Clinical implications of metagenomics next-generation sequencing (mNGS) in sepsis have not been fully evaluated. This study aimed to determine the diagnostic, therapeutic, and prognostic impacts of mNGS in sepsis.

This multicenter prospective study was conducted at 19 sites in China from 2020 to 2021, and 859 adult patients hospitalized with sepsis were enrolled. The advantages, challenges, knowledge gaps and privacy risks of mNGS were carefully introduced to all participants, and participants chose on their own to either receive conventional microbiological test (CMT) alone (conventional-test-only group, n = 394) or receive mNGS test along with CMT (combined test group, n = 465). For prognostic analysis, the primary endpoint was 28-day mortality. Secondary endpoints included 7-day mortality and average per-day hospital cost. Inverse probability of treatment weighting was used to balance covariates between groups. Concurrent CMT and mNGS results from patients in the combined test group were used for diagnostic analyses. Therapeutic impact of mNGS was evaluated based on subsequent antibiotic adjustment.

Compared with composite reference standard, the positive percent agreement of mNGS among infected site samples was significantly higher than that of CMT (92.0% [95% CI, 88.7 to 94.5] vs. 51.1% [95% CI, 45.9 to 56.2], p < 0.001), while the negative percent agreement of mNGS was inferior to that of CMT (39.6% [95% CI, 29.5 to 50.4] vs. 69.2% [95% CI, 58.7 to 78.5], p < 0.001). The mNGS test identified causal microbes in 344 (74.0%) patients, and concomitant antibiotic changes occurred in 136 patients (29.2%). Death by day 7 occurred in 24 of 465 (5.2%) patients in the combined test group and in 34 of 394 (8.6%) patients in the conventional-test-only group (hazard ratio, 0.44 [95% CI, 0.26 to 0.77], p = 0.004). However, no significant difference in 28-day mortality was observed between two study groups (hazard ratio, 0.82 [0.56 to 1.20], p = 0.300).

The mNGS test of infected site samples exhibited 40% higher pathogen detection rate than CMT in patients with sepsis, which led to improved etiological diagnosis and tailored antibiotic therapy. Additional use of mNGS halved the risk of early death in 7 days, but did not improve 28-day survival in patients with sepsis.

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The aim of this study was to evaluate the clinical value of metagenomic next-generation sequencing (mNGS) of blood samples for identifying Pneumocystis jirovecii pneumonia (PJP) in patients with human immunodeficiency virus (HIV).

A total of 76 people with HIV (PWH) with suspected lung infections were enrolled in the study. The patients were divided into two groups: the PJP group and the non-PJP group.All patients underwent pulmonary computed tomography scans, and blood or respiratory tract specimens were subjected to mNGS and conventional microbiological tests. Patient characteristics were collected from their medical records.

Thirty patients were diagnosed with PJP and 46 were confirmed to have non-P jirovecii (Pj) infectious pneumonia. mNGS was conducted on bronchoalveolar lavage fluid samples from 25 patients and on blood samples from 59 patients. Twenty-one of 22 (95.5%) blood samples from the PIP group contained sequences of Pi, with the number of specific reads for circulating Pj sequences ranging from 2 to 2035. In the non-PJP group, 4 blood samples exhibited low Pj sequences, ranging from 1 to 2 reads. The sensitivity and specificity for blood samples were 95.5% (95% confidence interval [CI], 91.2%-98.4%) and 90.0% (95% Cl, 89.5%-100%), respectively.

Our study indicates that mNGS of blood samples exhibits high sensitivity and specificity for diagnosing PJP in PWH. Caution should be exercised when interpreting low Pj mNGS read counts in blood samples; the definitive diagnosis of PJP relies on the synthesis of clinical data with Pj mNGS results. Further studies are necessary to validate this finding.

Interpreting the results of metagenomic next-generation sequencing (mNGS) presents a significant challenge in both clinical and laboratory contexts.

A retrospective analysis was conducted to validate mNGS findings, with a particular emphasis on Mycobacterium tuberculosis, Mycoplasma pneumoniae, and Pneumocystis jirovecii as representative pathogens, examined from both clinical and laboratory perspectives.

Based on a comprehensive clinical analysis, the mNGS demonstrated detection accuracies for M. tuberculosis, M. pneumoniae, and P. jirovecii of 87.0% (60 out of 69; 95% confidence interval [CI], 77.04%-92.99%), 97.6% (81 out of 83; 95% CI, 91.63%-99.34%), and 78.9% (45 out of 57; 95% CI, 66.72%-87.53%), respectively. Conversely, when incorporating laboratory confirmation from a variety of detection methodologies, the accuracy rates for mNGS in identifying M. tuberculosis, M. pneumoniae, and P. jirovecii were 92.7% (51 out of 55; 95% CI, 82.74%-97.14%), 82.3% (51 out of 62; 95% CI, 70.96%-89.80%), and 83.9% (26 out of 31; 95% CI, 67.36%-92.91%), respectively. Additionally, our analysis revealed no statistically significant difference in read counts and relative abundances between mNGS results deemed clinically as false positives and those considered true positives (P < 0.05).

In contemporary clinical practice, the detection of positive results from mNGS is notably high from both laboratory and clinical standpoints. Nonetheless, the interpretation of results with low read counts presents significant challenges for both clinical and laboratory environments under current conditions.

Accurate diagnosis of Pneumocystis jirovecii pneumonia (PJP) in HIV patients remains challenging. This study compares metagenomic next-generation sequencing (mNGS) with PCR, GMS staining, and serum β-D-glucan (BG) assays for PJP detection and co-infection identification.

BALF samples from 34 HIV-positive PJP patients and 50 non-PJP controls were analyzed. Diagnostic performance metrics (sensitivity, specificity, NPV, AUC) and co-pathogen profiles were evaluated for mNGS versus conventional methods.

mNGS and PCR both achieved 100% sensitivity. mNGS showed higher specificity (91.3% vs. 88%) and AUC (0.898 vs. 0.940 for PCR). Co-infections were detected in 67.6% of PJP cases by mNGS, including cytomegalovirus (41.2%), Epstein-Barr virus (29.4%), and non-tuberculous mycobacteria (14.7%). GMS and BG assays exhibited lower sensitivity (64.7% and 76.5%, respectively).

mNGS offers superior specificity, accuracy, and co-infection detection compared to traditional methods. Its high NPV (100%) supports clinical utility in ruling out PJP. While resource-intensive, mNGS is a promising first-line diagnostic tool for HIV-associated PJP, particularly in polymicrobial infection settings.

