Emergence and Rapid Diagnosis of Talaromyces marneffei Infections in Renal Transplant Recipients by Next-Generation Sequencing.

In the last few years, next-generation sequencing (NGS) has emerged as a technology for laboratory diagnosis of many culture-negative infections and slow-growing microorganisms. In this study, we describe the use of metagenomic NGS (mNGS) for rapid diagnosis of T. marneffei infection in a 37-year-old renal transplant recipient who presented with chronic pneumonia syndrome. Bronchoalveolar lavage for mNGS was positive for T. marneffei sequence reads. Prolonged incubation of the bronchoalveolar lavage revealed T. marneffei colonies after 6 days of incubation. Analysis of 23 cases of T. marneffei infections in renal transplant recipients from the literature revealed that the number of cases ranged from 1 to 4 cases per five years from 1990 to 2020; but increased rapidly to 9 cases from 2021 to 2023, with 7 of them diagnosed by NGS. Twenty of the 23 cases were from T. marneffei-endemic areas [southern part of mainland China (n = 9); Hong Kong (n = 4); northeastern India (n = 2); Indonesia (n = 1) and Taiwan (n = 4)]. For the 3 patients from non-T. marneffei-endemic areas [United Kingdom (n = 2) and Australia (n = 1)], they had travel histories to China and Vietnam respectively. The time taken for diagnosis by mNGS [median 1 (range 1 to 2) day] was significantly shorter than that for fungal culture [median 6 (range 3 to 15) days] (P = 0.002). mNGS is useful for picking up more cases of T. marneffei infections in renal transplant recipients as well as providing a rapid diagnosis. Talaromycosis is an emerging fungal infection in renal transplant recipients.

Rapid Diagnosis of Pneumocystis jirovecii Pneumonia and Respiratory Tract Colonization by Next-Generation Sequencing.

OBJECTIVES: 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. METHODS: We examined the NGS results for P. jirovecii in respiratory samples collected from patients and analysed their clinical, radiological and microbiological characteristics. RESULTS: 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-ß-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. CONCLUSION: 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.

Multifunctional Proximity Labeling Strategy for Lipid Raft-Specific Sialic Acid Tracking and Engineering.

Lipid raft-specific glycosylation has been implicated in many biological processes, including intracellular trafficking, cell adhesion, signal transduction, and host-pathogen interactions. The major predicament in lipid raft-specific glycosylation research is the unavailability of tools for tracking and manipulating glycans on lipid rafts at the microstructural level. To overcome this challenge, we developed a multifunctional proximity labeling (MPL) platform that relies on cholera toxin B subunit to localize horseradish peroxidase on lipid rafts. In addition to the prevailing electron-rich amino acids, modified sialic acid was included in the horseradish peroxidase-mediated proximity labeling substrate via purposefully designed chemical transformation reactions. In combination with sialic acid editing, the self-renewal of lipid raft-specific sialic acid was visualized. The MPL method enabled tracking of lipid raft dynamics under methyl-ß-cyclodextrin and mevinolin treatments; in particular, the alteration of lipid rafts markedly affected cell migration. Furthermore, we embedded functional molecules into the method and implemented raft-specific sialic acid gradient engineering. Our novel strategy presents opportunities for tailoring lipid raft-specific sialic acids, thereby regulating interactions associated with lipid raft regions (such as cell-virus and cell-microenvironment interactions), and can aid in the development of lipid raft-based therapeutic regimens for tumors.

First-line crizotinib therapy is effective for a novel SEC31A-anaplastic lymphoma kinase fusion in a patient with stage IV lung adenocarcinoma: a case report and literature reviews.

