Case Report: Diagnosis of Hemolytic Anemia from Babesia and Secondary Multi-Pathogen Pneumonia Using a Metagenomic Next-Generation Sequencing Approach.

Babesiosis, as a vector-borne infectious disease, remains relatively rare and is prone to being overlooked and misdiagnosed. Therefore, understanding the epidemiological characteristics and clinical manifestations of babesiosis is crucial for the prompt detection and treatment of the disease. We reported a 63-year-old male patient presenting with spontaneous fever and chills. Laboratory investigations revealed erythrocytopenia, reduced hemoglobin levels, and increased reticulocytes and total bilirubin. Bone marrow examination indicated vigorous cell proliferation, a decreased granulocyte to red cell ratio, and predominant erythroid cell proliferation, with a higher prevalence of intermediate and late-stage juvenile granulocyte and erythroid cells. Initial treatment focused on hemophagocytic syndrome triggered by Epstein-Barr virus infection yielded unsatisfactory results, leading to secondary multiple pulmonary infections. Metagenomic next-generation sequencing (mNGS) of sputum samples pointed to hemolytic anemia induced by Babesia infection, which was subsequently confirmed through peripheral blood smear analysis. The patient responded well to prompt administration of atovaquone and azithromycin, with symptoms resolving and laboratory parameters normalizing. Hemolytic anemia resulting from babesiosis should be distinguished from hemophagocytic syndrome caused by Epstein-Barr virus and other hematologic conditions. mNGS represents an efficient technique for Babesia detection.

Meta-genomic next-generation sequencing in the diagnosis of brucellosis: Five cases from a non-endemic area.

Metagenomic next-generation sequencing (mNGS) in diagnosis of human brucellosis is comparatively unexplored. This report details five human brucellosis cases diagnosed using mNGS based on Illumina sequencing platform, comprising three females and two males, four with epidemiological exposure. In cases 1 and 2, plasma mNGS results showed one positive and one negative for Brucella melitensis, and subsequent blood cultures were both positive. Cases 3, 4 and 5 involved spinal brucellosis, some with paravertebral abscesses. mNGS from infectious tissue samples successfully detected Brucella, with read counts ranging between 30 and 1314, yet cultures were negative in cases 4 and 5. Following antibiotic and surgical treatments, all patients showed clinical improvement. This report shows mNGS testing enhances the detection sensitivity of brucellosis diagnosis.

What is this summary about? Brucella is a type of bacteria that can infect humans and animals. It causes a disease called brucellosis. Symptoms of brucellosis include fever and fatigue, among others. Meta-genomic next-generation sequencing (mNGS) is a tool for sequencing the DNA of bacteria. In this report, we use mNGS to diagnose human brucellosis in five cases.What were the results? Brucella was found in the blood of two infected people, but mNGS found Brucella in only one. Of three people with Brucella infection of the spine, mNGS found Brucella in the infected tissue but Brucella was only cultured in one case. Following antibiotic and surgical treatments, all five patients showed improvement of their symptoms.What do the results of the study mean? mNGS is a relatively rapid and effective diagnostic method that can improve the detection of Brucella in brucellosis.

Severe community­acquired pneumonia caused by Legionella gormanii in combination with influenza A subtype (H1N1) virus in an immunocompetent patient detected by metagenomic next­generation sequencing: A case report.

Legionella pneumonia is an atypical form of pneumonia caused by Legionella gormanii that can also lead to multiple organ diseases, including acute respiratory distress syndrome and multiple organ dysfunction syndrome. Legionella gormanii requires a long incubation period for culture in clinical practice using BCYE medium. The specificity of serum for serological detection is low, resulting in a relatively high rate of missed Legionella diagnoses. Contracting the H1N1 virus can lead to the misdiagnosis of Legionella gormanii. Metagenomic next-generation sequencing (mNGS) is a novel tool that can rapidly and accurately identify potential Legionella gormanii strains. A severe case of community-acquired pneumonia in a 79-year-old patient was reported. The patient was diagnosed with Legionella gormanii and influenza A subtype (H1N1) virus using mNGS at The First Affiliated Hospital, Zhejiang University School of Medicine. After anti-Legionella and antiviral therapy, the number of reads identifying Legionella gormanii in bronchoalveolar lavage fluid using mNGS decreased from 665 to 112 as the patient's condition gradually improved. A search of PubMed revealed few reports of Legionella gormanii in association with the influenza A subtype (H1N1) virus. Patients with severe pneumonia caused by Legionella and influenza A subtype H1N1 virus infections should be screened early for infections using methods such as mNGS. This approach enables early and precise treatment, simplifying the administration of antibiotics and enhancing patient outcomes.