To analyze independent risk factors and the distribution of pathogenic bacteria associated with pulmonary infection (PI) in patients with non-small cell lung cancer (NSCLC); and to explore the utility of peripheral blood-related inflammatory markers in the diagnosis of PI after comprehensive treatment of NSCLC.

We retrospectively analyzed patients who were first diagnosed with NSCLC and received anti-tumor therapy from January 1, 2022 to December 31, 2022. There were 150 males and 50 females enrolled, and with a median age of 63 years. They were divided into PI (n=59) and non-PI (n=141) groups. The patients' characteristics, treatment histories, and peripheral blood inflammatory indicators of the two groups were compared.

Eighty-two strains of pathogens were isolated from 59 patients with NSCLC complicated by PI, of which 35 (42.7%) were Gram-negative and 29 (35.4%) were Gram-positive bacteria, 15 (18.3%) were fungi, and 3 (3.7%) were viruses. Logistic regression analysis revealed that ≥ 4 cycles of chemotherapy, radiotherapy, and higher neutrophil count before antineoplastic treatment were independent risk factors for PI in patients with NSCLC (P<0.05). C-reactive protein (CRP) and CRP-to-albumin ratio (CAR) exhibited high area under the curve values of 0.807 and 0.791, respectively. CRP demonstrated high sensitivity (Cutoff Value:10.095, P<0.001), whereas CAR had high specificity (Cutoff Value:0.747, P<0.001) for the early diagnosis of PI in patients with NSCLC.

Gram-negative bacteria were the main pathogens causing PI in patients with NSCLC. Greater than or equal to 4 cycles of chemotherapy, radiotherapy, and elevated neutrophil count before antineoplastic treatment were independent risk factors for PI in patients with NSCLC. Elevated CRP levels and higher CAR demonstrated a certain diagnostic utility for PI in patients with NSCLC after antitumor treatment.

Targeted next-generation sequencing (tNGS) has become a trending tool in the field of infection diagnosis, but concerns are also raising about its performance compared with metagenomic next-generation sequencing (mNGS). This study aims to explore the clinical feasibility of a tNGS panel for respiratory tract infection diagnosis and compare it with mNGS in the same cohort of inpatients.

180 bronchoalveolar lavage fluid samples were collected and sent to two centers for mNGS and tNGS blinded tests, respectively. The concordance between pathogen reports of both methods and the clinical significance among samples with/without known etiology was further evaluated.

Overall, both methods displayed high agreement on pathogen reports, as the average percent agreement reached 95.29%. But tNGS presented a slightly higher detection rate per species than mNGS (PWilcoxon=1.212e-05; standard mean difference = 0.2887091), as detection rates for 32 out of 48 species were higher than those of mNGS. Due to limitations of panel coverage, tNGS identified 28 fewer species than mNGS, among which only 3 were considered clinically relevant. In reference to composite reference standard, accuracy, sensitivity, and specificity combining both tNGS and mNGS reached 95.61%, 96.71%, and 95.68%, respectively, while positive prediction value (PPV) was low at 48.13%, which was caused by low agreement regarding opportunistic pathogens. tNGS and mNGS improved the etiology identification in 30.6% (55/180) and 33.9% (61/180) cases, respectively.

Collectively, tNGS presented a similar overall performance in pathogen identification compared to mNGS, but outperformed in some pathogens. This study also demonstrated that deployment of tNGS significantly improves etiology identification in routine practice and provides hints for clinical decisions. The low agreement between clinical diagnosis and NGS reports towards opportunistic pathogens implies that adjudication is essential for report interpretation. Finally, We proposed tNGS as a diagnosis option in clinical practice due to its cost-efficiency.

Next-generation sequencing (NGS) is a promising diagnostic tool for pathogens diagnosis. The aim of this study is to evaluate the application of NGS-based antimicrobial therapy on clinical outcomes in severe community-acquired pneumonia (SCAP) patients with empiric antimicrobial therapy failure.

We performed a multi-center, retrospective cohort of SCAP patients with initial empiric therapy failure. Propensity score (PS) matching was used to obtain balance among the baseline variables in NGS group (n = 82) and conventional group (n = 82). We compared the diagnostic performance of NGS with conventional microbial culture. We also compared the impact of NGS-based antimicrobial therapy on the prognosis of patients with empirical antimicrobial therapy failure.

The positive rate of NGS was higher than that of conventional pathogen detection methods (92.6% vs. 74.7%, P = 0.001). Compared to the conventional group, the NGS group has a considerably higher modifications rate of antibiotic treatment (73.2% vs. 54.9%, P = 0.015). The mortality of NGS group was significantly lower than that of conventional group (28.0% vs. 43.9%, P = 0.034). Moreover, the detection of NGS can significantly shorten the ventilation time (P = 0.046), and reduce the antibiotic cost (P = 0.026).

NGS is a valuable tool for microbial identification of SCAP patient with initial empiric therapy failure.

Anti-melanoma differentiation-associated gene 5 antibody-positive dermatomyositis (anti-MDA5+DM) patients are associated with considerable mortality, and opportunistic infections including Pneumocystis jirovecii pneumonia (PJP)is the main cause. This study was to identify clinical characteristics, risk factors, and prognostic factors of PJP diagnosed by bronchoalveolar lavage fluid (BALF) metagenomic next-generation sequencing (mNGS) in anti-MDA5+ DM patients.

In this retrospective observational study, all patients admitted with suspected pneumonia were detected for mNGS in BALF. The demographics, comorbidities, laboratory parameters, and treatments of the patients were compared and analyzed in both groups to identify the potential risk factors for PJP and death via Logistic regression and Cox proportional hazards regression, respectively.

Overall, 92 patients were included in this study, 46(50.0%) were defined as PJP+ group, and the other 46 (50.0%) as PJP- group, and 31(67.4%) PJP occurred in the first 3 months. Increased neutrophil-lymphocyte ratio (NLR) and CRP were independent risk factors for PJP occurrence, while trimethoprim-sulfamethoxazole (TMP/SMZ) prophylaxis was an independent protective factor (all p<0.05). The three-months mortality rate was higher in the PJP+ group compared to PJP- group (43.5% vs 23.9%, p=0.047). Rapidly progressive interstitial lung disease (RPILD) was a main predictor of mortality in anti-MDA5+DM patients with PJP, whereas glucocorticoid use was a significant protective factor.

