Comparison of metagenomic next-generation sequencing and conventional culture for the diagnostic performance in febrile patients with suspected infections.

BACKGROUND: Timely and accurate identification of pathogens is crucial for appropriate treatment and prognosis of infectious diseases. As an increasingly popular pathogen detection method, the performance of metagenomic next-generation sequencing (mNGS) in detecting pathogens in febrile patients with suspected infection requires further exploration. METHODS: This study included 368 febrile patients with suspected infections who were admitted to the Infectious Disease Department of Qilu Hospital, Shandong University between January 5, 2021 and April 14, 2023. Both mNGS testing and conventional culture were performed in all patients. Clinical data of enrolled patients were collected, and the diagnostic performances of mNGS and culture were compared. RESULTS: Of the 368 enrolled patients, 231 were finally diagnosed with infection and 137 were with diseases other than infection. The sensitivity (58.01% vs. 21.65%, p < 0.001) and negative predictive value (54.67% vs. 42.9%) of mNGS were superior to those of culture. In contrast, the culture exhibited higher specificity (99.27% vs. 85.40%, p < 0.001) and positive predictive value (98.84% vs. 87.01%) than mNGS. Among infected patients with positive mNGS results, 64 received adjusted antibiotic therapy including treatment transitions, antibiotic downgrading, and combination therapy. Among them, 9 had additional antifungal drugs and 21 patients had a treatment turning point based on the mNGS results and these patients recovered and discharged due to timely antibiotic adjustment. Both positive rates of puncture fluid mNGS and tissue mNGS were higher than those of culture in the patients who had prior antibiotic use, and this difference was statistically significant (p = 0.000). CONCLUSION: mNGS is more sensitive and accurate than traditional culture, making it ideal for identifying pathogens and screening infectious diseases, especially for those with uncultivated or difficult-to-cultivate species. Early diagnosis allows for prompt treatment with targeted antibiotics, and mNGS is recommended when samples are limited.

Identification of mixed anaerobic infections after inguinal hernia repair based on metagenomic next-generation sequencing: A case report.

Infection following inguinal hernia repair (IHR) is uncommon. Rational use of antibiotics can significantly improve the prognosis of patients. However, accurately identifying the pathogen involved is usually challenging. This case report describes a patient who developed intermittent fever after undergoing open preperitoneal tension-free repair of a bilateral inguinal hernia. The scrotal fluid specimen was cultured and subjected to metagenomic next-generation sequencing (mNGS). Culture revealed the presence of Enterococcus faecalis (a facultative anaerobe). However, mNGS detected E. faecalis along with multiple anaerobic bacteria including Bacteroides thetaiotaomicron, Parabacteroides distasonis, and Levyella massiliensis. The patient was finally diagnosed with a mixed infection of E. faecalis and multiple anaerobes, and his condition was effectively controlled after timely adjustment of the antibiotic regimen. Treating postoperative infections with multiple concurrent conditions can be challenging. mNGS is valuable for the accurate diagnosis and treatment of infections, as it not only can further verify the culture results, but also assist clinicians in ruling out pulmonary infection caused by hematogenous dissemination after IHR in patients.

Relationship between vaccine hesitancy and vaccination behaviors: Systematic review and meta-analysis of observational studies.