Sex-specific differences in the clinical profile among psychiatric patients with pulmonary Embolism: a hospital-based retrospective study.

BACKGROUND: Pulmonary embolism (PE) is a severe and life-threatening complication of venous thromboembolism. However, there is a lack of systematic studies on differences between female and male PE patients. This paper aimed to compare the sex-specific differences in clinical characteristics and laboratory indicators in psychotic patients with PE. METHODS: This retrospective study enrolled psychiatric patients with PE from June 2018 to June 2022 at Shenzhen Kangning Hospital (Shenzhen Mental Health Center). Demographic characteristics, factors associated with PE, and laboratory indices were collected to assess sex-specific differences. RESULTS: Of the 168 patients, 87 (51.8%) were female and 81 (48.2%) were male, with a mean age of 58 years for females and 46 years for male patients. The male group had higher ratio of hyperprolactinemia, more patients using antipsychotic medications, higher D-dimer levels at PE onset, greater D-dimer difference, and a higher rate of D-dimer elevation than the female group (p < 0.05). Female patients were significantly older, exhibited a higher prevalence of diabetes, and had a greater number of patients taking antidepressants and hypnotics/sedatives than male patients (p < 0.05). Schizophrenia spectrum disorders were more prevalent in male patients, while female patients had a higher incidence of mood disorders (p < 0.05). Among patients aged < 45 years, the male group had higher D-dimer levels at PE onset and greater D-dimer difference (p < 0.05). Among all 112 patients aged ≥ 45 years, male patients were more likely than female patients to have respiratory tract infections, higher D-dimer levels at PE onset, greater D-dimer difference, and a higher rate of D-dimer elevation (p < 0.05). The multiple linear regression analysis indicated that hyperprolactinemia and the use of first-generation antipsychotics (FGAs) were associated with D-dimer levels at PE onset in male patients, while the time of PE onset and protective restraints were associated with D-dimer levels at PE onset in female patients (p < 0.05). CONCLUSION: PE-associated clinical features differ between male and female patients. These differences may imply that the processes and mechanisms of PE onset are sex specific. Male patients are more likely to have respiratory tract infections and higher D-dimer levels at PE onset than female patients. The use of FGAs may be associated with increased D-dimer in male psychiatric patients, while protective restraints may be associated with increased D-dimer in female psychiatric patients.

Clinical usefulness of metagenomic next-generation sequencing for Talaromyces marneffei diagnosis in China: a retrospective study.

PURPOSE: Metagenomic next-generation sequencing (mNGS) has been widely used in the diagnosis of infectious diseases. However, studies on Talaromyces marneffei detection using mNGS remain scarce. Therefore, this study aimed to explore the diagnostic performance of mNGS in T. marneffei. METHODS: Between March 2021 and June 2023, patients who were discharged with a final diagnosis of talaromycosis, or confirmed T. marneffei infection by mNGS, culture or pathological examination were included in the study. Culture and mNGS were performed simultaneously for all patients. Clinical data were retrieved for analysis. RESULTS: A total of 78 patients were enrolled, with 40 in the talaromycosis group and 38 in the suspected-talaromycosis group. In the talaromycosis group, mNGS showed a higher positivity rate(40/40, 100.0%) compared to culture(34/40, 85.0%)(P = 0.111). All patients in the suspected-talaromycosis group tested negative via culture, while mNGS yielded positive results. The T. marneffei reads in the talaromycosis group were significantly higher than in the suspected-talaromycosis group (4399 vs. 28, P < 0.001). In the suspected-talaromycosis group, of the four patients with low reads who did not receive antifungal therapy, one died and one lung lesion progressed; most patients(31/34, 91.2%) recovered after receiving appropriate antifungal therapy. CONCLUSION: mNGS proves to be a rapid and highly sensitive method for detecting T. marneffei. Higher reads of T. marneffei correspond to a higher likelihood of infection. However, cases with low reads necessitate a comprehensive approach, integrating clinical manifestations, laboratory tests, and imaging examinations to confirm T. marneffei infection.

Clinical Performance of Self-Collected Purified Water Gargle for Detection of Influenza a Virus Infection by Real-Time RT-PCR.