PJP has high prevalence and mortality in anti-MDA5+DM, while TMP/SMZ prophylaxis significantly reduces PJP risk. Mortality in PJP+ patients is primarily concentrated within the first 3 months, associated with RPILD. Early intervention with corticosteroids and prophylactic measures are crucial in reducing mortality.

Early and accurate identification of pathogens in pulmonary infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is critically important. The clinical usefulness of metagenomic next-generation sequencing (mNGS) in the diagnosis of pulmonary infections after allo-HSCT remains under discussion. This multicenter retrospective study was conducted to compare mNGS and conventional microbiological tests (CMTs) in identifying the pathogens of pulmonary infections in allo-HSCT recipients. One hundred forty allo-HSCT recipients with suspected pulmonary infections who underwent bronchoscopy were included. mNGS and CMTs performed on bronchoalveolar lavage fluid specimens showed 71.4% positivity on mNGS compared to 55.0% positivity on CMTs. mNGS identified 182 pathogens, including bacteria (n = 88), fungi (n = 35) and viruses (n = 59), compared to 106 pathogens detected by CMTs (bacteria, n = 31; fungi, n = 24; viruses, n = 51). Pulmonary infection was finally diagnosed in 98 patients, including 22 bacterial, 7 fungal, 18 viral, and 48 mixed infections and 3 infections with an unknown pathogen. Mixed infections were identified in 50.5% of the patients with pulmonary infection. The sensitivity of mNGS and CMTs for diagnosing pulmonary infections was 88.8% and 69.4%, respectively (P = .001), and the specificity were 81.0% and 85.7%, respectively (P = .688). Our findings suggest that mNGS may be a promising technology for diagnosing pulmonary infections in allo-HSCT recipients.

This study aimed to identify the risk factors and construct the diagnostic model associated with pneumocystis pneumonia (PCP) in pediatric patients.

This retrospective observational study analyzed 34 cases of PCP and 51 cases of other types of pneumonia treated at Children's Hospital Affiliated to Shandong University between January 2021 and August 2023. Multivariate binary logistic regression was used to identify the risk factors associated with PCP. Receiver operating characteristic curves and calibration plots were constructed to evaluate the diagnostic model.

Twenty clinical variables significantly differed between the PCP and non-PCP groups. Multivariate binary logistic regression analysis revealed that dyspnea, body temperature>36.5°C, and age<1.46 years old were risk factors for PCP. The area under the curve of the diagnostic model was 0.958, the P-value of Hosmer-Lemeshow calibration test was 0.346, the R2 of the calibration plot for the actual and predicted probability of PCP was 0.9555 (P<0.001), and the mean Brier score was 0.069. In addition, metagenomic next-generation sequencing revealed 79.41% (27/34) and 52.93% (28/53) mixed infections in the PCP and non-PCP groups, respectively. There was significantly more co-infection with cytomegalovirus and Streptococcus pneumoniae in the PCP group than that in the non-PCP group (p<0.05).

Dyspnea, body temperature>36.5°C, and age<1.46 years old were found to be independent risk factors for PCP in pediatric patients. The probability of co-infection with cytomegalovirus and S. pneumoniae in the PCP group was significantly higher than that in the non-PCP group.

Chlamydia psittaci pneumonia (CPP) exhibits similar characteristics as of COVID-19 with respect to clustering outbreaks and onset symptoms. This study is aimed at exploring the different clinical manifestations of both pneumonias to establish a simple nomogram to distinguish them. This multicenter, retrospective, case-control study compared two independent cohorts of patients with CPP or COVID-19. The risk factors of CPP were analyzed using multivariate logistic regression, which was used to establish the nomogram. Both patients with CPP and COVID-19 exhibited similar clinical symptoms. As compared to patients with COVID-19, a higher proportion of patients with CPP had nervous system symptoms. Patients with CPP had higher inflammatory indicators, creatine kinase, and lower lymphocyte and albumin. They also had lower proportions of ground-glass opacity and bilateral lung involvement than COVID-19 patients. Furthermore, patients with CPP had higher 30 day mortality as well as higher rates of severe pneumonia, septic shock, and ICU admission. Multivariate logistic regression showed that nervous system symptoms, lymphocytes, creatine kinase, bilateral lung lesions, and ground-glass opacity were risk factors for CPP. Incorporating these five factors, the nomogram achieved good concordance index of 0.989 in differentiating CPP from COVID-19, and had well-fitted calibration curves. Despite similar clinical characteristics, nervous system symptoms, lymphocyte, creatine kinase, lesions in bilateral lungs, and ground-glass opacity may help in differentiating the pneumonias. These were combined into a clinically useful nomogram for rapid and early identification of CPP to avoid misdiagnosis and help in the decision-making process.

This study aimed to assess the diagnostic values of bronchoalveolar lavage fluid (BALF) real-time polymerase chain reaction (PCR) and BALF metagenomic next-generation sequencing (mNGS) for Pneumocystis jirovecii pneumonia (PJP) in patients infected with human immunodeficiency virus (HIV).

A total of 99 HIV-infected PJP patients and 61 HIV-infected patients diagnosed with non-PJP pneumonia between March 2019 and December 2022 were enrolled. P. jirovecii and multiple other co-pathogens detected in BALF by mNGS were analyzed. The clinical final diagnosis was employed as a benchmark. We compared the diagnostic performance of mNGS in PJP with serum BDG and BALF real-time PCR. The mixed infections detected by mNGS and modifications of antimicrobial treatment were also analyzed.

The sensitivity of mNGS test of BALF samples reached 85.86%, which was significantly higher than serum BDG (39.39%, P < 0.001). The sensitivity of BALF P. jirovecii PCR (84.85%) was similar with mNGS (P > 0.05). The specificity of mNGS (100%) was also same as PCR (100.0%), and superior to serum BDG (88.52%, P < 0.001). Besides, mNGS performs remarkably well in identifying co-pathogens of PJP patients infected with HIV. In addition to P. jirovecii, 82 cases (82.83%) of other co-pathogens were identified based on mNGS. Moreover, thirty-four patients (34.34%) increased therapeutic dose of trimethoprim-sulfamethoxazole (TMP-SMZ) based on BALF P. jirovecii PCR. Based on the mNGS results, initial antimicrobial treatment was modified in 86.87% (86/99) of PJP patients.