Anaplastic lymphoma kinase (ALK) fusion was found in 3-7% of all patients with nonsmall cell lung cancer. The efficacy of ALK-tyrosine kinase inhibitor (ALK-TKI) in EML4-ALK has been extensively studied, whereas little evidence is available on its efficacy in rare ALK fusions. Here, we report the performance of crizotinib in a 50-year-old male lung adenocarcinoma patient with a novel rare SEC31A-ALK fusion. Computed tomography (CT) scan revealed multiple patchy high-density shadows in both lungs. The larger ones are located near the spine in the right lung lower lobe (55 × 34 mm) and the left hilar region (45 × 26 mm), with multiple enlarged mediastinal and axillary lymph nodes. Biopsy by bronchoscopy revealed invasive adenocarcinoma. The pathological stage of T4N3M1b (clinical stage: IVA) was confirmed. Next-generation sequencing revealed SEC31A: exon20~ALK: exon20 fusion, ABCB1 amplification, FGF19 amplification, DAXX p.S213L, MUTYH p.R19*(germline mutation and pathogenic) with tumor mutational burden at 3.2 mutations/Mb, microsatellite stable, proficient mismatch repair and PD-L1 positive [immunohistochemistry, tumor proportion score(TPS) 1-49% (TPS = 25%)]. Based on these findings, crizotinib was recommended for the first-line treatment at 250 mg twice daily. The first CT assessment after 2-month therapy showed partial response (PR) for the two larger lesions, multiple shadows and nodules in both lungs and the mediastinal and axillary lymph nodes. Crizotinib at 250 mg twice a day was applied in the following 9 months. Assessment at every 3 months (up to 1-year after diagnosis) showed further absorption for all lesions (continuous PR). We reported a novel rare ALK fusion SEC31A: EXON20~ALK: exon20 and showed the effectiveness of crizotinib against the fusion. This study provided strong evidence for the efficacy of ALK-TKI for rare ALK fusion.

Cutaneous Mycobacterium szulgai infection in a patient with Cushing's syndrome: a case report and literature review.

BACKGROUND: Opportunistic infection is an under-recognized complication of Cushing's syndrome, with infection due to atypical mycobacterium rarely reported. Mycobacterium szulgai commonly presents as pulmonary infection, with cutaneous infection seldom reported in the literature. CASE PRESENTATION: 48-year-old man with a newly-diagnosed Cushing's syndrome secondary to adrenal adenoma presented with a subcutaneous mass on the dorsum of his right hand, was diagnosed with cutaneous Mycobacterium szulgai infection. The most likely source of the infection was through minor unnoticed trauma and inoculation from a foreign body. The patient's Cushing's syndrome, high serum cortisol levels and secondary immune suppression facilitated mycobacterial replication and infection. The patient was successfully treated with adrenalectomy, surgical debridement of cutaneous lesion, and a combination of rifampicin, levofloxacin, clarithromycin, and ethambutol for 6 months. There were no signs of relapse one year after cessation of anti-mycobacterial treatment. A literature review on cutaneous M. szulgai infection to further characterize the clinical characteristics of this condition, identified 17 cases of cutaneous M. szulgai infection in the English literature. Cutaneous M. szulgai infections with subsequent disease dissemination are commonly reported in immunocompromised hosts (10/17, 58.8%), as well as in immunocompetent patients with a history of breached skin integrity, such as invasive medical procedures or trauma. The right upper extremity is the most commonly involved site. Cutaneous M. szulgai infection is well controlled with a combination of anti-mycobacterial therapy and surgical debridement. Disseminated infections required a longer duration of therapy than localized cutaneous infections. Surgical debridement may shorten the duration of antibiotics. CONCLUSIONS: Cutaneous M. szulgai infection is a rare complication of adrenal Cushing's syndrome. Further studies are needed to provide evidence-based guidelines on the best combination of anti-mycobacterial and surgical therapy for managing this rare infective complication.

Usefulness of next-generation sequencing for laboratory diagnosis of rickettsiosis.

Rickettsiosis includes a diversity of culture-negative non-specific systemic infections. Laboratory diagnosis of rickettsiosis is often not easy. In this 12-month study, six patients with a variety of rickettsia infections of the spotted fever group, typhus group and scrub typhus were diagnosed directly or indirectly by metagenomic next-generation sequencing (mNGS). The patient with Japanese spotted fever was rapidly made when mNGS analysis of the patient's blood revealed Rickettsia japonica sequences. For the two patients with Rickettsia felis chest infections, the bacterium was detected in the bronchoalveolar lavage of one case and lung biopsy of the other. Both patients had underlying malignancies, carcinoma of the breast and carcinoma of the lung respectively, and were on chemotherapy with immunosuppressive effect. For the remaining three patients who presented over a period of 13 weeks, all had fever, headache and the typical eschar. They also had increased serum transaminases and responded promptly to doxycycline. However, the Weil-Felix test results of all three patients were negative. Since we considered the three cases typical of rickettsiosis, we submitted their serum samples for mNGS analysis. Results showed that Orientia tsutsugamushi sequences were present in the serum of one case. In view of the positive mNGS results, we repeated the Weil-Felix test for the residual sera of all three patients and it revealed that those of the other two cases showed OX-19 titers of 1:640 and 1:160 respectively, inferring that these two patients probably had rickettsiosis of the typhus group. As for the patient positive for O. tsutsugamushi sequences, we also detected IgM for O. tsutsugamushi in the serum, which double confirmed that it was a case of scrub typhus. mNGS is an important molecular tool and can complement serology for laboratory diagnosis of rickettsiosis.