Purpose: Self-collected specimens are increasingly being used as alternatives to swab-based methods for the detection of respiratory viruses. While saliva is well accepted, gargle specimens are a potential alternative with characteristics that are more favorable for laboratory handling. This study assessed the performance of gargle specimens in the detection of influenza A viruses (IAVs). Patients and Methods: We performed a prospective head-to-head comparison between combined nasopharyngeal and oropharyngeal swabs (NPS&OPS) and purified water gargle (PWG) among adult outpatients with febrile respiratory symptoms to detect IAVs using real-time RT-PCR during two influenza seasons. Results: During study periods 1 (July 13 to 26, 2022, H3N2 predominated) and 2 (February 25 to March 10, 2023, H1N1 pdm09 predominated), a total of 459 patients were recruited. The overall agreement between the NPS&OPS and PWG was 85.0% (390/459, κ = 0.697), with 88.0% in period 1 and 82.6% in period 2. The detection rate of IAVs in PWG (51.6%, 237/459) was lower than that in NPS&OPS (62.3%, 286/459) (p < 0.0001). The overall sensitivity and specificity were 96.6% (93.7-98.3%) and 100% (97.1-100%) in NPS&OPS and were 80.1% (75.0-84.4%) and 100% (97.1-100%) in PWG, respectively. Among the 227 pairs of concordant positive specimens, cycle threshold (Ct) values were significantly lower in NPS&OPS than in PWG (median Ct values: 24.2, 28.2, p < 0.0001). Conclusion: Although self-collected PWG specimens offer acceptable performance for IAVs molecular testing, NPS&OPS remain a reliable option. Given the convenience of collection, nonviscous gargles are recommended for viral detection during emergencies or under specific conditions.

Molecular rapid diagnostic testing for bloodstream infections: Nanopore targeted sequencing with pathogen-specific primers.

BACKGROUND: Nanopore sequencing, known for real-time analysis, shows promise for rapid clinical infection diagnosis but lacks effective assays for bloodstream infections (BSIs). METHODS: We prospectively assessed the performance of a novel nanopore targeted sequencing (NTS) assay in identifying pathogens and predicting antibiotic resistance in BSIs, analyzing 387 blood samples from December 2021 to April 2023. RESULTS: The positivity rate for NTS (69.5 %, 269/387) nearly matches that of metagenomic next-generation sequencing (mNGS) (74.7 %, 289/387; p = 0.128) and surpasses the positivity rate of conventional blood culture (BC) (33.9 %, 131/387; p < 0.01). Frequent pathogens detected by NTS included Klebsiella pneumoniae (n = 54), Pseudomonas aeruginosa (n = 36), Escherichia coli (n = 36), Enterococcus faecium(n = 30), Acinetobacter baumannii(n = 26), Staphylococcus aureus(n = 23), and Human cytomegalovirus (n = 37). Against a composite BSI diagnostic standard, NTS demonstrated a sensitivity and specificity of 84.0 % (95 % CI 79.5 %-87.7 %) and 90.1 % (95 % CI 81.7 %-88.5 %), respectively. The concordance between NTS and mNGS results (the percentage of total cases where both either detected BSI-related pathogens or were both negative) was 90.2 % (359/387), whereas the consistency between NTS and BC was only 60.2 % (233/387). In 80.6 % (50/62) of the samples with identical pathogens identified by both NTS tests and BCs, the genotypic resistance identified by NTS correlated with culture-confirmed phenotypic resistance. Using NTS, 95 % of samples can be tested and analyzed in approximately 7 h, allowing for early patient diagnosis. CONCLUSIONS: NTS is rapid, sensitive, and efficient for detecting BSIs and drug-resistant genes, making it a potential preferred diagnostic tool for early infection identification in critically ill patients.

Sepsis and Hepatapostema Secondary to Chromobacterium Violaceum Infection on Lower Limb Skin: A Case Report.