BALF mNGS and real-time PCR are two powerful techniques for rapid diagnosis of PJP with high specificity and sensitivity. Moreover, the benefit of mNGS is that it may identify other organisms besides PJP and it may benefit proper and prompt treatment.

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.

Timely adjustments of antibiotic and corticosteroid treatments are vital for patients with diffuse parenchymal lung diseases (DPLDs). In this study, 41 DPLD patients with negative metagenomic next-generation sequencing (mNGS) results who were responsive to corticosteroids were enrolled. Among these patients, about 26.8% suffered from drug-induced DPLD, while 9.8% presented autoimmune-related DPLD. Following the report of the negative mNGS results, in 34 patients with complete antibiotics administration profiles, 79.4% (27/34) patients discontinued antibiotics after receiving negative mNGS results. Moreover, 70.7% (29/41) patients began or increased the administration of corticosteroid upon receipt of negative mNGS results. In the microbiota analysis, Staphylococcus and Stenotrophomonas showed higher detection rates in patients with oxygenation index (OI) below 300, while Escherichia and Stenotrophomonas had higher abundance in patients with pleural effusion. In summary, our findings demonstrated the clinical significance of mNGS in assisting the antibiotic and corticosteroid treatment adjustments in corticosteroid-responsive DPLD. Lung microbiota may imply the severity of the disease.

Metagenomic next-generation sequencing (mNGS) excels in diagnosis of infection pathogens. We aimed to evaluate the performance of mNGS for the diagnosis of Pneumocystis jirovecii pneumonia (PJP) in non-HIV infected children.

Totally 36 PJP children and 61 non-PJP children admitted to the pediatric intensive care unit from March 2018 to December 2021 were retrospectively enrolled. Clinical features of PJP children were summarized. 1,3-β-D glucan (BDG) test and bronchoalveolar lavage fluid (BALF) mNGS were used for evaluation of PJP diagnostic performance. Antimicrobial management modifications for PJP children after the mNGS results were also reviewed.

Pneumocystis jirovecii was detected in all PJP children by mNGS (36/36), and the sensitivity of mNGS was 100% (95% confidence interval [CI]: 90.26-100%). The sensitivity of BDG was 57.58% (95% CI: 39.22-74.52%). Of the 26 (72.2%) PJP patients with mixed infection, twenty-four (66.7%) were detected by BALF-mNGS. Thirteen patients (36.1%) had their antimicrobial management adjusted according to the mNGS results. Thirty-six PJP children included 17 (47.2%) primary immunodeficiency and 19 (52.8%) secondary immunodeficiency, of whom 19 (52.8%) survived and 17 (47.2%) died. Compared to survival subgroup, non-survival subgroup had a higher rate of primary immunodeficiency (64.7% vs. 31.6%, P = 0.047), younger age (7 months vs. 39 months, P = 0.011), lower body weight (8.0 kg vs. 12.0 kg, P = 0.022), and lower T lymphocyte counts.

The mortality rate of PJP in immunosuppressed children without HIV infection is high and early diagnosis is challenging. BALF-mNGS could help identify PJP and guide clinical management.

Pneumocystis jirovecii pneumonia (PJP) is a life-threatening infection in immunocompromised individuals. Immune checkpoint inhibitor (ICI) has brought significant survival benefit in lung cancer patients. Although the few studies showed there was high mortality in PJP patients with ICI use, these studies had no comparative control groups.

A retrospective study was conducted to compare the mortality in PJP patients with lung cancer between those treated with ICI and a concurrent control group treated without ICI.

A total number of 20 non-human immunodeficiency virus (HIV) patients with confirmed PJP and co-existing lung cancer were included in the current study, and classified into ICI group (n=9) and non-ICI group (n=11).There was a clear trend to a shorter onset of PJP in ICI group than non-ICI group (118.9 ± 60.9 vs 253.0 ± 185.1 days), although without statistical significance (p=0.053). Bronchoscopic alveolar lavage fluid were collected from all patients and used to identify Pneumocystis jirovecii. In both groups, metagenomics next-generation sequencing (mNGS) were the most used diagnostic techniques. Within 28 days after the onset of PJP, mortality was significantly higher in the ICI group than non-ICI group (33.3% vs 0, p=0.042).

Lung cancer patients with ICI use had a higher mortality rate after PJP infection than patients without ICI use. Prospective studies with larger sample size and a multi-center design are warranted to further verify the present results.

Interstitial lung disease (ILD) is a new risk category for pneumocystis pneumonia (PCP) with a high mortality rate. The definite diagnostic criteria of PCP in ILD patients have not been established until now. The aims of this study were to identify potential risk factors of PCP in patients with ILD, and to evaluate the performance of metagenomic next-generation sequencing (mNGS), CD4 + T cell count, (1-3)-β-D-Glucan (BG) and lactate dehydrogenase (LDH) in the diagnosis of PCP in ILD patients.

This is a retrospective, single-center, case-control study. ILD patients who underwent mNGS from December 2018 to December 2022 were included in the study. Based on the diagnosis criteria of PCP, these patients were divided into PCP-ILD and non-PCP-ILD groups. The potential risk factors for PCP occurrence in ILD patients were analysed via logistic regression. The diagnostic efficacy of mNGS was compared with serological biomarkers.

92 patients with ILD were enrolled, 31 of which had a definite PCP and were assigned to the PCP-ILD group while 61 were to the non-PCP-ILD group. The infection rate of PJ in ILD patients was 33.7% (31/92). The history of glucocorticoid therapy, CD4 + T cell count, BG level and traction bronchiectasis on HRCT were associated with PCP occurrence in ILD patients. LDH level did not reach statistical significance in the logistic regression analysis. mNGS was confirmed as the most accurate test for PCP diagnosis in ILD patients.