Usenamine A triggers NLRP3/caspase-1/GSDMD-mediated pyroptosis in lung adenocarcinoma by targeting the DDX3X/SQSTM1 axis.

BACKGROUND: Usenamine A (C18H17NO6) is a newly developed, natural anticancer drug that reportedly exerts low toxicity. The therapeutic efficacy and underlying mechanisms of usenamine A in lung adenocarcinoma (LUAD) remain poorly understood. We aimed to explore the therapeutic effects and molecular mechanisms through which usenamine A inhibits LUAD tumorigenesis. METHODS: We used LUAD cell lines H1299 and A549 in the present study. CCK-8 and colony formation assays were performed to analyze cell proliferation. Cell migration, invasion, and apoptosis were evaluated using wound-healing, transwell, and flow cytometric assays, respectively. Levels of reactive oxygen species were measured using a DCFH-DA probe. Inflammatory factors (lactate dehydrogenase, interleukin [IL]-1ß, and IL-18) were detected using enzyme-linked immunosorbent assays. Western blotting was performed to determine the expression of NOD-like receptor pyrin 3 (NLRP3)/caspase-1/gasdermin D (GSDMD) pathway-related proteins. Pyroptosis was detected using transmission electron microscopy. The interaction and co-localization of DDX3X and sequestosome 1 (SQSTM1) were identified using co-immunoprecipitation and immunofluorescence assays, respectively. For in vivo assessment, we established a xenograft model to validate the usenamine A-mediated effects and mechanisms of action in LUAD. RESULTS: Usenamine A inhibited the proliferation, migration, and invasion of LUAD cells. Furthermore, usenamine A induced NLRP3/caspase-1/GSDMD-mediated pyroptosis in LUAD cells. Usenamine A upregulated DDX3X expression to trigger pyroptosis. DDX3X interacted with SQSTM1, which is responsible for inducing pyroptosis. In vivo, usenamine A suppressed LUAD tumorigenesis by triggering NLRP3/caspase-1/GSDMD-mediated pyroptosis via the upregulation of the DDX3X/SQSTM1 axis. CONCLUSIONS: Usenamine A was found to induce NLRP3/caspase-1/GSDMD-mediated pyroptosis in LUAD by upregulating the DDX3X/SQSTM1 axis.

Post-COVID-19 Pandemic Rebound of Macrolide-Resistant Mycoplasma pneumoniae Infection: A Descriptive Study.

The rebound characteristics of respiratory infections after lifting pandemic control measures were uncertain. From January to November 2023, patients presenting at a teaching hospital were tested for common respiratory viruses and Mycoplasma pneumoniae using a combination of antigen, nucleic acid amplification, and targeted next-generation sequencing (tNGS) tests. The number and rate of positive tests per month, clinical and microbiological characteristics were analyzed. A rapid rebound of SARS-CoV-2 was followed by a slower rebound of M. pneumoniae, with an interval of 5 months between their peaks. The hospitalization rate was higher, with infections caused by respiratory viruses compared to M. pneumoniae. Though the pediatric hospitalization rate of respiratory viruses (66.1%) was higher than that of M. pneumoniae (34.0%), the 4094 cases of M. pneumoniae within 6 months posed a huge burden on healthcare services. Multivariate analysis revealed that M. pneumoniae-infected adults had more fatigue, comorbidities, and higher serum C-reactive protein, whereas children had a higher incidence of other respiratory pathogens detected by tNGS or pathogen-specific PCR, fever, and were more likely to be female. A total of 85% of M. pneumoniae-positive specimens had mutations detected at the 23rRNA gene, with 99.7% showing A2063G mutation. Days to defervescence were longer in those not treated by effective antibiotics and those requiring a change in antibiotic treatment. A delayed but significant rebound of M. pneumoniae was observed after the complete relaxation of pandemic control measures. No unusual, unexplained, or unresponsive cases of respiratory infections which warrant further investigation were identified.