BACKGROUND: Vaccination is crucial for prevention of infectious diseases, and identification of the impact of vaccine hesitancy on vaccination programs is crucial for early intervention and formulation of policies to alleviate vaccine hesitancy. The aim of this systematic review was to explore the relationship between vaccine hesitancy and negative vaccination behavior globally. METHODS: We searched for observational studies in various databases. We conducted a meta-analysis using pooled odds ratios (OR) and 95 % confidence intervals (CI), performed meta regression and subgroup analysis to explore the role factors such as location and individual characteristics on the association between vaccine hesitancy and vaccination behavior. RESULTS: A total of 46 articles were included in systematic analysis and 34 articles were included in the meta-analysis. The systematic analysis comprised 162,601 samples, whereas the meta-analysis included 147,554 samples. The meta-analysis showed that a higher rate of vaccine hesitancy was associated with an increased likelihood of adverse vaccination behaviors (all adverse behaviors: OR = 1.50, 95 % CI, 1.33-1.70, P < 0.001; unvaccinated: OR = 1.48, 95 % CI, 1.29-1.70, P < 0.001; vaccine delay: OR = 2.61, 95 % CI, 1.97-3.44, P < 0.001). The meta-regression results indicated that the heterogeneity observed was mainly from sample selection methods, age of vaccinees and the health status of participants. The results showed that parents of minor vaccinees or without high-risk health status had a higher association between vaccine hesitancy and vaccine uptake compared with populations exposed to higher health risks or adult vaccinees. CONCLUSION: The findings provide evidence on the association between vaccine hesitancy and adverse vaccination behaviors. The results showed that these population-specific factors should be considered in future research, and during formulation of interventions and implementation of policies to improve vaccination uptake.

Metagenomic-based pathogen surveillance for children with severe pneumonia in pediatric intensive care unit.

Background: Pneumonia is a significant cause of morbidity and mortality in children. Metagenomic next-generation sequencing (mNGS) has the potential to assess the landscape of pathogens responsible for severe pulmonary infection. Methods: Bronchoalveolar lavage fluid (BALF) samples of 262 children with suspected pulmonary infections were collected from April 2019 to October 2021 in the Pediatric Intensive Care Unit (PICU) of Guangdong Women and Children Hospital. Both mNGS and conventional tests were utilized for pathogen detection. Results: A total of 80 underlying pathogens were identified using both mNGS and conventional tests. Respiratory syncytial virus (RSV), Staphylococcus aureus and rhinovirus were the most frequently detected pathogens in this cohort. The incidence rate of co-infection was high (58.96%, 148/251), with bacterial-viral agents most co-detected. RSV was the main pathogen in children younger than 6 months of age, and was also commonly found in older pediatric patients. Rhinovirus was prevalent in children older than 6 months. Adenovirus and Mycoplasma pneumoniae were more prevalent in children older than 3 years than in other age groups. Pneumocystis jirovecii was detected in nearly 15% of children younger than 6 months. Besides, influenza virus and adenovirus were rarely found in 2020 and 2021. Conclusions: Our study highlights the importance of using advanced diagnostic techniques like mNGS to improve our understanding of the microbial epidemiology of severe pneumonia in pediatric patients.

Clinical features of varicella-zoster virus caused neurological diseases detected by metagenomic next-generation sequencing.

Clinical presentation of central nervous system (CNS) infections caused by varicella-zoster virus (VZV) is highly sophisticated, making identification challenging. We retrospectively reported 18 cases of VZV neurologic disease confirmed by metagenomic next-generation sequencing (mNGS). The detection rate of mNGS was higher than that of PCR assay (100 vs 66.7%, p < 0.05) and serum IgM antibody (100 vs 68.8%, p < 0.05) measurement. Of the 18 cases, five patients were diagnosed with acute meningitis, three with acute meningitis combined with facial neuritis, three with acute meningitis combined with polycranial neuritis, and the remaining seven with various clinical diagnoses. Typical clinical symptoms included headache (15), fever (9), and rash (11). Cranial or spinal MRI showed abnormalities in 12 patients, and 17 patients had obvious neurological symptoms. The predominant genotype of VZV in this study was genotype J (100%, 10/10). All patients were treated with acyclovir/penciclovir and dexamethasone, 16 recovered and 2 died. Our study highlights the good performance of mNGS in diagnosing CNS infection caused by VZV. It could provide additional diagnostic evidence in patients with diverse clinical spectrum and variable manifestations.

Liver abscess complicated with multiple organ invasive infection caused by hematogenous disseminated hypervirulent Klebsiella pneumoniae: A case report.