ILD is a new risk group of PCP with high PCP prevalence. Clinicians should pay close attention to the occurrence of PCP in ILD patients who possess the risk factors of previous glucocorticoid therapy, decreased CD4 + T cell count, increased BG level and absence of traction bronchiectasis on HRCT. mNGS showed the most excellent performance for PCP diagnosis in ILD patients. Peripheral blood CD4 + T cell count and BG level are alternative diagnostic methods for PCP in ILD patients. However, the diagnostic value of serum LDH level was limited in ILD patients.

To describe the epidemiology of Pneumocystis jirovecii pneumonia and colonization diagnosed by next-generation sequencing (NGS) and explore the usefulness of the number of P. jirovecii sequence reads for the diagnosis of P. jirovecii pneumonia.

We examined the NGS results for P. jirovecii in respiratory samples collected from patients and analysed their clinical, radiological and microbiological characteristics.

Among 285 respiratory samples collected over a 12-month period (January to December 2022), P. jirovecii sequences were detected in 56 samples from 53 patients. Fifty (94.3%) of the 53 patients were HIV-negative. Following our case definitions, 37 (69.8%) and 16 (30.2%) of the 53 patients had P. jirovecii infection and colonization respectively. P. jirovecii infection was associated with presence of underlying disease with immunosuppression (94.6% vs 18.8%, P < 0.05), positive serum 1,3-β-D-glucan (41.2% vs 0%, P < 0.01) and higher number of P. jirovecii sequence reads (P < 0.005). In contrast, P. jirovecii colonization was associated with the male sex (93.8% vs 54.1%, P < 0.01), another definitive infectious disease diagnosis of the respiratory tract (43.8% vs 2.7%, P < 0.001) and higher survival (100% vs 67.6%, P < 0.01). Although P. jirovecii pneumonia was associated with higher number of P. jirovecii reads in respiratory samples, only a sensitivity of 82.14% and a specificity of 68.75% could be achieved.

Detection of P. jirovecii sequences in respiratory samples has to be interpreted discreetly. A combination of clinical, radiological and laboratory findings is still the most crucial in determining whether a particular case is genuine P. jirovecii pneumonia.

Lymphoma is complicated by intricate infections, notably Pneumocystis jirovecii pneumonia (PJP), marked by rapid progression, respiratory failure, and high mortality. Rapid diagnosis of PJP and effective administration of the first-line treatment trimethoprim-sulfamethoxazole (TMP-SMX) are important. For patients intolerant to TMP-SMX, selecting appropriate alternatives is challenging, necessitating careful decisions to optimize diagnosis and treatment. We present a lymphoma case complicated by PJP, illustrating medication adjustment until a positive response was observed.

A 41-year-old male patient with lymphoma presented with a week-long history of fever, fatigue, cough, sputum, chest tightness, and exertional dyspnea, unresponsive to treatment. Routine laboratory examinations revealed no pathogenic bacteria. PJ and Mycobacterium tuberculosis (MTB) were detected in bronchoalveolar lavage fluid (BALF) using metagenomic next-generation sequencing (mNGS). On Day 1 of admission, meropenem, TMP-SMX, and rifampicin+isoniazid+levofloxacin were administered. However, the patient developed drug-induced hepatotoxicity and gastrointestinal adverse reactions after six days of treatment. After a multidisciplinary team discussion, anti-tuberculosis therapy was stopped because of insufficient evidence of tuberculosis infection. A reduced dose of TMP-SMX with micafungin was used for PJP; however, symptoms persisted and repeated computed tomography showed extensive deterioration of bilateral pulmonary plaques. The PJP regimen was modified to include a combination of TMP-SMX and caspofungin. Due to the high fever and elevated infection indices, the patient was treated with teicoplanin to enhance the anti-infection effects. By Day 13, the patient's temperature had normalized, and infection control was achieved by Day 30. CT revealed that the infection in both lung lobes fully resolved. Subsequently, lymphoma treatment commenced.

BALF-NGS facilitates early and rapid diagnosis of PJP. mNGS reads of MTB bacillus <5 may indicate a bacterial carrier state, warranting other detection techniques to support it. There is insufficient evidence for using TMP-SMX with micafungin to treat PJP; however, TMP-SMX combined with caspofungin is suitable.

Studies have confirmed that the lung microbiome of lung transplant recipients is altered and serves as a prognostic indicator for long-term mortality. Other studies reported that the lung microbiome affects host immunity and the transcriptome. However, the lung microbiome composition at the early post-transplant period following lung transplantation is unclear, and the relationship of the lung microbiome with pulmonary immunity and the host transcriptome is also not well understood.

We hypothesize that changes in the lung microbiome composition in the early post-transplant period may have a predictive value for perioperative outcomes following lung transplantation and that the lung microbiome is correlated with pulmonary immunity and the host transcriptome. Thus, this prospective study aimed at observing the lung microbiome composition in the early post-transplant period and the impact of the lung microbiome on pulmonary cytokines and the host transcriptome. Our findings will help us gain a comprehensive understanding of the distribution and significance of the lung microbiome in the early post-transplant period.

An observational study was conducted to identify the lung microbiome and the host transcriptome characteristics using next-generation sequencing. Luminex was employed for quantifying alveolar cytokines. Spearman's correlation analysis was utilized to assess the impact of the lung microbiome on pulmonary immunity and differentially expressed genes in patients who died perioperatively after lung transplantation.

Patients with poor perioperative outcomes showed an increase in Mycoplasma and Arcobacter, a decrease of Gemella, and increased interleukin (IL)-10, IL-1β, and tumor necrosis factor (TNF)-α concentration. The lung microbiome correlates with lung immunity in lung transplant recipients. In the death group, the function of differentially expressed genes is associated with cell apoptosis, and promoting TNF production is upregulated. The lung microbiome is related to differentially expressed genes between the two groups.

The lung microbiome and cytokines can be considered as potential biomarkers for early prognosis in lung transplant recipients. The lung microbiome is associated with both lung immunity and differentially expressed genes in lung transplant recipients.

Infection is the main cause of death for patients after allogeneic hematopoietic stem cell transplantation (HSCT). However, pathogen profiles still have not been reported in detail due to their heterogeneity caused by geographic region.

To evaluate the performance of metagenomic next-generation sequencing (mNGS) and summarize regional pathogen profiles of infected patients after HSCT.

From February 2021 to August 2022, 64 patients, admitted to the Department of Hematology of The First Hospital of Jilin University for HSCT and diagnosed as suspected infections, were retrospectively enrolled.

