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Due to the heterogeneity of the human gut environment, the gut microbiota is complex and diverse, and has been insufficiently explored. In this study, one fresh fecal sample was cultured using 12 commercial or modified media and incubation of culture plates anaerobically and aerobically, the conventional experienced colony picking (ECP) was first used to isolate the colonies and obtain pure culture strains. On this basis, all the colonies grown on the culture plates were collected for culture-enriched metagenomic sequencing (CEMS), and the original sample was also subjected to direct culture-independent metagenomic sequencing (CIMS), the study compared the effects of three methods for analyzing the microbiota contained in the sample. It was found that compared with CEMS, conventional ECP failed to detect a large proportion of strains grown in culture media, resulting in missed detection of culturable microorganisms in the gut. Microbes identified by CEMS and CIMS showed a low degree of overlap (18% of species), whereas species identified by CEMS and CIMS alone accounted for 36.5% and 45.5%, respectively. It suggests that both culture-dependent and culture-independent approaches are essential in revealing gut microbial diversity. Moreover, based on the CEMS results, growth rate index (GRiD) values for various strains on different media were calculated to predict the optimal medium for bacterial growth; this method can be used to design new media for intestinal microbial isolation, promote the recovery of specific microbiota, and obtain new insights into the human microbiome diversity. This is among the first studies on CEMS of the human gut microbiota.

A case of Rickettsia sibirica subspecies sibirica BJ-90 infection in China was identified by metagenomic analysis of an eschar biopsy specimen and confirmed by nested PCR. Seroprevalence of spotted fever group Rickettsia was ≈17.4% among the local population. This report highlights the threat of rickettsioses to public health in the Qinghai-Tibet Plateau.

Background Acute pancreatitis (AP) is an inflammatory disorder that causes a considerable economic health burden. While the overall mortality is low, around 20% of patients have a complicated course of disease resulting in increased morbidity and mortality. There is an emerging body of evidence that the microbiome exerts a crucial impact on the pathophysiology and course of AP. For several decades multiple clinical and laboratory parameters have been evaluated, and complex scoring systems were developed to predict the clinical course of AP upon admission. However, the majority of scoring systems are determined after several days and achieve a sensitivity around 70% for early prediction of severe AP. Thus, continued efforts are required to investigate reliable biomarkers for the early prediction of severity in order to guide early clinical management of AP patients. Methods We designed a multi-center, prospective clinical-translational study to test whether the orointestinal microbiome may serve as novel early predictor of the course, severity and outcome of patients with AP. We will recruit 400 AP patients and obtain buccal and rectal swabs within 72 h of admission to the hospital. Following DNA extraction, microbiome analysis will be performed using 3rd generation sequencing Oxford Nanopore Technologies (ONT) for 16S rRNA and metagenomic sequencing. Alpha- and beta-diversity will be determined and correlated to the revised Atlanta classification and additional clinical outcome parameters such as the length of hospital stay, number and type of complications, number of interventions and 30-day mortality. Discussion If AP patients show a distinct orointestinal microbiome dependent on the severity and course of the disease, microbiome sequencing could rapidly be implemented in the early clinical management of AP patients in the future. Trial registration : ClinicalTrials.gov Identifier: NCT04777812

Purpose: Recent studies had shown that gut microbiota played a significant role in the development of the immune system and may affect the course of airway allergic disease. We conducted this study to determine unique gut microbial associated with allergic disease in children by shotgun gene sequencing. Methods: We collected fecal samples from children with allergic asthma (n = 23) and allergic rhinitis (n = 18), and healthy control (n = 19). The gut microbiota of specimens was analyzed by high-throughput metagenomic shotgun gene sequencing. Results: The intestinal microbiota of children with allergic asthma and allergic rhinitis was characterized by increased microbial richness and diversity. Simpson and Shannon were significantly elevated in children with allergic asthma. Principal coordinates analysis (PCoA) showed that the gut microbial communities cluster patterns of children with asthma or rhinitis were significantly different from those of healthy controls. However, no significant difference was found between asthma group and rhinitis group At the phylum level, higher relative abundance of Firmicutes was found in the allergic rhinitis group and allergic asthma group, while the level of Bacteroidetes was significantly lower. At the genus level, Corynebacterium, Streptococcus, Dorea, Actinomyces, Bifidobacterium, Blautia, and Rothia were significantly enriched in the allergic asthma group. Finally, a random forest classifier model selected 16 general signatures to discriminate the allergic asthma group from the healthy control group. Conclusion: In conclusion, children in the allergic rhinitis group and allergic asthma group had altered gut microbiomes in comparison with the healthy control group. Compared to healthy children, the gut microbiome in children with allergic diseases has higher pro-inflammatory potential and increased production of pro-inflammatory molecules. Keywords: childhood, allergic asthma, allergic rhinitis, gut microbiome, metagenomic sequencing

