sequencing in Detection of Pathogen in Bronchoalveolar Lavage Fluid and Sputum Samples of Patients with Pulmonary Infection.

Objective To uncover the application value of metagenomic next-generation sequencing (mNGS) in the detection of pathogen in bronchoalveolar lavage fluid (BALF) and sputum samples. Methods Totally, 32 patients with pulmonary infection were included. Pathogens in BALF and sputum samples were tested simultaneously by routine microbial culture and mNGS. Main infected pathogens (bacteria, fungi, and viruses) and their distribution in BALF and sputum samples were analyzed. Moreover, the diagnostic performance of mNGS in paired BALF and sputum samples was assessed. Results The pathogen culture results were positive in 9 patients and negative in 13 patients. No statistical differences were recorded on the sensitivity (78.94% vs. 63.15%, p = 0.283) and specificity (62.50% vs. 75.00%, p = 0.375) of mNGS diagnosis in bacteria and fungus in two types of samples. As shown in mNGS detection, 10 patients' two samples were both positive, 13 patients' two samples were both negative, 7 patients were only positive in BALF samples, and 2 patients' sputum samples were positive. Main viruses mNGS detected were EB virus, human adenovirus 5, herpes simplex virus type 1, and human cytomegalovirus. Kappa consensus analysis indicated that mNGS showed significant consistency in detecting pathogens in two samples, no matter bacteria (p < 0.001), fungi (p = 0.026), or viruses (p = 0.008). Conclusion mNGS showed no statistical differences in sensitivity and specificity of pathogen detection in BALF and sputum samples. Under certain conditions, sputum samples might be more suitable for pathogen detection because of invasiveness of BALF samples.

Broad range detection of viral and bacterial pathogens in bronchoalveolar lavage fluid of children to identify the cause of lower respiratory tract infections

BackgroundKnowledge on the etiology of LRTIs is essential for improvement of the clinical diagnosis and accurate treatment. Molecular detection methods were applied to identify a broad range of bacterial and viral pathogens in a large set of bronchial alveolar lavage (BAL) fluid samples. The patterns of detected pathogens were correlated to the clinical symptoms.MethodsBAL fluid samples and clinical data were collected from 573 hospitalized children between 1 month and 14 years of age with LRTIs, enrolled from January to December 2018. Pathogens were detected using standardized clinical diagnostics, with a sensitive, high-throughput GeXP-based multiplex PCR and with multiplex qPCR. Data were analyzed to describe the correlation between the severity of respiratory tract disease and the pathogens identified.ResultsThe pathogen detection rate with GeXP-based PCR and multiplex qPCR was significantly higher than by clinical routine diagnostics (76.09% VS 36.13%,χ2 = 8.191, P = 0.004). The most frequently detected pathogens in the BAL fluid were human adenovirus (HADV)(21.82%), Mycoplasma pneumoniae (20.24%), human rhinovirus (13.96%), Streptococcus pneumoniae (8.90%) and Haemophilus influenzae (8.90%). In 16.4% of the cases co-detection with two or three different pathogens was found. Viral detection rates declined with age, while atypical pathogen detection rates increased with age. Oxygen supply in the HADV and Influenza H1N1 infected patients was more frequent (49.43%) than in patients infected with other pathogens.ConclusionBroad range detection of viral and bacterial pathogens using molecular methods is a promising and implementable approach to improve clinical diagnosis and accurate treatment of LRTI in children.