Hypervirulent Klebsiella pneumoniae (hvKp) causes increasing infections in healthy individuals from the community. In severe cases, it can cause multiple organ infection with invasive metastasis of blood sources, seriously threatening the patients' life. Rapid and accurate diagnosis of the pathogen becomes the key to timely antibiotic treatment to improve the prognosis. This article reports a case of liver abscess complicated with multiple organ invasive infection caused by hematogenous-disseminated hvKp. K. pneumoniae was identified by culture and metagenomic next-generation sequencing (mNGS) using blood and liver abscess drainage fluid. The isolates from the two samples were subsequently identified with high homology (99.999%) by whole genome sequencing. In addition, multiple virulence genes were detected in the two isolates and the string test was positive, indicating hvKp with hypermucoviscosity phenotype. Multiple antibiotic treatments were given. The conditions of the patient were stable but the temperature remained high. Surgical drainage treatment was performed, and the patient's body temperature immediately dropped to normal. He finally recovered after 6 months of follow-up. mNGS using body fluids can facilitate the rapid diagnosis of pathogens. For hvKp infection, choosing a better antibiotic therapy and receiving surgical drainage can significantly improve the prognosis of the patient.

Disseminated Talaromyces marneffei infection after renal transplantation: A case report and literature review.

We reported a 31-year-old man who received renal transplantation for more than 2 years. He was admitted to our hospital on 9 March 2022 due to intermittent diarrhea accompanied by leukopenia for more than 1 month. The patient successively developed high fever, cough, anemia, weight loss, gastrointestinal bleeding, and liver function impairment. Computed tomography (CT) revealed a slight inflammation in the lower lobes of both lungs, enlargement of the lymph nodes in the retroperitoneal and the root of mesenteric areas, and hepatosplenomegaly. Talaromyces marneffei was detected by metagenomics next-generation sequencing (mNGS) in blood and bronchoalveolar lavage fluid, and the pathogen was subsequently verified by blood culture. After endoscopic hemostatic therapy and antifungal therapy with voriconazole and amphotericin B cholesteryl sulfate complex, the patient was successfully discharged. Oral voriconazole was given regularly after discharge. Diarrhea, fever, enlargement of the lymph nodes, and endoscopic evidence of erosion may indicate intestinal T. marneffei infection. Although the mortality of T. marneffei infection after renal transplantation is very high, timely and effective antifungal therapy with amphotericin B cholesteryl sulfate complex is still expected to improve its prognosis.

Diagnosis of Neurological Infections in Pediatric Patients from Cell-Free DNA Specimens by Using Metagenomic Next-Generation Sequencing.

Central nervous system (CNS) infections can cause significant morbidity and mortality, especially in children. Rapid and accurate pathogenic detection in suspected CNS infections is essential for disease control at the early stage of infection. To evaluate the performance of metagenomic next-generation sequencing (mNGS) of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) in pediatric patients, we retrospectively analyzed the efficiency of cfDNA mNGS in children with CNS infections (n = 257) or noninfectious neurological disorders (n = 81). The CSF samples of 124 random subjects were used to evaluate the accuracy between mNGS of cfDNA and whole-cell DNA (wcDNA). In total, cfDNA mNGS detected a wide range of microbes with a detection rate of 71.0%, and the sensitivity and total coincidence rate with clinical diagnosis reached 68.9% and 67.5%, respectively. Compared with wcDNA mNGS, cfDNA mNGS had a higher efficacy in detecting viruses (66 versus 13) and Mycobacterium (7 versus 1), with significantly higher reads per million. The dominant causative pathogens were bacteria and viruses in CNS infections, but these presented with different pathogen spectra in different age categories. The best timing for the mNGS test ranged from 1 to 6 days after the start of anti-infection therapy, and the earlier mNGS started, the better was identification of bacterial CNS infections. This study emphasized that cfDNA mNGS had overall superiority to conventional methods on causative pathogen detection in pediatric CNS infections, and it was even better than wcDNA mNGS. Furthermore, research needs to be better validated in large-scale clinical trials to improve the clinical applications of cfDNA mNGS. IMPORTANCE Our study emphasized that cfDNA mNGS had overall superiority to conventional methods on causative pathogen detection in CNS-infected children, and it was even better than wcDNA mNGS. cfDNA mNGS detected a wide range of pathogens with a high total coincidence rate (67.5%) against clinical diagnosis. The best timing for cfDNA mNGS detection ranged from 1 to 6 days, rather than 0 days, after the start of empirical anti-infection therapy. The earlier mNGS started, the better the identifications of bacterial CNS infections. To the best of our knowledge, this research is the first report evaluating the clinical utility of mNGS with different methods (cfDNA versus wcDNA) of extracting DNA from CSF specimens in diagnosing pediatric CNS infections. Meanwhile, this is the largest cohort study that has evaluated the performance of mNGS using cfDNA from CSF specimens in pediatric patients with CNS infections.