Background: Chromobacterium violaceum (C. violaceum) is a Gram-negative bacterium capable of causing severe infections in both humans and specific animals. Despite its infrequency, C. violaceum infections exhibit a notably high mortality rate. The timely and precise detection of this pathogen stands as a critical factor in achieving effective diagnosis and treatment. Traditional diagnostic approaches possess limitations, particularly in terms of their time-consuming nature and the range of pathogens they can identify. Metagenomic next-generation sequencing (mNGS) testing has emerged as a highly promising diagnostic tool for infectious diseases. Methods: Within this case report, we present a patient who developed a C. violaceum infection subsequent to a lower limb infection, leading to the progression of sepsis, a liver abscess, septic shock, multi-organ dysfunction, and altered mental status. Samples of the patient's blood and tissue from the lower limb skin are collected, and the infection is swiftly diagnosed through mNGS, allowing for the immediate initiation of suitable treatment. Results: The mNGS results revealed the patient's infection with C. violaceum. Subsequent conventional bacterial culture results were concordant with the mNGS findings. Following comprehensive management measures, including prompt and effective anti-infective treatment, the patient achieved cure and was successfully discharged. Conclusion: This case underscores the significance of employing advanced diagnostic methodologies like mNGS for the early detection of uncommon pathogens such as C. violaceum. The expedited diagnosis and timely intervention hold the potential to substantially enhance patient outcomes in cases of severe infections instigated by this bacterium.

Lemierre Syndrome: Report of a Case with an Innovative Diagnostic Method and Literature Review.

Objective: To understand the clinical features, diagnosis and treatment of Lemierre syndrome (LS), a high-risk and low-prevalence infectious disease. Methods: We present the severe LS case that was diagnosed using metagenomic next-generation sequencing (mNGS) in our hospital, and systematically summarized the diagnosis and treatment strategies of patients that reported LS from 2006 to 2022. Results: The 24-year-old patient in our hospital suffered from cranial nerve paralysis, a neurological complication rarely seen in LS cases. The causative agent (Fusobacterium necrophorum, Fn) of this patient was only detected by mNGS tests, and the reads number of Fn detected by plasma mNGS tests was decrease as the patients gradually improved, indicating plasma mNGS is valuable in monitoring treatment efficacy. Although most of the cases retrieved from the literature showed typical symptoms, such as a history of sore throat, septic emboli, and internal jugular vein thrombosis, clinical manifestations were still relatively heterogeneous (eg, diversity of predisposing factors and pathogens, differences in pulmonary imaging features). Conclusion: We summarized the clinical presentation, diagnosis, treatment, and regression of 17 symptomatic cases reported LS to provide clinicians with knowledge about this rare but fatal disease. mNGS assays should be considered as early as possible to identify the responsible pathogens for acute and critically ill patients with suspected infections in order to implement accurate and effective treatment.

Applicability of Bronchoalveolar Lavage Fluid and Plasma Metagenomic Next-Generation Sequencing Assays in the Diagnosis of Pneumonia.

Background: Metagenomic next-generation sequencing (mNGS) provides innovative solutions for predicting complex infections. A comprehensive understanding of its strengths and limitations in real-world clinical settings is necessary to ensure that it is not overused or misinterpreted. Methods: Two hundred nine cases with suspected pneumonia were recruited to compare the capabilities of 2 available mNGS assays (bronchoalveolar lavage fluid [BALF] mNGS and plasma mNGS) to identify pneumonia-associated DNA/RNA pathogens and predict antibiotic resistance. Results: Compared to clinical diagnosis, BALF mNGS demonstrated a high positive percent agreement (95.3%) but a low negative percent agreement (63.1%). Plasma mNGS revealed a low proportion of true negatives (30%) in predicting pulmonary infection. BALF mNGS independently diagnosed 65.6% (61/93) of coinfections and had a remarkable advantage in detecting caustic, rare, or atypical pathogens. Pathogens susceptible to invasive infection or bloodstream transmission, such as Aspergillus spp, Rhizopus spp, Chlamydia psittaci, and human herpesviruses, are prone to be detected by plasma mNGS. BALF mNGS tests provided a positive impact on the diagnosis and treatment of 128 (61.2%) patients. Plasma mNGS, on the other hand, turned out to be more suitable for diagnosing patients who received mechanical ventilation, developed severe pneumonia, or developed sepsis (all P < .01). BALF mNGS was able to identify resistance genes that matched the phenotypic resistance of 69.4% (25/36) of multidrug-resistant pathogens. Conclusions: Our data reveal new insights into the advantages and disadvantages of 2 different sequencing modalities in pathogen identification and antibiotic resistance prediction for patients with suspected pneumonia.

Rapid diagnosis of Aspergillus fumigatus endocarditis using mNGS assay: A case report and review of the literature.