A total of 38 patients were diagnosed as having infections, including bloodstream (n =17), pulmonary (n =16), central nervous system (CNS) (n =4), and chest (n =1) infections. Human betaherpesvirus 5 (CMV) was the most common pathogen in both bloodstream (n =10) and pulmonary (n =8) infections, while CNS (n =2) and chest (n =1) infections were mainly caused by Human gammaherpesvirus 4 (EBV). For bloodstream infection, Mycobacterium tuberculosis complex (n =3), Staphylococcus epidermidis (n =1), and Candida tropicalis (n =1) were also diagnosed as causative pathogens. Furthermore, mNGS combined with conventional tests can identify more causative pathogens with high sensitivity of 82.9% (95% CI 70.4-95.3%), and the total coincidence rate can reach up to 76.7% (95% CI 64.1-89.4%).

Our findings emphasized the importance of mNGS in diagnosing, managing, and ruling out infections, and an era of more rapid, independent, and impartial diagnosis of infections after HSCT can be expected.

Pneumocystis jirovecii pneumonia (PJP) is a life-threatening opportunistic infection in immunocompromised children with systemic lupus erythematosus (SLE). Prophylaxis against PJP in high-risk children is crucial, but the risk factors for PJP in children with SLE are not adequately characterized. This study sought to identify the risk factors for PJP in long-term glucocorticoid-treated pediatric SLE patients.

This study encompassed 71 treatment episodes involving 64 children with prolonged (≥4 weeks) high-dose (≥20 mg/d prednisone) steroid regimens. Fourteen treatment episodes involved the PJP, whereas others did not. Risk factors for PJP were assessed through Cox regression. The predictive value of these factors was evaluated using receiver operating characteristic curves. The incidence of PJP in different risk groups was compared using the Kaplan-Meier method.

The creatinine (hazard ratio, 1.009; 95% confidence interval [CI], 1.001-1.017; p = 0.021) and the lowest lymphocyte count (hazard ratio, 0.007; 95% CI, 0.000-0.373; p = 0.014) were independent risk factors for PJP in children with SLE. The receiver operating characteristic curve showed that using creatinine greater than 72.5 μmol/L and the lowest lymphocyte count less than 0.6 × 109/L as risk predictors for PJP resulted in an area under the curve value of 0.934 (95% CI, 0.870-0.997; p < 0.001). The study revealed a significant increase in PJP prevalence (p < 0.001) in children with elevated creatinine levels and low lymphocyte count.

Elevated levels of creatinine and decreased lymphocyte count are identified as distinct risk factors for PJP in children with SLE who receive prolonged high-dose steroid therapy.

To compare different criteria of Metagenomic Next-Generation Sequencing (mNGS) in bronchoalveolar lavage fluid (BALF) for diagnosing invasive pulmonary aspergillosis (IPA).

We compared the diagnostic agreement and performances of six BALF mNGS-derived criteria (SDSMRN>1, SDSMRN≥3, SMRN≥10, SMRN≥50, RPM ratio≥10, and relative abundance of genus>30 %) in pneumonia patients.

A total of 115 patients were analyzed, with 28 identified with IPA. Diagnostic agreement among the six mNGS-derived criteria was moderate, with a Cohen's kappa of 0.577(P < 0.001). mNGS-derived criteria had low sensitivity ranging from 21.4 % to 57.1 % and high specificity from 88 % to 92 %. The optimal threshold of SDSMRN, SMRN, RPM ratio, and relative abundance of genus for diagnosing IPA were 5, 0.25, 8, and 20 %, respectively. Although using the optimal threshold, the sensitivity of mNGS is lower than 50 %.

All mNGS-derived criteria had low sensitivity for diagnosing IPA. A combination of mNGS and conventional mycological tests may be the best diagnostic strategy.

Pulmonary trichomoniasis is considered a neglected disease due to failures in recognizing it, stemming from insensitive microbial methods and a lack of specific clinical features. This study aims to analyze the clinical implications of trichomonads detected in bronchoalveolar lavage fluid (BALF) by metagenomic next-generation sequencing (mNGS).

This multicenter retrospective study included patients diagnosed with pneumonia, admitted to three tertiary hospitals in China from July 2018 to September 2022, with trichomonads detected in BALF through mNGS. The analysis covered demographics, comorbidities, symptoms, laboratory findings, mNGS results, clinical treatment, and outcomes of these patients.

A total of 17 patients were enrolled, comprising 14 males and 3 females. Trichomonas tenax and Trichomonas vaginalis were detected by mNGS in BALF samples of 15 and 2 patients, respectively. Patients were categorized into two groups based on the presence of risk factors for trichomonad infection, including immunocompromised conditions, uncontrolled diabetes mellitus, oral/periodontal diseases, and aspiration. Among 11 patients with risk factors (Case 1-11), 4 received nitromidazoles as part of comprehensive treatment, achieving a 100% treatment success rate. The remaining 7 patients, who did not receive nitromidazoles, had only one achieving relief after broad-spectrum antimicrobial therapy, resulting in a 14.3% treatment success rate. For the 6 patients without any risk factors for trichomonad infection (Case 12-17), none received nitromidazoles during hospitalization. However, 4 out of these 6 patients (66.7%) eventually recovered.

mNGS proves to be an efficient tool for detecting trichomonads in BALF samples. Comprehensive analysis of clinical features and laboratory indicators is essential to distinguish between infection and colonization of trichomonads. Pulmonary trichomoniasis should not be overlooked when trichomonads are detected in BALF from patients with risk factors.

Objectives: To investigate the value of metagenomic next-generation sequencing (mNGS) in the diagnosis of Pneumocystis jirovecii pneumonia (PJP) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: The data of 98 patients with suspected pulmonary infection after allo-HSCT who underwent pathogen detection from bronchoalveolar lavage fluid between June 2016 and August 2023 at Nanfang Hospital were analyzed. The diagnostic performance of mNGS, conventional methods, and real-time quantitative polymerase chain reaction (qPCR) for PJP were compared. Results: A total of 12 patients were diagnosed with PJP, including 11 with a proven diagnosis and 1 with a probable diagnosis. Among the patients with a proven diagnosis, 1 was positive by both conventional methods and qPCR, and 10 were positive by qPCR only. Pneumocystis jirovecii was detected by mNGS in all 12 patients. The diagnostic sensitivity of mNGS for PJP was 100%, which was greater than that of conventional methods (8.3%, P=0.001) and similar to that of qPCR (91.6%, P=1.000) . A total of 75% of the patients developed mixed pulmonary infections, and cytomegalovirus and Epstein-Barr virus were the most common pathogens. Mixed infection was detected in eight patients by mNGS and in five patients by qPCR, but not by conventional methods (P=0.008) . Conclusions: mNGS had good sensitivity for diagnosing PJP after allo-HSCT and was advantageous for detecting mixed infectious pathogens; therefore, mNGS might be an effective supplement to regular detection methods and qPCR.