Case report: Occult Listeria monocytogenes invasion leading to prosthetic hip joint infection in a patient with rheumatoid arthritis taking tofacitinib.

It has been suggested that targeted therapy may potentially increase the risk of listeriosis. However, no reported cases of Listeria monocytogenes prosthetic joint infection have been documented during Janus Kinase (JAK) pathway inhibitor use. Herein, we present a 70-year-old female with rheumatoid arthritis who had undergone bilateral hip joint replacement and subsequently developed Listeria monocytogenes prosthetic joint infection following tofacitinib therapy. We suggest that the use of tofacitinib may potentially heighten susceptibility to listeriosis in patients afflicted with rheumatoid arthritis.

Nucleophosmin promotes lung adenocarcinoma cell proliferation, migration and invasion by activating the EGFR/MAPK signaling pathway.

Lung adenocarcinoma (LUAD) is the main cause of death globally. The present study investigated the prognostic value and functional verification of nucleophosmin (NPM1) in LUAD. LUAD and normal samples from The Cancer Genome Atlas were analyzed to identify whether NPM1 is associated with LUAD prognosis. NPM1 protein expression level was verified by western blotting. Cell proliferation, migration and invasion were detected by Cell Counting Kit­8, wound healing and Transwell assays, respectively. EGFR/MAPK pathway­related proteins [phosphorylated (p)­EGFR/EGFR, p­MEK/MEK, and p­ERK/ERK] expression was measured through western blotting. A xenograft tumor mice model was constructed to perform the in vivo verification. NPM1 was upregulated in LUAD cells, and high­level NPM1 indicated poor prognosis in patients with LUAD. In vitro experiments revealed that NPM1 knockdown inhibited LUAD cell proliferation, migration and invasion. Moreover, protein expression of p­EGFR/EGFR, p­MEK/MEK and p­ERK/ERK was reduced with the NPM1 silencing. Furthermore, EGF, an activator of the EGFR/MAPK pathway, reversed the effects of NPM1. In vivo experiments showed that NPM1 knockdown inhibited tumor growth and protein levels of p­EGFR/EGFR, p­MEK/MEK and p­ERK/ERK. NPM1 is related to the poor prognosis of LUAD and promotes the malignant progression of LUAD by activating the EGFR/MAPK pathway. This discovery provides a new potential therapeutic target for the diagnosis and treatment of LUAD.

Diverse and atypical manifestations of Q fever in a metropolitan city hospital: Emerging role of next-generation sequencing for laboratory diagnosis of Coxiella burnetii.

Although Q fever has been widely reported in the rural areas of China, there is a paucity of data on the epidemiology and clinical characteristics of this disease in large metropolitan cities. In this study, we profile the epidemiology and clinical manifestations of Q fever from a tertiary hospital in Shenzhen, a Southern Chinese metropolitan city with a large immigrant population from other parts of China. A total of 14 patients were confirmed to have Q fever during a nine-year-and-six-month period, five of whom were retrospectively diagnosed during case review or incidentally picked up because of another research project on unexplained fever without localizing features. Some patients had the typical exposure histories and clinical features, while a few other patients had rare manifestations of Q fever, including one with heart failure and diffuse intracapillary proliferative glomerulonephritis, a patient presenting with a spontaneous bacterial peritonitis-like syndrome, and another one with concomitant Q fever and brucellosis. Using a combination of clinical manifestation, inflammatory marker levels, echocardiographic findings and serological or molecular test results, nine, three and two patients were diagnosed to have acute, chronic and convalescent Q fever, respectively. Seven, five and two patients were diagnosed to have Q fever by serological test, nested real-time PCR and next-generation sequencing respectively. Diverse and atypical manifestations are associated with Q fever. The incidence of Q fever is likely to be underestimated. Next-generation sequencing is becoming an important diagnostic modality for culture-negative infections, particularly those that the physicians fail to recognize clinically, such as Q fever.