We performed nanopore-based metagenomic screening on 885 ticks collected from 6 locations in Mongolia and divided the results into 68 samples: 23 individual samples and 45 pools of 2-12 tick samples each. We detected bacterial and parasitic pathogens Anaplasma ovis, Babesia microti, Coxiella burnetii, Borrelia miyamotoi, Francisella tularensis subsp. holarctica and novicida, Spiroplasma ixodetis, Theileria equi, and Rickettsia spp., including R. raoultii, R. slovaca, and R. canadensis. We identified the viral pathogens Crimean-Congo hemorrhagic fever virus (2.9%), recently described Alongshan virus (ALSV) (2.9%), and Beiji nairovirus (5.8%). We assembled ALSV genomes, and maximum-likelihood analyses revealed clustering with viruses reported in humans and ticks from China. For ALSV, we identified surface glycoprotein markers associated with isolates from Asia viruses hosted by Ixodes persulcatus ticks. We also detected 20 virus species of unknown public health impact, including a near-complete Yanggou tick virus genome. Our findings demonstrate that nanopore sequencing can aid in detecting endemic and emerging tickborne pathogens.

Abstract Background Fecal specimens are critical for disease screening, diagnosis, and gut microbiome research. For domestic cats, lubricants are often necessary to obtain a sufficient quantity of sample. However, the effect of lubrication on feline microbiome analysis has not been assessed. Objectives To evaluate if lubrication using mineral oil during cat feces sample collection affects the DNA extraction, metagenomic sequencing yield, and the microbial composition and diversity in subsequent gut microbiome analyses. Animals Eight 6-year-old male, neutered, domestic short-haired cats housed in a research facility. Methods Cohort study. The gut microbiomes were investigated for fecal sample collection with and without lubrication using whole-genome shotgun metagenomic sequencing. Results Fecal specimens were collected using a fecal loop under sedation without lubrication and with mineral oil lubrication. There were no significant differences between the 2 groups in the microbial DNA yield in ng/mg fecal sample (75.75 [25.8-125.7] vs 60.72 [33.49-87.95], P = .95), metagenomic sequencing yield in Gbp (10.31 [6.29-14.32] vs 13.53 [12.04-15.02], P = .2), proportion of host contamination (0.1 [0.02-0.18] vs 0.15 [0-0.3], P = .84), relative taxonomy abundance (P > .8), or the number of microbial genes covered (408 132 [341 556-474 708] vs 425 697 [358 505-492 889], P = .31). Conclusions and Clinical Importance Fecal sampling with mineral oil lubrication did not change the microbial DNA extraction yield, metagenomic sequencing yield, level of host contamination, the microbial composition and diversity in subsequent gut microbiome analyses. Here we reported a proven cat-friendly protocol for fecal sample collection in clinical and research setting for gut microbiome analyses.

A 56-year-old man receiving rituximab who had months of neurologic symptoms was found to have Jamestown Canyon virus in cerebrospinal fluid by clinical metagenomic sequencing. The patient died, and postmortem examination revealed extensive neuropathologic abnormalities. Deep sequencing enabled detailed characterization of viral genomes from the cerebrospinal fluid, cerebellum, and cerebral cortex.