Fungal endocarditis is caused mainly by Candida albicans and Aspergillus spp. and was first reported in the 1950s. Natural-valve endocarditis caused by Aspergillus is relatively uncommon. In this case, a 56-year-old male patient was admitted to the hospital on account of a cough accompanied by chills and fever and ineffective self-medication. Infective endocarditis was initially suspected based on echocardiography (indicating right atrial growth) and clinical manifestations. However, routine pathogen detections were always negative. The patient's condition was identified as Aspergillus fumigatus endocarditis (AFE) and was treated with targeted therapy, considering the detection of significant AFE sequences in the blood through metagenomic next-generation sequencing (mNGS). On this basis, the paper further summarizes the clinical manifestations, diagnosis, treatments, and outcomes of AFE endocarditis cases reported in recent years, aiming to provide a reference to better understand this rare infective disease and guide medical practitioners in choosing the right diagnostic and therapeutic strategy.

ddPCR provides a sensitive test compared with GeneXpert MTB/RIF and mNGS for suspected Mycobacterium tuberculosis infection.

Introduction: The Metagenomics next-generation sequencing (mNGS) and GeneXpert MTB/RIF assay (Xpert) exhibited a sensitivity for tuberculosis (TB) diagnostic performance. Research that directly compared the clinical performance of ddPCR analysis, mNGS, and Xpert in mycobacterium tuberculosis complex (MTB) infection has not been conducted. Methods: The study aimed to evaluate the diagnostic performance of ddPCR compared to mNGS and Xpert for the detection of MTB in multiple types of clinical samples. The final clinical diagnosis was used as the reference standard. Results: Out of 236 patients with suspected active TB infection, 217 underwent synchronous testing for tuberculosis using ddPCR, Xpert, and mNGS on direct clinical samples. During follow-up, 100 out of 217 participants were diagnosed with MTB infection. Compared to the clinical final diagnosis, ddPCR produced the highest sensitivity of 99% compared with mNGS (86%) and Xpert (64%) for all active MTB cases. Discussion: Twenty-two Xpert-negative samples were positive in mNGS tests, which confirmed the clinical diagnosis results from ddPCR and clinical manifestation, radiologic findings. Thirteen mNGS-negative samples were positive in ddPCR assays, which confirmed the clinical final diagnosis.ddPCR provides a higher sensitive compared to Xpert and mNGS for MTB diagnosis, as defined by the high concordance between ddPCR assay and clinical final diagnosis.

Case Report: Non-negligible Epstein-Barr virus-associated posttransplant lymphoproliferative disorders in a lung transplant recipient.

Background: Posttransplant lymphoproliferative disorders (PTLDs) are uncommon but serious complications in patients following solid organ transplantation. Primary Epstein-Barr virus (EBV) infection is a risk factor for the development of PTLD, especially early-onset PTLD, in EBV-negative recipients. To date, however, there are no specific guidelines on the threshold of EBV-DNA load for therapeutic intervention, the source for measurement (e.g., blood, bronchoalveolar fluid), or the use of antiviral agents as prophylaxis for early PTLD prevention in EBV-mismatched patients. Methods: The present study describes a 56-year-old male lung transplant recipient diagnosed with EBV-associated PTLD. Results: This patient had a history of invasive fungal disease and Mucor and Aspergillus fumigatus infections in the early post-transplant period, necessitating antifungal therapy throughout the course of the disease. The patient was EBV-positive 15 days after transplantation, with lung CT showing multiple bilateral nodules of varying sizes beginning 98 days after transplantation. A lung biopsy showed PTLD, and next-generation sequencing (NGS) revealed EBV. This patient, however, did not receive any antiviral therapy for early PTLD prevention or any PTLD-related treatment. He died 204 days after lung transplantation. Conclusion: The present study describes a lung transplant recipient who developed EBV-associated PTLD, a non-negligible disease, after solid organ transplantation. Monitoring EBV-DNA load is important, as a sudden increase may be a sensitive indicator of PTLD. An earlier diagnosis may increase the likelihood of successful treatment.

Diagnostic efficiency of metagenomic next-generation sequencing for suspected infection in allogeneic hematopoietic stem cell transplantation recipients.

Introduction: 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). Methods: 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. Results: 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). Conclusion: 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.

Secondary Infection Surveillance with Metagenomic Next-Generation Sequencing in COVID-19 Patients: A Cross-Sectional Study.

