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.

UNASSIGNED: Targeted next-generation sequencing (tNGS) has emerged as a rapid diagnostic technology for identifying a wide spectrum of pathogens responsible for pulmonary infections.
UNASSIGNED: Sputum samples were collected from patients unable or unwilling to undergo bronchoalveolar lavage. These samples underwent tNGS analysis to diagnose pulmonary infections. Retrospective analysis was performed on clinical data, and the clinical efficacy of tNGS was compared to conventional microbiological tests (CMTs).
UNASSIGNED: This study included 209 pediatric and adult patients with confirmed pulmonary infections. tNGS detected 45 potential pathogens, whereas CMTs identified 23 pathogens. The overall microbial detection rate significantly differed between tNGS and CMTs (96.7% vs. 36.8%, p < 0.001). Among the 76 patients with concordant positive results from tNGS and CMTs, 86.8% (66/76) exhibited full or partial agreement. For highly pathogenic and rare/noncolonized microorganisms, tNGS, combined with comprehensive clinical review, directly guided pathogenic diagnosis and antibiotic treatment in 21 patients. This included infections caused by Mycobacterium tuberculosis complex, certain atypical pathogens, Aspergillus, and nontuberculous Mycobacteria. Among the enrolled population, 38.8% (81/209) of patients adjusted their treatment based on tNGS results. Furthermore, tNGS findings unveiled age-specific heterogeneity in pathogen distribution between children and adults.
UNASSIGNED: CMTs often fall short in meeting the diagnostic needs of pulmonary infections. This study highlights how tNGS of sputum samples from patients who cannot or will not undergo bronchoalveolar lavage yield valuable insights into potential pathogens, thereby enhancing the diagnosis of pulmonary infections in specific cases.

Metagenomic next-generation sequencing (mNGS) is widely used as a more promising technology than conventional tests. However, its clinical utility in the context of bronchoalveolar lavage fluid (BALF) samples for discriminating between non-severe and severe pneumonia is not well established. Thus, this study aimed to investigate the diagnostic performance of mNGS on BALF samples from 100 individuals suspected of pneumonia, and compared it with conventional microbiological tests (CMT) of BALF samples and the final clinical diagnosis. Twenty-seven cases of non-severe pneumonia and 73 cases of severe pneumonia patients were finally clinically diagnosed. Among 100 cases, diagnostic performance of mNGS and culture showed a significant difference; 65 cases had the same sample types, of which 25 cases were diagnosed as positive by mNGS only (38.46%) and 1 was diagnosed as positive by culture only (1.54%). Moreover, 24 cases were diagnosed positive in both mNGS and culture (36.92%) and 15 cases tested negative in both mNGS and culture (23.08%). Among 35 cases, 28 out of 35 cases were diagnosed as positive by mNGS, while only 4 out of 35 cases were diagnosed as positive by the indirect immunofluorescence method (IIFT). In addition, the positive rate of mNGS was higher than that of culture in cases regardless of prior antibiotic exposure. Mixed pathogens were found to be significantly more prevalent in severe pneumonia patients than in non-severe pneumonia patients. Importantly, among 38 cases who were diagnosed solely by mNGS, 25 patients experienced an improved outcome after physicians changed the therapy according to the mNGS results. In conclusion, the results showed that mNGS of BALF represents a potentially effective tool for detection of mixed pathogens in severe pneumonia.

Immunosuppression predisposes allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients to infection. Prompt and accurate identification of pathogens is crucial to optimize treatment strategies. This multi-center retrospective study aimed to assess the ability of metagenomic next-generation sequencing (mNGS) to detect causative pathogens in febrile allo-HSCT recipients and examined its concordance with conventional microbiological tests (CMT).
We performed mNGS and CMT on samples obtained from 153 patients with suspected infection during allo-HSCT. Patients were grouped based on their neutropenic status at the time of sampling.
The mNGS test was more sensitive than CMT (81.1% vs. 53.6%, P<0.001) for diagnosing clinically suspected infection, especially in the non-neutropenia cohort. mNGS could detect fungi and viruses better than bacteria, with a higher sensitivity than CMT. Immune events were diagnosed in 57.4% (35/61) of the febrile events with negative mNGS results, and 33.5% (48/143) with negative CMT results (P=0.002). The treatment success rate of the targeted anti-infection strategy was significantly higher when based on mNGS than on empirical antibiotics (85% vs. 56.5%, P=0.004).
The mNGS test is superior to CMT for identifying clinically relevant pathogens, and provides valuable information for anti-infection strategies in allo-HSCT recipients. Additionally, attention should be paid to immune events in patients with negative mNGS results.

UNASSIGNED: To evaluate the value and challenges of real-world clinical application of metagenomic next-generation sequencing (mNGS) for bronchoalveolar lavage fluid (BALF) in HIV-infected patients with suspected multi-pathogenic pneumonia.
UNASSIGNED: Fifty-seven HIV-infected patients with suspected mixed pneumonia who were agreed to undergo the bronchoscopy were recruited and retrospectively reviewed the results of mNGS and conventional microbiological tests (CMTs) of BALF from July 2020 to June 2022.
UNASSIGNED: 54 patients were diagnosed with pneumonia including 49 patients with definite pathogens and five patients with probable pathogens. mNGS exhibited a higher diagnostic accuracy for fungal detection than CMTs in HIV-infected patients with suspected pulmonary infection. The sensitivity of mNGS in diagnosis of pneumonia in HIV-infected patients was much higher than that of CMTs (79.6% vs 61.1%; P < 0.05). Patients with mixed infection had lower CD4 T-cell count and higher symptom duration before admitting to the hospital than those with single infection. The detection rate of mNGS for mixed infection was significantly higher than that of CMTs and more co-pathogens could be identified by mNGS. The most common pattern of mixed infection observed was fungi-virus (11/29, 37.9%), followed by fungi-virus-bacteria (6/29, 20.7%) coinfection in HIV-infected patients with multi-pathogenic pneumonia.
UNASSIGNED: mNGS improved the pathogens detection rate and exhibited advantages in identifying multi-pathogenic pneumonia in HIV-infected patients. Early performance of bronchoscopy and mNGS are recommended in HIV-infected patients with low CD4 T cell counts and long duration of symptoms. The most common pattern of mixed infection observed was fungi-virus, followed by fungi-virus-bacteria coinfection in HIV infected patients with multi-pathogenic pneumonia.