The incidence of Pneumocystis jirovecii pneumonia (PJP) is increasing.

108 patients were analysed retrospectively at the Wuhan Union Hospital. The patients were classified into the PJP group or the P. jirovecii colonisation (PJC) group based on clinical diagnosis. Clinical data included demographics, laboratory examinations, treatment, and outcomes.

A notable difference in the fungal load was seen between two groups, with median reads of 3215.79 vs. 5.61 in two groups, respectively (P<0.001). The optimal threshold value for discriminating P. jirovecii infection between colonisation for mNGS was six, and serum (1,3)-β-D-glucan (BDG) was 47.6 pg/mL. Besides, the positive detection rate of mNGS for co-pathogens in PJP patients was significantly higher than that of culture (88.16% vs. 22.37%, P<0.0001). Epstein-Barr virus and cytomegalovirus were the most common pathogens of co-infection in PJP patients. The antibiotic therapy in PJP patients was adjusted according to the mNGS results, of which seventeen (22.37%) were downgraded, 38 (50.0%) patients were upgraded, and 21 (27.63%) were unchanged. And almost all patients showed significant improvement in C-reactive protein.

mNGS is a promising and valuable technique with good performance for differentiating P. jirovecii infection and colonisation, the detection of pathogens, and antibiotic treatment.

The identification of Aspergillus by metagenomic next-generation sequencing (mNGS) remains a challenging task due to the difficulty of nucleic acid extraction. The objective of this study was to determine whether mNGS could provide an accurate and efficient method for detecting invasive pulmonary aspergillosis (IPA) in immunocompromised patients (ICP).

A total of 133 ICP admitted to the ICU between January 2020 and September 2022 were enrolled in the study, of which 46 were diagnosed with IPA and 87 were non-IPA cases. The bronchoalveolar lavage fluid (BALF) was analyzed for the presence of Aspergillosis and other co-pathogens using mNGS, and its diagnostic performance was compared to conventional microbial tests (CMTs) that included smear, cultures, serum and BALF galactomannan (GM) test. Clinical composite diagnosis was used as the reference standard.

mNGS had a sensitivity, specificity, and accuracy of 82.6%, 97.7%, and 92.5%, respectively, in diagnosing IPA. These findings were comparable to those of the combination of multiple CMTs. Interestingly, the sensitivity of mNGS was superior to that of any single CMT method, as demonstrated by comparisons with smears (8.7%, P < 0.001), culture (39.1%, P < 0.001), serum GM (23.9%, P < 0.001) and BALF GM (69.6%, P = 0.031). mNGS was capable of accurately distinguish strains of Aspergillus genus, with a consistency of 77.8% with culture. Furthermore, mNGS also identified A. fumigatus, A. flavus, A. terrestris, A. oryzae and Mucor spp. in culture-negative cases. The sequencing reads of Aspergillus by mNGS exhibited extensive variation, ranging from 11 to1702. A positive correlation was observed between the optical density index of BALF GM and unique reads by mNGS (r = 0.607, P = 0.001) in BALF-GM positive patients. Notably, mNGS was able to diagnose 35 out of 37 cases with mixed infection, with P. jirovecii and cytomegalovirus being the most common co-pathogens.

mNGS presents a feasible and remarkably sensitive approach for detecting Aspergillus in ICP, thereby serving as a valuable adjunctive tool to CMT. Furthermore, mNGS's ability to accurately identify fungal species and co-pathogens can assist in guiding appropriate antimicrobial therapy.

Previous studies achieved low microbial detection rates in lymphoma patients with interstitial pneumonia (IP) after chemotherapy. However, the metagenomic next-generation sequencing (mNGS) is a comprehensive approach that is expected to improve the pathogen identification rate. Thus far, reports on the use of mNGS in lymphoma patients with chemotherapy-related IP remain scarce. In this study, we summarized the microbial detection outcomes of lymphoma patients with chemotherapy-related IP through mNGS testing of bronchoalveolar lavage fluid (BALF).

Fifteen lymphoma patients with chemotherapy-related IP were tested for traditional laboratory microbiology, along with the mNGS of BALF. Then, the results of mNGS and traditional laboratory microbiology were compared.

Of the 15 enrolled patients, 11 received rituximab and 8 were administered doxorubicin hydrochloride liposome. The overall microbial yield was 93.3% (14/15) for mNGS versus 13.3% (2/15) for traditional culture methods (P ≤ 0.05). The most frequently detected pathogens were Pneumocystis jirovecii (12/15, 80%), Cytomegalovirus (4/15, 26.7%), and Epstein-Barr virus (3/15, 20%). Mixed infections were detected in 10 cases. Five patients recovered after the treatment with antibiotics alone without glucocorticoids.

Our findings obtained through mNGS testing of BALF suggested a high microbial detection rate in lymphoma patients with IP after chemotherapy. Notably, there was an especially high detection rate of Pneumocystis jirovecii. The application of mNGS in patients with chemotherapy-related IP was more sensitive.

Recent studies have shown that the lung microbiota is altered in critically ill patients and predicts clinical outcomes. Primary graft dysfunction (PGD) is a common complication and a leading cause of death within 1 month of lung transplantation, but the clinical significance of changes in the lung bacterial community during PGD is unclear. The aim of this study was to determine the contribution of the lung microbiota to the development and course of severe PGD.

We conducted a retrospective study to characterize the lung microbiota of 32 lung transplant patients with combined PGD using next-generation sequencing of bronchoalveolar lavage samples. The relationship between lung flora dysbiosis and lung immunity in PGD was assessed by quantification of alveolar cytokines. The contribution of microbiota characteristics to patient outcomes was assessed by estimating overall survival.