Kikuchi-Fujimoto disease complicated by aseptic meningitis and hemophagocytosis successfully treated with intrathecal dexamethasone.

Kikuchi-Fujimoto disease (KFD) is thought to be a self-limited disease featuring fever and cervical lymphadenopathy; most cases having a favorable outcome. Severe disease and death are occasionally reported. Here we report a case of KFD complicated by hemophagocytosis and aseptic meningitis. The symptoms and laboratory parameters improved after systemic glucocorticoids, intravenous immunoglobulin and one dose of intrathecal dexamethasone. Clinicians should aware of this disease and make early diagnosis by lymph node biopsy to avoid over-treatment.

Genome­wide identification of long noncoding RNAs in CCl4­induced liver fibrosis via RNA sequencing.

Liver fibrosis occurs as a result of chronic liver lesions, which may subsequently develop into liver cirrhosis and hepatocellular carcinoma. The involvement of long noncoding RNAs (lncRNAs) in liver fibrosis is being increasingly recognized. However, the exact mechanisms and functions of the majority of lncRNAs are poorly characterized. In the present study, the hepatotoxic substance carbon tetrachloride (CCl4) was employed to induce liver fibrosis in an animal model and agenome­wide identification of lncRNAs in fibrotic liver tissues compared with CCl4 untreated liver tissues was performed using RNA sequencing. Sprague­Dawley rats were treated with CCl4 for 8 weeks. Histopathogical alterations were observed in liver tissues, and serum levels of alanine aminotransferase, aspartate aminotransferase, transforming growth factor­ß1 and tumor necrosis factor­α were significantly higher, in the CCl4­treated group compared with the CCl4 untreated group. RNA sequencing of liver tissues demonstrated that 231 lncRNAs and 1,036 mRNAs were differentially expressed between the two groups. Furthermore, bioinformatics analysis demonstrated that the differentially expressed mRNAs were predominantly enriched in 'ECM­receptor interaction', 'PI3K­Akt signaling pathway' and 'focal adhesion' pathways, all of which are essential for liver fibrosis development. Validation of 12 significantly aberrant lncRNAs by reverse transcription­quantitative polymerase chain reaction indicated that the expression patterns of 11 lncRNAs were consistent with the sequencing data. Furthermore, overexpression of lncRNA NR_002155.1, which was markedly downregulated in CCl4­treated liver tissues, was demonstrated to inhibit HSC­T6 cell proliferation in vitro. In conclusion, the present study determined the expression patterns of mRNAs and lncRNAs in fibrotic liver tissue induced by CCl4. The identified differentially expressed lncRNAs may serve as novel diagnostic biomarkers and therapeutic targets for liver fibrosis.

Effect of Dahuang Zhechong pills on long non-coding RNA growth arrest specific 5 in rat models of hepatic fibrosis.

OBJECTIVE: To investigate the involvement of growth arrest specific 5 (GAS5), a long non-coding RNA, in the anti-hepatic fibrosis process induced by Dahuang Zhechong pill (DHZCP) in rats. METHODS: Thirty adult rats were divided into three groups, including a control group, a CCl4-induced fibrosis group and a DHZCP-treated fibrosis group. Hematoxylin-eosin and Masson staining were used for histopathological study. Serum enzymes, cytokines and cell proliferation were assayed using commercially available kits. A GAS5 lenti-virus vector was constructed to further investigate the role of GAS5 in the anti-hepatic fibrosis effect of DHZCP in rats. RESULTS: Our results revealed that the proliferation of hepatic stellate cells cultured in serum derived from rats treated with DHZCP was significantly decreased, compared with cells treated with serum from the untreated rats. DHZCP alleviated the CCl4-induced hepatic fibrosis. Additionally, DHZCP can restore the expression of GAS5, which was significantly decreased in the CCl4-induced group, and markedly suppress the expression of p-p38 and p-Erk induced by CCl4, but not p-Jnk. Cell proliferation was significantly arrested when cells overexpressed GAS5. Thus, DHZCP can inhibit the expression of p-p38 and p-Erk, while GAS5 can only inhibit the expression of p-Erk. CONCLUSIONS: DHZCP can alleviate hepatic fibrosis by increasing the expression of GAS5 to suppress p-Erk and regulating other factors to inhibit p-p38.