A New Allele of the SPIKE1 Locus Reveals Distinct Regulation of Trichome and Pavement Cell Development and Plant Growth.

The single-celled trichomes of Arabidopsis thaliana have long served as an elegant model for elucidating the mechanisms of cell differentiation and morphogenesis due to their unique growth patterns. To identify new components in the genetic network that governs trichome development, we carried out exhaustive screens for additional Arabidopsis mutants with altered trichome morphology. Here, we report one mutant, aberrantly branched trichome1-1 (abt1-1), with a reduced trichome branching phenotype. After positional cloning, a point mutation in the SPIKE1 (SPK1) gene was identified in abt1-1. Further genetic complementation experiments confirmed that abt1-1 is a new allele of SPK1, so abt1-1 was renamed as spk1-7 according to the literatures. spk1-7 and two other spk1 mutant alleles, covering a spectrum of phenotypic severity, highlighted the distinct responses of developmental programs to different SPK1 mutations. Although null spk1 mutants are lethal and show defects in plant stature, trichome and epidermal pavement cell development, only trichome branching is affected in spk1-7. Surprisingly, we found that SPK1 is involved in the positioning of nuclei in the trichome cells. Lastly, through double mutant analysis, we found the coordinated regulation of trichome branching between SPK1 and two other trichome branching regulators, ANGUSTIFOLIA (AN) and ZWICHEL (ZWI). SPK1 might serve for the precise positioning of trichome nuclei, while AN and ZWI contribute to the formation of branch points through governing the cMTs dynamics. In summary, this study presented a fully viable new mutant allele of SPK1 and shed new light on the regulation of trichome branching and other developmental processes by SPK1.

Isolation and identification of two new alleles of STICHEL in Arabidopsis.

Plant trichome has long been selected as a model system to study the regulatory mechanism during cell development due to its unique growth characteristics. In an effort to explore new components that contribute to trichome development, we performed large-scale forward genetic screening in Arabidopsis, and found two recessive mutants with significant trichome branching defects, both of which display one branched trichomes. After map-based cloning, genetic complementation experiments, we confirmed that they are new alleles of STICHEL (STI) that has been reported to act as a key regulator of trichome branching. The predicted product of STI gene is comprised by three functional domains including a large domain with sequence similarity to eubacterial DNA polymerase III γ-subunit in the middle, two PEST domains in the N terminal, and two nuclear localization signals (NLS) at the very N terminal and C terminal, respectively. Our biochemistry and molecular analysis indicated that a PEST domain in the N-terminal could be important for STI functioning in regulating trichome branching. It can directly interact with BRACHLESS TRICHOME (BLT), an important linker of cell shape and endoreplication. Because the previously data showed that the function of STI in regulating trichome branching was not linked to DNA replication and actin or microtubule cytoskeleton configuration, our findings of the direct interaction domains between STI and BLT veiled the probable roles of STI in cell morphogenesis.