Background: Metagenomic next-generation sequencing (mNGS) is a promising tool for improving antimicrobial therapy and infection control decision-making in complex infections. Secondary infection surveillance using mNGS in COVID-19 patients has rarely been reported. Methods: Respiratory pathogen and antibiotic resistance prediction were evaluated by BALF mNGS for 192 hospitalized COVID-19 patients between December 2022 and February 2023. Results: Secondary infection was confirmed in 83.3% (160/192) of the COVID-19 patients, with bacterial infections (45%, 72/160) predominating, followed by mixed bacterial and fungal infections (20%, 32/160), and fungal infections (17.5%, 28/160). The incidence of bacterial or viral secondary infection was significantly higher in patients who were admitted to the ICU, received mechanical ventilation, or developed severe pneumonia (all p<0.05). Klebsiella pneumoniae (n=30, 8.4%) was the most prevalent pathogen associated with secondary infection followed by Acinetobacter baumannii (n=29, 8.1%), Candida albicans (n=29, 8.1%), Aspergillus fumigatus (n=27, 7.6%), human herpes simplex virus type 1 (n=23, 6.4%), Staphylococcus aureus (n=20, 5.6%) and Pneumocystis jiroveci (n=14, 3.9%). The overall concordance between the resistance genes detected by mNGS and the reported phenotypic resistance in 69 samples containing five clinically important pathogens (ie, K. pneumoniae, A. baumannii, S. aureus, P. aeruginosa and E. coli) that caused secondary infection was 85.5% (59/69). Conclusion: mNGS can detect pathogens causing secondary infection and predict antimicrobial resistance for COVID19 patients. This is crucial for initiating targeted treatment and rapidly detect unsuspected spread of multidrug-resistant pathogens.

PCR cloning Intermediated Gibson assembly (PIG) for Constructing DNA Repair Templates in CRISPR-Cas9 Based Gene Editing.

The CRISPR-Cas9 gene-editing system has revolutionized genome engineering, allowing precise modifications to be made in a wide range of organisms. One significant challenge associated with CRISPR-Cas9 mediated gene editing is the construction of DNA repair templates containing homology arms, a screenable marker and a tag sequence of interest. Here, we present an efficient, two-step strategy to generate DNA repair templates in approximately one week, facilitating rapid and precise genome engineering applications.

Diagnostic Performance of Metagenomic Next-Generation Sequencing in Central Nervous System Cryptococcosis Using Cerebrospinal Fluid.

Purpose: Metagenomic next-generation sequencing (mNGS) has been widely used to diagnose infectious diseases. However, there are few studies on its diagnostic performance in the central nervous system (CNS) cryptococcosis. This study examined the diagnostic efficacy of mNGS in identifying Cryptococcus spp. in cerebrospinal fluid (CSF) samples. Patients and Methods: From March 2021 to March 2023, 290 patients with suspected CNS infection were recruited from the First Affiliated Hospital, School of Medicine, Zhejiang University, and 74 patients were ultimately included in the study. Lastly, 22 patients with CNS cryptococcosis were included. Of these patients, 25 CSF samples were enrolled. The diagnostic performance of conventional assays [including India ink, cryptococcal antigen (CrAg) testing, and culture] and mNGS was evaluated for CNS cryptococcosis. Results: In the 25 samples collected, the coincidence rates of mNGS with India ink, CrAg, and culture were 64.0% (16/25), 80.0% (20/25), and 80.0% (20/25), respectively. Without antifungal drug exposure, the coincidence rates were increased to 66.7% (10/15), 100.0% (15/15), and 93.3% (14/15), respectively. The coincidence rates after antifungal therapy were all decreased to 60.0% (6/10), 50.0% (5/10), and 60.0% (6/10), respectively. Moreover, in the 25 samples, the sensitivity of mNGS reached 80.0%, and of India ink, CrAg testing, and culture were 68.0, 100.0, and 60.0%, respectively. The mNGS showed an excellent positive rate (100.0%) in the 15 samples collected without antifungal drug exposure, which was significantly higher than the antifungal drug-exposed group (n = 10) (50.0%) (P = 0.005). The reads of Cryptococcus spp. before antifungal therapy were significantly higher than after it (median, 25,915 vs 2, P = 0.008). Conclusion: mNGS is an effective tool for diagnosing CNS cryptococcosis using CSF; however, its sensitivity decreases considerably in patients who have been effectively treated with antifungal drugs.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3 (Bromodomain and WD repeat-containing protein 3), a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased H3K4me1 (H3 lysine 4 monomethylation) levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels but also causes a decrease in H3K4me3 (H3 lysine 4 trimethylation) levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific lysine demethylase 5 (KDM5/Lid), an enzyme that removes tri- and dimethyl marks from H3K4. Moreover, analysis of ChIP-seq (chromatin immunoprecipitation sequencing) data revealed that BRWD3 and KDM5 are significantly colocalized throughout the genome and H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

Diagnosis of Non-Tuberculous Mycobacterial Pulmonary Disease by Metagenomic Next-Generation Sequencing on Bronchoalveolar Lavage Fluid.