UNASSIGNED: Metagenomic next-generation sequencing (mNGS) has been widely studied, due to its ability of detecting all the microbial genetic information unbiasedly in a sample at one time and not relying on traditional culture. However, the application of mNGS in the diagnosis of clinical pathogens remains challenging.
UNASSIGNED: From December 2019 to March 2021, 134 specimens including Broncho alveolar lavage fluid (BAFL), blood, sputum, cerebrospinal fluid (CSF), bile, pleural fluid, pus, were continuously collected in The First Hospital of Qinhuangdao, and their retrospective diagnoses were classified into infectious disease (128, 95.5%) and noninfectious disease (6, 4.5%). The pathogen-detection performance of mNGS was compared with conventional microbiological tests (CMT) and culture method. In addition, the antibiotic resistance genes (ARGs) and evolutionary relationship of common drug-resistant A. baumannii were also analyzed.
UNASSIGNED: Compared with CMT and culture methods, mNGS showed higher sensitivity in pathogen detection (74.2% vs 57.8%; P < 0.001 and 66.3% vs 31.7%; P < 0.001, respectively). Importantly, for cases that mNGS-positive only, 18 (35%) cases result in diagnosis modification, and 7 (23%) cases confirmed the clinical diagnosis. In 17 cases that A. baumannii were both detected in mNGS and culture, ade genes were the most frequently detected ARGs (from 13 cases), followed by sul2 and APH(3\")-Ib (both from 12 cases). High consistency was observed among these ARGs and the related phenotype (100% for ade genes, 91.6% for sul2 and APH(3\")-Ib). A. baumannii strains were classified into three groups, and most were well-clustered. It suggested those strains may be the epidemic strains.
UNASSIGNED: In our study, mNGS had a higher sensitivity than CMT and culture method. And the result of ARGs frequency and cluster analysis of A. baumannii was of great significance to the anti-infective therapy.

BACKGROUND: To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) in immunocompromised patients for the diagnosis of suspected pneumonia in comparison with that of conventional microbiological tests (CMTs).
METHODS: Sixty-nine immunocompromised patients with suspected pneumonia received both CMTs and mNGS of BALF were analyzed retrospectively. The diagnostic value was compared between CMTs and mNGS, using the clinical composite diagnosis as the reference standard.
RESULTS: Sixty patients were diagnosed of pneumonia including fifty-two patients with identified pathogens and eight patients with probable pathogens. Taking the composite reference standard as a gold standard, 42 pathogens were identified by CMTs including nine bacteria, 17 fungi, 8 virus, 6 Mycobacterium Tuberculosis, and two Legionella and 19(45%) of which were detected by BALF culture. As for mNGS, it identified 76 pathogens including 20 bacteria, 31 fungi, 14 virus, 5 Mycobacterium Tuberculosis, four Legionella and two Chlamydia psittaci. The overall detection rate of mNGS for pathogens were higher than that of CMTs. However, a comparable diagnostic accuracy of mNGS and CMTs were found for bacterial and viral infections. mNGS exhibited a higher diagnostic accuracy for fungal detection than CMTs (78% vs. 57%, P < 0.05), which mainly because of the high sensitivity of mNGS in patients with Pneumocystis jirovecii pneumonia (PJP) (100% vs. 28%, P < 0.05). Nineteen patients were identified as pulmonary co-infection, mNGS test showed a higher detection rate and broader spectrum for pathogen detection than that of CMTs in co-infection. Moreover, Pneumocystis jirovecii was the most common pathogen in co-infection and mNGS have identified much more co-pathogens of PJP than CMTs.
CONCLUSIONS: mNGS of BALF improved the microbial detection rate of pathogens and exhibited remarkable advantages in detecting PJP and identifying co-infection in immunocompromised patients.

Infections are the major cause of morbidity and mortality in patients with primary immunodeficiency disease (PID). Timely and accurate microbiological diagnosis is particularly important in these patients. Metagenomic next-generation sequencing (mNGS) has been used for pathogen detection recently. However, few reports describe the use of mNGS for pathogen identification in patients with PID.
To evaluate the utility of mNGS for detecting pathogens in patients with PID, and to compare it with conventional microbiological tests (CMT).
This single center retrospective study investigated the diagnostic performance of mNGS for pathogens detection in PID patients and compared it with CMT. Sixteen PID patients with suspected infection were enrolled, and medical records were analyzed to extract detailed clinical characteristics such as gene variation, immune status, microbial distribution, time-consuming of mNGS and CMT, treatment, and outcomes.
mNGS identified pathogenic microbe in 93.75% samples, compared to 31.25% for culture and 68.75% for conventional methods, and detected an extra 18 pathogenic microorganisms including rare opportunistic pathogens and Mycobacterium tuberculosis. Pathogen identification by mNGS required 48 hours, compared with bacterial culture for 3-7 days and even longer for fungus and Mycobacterium tuberculosis culture.
mNGS has marked advantages over conventional methods for pathogenic diagnosis, particularly opportunistic pathogens and mixed infections, in patients with PID. This method might enable clinicians to make more timely and targeted therapeutic decisions, thereby improving the prognosis of these patients.