ObjectiveTo assess the diagnostic efficacy of metagenomic next generation sequencing (mNGS) for proven invasive pulmonary aspergillosis (IPA).MethodsA total of 190 patients including 53 patients who had been diagnosed with proven IPA were retrospectively analyzed. Using the pathological results of tissue biopsy specimens as gold standard, we ploted the receiver operating characteristic (ROC) curve to determine the optimal cut-off value of mNGS species-specific read number (SSRN) of Aspergillus in bronchoalveolar lavage fluid (BALF)for IPA. Furthermore, we evaluated optimal cut-off value of mNGS SSRN in different populations.ResultsThe optimal cut-off value of Aspergillus mNGS SSRN in BALF for IPA diagnosis was 2.5 for the whole suspected IPA population, and 1 and 4.5 for immunocompromised and diabetic patients, respectively. The accuracy of mNGS was 80.5%, 73.7% and 85.3% for the whole population, immunocompromised and diabetic patients, respectively.ConclusionsThe mNGS in BALF has a high diagnostic efficacy for proven IPA, superioring to Aspergillus culture in sputum and BALF and GM test in blood and BALF. However, the cut-off value of SSRN should be adjusted when in different population.

IntroductionDental caries is one of the most common and costly biofilm-dependent oral diseases in the world. Streptococcus mutans is the major cariogenic pathogen of dental caries. S. mutans synthesizes extracellular polysaccharides by autologous glucosyltransferases, which then promotes bacterial adhesion and cariogenic biofilm formation. The S. mutans biofilm is the principal target for caries treatment. This study was designed to explore the antibacterial activity and mechanisms of areca nut essential oil (ANEO) against S. mutans.MethodsThe ANEOs were separated by negative pressure hydro-distillation. The Kirby-Bauer method and broth microdilution method were carried out to evaluate the antibacterial activity of different ANEOs. The antibacterial mechanism was revealed by crystal violet staining, XTT reduction, microbial adhesion to hydrocarbon test, extracellular polysaccharide production assay, glucosyltransferase activity assay, lactate dehydrogenase leaking, propidium iodide staining and scanning electron microscopy (SEM). The cytotoxicity of ANEOs was determine by MTT assay.ResultsThe ANEOs separated at different temperatures exhibited different levels of antibacterial activity against S. mutans, and the ANEO separated at 70°C showed the most prominent bacteriostatic activity. Anti-biofilm experiments showed that the ANEOs attenuated the adhesion ability of S. mutans by decreasing the surface hydrophobicity of the bacteria, prevented S. mutans biofilm formation by inhibiting glucosyltransferase activity, reducing extracellular polysaccharide synthesis, and reducing the total biofilm biomass and activity. SEM further demonstrated the destructive effects of the ANEOs on the S. mutans biofilm. Cell membrane-related experiments indicated that the ANEOs destroyed the integrity of the cell membrane, resulting in the leakage of lactic dehydrogenase and nucleic acids. SEM imaging of S. mutans cell showed the disruption of the cellular morphology by the ANEOs. The cytotoxicity assay suggested that ANEO was non-toxic towards normal oral epithelial cells. DiscussionThis study displayed that ANEOs exerted antibacterial activity against S. mutans primarily by affecting the biofilm and disrupting the integrity of the cell membrane. ANEOs has the potential to be developed as an antibacterial agent for preventing dental caries. Additionally, a new method for the separation of essential oil components is presented.