Purpose: Metagenomic next-generation sequencing (mNGS) has been extensively used in the diagnosis of infectious diseases but has rarely been applied in non-tuberculous mycobacterial pulmonary disease (NTMPD). This study analyzed the diagnostic performance of mNGS in bronchoalveolar lavage fluid (BALF) samples to identify non-tuberculous mycobacteria (NTM). Patients and Methods: A total of 231 patients with suspected NTMPD were recruited from the First Affiliated Hospital, School of Medicine, Zhejiang University, from March 2021 to October 2022. A total of 118 cases were ultimately included. Of these patients, 61 cases were enrolled in the NTMPD group, 23 cases were enrolled in the suspected-NTMPD group, and 34 cases were enrolled in the non-NTMPD group. The diagnostic performance of traditional culture, acid-fast staining (AFS), and mNGS for NTMPD was assessed. Results: Patients in the NTMPD group had a higher proportion of bronchiectasis (P=0.007). Among mNGS-positive samples in the NTMPD group, a significantly higher reads number of NTM was observed in AFS-positive patients [61.50 (22.00, 395.00) vs 15.50 (6.00, 36.25), P=0.008]. Meanwhile, mNGS demonstrated a sensitivity of 90.2%, which was far superior to AFS (42.0%) and culture (77.0%) (P<0.001). The specificity of mNGS in detecting NTM was 100%, which was the same as that of traditional culture. The area under the receiver operating characteristic curve of mNGS was 0.951 (95% CI 0.906-0.996), which was higher than that of culture (0.885 [95% CI 0.818-0.953]) and AFS (0.686 [95% CI 0.562-0.810]). In addition to NTM, other pulmonary pathogens were also found by mNGS. Conclusion: mNGS using BALF samples is a rapid and effective diagnostic tool for NTMPD, and mNGS is recommended for patients with suspected NMTPD or NTM coinfected pneumonia.

Dynamic associations between the respiratory tract and gut antibiotic resistome of patients with COVID-19 and its prediction power for disease severity.

The antibiotic resistome is the collection of all antibiotic resistance genes (ARGs) present in an individual. Whether an individual's susceptibility to infection and the eventual severity of coronavirus disease 2019 (COVID-19) is influenced by their respiratory tract antibiotic resistome is unknown. Additionally, whether a relationship exists between the respiratory tract and gut ARGs composition has not been fully explored. We recruited 66 patients with COVID-19 at three disease stages (admission, progression, and recovery) and conducted a metagenome sequencing analysis of 143 sputum and 97 fecal samples obtained from them. Respiratory tract, gut metagenomes, and peripheral blood mononuclear cell (PBMC) transcriptomes are analyzed to compare the gut and respiratory tract ARGs of intensive care unit (ICU) and non-ICU (nICU) patients and determine relationships between ARGs and immune response. Among the respiratory tract ARGs, we found that Aminoglycoside, Multidrug, and Vancomycin are increased in ICU patients compared with nICU patients. In the gut, we found that Multidrug, Vancomycin, and Fosmidomycin were increased in ICU patients. We discovered that the relative abundances of Multidrug were significantly correlated with clinical indices, and there was a significantly positive correlation between ARGs and microbiota in the respiratory tract and gut. We found that immune-related pathways in PBMC were enhanced, and they were correlated with Multidrug, Vancomycin, and Tetracycline ARGs. Based on the ARG types, we built a respiratory tract-gut ARG combined random-forest classifier to distinguish ICU COVID-19 patients from nICU patients with an AUC of 0.969. Cumulatively, our findings provide some of the first insights into the dynamic alterations of respiratory tract and gut antibiotic resistome in the progression of COVID-19 and disease severity. They also provide a better understanding of how this disease affects different cohorts of patients. As such, these findings should contribute to better diagnosis and treatment scenarios.