Patients diagnosed with PGD grade 3 showed a reduction in alpha diversity, driven by a significant increase in the abundance of the genera Modestobacter, Scardovia and Selenomonas, and a reduction in the proportion of the genera Klebsiella and Oribacterium. Alpha diversity of the lung microbiota in PGD3 patients was negatively correlated with BALF interleukin (IL)-2 (r = -.752, p < .05). In addition, bacterial diversity in the lung microbiota of non-survivors was lower than that of survivors (p = .041).

There is variation in the lung microbiota of PGD grade 3 patients and dysbiosis of the lung microbiota is associated with lung immunity. The lung microbiota has potential in the diagnosis and treatment of PGD grade 3.

To identify potential risk factors and prognostic factors of Pneumocystis jirovecii pneumonia (PJP) infection in anti-melanoma differentiation-associated gene 5 antibody-positive DM (anti-MDA5+ DM) patients, and to evaluate the diagnostic performance of metagenomic next-generation sequencing (mNGS).

Anti-MDA5+ DM patients who underwent mNGS or real-time PCR for PJP detection were recruited. The potential risk factors for PJP occurrence and death were analysed via Logistic regression and Cox proportional hazards regression, respectively. The diagnostic efficacy of mNGS was compared with the conventional methods.

91 patients were enrolled and 44 were assigned to PJP+ group. The PJP detection rate was 48.4%. PJP often occurred in the first 3 months (68.2%) of the disease; this period also showed the highest mortality rate (20.5%). Fever and increased lactate dehydrogenase (LDH) were independent risk factors for PJP occurrence, while trimethoprim-sulfamethoxazole (TMP/SMZ) prophylaxis was an independent protective factor (all P < 0.05). Older age and increased LDH were predictors for mortality in patients with anti-MDA5+ DM and PJP (all P < 0.05). In addition, we found that mNGS had a sensitivity of 100.0% and specificity of 90.0% in diagnosing PJP, with the highest area under the curve of 0.95 (P < 0.001).

PJP has high prevalence and mortality in anti-MDA5+ DM. It is crucial for clinicians to identify high-risk patients and promptly institute TMP/SMZ to prevent PJP. mNGS is the preferred approach for pathogen detection in anti-MDA5+ DM when PJP is suspected.

Pneumocystis pneumonia (PCP) is a growing concern as the immunocompromised population expands. Current laboratory approaches are limited. This systematic review aimed to evaluate metagenomic next-generation sequencing (MNGS) tests' performance in detecting PCP.

Five databases were searched through December 19, 2022, to identify original studies comparing MNGS with clinically diagnosed PCP. To assess the accuracy, symmetric hierarchical summary receiver operating characteristic models were used.

Eleven observational studies reporting 1442 patients (424 with PCP) were included. Six studies focused exclusively on recipients of biologic immunosuppression (none with HIV-associated immunosuppression). Six were exclusively on bronchoalveolar lavage, while 1 was on blood samples. The sensitivity of MGNS was 0.96 (95% CI, 0.90-0.99), and specificity was 0.96 (95% CI, 0.92-0.98), with negative and positive likelihood ratios of 0.02 (95% CI, 0.01-0.05) and 19.31 (95% CI, 10.26-36.36), respectively. A subgroup analysis of studies exclusively including bronchoalveolar lavage (BAL) and blood samples demonstrated a sensitivity of 0.94 (95% CI, 0.78-0.99) and 0.93 (95% CI, 0.80-0.98) and a specificity of 0.96 (95% CI, 0.88-0.99) and 0.98 (95% CI, 0.76-1.00), respectively. The sensitivity analysis on recipients of biologic immunosuppression showed a sensitivity and specificity of 0.96 (95% CI, 0.90-0.98) and 0.94 (95% CI, 0.84-0.98), respectively. The overall confidence in the estimates was low.

Despite the low certainty of evidence, MNGS detects PCP with high sensitivity and specificity. This also applies to recipients of biologic immunosuppression and tests performed exclusively on blood samples without the need for BAL. Further studies are required in individuals with HIV-associated immunosuppression.

Metagenomic next-generation sequencing (mNGS) is a powerful method for pathogen detection in various infections. In this study, we assessed the value of mNGS in the pathogen diagnosis and microbiome analysis of pneumonia in pediatric intensive care units (PICU) using bronchoalveolar lavage fluid (BALF) samples.

A total of 104 pediatric patients with pneumonia who were admitted into PICU between June 2018 and February 2020 were retrospectively enrolled. Among them, 101 subjects who had intact clinical information were subject to parallel comparison of mNGS and conventional microbiological tests (CMTs) for pathogen detection. The performance was also evaluated and compared between BALF-mNGS and BALF-culture methods. Moreover, the diversity and structure of all 104 patients' lung BALF microbiomes were explored using the mNGS data.

Combining the findings of mNGS and CMTs, 94.06% (95/101) pneumonia cases showed evidence of causative pathogenic infections, including 79.21% (80/101) mixed and 14.85% (15/101) single infections. Regarding the pathogenesis of pneumonia in the PICU, the fungal detection rates were significantly higher in patients with immunodeficiency (55.56% vs. 25.30%, P =0.025) and comorbidities (40.30% vs. 11.76%, P=0.007). There were no significant differences in the α-diversity either between patients with CAP and HAP or between patients with and without immunodeficiency. Regarding the diagnostic performance, the detection rate of DNA-based BALF-mNGS was slightly higher than that of the BALF-culture although statistically insignificant (81.82% vs.77.92%, P=0.677) and was comparable to CMTs (81.82% vs. 89.61%, P=0.211). The overall sensitivity of DNA-based mNGS was 85.14% (95% confidence interval [CI]: 74.96%-92.34%). The detection rate of RNA-based BALF-mNGS was the same with CMTs (80.00% vs 80.00%, P>0.999) and higher than BALF-culture (80.00% vs 52.00%, P=0.045), with a sensitivity of 90.91% (95%CI: 70.84%-98.88%).

mNGS is valuable in the etiological diagnosis of pneumonia, especially in fungal infections, and can reveal pulmonary microecological characteristics. For pneumonia patients in PICU, the mNGS should be implemented early and complementary to CMTs.

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 ≤ 7 days) 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 > 7 days).

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.