BackgroundThe incidence of nephrolithiasis is increasing rapidly worldwide. Calcium oxalate is the most common constituent, contributing to approximately 80% of all kidney stones. The gut microbiome, through its oxalate-degrading ability, may play a role in decreasing morbidity due to urinary calculus. Fecal microbiome transplantation (FMT) has been reported to be effective in restoring the gastrointestinal microbial community in different conditions. The transplantation of whole communities that have oxalate-degrading function may be a more effective strategy than the transplantation of isolated strains.MethodsFMT was carried out in male guinea pigs and male Sprague–Dawley laboratory rats (SDRs). Fresh feces were collected from guinea pigs housed in metabolic cages. SDRs were divided into four groups: two groups received standard rat chow (SC) (groups SC and SC + FMT), and two groups were fed a 5% potassium oxalate diet (OD) (groups OD + phosphate-buffered saline (PBS) and OD + FMT). On day 14, groups OD + PBS, OD + FMT, and SC + FMT received either PBS or guinea pig feces by esophageal gavage. The composition of the microbiota of guinea pigs and SDRs was analyzed using a 16S rRNA gene sequencing approach. Biochemical analysis of urine samples from SDRs revealed the presence of calcium oxalate (CaOx) crystals, which were presumed to originate from kidney stones. Renal function was examined using real-time PCR analysis and immunohistochemical staining for renin, angiotensin-converting enzyme, and osteopontin (OPN) expression.ResultsFMT resulted in a gut microbiota that was a mixture of guinea pig and SDR bacteria. A microbial network involving Muribaculaceae, Lactobacillus, and Bifidobacterium was activated by FMT in group OD + FMT. As a result, urinary oxalate, calcium, uric acid, creatinine and urea in urine samples were reduced significantly. Similarly, significant reduction of uric acid and blood urea nitrogen to creatinine ratio in serum samples was observed (p < 0.05). Microscopic observations revealed a high CaOx crystal score (4+) in the kidneys of rats in group OD + PBS, whereas a lower score (2+) was observed in the rats in group OD + FMT. Up-regulation of OPN and down-regulation of renin were also associated with FMT.ConclusionA microbial network involving Muribaculaceae and other oxalate-degrading bacteria achieved by FMT was capable of reducing urinary oxalate excretion and CaOx crystal deposition in the kidney through increasing intestinal oxalate degradation. FMT may exert a renoprotective function in oxalate-related kidney stones.

Worldwide, lower respiratory tract infections (LRTI) are an important cause of hospitalization in children. Due to the relative limitations of traditional pathogen detection methods, new detection methods are needed. The purpose of this study was to evaluate the value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) samples for diagnosing children with LRTI based on the interpretation of sequencing results. A total of 211 children with LRTI admitted to the First Affiliated Hospital of Guangzhou Medical University from May 2019 to December 2020 were enrolled. The diagnostic performance of mNGS versus traditional methods for detecting pathogens was compared. The positive rate for the BALF mNGS analysis reached 95.48% (95% confidence interval [CI] 92.39% to 98.57%), which was superior to the culture method (44.07%, 95% CI 36.68% to 51.45%). For the detection of specific pathogens, mNGS showed similar diagnostic performance to PCR and antigen detection, except for Streptococcus pneumoniae, for which mNGS performed better than antigen detection. S. pneumoniae, cytomegalovirus and Candida albicans were the most common bacterial, viral and fungal pathogens. Common infections in children with LRTI were bacterial, viral and mixed bacterial-viral infections. Immunocompromised children with LRTI were highly susceptible to mixed and fungal infections. The initial diagnosis was modified based on mNGS in 29.6% (37/125) of patients. Receiver operating characteristic (ROC) curve analysis was performed to predict the relationship between inflammation indicators and the type of pathogen infection. BALF mNGS improves the sensitivity of pathogen detection and provides guidance in clinical practice for diagnosing LRTI in children.

ObjectiveThe aim of this study is to report an isolated pleural cryptococcosis with pleural effusion as the only manifestation, confirmed by pleural biopsy in a patient with thymoma combined with myasthenia gravis, who developed pleural effusion of unknown origin after long-term glucocorticoids and tacrolimus therapy.MethodsPathological examination of the right pleural biopsy tissue from a patient with unexplained recurrent pleural effusion was implemented. Morphological analysis of the fungal component and metagenomic next-generation sequencing (mNGS) on the pleural tissue were performed.ResultsA biopsy specimen of the right pleura revealed numerous yeast-like organisms surrounded by mucous capsules and Cryptococcus neoformans was detected by mNGS with a species-specific read number (SSRN) of 4, confirming the diagnosis of pleural cryptococcosis. Pleural effusion was eliminated with amphotericin B and fluconazole, and healthy status was maintained at the time of review 1 year later.ConclusionCryptococcosis, manifested by simple pleural effusion, is extremely rare, but when repeated pleural effusion occurs in immunocompromised patients or in patients with malignant tumors, the possibility of cryptococcosis should be treated with high vigilance and pleural biopsy is recommended if necessary in order to confirm the diagnosis.