The purpose of this study was to evaluate the ability of metagenomic next-generation sequencing (mNGS) diagnosing prosthetic joint infection (PJI) using tissue from hip/knee rapidly and precisely, especially in patients who had received antibiotic treatment within the preceding two weeks. From May 2020 to March 2022, 52 cases with suspected PJI were enrolled. mNGS was performed on surgical tissue samples. The sensitivity and specificity of mNGS in diagnosis was evaluated using culture in conjunction with MSIS criteria. This study also looked at how antibiotic use affected culture and mNGS efficacy. According to MSIS criteria, 31 of the 44 cases had PJI, and 13 were classified in the aseptic loosening group. Sensitivity, specificity, positive/negative predictive value (PPV/NPV), positive/negative likelihood ratio (PLR/NLR), and area under the curve (AUC) of mNGS assay were 80.6% (71.9-91.8%), 84.6 (73.7-97.9%), 92.6 (84.2-98.7%), 64.7 (58.6-74.7%), 5.241 (4.081-6.693), 0.229 (0.108-0.482) and 0.826 (0.786-0.967), respectively, with MSIS as a reference. When MSIS was used as a reference, the results of culture assay were 45.2% (40.8-51.5%), 100 (100.0-100.0%), 100 (100.0-100.0%), 43.3 (39.1-49.5%), +∞, 0.548 (0.396-0.617) and 0.726 (0.621-0.864), respectively. The AUC values for mNGS and culture were 0.826 and 0.731, respectively, and the differences were insignificant. mNGS demonstrated higher sensitivity than culture in PJI subjects who had previously received antibiotic treatment within 2 weeks (69.5% vs 23.1%, P = 0.03). In our series, mNGS yield a higher sensitivity for diagnosis and pathogen detection of PJI compared to microbiological culture. Additionally, mNGS is less affected by prior antibiotic exposure.

Disseminated infection caused by Streptococcus constellatus was seldom occurred. We reported a case of Streptococcus constellatus infection, presenting as multiple pulmonary cavities, thoracic wall abscess and vertebral destruction. The 37-year-old male had recurrent fever, chest wall swelling and pain, and lower limb numbness, he had weak physical condition and previously suffered from poorly controlled diabetes and severe periodontal disease for 3 years. Definite diagnosis of Streptococcus constellatus infection was made by metagenomic next-generation sequencing (mNGS) in abscess drainage fluid. Systemic antibiotics and thoracic wall drainage were given, and the pulmonary cavity and the thoracic intermuscular abscess were significantly decreased. Few to no study reported the disseminated infection (pulmonary cavities, thoracic wall abscess and vertebral destruction) caused by Streptococcus constellatus. This case report highlighted the importance of mNGS for accurate diagnosis, as well as the timely drainage and antibiotics for effective treatment of Streptococcus constellatus infection. Keywords: Streptococcus constellatus, pulmonary cavity, thoracic wall abscess, vertebral destruction, metagenomic next-generation sequencing

( ) is a pathogen that is seldom implicated in community-acquired pneumonia and is rarely linked to severe pneumonia. Reports of severe pneumonia accompanied by Guillain-Barre syndrome (GBS) are scarce. Tetracyclines are the preferred therapeutic approach for psittacosis. Omadacycline, a novel tetracycline, demonstrates strong antibacterial efficacy against typical bacteria and atypical pathogens, including . However, its application in the treatment of psittacosis pneumonia remains constrained. A 77-year-old female patient was admitted to the hospital presenting with symptoms of fever, low back pain, and headache. The diagnosis of was established through the utilization of metagenomic next-generation sequencing (mNGS). Initial administration of moxifloxacin, meropenem, piperacillin-tazobactam, and doxycycline proved to be ineffective. Subsequent omadacycline leaded to the successful resolution of fever and dyspnea. However, after the endotracheal tube was removed, the patient experienced a rapid decline in symmetrical limb strength, leading to a diagnosis of GBS based on clinical manifestations, cerebrospinal fluid analysis, and electromyography. Following a 5-day course of immunoglobulin therapy and nutritional nerve treatment, the patient's condition ameliorated, culminating in an uncomplicated discharge. This case provides evidence supporting the potential use of omadacycline as a therapeutic option for the treatment of severe pneumonia. The utilization of mNGS technology is of paramount importance in the prompt identification of uncommon pathogens, including . Nevertheless, the occurrence of GBS should be taken into consideration when pneumonia is accompanied by symmetrical limb weakness. These findings have important implications for the diagnosis, treatment, and management of patients with pneumonia.